Biodegradation and Transport of Crude Oil 

in Sand and Gravel Beaches of Arctic Alaska 

Principal Investigator 

Dr. Silke Schiewer 

Graduate Students 

Anna Iverson 

Priyam Sharma 

Department of Civil & Environmental Engineering 

University of Alaska Fairbanks 

FINAL REPORT 

July 2015 

OCS Study BOEM 2015-041 

BOEM ll~f. 

U H IVCRS IJV or 

ALASKA 

F A IR &AH KS 
Contact Information: 

email: CMl@alaska.edu 

phone: 907.474.6782 

fax: 907.474.7204 

Coastal Marine Institute 

School of Fisheries and Ocean Sciences 

University of Alaska Fairbanks 

P. 0. Box 757220 

Fairbanks. AK 99775-7220 

This study was funded in part by the U.S. Dcpartment or the Interior, Bureau of Ocean Energy Management (BOEM) through 

Cooperative Agreement M 14AC00020 between BOEM. Alaska Outer Continental Shelf Region. and the Uni versity of Alaska 

Fairbanks. This report. OCS Study BOEM 2015-041. is available through the Coastal Marine Institute. select federal ckpository 

I ibraries and elt:ctronical ly from http://www.boem.gov/Environmental-Stewardsh ip/Envi ronmental-Studies/ AlaskaRegion/1 ndex.aspx. 

The views and conclusions contained in this document arc: those or the authors and should not be interpreted as representing the 

opinions or policies or the U.S. Government. Mention of trade names or commcrcial products does not constitute their 

endorsement by the U.S. Government. 
Biodegradation and Transport of Crude Oil 

in Sand and Gravel Beaches of Arctic Alaska 

Principal Investigator 

Dr. Silke Schiewer 

Graduate Students 

Anna Iverson 

Priyam Sharma 

Department of Civil & Environmental Engineering 

University of Alaska Fairbanks 

FINAL REPORT 

July 2015 

OCS Study BOEM 2015-041 

BOEM Buruuos Oa~ ENI~, Mt.."U(,(w,,-.,1 

ll~f 

U tflV[R S ITY or 

ALASKA 

F A I RBA N KS 

CONTENTS 

LIST OF FIGURES ........................................................................................................................... v 

LIST OF TABLES ............................................................................................................................. vi 

LIST OF ACRONYMS ..................................................................................................................... vii 

ABSTRACT ....................................................................................................................................... ix 

INTRODUCTION ............................................................................................................................. 1 

Background ........................................................................................................................................ 1 

Oil exploration in the Arctic ......................................................................................................... 1 

Oil spill management in the Arctic ............................................................................................... 1 

Oil spill cleanup methods .............................................................................................................. 1 

Objectives .......................................................................................................................................... 2 

Hypotheses ......................................................................................................................................... 3 

Microcosm experiments ................................................................................................................ 3 

Mini-column experiments ............................................................................................................. 4 

LITERATURE REVIEW .................................................................................................................. 4 

Factors affecting biodegradation ........................................................................................................ 4 

Role of temperature ....................................................................................................................... 4 

Role of nutrients ............................................................................................................................ 5 

Role of seawater salinity ............................................................................................................... 6 

Role of crude oil characteristics .................................................................................................... 6 

Role of evaporation ....................................................................................................................... 7 

Role of sediment characteristics ................................................................................................... 7 

Environmental Sensitivity Index ................................................................................................... 8 

METHODS ........................................................................................................................................ 8 

Sediment sampling ............................................................................................................................. 8 

Microcosm experiment ...................................................................................................................... 9 

Analytical methods ............................................................................................................................ 12 

Titration to determine CO2 production ......................................................................................... 12 

Gas chromatography/flame ionization detection .......................................................................... 12 

Gas chromatography/mass spectroscopy ...................................................................................... 13 

Most probable number .................................................................................................................. 13 

Mini-column studies .......................................................................................................................... 15 

Construction of mini-columns ...................................................................................................... 15 

Experimental design ...................................................................................................................... 15 

Methodology for mini-column experiments ................................................................................. 16 

Wave tank study ................................................................................................................................. 18 

111 
RESULTS AND DISCUSSION ........................................................................................................ 18 

Microcosn1 experiments ..................................................................................................................... 18 

Carbon dioxide production in microcosms at 20°C ...................................................................... 19 

Volatilization in microcosms at 20°C ........................................................................................... 20 

Hydrocarbons remaining in sediments in microcosms at 20°C ...................................... .............. 21 

Carbon dioxide production in microcosms at 3°C ........................................................................ 21 

Volatilization in microcosms at 3°C ............................................................................................. 23 

Hydrocarbons remaining in sediments in microcosms at 3 °C ...................................................... 23 

Carbon dioxide production in microcosms at both temperatures ................................................. 26 

Volatilization in microcosms at both temperatures ...................................................................... 27 

Hydrocarbons remaining in sediments in microcosms at both temperatures ............................... 27 

Mass balance of crude oil in microcosms at both temperatures ................................................... 28 

Mini-colunm experiments .................................................................................................................. 29 

Crude oil movement in sandy gravel-sediment ............................................................................ 30 

Carbon dioxide release in sandy gravel-sediment ........................................................................ 31 

Effect of fertilization & temperature in sandy gravel ................................................................... 33 

Crude oil movement in pebble sediment ...................................................................................... 34 

Carbon dioxide release in pebble sediment .................................................................................. 36 

Effect of fertilization & temperature in pebble sediment ............................................................. 3 7 

Respiration .................................................................................................................................... 39 

Comparison of sediment types at 20 °C ........................................................................................ 39 

Comparison of sediment types at 3°C .......................................................................................... .41 

CONCLUSIONS ................................................................................................................................ 43 

Microcosms ................................................................................................................................... 43 

Mini-columns ................................................................................................................................ 45 

RECOMMENDATIONS ................................................................................................................... 46 

ACKNOWLEDGEMENTS ............................................................................................................... 46 

REFERENCES .................................................................................................................................. 47 

APPENDIX ........................................................................................................................................ 50 

IV 
LIST OF FIGURES 

Figure 1. Sampling locations .......................................................................................................................... 9 

Figure 2. Illustration of microcosm containing sediments with crude oil... .................................................... 10 

Figure 3. MPN plate with crude oil as a carbon source .................................................................................. 14 

Figure 4. Column design used in the experiment.. .......................................................................................... 15 

Figure 5. Mini-column experimental set-up ................................................................................................... 16 

Figure 6. Wave tank schematic ....................................................................................................................... 18 

Figure 7. Cumulative CO2 production at 20°C ............................................................................................... 19 

Figure 8. Cumulative CO2 production due to crude oil at low salinity and 20°C .......................................... 20 

Figure 9. Amount of volatile compounds released per week from the crude oil at 20°C ............................... 20 

Figure 10. Total petroleum hydrocarbons remaining in sediments at 20°C ................................................... 21 

Figure 11. Percentage of TPH removal from sediments over 6 weeks at 20°C .............................................. 22 

Figure 12. Cumulative CO2 production at 3°C ............................................................................................... 23 

Figure 13. Cumulative CO2 production due to crude oil for low salinity at 3°C ............................................ 23 

Figure 14. Amount of volatile compounds released per week from the crude oil at 3°C ............................... 24 

Figure 15. Total petroleum hydrocarbons remaining in sediments at 3°C ..................................................... 25 

Figure 16. Percentage of TPH removal from sediments over 6 weeks at 3°C ................................................ 26 

Figure 17. Cumulative CO2 produced in 6 weeks at 20°C and 3°C ............................................................... 26 

Figure 18. Percentage of volatile compounds released during 6 weeks at 20°C and 3°C .............................. 27 

Figure 19. Percentage of TPH removal from sediments over 6 weeks at 20°C and 3°C ................................ 27 

Figure 20. Mass balances of crude oil at 20°C and 3°C after 6 weeks. Graph shows fractions mineralized 

(CO2), volatilized (VOC) and remaining in soil (TPH) .................................................................................. 29 

Figure 21. TPH concentration at different depths after 3-12 days in sandy-gravel at 20°C ........................... 31 

Figure 22. TPH concentration at different depths after 3-18 days in sandy-gravel at 3°C ............................. 31 

Figure 23. Cumulative CO2 released from sandy-gravel at 20°C ................................................................... 32 

Figure 24. Cumulative CO2 released from sandy-gravel at 3°C ..................................................................... 32 

Figure 25. Effect of temperature on TPH concentrations in samples with no fertilizer and with liquid 

fertilizer ........................................................................................................................................................... 33 

Figure 26. Effect of temperature on release of CO2 from sandy-gravel with liquid fertilizer ....................... 34 

Figure 27. TPH concentration at different depths after 3-18 days in pebble sediment at 20°C ...................... 35 

Figure 28. TPH concentration at different depths after 3-18 days in pebble sediment at 3°C ........................ 35 

Figure 29. Cumulative CO2 released from pebble sediment at 20 °C ............................................................ 37 

V 
Figure 30. Cumulative CO2 released from pebble sediment at 3°C ............................................................... 37 

Figure 31. Temperature effect on crude oil movement in pebble sediment with no fertilizer. ....................... 38 

Figure 32. Temperature effect on crude oil movement in pebble sediment with liquid fertilizer ................... 38 

Figure 33. Temperature effect on release of CO2 from pebble sediment with ferti lizer ................................ 39 

Figure 34. Impact of sediment type on crude oil movement at 20°C with no fertilizer and with liquid 

fertilizer ........................................................................................................................................................... 40 

Figure 35. Impact of sediment type on CO2 release at 20°C with liquid fertilizer ......................................... 41 

Figure 36. Impact of sediment type on crude oil movement at 3°C with no fe1ti lizer, with liquid fertil izer, 

and \Vith solid fertilizer ................................................................................................................................... 42 

Figure 37. Impact of sediment type on CO2 release at 3°C with fe1tilizer ..................................................... 43 

Figure A I. MPN of crude oi I degraders from 20°C experiments ................................................................... 50 

Figure A2. MPN plate for the C I S2 and C2S I samples at 20°C with crude oil as C source ......................... 51 

Figure A3. MPN plate for C2S 1 and C2S2 samples at 3°C with diesel as C source ..................................... 52 

Figure A4. Addition of pebble sediment ......................................................................................................... 52 

Figure AS. Addition of sandy-gravel sediment... ............................................................................................ 53 

Figure A6. Waterline on sediments ................................................................................................................. 53 

Figure A 7. Wave tank in action ...................................................................................................................... 54 

Figure AS. Sequence of steps in wave tank experiment ................................................................................. 54 

LIST OFT ABLES 

Table I. Sampling location coordinates .......................................................................................................... 9 

Table 2. Experimental parameters for microcosms at 20'C and 3'C .............................................................. 11 

Table 3. Microcosm sampling schedule .......................................................................................................... 12 

Table 4. Sampling schedule for replicate microcosm jars .............................................................................. 12 

Table 5. Conditions in each column experiment... .......................................................................................... 16 

Table 6. Column flush cycle ........................................................................................................................... 17 

Table A 1. MPN va lues of number of microbes per gram of soil at 20°C ....................................................... 50 

VI 
LIST OF ACRONYMS 

API 

BOEM 

co 

Cl 

C2 

ESI 

GC/FID 

GC/MS 

MPN 

PAH 

PVC 

Sl 

S2 

UAF 

voe 

WERC 

TPH 

American Petroleum Institute 

Bureau of Ocean Energy Management 

no crude oil added 

1 mL crude oil added 

5 mL crude oil added 

Environmental Sensitivity Index 

Gas Chromatography with Flame Ionization Detector 

Gas Chromatography with Mass Spectrometry 

Most Probable Number 

Polycyclic Aromatic Hydrocarbons 

Polyvinylchloride 

Low salinity of 30 g/L 

High salinity of 3 5 g/L 

University of Alaska Fairbanks 

Volatile Organic Compounds 

Water and Environmental Research Center 

Total Petroleum Hydrocarbons 

Vil 
viii 
ABSTRACT 

Offshore oil production along Alaska's arctic coast is expected to increase in coming years. 

While this will create large economic benefits for the state, crude oil spills may occur. Oil spills 

reaching the shoreline could create adverse ecological effects, so it is important to understand 

methods, such as bioremediation, that might expedite oil removal. Mass transfer processes play 

an important role in determining the fate of crude oil along shorelines. Biodegradation and mass 

transfer processes are strongly dependent on temperature and sediment material. It is, therefore, 

necessary to study how the beach matrix, temperature, and nutrient addition affect the fate of 

hydrocarbons. 

The effect of environmental conditions on the rate of crude oil removal was studied in the 

laboratory, simulating oil spills at arctic seashores. Laboratory microcosms were set up 

containing beach sediments collected from Barrow, spiked with North Slope Crude. The 

microcosms were spiked with a standard concentration of nutrients and incubated at varying 

temperatures (3°C vs. 20°C), salinities (30 vs. 35 g/L), and crude oil concentrations (1 vs. 5 

mL/kg). Respiration rates (breakdown of hydrocarbons to CO2), hydrocarbons remaining in the 

sediment (GC/FID), and hydrocarbons volatilized and sorbed to activated carbon (GC/MS) were 

measured. Mini-column experiments to simulate the transport of oil through the sediment profile 

were conducted for two different sediment types (sandy-gravel versus pebble) obtained from 

Barrow and two different temperatures (20° and 3° Celsius). 

In all microcosms, higher respiration rates by naturally occurring microorganisms were observed 

at 20°C compared to 3°C. Surprisingly, the release of volatile organic compounds (VOC) was 

similar at both temperatures, for different crude oil concentration and salinities. Regardless of 

temperature, increased salinity had a positive impact on the rate of crude oil removal. At higher 

crude oil dosages, a larger amount of volatiles was released; however, CO2 production did not 

significantly increase with the contaminant concentration. A mass balance was established that 

showed that only a small fraction of the crude oil was mineralized, approximately 40% was 

volatilized and most TPH remained in the sediment. 

Mini-column data showed that the amount of crude oil pooling and its location was dependent on 

sediment structure and fertilizer addition. In sandy gravel sediment, TPH pooled in the middle of 

the column, six inches below the surface. In pebble sediment, the highest TPH concentrations 

initially occurred at the top and then at the bottom of the column. Overall, less TPH remained in 

pebble sediment compared to sandy gravel. Apparently hydrocarbons had been washed out more 

easily from pebble sediment. Both sediments had higher CO2 production at higher temperatures, 

with the highest respiration found in sandy-gravel, i.e., more biodegradation occurs in sandygravel sediment. While CO2 releases were slightly higher in sediments with the addition of 

fertilizer; overall the application of fertilizer did not have a significant impact on CO2 release. 

The results of this study will assist decision-makers in choosing effective spill response 

strategies for future crude oil spills in Arctic shorelines. 

IX 
·':r',,. 
INTRODUCTION 

Background 

Oil exploration in the Arctic 

The expected increase in offshore oil production in Alaska, combined with the potential opening 

of the Northwest Passage in the coming years, could lead to an increase in barge and tanker 

traffic through the Arctic. Increased exploratory drilling in the Chukchi and Beaufort Seas can 

increase the risk of oil spills in Alaska's arctic marine waters. While oil production provides 

economic benefits to local communities and the state of Alaska, related activities have the 

potential to adversely affect ecosystems, as well as the livelihood of local residents, especially 

when it is based on subsistence fishing. The commercial benefits of increased oil revenues must 

be weighed against the potential environmental costs of oil spills. Due to its remoteness and 

harsh weather, Alaska's Arctic coast has limited infrastructure such as ports, roads and airports 

(CRRC 2010), which makes oil spill response more difficult. Therefore, it becomes important to 

investigate which oil spill response strategies might be most effective in such conditions. 

Oil spill management in the Arctic 

Overall management of oil spills is increasing m complexity and magnitude worldwide 

(Othumpangat and Castranova 2014). The most effective response strategy for a specific spill 

depends on a number of factors, such as type of oil (viscosity, composition), geology, amount of 

turbulent energy, temperature, sea and air currents, sensitivity of biological communities and 

water salinity (EPA 2014, Owens and Lee 2003). Further, rapid removal of spilled oil is 

important to reduce the harmful effects of oil spills on sensitive habitats (EPA 2014). For any oil 

response, the oil company and other oil spill response organizations should have a generalized 

protocol follow (Sydnes and Sydnes 2013 ). 

Clean-up of oil spills in Arctic waters poses significant challenges because of harsh conditions. 

Inadequate infrastructure and poor weather can delay the arrival of vessels, equipment, and other 

supplies, and the presence of ice can confound response efforts by interfering with mechanical 

oil removal (PEW Charitable Trust 2013). It is critical to identify technologies and recovery 

techniques specifically tailored to oil spill clean-up in Arctic waters. 

Oil spill cleanup methods 

There are mechanical, chemical and biological methods for oil spill response. Mechanical 

methods include the use of skimmers, sorbent barriers, and inflatable booms, the latter most 

frequently used to concentrate oil on the water surface for easier recovery. Skimmers draw the 

oil from the water surface, and sorbent materials absorb the oil (EPA 2014 ). Chemical methods 

include the use of dispersants that break down the oil into tiny droplets, creating more surface 

area for degrading microbes (EPA 2014 ). Herders are chemicals that thicken oil spills so they 

can undergo in situ burning. Biological methods rely on microbial degradation for removing the 

spilled oil. Biodegradation use as an oil removal process is well documented in the literature 
(Brakstad and Bonaunet 2006) and can be an affordable and environmentally beneficial approach 

for removing hydrocarbons (Prince et al. 2003). 

Biodegradation is the breakdown of complex compounds into simple molecules by microbes. 

Bioremediation is a specific case of biodegradation, where the latter is used as an engineered 

method to degrade undesired complex compounds. To accelerate biodegradation, bioremediation 

strategies may include the addition of nutrients or electron acceptors to accelerate the growth and 

metabolic rate of micro-organisms (biostimulation) and/or the addition of oil-degrading microbes 

at the contaminated region (bioaugmentation). 

Bioremediation can be a cost-effective and safe technique for cleaning up crude oil contaminated 

sediments, even in Arctic and subarctic environments. A combination of mechanical removal and 

bioremediation, enhanced by nutrient addition, has shown potential in clean-up effo11s in the 

past; for example, after the Exxon Valdez oil spill (Wrabel and Peckol 2000). In situ 

bioremediation offers a promising alternative in remote locations and other areas where 

mechanical removal is not feasible. However, previous studies have also shown that 

bioremediation was not effective for heavy crude oil or in low-temperature environments, so 

further research is necessary to evaluate conditions under which bioremediation of crude oil can 

be effective in cold climates. Heiser ( 1999) demonstrated that, even at low temperatures, oil spil I 

bioremediation can be successful if there is a nutrient supply. Several researchers have concluded 

that nutrient supply and adjustment of pH, oxygen, and soil moisture levels can increase the oil 

biodegradation rates in Alaskan soils (Rice 2007, Heiser 1999, Hore! and Schiewer 2009). 

Limited research has been conducted on crude oil degradation at low temperatures in soil and on 

the effect of salinity (Minai-Tehrani et al. 2009). 

Arctic shorelines have not been exposed to a major oil spill, so little is known about 

biodegradation of crude oil in that environment. Therefore, it is essential to study the rate of 

crude oil biodegradation in arctic seashore sediments. However, sometimes results of small-scale 

laboratory studies cannot directly be scaled up due to heterogeneity and concentration gradients 

in larger settings (Hore] and Schiewer 2015). 

Objectives 

The objective of this study was to investigate the combined effects of varying temperatures, 

crude oil concentrations and salinities on the fate of crude oil biodegradation in laboratory 

experiments simulating environmental conditions along Alaska's arctic shore. This project 

focused on evaluating the potential for crude oil biodegradation in an oil spill response along 

Arctic shorelines and providing a better understanding of how crude oil interacts with the 

shoreline sediment. Mass transfer processes play an impo11ant role in determining the fate of 

crude oil along shorelines, and the diffusion and dispersion of oil, and its viscous properties, are 

strongly temperature dependent. Penetration of oil also depends on the sediment characteristics 

(Yang et al. 2009). Additionally, nutrients added to stimulate bioremediation may be washed out 

by waves and tides. Therefore, it is necessary to study how factors such as the beach matrix and 

temperature affect hydrocarbon and nutrient distribution. 

2 
Biodegradation was studied in Barrow beach sediments in laboratory microcosms as a proxy for 

an actual oil spill bioremediation on the Arctic shoreline. Typical sandy-gravel beach sediments 

obtained from Barrow were spiked with crude oil as the contaminant, amended with nutrients, 

and incubated to determine the extent to which crude oil was degraded, volatilized or remained 

in the sediments. Microcosm experiments were designed to examine the role of varying 

temperature, crude oil concentration and salinities on the fate of crude oil and the degradation 

rate. 

In a laboratory setting, mini-columns were used to study mass transfer, simulating shoreline 

conditions of the Chukchi and Beaufort Seas. The effectiveness of fertilizer addition (both solid 

and liquid) was examined to determine if nutrient addition had a significant impact on CO2 

production. Studies were done at two temperatures (3°C, as typical for the arctic summer, and 

20°C, as typical for temperate regions), to determine if temperature has a significant effect on oil 

movement and degradation. Crude oil fate was determined by tracking its movement through the 

sediment profile (top, middle, and bottom) for two different sediment types to determine whether 

petroleum hydrocarbons were washed out, converted to CO2 or volatilized. 

Identifying where oil might pool and estimating the depth to which it might penetrate shoreline 

sediments are important considerations in research on microbial degradation in the Arctic. 

Furthermore, the ability to predict how an environment will react to an oil spill under specific 

conditions will help inform response planning and assist decision-makers in choosing effective 

spill response strategies should crude oil spills occur on Arctic shorelines. 

Hypotheses 

Microcosm experiments 

1. Crude oil is degraded by indigenous microbes present in the Barrow sediments (i.e., 

biostimulation or inoculation with microbes is not required). 

2. Mineralization of crude oil increases with increasing contaminant concentration in 

absolute terms, but relative mineralization percentages will be lower for higher crude oil 

concentrations. 

3. Increasing crude oil concentrations lead to higher volatilization in absolute terms. 

4. For higher crude oil concentrations, higher quantities of crude oil will remain in the 

sediment. 

5. Biodegradation is faster at higher temperatures, but even at low temperatures measurable 

degradation (CO2 production) will take place over the course of several months. 

6. Overall hydrocarbon removal from the soil will be greater at higher temperatures. 

7. Volatilization is greater at higher temperatures. 

8. Higher salinity will have a positive impact on hydrocarbon degradation rates (CO2 

production). 

3 
Mini-column experiments 

9. Remaining crude oil concentrations will be lower m pebble versus sandy-gravel 

sediment. 

10. Oil pooling should be visible in sandy-gravel, but not pebble sediments. 

11. Lower temperatures should inhibit crude oil movement, causmg higher TPH 

concentrations, especially in the upper layers. 

12. Higher temperatures will allow for higher CO2 production. 

13. The addition of fertilizer should have a positive influence on CO2 production. 

14. Solid fertilizer will increase CO2 production more than liquid fertilizer. 

LITERATURE REVIEW 

Factors affecting biodegradation 

The rate of hydrocarbon biodegradation depends on many different factors such as nutrient 

availability, microbial growth, oxygen, water content, sediment type, temperature, hydrocarbon 

type, pH-value and bioavailability of contaminants (Margesin 2000). For any oil spill, all 

environmental factors influence the degradation rate cumulatively. Bioremediation accelerates 

the natural attenuation by optimizing the limiting environmental conditions present at a spill site 

(Margesin 2000). Some of the factors particularly relevant for the present research will be 

discussed in the following subsections. 

Role of temperature 

Temperature plays an important role in the process of biodegradation and bioremediation. 

Different organisms have different optimal temperatures, depending on the environment in which 

they typically occur. However, even organisms found in areas with cold climates, such as 

Alaska, often show higher metabolism at room temperature than at lower temperatures. This 

suggests that bioremediation is less effective at lower temperatures. Biodegradation typically 

follows the Arrhenius relationship, where metabolic activity decreases with a decrease in 

temperature (Heiser 1999, Yang et al. 2009). The Arrhenius relationship can also be applied to 

microbial community systems, where microbial growth increases with an increase in 

temperature. A study conducted by Yang et al. (2009) showed that heavy fuel biodegradation in 

the North Sea was four times faster at l 8°C than at 4°C. However, while microbial activity at low 

temperatures slows down, it does not stop (Yang et al. 2009, Aislabie et al. 2006). Laboratory 

experiments have shown that, though the microbial activity does not cease in a cold environment, 

the rate of mineralization is still higher in warmer environments (Aislabie et al. 2006, Horel and 

Schiewer 2009, Schiewer and Niemeyer 2006). Even at low temperature, there are hydrocarbondegrading microorganisms that occur and can survive solely on hydrocarbon products (Rike et al. 

2003, Heiser 1999, Yang et al. 2009). 

4 
Crude oil is a complex mixture of different hydrocarbons, and its properties depend on the 

surrounding temperature (Heiser 1999, Yang et al. 2009). Temperature is a critical parameter for 

bioremediation as it affects the rates of hydrocarbon degradation, microbial growth and mass 

transfer of substrate in cold soils (Yang et al. 2009). Additionally, the rates of volatilization are 

significantly reduced as temperatures decrease (Paudyn et al. 2008). 

Role of nutrients 

Microbial growth would not be possible without suitable nutrients present in the system. For 

growth to occur, microbes require a source of carbon (main substrate, e.g. hydrocarbon), sources 

of nitrogen and phosphorus (nutrients), and electron donors/acceptors (Yang et al. 2009). In 

aerobic respiration, heterotrophic microorganisms use oxygen as a terminal electron acceptor 

(Rike et al. 2003). Nitrogen and phosphorous are often limiting nutrients in Arctic soils; 

therefore, nutrient supplementation enhances the degradation of hydrocarbons. The addition of a 

commercial fertilizer such as 20:20:20 has been demonstrated to increase the mineralization of 

the majority of crude oil alkanes in Arctic soils (Aislabie et al. 2006). Amending sandy-gravel 

soil (from Barrow, AK) with 50-100 mg N/kg increased hydrocarbon degradation, whereas 200 

mg N/kg inhibited the degradation activity (Aislabie et al. 2006). This inhibition of soil microbial 

activity could be due to nitrite toxicity (Yang et al. 2009). Redfield stoichiometry states that the 

desired ratio of C, N, P and K is 100: 15: 1: 1; therefore, it is important to supply nutrients in the 

form of N and P as they often become limiting when the contaminant functions as a carbon 

source (Yang et al. 2009). 

The addition of nutrients to oiled sites facilitates faster microbial growth and hydrocarbon 

degradation. A field study by Margesin (2000) was conducted on oil-contaminated soil at an 

alpine skiing resort. The contamination had resulted from motor vehicles oil leakage and storage 

tank rupture. Results showed that fertilization led to a 42% reduction in hydrocarbon 

contamination, whereas natural attenuation led to a reduction of only 14% (Margesin 2000). 

Similar studies at low temperatures have demonstrated a positive relationship between 

degradation of oil and nutrient supply. For example, addition of fertilizer in a cold alpine soil 

contaminated with diesel fuel showed 43% decontamination in 30 days (Heiser 1999). 

The addition of nutrients can have a significant effect on the bioremediation of oil. Various 

studies have investigated the combination of nutrients that would result in the maximum 

microbial productivity. Braddock et al. ( 1997) found that phosphorus and nitrogen have the 

greatest effect on petroleum biodegradation by microbial communities. Mohn and Stewart (2000) 

found that adding phosphorous and nitrogen increased microbial activity in mineralizing 

petroleum products. However, the biodegradation rate was not increased for hydrocarbons with 

higher molecular weights. 

Braddock et al. ( 1997) showed that different rates of nutrient addition to soil samples affect 

microbial activity. They found that nitrogen was the most important nutrient to stimulate 

microbial activity; however, nutrient addition over the 400 mg N/kg soil threshold caused 

inhibition. This inhibition was assumed to be caused by reduced water availability due to 

5 
osmotic effects. Furthermore, the soil may have contained less moisture due to the lower 

precipitation rates and higher permafrost level at the study sites. Reduced productivity may have 

followed a decrease in carbon in the soil and a change in salinity. 

Role of seawater salinity 

The Beaufort Sea, Chukchi Sea, and the North Aleutian Basin have water temperatures and 

salinity levels ranging from 5-10°C and 10-24 %0 in the summer (CRRC 2010). 

The interaction of oil and minerals (present either in the sediments, soil or rocks) is an important 

factor in the clean-up of an oil spill. Recent studies have confirmed that saline seawater enhances 

the formation of oil-mineral aggregates (OMA), which help in the degradation of oil and increase 

biodegradation at higher salinities. Owens and Lee (2003) found that some OMA formation still 

occurs under low saline conditions. 

Salinity is a major factor that affects microbial activity in the marine environment (Thavasi et al. 

2007). Thavasi et al. (2007) found that increased levels of salinity (33-282 g/L) decreased 

hydrocarbon degradation. A study was conducted by Diaz et al. (2002) where a bacterial 

consortium was immobilized on polypropylene fibers to degrade the oil in saline water. The 

immobilized cells showed higher hydrocarbon degradation rates than non-immobilized cells 

(Diaz et al. 2002). This is a new approach to bioaugmentation, in this case, immobilizing 

microbes to increase the efficiency of degradation. High salinity can make the conventional 

bioremediation of oil difficult in crude oil contaminated water (Diaz et al. 2002). 

Minai-Tehrani et al. (2009) observed a positive correlation between salinity and the rate of 

phenanthrene mineralization; 1 0g/L and 30 g/L NaCl facilitated degradation of P AHs in soil, 

whereas 50g/L NaCl inhibited the microbial activity. Thavasi et al. (2007) found the strain of the 

common Gram-negative bacterium Pseudomonas aeruginosa exhibited maximum 

biodegradation activity and growth at 35 g/L salinity. 

Most of the above experimental studies used NaCl to prepare saline water. However, one should 

note actual seawater contains other constituents in addition to N aCI. Results from different 

experimental studies discussed above concur that salinity does have a significant effect on oil 

biodegradation; however, results differ as to what salinity level leads to the highest degradation 

rates. 

Role of crude oil characteristics 

Crude oil is a complex mixture of hydrocarbon compounds including alkanes, cycloalkanes and 

aromatic hydrocarbons (Yang et al. 2009, API 2011). The American Petroleum Institute (API) 

submitted a report to the Environmental Protection Agency (EPA) in 2011 that divided crude oil 

into three categories: light, medium and heavy. Crude oil is classified by its density, common 

measured as API gravity. The API gravity calculation is API=141.5/specific gravity - 131.5. 

Light crude has an API gravity ~33°, heavy crude has an API gravity :S28°, and medium crude 

has values between these grades. 

6 
The rate of crude oil biodegradation depends on the composition of hydrocarbon compounds 

present in the crude. Polycyclic aromatic hydrocarbons (P AHs) take longer than aliphatic 

compounds to degrade at cold temperatures (Yang et al. 2009). In decreasing order, the 

biodegradability of compounds is n-alkanes, branched-chained alkanes, branch alkenes, n-alkyl 

aromatics, monoaromatics, cyclic alkanes and P AHs (Yang et al. 2009). 

The crude oil concentration plays an important role in the act of degradation. Thavasi et al. 

(2007) conducted a biodegradation study with substrate ( crude oil) concentration varying from 

0.1 % - 4.5% in water. Maximum degradation activity occurred at a substrate concentration of 2% 

in water samples. This experiment revealed the significance of oil concentration on its 

biodegradation. 

Residual crude oil is no longer degradable and contains mainly P AH and asphaltenes. A study by 

Fernandez-Alvarez et al. (2006) at Sorrizo beach, which was affected by the Prestige oil spill, 

monitored the rate of biodegradation of weathered fuel oil that remained after initial 

volatilization and some microbial degradation. Neither biostimulation nor bioaugmentation 

increased degradation of the residual fuel oil; however, the introduction of biodiesel increased 

the degradation of the weathered oil (Fernandez-Alvarez et al. 2006). 

The role of evaporation 

Weathering of oil in the water column includes surface evaporation, droplet formation, 

biodegradation and other environmental processes (Wrabel and Peckol 2000, Brakstad and 

Bonaunet 2006). Wrabel and Peckol (2000) found that natural attenuation proceeded mainly by 

evaporation, with little microbial degradation, when there was low nutrient availability at an oilspill site; 25-30% more n-alkanes were lost with the addition of nutrients (N and P). Low 

temperature usually results in reduced evaporation of volatiles and a delayed start of 

biodegradation (Heiser 1999, Yang et al. 2009, Aislabie et al. 2006, Margesin 2000). The 

volatilization of short-chain alkanes is higher at higher temperatures. 

The role of sediment characteristics 

Shoreline particle size and distribution of the sediment can have a large impact on the movement 

of oil. Higher porosity sediments contain a larger amount of voids allowing fluids to travel more 

freely through the sediment. Sand sediment presents a much greater resistance to fluid flow 

through porous media because sand grains can be more neatly packed resulting in much smaller 

pore sizes. The ability for oil to be retained in soil is inversely related to its ability to penetrate 

the sediment (Harper 1978). Additionally, the sediment particle size can influence how many 

microbes are present in the soil. If sediment has large grain size (such as pebbles), there is less 

surface area for organic matter and moisture to be retained, making it a harsh environment for 

microbes to survive. 

Sediment characteristics are also an important factor in determining the fate of crude oil in 

sediments. Every beach is different in terms of climate, grain size distribution and biological and 

chemical characteristics. The components of the beach sediments govern the possible effects of 

7 
oil on the shoreline (EPA 2014). The eastern Beaufort Sea coastal sediments contain a laroe 

t> 

amount of organic carbon due to river inputs and coastal erosion of peat. However, the fate of 

this organic matter in the sediments is still unknown (CRRC 2010). During storms, these 

sediments can be redistributed. In the Barrow area, separate zones of mostly pebbles alternate 

with those that are mostly sand. If an oil spill occurs on such a beach, oil could penetrate up to 50 

cm depth (pooling there) and storms can erode some surface-spilled oil (NOAA 2002). 

Oil movement deeper into the sand renders degradation difficult due to limited availability of 

oxygen (EPA 2014). Tilling has been used as an oil spill response method on oiled beaches. 

Tilling allows escaped oil to materialize on the surface where microbes can readily degrade the 

oil because of oxygen availability. Tilling accelerates physical, chemical and biological 

processes that would be absent or slower under natural conditions (Owens and Lee 2003). An 

EPA study (2014) was conducted on a soil plot where IF-30 intermediate fuel was used as a 

contaminant, and the role of tilling and fertilizer addition was monitored. The tilled sediment 

with fertilizer showed the maximum oil degradation compared to untilled and unfertilized 

sediment. 

Environmental Sensitivity Index 

Since coastal environments respond differently to oil spills, the National Oceanic and 

Atmospheric Administration (NOAA) developed an Environmental Sensitivity Index (ESI) that 

classifies susceptibility to oil spills on the basis of three factors: shoreline classification, 

biological resources and human use resources (NOAA 2002). The ESI shoreline classifications 

range from I (high levels of physical energy and low biological activity) to 10 (sheltered 

shorelines, high biological activity). By looking at substrate type, grain size, wave action, tidal 

currents and river currents, a prediction of behavior and persistence of oil in inte11idal habitat can 

be made. If a spill were to occur, responders could then look up the affected shoreline's 

characteristics and determine its ESI number; which gives the responder a general idea of how 

oil will react in the environment, and what potential problems could arise. ESI maps have been a 

vital part of oil spill response and planning since 1979. 

The North Slope contains shorelines that fall under ESI 1, 4 and 5. This research will focus on 

sediments typical of ESI 4 and 5 areas, coastlines predominantly composed of sand and gravel 

sediment (NOAA 2002). Much of the Chukchi Sea and Beaufort Sea region falls under the ESI 

5B category, which is characterized by mixed sand with at least 20% gravel, an intermediate 

slope of 8-15 degrees, and low fauna and epifauna populations (NOAA 2002). 

METHODS 

Sediment sampling 

Sediment samples were collected from July 22 - 25, 2013 at a four beach locations near the city 

of Barrow, the local hub of Alaska's northern coast at the intersection of the Beaufort and 

Chukchi Seas. Four 10-gallon buckets were filled from the top 60 cm of beach sediment (roughly 

30 kg per bucket). Samples were collected at four different locations (Figure 1, Table 1 ). Two 

8 
buckets were collected along the Beaufort Sea/Elson Lagoon, where the sediment had a more 

sandy-gravel composition ( 4-ESI). Two buckets were collected a long the Chukchi Sea; these 

sediments were primarily composed of pebble material (5-ESI). The samples were transported 

back to the laboratory and kept at 4°C. Water sampl es were collected from the sea adjacent to 

each sampling site and returned to the laboratory where salinity (30 g/L) was determined by 

salinity meter. 

A sandy gravel sample with some pebbles collected at 71 °2 I '39.80"N, l 56°2 l '47.90"W was used 

fo r the microcosms. One bucket of each ES! type was used to determine the fate of crude oil for 

a variety of conditions both in mini-column and wave tank studies. The two sediment types were 

classified as sandy-gravel and pebble, respectively. 

Figure I. Sampling locations. 

Table 1. Sampling location coordinates. 

Sample Number Latitude 

Sandy-Gravel I 71 °2 I '34.91 "N 

Sandy-Gravel 2 7 1 °2 1 '39.80"N 

Pebble I 71 °23'2.99"N 

Pebble 2 71 ° 17' 11.00"N 

Microcosm experiment 

Longitude 

156°2 1 '42. 74"W 

156°2 1 '47.90"W 

156°29'7. 13"W 

I 56°48'29.12"W 

The microcosm experiment was designed to approximate the environmental conditions of 

Barrow. Clear canning j ars were filled with 1 kg of Barrow sediments, characterized using the 

ASTM C 136-06 method. The sediment material was mixed to ensure that each jar received 

approximately the same material. Approximately 10 kg of sediment was autoclaved fo r use in the 

9 
sterile control microcosms. To evaluate biodegradation by the naturally present microorganisms, 

sediment used in the experimental microcosms was not sterilized. No inoculation was performed. 

Figure 2 below shows the experimental setup for this study. In each microcosm, a "tea bag" (one 

side open) made from mosquito netting, with a size of I 0x8 cm, was fi lled with 5 g of coconut 

shell activated carbon and suspended with a string in the jar's headspace to trap the volatile 

compounds released from the crude oil. Each jar had a metal loop stand, prepared from a 12-inch 

steel wire inserted into the sediment, to suspend a beaker filled with 20 mL of I N NaOH 

solution in the headspace. Each jar received IO mL of nutrient solution prepared from fertilizer 

with an N:P:K ratio of 20:20:20; the nitrogen fraction consisted of 20% ammonia, 30% nitrate, 

and 50% urea nitrogen. Each microcosm received a total nitrogen concentration of 300 mg/kg 

sediment. 

2 L sealed container 

5 g acti,·ated carbon 

20 mL of l N NaOH solution in beaker 

0. l or 5 mL crnde oil 

l kg sediment with :-00 mg N as fertilizer 

Figure 2. Illustration of microcosm containing sediments with crude oil. 

Quadruple microcosms were set up to represent seven different conditions, three as controls and 

four experimental, with varying crude oil concentrations and salinities as specified in Table 2. 

Twenty-eight jars were prepared for each temperature regime (20°C and 3°C). Microcosm jars 

were tightly sealed and placed inside a fume hood for 6 weeks for the 20°C series and kept in a 

refrigerator for 9 weeks for the 3°C series. 

Table 2. Experimental parameters fo r microcosms at 20°C and 3°C. 

Type Setup ID Crude Oil Salinity (g/L) Sterilized No. replicate 

C0S I 0 30 4 

0 

b C0S l 0 30 yes 4 C: 

0 

u CISl 30 yes 4 

(13 

.... Cl Sl 30 4 

C: CIS2 35 4 Q.) 

E 

v C2Sl 5 30 4 

a. 

X 

w C2S2 5 35 4 

10 
Experiments were performed at two different salinities. Sediments brought from Barrow had 

been naturally saturated with ocean water of a salinity of 30 g/L. This material was used for S 1 

(low salinity of 30 g/L) experiments. For the high salinity (S2) experiments, sediment was 

flushed with artificial seawater (35 g/L salinity prepared using Instant Ocean Aquarium Salt) 

before filling the jars. 

Either 1 or 5 mL of crude oil was pipetted onto the sediment surface. The specific gravity of 

crude oil was measured at 0.87 at 20°C and 3°C. Therefore, the initial concentration of added 

crude oil was 870 mg/kg for 1 mL of crude oil added and 4350 mg/kg for 5 mL of crude oil. 

After addition of crude oil and fertilizer, the sediment was mixed vigorously with a spoon as a 

proxy for sediment tilling. 

The experiment was designed to establish a mass balance for the initially present crude oil 

including volatilization of short chained hydrocarbons, crude oil remaining in the sediment, and 

crude oil mineralized ( converted to carbon dioxide). The following parameters were used to 

evaluate the results: 

1. CO2 produced and captured in NaOH solution was quantified by titration with HCI. 

2. Volatiles released from the crude oil and captured by activated carbon were assessed via 

gas chromatography/mass spectrophotometry (GC/MS). 

3. Crude oil remaining in the sediments was determined by gas chromatography/flame 

ionization detector (GC/FID). 

4. Number of microbes present in the sediments was calculated by using the most probable 

number (MPN) method. 

The sampling frequencies for the above parameters are shown in Table 3. Additionally, each 

temperature series had four replicate microcosm jars (A-D) for each environmental condition, 

and these were sacrificed for sampling every two or three weeks (Table 4). 

Table 3. Microcosm sampling schedule. 

Parameter Medium Sampled Temperature Sample Size Frequency Duration 

MPN Sediment 20°c 1 g Every two weeks 6 weeks 

Crude Oil Sediment 20°c 10 g Every two weeks 6 weeks 

Volatile Activated Carbon 20°c 5g Once per week 6 weeks 

CO2 NaOH 20°c 20mL Once daily Week 1-3 

CO2 NaOH 20°c 20mL Every two days Week 4-6 

MPN Sediment 3°c 1 g Every three weeks 9 weeks 

Crude Oil Sediment 3°c IO g Every three weeks 9 weeks 

Volatile Activated Carbon 3°c 5g Once per week 9 weeks 

CO2 NaOH 3°c 20mL Once daily Week 1-3 

CO2 NaOH 3°c 20mL Every two days Week4-9 

I I 
Table 4. Sampling schedule for replicate microcosm jars. 

Replicate D Replicate C 

All 20°C microcosms after 2 weeks after 4 weeks 

All 3°C microcosms after 3 weeks after 6 weeks 

Analytical methods 

Titration to determine CO2 production 

Replicate B 

after 6 weeks 

after 9 weeks 

Replicate A 

after 6 weeks 

after 9 weeks 

Carbon dioxide evolution, a proxy of microbial activity, was measured as described by Hore) and 

Schiewer (2009). For microcosms, every day or every second day the beaker was removed; since 

microbial activity decreases with time, titrations were conducted every two days from the third 

week onwards. For mini-columns, the NaOH-filled balloon was removed at the end of the 

experiment. Excess BaCI2 and l % phenolphthalein as a color indicator were added to the NaOH 

solution. A Metrolun titrino was used for conducting the titrations. 1 N HCI solution was titrated 

until the solution changed from pink to clear. The mass of carbon dioxide released in a day or 

two days describes the rate of microbial activity and was calculated from the following equation: 

Vtitrant(mL) X Cacid (M) X MWco2 = mco2(mg) 

Gas chromatography/flame ionization detection 

The remaining crude oil in the sediments was determined using gas chromatography/fl ame 

ionization (GC/FID). Triplicate 10 g sediment samples from each jar were stored at-80°C until 

analysis. Crude oil was extracted from sediment samples via 25 mL methylene chloride. Twentyfive μl of D-5 nitrobenzene was used as an internal standard and 250 μl D-8 naphthalene as a 

surrogate. The standard concentration for D-5 nitrobenzene was 2500 mg/L and 2190 mg/L for 

D-8 naphthalene. The total petroleum hydrocarbons (TPH) in the sediments were computed 

using a modified AK 102 and AK l 03 methodology developed by the Alaska Department of 

Environmental Conservation (ADEC 2002a,b,c). 

We used Agilent Technologies, Inc., 6890N Network GC coupled with flame ionization detector 

with column parameters 30 m by 250 μm by 0.25 μm. The TPH method used a pulsed splitless 

injection with hydrogen or helium as gas carriers. We replaced hydrogen with helium halfway 

through the experiment because helium is less flammable (pressure 20 psi, flow 12.4 mL/min, 

average velocity 15.2 cm/sec). The initial oven temperature was 40°C and increased to 350°C 

over 34.50 minutes. 

The calibration standards were prepared over the range of 500-5000 mg/L for the microcosms 

and 250-2500 mg/L for the mini-columns. The standard concentrations were based on the 

theoretical initial crude oil concentration of 870 mg/kg for C I and 4350 mg/kg for C2 in the 

microcosms and a dosage of 2 mL, i.e., 1,740 mg of crude to each column. The density of the 

crude oil was found to be 0.8672 glee (20°C) and 0.8725g/cc (3°C), with a viscosity of 43.58 cP 

(20°C) and I 03.92 cP (3°C). 

12 
The total area of the chromatogram was taken into account when calculating the concentration of 

crude oil present in the sediments for days 0, 14, 28 and 42 at 20°C and for days 0, 21, 42 and 63 

at 3°C. 

Gas chromatography/mass spectroscopy 

For microcosms, volatile compounds released by crude oil were trapped in 5 g of activated 

carbon suspended in a mesh bag in the jar. In weekly intervals, the activated carbon was removed 

from the microcosms. To extract hydrocarbons from the activated carbon, 0.22 g of activated 

carbon was put in a GC vial and 1.5 mL of carbon disulfide was added with 25 μl of D-5 

Nitrobenzene as an internal standard to verify the efficiency of the extraction. The concentration 

of the internal standard was 2500 mg/L. 

For mini-columns, 0.5 g of activated carbon was measured into individual GC vials; the vials 

were labeled and stored at -80°C. When the samples were ready for analysis, the vials were 

allowed to defrost. One mL of carbon disulfide was added to each vial. Five μL of the internal 

standard of 2500 mg/L of heating fuel in carbon disulfide was added to each vial. The vials were 

immediately placed on the GC-MS for analysis. 

To determine the gasoline range organics for microcosms and mini-columns, we modified the 

AK 102 (ADEC 2002) method. The GC-MS used was an Agilent Technologies 6890N Network 

with a JW 123-1062 and a 60 m by 250 μm by 0.25 μm column. The volatile organic compounds 

(VOC) method uses a splitless injection with helium as the carrier gas (pressure 9 psi, flow 1.6 

mL/min, average velocity 3.2 cm/sec). The oven was set at an initial temperature of 150°C and 

increased to 350°C, over 16.50 minutes. 

A calibration curve was established using standards over a range from 250 to 5000 mg/L. The 

total area of the gasoline range was used, and the concentration of the released volatiles was 

calculated based on the calibration curve. The D-5 Nitrobenzene had an affinity for the activated 

carbon that was used in this study and this compromised the recovery efficiency for the volatiles. 

The same procedure and data analysis were followed for experiments at both 20°C and 3 °C. 

When analyzing samples from mini-columns, the GC/MS encountered some technical 

difficulties (samples were skipped over and not analyzed or only analyzed for an insufficient 

time period) leaving over a quarter of the data unusable. Therefore, no results are available for 

volatiles in the mini-columns. 

Most probable number 

Crude oil is degraded by microbes present in the sediments. The number of crude oil-degrading 

microorganisms was calculated by using the most probable number (MPN) method. This 

technique follows a standard protocol where triplicate 1 gram sediment samples from each jar 

were taken in a falcon tube. Ten mL of 1 % sodium pyrophosphate solution along with 3-4 grams 

of sterile glass beads were added to those falcon tubes. This mixture was put on the shaker table 

for 1 hour, after which the tube was allowed to stand for half an hour. Then, in each well of 96-

13 
well microtiter plates, 180 μl of Bushnell growth medium, 20 μI of microbial suspension (after 

1.5 hours) and 5 μl of filtered crude oil (carbon source) was added. Three replicates (i.e., 3 rows) 

were performed for each sample, each row with increasing dilution from left to right; including 

one control row without a carbon source and one control row containing no microbial suspension 

(Figure 3). The 96-well plates were incubated for 14 days at room temperature. Five μI of 2-(4-

iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium (INT) dye was added to each well on the 

I 5th day, and again the plates were incubated for 24 hours in the dark. Positive growth as 

indicated by a change in color (pink) was noted on the I 6th day. The experimental procedure was 

followed for 20°C. 

The observed data were entered into the EPA MPN calculator software to determine the number 

of microbes present in the sediments. The EPA MPN calculator provides a specific concentration 

in MPN/mL based on the number of positive wells. 

However, there was some inconsistency in recording the color change on day I 6, as the crude oil 

formed a dark layer on top of each well, hindering the correct color change identification. 

Therefore, the carbon source and incubation temperature were changed for low-temperature 

experiments. 

For the experimental study at 3°C, diesel was used as the carbon source. Since diesel is clear in 

color, it was expected that this would enable better visual inspection. Wrenn and Venosa ( 1996) 

demonstrated success using separate carbon sources to provide alkanes and PAHs as an 

alternative to using only crude oil. The incubation temperature was kept at I 0°C as this was 

expected to result in a reasonable number of microbes for the reading on day 16. 

Unfortunately, no reasonable MPN results were obtained due to low-temperature dormancy of 

the microbes and poor conditions for visual inspection. Therefore, the MPN results will not be 

discussed in the results section but are included in the Appendix. 

No C source 

No microbes 

Figure 3. MPN plate with crude oil as a carbon source. 

14 
Mini-column studies 

The purpose of these experiments was to study biodegradation and transport of crude oil through 

the sediment. Mini-columns were filled with sediment, crude oil was added, and several flushing 

cycles were performed to simulate tidal action. After different experimental durations, the 

petroleum hydrocarbons at different depths within the column were measured. The amount of 

hydrocarbons evaporated was determined by measuring volatiles collected in activated carbon, 

and the amount of CO2 released was determined by titrations of NaOH. Temperature, sediment 

type, and the addition of liquid or solid ferti lizer varied for the experiment. 

Construction of mini-columns 

Mini-columns were constructed using PVC pipe, as shown in Figure 4. A I1⁄2 inch ABS PVCpipe was cut into 18-inch sections, each with an ABS adapter and a threaded plug fitti ng attached 

to each end. A threaded 1⁄4 in. hole was drilled into the plug fitting and a barbed nylon national 

pipe thread was tlu·eaded into place. ABS cement was then used to seal the adapter fitting and 

barbed nylon into place to ensure no water could leak out. 

Figure 4. Column design used in the experiment. 

Experimental design 

Two preliminary experiments were performed to determine whether the frequency or number of 

flushes was most influential in moving crude oil through the profil e. The first experiment 

involved varying the time between each flush, with the same number of flushes (six) in each 

experiment. Tlu·ee setups were compared; the first was a 6-hour study where the system was 

flushed every hour. The second was over 48 hours, and the system was flushed every 8 hours. 

The latter took 72 hours, and the system was flushed every 12 hours. The second experiment 

varied the number of flushes using a standard time of 12 hours between flushes to reflect ocean 

tidal cycles. As a 3-day trial had already been run, two additional studies were run for durations 

of 6 and 12 days, with a total of 12 and 24 flushes, respectively. It was concluded that the 

number of flushes had a greater impact on the overall movement of the oil than did time between 

flushing 

The titration data showing the amount of CO2 released produced interesting results. There was no 

difference in the release of CO2 between the control column and the columns with crude oil 

added. Two possible explanations for the relati vely high respiration in the control and the low 

15 
respiration in columns with crude oil are (I) a carbon source may have been present in the soil, 

and (2) the liquid fertilizer was rinsed out too fast to have an impact. For these reasons, it was 

decided to conduct additional experiments of 18-day duration and with solid fertilizer as shown 

in Table 5. These experiments were run for 3, 6, 12 and 18 days, with flushes every 12 hours. 

Methodology for mini-column experiments 

The mini-column experiment is schematically represented in Figure 5. Columns representing 

seven different environmental conditions (Table 5) were used in each experimental run, with 

duplicate columns for all experimental conditions except for the control. Experiments were 

performed at both 3°C and 20°Cwith durations as specified in Table 6. 

() 

,.._, I 

Otl•dS.dtfflt;t'rt I 

w,a,Orc~J 6 

Saturate 

Sediment 

6 

6 

6 

0 

Add Crude Oil 

(Rush with S.,j 

watt t) 

0 1 

Sit 

(12hrs) 

Flush With 

Seo Wotcr 

6 

6 

6 

l o 

(Repeat 

Steps iHS) 

Figure 5 . Mini-column experimental set-up. 

Table 5 . Conditions in each column experiment. 

PVC Oil Liquid Solid NaOH Activated 

Pipe Fertilizer Fertilizer Carbon 

Yes No No Yes Yes 

2 Yes No No Yes Yes 

3 Yes Yes No Yes Yes 

4 Yes Yes No Yes Yes 

5 Yes No Yes* Yes Yes 

6 Yes No Yes* Yes Yes 

7 No No No Yes Yes 

*Note: no solid fertilizer was used for the 20°C sandy-gravel study. 

16 
The procedure for the mini-column experiment follows: 

1. Seven PVC pipes were filled with enough sediment mixture to make 12-inch sections and 

placed on a shaker table to pack the column down to simulate ocean beaches. 

2. The sediment was saturated from the top with water. Once saturated the bottom of the 

column was opened to drain excess water. 

3. Two mL of crude oil was introduced from the top to PVC pipes 1, 2, 3, 4, 5 and 6. 

4. Twenty mL of fertilizer (N=300 mg/mL) was then added to column 3 and 4. The fertilizer 

solution was prepared by dissolving a solid 20-20-20 fertilizer (20% nitrogen from 20% 

ammonia, 30% nitrate, and 50% urea) in water. 

5. 0.5 g of solid fertilizer of the same type was added to column 5 and 6 to achieve a dosage 

of 600 mg/ of nitrogen. PVC pipe 7 was used as a control with no fertilizer or oil added. 

6. Fifty mL of artificial sea water (prepared using Instant Ocean Aquarium Salt to 

salinity=30 g/L) was added from the top and allowed to drain out completely. Once 

drained, the bottom valve was shut allowing no more water or oil to exit the column. 

7. A "tea bag" filled with ~ 1.5 g of activated carbon was suspended in the tube air space. 

The same "tea bag" would be used for the entire course of each experiment, to ensure that 

the final reading would be total volatiles released over the specific time frame. 

8. A clear balloon filled with 20 mL of 1 N NaOH solution was attached to the cap of the 

PVC-pipe to ensure that no air could escape. The same NaOH was used for the entire 

experiment and, after the 18 day experiment, it was clear from titrations that the NaOH 

was still able to absorb CO2, so no loss of CO2 was believed to occur. 

9. The top of the column was sealed using plumbers tape wrapped around the thread cap 

tightly fitted to the top of the column so no air could escape. 

10. After allowing the system to sit for 12 hours, the NaOH balloon and activated carbon 

were removed. Steps 7-12 were repeated for the remaining flush cycles with experimental 

durations of 3, 6, 12 or 18 days (Table 6). 

Table 6. Column flush cycle. 

Hours between flushes Number of Flushes 

12 6 

12 12 

12 24 

12 36 

Number Days 

3 

6 

12 

18 

Total petroleum hydrocarbon remaining in the sediment, released CO2, captured in NaOH 

solution (indicating hydrocarbon mineralization) and volatilized hydrocarbons captured in 

activated carbon were analyzed (see above: Analytical methods). After the last cycle, the bottom 

end of the mini-column PVC pipe was opened and allowed to sit for 30 minutes. The NaOH 

balloon and activated carbon were removed for analysis. One composite sediment sample of 

approximately 10 g was taken from the top, middle and bottom of each column. The samples 

were stored in amber vials at -80°C until analysis. 

17 
Wave tank study 

A wave tank was used in an observational study to simulate how crashing waves on the shore 

affect the movement of the oil through the sediment horizon. A 5 x 1.5 x2 ft. Plexiglas tank was 

fitted with a divider so sandy-gravel and pebble sediment types could be evaluated 

simultaneously under identical conditions. Sediment was placed into the tank creating a slope of 

approximately 30 degrees. The sediment was approximately 12 inches high and extended 20 

inches on the tank's bottom (Figure 6). 

60in 

20in 

Figure 6. Wave tank schematic. 

The tank was filled with 10 gallons of artificial salt water mixture. After allowing the water to 

saturate the sediment (~ 30 min), a wave-maker (Jebao WP-40, 900 to 3400 GPH) was used to 

generate a consistent wave pattern. Once the wave generation stabilized, 20 mL of crude oil was 

added to one end of the tank, and 5 mL was added straight to the shoreline. The 20°C and 3°C 

experiments were conducted over three day periods with continuous wave action. At the end of 

the study, the wave simulator was turned off. After 30 min, the water was slowly drained from 

the tank. Samples were collected but could not be analyzed due to water saturation; drying them 

would have resulted in too much hydrocarbon loss. Therefore, this study was strictly 

observational, and results are presented in the Appendix. 

RES UL TS AND DISCUSSION 

Microcosm experiments 

The rate of crude oil degradation for different salinities and crude oil concentration was assessed 

as carbon dioxide production, loss of volatile compounds, and quantity of crude oil remaining in 

the sediments. Microcosms were run for six weeks for 20°C treatments, and nine weeks fo r 3°C 

treatments; all used subsamples of the same sandy-gravel sediment. Seven condition scenarios (3 

controls and 4 experimental) were executed for each temperature and experimental duration. 

Condition variables are described by the following abbreviations: Co specifies that no crude oil 

was added, C 1 stands fo r a low crude oil concentration (I mL/kg), C2 indicates high crude oil 

concentration (5 mL/kg), S 1 refers to low salinity (30 g/L, as present in Beaufo rt and Chukchi 

sea) and S2 refers to a high salinity (35 g/L, as common worldwide) (Table 2). Abbreviation 

pairs represent condition combinations. 

18 
Carbon dioxide production in microcosms at 20°C 

Measuring carbon dioxide production, i.e., respiration, is a primary method to determine the rate 

of biodegradation of hydrocarbons over time. Figure 7 displays the total CO2 produced in each 

jar at different conditions over a period of 6 weeks, allowing the following observations. 

I. Respiration for CoS I sterile was higher than fo r CoS I. Also, the series for C I SI sterile 

and CI SI overlapped. As further discussed in Appendix A, sterilization of sediments was 

not effective, the MPN of hydrocarbon degraders in "sterile" microcosms were of similar 

magnitude as for unsterilized ones. Due to the lack of a biological hood and ineffective 

sterilization, the sediments in both C I S I sterile and C I S I jars were exposed to similar 

conditions ( I mL of crude oil, low salinity). Therefore, the CO2 production was similar in 

both cases. 

2. Respiration for C I S2 was higher than Jar CI SI, and similarly C2S2 produced more CO2 

compared to C2S I. This indicates higher salinity had a positive impact on CO2 

production or increased microbial activity. 

3. The controls without any crude oil addition (Co) had a relatively high respiration though 

lower than for C l and C2. This suggests another carbon source (beyond the added crude 

oil) was present in the sediment samples and only part of the CO2 production can be 

attributed to crude oil degradation. This was adj usted for by subtracting the CO2 

production of the corresponding control microcosms (Co) from the Cl or C2 microcosm 

with otherwise same conditions. 

00 

E 

'tl • 

., 

u 

::, 

'tJ 

0 

a. 

"' 0 

u 

., " > 

:i " 

E 

::, 

u 

1000 

900 

800 -

700 

600 

500 

400 

300 

200 

100 

0 

0 

1 ml crude 

- ClSl 

- ·- C1S2 

10 

5 ml crude 

- C2S1 

- · C2S2 

20 

no crude 

- CoSl 

······· CoSlsteri 

30 40 

Time, days 

Figure 7. Cumulative CO2 production at 20°C. 

so 

Figure 8 shows the CO2 production due to crude oil degradation at low salinity (CI S I sterCoS I ster, C I S 1-CoS I, and C2S 1-CoS 1 ). As depicted in Figure 8, C2 showed higher CO2 

production than C l and C l sterile. This shows that the higher the concentration of crude oil, the 

higher the rate of CO2 production and thus the rate of biodegradation. 

19 
300 ········· C151steri - - - C151 --C251 t>D 

E 250 

c 

0 

·z 200 u -- ::, 

"C 

0 150 a. 

0 

u 100 ············ QI 

> ·z 

111 so 

:i 

E 

::, 0 u 

0 10 20 30 40 so 

Time, days 

Figure 8. Cumulative CO2 production due to crude oi l at low salinity and 20°C. 

Volatilization in microcosms at 20°C 

Volatilization was measured as one component to establish a mass balance for the added 

hydrocarbons. The activated carbon included in the experimental setup acted as a sink, as a 

proxy for dispersal volatilized hydrocarbon compounds in the air. Figure 9 shows the amount of 

volatile compounds released per week from the crude oil during the 6 week incubation time. 

1200 ----c151 - : -CoSl 

- c- (152 ---+--- Co51steri - l.! • C252 

1000 t>D 

E 

c· 800 0 

·z; 

111 

N 600 ·z; 

111 

0 

> 400 > 

32 

QI 

QI 

3 200 

0 

0 10 20 30 40 50 

Time, days 

Figure 9. Amount of volatile compounds released per week from the crude oil at 20°C. 

The following observations can be made from Figure 9: 

1. The amount of volatiles released significantly increased with the amount of crude oil 

present in the sediments. 

2. Volatilization for microcosms without crude oil addition (CoSl and CoS I sterile) was 

approximately zero for the first four weeks. Since there was no crude oil present in the 

sediments, no volatiles were released from the sediments. However volatilization 

increased after 30 days to about IO mg/week. This could either be due to some volatile 

compounds originating from crude oil stored in the fume hood during the experiment, due 

to partial degradation of organic compounds creating more volatile products, or due to 

measurement error since such small concentrations could not be reliably dete rmined. 

20 
3. C I SI sterile, CI SI and C I S2 initially contained the same amount of crude oil and 

consequently showed very similar release of volatiles, which was only substantial over 

the first week and rapidly declined thereafter. 

4. C2Sl and C2S2 also showed similar volatilization, as refl ected in similar amounts of 

hydrocarbons being present. Volati lization was very high in the initial week and rapidly 

declined over the course of the experiment. 

5. Salinity had no significant impact on volatilization. 

Hydrocarbons remaining in sediments in microcosms at 20°C 

Total petroleum hydrocarbons present in the sediments declined over time at 20°C as illustrated 

in Figure I 0. 

---;;;;.-- 051 --Co51 - c251 

5000 Cl52 ···-r··· Co5lsteri - C252 - 4500 --~ -- Cl5lsteri 

Jf 4000 1 ' ' -- ' 3500 

C: 

., 3000 

E 

:;; 

5: 2500 

·= 2000 

"" C 1500 ·c 

·;;; 

E 1000 - ·- ..:..- •- · - · :c 500 c.. 

I0 

0 10 20 30 40 50 

Time, days 

Figure 10. Total petroleum hydrocarbons remaining in sediments at 20°C. 

Total petroleum hydrocarbon changes showed the following trends: 

I. The CoS I control and CoS 1 sterile series coincided, which can be explained by the fact 

that sterilization was not successfully executed. 

2. CI SI sterile, CI SI , and C I S2 also showed very similar results. Again, the fact that 

sterilization was not effective explains the similar behavior of the "sterile" set-ups. 

Salinity did not show a significant impact on TPH removal at low crude oil 

concentrations. 

3. C2S2 and C2S I initially showed a difference in the amount of crude oil measured 

initially though the same amount of crude oil was added for both. This could be either a 

measurement error or an error when adding crude oil. TPH in C2S2 declined sharply and 

then showed similar values as for C2S 1, which makes it more likely that a measurement 

e1Tor on day I occurred. 

Hydrocarbon data showed a similar declining trend over time as volatile compounds in Figure 9. 

Comparing Figures 8 through I 0, the same result was observed; maximum oil remova l was 

2 1 
observed at high crude oil concentration and high salinity. The percentage of crude oil removal 

from the sediments over 6 weeks was: 

% removal= 100 x (TPHdayo-TPHday42)/TPHdayO 

The removal percentages shown in Figure 11 increase with increasing concentration and salinity, 

following the same trends as discussed above. 

50 

.,, 40 GI 

E 

=a 

30 

E 

2 

:: 20 

i 

I 10 

:z: 

a. 

0 

-n 

! : 

l 

,b c,'\- c,'l, 

c,""~ v'\. v'\. 

v'\i 

Sample Set up ID 

1 

f?'l, cY 

Figure 11. Percentage ofTPH removal from sediments over 6 weeks at 20°C. 

Carbon dioxide production in microcosms at 3°C 

The cumulative respiration over time is shown in Figure 12, and the following observations can 

be made from this figure. 

1. CoS 1 and CoS 1 sterile series (controls) are overlapping, which was due to ineffective 

sterilization. Both show substantial CO2 production, in the same order of magnitude as 

other experiments with crude oil addition, which suggests another carbon source was 

present in the sediments. This matches observations made at 20°C. This can also be 

confirmed by data shown in Figure 15, where organic carbon was found in the sediments, 

even when no crude oil had been added. 

2. The low crude oil concentration series ClSl and ClS2 overlapped (no effect of salinity) 

and show the highest CO2 production of all setups at 3°C. 

3. All microcosms at the lower crude oil concentration, even the C 1 S 1 sterile control 

showed higher CO2 production than C2S2 and C2S 1. This is unusual, typically CO2 

production increases with higher substrate (hydrocarbon) concentration. This could be 

because the microbes were not able to break down the complex compounds of crude into 

CO2. Inhibition or toxicity may have occurred at the higher crude oil concentration. 

4. C2S2 showed higher CO2 production compared to low salinity C2S 1. Apparently higher 

salinity had a positive impact on CO2 production for C2, as observed at 20°C. 

22 
800 

"° 700 E 

"g 600 u 

:::, 

"g 500 

a. 

0400 

u 

300 

·;:; 

"' :5 200 

E 

:::, 

u 100 

0 

0 

--ClSl 

- · - · - C1S2 

-----· ClSlsteri 

--c2s1 

- · - C2S2 

- CoSl 

········· CoSlsteri 

- -· - .-- -. -. - . - . - ..... 

10 20 30 40 so 60 70 

Time, days 

Figure 12. Cumulative CO2 production at 3°C. 

Figure 13 shows the CO2 production due crude oil mineralization for different concentrations at 

low salinity. From C 1 and C2 respiration values, the CO2 production for the control without 

crude oil was subtracted to calculate the respiration due lo crude oil mineralization. High crude 

oil concentrations (C2) clearly led to lov,1er CO2 production than for low crude oil concentration 

(C 1 ). At 3°C, the higher crude oil concentration apparently inhibited the process of CO2 

production. Some initial negative va lues (shown as zero) were calculated since Co initially had 

higher respiration than C l or C2 in some cases. A possible explanation could be that crude oil 

hindered oxygen supply. 

180 

00 160 

E 

c:.- 140 0 

.t: 120 ::s 

"C 

e 100 c.. 

0 80 u 

QJ 

-~ 60 

"' :i 40 E 

::s 

u 20 I 

0 

········· Cl st er - - - Cl exp --C2 exp 

. 

, , ---------~ I --

. / 

I II 

I' 

, I 

I \ • 

I 

j•·· 

...... -··············································· 

20 40 

Tim e, days 

60 80 

Figure 13. Cumulative CO2 production due to crude oil for low salinity at 3°C. 

Volatilization in microcosms at 3°C 

The following observations can be made based on Figure 14, which presents the amount of 

volatiles released from crude oil over 9 weeks at 3 °C. 

23 
I. CoS I control and CoS 1 sterile volatilization values were insignificant for the first four 

weeks. Since there was no crude oil present in the sediments, no volatiles were released 

from the sediments. 

2. In all setups with I mL crude oil (C ISI sterile, CISI and C1S2), volatile compounds 

released declined over time, with similar volatilization values for all tlu·ee series. 

3. Similarly, the two setups with the higher crude oil concentration, C2S I, and C2S2, 

showed initially high volatilization with a decreasing trend, though some fluctuations 

were observed especially around day 21. 

4. Salinity had no significant impact on volatilization. 

5. The amount of volatiles released increased with the amount of crude oil present. 

tlO 

800 

700 

':_ 600 C 

.g 500 11) 

400 ·.;::; 

300 

> 

..2:-200 

.... 100 

:i: 

0 

- - - c151 

- r - ( 152 

-.--- - C15l ster 

10 20 

-+-:- Co51 - C251 

+ Co51ster - 6 -· C252 

30 

Time, days 

40 so 60 

Figure 14. Amount of volatile compounds released per week from the crude oil at 3°C. 

Hydrocarbons remaining in sediments in microcosms at 3°C 

Total petroleum hydrocarbons present in the sediments at 3°C declined over time as illustrated in 

Figure 15. For data obtained at different crude oil concentrations, the following observations 

were made. 

1. TPH in the CoS 1 control and CoS 1 sterile were very low as expected. The small amount 

of TPH measured may be due to measurement error or other carbon compounds 

interfering with the measurement, as similarly observed fo r 20°C. 

2. All setups at the lower crude oil concentration, i.e., C I SI sterile control, CI SI, and C 1 S2 

showed very similar and quite low TPH values, with little change over time. Salinity did 

not show a significant impact at low crude oil concentrations. Sterilization also showed 

no effect. 

3. C2S2 and C2S I, which had the same higher concentration of crude oil, initially showed a 

difference; however, TPH values for C2S2 declined sharply, eventually approaching 

24 
C2S 1 values, which tapered off slowly over time. Since volatilization and respiration data 

for both microcosms were comparable, it appears that both microcosms did indeed 

contain the same amount of fuel and sampling bias or measurement error likely caused 

the initial difference between C2S 1 and C2S I. 

--i=- ClSl 

- I - C1S2 

6000 l - -'--- ClSlsteri 

£ .. ~ - - · - · - - - - - ~ -, 

-= 5000 C OD 

- -" ro ---.. 

e4ooo 

; ~=--

E3ooo "'C =c C 0/ 

; "' 2000 a: 

0 

' ' 

-CoSl - C2S1 

... ...:.. ... CoSlsteri - ,· - C2S2 

' ' ' ' 

0 +, - - ~ -~- ~ --~ - ~ --~-~ 

0 10 20 30 40 

Time, days 

50 60 70 

Figure 15. Total petroleum hydrocarbons remaining in sediments at 3°C. 

The percentage of TPH reduction in sediments, calculated by dividing the measured TPH after 9 

weeks by the theoretical initial amount (I mL or 5 mL of crude per kg of soil) is shown in Figure 

16, allowing the following observations: 

1. The percentage of TPH removal increased with increasing TPH levels. 

2. TPH in CoS I control and CoS I sediments shO\,ved little reduction of the already very low 

carbon levels. 

3. The C I SI "sterile" control showed comparable results to C I S I and C I S2. 

4. Salinity did not have a significant impact on TPH removal at the lower crude oil 

concentration. The maximum TPH reduction over 9 weeks occurred in C2S2, which 

exceed the percent reduction for C2S I. This shows the significant positive impact of 

salinity on the rate of crude oil degradation at higher crude oil concentration. 

5. C2S2 showed lower CO2 production compared to C I S I. On the other hand, C2S2 

revealed the maximum TPH reduction percentage at 3°C. This means that crude oil 

removal was not directly linked to mineralization, requiring other explanations. It is 

possible that fo r this setup more crude oil compounds removed from the soil were 

converted to other compounds or biomass rather than to CO2. A mass balance has to be 

established in order to better understand the fate of the crude oil. 

25 
70 

"' I 60 

E 

'5 50 

e: 

E 40 

_g 

ni 30 > 0 

E 20 

10 ... 

* 0 

Sample Set up ID 

Figure I 6. Percentage of TPH removal from sediments over 6 weeks at 3 °C. 

Carbon dioxide production in microcosms at both temperatures 

Since 20°C experiments were conducted for 6 weeks and 3°C studies were carried out for 9 

weeks, the results for both temperatures will be compared over 6 weeks. The following 

observations can be made from Figure 17, which shows the cumulative CO2 production. 

1. For each set of conditions, the CO2 production at 20°C was higher than at 3°C. These 

results are consistent with Arrhenius principle, according to which biological activity 

slows down at low temperatures. Therefore, more time will be required for the microbes 

to mineralize crude oil at lower temperatures. 

2. For both temperatures and both crude oil concentrations (C IS I vs. ClS2, and C2S l vs. 

C2S2), respiration for S2 was slightly higher than for S 1. 

3. At 20°C, respiration was a little higher for C2 than for the corresponding C 1 microcosms. 

However, at 3°C respiration in C2 was lower than in Cl. This indicates that higher crude 

concentration inhibits the conversion of crude to CO2 at 3°C. 

4. For both temperatures, CI S 1 sterile and CI SI showed similar values, suggesting 

incomplete sterilization. 

1000 2o·c 3•c 

900 .., 

E 800 

"O 700 1:: 

::, 

"C 600 e 

C. 

0 

u 

SOO 

., 

., > 400 

.. 300 ;; 

E 200 ::, 

u 

100 

0 

CoSl CoSl ster ClSl ster ClSl C1S2 C2S1 C2S2 

Figure 17. Cumulative CO2 produced in 6 weeks at 20°C and 3°C. 

26 
Volatilization in microcosms al both temperatures 

Figure 18 compares volatilization at both temperatures. The percentage volatilized was 

calculated as described above. According to literature, volatilization is slower at low 

temperatures. However, the data in the figure below show comparable volatilization percentages 

for both temperatures, in some cases even higher volatilization at the lower temperature. 

80 

ri" 

60 

I 

I I 

0 

ClSl ster ClSl 

fl 

I 

C152 

sample ID 

2o·c 3•c 

I r.EI rt· 

C251 C252 

Figure 18. Percentage of volatile compounds released during 6 weeks at 20°C and 3°C. 

Hydrocarbons remaining in sediments in microcosms at both temperatures 

Figure 19 compares the percentage of total petroleum hydrocarbons removed from the sediments 

at 20°C and 3°C. Similarly high removal percentages were achieved at 3°C as observed at 20°C. 

At both temperatures, C2S2 showed the highest percentage decline of carbon in sediments, 

which confirms the initial hypothesis that better removal is achieved at high salinity. Removal at 

3°C exceeded 50% after 6 weeks. 

60 

E 50 

0 

... 

.... 

ro 40 

> C: 0 Q) 

E E 30 

Q) · - ... ""C 

::c 20 

a. 

I10 

0 

20°c 3°c 

I 

I 

I 

I 

ClSlste r ClSl C1S2 C2S1 C2S2 

Figure 19. Percentage of TPH removal from sediments over 6 weeks at 20°C and 3°C. 

27 
Mass balance ofcrude oil in microcosms at both temperatures 

A mass balance was made for the carbon initially present as crude oil, based on carbon recovery 

in the form of carbon dioxide, volatile compounds, total petroleum hydrocarbons present in the 

sediments, and an unidentified "other" being the difference between the above three categories 

and 100%. 

The carbon from crude oil converted to CO2 was calculated after subtracting the Co control 

values from the respiration for C I and C2 samples. Since Co had some carbon content initially, 

Cl and C2 should have the same initial natural carbon content contributing towards CO2 release. 

To relate the mass of hydrocarbons consumed to the amount of CO2 produced in the experiment, 

the following generic stoichiometric equation for mineralization of hydrocarbons applies 

(Cunningham 2004). 

For icosane, C20H42 as a representative TPH for the current study, the above equation can be 

written as: 

This means the mineralization of 282 mg of hydrocarbons (1 mole icosane) leads to the 

production of 880 mg of carbon dioxide (20 moles), i.e., 0.32 mg crude were consumed per mg 

of CO2 produced. In order to calculate what percentage of crude oil was converted to CO2 the 

following equations were used: 

9 crude m ineralized 

m cr,ude min er alized = (mco2 - mcoz Co) X 0.32 CO f crud 9 2 rom e 

o/c f d t d t Co 100 mc,·ude m ing.ralized o o cru e conv er e o 2 = x 

m cruds added 

For the amount of crude initially present (mcmde added), the theoretical value based on the volume 

of crude oil added was used, i.e., 870 mg for Cl and 4350 mg for C2. 

The following equation was used to calculate the % of carbon recovered as Volatile Organic 

Compounds (VOC): 

% of cruderecov ered asVOC = 100 X m volatiles 

m c r uds added 

The percentage of Total Petroleum Hydrocarbons (TPH) rema111111g 111 the sediment was 

calculated as: 

TPHfinal % of crude remaing asTPH = 100 X ----'---

TPH init ial 

These mass balance percentages provide a better understanding of what is actually happening 

and how much CO2, VOC, and TPH were recovered under different environmental conditions. 

28 
Mass balances for both temperatures are compared m Figure 20, allowing the following 

observations: 

I. Mineralization (CO2) was the smallest of the fractions shown in the mass balance. The 

mineralization percentage at 20°C was consistently higher than at 3°C. This conforms to 

the common observation that microbial activity increases with temperature. At the higher 

crude oil dosage, a smaller percentage was minerali zed. 

2. Volatilization was quite high. The percentage volatilized was a little higher at the lower 

crude oil dosage. According to the literature, VOC release should slow down at low 

temperature. However most microcosms, especially C I S I sterile, showed higher 

volatilization at 3°C than at 20°C. VOC releases in other microcosms were similar for 

both temperatures. 

3. The remaining TPH percentage did not show a clear trend with respect to temperature. 

4. At the lower crude oil concentration, recovery was> I 00 %, which may have been due to 

the presence of some naturally occurring carbon source. The impact of that naturally 

present carbon was less noticeable in the mass balance for the higher crude oil 

concentration. 

160 -

"' Ill 140 "C 

cu 

cii 120 

> 

0 

100 ... 

C 

_g 80 ... 

Ill 

u 60 Ill 

·.;:; 

:~ 40 

-0 20 

0 

TPH-cont 

fi,,J 

20°c 3°c 20°c 3°c 2o·c 3•c 20°c 3°c 20°c 3°c 

ClSl st eri ClSl C152 C251 C252 

Figure 20. Mass balances of crude oil at 20°C and 3°C after 6 weeks. The graph shows fractions 

mineralized (CO2) , volatilized (VOC) and remaining in soil (TPH). Red line is at I 00%. 

Mini-column experiments 

The purpose of these experiments was to understand the fate of crude oil in different shoreline 

substrates. After describing results for sandy-gravel and pebble sediment, respectively, a 

comparison of sandy-gravel versus pebble was presented. In each case, two temperatures and 

different fertilizer types were compared to determine their effect on the fate of the crude oil. 

29 
For all of the above conditions ( different sediment types, temperatures, and fe11ilizer 

application), the crude oil degradation and transport were evaluated by measuring three 

parameters: the concentration of crude oil throughout the column profile, the CO2 production, 

and the release of hydrocarbon volatiles ( data not shown due to analytical problems). Studies 

perfom1ed over 3, 6, 12, and 18 days were compared to evaluate hydrocarbon movement in the 

sediment over time. 

It should be noted that fo r all graphs showing the variation of crude oil concentration, the control 

columns are not inc luded because the hydrocarbon concentration was consistently very low 

(approx. 50 mg/kg) for all experiments. 

Crude oil movement in sandy-gravel sediment 

The sandy-gravel sediment was composed of coarse grain sand, with some gravel. The sandygravel sediment was found to have a porosity of 0.359 with an expected oil penetration of 25 cm 

(NOAA 2004). Figures 21 and 22 show the average crude oi l concentration at different places in 

the column for the 20°C and 3°C studies, respectively. 

At 20°C, it is notable that all sandy-gravel columns follow the same general trend. A much 

higher concentration of crude oil resided in the middle section of the column, about 6 inches 

below the surface. This result is consistent with the classification as ESI 4, which has an 

expected maximum oil penetration of 25 cm (NOAA 2002). 

For most days, a higher concentration of crude oil was noted in the columns with no fertilizer 

added. The crude oil concentration in the column with no ferti lizer showed a consistent 

downward trend over time for the top and middle of the column, with concentrations consistently 

highest in the middle. 

The liquid fertilizer fo llows the same trend with highest concentrations observed in the middle of 

the column followed by the bottom of the column and lowest concentrations at the top. The 

increase in TPH at the top of the column from day 3 to day 6 must have been a sampling error on 

either day 3 or 6. There was an initial decline in TPH from O to 3 days, but from 3 to 12 days a 

very little additional decline in crude oil concentration is seen. Therefore, it appears that the 

addition of ferti li zer does not have a significant impact on the degradation of crude oil after 3 

days. This can be attributed to the fact that the fertilizer was in liquid form and most li kely 

washed through the entire column by day 3. 

Crude oil concentrations at 3°C did not follow the clear trend as seen at 20°C. In unfertilized 

columns, the maximal crude oil concentration occurred in the middle of the column. It also 

appears that past day 6, there was no significant decrease in crude oil concentration at the top of 

the colunm, with nearly identical concentrations on day 6, 12, and 18, indicating that no fu11her 

movement from the top of the column occurred tlu·ough the sediment. Over time, the average 

crude oil concentrations slowly decreased. Although there is a downward trend, removal did not 

occur to the same extent as at 20°C. 

30 
j 

900 

800 

700 

.§. 600 

6 500 

E 400 .,, 

> 

300 

:, 

" 200 ] 100 

-

nm No Fertilizer 

Top 

n na 

Liquid 

Fertilizer 

0 3 0 6 11 12 

- - -

- - ~ i - No Fertilizer Liquid No Fenllizer Liquid 

Fertilizer Fertilizer 

Middle Bottom 

Figure 2 1. TPH concentration at different depths after 3-1 2 days in sandy-gravel at 20°C. 

500 

j450 

..... .., 

.§. 400 

8 350 

-e 

300 0 

t2so 

::c 

E 200 ::, 

150 

100 

13 so 

0 

D 3 D 6 11 12 18 

No Liquid Solid No Liquid Solid No Liquid Solid 

Fertilizer Fertilizer Fertilizer Fertilizer Fertilizer Fertilizer Fertilizer Fertilizer Fertilizer 

Top Middle Bottom 

Figure 22. TPH concentration at different depths after 3-18 days in sandy-gravel at 3°C. 

In columns with liquid or solid fertilizer, either the top or the middle of the co lumn showed the 

highest TPH concentrations. Similar to 20°C the crude oil concentrations decreased over time, 

but it does not appear that fertilizer addition contributes to oil movement. With solid fertilizer, 

average TPH concentrations decrease at a very slow rate. The concentration at the top of the 

column remains consistently high. By day 18, the columns with solid fertilizer had the highest 

crude concentration at any level. 

Carbon dioxide release in sandy-gravel sediment 

Figures 23 and 24 show the average cumul ati ve amounts o f CO2 that were released from each of 

the co lumns over 3, 6, 12, and 18 days for 20°C and 3°C, respectively. Respiration data at 20°C 

fol lowed the generally expected trend, with a steady increase in CO2 produced over time. Day 3 

and 6 show very similar respiration fo r all 3 conditions, i.e., there was no noticeable effect o f 

crude oil or nutrient addition. The similarly high CO2 release without crude oil addition indicates 

that there must have already been a carbon source present in the so il; a similar finding was made 

by Sharma, 2015. For the first 6 days, the microbes present in the soil may still have been 

31 
utilizing the original carbon source and had not yet begun to degrade the crude oil. By day 12, 

the original carbon source may have been used up, and the microbes moved on to the crude oil 

allowing them to release an additional 20-40 mg of CO2. It is interesting to note that the liquid 

fertilizer does not appear to have as positive an impact on the release of CO2, as was expected. 

As mentioned before, this could be due to the fertilizer being flushed out of the system early on, 

and therefore no longer being present by day 12 when crude oil utilization started. It should be 

mentioned that throughout the experiment, the columns were flushed with sea water, which 

means that along with a higher salinity, there were also nutrients present in the water. So it is 

possible that the simple addition of sea water to the system was enough to stimulate the 

microbes. It is still unclear why the columns with no fertilizer released so much more CO2. 

120 

100 

; 80 ,. 

.. 60 0~ 

u 

.. 

E 40 

20 

0 

3 Day 6 Day 12 Day 

No Fertilizer liquid Fertilizer Control 

Figure 23. Cumulative CO2 released from sandy-gravel at 20°C. 

" CII 

120 

100 

80 CII 

Qi 

a: 60 N 

0 

u 40 Ill) 

E 

20 

0 

3 Day 6 Day 12 Day 18 Day 

• I I I 

No Fertilizer Solid Fertilizer liquid Fertilizer Control 

Figure 24. Cumulative CO2 released from sandy-gravel at 3°C 

The CO2 production from sandy-gravel at 3°C showed a similar trend as in the 20°C study, 

where independent of fertilization and crude oil addition almost the same amount of CO2 was 

released fo r day 3 and 6 (in the 20°C study a nearly identical release was also seen at day 12). It 

was not until day 18 that a signifi cant spike in CO2 release due to crude oil degradation was seen. 

This longer lag time can be attributed to the lower temperature. It is known that as temperature 

decreases, microbes slow down their metabolic rate and, therefore, do not need to consume their 

energy source at as quickly (Hore!, 2009). While it is higher than on day 12, it is important to 

32 
note that the control on day 18 was nearly 40 mg less than the columns that contained crude oil. 

This indicates that somewhere between the 12th and 18th day, the microbes finally depleted the 

sediments original carbon source and moved on to the crude oil. Similar to the 20°C study, the 

addition of fertilizer does not seem to play a significant role in the release of CO2. While, at day 

18, we do see a slightly higher release with the addition of solid and liquid ferti lizer, it is by less 

than 15 mg higher than in the unfe rtilized column. As mentioned earlier it is possible that the sea 

water is contributing sufficient nutrients to the mi crobes, and that the addition of fertilizer would 

be unnecessary. 

Effect of fertilization and temperature in sanely-gravel 

Figure 25 shows crude oil concentrations for varying temperatures, with no fertilizer addition (A) 

and with liquid fertilizer (B), respectively. The temperature seems to have a significant effect on 

TPH degradation and CO2 production. For both figures, it appears that more crude oil was 

recovered from the soil at the higher temperature. 

900 

oo 

..:,: 800 

-"" .§. 700 

"' <: 

0 600 

-e 

.. 

e 500 

-c 

> 

:,: 400 

E 

:, 

.. 300 

e 

.; 200 a. 

iii 

.. 100 

0 

A 

800 

~ 700 

-.. 

E 

~ &oo 

-f 500 

i 400 :,: 

§ 300 .. 

0 

200 a. 

100 

0 

B 

0 in 6 in 

3 Day 

' 

n 

0in Gin 

3 Day 

3 C D 20C 

0 in 6 In 12 In 0 in 6 In 12 in 

6 Day 12Day 

D 20C 

-

nl 

-l 

' rl- -

- "" .... -

I n 

12 in I O in Gin 12in 

I 

0in 6in 12in 

G Day 12 Day 

Figure 25. Effect of temperature on TPH concentrations in samples without (A) and with (B) 

liquid fertilizer. 

-,-, 

.).) 
At 20°C, there is a fairly clear trend of crude oil moving through the column, with only little 

TPH present in the top layer after day 6. TPH concentrations overall decreased with time but 

were nevertheless on average still higher than at 3°C. At the lower temperature, the concentration 

after 3 days was already relatively low and decreased only slightly over time. One explanation 

for this apparentl y quick loss of TPH in the first three days at 3°C could be that an initial 

movement of crude oil tlu·ough the system led to the removal of crude oil, but, considering the 

lower viscosity at low temperatures, that situation seems unlikely. Another possible explanation 

could be that the 20°C samples were extracted and analyzed within a week of the completion of 

the experiment. Whereas, the 3°C samples were stored in the -80°C freezer for about three weeks 

(up to 31 days), before extraction and then due to machine malfunction had to be stored for 

another four weeks (up to 40 days) before analysis could be completed. While extraction and 

analysis were both within the required time frames, they did have a slightly lower crude oil 

recovery from the soil, but still within the recommended recovery rate. 

Figure 26 shows how the temperature affected the release of CO2 in sandy-gravel sediment. For 

any time period, there was a higher release at 20°C than at 3°C. It is normal for mi crobes to be 

more active at higher temperatures than at lower temperatures. Nevertheless, this is interesting as 

the native microorganisms would be more adapted to low temperatures and perform well. The 

majority of the Arctic rarely reaches air temperatures of 20°C, and the average summer 

temperature is around 3°C. It can be extrapolated from the data that if an 18 day study at 20°C 

had been completed, the CO2 release would have been greater than the 110 mg released at 3°C. 

12 Day 

6 Day 

3 Day 

3 C Fertilizer 20 C Fertilizer 

o w m w ro w 

CO2 released, mg 

Figure 26. Effect of temperature on the release of CO2 from sandy-gravel with liquid fertili zer. 

Crude oil movement in pebble sediment 

The pebble sediment used was characteristic of ESI 5 sediments, which are comprised of a 

mixture of pebbles with some sand. The pebble sediment porosity was determined to be 0.307, a 

medium to high permeability with a high chance of oil penetration up to 50 cm (NOAA 2002). 

Figures 27 and 28 show the average crude oil concentration at different locations in the column 

for the 20°C and 3°C studies, respectively. A similar trend is seen at both temperatures, with a 

higher crude oil concentration at the top (0 in) and the bottom (12 in), and the lowest 

concentration in the middle. 

34 
- 700 bl) 

~600 

.§. 

.,, 500 

8 

-e 400 

"' 

300 "t:I 

> 

:I: 

E 200 ::, 

100 ... 

QI 

0.. 0 

ii 

... .. 

i!l 

'f 

QI 

... 

0 

z 

.. 

QI 

-~ 

·.; 

ai 

... 

"C 

':i 

Cl" 

:.::; 

Top 

.. 

QI 

N 

'f 

QI 

u.. 

0 

z 

0 3 1:1112 18 

ai ai 

-~ N 

... ai .. QI QI N -~ N 

.. 'f ai QI 

'f -.;; 'f QI ai QI 

... ... ... ... ... "C 

':i 

Cl" ] :.::; 

0 "t:I 

z ':i 

Cl" ] :.::; 

Middle Bottom 

Figure 27. TPH concentration at different depths after 3 - 18 days in pebble sediment at 20°C. 

800 

-;;; D 3 D 6 Ci1 12 18 

700 -

bl) 

.§. 600 

"' C 

0 500 

-e 

"' u 400 T 0 

.. 

"t:I 300 > 

:I: 

E 200 ::, 

QI 100 

.... 

QI 0 0.. 

ii .. .. QI QI ai 

-~ N 

'f 'f 'f 

ai .. ai .. .. .. QI QI QI QI N .!::! N N .!::! N 

'f 'f 'f 'f 'f 'f QI QI QI QI QI QI QI QI QI ... u.. u.. u.. u.. u.. u.. u.. u.. 

0 "t:I 

z ':i 

Cl" ] :.::; 

0 "C 0 "t:I 

2 ':i 2 ':i Cl" Cl" :.::; :.::; 

Top M iddle Bottom 

Figure 28. TPH concentration at different depths after 3 -18 days in pebble sediment at 3°C. 

At 20°C, it appears that the movement of crude oil is consistent with the ESI 5 rating. Already on 

day 3, a high concentration of crude oil was observed not only at the top but also at the bottom of 

the column, indicating that significant transport had occurred during that short time period, i.e., 

the crude oil moved freely through the column. By day 6, the concentration at all levels, but 

especially at the top had decreased, indicating that, the majority of crude oil ( ~ 75 mg/kg remain) 

had been removed from both the top and middle layers of the column. This removal could have 

been due to volatilization and/or biodegradation (discussed below). The upper two layers showed 

a further slight decrease in concentration over the next I 2 days, with a residual concentration of 

approximately 50 mg/kg. At the bottom of the column, a relatively high concentration was still 

35 
present at day 6 and 12, indicating some pooling may have occurred. By day 18, the entire 

column exhibited the same low crude oil concentration, i.e., the majority of crude oil was 

removed from the system, and the soils approached the TPH concentrations in the control 

without fuel addition ( approx. 50 mg/kg). TPH removal from the bottom layer may have been 

due to crude oil slowly being washed out of the column over time. In situ, crude oil may be 

washed back out into the ocean. 

The same trend is seen at 3 °C. The top and bottom of the column had a very high concentration 

at day 3. At the top, this concentration dropped by day 6 to about 50 mg/kg and remained at that 

level for the remainder of the study. After three days, the middle of the column showed 

concentrations ( 100 mg/kg) that were lower than at the top or bottom. Over the next 12 days, 

concentrations slowly decreased to a final concentration of 50 mg/kg. By day 18, this pooling 

effect was no longer exhibited, and the entire column exhibited the same generally low 

concentration of 50 mg/kg which is consistent with the control concentration. This means, at the 

lower temperature, crude oil had been largely removed from the column over the 18-day 

duration. As in the 20°C pebble study, the addition of fertilizers did not cause a large effect on 

the movement of crude oil through the column. However, at 3°C, it appears the solid fertilizer 

enabled TPH removal at a slightly faster rate. The liquid fertilizer also had this impact, but not to 

the same degree as the solid fertilizer. 

Carbon dioxide release in pebble sediment 

Figures 29 and 30 show the amount of CO2 that was released (average of duplicate columns) 

over 3, 6, 12, and 18 days for 20°C and 3°C, respectively. Respiration data at 20°C followed the 

general trend that was seen in the sandy-gravel sediment. Compared to sandy-gravel, where for 

the first 6 days all conditions showed comparable results (i.e., no effect of fertilizer or crude oil), 

we see this trend at day 12 for the pebble sediment. Only on day 18, the effect of crude oil 

mineralization on overall CO2 production becomes noticeable. Lower microbial numbers present 

in the pebble sediment offers one explanation for this consistent release extending as far as day 

12. A lower microbial population could be sustained by the original carbon source for a greater 

amount of time and take longer before utilizing the crude oil. The reason why fewer microbes 

might be present has to do with the structure of the sediment. Due to the greater porosity there is 

a larger amount of air space in the sediment and a smaller surface area of the sediment grains. 

Microbes, in general, favor lower porosity sediments, where they can be in contact with a greater 

amount of resources. If large gaps exist, the microbes additionally encounter the risk of being 

flushed out of the system. 

The addition of fertilizer does appear to have had a fairly significant effect on the release of CO2. 

Over time, the addition of both liquid and solid fertilizer increased the amount of CO2 being 

produced. It appears that the solid fertilizer was more effective than the liquid fertilizer. By day 

6, the columns with fertilizer released almost 20 mg (solid) and 10 mg (liquid) more CO2. At day 

18 the fertilized columns released 40 mg (solid) and 20 mg (liquid) respectively more than the 

unfertilized column, showing that fertilizer, in fact, facilitates CO2 production. 

36 
OJ 

180 

160 

140 

::l 120 OJ 

100 

o"' 80 u 

f 60 

40 

20 

0 I 

3 Day 6 Day 12 Day 18 Day 

I 

:r., I 

I I 

No Fertilizer Solid Fertilizer Liquid Fertilizer Control 

Figure 29. Cumulati ve CO2 released from pebble sediment at 20 °C. 

140 

120 

100 -

"' OJ 

-.; 80 - «: 

N 8 60 -

.. 

E 40 

20 

0 

3 Day 6 Day 12 Day 18 Day 

-1-

No Fertilizer Solid Fertilizer Liquid Fertilizer Control 

Figure 30. Cumul ative CO2 released from pebble sediment at 3°C. 

The CO2 release from pebble sediment at 3°C shows a similar trend as at 20°C, with nearly 

identical CO2 release for day 3, 6 and 12. On day 18, a much larger release of CO2 was observed 

in the columns that contained crude oil. It is interesting that both the 20°C and 3°C studies have 

nearl y identical releases for the first I 2 days. However, once the original carbon content was 

apparently depleted (around day I 2), and the microbes moved on to the crude oil, we see that the 

release of CO2 is much lower at 3°C than at 20°C. Apparently CO2 production based on the 

original carbon source was independent of temperature. However, as soon as crude o il 

mineralization commenced, the temperature began to show an effect. 

The addition of fertilizer had some impact, with a roughly 20 mg more released in the columns 

with fe11ilizer. At day 6 and 12 both fertilizers had minimal effect. By day 18 it appears that, 

unlike the 20°C study, the liquid fertilizer had a slightly larger impact on the release of CO2. 

Despite both fertilizers having a positive effect on the release of CO2, it was only to a very small 

degree. Further experiments on mineralization rates past day 18 would be needed to determine if 

fertilizer causes a significant increase that would make it a cost-e ffective option. 

Effect of fertilization and temperature in pebble sediment 

Just as in the sandy-gravel study, a higher loss of crude oil was observed in the 20°C study 

compared to the 3°C study, although the trends are almost identical. The biggest difference 

37 
between the two temperatures was seen at day 3. However, by day 6 there was no real 

discernable difference between the two studies. It appears that crude oil movement in pebble 

sediment is not greatly affected by the change in viscosity and available pore size as was seen in 

the sandy-gravel studies. This makes sense, as pebble sediments generally have a much larger 

pore size, which allows water to drain quickly thrnugh the system. 

In an environment with no fertili zer addition (Figure 31 ), the TPH concentrations in the sediment 

are generally slightly lower at 20°C than at 3°C. This means at the warmer temperature a slightly 

greater loss of crude oil from the system takes place, especially during the first days; however, 

there was no significant difference between TPH at 3°C and 20°C. In contrast to the sandygravel sediment, a much small er difference between the two temperatures was observed. Pebble 

sediment has a much larger pore space, which allows for the crude oil to penetrate more freely. It 

is apparent that temperature had no significant impact on the movement of crude oil. Figure 32 

shows the movement of oil in the presence of ferti lizer. Just as with the previous figure, 

temperature does not appear to have a significant impact on the movement of crude oil. 

700 

0 20( 

600 

1-

-.. 500 

.§_ 

"' C 

0 400 ..Q 

;. 

2 300 

"' :,: 

E 200 ::, 

.. 

0 

:, 100 

C. " 

;; 

;§ 0 

Din 

Figure 31. Temperature effects on crude oil movement in pebble sediment with no fertilizer. 

700 

j 

020 C Liquid 3 C Liquid 

- 600 O20C Solid 00 3 C Solid 00 

.§. 

.,, 

g 500 

-E 

.. 400 ,, 

>-

::c 300 E 

::, 

.. 

e 200 

; 

0. 

]! 100 

0 

o In 6 In 12 In o In 6 In 12 In o In 6 In 12 In O In 6 In 12 In 

3 Day 6 Day 12Day 18Day 

Figure 32. Temperature effects on crude oil movement in pebble sediment with fertilizer. 

38 
Respiration 

Figure 33 illustrates the release of CO2 and its dependency on temperature. The graph shows 

CO2 production for solid fe rtilizer (light lines) and liquid fertilizer (dark lines) as a function of 

time. For the first six days, the release of CO2 is nearly identical for all temperatures and 

ferti lizers. At day 12, we begin to see a significantly higher release at 20°C. Recall that it was 

concluded that, at day 12, the organisms had finally consumed the originally present natural 

carbon source and had moved on to the crude oil. This caused a significant increase in CO2 

release, especially at 20°C. At day 18, it was very clear that the warmer temperature showed a 

much greater release of CO2 over time. 

This trend makes sense because microbes require a higher amount of substrate at warmer 

temperatures and more readily degrade contaminants due to increased metabolic rates. It can be 

concluded that over the course of the first 12 days, as long as respiration was due to the natural 

carbon source, the temperature had no significant impact. However, once the microbes had to 

move to a new foreign substrate, the higher temperature led to a faster mineralization rate. 

18 Day 

iii :c;rn 

12 Day 

QI 

E 

i= 

6 Day 

.~;;;;~ 

3 Day 

0 

El 

so 

!S!!S!S!I ;m: 51 

3 Solid Fertilizer 

20 Solid Fertilizer 

3 Liquid Fertilizer 

!!! 20 Liquid Fertilizer 

100 

CO2 released, mg 

150 200 

Figure 33. Temperature effects on the release of CO2 from pebble sediment with fertilizer. 

Comparison of sediment types at 20 °C 

Figures 34 A and B show the distribution of crude oil in the column at 20°C, for both sediment 

types with no fertilizer addition. As already discussed above, the sandy-gravel sediment 

accumulated oil in the middle of the column, whereas pebble sediment showed a higher 

concentration initially at the top and bottom of the column. In a natural environment, it appears 

that crude oil would remain in the sandy-gravel sediment for a much longer time frame. By day 

6, the oil concentrations in pebble sediment have decreased significantly, where sandy-gravel 

contained 700 mg/kg or 10 times as much crude oil as the pebble sediment in the middle section. 

39 
By day 12, the TPH values for sandy-gravel become closer to pebble sediment in the top and 

bottom sections of the columns but were still significantly higher in the middle. 

Figure 34B shows the movement of crude oil at 20°C in the presence of liquid fertilizer. The 

same general trend as described above is seen. But it should be noted that the initial 

concentration on day 3 at the top is significantly different. There is a surprisingly large amount 

of crude oil located on the top of the pebble compared to the sandy-gravel. 

900 

-;. 

800 

"" .S 700 

"' C 

_g 600 

ii 

500 

"C 

400 

E al 300 

°g 200 ., 

II. 

"! 100 

0 

A 

800 

j 

';;. 700 

.s 

600 

..Q 

ii 500 

"C 

400 

E 

al 300 

g 

:_ 200 

100 

0 

B 

D Sandy Pebble 

Din 6in 12in 

3 Day 

D Sandy Pebble 

Figure 34. Impact of sediment type on crude oil movement at 20°C without (A) fertilizer and 

with (B) liquid fertilizer. 

Figure 35 shows how the release of CO2 was impacted by the type of sediment. For the first 3 

days, the pebble sediment showed a higher release of CO2, which is surprising. This could be 

related to the fact that at 20°C, a higher concentration of oil is found at the top layer during the 

first 3 days. However, microbes should still be consuming their natural carbon source and the 

crude oil should not be having a significant impact at this time, so the cause of the higher 

respiration in pebble sediment is not clear. By day 6, a much higher CO2 release occurred for the 

sandy-gravel. One explanation for this could be that higher concentrations of microbes are often 

40 
present in finer grained material. Additionally, the concentration of crude oil in sandy-gravel was 

higher, i.e., an increased amount of carbon source was available to the microbes. 

12 Day 

6 Day 

3 Day 

0 20 40 60 80 

CO2 released, mg 

Sandy Gravel Pebble 

Figure 35. Impact of sediment type on CO2 release at 20°C with liquid fertilizer. 

Comparison of sediment types at 3°C 

Figures 36A and B show the crude oil distribution at 3°C, without and with liquid fertilizer 

addition, respectively. A similar trend as seen at 20°C is shown. There is higher crude oil 

concentration for pebble sediment over the first 3 days. However, there is a smaller difference 

between the concentration levels over time. From day 6 on, the crude oil concentration at the top 

and middle in sandy-gravel was a multiple of that for pebble. Sandy-gravel and pebble shO\,ved 

similar concentrations at the bottom of the columns, with a decreasing trend over time. 

Different results were observed with the addition of solid fertilizer. Unlike previous results, 

Figure 36C shows that, after 3 days, sandy-gravel had a much higher TPH concentration on the 

top than pebble. Concentrations in pebble were extremely low after the first 3 days in the top and 

middle sections of the column, with the highest crude oil concentrations at the bottom of the 

column, where it also reached background (2 mg) levels after 18 days. By day 6 similar trends as 

for the other ferti lization regimes are shown, with lower concentrations in pebble sediment, and 

TPH values in sandy-gravel remaining relatively high in the top and middle sections. 

4 1 
700 

j 

600 

.§. 

"' 500 g 

.c 

"' 400 

-i; 

300 

E 

::, 

Cl.I 200 

.. 

Q,I 

c. 100 

l! 

0 

A 

450 

j 400 

-l>O .§_ 350 

300 0 

250 u 

0 

200 

::c 

E 150 ::, 

Q,I 

o 100 

.ti 

Q,I 

c. 50 

0 

B 

500 

]i 450 

-l>O 

.§. 400 

350 0 

300 

250 "t:I 

> 

::c 200 

E 

150 

'al 100 

C. 

C 

50 

0 

D Sandy Pebble 

0in Gin 12in 0in Gin lZin 0in Gin lZin 0in Gin lZin 

3 Day G Day 12 Day 18Day 

D Sandy Pebble 

0in Gin 12in 0in Gin lZln 0in Gin 12in 0in Gin 121n 

3Day G Day 12 Day 18Day 

D Sandy Pebble 

Oin Gin 12in 0in Gin 12in 0ln Gin lZin 0in Gin lZin 

3 Day G Day 12 Day 18 Day 

Figure 36. Impact of sediment type on crude oil movement at 3°C without fertili zer (A), with 

liquid fertilizer (B), and with solid fe11ilizer (C). 

42 
Figure 37 shows the release of CO2 in fertilized sediment at 3°C. Similar to the CO2 release at 

20°C, for the first 3 days, the pebble sediment showed a higher release of CO2. However unlike 

20°C, at 3°C the pebble crude oil concentration was not significantly greater than the one in 

sandy-gravel, so a highe r concentration of crude oil could not explain this higher release. By day 

6, the two sediments had released nearly identical rates and over the next 12 days there was a 

higher release of CO2 in the sandy-gravel sediment. While it does appear that sandy-gravel had a 

higher release of CO2, the difference was less than 20 mg. The effectiveness of the different 

fertilizer types is also interesting. In sandy-gravel sediment, it appears the solid fertilizer had a 

slight edge, where the liquid fertilizer seemed more effective for pebble sediments. While it 

appears that each sediment has a slight preference in type of fertilizer applied, it did not appear to 

have a significant impact. 

3 Day 

0 20 40 60 

Sandy Gravel Solid 

Sandy Gravel Liquid 

D Pebble Solid 

D Pebble liquid 

80 100 

CO2 released, mg 

120 

Figure 37. Impact of sediment type on CO2 release at 3°C with fertilizer. 

CONCLUSIONS 

Microcosms 

Controls without crude oil addition 

1. There was some carbon naturally present in the Barrow sediments. Therefore, CoS 1 and 

CoS 1 sterile, both of which had no crude oil added, showed substantial CO2 release. CoS I 

released 560 mg CO2 at 20°C and 522 mg CO2 at 3°C. 

Effect of microbes 

I. Crude oil degradation by naturally present microbes occurred, as evident from CO2 

production, confirming hypothesis I. 

2. The CoS I sterile control released a similar quantity of carbon dioxide, i.e., 648 mg CO2 

at 20°C and 516 mg CO2 at 3°C as the unsterilized control. This shows that sterilization 

was not properly executed. It is advised that a biological hood should be used for the 

sterile microcosms to prevent cross contamination. 

43 
Effect of crude oil concentration 

I. Samples with higher crude oil concentration did not produce a significantly higher 

amount of CO2 at either temperature. At 3 °C, mineralization was even somewhat lower 

for C2 than for C 1, showing that complete mineralization is difficult in higher 

contamination levels. Hypothesis 2a, which stated that a higher crude oil concentration 

would increase the rate of mineralization was therefore proven wrong. In the mass 

balance, relative mineralization percentages were lower for higher crude oil 

concentrations confirming the initial hypothesis 2b. 

2. VOC release was higher for C2 than for CI, i.e., more volatilization took place at higher 

crude oil dosages, confirming hypothesis 3. However, the volatilization percentage was 

lower for C2. 

3. At higher crude oil dosages, more TPH remained in the sediments, both in absolute and 

relative terms, confirming hypothesis 4. 

Effect of temperature 

1. Temperature played an important role in mineralization of crude oil. Higher CO2 

production was observed at 20°C, than at 3°C, confirming hypothesis 5. 

2. Also, the amount of TPH remaining in the sediment was higher at the lower temperature, 

especially for high fuel dosages, confirming hypothesis 6 that higher temperatures 

increase the microbial activity and lead to better crude oil removal. 

3. Surprisingly, similar VOC production was noted for both temperatures, contradicting 

hypothesis 7; literature describes that volatilization is lower at low temperatures. 

Effect of Salinity 

I. According to hypothesis 8, salinity has a positive impact on hydrocarbon degradation 

rates, which was confirmed by the results at 20°C and 3°C. At both temperatures, S2 

samples displayed higher respiration and volatilization as well as lower TPH. Literature 

supports that at a high temperature increased salinity leads to higher oil degradation rates. 

Based on the present research it can be concluded that the same effect can be seen at 

lower temperatures. 

Mass balance 

I. According to the mass balance, only a small fraction was mineralized, most TPH removal 

was due to volatilization rather than biodegradation. 

2. Overall it can be concluded that environmental factors like temperature, crude oil volume 

and salinity all impact the rate of crude oil degradation in laboratory experiments and are 

expected to do so in real oil spill accidents. 

44 
Mini-columns 

The experiments showed that the movement of crude oil is overall not dependent on temperature. 

Rather, sediment type had a significant impact on the fate of the crude oil. While fertilizer 

application can increase CO2 production, this may not be at a significant enough level to justify 

the cost of application. The following summary can be made for each sediment type and each 

temperature. 

Sandy-gravel sediment 

1. TPH persisted 6 inches below the surface for first 12 days 

2. Overall lower oil concentrations at 3 °C 

3. By day 12, TPH concentrations were roughly equal at both temperatures 

4. Higher crude oil retention than in pebble sediment (at both temperatures) 

5. Higher respiration than in pebble sediment, i.e., more biodegradation in sandy-gravel 

6. Higher CO2 release at 20°C 

Pebble Sediment 

1. TPH persists at the top and bottom of column 

2. Nearly identical TPH movement for both temperatures. By day 18, TPH concentrations 

were roughly equal at both temperatures 

3. Overall much lower TPH levels than in sandy-gravel 

4. Higher CO2 released at 20°C starting at day 12 

5. Liquid Fertilizer was most effective after day 12 

Temperature at 20° C 

1. No large difference between sediment types 

2. Liquid fertilizer is more effective than solid fertilizer in Sandy-Gravel 

3. Solid fertilizer is more effective than liquid fertilizer in Pebble 

Temperature at 3° C 

1. Pebble sediment had initial higher CO2 release 

2. Sandy-gravel had significantly higher CO2 release after day 3 

3. Neither sediment-type nor fertilizer-type had a significant impact on CO2 release 

45 
RECOMMENDATIONS 

While this experiment has provided some beneficial insight on how fe1tilizer and temperature 

can affect the fate of crude oil on an Arctic shoreline, frnther research would he lp explain the 

observed results and address open questions. 

To better understand the influence of salinity, crude oil concentration, and temperature, 

experiments with longer incubation periods could be performed. A longer timeframe will help to 

determine what quantities of TPH would remain in the sediments in the long run. 

Nutrient addition in regular time intervals might help maintain the microbial population in the 

exponential growth phase. This could increase the rate of degradation. 

A different technique should be used for monitoring the number of microbes present in the 

sediments to provide an accurate number. 

It would be useful to perform experiments with saturated versus unsaturated sediments and 

investigate the effect of saturation level on oxygen supply (aerobic vs. anoxic) and resulting 

degradation rates. 

Additional wave tank studies and the ability to collect and analyze the sediment would greatly 

help to show how the swash and backwash affect the crude oil movement. 

Larger columns that extend at least 50 cm (the proposed depth that ESI 5 sediment will penetrate 

to) would be extremely useful. 

Future studies should measure the quantities of nutrients washed out, and if other types of slow 

release fertilizer would be more effective. 

ACKNOWLEDGEMENTS 

This research was funded through the University of Alaska Coastal Marine Institute and the 

Bureau of Ocean Energy Management cooperative agreement number Ml 4AC00020. Flint Hills 

provided the crude oil. Financial suppo1t for the initial phase of this research was provided by a 

USGS NIWR grant and funds for supplies from the UAF Water and Enviro1m1ental Research 

Center (WERC). WERC lab manager Shane Billings helped MS students, Anna Iverson and 

Priyamvada Sharma, with analytical methods and data analysis. Andy Chamberlain helped with 

collecting seashore sediments from BmTow, Alaska. 

46 
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49 
APPENDIX 

MPN results 

MPN results were obtained using the EPA MPN calculator after visual identification of positive 

wells (Table A.1 ). As shown in Figure A.1 , the data do not show a clear trend over time and do 

not fo llow the growth kinetics anticipated from the review of prior studies. 

The "sterile" microcosms contained similar quantities of hydrocarbon degraders as the other 

microcosms. This means sterilization by autoclaving had no lasting effect. Either the autoclaving 

was not effective in the first place, and/or microbes present in the ambient air colonized the 

microcosms. The latter is quite likely since no biological hood was available for these 

experiments. During daily CO2 measurements, microcosms had to be opened, whi ch could have 

allowed microbes to enter the sterile microcosms. 

Table Al. MPN values of number of microbes per gram (MPN/g) of soil at 20°C. 

Days CoSl con CoSl ster ClSl ster ClSl exp C1S2 exp C2S1 exp C2S2 exp 

14 0 370 11 989 1053 466 9328 0 

28 

42 

0 

0 

466 

105 

1000000 

100000 

E 

-10000 

1000 

11 9893 

7344 

734 

10532 

-e-C2S1 exp -El · C2S2 exp 

ClSl exp - t:,- C1S2 exp 

- -A--ClSl ster ---::.:--- CoSl ster 

11 9893 

1894 

7344 

1913 

100 -1---~----r----.-------r~ --~ 

20 C 

0 10 20 30 

Days 

40 so 

Figure Al. MPN of crude oil degraders from 20°C experiments. 

360 

310 

50 
A fu rther potential error source was the difficulty of identifying the color change indicating 

positive samples. Figure A.2 shows an MPN plate using crude oil as a carbon source at 20°C. 

Due to the presence of a dark surface layer of crude oil, it was difficult to visually identi fy the 

positive wells. 

Therefore, diesel was used as a carbon source for 3°C study. An example of a well plate using 

sampl es from 3°C experiments incubated at 10°C with diesel as a carbon source is shown in 

Figure A.3. A temperature of 10°C was used to incubate well plates from the 3°C experimental 

study because incubation at 3°C may not have resulted in color change w ithin the incubation 

period. However, even at 10°C, only one well developed a pink color (i.e., positive for diesel 

degraders) which is not a good representative of the number of microbes in the sediments. 

Therefore, no MPN can be reported for the 3°C experiments. 

The absence of color change in wells could be due to the lower temperature where the microbes 

would not yet degrade the diesel in the Bushnell medium since lag phases are typically longer at 

low temperature (Hore! 20 I I). It is likely that microbes were inactive during the incubation 

period, because after their incubation at low temperature, these plates were moved to a hazardous 

waste cabinet at room temperature, where all wells changed to a pink color within a fe'vv days. 

This shows that microbes must have been present, and those microbes became active after being 

discarded, consumed the diesel, and caused a color change. Therefore, no data are available for 

3°C. For future studies, it is recommended to change the protocol, possibly by using longer 

incubation periods or higher incubation temperatures, to ensure that microbes become acti ve 

during incubation. 

0 (..,.Sz. c:-.p .,,._ "J C ,. .s, c.-; p ct. 

Figure A2. MPN plate fo r the CI S2 and C2S l samples at 20°C with crude oil as C source. 

51 
Figure A3. MPN plate for C2S 1 and C2S2 samples at 3°C with diesel as C source. 

Wave tank photographs 

Figures A4-A8 show the wave tank experiment. 

Figure A4. Addition of pebble sediment 

52 
Figure AS. Addition of sandy-gravel sediment 

Figure A6. Waterline on sediments. 

53 
Figure A 7. Wave tank in action. 

Figure A8. Sequence of steps in wave tank experiment (a) Sediment before water add ition, (b) 

sediment with water, and (c) sediment with water and crude oil. 

54 
BOEM 8 u1.u.u Of OcCA.'l ENERGY M .1.NAGtMrnr 

The Department of the Interior Mission 

As the Nation's principal conservation agency, the Department of the Interior has 

responsibility for most of our nationally owned public lands and natural resources. This 

includes fostering the sound use of our land and water resources, protecting our fish, 

wildlife and biological diversity; preserving the environmental and cultural values of our 

national parks and historical places; and providing for the enjoyment of life through 

outdoor recreation. The Department assesses our energy and mineral resources and 

works to ensure that their development 1s in the best interests of all our people by 

encouraging stewardship and citizen participation in their care. The Department also 

has a major responsibility for American Indian reservation communities and for people 

who live in island communities. 

The Bureau of Ocean Energy Management 

The Bureau of Ocean Energy Management (BOEM) works to manage the exploration 

and development of the nation's offshore resources in a way that appropriately balances 

economic development, energy independence, and environmental protection through oil 

and gas leases, renewable energy development and environmental reviews and studies.