United States
Environmental Protection
Agency
EPA/600/R-17/273 | July 2017 j www.epa.gov/research
Characterization and Behavior of Cold
Lake Blend and Western Canadian
Select Diluted Bitumen Products
Office of Research and Development
National Risk Management Research Laboratory
Land and Materials Management Division
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July 2017
Characterization and Behavior of Cold Lake Blend and
Western Canadian Select Diluted Bitumen Products
by
Robyn N. Conmy, Ph.D.
U.S. EPA/National Risk Management Research
Laboratory/Land and Materials Management Division,
Cincinnati, OH 45268
Mace G. Barron, Ph.D.
U.S. EPA/National Health and Environmental Effects Research
Laboratory/Gulf Ecology Division, Gulf Breeze, FL 32561
Jorge SantoDomingo, Ph.D.
U.S. EPA/National Risk Management Research
Laboratory/Water Systems Division, Cincinnati, OH 45268
Ruta Deshpande, M.Sc.
Pegasus Technical services, Inc. Contractor to U.S.
EPA/National Risk Management Research Laboratory,
Cincinnati, OH 45268
Contract Number EP-C-15-010
Project Officer: Robyn N. Conmy
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July 2017
Notice/Disclaimer
The U.S. Environmental Protection Agency, through its Office of Research and Development,
funded and conducted the research described herein under approved Quality Assurance Project
Plans. It has been subjected to the Agency's peer and administrative review and has been
approved for publication as an EPA document. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
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Foreword
The U.S. Environmental Protection Agency (US EPA) is charged by Congress with protecting the
Nation's land, air, and water resources. Under a mandate of national environmental laws, the
Agency strives to formulate and implement actions leading to a compatible balance between
human activities and the ability of natural systems to support and nurture life. To meet this
mandate, US EPA's research programs are providing data and technical support for solving
environmental problems today and building a science knowledge base necessary to manage our
ecological resources wisely, understand how pollutants affect our health, and prevent or reduce
environmental risks in the future.
The National Risk Management Research Laboratory (NRMRL) within the Office of Research
and Development (ORD) is the Agency's center for investigation of technological and management
approaches for preventing and reducing risks from pollution that threaten human health and the
environment. The focus of the Laboratory's research program is on methods and their cost-
effectiveness for prevention and control of pollution to air, land, water, and subsurface resources;
protection of water quality in public water systems; remediation of contaminated sites, sediments
and ground water; prevention and control of indoor air pollution; and restoration of ecosystems.
NRMRL collaborates with both public and private sector partners to foster technologies that reduce
the cost of compliance and to anticipate emerging problems. NRMRL's research provides solutions
to environmental problems by: developing and promoting technologies that protect and improve
the environment; advancing scientific and engineering information to support regulatory and
policy decisions; and providing the technical support and information transfer to ensure
implementation of environmental regulations and strategies at the national, state, and community
levels.
Cynthia Sonich-Mullin, Director
National Risk Management Research Laboratory
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Acknowledgements
July 2017
We thank the staff and management of Pegasus Technical Services and Hydrosphere for technical
and administrative support. In addition to the authors, contributors to this research and report
include Edith Holder, Robert Grosser, Devi Sundaravadivelu, Craigs Watts, Peter Meyer, Michael
Elk, Vikram Kapoor, Stephen Techtmann, Raghu Venkatapathy, and Marilyn Dapper. This
research was supported in part by an appointment to the ORISE participant research program
supported by an interagency agreement between the U.S. EPA and the U.S. Department of
Energy. Research was funded through the Oil Spill Liability Trust Fund. The conclusions may not
necessarily reflect the views of the EPA and no official endorsement should be inferred. This
research supports the Agency's regulatory authority under the Clean Water Act and the Oil
Pollution Act.
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July 2017
Table of Contents
Notice/Disclaimer ii
Foreword iii
Acknowledgements iv
List of Figures vii
List of Tables ix
Acronyms and Abbreviations xi
Executive Summary xiii
1. Introduction 16
2. Methods 22
2.1. Diluted Bitumen Dispersion Effectiveness 22
2.1.1. Baffled Flask Test Approach 22
2.1.2. Analysis of Extracts 23
2.1.3. Droplet Size Distribution 23
2.2. Diluted Bitumen Biodegradation Study 24
2.2.1. Chemicals and Reagents 24
2.2.2. Media 24
2.2.3. Microbial Enrichment 24
2.2.4. Microcosm Design 24
2.2.5. Oil Extraction and Analysis 25
2.2.6. DNA Extraction and Sequencing 26
2.3. Diluted Bitumen Toxicity to Standard Aquatic Test Species 26
2.3.1. Test Organisms, Conditions and WAF Preparation 26
2.3.2. Analytical Chemistry 27
2.3.3. Statistical Analyses 28
2.4. Quality Assurance Summary 30
3. Results and Discussion 31
3.1. Chemical and Physical Characterization of Source Oil 31
3.2. Dispersion Effectiveness 33
3.3. Dilbit Biodegradation 34
3.3.1. Alkane and PAH Biodegradation by Kalamazoo River Cultures 34
3.3.2. Dilbit Biodegradation by Anderson Ferry Culture 35
3.3.3. Microbial Community Structure during Dilbit Biodegradation 37
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July 2017
3.4. Toxicity of Dilbit to Standard Aquatic Species 45
3.4.1. Oil and WAF Chemistry 45
3.4.2. Acute and Chronic Dilbit Toxicity 46
4. Report Summary 52
5. References 53
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List of Figures
Figure 1.1. Saturate-Aromatic-Resin-Asphaltene (SARA) composition for different oils. Cold Lake Blend
and Western Canadian Select are specific diluted bitumen products. Source: National Academies of
Sciences, Engineering, and Medicine. Spills of Diluted Bitumen from Pipelines: A Comparative Study
of Environmental Fate, Effects, and Response 17
Figure 1.2. Existing and proposed Canadian and U.S. oil pipelines for dilbit transport. Source: Canadian
Association of Petroleum Producers, also found at https://www.nap.edu/read/21834/chapter/3ffl2.
19
Figure 2.1. Acute and chronic test species: Ceriodaphnia dubia (C. dubia ; top left); Pimephales promelas
(fathead minnow; top right); Americamysis bahia (mysid; bottom left); Menidia beryllina (inland
silverside; bottom right) 27
Figure 3.1. Hydrocarbon composition for Cold Lake Blend (CLB) and Western Canadian Select (WCS)
dilbits. Source: Deshpande et a!., 2017 32
Figure 3.2. Droplet size distribution and particle concentration of Cold Lake Blend (CLB) and Western
Canadian Select (WCS) dilbit dispersed in water with and without chemical dispersants (COR:
Corexit 9500; FIN: Finasol OSR 52; NONE: no dispersant) 34
Figure 3.3. Alkanes, PAH and TPH concentration at 25 and 5 °C. KRE = Kalamazoo River Enrichment; AFE
= Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select. Data
Sources: Deshpande, 2016; Deshpande et a!., 2017 40
Figure 3.4. Alkanes, PAH and TPH percent removal at 25 and 5 °C. Figure 3.2. Alkanes, PAH and TPH
concentration at 25 and 5 °C. KRE = Kalamazoo River Enrichment; AFE = Anderson Ferry
Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select. Data Sources: Deshpande,
2016; Deshpande et al., 2017.Data Sources: Deshpande, 2016; Deshpande et al., 2017. 41
Figure 3.5. Percent removal of individual PAH compounds. Source: Deshpande etaL, 2017. 43
Figure 3.6. Microbial Community Structure Analysis during oil biodegradation experiments. KRE =
Kalamazoo River Enrichment; AFE = Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS =
Western Canadian Select; PBC = Prudhoe Bay Crude. Data Sources: Deshpande, 2016; Deshpande
et al., 2017. 44
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July 2017
Figure 3.7. Concentration of individual PAH analytes and alkyl homologs in water accommodated
fractions (WAF) of fresh (unweathered) and weathered Cold Lake Blend (CLB) and Western
Canadian Select (WCS) dilbits. FW = freshwater; SW = saltwater. Source: Barron et ai, 2017 46
Figure 3.8. Percent survival of four aquatic species exposed to water accommodated fractions of fresh
and weathered dilbits: Cold Lake Blend (CLB), weathered CLB (wCLB), Western Canadian Select
(WCS), and weathered WCS (wWCS). Test concentrations: BTEX (mg/L); PAH (ug/L); TPH (mg/L).
Test species: Ceriodaphnia (Ceriodaphnia dubia); mysid (Americamysis bahia); fathead minnow
(Pimephales promelas); inland silverside (Menidia beryllina) 49
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List of Tables
Table 1.1 Trace element concentrations in various heavy crude oil types. Source: American Petroleum
Institute; 2011 White Paper; Pipeline transportation of diluted bitumen form the Canadian Oil
Sands 18
Table 2.1. Summary of Biodegradation Experimental Design 25
Table 2.2. Summary of acute and chronic test conditions for four aquatic species: Ceriodaphnia
(Ceriodaphnia dubia), mysid (Americamysis bahia), fathead minnow (Pimephales promelas), and
inland silverside (Menidia beryllina). Data and Table Source: Barron et al., 2017 29
Table 3.1. Physical and chemical properties of CLB and WCS diluted bitumens. Characterization was
conducted by Maxxam Analytical International Corp. (Petroleum Technology Center, Edmonton,
Canada) 32
Table 3.2. Dispersant Effectiveness using the BFT (Baffled Flask Test) expressed by the LCL95 for CLB and
WCS using Corexit 9500 and Finasol OSR 52 at 5 and 25 °C 33
Table 3.3 Percent removal and first order rate constants for total alkanes, total PAHs and TPH
degradation. KRE = Kalamazoo River Enrichment; AFE = Anderson Ferry Enrichment; CLB = Cold
Lake Blend; WCS = Western Canadian Select; PBC = Prudhoe Bay Crude. Data Sources: Deshpande,
2016; Deshpande et al., 2017. 39
Table 3.4. Oil biodegradation rate comparison matrix. KRE = Kalamazoo River Enrichment; AFE =
Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select. Red box
represents significant difference between treatment and green box represents no significant
difference based on ANOVA p values. Data Sources: Deshpande, 2016; Deshpande et al., 2017..... 42
Table 3.5. Hydrocarbon concentrations1 and percent survival of four species in 100% water
accommodated fractions (WAF) of two unweathered and weathered dilbits2 at 24 hours and test
end (48 hr invertebrates; 96 hr fish). Source: Table generated from Barron et al., 2017 data 45
Table 3.6. Acute toxicity of fresh and weathered Cold Lake Blend and Western Canadian Select dilbit to
four species as three measures of hydrocarbon exposure.1 Data source: Barron et al., 2017 48
Table 3.7. Short-term chronic toxicity of two weathered dilbits to C. dubia and A. bahia as three
measures of hydrocarbon exposure.1 Source: Barron et al., 2017. 50
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July 2017
Table 3.8. Acute toxicity of Alaskan North Slope crude oil to four species as three measures of
hydrocarbon exposure.1 Data source: Barron et ai, 2017 51
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July 2017
Acronyms and Abbreviations
AFE
Anderson Ferry Enrichment
ANOVA
Analysis of Variance
ANS
Alaskan North Slope Crude Oil 521
API
American Petroleum Institute
Bbl
Billion Barrels
BFT
Baffled Flask Test
BTEX
Benzene-Toluene-Ethylbenzene-Xylene
Cryo
5°C microbial cultures
CLB
Cold Lake Blend
CHY
Chrysenes
DCM
Dichloromethane
DE
Dispersion Effectiveness
Dilbit
Diluted Bitumen
DOR
Dispersant-to-Oil Ratio
DSD
Droplet Size Distribution
EPA
Environmental Protection Agency
FID
Flame Ionization Detector
GC
Gas Chromatography
HMW
High Molecular Weight
IC25
25% Inhibition Concentration
KRE
Kalamazoo River Enrichment
LC20
Lethal Concentration to kill 20% of the population
LC50
Lethal Concentration to kill 50% of the population
LCL95
Lower Confidence Level 95 %
LDR
Linear Dynamic range
LISST
Laser In Situ Scattering and Transmissometry
Meso
25°C microbial cultures
MRM
Multi Reaction Monitoring
MSD
Mass selective Detector
NELAC
National Environmental Laboratory Accreditation Conference
NAP
Naphthalenes
NAS
National Academies of Science, Engineering & Medicine
NOEC
No Observable Effect Concentration
ORD
Office of Research and Development
PAH
Polycyclic Aromatic Hydrocarbons
PBC
Prudhoe Bay Crude
PHE
Phenanthrenes
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PHMSA
QAPP
SARA
SCO
SOP
Synbit
TPH
WAF
WCS
July 2017
Pipeline and Hazardous Materials Safety Administration
Quality Assurance Project Plan
Saturates-Aromatics-Resins-Asphaltenes
Synthetic Crude Oil
Standard Operating Procedure
Synthetic Bitumen
Total Petroleum Hydrocarbons
Water Accommodated Fraction
Western Canadian Select
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July 2017
Executive Summary
Unconventional diluted bitumen (dilbit) oil products present an increasing environmental
concern because of extensive transport in North America, recent spills into aquatic habitats, and
limited understanding of environmental fate and toxicity. Dilbits are blends of highly weathered
bitumen and lighter diluent oils that contain higher concentrations of asphaltenes (>10%) and
lower levels of saturates (~40%). They have unique properties, including high adhesion, and the
potential for rapid weathering, sinking and associating with sediments. Information on dilbit
biodegradation, toxicity, dispersion and fate is limited and warrants further study, particularly
given diversity in dilbit types and weathering state. Recent reviews produced in collaboration by
government agencies, the National Academies of Science, academics and industry highlight the
pressing need to better understand the behavior and potential impacts of dilbit spills over land
and water. To address knowledge gaps pertaining to the behavior, fate and effects of select
fractions of spilled diluted bitumen, this report summarizes research conducted within the US
EPA ORD on the chemical characterization, dispersion effectiveness, biodegradation and toxicity
of two types of diluted bitumen- Western Canadian Select and Cold Lake Blend. These studies
are also being published in Deshpande, 2016; Deshpande et a I, 2017; Barron et al., 2017. The
objectives of this research were to:
1. Characterize Cold Lake Blend (CLB) and Western Canadian Select (WCS) dilbit products
(unweathered) via chemical and physical properties and dispersion effectiveness.
2. Evaluate the biodegradation of alkanes and aromatics within CLB and WSC dilbit products
as function of two temperatures (5 and 25 °C) using two freshwater cultures: one
consortium acclimated to dilbit (Kalamazoo River Enrichment, KRE) and the other
consortium enriched on soil contaminated with conventional hydrocarbons from the Ohio
River (Anderson Ferry Enrichment, AFE). An analysis of microbial community structure via
genomic sequencing was also conducted.
3. Determine the acute and sublethal toxicity of unweathered and weathered CLB and WCS
dilbit to standard aquatic test organisms: the freshwater invertebrate Ceriodaphnia
dubia, the freshwater fish Pimephales promelas (fathead minnow), the saltwater
invertebrate Americamysis bahia (mysid), and the saltwater fish Menidia beryllina (inland
silverside). Conventional slow-stir water accommodated fractions (WAF) and static or
static renewal methods were used to allow comparison to the broader literature on the
toxicity of oil products.
This research will allow for better informed risk assessments and improved emergency response
during dilbit spills in aquatic and terrestrial environments.
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July 2017
Results of our study suggest that select fractions of Western Canadian Select (WCS) and Cold Lake
Blend (CLB) dilbit can be efficiently biodegraded, but under similar conditions, conventional
crude oil (Prudhoe Bay Crude; PBC) was eliminated more effectively due to the higher content of
lighter hydrocarbons. The rates of alkane and polycyclic aromatic hydrocarbons (PAH)
degradation were comparable between the two dilbit products, but the extent of degradation
was greater for conventional crude oil owing to the higher concentrations of lighter alkanes.
The potential of microbial enrichment to degrade crude oil was highly influenced by temperature
as well as the composition. Lower degradation rates were achieved at the lower temperature,
consistent with previous studies. Extended acclimation period and slower rates of degradation
at lower temperatures may be due to lower solubilities and crystallization of hydrocarbons, and
lower metabolic rates. As per the results of genomic sequencing, well-known oil degraders
metabolized select fractions of both the dilbit types, but their performance varied. All the
enrichments metabolized select fractions of the PBC as well WCS dilbit, but the nature and extent
of the degradation was distinct. Kalamazoo River enrichment (KRE) meso culture was the most
effective among all, as it completely removed alkanes and PAHs. Anderson Ferry enrichment
(AFE) performed differently at two temperatures; where an acclimation period of 8 days was
observed at 5 9C while there was no lag at 25 °C. KRE meso culture as well as AFE culture at both
the temperatures degraded alkanes completely while they were not able to metabolize heavier
fractions of the oil (C2-4 homologues of 3 ring compounds and 4 ring compounds). Differences in
the microbial activities can be explained by their composition, where the two cultures were a
mixture of various microbial species and their composition was diverse. Diversity in the
composition can be explained by difference their origin (Kalamazoo River vs. Ohio River) as well
as the carbon source (dilbit vs conventional crude) on which they were enriched.
The aquatic effects were examined for select fractions of dilbit using four standard aquatic
species. The results for these fractions indicate that dilbits can have similar acute and sublethal
toxicity as crude oils and other petroleum products. Water accommodated fractions of the dilbits
were characterized for total petroleum hydrocarbons (TPH), PAHs, and monoaromatics
(benzene-toluene-ethylbenzene-xylene; BTEX). Acute toxicity of unweathered and weathered
dilbits were similar for all four species ranging from 4 to 16 mg/L TPH, 8 to 40 ug/L total PAHs,
and 0.7 to 16 mg/L BTEX. Weathered dilbits were sublethally toxic (impaired growth and
reproduction) at 0.8 to 16 ug/L TPH and they can have similar acute and short term toxicity as
other oils.
Results of this work serve to gain insight on the behavior, fate and potential hazards of select
fractions of spilled diluted bitumen in the environment. Such research is needed to better
prepare emergency responders for remediation options and to understand the persistence of
dilbit in water. Increasing the knowledge base of dilbit has application in the risk assessments of
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July 2017
spills, and allows comparison of the relative hazards of dilbit products to conventional oil
products. Further research is needed on other fractions of dilbit, especially the asphaltenes, as
well as long term toxicity.
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1. Introduction
Oil sands deposits consist of a mixture of oil, sand, clay and water (Yang et al., 2011). They are formed
when petroleum reserves do not exceed 80 deg C, thus impeding pasteurization. Over geologic time,
lighter, soluble petroleum compounds are evaporated and biodegraded as the oil cooled. This results in
residual high molecular weight (HM W) oil compounds of high viscosity, sulfur and metal content, organic
acids and asphaltenes (Lin et al., 1989; Crosby et al., 2013), termed bitumen. Bitumen is a heavy sour oil
with a gas-free viscosity > 10,000 cSt (WEC, 2010) that can be either more or less than the density of
pure water. Oil sands bitumen is found throughout the world, with the largest reservoir in Alberta,
Canada. Alberta's reserves are estimated at 166 billion Barrels (bbl) and production of 2.3 million barrels
per day (bbl/d) (Alberta Energy Regulator Agency, 2014), making it the third largest oil reserve in the
world behind Venezuela and Saudi Arabia. The extraction of bitumen occurs via two ways. Surface
mining is sufficient for deposits less than 75 m from the surface, where oil is separated from sand and
water via centrifuge prior to preparation for transport. For deeper deposits, which is nearly 80% of the
remaining reserves (Energy Information Administration, 2013; Canadian Energy Pipelines Association,
2013; Alberta Energy, 2016), in situ recovery is used. This unconventional technique is a method for using
steam and solvent injection to separate and pump bitumen to the surface.
Due to the high viscosity of bitumen, it is difficult to transport through pipelines. The produced material
does not meet transmission pipeline specifications, such as the Maximum Operating Pressure
requirements administered by PHMSA (Pipeline and Hazardous Materials Safety Administration) in the
U.S.A. Thus, for pipeline transport, bitumen must be diluted with diluent such as natural gas
condensates, or lower density crude oils to form Diluted Bitumen, commonly known as dilbit (CAPP,
2012). Bitumen can also undergo an upgrading process to form a light, sweet 'synthetic' crude oil (SCO)
that is then diluted to produce a synthetic bitumen (synbit). The mixture of bitumen and diluent varies
but has a general target ratio of 70% bitumen to 30% diluent, allowing dilbits to be transported at
comparable pipeline pressures as conventional heavy crudes (Canadian Government, 2013; Canadian
Energy Pipelines Association, 2013; American Petroleum Institute, 2012). Table 1.1 shows how dilbits
compare to heavy conventional crudes in North America. Although dilbits differ from conventional crude
oils in adhesion properties and asphaltene / resin content, recent studies have shown that their
corrosivity is akin to conventional crudes and considered to be low (Alberta Innovates, 2011; Penspen,
2013). Specific dilbit (Cold Lake Blend and Western Canadian Select) composition in terms of weight
percent of saturates, aromatics, resins and, asphaltenes (SARA) as compared to other crudes is
represented in Figure 1.1. Illustrated is that dilbit has higher concentrations of resins and asphaltenes,
yielding it more viscous and denser as compared to other petroleum products. The average dilbit density
ranges between 824 and 941 kg/m3 at 15 °C with average API (American Petroleum Institute) gravity
values between 18-39 (POLARIS, 2013).
Bitumen oil products are known as unconventional oils, in part because of extraction and transport
preparation, but also because until recently they were not commonly extracted. In North America
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bitumen products have been transported in small percentages for the past four decades (NAS, 2016).
Improvements in extraction technologies, increased global demand and rising oil prices from the early
2000's led to an increase in dilbit production, (CAPP, 2015). Subsequently, shipment of dilbit via rail,
pipeline and tanker has increased in North America over the last decade, where the U.S. imports 1.2
million barrels per day. Evidence of this can be gathered from mapping the existing and proposed dilbit
pipelines, illustrated in Figure 1.2.The primary pipelinesareTransCanada, Keystone, Enbridgeand Kinder
Morgan.
In the North America there have been three major dilbit spill incidents reported in the past 10 years. In
2007 a Trans Mountain Pipeline operated by Kinder Morgan Canada Inc. was punctured in Burnaby,
British Columbia, Canada, resulting in a discharged volume of 224,000 liters of Albian Heavy (blend of
synthetic crude oil and bitumen) that impacted soils, storm drains, sewer lines and 15 km of shoreline /
marine waters of Burrard Inlet (Transportation Safety Board of Canada, 2007). The emergency response
and remediation minimized the short and long-term effects of the spill, where approximately 218,000
liters of oil were recovered by skimming, booming and flushing of shorelines (Stantec Consulting Ltd.,
2012). In July 2010, the Enbridge pipeline ruptured, releasing 3,320,000 liters of dilbit (Cold Lake and
MacKay River Blends) into Talmadge Creek and Kalamazoo River, near Marshall, Michigan. Presence of
floating, submerged and sunken oil was reported, and approximately 700,000 liters of oil still remains in
the river submerged, bound to sediment for which dredging is being used (USEPA, 2013a). Most recently,
a dilbit spill occurred in March 2013 near the suburban area of Mayflower, Arkansas. The ExxonMobil's
Pegasus pipeline spill caused the release of 800,000 liters of Wabasca Heavy crude, which is a blend of
bitumen and condensate (www.phmsa.dot.gov).
100%
ono/
¦ Saturates Aromatics ¦ Resins BAsphaltenes
30/o
ono/
¦
¦
m
m
oU/o
~7(\%
m
/\J/o
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¦
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ah0/.
¦
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ju/o
20%
1 n%
¦
j.u/0
no/
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Light Crude Medium Crude Heavy Crude Bitumen Cold Lake Western
Blend Canadian
Select
Figure 1.1. Saturate-Aromatic-Resin-Asphaltene (SARA) composition for different oils. Cold Lake Blend and
Western Canadian Select are specific diluted bitumen products. Source: National Academies of Sciences,
Engineering, and Medicine. Spills of Diluted Bitumen from Pipelines: A Comparative Study of Environmental
Fate, Effects, and Response.
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Table 1.1 Trace element concentrations in various heavy crude oil types. Source: American Petroleum
Institute; 2011 White Paper; Pipeline transportation of diluted bitumen form the Canadian Oil Sands.
Location
Crude Name
API
Gravity
Sulfur %
wt
Vanadium
(PPm)
Nickel
(ppmj
Mercury
(ppm)
Lead
(ppm)
Canada
Bow River Heavy*
26.7
2.1
54
21
—
—
Western Canadian
Select1
20.8
3.4
134
56
—
—
Cold Lake Blend"
22.6
3.6
169
65
Wainwright-Kinsella "
23,1
1.6
80
40
California
California API 15
13.2
5.5
266
111
3
California API 11
10.3
3.3
245
106
bdi
3
Hondo
19.8
4.3
196
75
bdl
bdl
Point Arguelfo Heavy
18.2
3.4
—
—
Santa Clara
22.1
2.9
193
77
bdi
bdl
Iran
Soroosh
18.1
3.3
101
35
—
—
Mexico
Maya
21.3
3.0
257
44
bdi
bdl
Nigeria
Focardos Blend
20.7
0.3
—
—
—
—
Venezula
Tia Juana Heavy
12.1
2.7
—
—
—
___
Lago Treco
22.8
2.6
—
—
—
—
Boscan
10.1
5.5
1320
117
bdl
bdl
Bacaquero
16.8
2.4
—
—
BCF24
23.5
2.0
_
...
bdi = below detection limits; — = no data reported
* = Conventional crude
** = Dii-bit
t= Made up of conventional and Oil-Bit streams as it is a special blend of various crude types
References:
Dude Monitor. 2011, Crudemonitor.ca. Website accessed 24 Jan 2011. Website: http://www.crudemonitor.ca/home.php-
Env.jonment Canada, 2011. Of! Properties Database. Website accessed 24 Jan 2011. Website: http://www.etc-
cte.ec.gc.ca/databases/oilprope.rties/.
18
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Canadian and U.S. Oil Pipeline?
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Figure 1.2. Existing and proposed Canadian and U.S. oil pipelines for dilbit transport. Source: Canadian
Association of Petroleum Producers, also found at https://www.nap.edu/read/21834/chapter/3tfl2.
Dilbits are a growing concern because of increasing transport in North America, recent high-profile spills
with considerable impacts to communities, unique properties, and limited information on environmental
fate and behavior in aquatic environments. Further, the composition of bitumen varies with the location
of the deposits and since different diluents can be used for dilution purposes, there is no uniformity in
the quality or the composition of dilbit. The specifications for diluents are vaguely stated in literature. It
is suggested that diluents should have a density of 650-750 kg/m3, maximum viscosity of 2 cSt, and no
more than of 0.5 weight % of sulfur. The most frequently used diluents are condensates, which are liquid
by-products of natural gas extraction processes containing mainly pentane and heavier hydrocarbons
(Canadian Energy Pipelines Association, 2013; Crosby et al., 2013; Canadian Government, 2013). Thus
hazard and risk assessment, and subsequent remediation options, remains complex because of the
diversity of dilbit products that can vary geographically and seasonally in composition (Canadian
Government, 2013; Polaris, 2013).
Dilbits can exhibit rapid environmental weathering with the loss of the diluent components (Canadian
Government, 2013; Polaris, 2013) This equates to uncertainty in knowing if and over what timeframe
they will sink or float as they weather. Recent studies have targeted the behavior of dilbit in water to
better understand sinking and floating scenarios. In a 13-day weathering experiment within a flume tank,
using filtered water, Access Western Blend was found to sink after 6 days, however Cold Lake Blend was
more resistant to weathering due to higher concentration of alkylated polycyclic aromatic hydrocarbons
(PAHs) and did not sink within 13 days (King et al., 2014). However, with unfiltered water and in the
presence of particles, sinking could have occurred earlier. Weathered dilbit has also been shown to be
19
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amenable to dispersion (Canadian Government, 2013) where dispersants were effective within the first
48 hours. Additionally, in situ burning can be considered within 96 hours of a spill, where the water is
calm, the oil is greater than 2 mm thick, and the spill can be contained, as per the spill response options
decision matrix presented in King et al., 2017. Although these studies are providing information on the
behavior of dilbit, knowledge gaps still exist regarding the effects of suspended particles, salinity, or
sunlight-induced oxidation on dilbit sinking.
Additionally, there are knowledge gaps with respect to dilbit biodegradation. Given that bitumen
deposits are themselves residues remaining after extensive anaerobic biodegradation, evaporation and
water washing of original crude oils (Lin et al., 1989; Yang et al., 2011; NAS, 2016), it has been posited
that a spill of diluted bitumen may be less susceptible to biodegradation than a comparable spill of light
or medium crude oil. This is supported by the presence of large unresolved complex mixtures in
chromatograms that serves as an indicator of extensive biodegradation and suggests that no further
degradation was likely (Yang et al., 2011; Crosby et al., 2013). Although resins and asphaltenes in the
bitumen are expected to remain recalcitrant over long time periods, any saturates and aromatics would
be expected to biodegrade more rapidly. A 30-day laboratory biodegradation experiment was conducted
using residual oil in sediment (19-20 months after the spill) from the Enbridge oil spill in the Kalamazoo
River (US EPA, 2013a), where approximately 25% of the total petroleum hydrocarbons (TPH) degraded,
mostly in the first 14 days. However, the decreasing rate of biodegradation over the 30-day period
suggested that the majority of the spilled oil would not degrade overtime scales of at least a few months
in spite of the experiment's favorable conditions for bacterial activity. This is consistent with the work of
King et al. (2014), where first-order rate constants were 0.0011 and 0.0014 d 1 for alkanes using Access
Western Blend and Cold Lake Blend; and for PAHs, the corresponding rate constants were 0.0011 and
0.0005 d"1. Similarly, Coblani et al. (2015) found that for diluted bitumen, with and without chemical
dispersants, alkanes were almost completely degraded for fresh and saltwater, whereas aromatics
persisted over a 42-day period. These limited studies highlight that dilbit biodegradation warrants
further study to better ascertain the biodegradation rates, environmental factors and compositional
variability (Wang and Fingas, 1996).
Compared to conventional crudes, middle distillates and heavy fuel oils, very little is known about the
toxicity of dilbits and synthetic crudes derived from bitumen (Dew et al. 2015). Previous studies have
reported the toxicity of bitumen, bitumen extracts, or process water from the Alberta tar sands region
of Canada (e.g., Colavecchia et al. 2004; Alharbi et al. 2016; Bauer et al. 2017). However, aquatic toxicity
data are extremely limited fordilbit, with only four published studies to date (Madison et al. 2015, 2017;
Alderman et al. 2016; Philibert et al. 2016). The need for dilbit toxicity data has been highlighted in a
number of comprehensive reviews (Dupuis and Ucan-Marin, 2015; NAS, 2016; Lee et al. 2015), including
the need for baseline toxicity for a range of dilbits for application in hazard assessment and comparison
to other oil products (Dew et al., 2015). Of the published dilbit toxicity studies, none have reported data
for standard aquatic species determined with conventional test methods. In the first study of dilbit
toxicity in fish, Access Western Blend dilbit caused developmental toxicity and oxidative stress in medaka
(Oryzias latipes) at total PAH concentrations of 10 to 100 ug/L (Madison et al., 2015). Alderman et al.
(2016) reported biomarker responses and impaired swimming performance in salmon early life stages
20
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exposed to 4 to 67 total PAHs from Cold Lake Blend. Philibert et al. (2016) reported similar
developmental toxicity, impaired avoidance behavior, and reduced swimming performance in zebrafish
[Danio rerio) exposed to dilbit and crude oil, where adverse effects were more strongly associated with
monoaromatics (15-20 mg/L) rather than PAHs (50-200 ug/L). In the most recent report of dilbit toxicity,
physically and chemically dispersed Cold Lake Blend caused developmental toxicity in medaka at
concentrations of 3 and 0.1 ug/L total PAHs, respectively (Madison et al. 2017).
Recent reviews produced in collaboration by government agencies, the National Academies of Science,
academics and industry highlight the pressing need to better understand the behavior and potential
impacts of dilbit spills over land and water. To address knowledge gaps pertaining to the behavior and
fate of spilled diluted bitumen, this report summarizes research conducted within the US EPA ORD on
the chemical characterization, dispersion effectiveness, biodegradation and toxicity of two types of
diluted bitumen- Western Canadian Select and Cold Lake Blend. These studies are being published in
Deshpande (2016); Deshpande et al (2017); Barron et al. (2017). The objectives of this research were to:
1. Characterize Cold Lake Blend (CLB) and Western Canadian Select (WCS) dilbit products
(unweathered) via chemical and physical properties and dispersion effectiveness.
2. Evaluate the biodegradation of alkanes and aromatics of CLB and WCS dilbit as function of two
temperatures (5 and 25 °C) using two freshwater cultures: one consortium acclimated to dilbit
(Kalamazoo River Enrichment, KRE) and other consortium enriched on soil contaminated with
conventional hydrocarbons from the Ohio River (Anderson Ferry Enrichment, AFE); and analysis of
microbial community structure via genomic sequencing.
3. Determine the acute and sublethal toxicity of unweathered and weathered CLB and WCS dilbits to
standard aquatic test organisms: the freshwater invertebrate Ceriodaphnia dubia, the freshwater
fish Pimephales promelas (fathead minnow), the saltwater invertebrate Americamysis bahia (mysid),
and the saltwater fish Menidia beryllina (inland silverside); using conventional slow-stir Water
accommodated fractions (WAF) and static or static renewal methods to allow comparison to the
broader literature on the toxicity of oil products.
Results of this work serve to gain insight on the behavior, fate and potential impacts of spilled diluted
bitumen in the environment. Such research is needed to better prepare emergency responders for
remediation options and the persistence of dilbit in water and sediments. Increasing the knowledge
base of dilbit has application in the risk assessments of spills, and allows comparison of the relative
hazards of dilbit products to conventional oil products.
21
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2. Methods
Experiments utilized Cold Lake Blend (CLB) and Western Canadian Select (WCS) dilbits obtained from
Crude Quality (Edmonton, Alberta, Canada). Subsamples were artificially weathered by nitrogen gas
stripping until no change in volume was observed to yield 'weathered' oil for this study. Alaskan North
Slope 521 (ANS) and Prudhoe Bay (PBC) crude oils were obtained from the US EPA laboratory stock and
tested without weathering.
2.1. Diluted Bitumen Dispersion Effectiveness
2.1.1. Baffled Flask Test Approach
The Baffled Flask Test (BFT) developed by the EPA (Sorial et al., 2004) provides a measure of dispersion
effectiveness for a given oil with and without a chemical dispersant. The test utilizes a 150-mL screw-
cap trypsinizing flask (an Erlenmeyer flask with baffles), modified by the placement of a glass stopcock
near its bottom so that a subsurface water sample can be removed without disturbing the surface oil
layer. Instant Ocean artificial seawater (Aquarium Systems; Mentor, OH) at salinity of 34 ppt (120 ml)
was placed in the baffled flask. 100 |aL oil was pipetted directly onto the seawater surface with an
Eppendorf repeater pipettor (set at stop 1 using a 5 mL tip). A volume of 4 |aL of chemical dispersant
was pipetted onto the center of the oil slick (pipettor set at stop 2 using a 100-|aL tip), ensuring that the
dispersant contacted the oil prior to the water, yielding a dispersant-to-oil (DOR) ratio of 1:25. The flask
was mixed on an orbital shaker set at a rotation speed of 250 rpm for 10 min., to receive moderate
turbulent mixing (Kaku et al., 2006). At the end of the mixing period, the flask was removed from the
shaker and allowed to remain stationary for a quiescent period of 10 minutes. A 30 mL sample volume
was collected without disturbing the flask contents in a 50 mL graduated cylinder by opening the
stopcock at the bottom of the baffled flask. The sample was transferred to separatory funnel for liquid-
liquid extraction; 3 times with 5 mL fresh Dichloromethane (DCM, pesticide quality). The BFT was
conducted with 6 replicates and method blanks.
Dispersed oil concentrations were analyzed by UV-visible absorption spectrophotometry (Shimadzu UV-
1800) using standard transmission-matched quartz 10-mm path length rectangular cells. Measurements
at 340, 370, and 400 nm wavelengths were collected (Fingas et al., 1987). Oil extraction standards in
DCM were prepared for each oil with and without dispersant by combining 2 mL oil with 18 mL DCM. A
volume of 80 |aL dispersant was added to the oil-dispersant stock solutions to give a DOR of 1:25. Stock
solution concentrations were determined by mass measurements after each addition. To generate a 6-
point calibration curve, a specific volume of the stock standard solution was added to 30 mL synthetic
seawater in a 125 mL separatory funnel. These stock solution volumes were adjusted so that the
absorbance readings fell within the linear dynamic range (LDR) of the spectrophotometer. Triplicate
liquid / liquid extractions of samples were then performed by using 5 mL of DCM for each extraction and
adjusting the final extract to 20 or 25 mL (adjusted to maintain the LDR).
22
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2.1.2. Analysis of Extracts
Absorbance at three discrete wavelengths (340, 370, and 400 nm) was used to calculate the area under
the absorbance vs. wavelength curve by applying the trapezoidal rule according to the following
equation:
(Abs34o + Abs37o)x30 (Abs37o + Abs4oo)x30
Area = - h- (1)
2 2
Area was then used to calculate the Total Oil Dispersed and then the percentage of oil dispersed (%OD),
based on the ratio of oil dispersed in the test system to the total oil added to the system, as follows:
Ay pa V
TotalOilDispersed(g) = x VDCM x -f- (2)
( ahbrationl urve Slope Vew
where: Vdcm = volume of DCM extract, Vtw = total volume of seawater in flask, and Vew = total volume of
seawater extracted, and
TotalOilDispersed
%ODd or %OD = i- (3)
Ponton
where: %ODd or%ODc =average %oil dispersed by chemical dispersant (d) or physically (c), p0jl =
density of the specific test oil, g/L, and V0ii = volume (L) of oil added to test flask (100 |aL = 10"4 L).
The reported dispersion effectiveness (DE) value is the lower 95% confidence level (LCL95) of the
independent replicates, calculated from:
LCL95d or LCL95c =x- tn_hl_a
00
\4nj
(4)
where: LCL95 is the lower 95% confidence level for chemically dispersed (d) and physically dispersed (c)
oil, X = mean dispersion effectiveness of the n = 4 replicates, s = standard deviation, tn_x x_a = 100 x (1 -
a)th percentile from the t-distribution with n-1 degrees of freedom, and a = 0.05. Prior to conducting
the statistical comparisons, the replicates within a given treatment were subjected to an outlier test, the
Grubb'sTest or Maximum Normal Residual test (Grubbs, 1969), and if an outlier (p < 0.05) was detected,
an additional replicate was analyzed to maintain the required four replicates.
2.1.3. Droplet Size Distribution
Dispersions generated with the BFTs were analyzed by a Laser In Situ Scattering and Transmissometry
sensor (LISST 100X; Sequoia Scientific) for droplet size distribution and particle concentration operated
in bench top mode. Data processing follows Li et al. (2009), where concentrations are normalized to the
maximum value to allow for comparison between dispersions.
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2.2. Diluted Bitumen Biodegradation Study
2.2.1. Chemicals and Reagents
Fresh CLB and WCS dilbits were used in the biodegradation experiments. Prudhoe Bay Crude (PBC) and
Alaskan North Slope 521 (ANS) were used to represent conventional crude oil. Mineral salts,
dichloromethane (DCM), and hexane were acquired from Fisher Scientific (Pittsburg, PA, USA).
2.2.2. Media
Bushnell Hass broth was used as freshwater media. It was prepared by dissolving the required amounts
of mineral salts in distilled water. The concentrations of each salt (expressed in g/L) were magnesium
sulfate (0.2), calcium chloride (0.02), monopotassium phosphate (1.0), dipotassium phosphate (1.0),
ammonium nitrate (1.0), and ferric chloride (0.05). This solution was autoclaved at 120 °Cfor 15 min in
batches of 1 L. The pH for this media was 7.
2.2.3. Microbial Enrichment
Cultures were enriched on hydrocarbon impacted sediments isolated from two different locations. One
set of sediments was collected from the Ohio River, downstream of fuel tanks at Anderson Ferry
(Cincinnati, Ohio, USA). The other batch of sediments was obtained from the dredging operations
following the Kalamazoo River En bridge Energy Spill. These cultures were named after the locations from
where they were acquired: Anderson Ferry (AFE) and Kalamazoo River (KRE) culture. AFE and KRE were
enriched on ANS and dilbit, respectively as the carbon source in Bushnell Haas broth for a month. KRE
mixed culture was enriched at both 5 (cryo) and 25 9C (meso), while the AFE was grown at 25 9C. After
30 days of incubation, cultures were centrifuged, washed with saline and mixed with 10% glycerol before
storing at -80 9C. On the day of experimental set up, enrichment stocks were thawed to room
temperature and re-suspended in sterile saline before use.
2.2.4. Microcosm Design
Experiments were set up to examine biodegradation of three types of oils at 5 and 25 9C using microbial
consortia obtained from different locations as summarized in Table 2.1. Sampling days at 25 9C occurred
at days 0, 2, 4, 8,12, 16, 20, 28, 35, 42, 54, and, 60. The 5 9C experiments were analyzed on days 0, 2, 4,
8, 16, 24, 32, 40, 48, 56, 62, and, 72. To account for any possible abiotic losses, triplicate killed controls
(KCs) containing 500 mg/L of sodium azide were also included and sampled at the end of each
experiment. Each flask containing 100 mL of broth was spiked with 0.07 g of oil and was inoculated with
0.5 mL of the culture. These microcosms were then placed on a rotary shaker and mixed at 200 rpm in
temperature controlled rooms for the duration of the experiments.
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Table 2.1. Summary of Biodegradation Experimental Design.
Test
Temperature
Treatment
Sampling
Events
Sample
Replicates
Total
Experimental
unit (EU)
1
5°C
Oil
12
3
36
2
5°C
Kill Control
1
3
3
Subtotal EU's
39
3
25°C
Oil
12
3
36
4
25°C
Kill Control
1
3
3
Subtotal EU's
39
Total EU's for one oil and one enrichment
78
2.2.5. OH Extraction and Analysis
On every sampling day, three flasks were sacrificed per treatment. Oil was extracted with DCM. Obtained
extracts were filtered through anhydrous sodium sulfate to remove any water and then concentrated to
reduce the volume under nitrogen. Residual hydrocarbons were measured with an Agilent 7890A Gas
Chromatograph with an Agilent 7000 mass selective detectortriple quadrupole. The chromatograph was
equipped with a general purpose low bleed capillary column (DB-5, 30 m X 0.25 mm and 0.25 |am film
thickness, J&W Scientific) and a split/splitless injection port operated in the splitless mode. Multi
Reaction Monitoring (MRM) mode was used for analyte detection. The targeted analytes included 28
alkanes ranging in carbon number from n-CIO to n-C35 plus pristane, and phytane, while the aromatics
targeted were the 2- ,3-, and 4-ring polycyclic aromatic hydrocarbons (PAHs) [naphthalenes (NAP),
phenanthrenes (PHE), fluorenes (FLU), dibenzothiophenes (DBT), naphthbenzothiophenes (NBT),
pyrenes (PYR), and chrysenes (CHY)] along with their alkylated homologs. Concentrations of individual
analytes were added together to calculate total alkanes and total PAH content. Total Petroleum
Hydrocarbons (TPH) were determined with a GC flame ionization detector (GC-FID) according to the EPA
Method 8015C.
First order biodegradation rate coefficients were determined by non-linear regression using GraphPad
Prism 7. An analysis of variance (ANOVA) was conducted to test the following null hypotheses: (1) no
differences exist in the biodegradability of select fractions of dilbit and conventional crude oil, (2) all the
enrichments metabolize select fractions of the crude in a similar fashion, and (3) temperature does not
affect biodegradation of crude.
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2.2.6. DNA Extraction and Sequencing
Samples were collected for molecular analysis on days 0, 2,16, 35, and 60 for 25 9C and on days 0, 2,16,
40, and 72 for 5 9C. On these sampling days, pellets of biomass were obtained by spinning 1.5 mL of
media. Following the manufacturer's instructions, DNA was isolated with the PowerLyzerTM PowerSoil®
DNA kit (MoBio Laboratories, Solana Beach, CA). The DNA extracts were then amplified with barcoded
primers 515F/806R. Polymerase chain reaction (PCR) conditions used were as follows: denaturing for 3
min at 95 °C, 35 cycles at 95 °C for 45 s, 50 °C for 60 s, and 72 °C for 90 s, followed by 10 min of final
primer extension at 72 °C. The PCR products were visualized on an agarose gel to confirm product sizes
and then pooled in an equimolar ration. Next generation sequencing was performed on an lllumina
MiSeq sequencer (lllumina, San Diego, CA) by using pair-end 250 bp kits at the Cincinnati Children's
Hospital DNA Core facility. Sequence reads were processed with MOTHUR vl.25.1.
2.3. Diluted Bitumen Toxicity to Standard Aquatic Test Species
2.3.1. Test Organisms, Conditions and WAF Preparation
Test organisms (Figure 2.1) were cultured in either moderately hard reconstituted synthetic fresh water
(C. dubia, P. promelas) or 20 parts per thousand (ppt) synthetic sea water (A. bahia) following standard
methods (USEPA 2002a; 2002b; 2002c). M. beryllina were obtained from a commercial supplier and
acclimated to 20 ppt culture water (USEPA 2002a). Neonatal C. dubia less than 24-hour old were used in
acute and chronic tests. Forthe chronic tests, they were also isolated within an eight-hour period (USEPA
2002b, method 1002.0). Larval A. bahia were used at 3 to 4 days old in acute tests (USEPA 2002a, method
2007.0), and when 7 days old in chronic tests (USEPA 2002c, method 1007.0). Larval P. promelas were
used in acute tests when 7 to 12 days old (USEPA 2002a, method 2000.0), and M. beryllina were used in
acute tests as 10 to 14 day old larvae (USEPA 2002a, method 2006.0).
Test methods were species specific and generally followed U.S. EPA effluent test guidelines as modified
for tests with petroleum (Table 2.2). Chambers were covered to minimize loss of volatile hydrocarbons,
but were not sealed to allow gas exchange. Static (no test solution renewal) acute tests were either 48
hours (C. dubia, A. bahia) or 96 hours (P. promelas, M. beryllina) that measured mortality and morbidity
(no response to gentle prodding). Chronic tests were 7 day exposures with periodic test solution
renewals that measured mortality and morbidity, and either reproduction (C. dubia) or growth (A.
bahia).
The CLB and WCS dilbits were artificially weathered by nitrogen gas stripping until no change in volume
was observed. WAFs (water accommodated fractions) were prepared in species-specific fresh or 20 ppt
salinity culture water in 4 liter sealed and covered glass jars following the standard slow-stir method
(e.g., Barron et al. 2003). Oil was added at either 25 or 50 g/L, stirred to achieve an approximately 20%
vortex for 18 hours, then settled for 6 hours. The aqueous phase was removed, serially diluted and used
in toxicity tests.
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Figure 2.1. Acute and chronic test species: Ceriodaphnia dubia (C. dubia ; top left); Pimephales promelas
(fathead minnow; top right); Americamysis bahia (mysid; bottom left); Menidia beryllina (inland silverside;
bottom right).
2.3.2. Analytical Chemistry
WAF samples were collected immediately after preparation and analyzed for BTEX, PAHs including alkyl
homologs, and TPH as total extracted hydrocarbons, BTEX samples were collected in 40 mL glass head
space vials and samples for analysis of PAHs and TPH were collected in 1 L glass jars. Samples were
extracted with dichloromethane and analyzed for oil components following SW-846 Method 3500C
(USEPA 2007a). Alkane and PAH concentrations were quantified using an Agilent 6890N Gas
Chromatograph (GC) with an Agilent 5975 mass selective detector (MSD) and an Agilent 7683 series
autosampler, equipped with a DB-5 capillary column by J&W Scientific (30 m, 0.25 mm I.D., and 0.25 [am
film thickness) and a splitless injection port following EPA Method 8270D (USEPA 2014). Alkanes
consisted of normal aliphatics ranging in carbon number from 10 to 35 as well as branched alkanes
(pristine and phytane). Aromatics included 2, 3 and 4 ring PAH compounds and their alkylated homologs
(i.e. Co-4 - naphthalenes, Co~4 -phenanthrenes, Co-3 - fluorenes, Co-4 dibenzothiophenes, Co-3 -
napthobenzothiophenes, C0-2 -pyrenes, Co 4- chrysenes). TPH in solvent extracts were determined with
an Agilent 7890B GC equipped with a flame ionization detector (FID) and 7693 autosampler following
EPA Method 8015C (USEPA 2007b). Concentrations of all the individual alkanes and PAHs were summed
up to measure total alkane and PAH concentrations. BTEX were measured using an Agilent 7890A GC
with a 5975C MSD with Triple Axis Detector and CombiPal autosampler (CTC Analytics) following EPA
Method 524.3 modified to perform head space analysis instead of purge and trap (USEPA 2009). All
reported hydrocarbon measurements met EPA method quality assurance and quality control
requirements, including precision, reproducibility and required detection limits.
27
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2.3.3. Statistical Analyses
Median and 20% lethal concentrations for acute tests (LC50/LC20) were calculated from a log-logistic
regression using the drc package in R Statistical Software (Ritz and Streibig 2005) as a percent WAF value.
Percent WAF values were then multiplied by initial test stock solution concentrations for BTEX, total PAH
(tPAH; sum of detected analytes and homolog groups), and TPH to determine lethal concentrations for
each test species, dilbit, and WAF loading level. The average TPH concentration measured in control
samples was subtracted from stock solution measurements before determining effective
concentrations. For 7 day chronic tests, no observed effect concentrations (NOEC) and 25% inhibition
(IC25) values were calculated using the geometric mean concentration of BTEX, PAH, and TPH in test
specific stock solution WAF samples. Relationships between species-specific dilbit toxicity and
concentrations of TPH, PAH, or BTEX were determined by linear regression analysis using r-squared and
p-value.
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Table 2.2. Summary of acute and chronic test conditions for four aquatic species: Ceriodaphnia {Ceriodaphnia
dubia), mysid (Americamysis bahia), fathead minnow (Pimephales promelas), and inland silverside (Menidia
beryllina). Data and Table Source: Barron et at, 2017.
Test Parameter
Acute
C. dubia
Acute
A.
bahia
Acute
P.
promela
Acute
M.
beryllina
Chronic
C.
dubia
Chronic
A.
bahia
Test method
EPA-821-R-
02-012
Method
2002
EPA-821-R-
02-012
Method
2007
EPA-821-R-
02-012
Method
2000
EPA-821-R-
02-012
Method
2006
EPA-821-R-
02-013
Method
1002
EPA-821-R-
02-014
Method
1007
Test type
Static
Static
Static
Static
Static
renewal
Static
renewal
Test duration
48 hours
48 hours
96 hours
96 hours
7 days
7 days
Salinity
NA
20 ± 2%o
NA
20 ± 2%o
NA
20 ± 2%o
Renewal
NA
NA
NA
NA
Daily
Daily
Temperature
25 ± 1 °C. Test temperatures must not deviate (maximum minus minimum temperature)
by more than 3 °C during the test.
Light quality
Ambient laboratory illumination
Light intensity
10-20 (E/m2/s)
Photoperiod
16 h light, 8 h darkness, with phase in/out period recommended
Test chamber size
30 mL
500 mL
1 L
1 L
30 mL
500 mL
Test solution
volume
20 mL
200 mL
200 ml
200 mL
20 mL
200 mL
Age of test
organism
<24 hours
1-5 days
1-14 days
9-14 days
<24 hours1
7 days
Organisms per
test chamber
5
10
10
10
1
5
Replicate
chambers per
concentration
4
3
3
3
10
8
Feeding regime
Refer to specific feeding procedures provided in each test method
Aeration
None, unless DO falls below 4.0 mg/L, then aerate all chambers. Rate: <100 bubbles/minute
Phys./ Chem.
Measurements
Daily temperatures were measured in one replicate for each test concentration.
Exposure test solutions analyzed daily for pH, dissolved oxygen, and conductivity / salinity.
Test conc.
5 exposure concentrations and a control
Test acceptability
>90%
control
survival
>90%
control survival
>90%
control
survival
>90%
control
survival
>80%
control
survival2
>80%
control
survival3
1. Released within an 8-hour period; 2. Minimum 15 young per surviving female; 3. Minimum weight of 0.2 mg per
adult in surviving controls.
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2.4. Quality Assurance Summary
This multidisciplinary research project was a collaborative effort of the EPA Office of Research and
Development (ORD) national research laboratories NRMRL and NHEERL. Research was conducted under
approved Quality Assurance Project Plans. The EPA Quality system is integral to this effort, providing
policy and procedures which are implemented in all aspects of the project to ensure that the data
generated from each discipline would be of a type and quality necessary and sufficient to achieve project
objectives.
The EPA Quality System encompasses management and technical activities related to the planning,
implementation, assessment and improvement of environmental programs that involve:
• the collection, evaluation and use of environmental data
• the design, construction and operation of environmental technology
Consistent with the requirements of the EPA Quality system, the participating EPA organizations have
implemented Quality Management Plans to define the specific processes and procedures that each EPA
organization uses to ensure implementation of the EPA Quality system.
To that end, the following Quality Assurance (QA) tools were implemented during the project:
• A systematic planning approach was implemented to develop acceptance or performance criteria
for all work covered by the EPA Quality System, defined in the Quality Assurance Project Plan
(QAPP). QAPPS were developed and approved for use by the EPA Quality staff for each project
effort, before any data collection activities were initiated in the field or laboratory. QAPPs that
were developed and implemented for this project are identified in the relevant sections of this
report and in the references section.
• Standard Operating Procedures (SOP) were implemented for all applicable field and laboratory
activities, to ensure consistency in the collection of samples, operation of environmental
technologies, and generation of environmental data in the field and in the laboratory.
• Appropriate training was provided for staff to ensure that quality-related responsibilities and
requirements as defined in the QAPPs are understood, and that SOPs are implemented for all
applicable activities. This ensures that research activities are conducted in a consistent and
reproducible manner, with the intent that research data will meet project data quality objectives
and/or acceptance criteria for usability to achieve the project objectives.
• Technical assessments were scheduled and performed by EPA and/or contractor quality staff to
verify that the QAPP requirements and SOPs were implemented during the project. A technical
Systems Assessment was performed by the contractor as required by the QAPP developed for
stage 1 of the project.
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• Data was reviewed and verified by research staff after collection, and validated by the project
leads to ensure that the type, quantity and quality was sufficient to reach conclusions stated in
this report and ultimately to achieve project objectives.
Furthermore, it is a requirement that all EPA Quality system elements "flow down" to the contractor
support entities. EPA Quality System specifications are incorporated to all applicable EPA-funded
agreements, and are defined in 48CFR46. An important element of this forcontracted analytical services
is certification by an independent accrediting organization such as the National Environmental
Laboratory Accreditation Conference (NELAC). This ensures that data is collected according to standard
procedures and methodologies under a quality system which is equivalent to ANSI/ASQC E4, which is
the basis of the EPA Quality system.
3. Results and Discussion
3.1. Chemical and Physical Characterization of Source Oil
WCS is composed of bitumen from Athabasca basin, whereas CLB is bitumen from the Cold Lake region.
Source bitumen from Athabasca is heavierthan Cold Lake because of the higher asphaltene content (~17
% versus ~11%, respectively; Figure 1.1) and hence it is blended with conventional crude and synthetic
crude oils along with the diluent to meet pipeline specifications (Oil Sands Magazine, 2017). Both WCS
and CLB are classified as unconventional heavy crude oil. The physical properties for both CLB and WCS
were similar to values previously reported (Alberta Innovates Energy, 2011; NRC, 2013; Canadian
Government, 2013). API gravity, density, and sulphur content of two dilbit products were nearly
identical, but CLB had higher viscosity and total acid number (Table 3.1). Relative proportion of total
alkanes and total PAHs were similar for both dilbit products. The equal saturate and aromatic content of
these dilbit products are consistent with the higher densities and viscosities when compared to
conventional crude oils. Distribution of n-alkanes for the two dilbits was similar, with concentrations
decreasing with the carbon chain length. Nevertheless, differences in branched alkane composition were
observed namely, n-C17/pristane and n-C18/phytane ratios for WCS were lower than in CLB (Figure
3.1A). Overall WCS exhibited 22.5% more total alkanes as compared to CLB. WCS presented higher
content of naphthalenes with phenanthrenes and dibenzothiophenes being more abundant in CLB. No
differences were noted in the concentrations of other PAH groups (Figure 3.IB). Higher quantities of
lower molecular weight hydrocarbons like alkanes and naphthalenes in WCS can be one of the reasons
for the lower viscosity as compared to CLB (NAS, 2016).
31
-------
Table 3.1. Physical and chemical properties of CLB and WCS diluted bitumens. Characterization was
conducted by Maxxam Analytical International Corp. (Petroleum Technology Center, Edmonton, Canada).
Properties
Cold Lake Blend
(CLB)
Western Canadian
Select (WCS)
API Gravity at 15 °C
0 API
21.7
22.1
Absolute Density at 15 °C
kg/m3
922.8
920.7
Total Acid Number (TAN)
mg KOH/g
1.03
0.86
Total Sulphur,
mass %
3.68
3.40
Kinematic Viscosity at 15 °C
cSt
230.7
199.7
Total Alkanes
jjg/g of dilbit
3100
4000
Total PAHs
(jg/g of dilbit
3540
3845
(A) Alkane Distribution
800
(B) PAH Distribution
I CLB
I WCS
n
n-
o
U)
c
d)
C
o
a
E
o
600
400 --
O)
=L
200
M4-U
I
u
wn
PAH Group Distribution
I;:;-:
CLB WCS
uhs Naphthalene
Phenanthrene
cxzza Fluorene
¦Mi Dibenzothiophene
Ha Naphthobenzothiophene
ttstaa Pyrene
XZZZZ Chysene
o o^'o^' ov o"W
Figure 3.1. Hydrocarbon composition for Cold Lake Blend (CLB) and Western Canadian Select (WCS) dilbits.
Source: Deshpande et a I., 2017.
32
-------
3.2. Dispersion Effectiveness
Fresh CLB and WCS dilbits were evaluated for dispersant effectiveness using the US EPA Baffled Flask
Test (BFT). LCL95 values of percent of oil dispersed using Corexit 9500 and Finasol OSR 52 chemical
dispersants are listed in Table 3.2. WCS was better dispersed compared to CLB, regardless of dispersant
or temperature. The low dispersion values of CLB also yielded higher variability with standard deviations
of approximately 3 for cold temperature and 14.5-17.0 for 25 °C. The size of the oil dispersed droplets
produced during BFTs are presented in Figure 3.2. Volume mean diameter was lower for dispersant
treatments and at warmer temperatures. Cold temperature appeared to have a greater influence on CLB
dispersion compared to WCS, suggesting that CLB may be less amenable to dispersion at cold
temperature.
Table 3.2. Dispersant Effectiveness using the BFT (Baffled Flask Test) expressed by the LCL95 for CLB and WCS
using Corexit 9500 and Finasol OSR 52 at 5 and 25 °C.
BFT Dispersant Effectiveness % (LCL95)
Temp
Cold Lake Blend
Western Canadian Select
(°C)
(CLB)
(WCS)
Corexit
Finasol
Corexit
Finasol
5
17.97
17.91
39.18
49.85
25
28.74
30.61
55.79
61.89
33
-------
¦ CLB+COR bCLB+FIN bCLB+NONE
¦ WCS+COR ¦ WCS+FIN DWCS+NONE
Particle Size Distri bution (um)
Figure 3.2. Droplet size distribution and particle concentration of Cold Lake Blend (CLB) and Western Canadian
Select (WCS) dilbit dispersed in water with and without chemical dispersants (COR: Corexit 9500; FIN: Finasol
OSR 52; NONE: no dispersant).
3.3. Dilbit Biodegradation
3.3.1. Alkane and PAH Biodegradation by Kalamazoo River Cultures
Time series removal of total alkanes by Kalamazoo River cultures (KRE) for both dilbits at 5 and 25 °C is
shown in Figure 3.3. At lower temperature, the first-order biodegradation rate coefficients were 0.52 ±
0.09 d 1 for CLB and 0.48 ± 0.10 d 1 for WCS, which were not significantly different (p = 0.9993) (Table
3.3). As expected, rates were higher (2-fold) at 25 °C, with coefficients of 1.13 ± 0.04 and 1.14 ± 0.01 d 1
for CLB and WCS, respectively over the course of the experiemnt. No significant difference between dilbit
products was observed at 25 °C either (p = 0. 9999). Figure 3.3 illustrates the removal of the sum of all
alkanes. As previously reported, linear alkanes are metabolized faster than /'so-alkanes (Pirnik et al.,
34
-------
1974; NAS, 1985; Singh et al., 2012). Our study is consistent with these findings, where the meso culture
degraded n-alkanes within the first two days with the /'so-alkanes being removed within 8 days. At 5 9C
the n-alkanes disappeared by day 8 while /'so-alkanes persisted for 40 days. The branched alkanes
degraded 3.5 times faster at 25 °C than at 5 °C, where degradation rate differences as a function of dilbit
type were statistically insignificant at both temperatures (p = 0.5605 (5 °C); p = 0.719 (25 °C)). First-order
coefficient rates for individual alkanes were not calculated for the 25 °C treatment as they tend to
degrade rapidly. Coefficients calculated for individual alkanes at 5 °C suggested that degradation rates
decreased with the increase in carbon number, and no significant difference was observed between
dilbit type. Over all, the Kalamazoo River meso culture (25 °C) removed 99.9% of the total alkanes by
day 12 (Figure 3.4), while the cryo culture (5 °C) needed more than 40 days for removal of total alkanes
owing to the persistence of branched alkanes.
Degradation of both CLB and WCS by the Kalamazoo River culture yielded comparable degradation rates
for PAHs, with no significant differences for cryo (p = 0.0944) or meso (p > 0.9999) cultures (Figure 3.3;
Table 3.3). At 5 °C, a lag period of 4 days was noted for both dilbits, which was not the case at 25°C. PAHs
were metabolized by the meso culture 2 and 2.5 times faster than at 5°C in the WCS and CLB microcosms,
respectively. At experiment completion, approximately 97.5% of total PAHs biodegraded at 25 °C, while
30% remained at 5 °C (Figure 3.4). Examination of specific aromatic groups (2-, 3-, and 4-rings and their
alkyl derivatives) as well as individual PAH compounds was conducted. Differences in the rate
coefficients of 2- and 3-ring PAHs (i.e., naphthalene (Co i-NAP), phenanthrene (Co-PHE), fluorene (Co-
FLU)) between the two dilbits at a given temperature were found to be statistically significant owing to
their dissimilar initial concentrations in the source oil. In contrast, the degradation rates of
napthbenzothiophenes (NBT), pyrene (PYR), and chrysene (CHY) for the two dilbits were comparable. In
the case of naphthalenes, complete removal was achieved at 25 °C by 12 d. At 5 °C, C0-3-NAP disappeared
within 40 d and the concentration of C4-NAP was reduced by 80-85% after 72 d (Figure 3.5). The meso
culture almost entirely degraded 3-ring aromatics (PHE, FLU, and DBT (dibenzothiophenes)), while 35-
40% of these analytes remained at 5 °C, which mainly comprised C3 and C4 types. NBT compounds
persisted at 5 °C, whereas at 25 °C, their concentration dropped by 92%. A significant decrease in 4-ring
PAH concentration was observed at 25 °C, while at 5 °C < 10% of PYR and CHY degraded. A
biodegradation rate comparison matrix (Table 3.4) was created to illustrate significant differences in
alkane and PAH biodegradation amongst oil types, cultures and temperatures.
3.3.2. Dilbit Biodegradation by Anderson Ferry Culture
Also investigated in this project was the degradation of WCS using two freshwater cultures: one culture
acclimated to dilbit (Kalamazoo River Enrichment, KRE) and other culture enriched on soil contaminated
with conventional hydrocarbons from the Ohio River near Cincinnati (Anderson Ferry Enrichment, AFE).
Degradation experiments were also conducted using Prudhoe Bay Crude (PBC) with both cultures for
comparison. This research is also published in Deshpande, 2016 and Deshpande et al., 2017. PBC is a
conventional crude oil exhibiting high aliphatic content with a concentration approximately 6 folds
greater than that for WCS and CLB. Thus PBC is not plotted in Figure 3.3 due to concentration scaling
issues, but appears in Figure 3.4 as percent removal for comparison to dilbits. The KRE culture nearly
35
-------
completely eliminated total alkanes at both the temperatures and for both the dilbits and the
conventional crude. Biodegradation rates were higher at warmer temperature, and the extent of
removal exceeded 99% by day 8 while it took 40 days to achieve the same level of degradation at 5 °C.
Similar to KRE, AFE was capable of metabolizing aliphatics completely at 25 °C, but the degradation rates
were lower for this enrichment. An acclimation period of 8 days was observed in case of AFE treatment
at 5 °C, however almost 98% of total alkanes disappeared by the end of the experiment and residual
alkanes mainly comprised of /'so-alkanes. (Figure 3.3). Biodegradation of branched alkanes showed that
rapid and complete removal of pristane and phytane was achieved at 25 °C. After a lag of 8 days, /'so-
alkanes started depleting at 5 °C and trace amounts of branched alkanes were noted at the end of the
experiment and the variability in three replicates was higher. At both the temperatures, removal was
faster using KME enrichment. Degradation patterns for both the oils were similar for AFE (p = 0.5) as well
as KRE treatments (p = 0.42).
The pattern of PAH degradation varied for each treatment (Figures 3.3 and 3.4). Total PAH content of
the conventional crude oil was almost twice of the dilbit aromatic content. KRE meso culture
metabolized almost 98% of the total PAHs at 25°C, whereas, at 5 °C experiment the extent of
biodegradation by KRE culture was observed to be between 75% and 85 %. Conversely, at higher
temperature, AFE culture degraded PAHs rapidly till day 20, after which no change in their concentration
was observed for WCS or PBC. Approximately 40 % of the initial PAH load remained in WCS while 20 %
of residual PAHs were observed in PBC treatments. Lower degradation rates were achieved at 5 °C, with
PAH depletion occurring after a 4-day acclimation period. AFE enrichment was able to metabolize 50%
PAH content of WCS and 78% in case of PBC.
PAH distribution between WCS and PBC was quite different. Naphthalenes accounted for 58% and 38%
of total PAHs in PBC and WCS, respectively. However, quantities of 3 and 4 ring compounds in PBC were
less than those for WCS. Biodegradation of individual PAHs and their alkylated homologues suggest that
naphthalenes diminished faster and to greater extent as compared to other PAHs, where C0-2 -NAP
approached below detectable limit for all treatments. Both cryo and meso KRE enrichments were able
to metabolize 100% of C3- NAP and almost 90-95% of C4- NAP. However, AFE culture did not exhibit
extensive degradation of C3-4-NAP for either WCS or PBC. Higher removal of the 3 ring compounds (PHE,
FLU, DBT) was noted at 25 °C for the KRE treatments, however, for the AF treatments, concentration
decreased until day 20 after which there was no apparent change. Almost 33% and 57% of the residual
compounds were seen in PBC and WCS respectively and this fraction mainly comprised of C2-4-PHE, C2-3-
FLU, and C1-3 DBT. At 5 9C, AFE metabolized C01-PHE, Co-FLU, and Co-DBT hydrocarbons, compared to
treatments with KRE cryo culture which also showed disappearance of C1-2-FLU, Co-DBT. No significant
change was observed in concentrations of heavier hydrocarbons (NBT, CHY, PYR) in treatments involving
AFE or KRE cultures. However, KRE meso culture was able to assimilate 80% of the 4 ring compounds in
both WCS and PBC.
Overall, the rate and extent of biodegradation by KRE and AFE was much lower at colder temperatures,
similar to previous observations (Atlas and Bartha, 1972; Margesin and Schinner, 2001; Venosa and
36
-------
Holder, 2007; Campo et al., 2013; Zhuang et al., 2016). This may be due to lower solubilities and
crystallization of hydrocarbons, and lower metabolic rates at lower temperatures. Along with the
temperature, microbial enrichment had a substantial influence on the degradation of both the crude
oils. Significant differences were noted in the nature and extent of hydrocarbon metabolism of the three
cultures. KRE meso culture degraded both the crude oils more efficiently. All cultures were able to
degrade alkanes, while for PAH degradation, KRE cryo culture was less effective than KRE meso culture.
The AFE culture behaved differently at 5 and 25 9C, taking almost 8 days to adapt to the colder
temperature before metabolizing the hydrocarbons. This difference in the microbial activities could be
explained by diversity in their community composition, differences in their origin (Kalamazoo River vs.
Ohio River), and varying carbon source (dilbit vs ANS) on which they were enriched. Although studies
have reported degradation of crude oils using aforementioned dominant microbial communities
(Margesin and Schinner, 2001; Cao et al., 2009; Rojo, 2009), AFE was less competent.
3.3.3. Microbial Community Structure during Dilbit Biodegradation
Consortia used in these experiments were isolated from two different locations and were enriched on
two different oils at different temperatures prior to the inoculation of the microcosm. Microbial
community structure analysis was done to characterize these enrichments and provide composition of
the three cultures - KRE meso, KRE cryo and AFE. Analysis of 279,807 16S rRNA gene sequences from all
the treatments showed that several bacterial groups were present in the Kalamazoo River meso and cryo
enrichments, with similar bacterial composition (Figure 3.6). On the phylum level, no significant
difference was observed. Proteobacteria was the dominating phyla in all the cultures, additionally,
Actinobacteria and Bacteroidetes were also present in both the KRE cultures. Oil degrading microbial
community on the genus level was markedly different between the KRE cultures and AFE. Acinetobacter
(72%) was the dominant genera in AFE microbial community, whereas high abundance of Pseudomonas
(13%, 17%), Rhodococcus (22%, 26.5%), and Hydrogenophaga (15%, 12%) was observed in KRE cryo and
meso cultures. Though several other genera were present in these consortia, their abundance was very
low as compared to the above mentioned genera.
For experiments using KRE cultures, at the start of experiments (day 0), 10 phyla were detected;
dominated by members of the Proteobacteria (74 ± 7 and 73 ± 3% for meso and cryo, respectively), the
Actinobaceria (24 ± 7 and 16 ± 4%), and the Bacteroidetes (3 ± 1 and 10 ± 1%). Most of the Proteobacteria
in the cryo enrichment was associated with beta-Proteobacteria (41 ± 3%), while in the meso
enrichment, gamma-Proteobacteria (41 ± 6%) was the most abundant class. A total of 190 different
genera were identified from the DNA extracts obtained at day 0. Pseudomonas, Rhodococcus, and
Hydrogenophaga were the prominent genera present in both enrichments, but their abundance varied.
For example, 21 ± 4 % of the sequences in the meso enrichment were linked to Pseudomonas versus 9 ±
1% in the cryo enrichment. Rhodocoocus represented 15 ± 4 and 19 ± 8 % of the sequences in cryo and
meso enrichments, respectively. Genera with abundance less than 1% were grouped together and
labeled as other. This group represented more than 150 different genera (e.g., Acinetobacter,
Pedobacter, Achromobacter, Aquabacterium). The microbial community structure of KRE cryo and meso
37
-------
enrichments changed considerably during experiments, however, there were no observable differences
when the two different oil types were compared. For example, by day 2, Pseudomonas sp. comprised up
to ~ 90% of the total community in meso enrichment, which represented more than a four-fold increase
from day 0. The same group decreased in abundance by day 16 (i.e., approximately 60%) followed by a
gradual increase. The community profile did not alter significantly from day 16 until the end of the
experiment (day 60) in either one of the meso treatments. Even though Pseudomonas dominated
microbial community in meso consortium throughout the time course, other genera like
Hydrogenophaga, Parvibaculum and unclassified members of Xanthomonadaceae and gamma-
Proteobacteria showed higher abundance as well. Unlike meso enrichment, no one single genus
dominated the cryo enrichment.
While there were no differences between the overall community profiles between two dilbits,
differences were noted when temperatures were taken into account. Specifically, no significant
differences were detected in observed species when treatments with two different dilbits were
compared (p = > 0.901). Nevertheless, statistical analysis showed significant differences in Shannon
diversity based on temperature (p < 0.001), although the difference in observed species was not
significant (p = 0.443). These results indicate that while similar number of taxa present at 5 and 25 °C,
their abundance and evenness varied. Multivariate analysis showed that for a given treatment similar
taxa were present and microbial community shifted with time. The changes in community structure were
more significant at 5 °C compared to 25 °C. PERMANOVA analysis suggested that there was no significant
difference in taxa composition between CLB and WCS (p = 0.953) whereas the when two temperatures
were compared, the taxa composition was statistically different (p = 0.001).
38
-------
Table 3.3 Percent removal and first order rate constants for total alkanes, total PAHs and TPH degradation.
KRE = Kalamazoo River Enrichment; AFE = Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western
Canadian Select; PBC = Prudhoe Bay Crude. Data Sources: Deshpande, 2016; Deshpande et at, 2017.
I
<°< >
Oil
I'.n riili mi-ill
1
II.
ki'llliiN ;il
(Hill Alk;
/. (>l ')
IKS
SI)
R2
<».'
o
Kl'llln\;il
I hI;iI l>.\
/. <>l ')
II
SI)
R2
<».'
O
Ri-nui\;il
1 l>l 1
/. (il ')
sill.
1. I'll II'
R:
5
CLB
KRE
99.62
0.52
0.09
1.00
65.56
0.05
0.02
1.00
19.75
0.04
0.03
0.83
5
WCS
KRE
99.92
0.48
0.10
1.00
74.19
0.07
0.03
0.99
26.18
0.06
0.01
0.97
5
PBC
KRE
99.68
0.56
0.08
1.00
85.31
0.15
0.09
0.99
56.56
0.06
0.01
0.97
5
WCS
AFE
98.01
0.44
0.23
1.00
55.74
0.04
0.03
0.98
25.83
0.01
0.01
0.94
5
PBC
AFE
99.23
0.24
0.09
1.00
79.18
0.03
0.03
0.98
44.47
0.07
0.02
0.96
25
CLB
KRE
99.97
1.13
0.04
1.00
97.56
0.14
0.03
0.98
69.18
0.07
0.02
0.97
25
WCS
KRE
99.95
1.14
0.06
1.00
97.26
0.16
0.01
1.00
64.32
0.11
0.04
0.96
25
PBC
KRE
99.89
1.53
0.10
1.00
98.47
0.24
0.04
1.00
82.51
0.11
0.06
0.97
25
WCS
AFE
99.18
0.90
0.04
1.00
60.88
0.18
0.06
0.98
25.17
0.12
0.02
0.96
25
PBC
AFE
99.85
1.26
0.09
1.00
84.46
0.31
0.05
1.00
52.21
0.16
0.03
0.96
39
-------
5000
5000
(A) Total Alkanes - 25°C
(B) Total Alkanes - 5°C
4000
-- 4000
CLB+KRE
WCS+KRE
WCS+AFE
3000
3000
2000
- 2000
1000 --
-- 1000
(D) Total PAHs - 5°C
(C) Total PAHs - 25°C
4000 z;
- 4000
"T 3000
-- 3000
V 2000
- 2000
1000
- 1000
(E) TPH - 25°C
(F) TPH - 5°C
600
-- 600
400
— 400
200
-- 200
0
20
40
60
0
20
40
60
Time (Days)
Figure 3.3. Alkanes, PAH and TPH concentration at 25 and 5 °C. KRE = Kalamazoo River Enrichment; AFE =
Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select. Data Sources:
Deshpande, 2016; Deshpande eta!., 2017.
40
-------
(A) Total Alkanes - 25 °C
(B) Total Alkanes - 5 °C
100 -
-- 100
80
-- 80
CLB+KRE
WCS+KRE
WCS+AFE
PBC+KRE
PBC+AFE
40 -
- 40
20 - I
-- 20
(C) Total PAHs - 25 °C
(D) Total PAHs - 5 °C
100 -
-- 100
80 --
- - 80
>
o
E 60 -
03
a:
©"
40
40
20
-- 20
(E) TPH -25 °C
(F) TPH - 5 °C
100 --
-- 100
80
60
60
40 -
40
20 --
-- 20
0
20
40
60
0
20
40
60
Time (Days)
Figure 3,4. Alkanes, PAH and TPH percent removal at 25 and 5 °C. Figure 3.2. Alkanes, PAH and TPH
concentration at 25 and 5 °C. KRE = Kalamazoo River Enrichment; AFE = Anderson Ferry Enrichment; CLB =
Cold Lake Blend; WCS = Western Canadian Select. Data Sources: Deshpande, 2016; Deshpande et a!.,
2017.Data Sources: Deshpande, 2016; Deshpande et a!., 2017.
41
-------
Table 3.4. Oil biodegradation rate comparison matrix. KRE = Kalamazoo River Enrichment; AFE = Anderson
Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select. Red box represents significant
difference between treatment and green box represents no significant difference based on ANOVA p values.
Data Sources: Deshpande, 2016; Deshpande et al., 2017.
Significant
difference
No Significant
Difference
Degradation Rate Comparison A - Total Alkanes P - Total PAH
Enrichment
WCS
42
-------
CLB - 5 °C
I WCS - 5 °C
I CLB - 25 °C
100 -
h- I— H
CD CO CO
D Q Q
¦A Csl
o o
WCS - 25 °C
ill
H H K
CD CO DO
z z
DO
z z
h i
>- >-
Z X
o o
>-
X
o
Figure 3.5. Percent removal of individual PAH compounds. Source: Deshpande eta!., 2017.
43
-------
100
100
d)
o
c
ra
T3
c
3
¦O
<
0)
o
c
ro
T3
c
-Q
<
I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I l I I I I I II I I I I II I I
0 2 163260 0 2 163260 0 2 163260 0 2 163260 0 2 163260 0 2 164072 0 2 164072 0 2 164072 0 2 164072 0 2 164072
CLB+KRE WCS+KRE PBC+KRE WCS+AFE PBC+AFE CLB+KRE WCS+KRE PBC+KRE WCS+AFE PBC+AFE
25 °C 5 °C
Pseudomonas
Dokdonella
Pedobacter
Acinetobacter
~
Dyadobacter
~
Polaromonas
Rhodococcus
Ferrugnibacter
~
Pseudomonadaceae Other
Hydrogenophaga
Flavobacterium
Pseudoxanthomonas
Parvibaculum
Frigoribacterium
Ralstonia
Sphingobium
HB2-32-21
Reyranella
Acidovorax
Janthinobacterium
Rhizobium
Alcaligenaceae Other
Legionella
Rhodanobacter
Arthrobacter
Microbacteriaceae Other
Sphingomonas
Bacteriovorax
Mucilaginibacter
Sphingopyxis
Brucellaceae Other
Mycobacterium
Stenotrophomonas
Gammaproteobacteria other
Mycoplana
Sulfuritalea
Caulobacter
Novosphingobium
Verrucomicrobium
Caulobacteraceae Other
PYR10d13 Other
Williamsia
~
Chryseobacterium
Ochrobactrum
z
Xanthomonadaceae other
~
Achromobacter
Oxalicibacterium
Other
Figure 3.6. Microbial Community Structure Analysis during oil biodegradation experiments. KRE = Kalamazoo
River Enrichment; AFE = Anderson Ferry Enrichment; CLB = Cold Lake Blend; WCS = Western Canadian Select;
PBC = Prudhoe Bay Crude. Data Sources: Deshpande, 2016; Deshpande et a!., 2017.
44
-------
3,4, Toxicity of Dilbit to Standard Aquatic Species
3,4,1. Oil and WMF Chemistry
Chemical analyses of the 100% WAF indicate that total PAH and alkane concentrations in unweathered
(fresh) and weathered CLB and WCS were generally similar (Table 3.5). Fresh CLB exhibited higher TPH
values (8.36-16.2 mg/L oil), whereas WCS was higher in PAH (18.0-40.0 |ag/L oil). CLB and WCS
demonstrated similar concentrations of individual PAH analytes and alkyl homologs, where WCS
exhibited a larger proportion of NO-4 Naphthalenes (Figure 3.7). Results indicate that Total PAH (17.7
mg/g) and alkane (50.5 mg/g) concentrations in the ANS reference oil were 2 to 10 fold higher than in
the dilbits. Mean TPH concentrations in 100% WAF were about 1.5 to 3 fold higher in CLB and WCS (5.9
to 16.2 mg/L) compared to the weathered dilbits (3.9 to 5.6 mg/L) (Table 3.5). Mean PAH concentrations
in 100% WAF were similar in fresh or weathered CLB and WCS, and ranged from 13.8 to 40 |-ig/L. Mean
BTEX concentrations in 100% WAF were about tenfold higher in CLB and WCS (4.6 to 11.4 mg/L) than in
the weathered dilbits (0.53 to 0.94 mg/L).
Table 3.5. Hydrocarbon concentrations1 and percent survival of four species in 100% water accommodated
fractions (WAF) of two unweathered and weathered dilbits2 at 24 hours and test end (48 hr invertebrates; 96
hr fish). Source: Table generated from Barron et at, 2017 data.
Oil
% Survival in 100% WAF
% Survival in 100% WAF
100% WAF concentration2
Test WAF1
Loading
(g/L)
24 hour
Test End
24 hour
Test End
BTEX
(mg/L)
PAH
(ug/L)
TPH
(mg/L)
FW WAF
C. dubia
C. dubia
P. promelas
P. promelas
CLB
50
25
80
100
65
85
73
83
73
80
7.24
11.4
27
20.5
16.2
11.1
CLB W
50
100
100
93.3
93.3
0.76
26.8
5.57
WCS
50
25
100
100
95
85
80
100
80
100
5.86
5.56
40
29.4
10.7
8.33
WCS W
50
100
85
100
100
0.94
34.9
4.69
SW WAF
A. Bahia
A. Bahia
M. beryllina
M. beryllina
CLB
50
25
0
10
0
3
6
47
3
47
5.66
5.09
20.7
13.8
9.88
8.36
CLB W
50
86.6
70
100
83.3
0.53
22.1
3.92
WCS
50
25
0
70
0
53
13.3
97
13.3
97
4.91
4.59
32.6
18
7.96
5.9
WCS W
50
100
100
100
93.3
0.7
28.7
3.93
1. CLB: Cold Lake Blend dilbit; CLB W: weathered CLB; WCS: Western Canadian Select dilbit; WCS W: weathered WCS; FW:
freshwater; SW: saltwater.
2. BTEX: benzene, toluene, ethylbenzene, xylenes; PAH
hydrocarbons.
polycyclic aromatic hydrocarbons; TPH: total petroleum
45
-------
10-0 -
r.5-
5.0-
>0".
2.5-
2.5-
to.o-
CLB
wcs
L
ll«B—
JUUu
ll*l"- *¦- ¦¦¦—
¦-rffTrMM1* . i ¦ ft
IMtt i Iff-rjMft i
¦n
CD
=r
¦
cn
1.
§
ILlal.—
0^c.r~r70—rfn'^O'-rina'-tNr^c^'-ryp'TO'-c iCTTo—rjr^r o—T^To—o;oo—CNe:'ro--c.>T?Q--fC>^o—c.cn*T
zzzzzLi^i^^^GCCGa^-^L^"-y>>X)uuaj zzzzzai.c.iiji.iJ»^aQQaQri-(-H-»>.V>.ooooo
22222QAQ.Q.C.
^ -r -r ¦*" -r f\ ft f|fl r
PAH
Figure 3.7. Concentration of individual PAH analytes and alkyl homologs in water accommodated fractions
(WAF) of fresh (unweathered) and weathered Cold Lake Blend (CLB) and Western Canadian Select (WCS)
dilbits. FW = freshwater; SW = saltwater. Source: Borron et a!., 2017.
3.4.2. Acute and Chronic Dilhit Toxicity
In general, the toxicity of WAF prepared from unweathered and weathered CLB and WCS was similar in
all four tested species. LC20 and LC50 values determined at loading rates of 25 or 50 g oil/L ranged from
3.6 to >16 mg/L TPH and 7.4 to >40 ug/L PAH (Table 3.6). Acute toxicity values based on measured BTEX
in WAF were lower in weathered CLB and WCS and were substantially more variable (>0.7 to >15.9 mg/L)
most likely due to depleted BTEX levels (Table 3.6). TPH concentrations were highly correlated and a
significant predictor of acute dilbit toxicity to three of the four test species (excluding C. dubia),
compared to BTEX and PAH, which were generally not significantly correlated with dilbit toxicity except
for mysids (Figure 3.8). Weathered CLB and WCS impaired growth [A. bahia) and reproduction (C. dubia)
at concentrations of 0.41 to 3.5 mg/L TPH, 5.7 to 16 ug/L PAH, and 0.0023 to 1.1 mg/L BTEX (Table 3.7).
Acute toxicity values of ANS in the four test species ranged from 2,1 to >5 mg/L TPH, 75 to >107 ug/L
46
-------
PAH, and >4.3 to >5.6 mg/L BTEX (Table 3.9). All acute and chronic tests met quality control and quality
assurance requirements, including control survival and water quality conditions (Table 2.2).
The toxicity of CLB and WCS determined in standard aquatic toxicity tests were generally similar to other
oil products based on both TPH and PAH-based measures of effect, including ANS, tested here. For
example, TPH-based LC50s for unweathered CLB and WCS determined in mysids (5.6-7.0 mg/L) and
menidia (5.9-8.3 mg/L) were consistent with average acute toxicity values for mysids (2.7 mg/L) and
menidia (7.5 mg/L) using conventional WAF preparation and toxicity test methods across a range of
crude oils (Barron et al. 2013). PAH concentrations impairing survival, growth or reproduction in short
term chronic studies with these four North American species (6 to >16 ug/L) were consistent with PAH
effect concentrations (3 to 200 ug/L) determined in developmental toxicity studies of dilbits with
zebrafish and medaka (Madison et al. 2015, 2017; Alderman et al. 2016; Philibert et al. 2016). PAH-based
LC50 values of CLB and WCS (9.8 to >40 ug/L) were also within the range of acute toxicity values of crude
oils for a diversity of aquatic species (30-150 ug PAH/L) (Bejarano et al., 2017). The weathered dilbits
were chronically toxic, but showed only limited acute toxicity, presumably due to depletion of BTEX.
Monoaromatics and napthalenes have long been considered the drivers of acute toxicity of petroleum,
including in a recent study of two crude oils and a dilbit (Philibert et al. 2016). Toxicity values were
reported for separate analyte groups (BTEX, PAHs), with the recognition that petroleum exposures are
mixtures of a group of diverse hydrocarbons that can contribute to toxicity. Consistent with the general
literature, effect levels of the dilbits were based on measured concentrations in 100% WAF and did not
reflect loss of hydrocarbons over time, thus actual effect levels would be lower than reported here
(Redman and Parkerton 2015).
47
-------
Table 3.6. Acute toxicity of fresh and weathered Cold Lake Blend and Western Canadian Select dilbit to four
species as three measures of hydrocarbon exposure.1 Data source: Barron etaL, 2017.
Cold Lake Blend
Western Canadian Select
Test Species
Weathered
State
Test
Endpoint
Oil
Loading
(g/L)
%
WAF
BTEX1
(mg/L)
tPAH1
(ug/L)
TPH1
(mg/L)
%
WAF
BTEX1
(mg/L)
tPAH1
(ug/L)
TPH1
(mg/L)
Cladoceran
(Ceriodaphnia
dubia)
Fresh
48 hr LC20
50
25
94.2
68.2
6.82
7.74
25.4
14.0
15.2
7.54
>100
>100
>5.86
>5.56
>40.0
>29.4
>10.7
>8.33
48 hr LC50
50
25
>100
70.7
>7.24
8.02
>27.0
14.5
>16.2
7.82
>100
>100
>5.86
>5.56
>40.0
>29.4
>10.74
>8.33
Weathered
48 hr LC20
50
>100
>0.76
>26.8
>5.57
>100
>0.94
>34.9
>4.69
48 hr LC50
50
>100
>0.76
>26.8
>5.57
>100
>0.94
>34.9
>4.69
Mysid
(Americamysis
bahia)
Fresh
48 hr LC20
50
25
67.8
55.2
3.84
2.81
14.1
7.63
6.7
4.61
67.8
91.1
3.33
4.18
22.1
16.4
5.37
5.37
48 hr LC50
50
25
70.5
70.9
3.99
3.61
14.6
9.80
6.97
5.93
70.5
>100
3.46
>4.59
23.0
>18.0
5.61
>5.90
Weathered
48 hr LC20
50
91.1
0.48
20.1
3.57
>100
>0.70
>28.7
>3.93
48 hr LC50
50
>100
>0.53
>22.1
>3.92
>100
>0.70
>28.7
>3.93
Fathead
minnow
(Pimephales
promelas)
Fresh
96 hr LC20
50
25
96.9
97.2
7.02
11.03
26.2
19.9
15.7
10.8
91.1
>100
5.34
>5.56
36.5
>29.4
9.78
>8.33
96 hr LC50
50
25
>100
>100
>7.24
>11.4
>27.0
>20.5
>16.3
>11.1
>100
>100
>5.86
>5.56
>40.0
>29.4
>10.7
>8.33
Weathered
96 hr LC20
50
>100
>0.76
>26.8
>5.57
>100
>0.94
>34.9
>4.69
96 hr LC50
50
>100
>0.76
>26.8
>5.57
>100
>0.94
>34.9
>4.69
Inland
silverside
(Menidia
beryllina)
Fresh
96 hr LC20
50
25
61.3
89.9
3.47
4.58
12.7
12.4
6.06
7.52
50.7
>100
2.49
>4.59
16.5
>18.0
4.04
>5.90
96 hr LC50
50
25
70.7
99.1
4.00
5.04
14.5
13.7
6.99
8.28
73.8
>100
3.62
>4.59
24.1
>18.0
5.87
>5.90
Weathered
96 hr LC20
50
>100
>0.53
>22.1
>3.92
>100
>0.7
>28.7
>3.93
96 hr LC50
50
>100
>0.53
>22.1
>3.92
>100
>0.7
>28.7
>3.93
1. BTEX: benzene, toluene, ethylbenzene, xylenes; tPAH: sum of detected polycyclic aromatic hydrocarbons and homolog
groups; TPH: total petroleum hydrocarbons.
48
-------
BTEX
PAH
TPH
y = -16.4x + 96.5
RZ = 0.791
p value = 0.018
y = -1.21x + 10.2
R2 = 0.038
p value = 0.711
\
O"
Qj
y = -16.4x + 147 03
R2 = 0.889
p value = 0.005
100 -
75-
50-
25-
o-
y = -1.26x + 92.5
R2 = 0.177
p value = 0.406
•X,
y = 0.480x + 71.5
R2 = 0.076
p value = 0.598
y = -2.03x + 105
R2 = 0.506
p value = 0.113
•
y = -12.2x + 99.9
y = -0.94x + 77.4
•
•\
y = -14.7x + 154
•
r2 = 0.473
R2 = 0.025 •
•
R2 = 0.776
p value = 0.131
p value = 0.766
p value = 0.020
y = -1.93x + 97.9
R2 = 0.449
p value = 0.146
y = 0.321x + 78.3
R2 = 0.035
p value = 0.722
i I II
o
CL
a
O"
CO
D"
CD
^2
TJ
O
3
fD_
y = -2.44x +111 Qj
2 co
R =0.784
p value = 0.019
0 10 20 30 40 0 10 20 30 40 0 10 20 30 40
Concentration
Oil
CLB
wCLB
WCS
wWCS
Figure 3.8. Percent survival of four aquatic species exposed to water accommodated fractions of fresh and
weathered dilbits: Cold Lake Blend (CLB), weathered CLB (wCLB), Western Canadian Select (WCS), and
weathered WCS (wWCS). Test concentrations: BTEX (mg/L); PAH (ug/L); TPH (mg/L). Test species:
Ceriodaphnia {Ceriodaphnia duhia); mysid (Americamysis bahia); fathead minnow (Pimephales promelas);
inland silverside (Menidia beryllino).
49
-------
Table 3.7. Short-term chronic toxicity of two weathered dilbits to C. dubia and A. bahia as three measures of
hydrocarbon exposure.1 Source: Barron etaL, 2017.
Test Species
Test Type
Dilbit
Endpoint2
%
WAF
BTEX1
(mg/L)
tPAH1
(ug/L)
TPH1
(mg/L)
Cladoceran
(Ceriodaphnia
dubia)
7-day survival
and
reproduction
Cold Lake
Blend
NOEC
25
0.088
5.99
1.67
IC25
52
0.185
12.5
3.49
Western
Canadian
Select
NOEC
6.25
0.0003
1.90
0.094
IC25
53
0.0024
16.0
0.789
Mysid
(Americamysis
bahia)
7-day survival
and growth
Cold Lake
Blend
NOEC
25
0.0015
3.67
0.262
IC25
39
0.0023
5.72
0.409
Western
Canadian
Select
NOEC
25
0.865
6.15
0.561
IC25
32
1.10
7.82
0.713
1. BTEX: benzene, toluene, ethylbenzene, xylenes; tPAH: sum of detected polycyclic aromatic hydrocarbons and
homolog groups; TPH: total petroleum hydrocarbons.
2. NOEC: no observed effect concentration; IC25: concentrations causing 25% inhibition.
50
-------
Table 3.8. Acute toxicity of Alaskan North Slope crude oil to four species as three measures of hydrocarbon
exposure.1 Data source: Barron et al., 2017.
Test Species
Test
Endpoint
% WAF
BTEX1
(mg/L)
tPAH1
(ug/L)
TPH1
(mg/L)
Cladoceran
LC20
96.1
5.4
103
4.83
(Ceriodaphnia
dubia)
LC50
>100
>5.6
>107
>5.03
Mysid
LC20
97.8
5.4
75.3
2.14
(Americamysis
bahia)
LC50
>100
>4.3
>77.0
>2.19
Fathead minnow
LC20
>100
>5.6
>107
>5.03
(Pimephales
promelas)
LC50
>100
>5.6
>107
>5.3
Inland silverside
LC20
>100
>4.3
>77.0
>2.19
(Menidia
beryllina)
LC50
>100
>4.3
>77.0
>2.19
1. BTEX: benzene, toluene, ethylbenzene, xylenes; PAH: polycyclic aromatic
hydrocarbons; TPH: total petroleum hydrocarbons.
51
-------
4. Report Summary
Dilbit presents an increasing environmental concern because of extensive transport in North America,
recent spills into aquatic habitats, and limited understanding of environmental fate and toxicity (Dupuis
and Ucan-Marin 2015, NAS 2016; Lee et al. 2015). Dilbit is a blend of highly weathered bitumen and
lighter diluent oils that contain higher concentrations of asphaltenes (>10%) and lower levels of saturates
(~40%) (NAS 2016), with unique properties, including high adhesion, and the potential for rapid
weathering, sinking and associating with sediments. Information on dilbit biodegradation, toxicity,
dispersion and fate is limited and warrants further study, particularly given the variety of types of dilbit
and variability in weathering states in the environment. This research will allow for more informed risk
assessments and improved emergency response during dilbit spills in aquatic and terrestrial
environments.
This research showed that select fractions of dilbit can be efficiently biodegraded, but under similar
conditions, conventional crude oil was degraded more effectively due to the higher content of lighter
hydrocarbons. The rates of alkane and PAH biodegradation were comparable for dilbit products, but the
extent of degradation was greater for PBC because of the higher concentrations of lighter alkanes. The
potential of microbial enrichment to degrade crude oil was highly influenced by temperature as well as
the composition. Lower degradation rates were achieved at the lower temperature. As per the results
of genomic sequencing, well-known oil degraders metabolized both the dilbit types, but their
performance varied. All the enrichments metabolized PBC as well WCS, but the nature and extent of the
degradation was distinct. KRE meso culture was the most effective among all, as it completely removed
alkanes and PAHs. AFE enrichment performed differently at two temperatures; an acclimation period of
8 days was observed at 5 9C while there was no lag at 25 °C. KRE meso culture as well as AFE culture at
both the temperatures degraded alkanes completely while they were not able to metabolize heavier
fractions of the oil (C2-4 homologues of 3 ring compounds and 4 ring compounds).
The aquatic effects data generated here for two dilbits to four standard aquatic species indicate that
dilbit products can have similar acute and short-term sublethal toxicity as crude oils and other petroleum
products, but information is still extremely limited. Acute toxicity of unweathered and weathered dilbit
products was similar in all four species, and weathered dilbit products were sublethally toxic at 0.8 to 16
ug/L total petroleum hydrocarbons. These levels are similar to toxicity reported for other aquatic species
and oil products. Additionally, for future research, the unique hydrocarbon composition of dilbit
consisting of both heavy and light components necessitates including additional analytes such as
asphaltenes, C5-C9 alkanes, and additional monoaromatics.
52
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5. References
Alberta Innovates Energy an Environment Solution (2011), Comparison of the Corrosivity of Dilbit and
Conventional Crude. 2480002; 29 pp.
Alberta Energy (2016) Facts and Statistics. http://www.energy.alberta.ca/OilSands 2016.
Alderman SL, F Lin, AP Farrell, CJ Kennedy, TE Gillis (2016) Effects of diluted bitumen exposure on juvenile sockeye
salmon: from cells to performance. Env Tox Chem 36:354-360.
Alharbi HA, G Morandi, JP Giesy, SB Wiseman (2016) Effect of oil sands process-affected water on toxicity of retene
to early life-stages of Japanese medaka (Oryzias latipes). Aquat Tox 176:1-9.
American Petroleum Institute (2012) Diluted Bitumen: What it is, pipeline transportation and impact on pipelines.
Presentation to the TRB Panel Investigating Diluted Bitumen's Impact on Oil Pipelines by Peter Lidiak, API Pipeline
Director. http://onlinepubs.trb.org/onlinepubs/Dilbit/Lidiak072312.pdf.
American Petroleum Institute (2011) Pipeline transportation of diluted bitumen from the Canadian Oil Sands,
White Paper, 3 pp.
Atlas, R. M., Bartha, R. (1972) Biodegradation of petroleum in seawater at low temperatures. Canadian Journal of
Microbiology, 18:1851-1855.
Barron, M.G., Carls, M.G., Short, J.W., Rice, S.D. (2003) Photoenhanced toxicity of aqueous phase and chemically
dispersed weathered Alaska North Slope crude oil to Pacific herring eggs and larvae. Environ Toxicol Chem.; 22(3):
650-60.
Barron, MG, Hemmer MJ, Jackson CR (2013) Development of aquatic toxicity benchmarks for oil products using
species sensitivity distributions. Integrat. Environ. Assess Manag 9:610-615.
Barron, M.G., Conmy, R.N., Holder, E.L., Meyer, P., Wilson, G.J., Principe, V.E., Willming, M.M. (2017; under
review) Toxicity of Cold Lake Blend and Western Canadian Select dilbits to standard aquatic test species.
Chemosphere.
Bauer AE, RA Frank, JV Headley, KM Peru, AJ Farwell, DG Dixon (2017) Toxicity of oil sands acid-extractable organic
fractions to freshwater fish: Pimephales promelas (fathead minnow) and Oryzias latipes (Japanese medaka).
Chemosphere 171:168-176.
Bejarano AC, WW Gardiner, MG Barron, JQ Word (2017) Relative sensitivity of Arctic species to physically and
chemically dispersed oil determined from three hydrocarbon measures of aquatic toxicity. Mar Poll Bull (in press).
Campo, P. (2013) Biodegradability of Corexit 9500 and dispersed South Louisiana crude oil at 5 and 25 C. Environ.
Sci. Technol, 47, 1960.
Canadian Association of Petroleum Producers (CAPP) (2012) Crude Oil Forecast, Markets & Pipelines; Appendix D.
CAPP; Canadian Association of Petroleum Producers (2015) Crude Oil Forecast, Markets & Transportation;
Publication Number 2015-0007.
Canadian Energy Pipelines Association (CEPA) (2013) State of the Art Report: Dilbit Corrosivity, 12671-RPT-001
REV 1.
53
-------
Canadian Government (2013) Properties, composition and marine spill behavior, fate and transport of two diluted
bitumen products from the Canadian Oil Sands. Federal Government Technical Report. Environment Canada,
Fisheries and Oceans Canada, Natural Resources Canada. November 30, 2013. ISBN 978-1-100-23004-7; 85pp.
Cao, B.; Nagarajan, K.; Loh, K. (2009) Biodegradation of aromatic compounds: current status and opportunities for
biomolecular approaches. Appl. Microbiol. Biotechnol., 85, 207-228.
Coblani, S. E.; Greer, C. W.; King, T. L.; Robinson, B. J.; Ryan, S. A.; Fortin, N.; Wohlgeschaffen, G. D.; Mason, J. A.;
Thamer, P. M.; Mclntyre, E. C.; Lee, K. (2015) In: In Field Studies to Monitor Indigenous Microbial Respiration to
Determine the Potential Biodegradation of Naturally and Chemically Dispersed Crude Oil, Condensate and Diluted
Bitumen; Poster presented at 38th AMOP Technical Seminar; Vancouver, Canada.
Colavecchia MB, SB Backus, PV Hodson, JL Parrott (2004) Toxicity of oil sands to early life stages of fathead
minnows (Pimephales promelas). Env Tox Chem 23:1709-1718.
Crosby, S.; Fay, R.; Groark, C.; Kani, A.; Smith, J. R.; Sullivan, T.; Pavia, R. (2013) Transporting Alberta Oil Sands
Products: Defining the Issues and Assessing the Risks. U.S. Dept. of Commerce, NOAA Technical Memorandum
NOSOR&R43, 153 pp.
Deshpande, R.S. (2016) Biodegradability of Diluted Bitumen. University of Cincinnati, Master's Thesis, Cincinnati,
OH, USA, 72 pp.
Deshpande, R.S., Sundaravadivelu, D., Campo-Moreno, P., Techmann, S. SantoDomino, J.W., Conmy, R.N. (2017,
in review) Microbial degradation of Cold Lake Blend and Western Canadian Select dilbits in freshwater. J.
Hazardous Materials.
Dew WA, A Hontela, SB Rood, GG Pyle (2015) Biological effects and toxicity of diluted bitumen and its constituents
in freshwater systems. J. Appl. Tox. 35:1219-1227.
Dupuis, A., and Ucan-Marin, F. (2015) A literature review on the aquatic toxicology of petroleum oil: An overview
of oil properties and effects to aquatic biota. DFO Can. Sci. Advis. Sec. Res. Doc. 2015/007. 52 pp.
Energy Information Administration. (2013). Company Level Imports. From U.S. Energy Information Administration:
http://www.eia.gov/petroleum/imports/companylevel/.
Fingas, M.F., Hughes, K.A., and Schweitzer, M.A. (1987) Dispersant Testing at The Environmental Emergencies
Technology Division, Proc. Tenth Arctic Marine Oilspill Program Technical Seminar, Edmonton, Alberta, Canada,
Environment Canada: 343-356.
Grubbs, F. (1969) Sample Criteria for Testing Outlying Observations, Annals of Mathematical Statistics, pp. 27-58.
Kaku, V.J., M.C. Boufadel, A.D. Venosa (2006) Evaluation of mixing energy in laboratory flasks used for dispersant
effectiveness testing. ASCE J. Environmental Eng. Div. 132(1): 93-101.
King, T. L.; Robinson, B.; Boufadel, M.; Lee, K. (2014) Flume tank studies to elucidate the fate and behavior of
diluted bitumen spilled at sea. Mar. Pollut. Bull., 83, 32-37.
King, T. L.; Robinson, B.; Boufadel, M.; Lee, K., Cui, F., Clyburne, J.A.C. (2017) An oil spill decision matrix in response
to surface spills of various bitumen blends. Environ. Sci.: Processes and Impacts. D0l:10.1039/C7EM00118E.
Lee, K, M Boufadel, B Chen, J Foght, P Hodson, S Swanson, A Venosa (2015) Expert Panel Report on the Behaviour
and Environmental Impacts of Crude Oil Released into Aqueous Environments. Royal Society of Canada, Ottawa,
ON. ISBN: 978-1-928140-02-3.
54
-------
Li, Z., Lee, K., King, T., Boufadel, M.C., Venosa, A.D. (2009) Evaluating crude oil chemical dispersion efficacy in a
flow-through wave tank under regular nonbreaking wave and breaking wave conditions, Mar. Pollut. Bull. 58 (5),
735-744.
Lin, L.H., Michael, G.E., Kovachev, G., Zhu, H., Philp, R.P., Lewis, C. A. (1989) Biodegradation of tar-sand bitumens
from the Ardmore and Anadarko Basins, Carter County, Oklahoma. Org. Geochem., 14, 511-523.
Madison BN, PV Hodson, VS Langlois (2015) Diluted bitumen causes deformities and molecular responses
indicative of oxidative stress in Japanese medaka embryos. Aquat Tox 165:222-230.
Madison BN, PV Hodson, VS Langlois (2017) Diluted bitumen toxicitiy to the early development of Japanese
medaka. Environ Poll 225:579-586.
Margesin, R., Schinner, F. (2001) Biodegradation and bioremediation of hydrocarbons in extreme environments.
Applied Microbiology and Biotechnology, 56, 650-663.
NAS (National Academies of Sciences, Engineering, and Medicine) (1985) Oil in the Sea: Inputs, Fates and Effects,
National Academies Press, Washington, D.C.
NAS (National Academies of Sciences, Engineering, and Medicine) (2016) Spills of Diluted Bitumen from Pipelines:
A Comparative Study of Environmental Fate, Effects, and Response, The National Academies Press, Washington,
D.C.
NRC (National Research Council) (2013) TRB Special Report 311: Effects of Diluted Bitumen on Crude Oil
Transmission Pipelines, Transportation Research Board, Washington, D.C.
Oil Sands Magazine (2017) Oil sands product stream, Western Canadian Select explained. Accessed 17.03.08;
http://www.oilsandsmagazine.com.
Penspen Integrity (2013) Dilbit Corrosivity Report, February 2013, p. 35.
Pirnik, M.P., R.M. Atlas, R. Bartha (1974) Hydrocarbon Metabolism by Brevibacterium erythrogenes: Normal and
Branched Alkanes, J. Bacteriol. 119: 868-878.
Philibert DA, CP Philibert, C Lewis, KB Tierney (2016) Comparison of diluted bitumen (dilbit) and conventional
crude oil toxicity to developing zebrafish. Env Sci Tech 50:6091-6098.
POLARIS Applies Science (2013) Report: A comparison of the properties of diluted bitumen crudes with other oils.
Redman AD, TF Parkerton (2015) Guidance for improving comparability and relevance of oil toxicity tests. Marine
Poll Bull 98:156-170.
Singh, S.N., B. Kumari, S. Mishra (2012) Microbial Degradation of Alkanes, in: N.S. Singh (Ed.), Microbial
Degradation of Xenobiotics, Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 439-469.
Sorial, G. A., A.D. Venosa, K.M. Miller, E. Holder, and D.W. King (2004) Oil spill dispersant effectiveness protocol -
Part I. Impact of operational variables. ASCE. J. Env. Eng. Div., 130(10): 1073-1084.
Rojo, F. (2009) Degradation of alkanes by bacteria. Environ. Microbiol., 11, 2477-2490.
Transportation Safety Board of Canada (2007) Pipeline Investigation Report. P07H0040.
USEPA (2002a) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and
marine organisms. Fifth edition. U.S. Environmental Protection Agency, Washington, DC. EPA/821/R-02/012.
55
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USEPA (2002b) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to
freshwater organisms. Fourth edition. U.S. Environmental Protection Agency, Washington, DC. EPA/821/R-
02/013.
USEPA (2002c) Short-term methods for estimating the chronic toxicity of effluents and receiving waters to marine
and estuarine organisms. Third edition. U.S. Environmental Protection Agency, Washington, DC. EPA/821/R-
02/014.
USEPA (2007a) SW-846, Method 3500C. Organic Extraction and Sample Preparation. U.S. Environmental
Protection Agency. February 2007. www.epa.gov/hw-sw846/sw-846-test-method-3500c-organic-extraction-and-
sample-preparation
USEPA (2007b) SW-846, Method 8015C. Nonhalogenated Organics by Gas Chromatography. U.S. Environmental
Protection Agency. February 2007. www.epa.gov/hw-sw846/sw-846-test-method-8015c-nonhalogenated-
organics-gas-chromatographv.
USEPA (2009) Method 524.3. Measurable of Purgeable Organic Compounds in Water by Capillary Column Gas
Chromatography/Mass Spectrometry. U.S. Environmental Protection Agency, Cincinnati, OH. EPA 815-B-09-009.
USEPA (2013a) Dredging Begins on Kalamazoo River, Enbridge Oil Spill, Marshall, Michigan, United States
Environmental Protection Agency.
USEPA (2013b) Environmental Response Team's Final Bench Scale/ Screening Level Oil Biodegradation Study;
Report Number 1597.
USEPA (2014) SW-846, Method 8270D. Semivolatile Organic Compounds by Gas Chromatography / Mass
Spectrometry. U.S. Environmental Protection Agency. July 2014. www.epa.gov/hw-sw846/sw-846-test-method-
8270d-semivolatile-organic-compounds-gas-chromatographymass-spectrometry.
Venosa, A. D., Holder, E. L. (2007) Biodegradability of dispersed crude oil at two different temperatures. Mar.
Pollut. Bull., 54, 545-553.
WEC (2010) World Energy Council; Survey of Energy Resources. Natural Bitumen and Extra-Heavy Oil; Chapter 4,
pp. 123-150. energy.usgs.gov/portals/0/Rooms/economics/text/WEC10NBEHO.pdf.
Yang, C., Wang, Z., Yang, Z., Hollebone, B., Brown, C.E., Landriault, M., Fieldhouse, B. (2011) Chemical Fingerprints
of Alberta Oil Sands and Related Petroleum Products. Environmental Forensics, 12, 173-188.
Zhuang, M.; Abulikemu, G.; Campo, P.; Platten III, W. E.; Suidan, M. T.; Venosa, A. D.; Conmy, R. N. (2016) Effect
of dispersants on the biodegradation of South Louisiana crude oil at 5 and 25 °C. Chemosphere, 144, 767-774.
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