the effects of diluted bitumen and the dispersant corexit...
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The Effects of Diluted Bitumen and the Dispersant
Corexit 9500A on Pacific Marine Organisms
by
Kassondra Nicole Rhodenizer
B.Sc., Acadia University, 2014
Project Submitted in Partial Fulfillment of the
Requirements for the Degree of
Master of Environmental Toxicology
in the
Department of Biological Sciences
Faculty of Science
© Kassondra Nicole Rhodenizer 2019
SIMON FRASER UNIVERSITY
Spring 2019
ii
Approval
Name: Kassondra Nicole Rhodenizer
Degree: Master of Environmental Toxicology
Title: The Effects of Diluted Bitumen and the Dispersant Corexit 9500A on Pacific Marine Organisms
Examining Committee: Chair: Rolf Mathewes Professor
Christopher Kennedy Senior Supervisor Professor
Vicki Marlatt Supervisor Assistant Professor
Jorgelina Muscatello External Examiner Senior Aquatic Scientist and Environmental Toxicologist Lorax Environmental Services Ltd.
Date Defended/Approved:
March 7, 2019
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Ethics Statement
iv
Abstract
Canada is expected to significantly increase the production and exportation of bitumen in
the next decade. Raw bitumen is diluted with natural-gas condensates to produce diluted
bitumen (dilbit), facilitating its flow through pipelines. Few data currently exist on dilbit
toxicity to Pacific marine species, either alone or in combination with recently approved
chemical dispersant Corexit 9500A. The current study investigated the toxicity of the
water-accommodated fraction (WAF) of dilbit, Corexit 9500A, and the chemically
enhanced water-accommodated fraction (CEWAF) of dilbit to representative marine
species of the west coast of Canada. Oil chemically dispersed by Corexit showed
evidence of higher toxicity than dilbit WAF to each test species including juvenile mysids
(Mysidopsis bahia), juvenile topsmelt (Atherinops affinis) and adults spot prawns
(Pandalus platyceros). Additionally, purple sea urchin (Strongylocentrous purpuratus)
fertilization showed high susceptibility to Corexit toxicity, both with and without dilbit
present, as nearly 100% of eggs exposed to Corexit remained unfertilized. Overall these
results suggest that the use of Corexit as a remediation technique may increase the toxic
impacts to Pacific marine species over those caused by a dilbit spill alone.
Keywords: Diluted bitumen, dilbit, Corexit 9500A dispersant, crude oil, polycyclic aromatic hydrocarbons, oil spill, toxicity
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Dedication
To my parents, who have always supported me through
my many changes in life direction
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Acknowledgements
This work was supported by grants from the National Contaminants Advisory
Group of by Fisheries and Oceans Canada to C.J. Kennedy. The author also
acknowledges Nautilus Environmental, an environmental toxicity consulting and testing
firm, for use of their laboratory facilities and all of their guidance and support while
conducting the acute toxicity tests. The author would also like to acknowledge Simon
Fraser University for hosting the facilities in which to conduct a large part of these
experiments. Acute toxicity experiments followed Environment Canada and USEPA
guidelines, while the spot prawns behavioural tests were adapted and modified by Kate
Mill. The following organizations are acknowledged for their supply of test materials: Nalco
Environmental Solutions LLC. for supply of Corexit 9500A; Environment Canada for
supply of summer blend dilbit from the Cold Lake region.
The author is deeply grateful for the members of Chris Kennedy's lab who gave a
helping hand on many of the included experiments, in particular Kate Mill, Vinicius
Azevedo, Jessica Banning and Jenna Keen. The author would also like to acknowledge
contributions from Charanveer Sahota and Henry Tran, undergraduate students who
provided much hands-on assistance within the lab, and also helped analyze many prawn
videos. The author also acknowledges Dr. Chris Kennedy for his support and guidance
during the graduate process. The author would also like to acknowledge Ian Bercovitz for
his statistical consultations.
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Table of Contents
Approval ............................................................................................................................... ii Ethics Statement ................................................................................................................. iii Abstract ............................................................................................................................... iv Dedication ............................................................................................................................ v Acknowledgements ............................................................................................................. vi Table of Contents ............................................................................................................... vii List of Tables ....................................................................................................................... ix List of Figures...................................................................................................................... xi List of Acronyms................................................................................................................. xii
Chapter 1. General Introduction ...................................................................................1 1.1. Diluted Bitumen (dilbit)................................................................................................1
1.1.1. Oil Industry in Canada ...................................................................................1 1.1.2. Dilbit Composition..........................................................................................2 1.1.3. Environmental Fate .......................................................................................3 1.1.4. Proposed Mechanisms of Action of Dilbit Constituents ................................4
Aliphatic Hydrocarbons ..................................................................................... 4 Aromatic Hydrocarbons .................................................................................... 5
1.1.5. Previous Dilbit Spills in the Environment ......................................................9 1.2. Use of Chemical Dispersants ...................................................................................10
1.2.1. Purpose and Effectiveness of Dispersants .................................................10 1.2.2. Corexit 9500A Composition ........................................................................11 1.2.3. Use of Corexit in Spill Scenarios .................................................................12 1.2.4. Environmental Fate .....................................................................................12 1.2.5. Proposed Mechanisms of Action of Corexit ................................................13 1.2.6. Corexit Toxicity ............................................................................................14 1.2.7. Oil Dispersed by Corexit..............................................................................16
Proposed Mechanisms of Action ..................................................................... 16 Toxicity of Dispersed Oil ................................................................................. 18
1.2.8. Previous Spills Remediated Using Corexit .................................................19 1.3. Juan de Fuca and Strait of Georgia Ecosystem.......................................................20
1.3.1. Burrard Inlet Representation .......................................................................20 1.3.2. Purple Sea Urchins (Strongylocentrous purpuratus) ..................................20 1.3.3. Mysid Shrimp (Mysidopsis bahia) ...............................................................21 1.3.4. Topsmelt (Atherinops affinis) ......................................................................21 1.3.5. Spot Prawn (Pandalus platyceros)..............................................................22
1.4. Objectives of Study ...................................................................................................23
Chapter 2. The Effects of Diluted Bitumen and the Dispersant Corexit 9500A on Pacific Marine Organisms .......................................................25
2.1. Introduction ...............................................................................................................25 2.2. Materials and Methods..............................................................................................27
2.2.1. Organisms ...................................................................................................27 2.2.2. Test Chemicals ............................................................................................28
Chemicals...................................................................................................... 28
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WAF and CEWAF .......................................................................................... 28 Water Analyses .............................................................................................. 29
2.2.3. Standardized Toxicity Tests ........................................................................30 Purple sea urchin (Strongylocentrous purpuratus) 20-min fertilization assay ....... 30 Mysid (Mysidopsis bahia) 48-h static renewal test ............................................ 31 Topsmelt (Atherinops affinis) 96-h static renewal test ....................................... 32
2.2.4. Spot Prawn (Pandalus platyceros) Experiments ........................................34 Exposure ....................................................................................................... 34 Behavioural Tests .......................................................................................... 34
Antennule Flicking .................................................................................... 34 Pre-Feeding and Feeding Behaviours ........................................................ 35
2.2.5. Statistical Analyses......................................................................................35 2.3. Results ......................................................................................................................37
2.3.1. Chemical Analyses ......................................................................................37 2.3.2. Effects of Dilbit and Corexit on Juvenile Topsmelt .....................................38 2.3.3. Effects of Dilbit and Corexit on Juvenile Mysids .........................................42 2.3.4. Effects of Dilbit and Corexit on Echinoderm Fertilization ...........................45 2.3.5. Effects of Dilbit and Corexit on Behaviour in Spot Prawns.........................47
Mortality During the 7-d Exposure ................................................................... 47 Behavioural Tests .......................................................................................... 47
Antennule Flicking .................................................................................... 47 Pre-feeding and Feeding Behaviours ......................................................... 48
2.4. Discussion .................................................................................................................51
Chapter 3. Extended Discussion ................................................................................55 3.1. Chemical Analyses ...................................................................................................55 3.2. Toxicity Tests ............................................................................................................55
3.2.1. Effects on Juvenile Topsmelt ......................................................................55 3.2.2. Effects on Juvenile Mysids ..........................................................................56 3.2.3. Effects on Echinoderm Fertilization ............................................................57 3.2.4. Effects on Spot Prawns ...............................................................................58
3.3. PAH Toxicity at Low Concentrations ........................................................................59 3.4. Limitations .................................................................................................................59
3.4.1. Measured Concentrations ...........................................................................59 3.4.2. Spot Prawn Behavioural Tests ....................................................................60
3.5. Applied Aspects of the Study....................................................................................61 3.6. Future Research .......................................................................................................62
References .....................................................................................................................63
Appendix Supplementary Tables ............................................................................78
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List of Tables
Table 1 Bioassay parameters for each species tested, including life stage, test duration, concentrations, replicates, individuals per replicate, volume used, temperature, photoperiod, salinity, feeding regimen, protocol and endpoints. .............................................................................33
Table 2 Nominal loadings of oil (mL/L) and Corexit (mg/L) with measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) in 100%v/v water-accommodated fraction (WAF) and chemically-enhanced water-accommodated fraction (CEWAF) solutions of diluted bitumen (dilbit) and mineral oil at various dispersant-oil ratios (DORs). .....................................................................38
Table 3 Mean percent survival of juvenile topsmelt (Atherinops affinis) based on nominal oil loading (%v/v) and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) following a 96-h exposure to dilbit water-accommodated fraction (WAF) and chemically enhanced water-accommodated fraction (CEWAF), mineral oil CEWAF, and Corexit. ...............................................................40
Table 4 24-h and 96-h LC50 (Lethal Concentration to 50 percent of the population) values for diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) by Corexit, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L), and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) for topsmelt (Atherinops affinis) juveniles. .............42
Table 5 Mean percent survival of juvenile mysids (Mysidopsis bahia) based on nominal oil loading (%v/v) and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) following exposures to the water-accommodated fractions (WAF) and chemically-enhanced water-accommodated fractions (CEWAF) of dilbit and mineral oil, and Corexit alone for 48 h. ......................................43
Table 6 48-h LC10 and LC50 values for diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) by Corexit, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L), and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) for mysid (Mysidopsis bahia) juveniles. .........................................................................................45
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Table 7 Concentrations inhibiting 20% fertilization (IC20) and 50% fertilization (IC50) after 20-min purple sea urchin (Strongylocentrous purpuratus) fertilization assay using diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically-enhanced water-accommodated fractions (CEWAF), and Corexit alone, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L) and measured total polycyclic aromatic hydrocarbon (TPAH) concentration (µg/L). ...............46
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List of Figures
Figure 1 Concentration-response relationship for juvenile topsmelt mortality after 96-h exposure to: a) diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent mortality of topsmelt vs. measured total polycyclic aromatic hydrocarbon (TPAH) concentrations [µg/L, log-scale]); b) dilbit chemically-enhanced water-accommodated fraction (CEWAF) (plotted as percent mortality of topsmelt vs. measured TPAH concentrations [µg/L, log-scale]); c) Corexit (plotted as percent mortality of topsmelt vs. nominal loading of Corexit [mg/L, log-scale]), and d) mineral oil CEWAF (plotted as percent mortality of topsmelt vs. nominal loading of Corexit [mg/L, log-scale]). ...........................................41
Figure 2 Concentration-response relationship for juvenile mysid mortality after 48 h of exposure to: a) diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent mortality of mysids vs. measured total polycyclic aromatic hydrocarbon (TPAH) concentrations [µg/L, log-scale]), and b) dilbit chemically-enhanced water-accommodated fraction (CEWAF) (plotted as percent mortality of mysids vs. measured TPAH concentrations [µg/L, log-scale]). .......................................................................................44
Figure 3 Concentration-response relationship for percentage of unfertilized echinoderm eggs after 20-min fertilization assay with exposure to diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent unfertilized vs. measured total polycyclic aromatic hydrocarbon [TPAH] concentrations [µg/L]). ..............................47
Figure 4 Graphical representation of mean antennule flicks (least squares mean) counted in the 2-min period after acclimatization to clean water, before the addition of the liquid food stimulus, between chemical groups including control, diluted bitumen (dilbit) water-accommodated fraction (WAF), dilbit chemically-enhanced water-accommodated fraction (CEWAF), Corexit, mineral oil WAF and mineral oil CEWAF calculated using a 2-factor completely randomized design (CRD) Analysis Of Variance (ANOVA). Error bars express standard error. Control data was not run in the 2-factor model but is shown here for comparison. .......................................48
Figure 5 Mean proportion of prawns at each chemical and concentration that ate solid food after the 7-d exposure, expressed as %v/v (ranging from 1.0%v/v to 100%v/v), for: a) diluted bitumen (dilbit) water-accommodated fraction (WAF); b) dilbit chemically-enhanced water-accommodated fraction (CEWAF); c) Corexit; d) mineral oil WAF, and e) mineral oil CEWAF. Error bars express standard error. Data for control prawns are expressed as 0.0 %v/v to allow visual expression on the log scale. Total number of prawns shown is N = 144...........................................................................50
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List of Acronyms
ANOVA Analysis Of Variance
BTEX Benzene Toluene Ethylbenzene and Xylene
CEWAF Chemically Enhanced Water-Accommodated Fraction
CI Confidence Interval
CLB Cold Lake Blend
CRD Completely Randomized Design
Dilbit Diluted Bitumen
DOR Dispersant-Oil Ratio
DOSS Dioctyl Sodium Sulfosuccinate
DWH Deepwater Horizon oil spill
EC50 Effective Concentration to 50 percent of the population
ER Estrogen Receptor
HMW High Molecular Weight
ICp Inhibiting Concentration for a (specified) percent effect
LC50 Lethal Concentration to 50 percent of the population
LMW Low Molecular Weight
MAH Monocyclic Aromatic Hydrocarbon
NA Naphthenic Acid
PAH Polycyclic Aromatic Hydrocarbon
SSD Species Sensitivity Distribution
TPAH Total Polycyclic Aromatic Hydrocarbons
TPH Total Petroleum Hydrocarbons
VOC Volatile Organic Compound
WAF Water-Accommodated Fraction
WCS Western Canadian Select
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Chapter 1. General Introduction
1.1. Diluted Bitumen (dilbit)
1.1.1. Oil Industry in Canada
Canada is one of the world’s largest oil-producing countries, with the majority of
production coming from the oil sands in Northern Alberta (Alsaadi et al., 2017). Northern
Alberta is also the largest producer of oil sands bitumen in the world, containing over 169
billion barrels of recoverable bitumen with current extraction methods (ERCB, 2012).
Ores that lie close to the surface are mined directly, whereas deeper reserves are heated
with steam for extraction (Environment Canada, 2013). Once extracted, raw bitumen is
combined with natural gas condensates or synthetic oils to reduce its high viscosity
before transport (Barron et al., 2018). This produces diluted bitumen (dilbit) and facilitates
its efficient flow through pipelines (Barron et al., 2018).
Bitumen extraction, production and exportation levels are expected to triple within
the next decade (Alsaadi et al., 2017). Major new pipelines for the transport of dilbit have
been proposed to accommodate a rise in production levels. Although the recent
proposals for the Northern Gateway and Energy East pipeline were rejected, other
proposed pipelines destined to carry dilbit have been approved, with the Kinder Morgan
pipeline expansion attracting the most public attention. In BC, the existing and proposed
pipelines will transport dilbit to coastal ports where it will then be shipped by marine
tankers to overseas markets, greatly enhancing the potential risks of a spill of dilbit into
the coastal marine environment (Environment Canada, 2013). Once dilbit enters aquatic
environments, it is very difficult to remediate, as it can disperse quickly from the release
site, as well as partition into different compartments of the environment (Environment
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Canada, 2013; Hua et al., 2018). Understanding the potential environmental impacts of
dilbit to Canada’s Pacific organisms is necessary to ensure that adequate regulation and
effective mitigation procedures are in place, should a spill occur.
1.1.2. Dilbit Composition
Dilbit blends are classified as heavy sour crudes (CEPA, 2013), formed by a
combination of raw bitumen mixed with natural gas condensates or synthetic oils as
diluents (Barron et al., 2018). The composition of these dilbit blends vary widely, as there
are a variety of diluents and blending ratios that can be used by producers depending on
the location and time of year (King et al., 2017a). The source of raw bitumen, as well as
the extraction method, also influence the dilbit composition (King et al., 2017a). Blending
ratios commonly fall between 20-30% diluent to 70-80% bitumen (King et al. 2017a;
Crosby et al. 2013). For dilbit to be transported in pipelines, it must meet pipeline
requirements of a density ranging from 915 to 940 kg/m3 (CEPA, 2013). Once diluted,
dilbit is transported via pipelines at a velocity of 1 – 2.5 m/s and at a temperature between
17 and 40°C (CEPA, 2013).
Dilbit composition is similar to that of conventional crude oils, containing
thousands of different compounds with varying densities and thermochemical properties.
For example, Strausz et al. (2011) found almost 6,000 different aromatic compounds in
a raw bitumen sample. Much like crude oil, dilbit contains many polycyclic aromatic
hydrocarbons (PAHs) which can be highly toxic to aquatic organisms (Dew et al., 2015;
Incardona et al., 2005; Finch et al., 2017). Although total PAH (TPAH) concentrations
tend to be lower in dilbit than in conventional crude oils, they have similar 3- to 5-ringed
alkyl PAH concentrations (Madison et al., 2015). Alkyl PAHs are of high concern because
they are generally more persistent, water-soluble and bioavailable than their parent non-
alkylated compounds (Barron et al., 2004).
Dilbit blends have greater density, viscosity and adhesion properties than
conventional crude oils (King et al., 2017b; Barron et al., 2018) and also contain a higher
level of resins and asphaltenes (King et al., 2017b; Barron et al., 2018). These
components have larger molecular structures and are the components that are likely to
3
become more dense through weathering and sink when combined with suspended
sediments (Dew et al., 2015). Dilbit also contains a number of monocyclic aromatic
hydrocarbons (MAHs), the most recognized of which are benzene, toluene, ethylbenzene
and xylene (BTEX). These compounds are also classified as volatile organic compounds
(VOCs) and readily volatilize after a spill (Almeda et al., 2013; Lee et al., 2015). Dilbit
differs from other crude oils with their higher prevalence of naphthenic acids (NAs), a
class of carboxylic acid derivatives of naphthenes (cyclic aliphatic hydrocarbons)
resulting from the biodegradation of mature petroleum (Clemente and Fedorak, 2005)
and generated by the bitumen extraction processes (Headley and McMartin, 2004). The
NAs compose 2-3% by weight of bitumen found in the Athabasca Oil Sands (Headley
and McMartin, 2004). Dilbit also contains a lower amount of saturates and naphthalenes
compared to crude oils (Madison et al., 2015).
1.1.3. Environmental Fate
Dilbit tends to behave similar to lighter oils during the early time frame of a spill,
and similar to heavier oils as the weathering process proceeds (Environment Canada,
2013; Madison et al., 2017). This is due to the evaporative loss of the lighter volatile
components of dilbit including BTEX that occurs soon after a spill (Madison et al., 2017).
This increases the density and viscosity of the remaining oil, which has a greater
percentage of high molecular weight (HMW) resins and asphaltenes (Environment
Canada, 2013). If waters are calm, floating material can be boomed and skimmed off the
surface (Environment Canada, 2013). King et al. (2017b) showed that at 15°C, all
bitumen blends would initially float after a spill at sea, even following evaporation,
photodegradation and weathering. Environment Canada (2013) found that dilbit products
dispersed throughout the water column by breaking wave conditions eventually resurface
as large oil droplets and form an oil slick. Even the most weathered products, which can
form water-in-oil emulsions (tarballs and tarmats) either floated or resurfaced from the
water column. Environment Canada (2013) suggests that typical marine water
temperatures that occur in Canada (0–15°C) are not sufficient to cause dilbit to sink, even
in combination with evaporation.
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When high mixing energy is present, particles with high solubilities can dissolve
in water to create a water-accommodated fraction (WAF). The majority of these water-
accommodated compounds are light molecular PAHs and their alkylated congeners
(Yang et al., 2018), although some monoaromatic hydrocarbons like BTEX may be
present in the WAF in the early stages (Philibert et al., 2016). These alkylated congeners,
particularly 4- to 6-ring alkyl PAHs, are more environmentally persistent than those with
3-rings and lower (King et al., 2017b). Additionally, smaller, neutrally-buoyant particles
may also become suspended in the water column (SL Ross Ltd, 2012). Environment
Canada (2013) laboratory studies have shown that particulate presence in the water
column, combined with high mixing energy, have the greatest impact on dilbit buoyancy.
Fresh and weathered dilbit can mix with fine- and medium-sized sediments, forming oil
particulate aggregates that sink in salt water (Hua et al., 2018). More highly weathered
oil mixes with fine- and medium- sized sediments to form free-floating tarballs, even in
low-energy wave conditions (Hua et al., 2018). However, studies that have evaluated
these phenomena have used very high mixing-energy, so it is unclear whether natural
processes would create enough disturbance to cause these particles to sink.
Furthermore, King et al. (2014) showed that although bitumen-containing products have
similar physical properties to start, their chemical composition causes them to behave
differently during weathering. This complicates potential remediation, as a blanket
remediation process is not appropriate for all dilbit blends. Models are therefore important
to determine whether a dilbit blend might sink after a spill (King et al., 2017b).
1.1.4. Proposed Mechanisms of Action of Dilbit Constituents
Aliphatic Hydrocarbons
Aliphatic hydrocarbons present in oil are not believed to directly contribute to
aquatic toxicity, as they are less bioavailable and are readily degraded in the environment
(Payne et al., 1995). Naphthenic acids (NAs) are the most concerning of the aliphatics,
and although most NAs are released into tailing ponds after bitumen extraction, some do
remain in dilbit which is transported in pipelines (Headley and McMartin, 2004). Toxicity
is associated with their surfactant properties, with toxicity likely occurring due to nonpolar
narcosis (Headley and McMartin, 2004; Tollefsen et al., 2012). Nonpolar narcosis is a
non-specific form of toxicity that is believed to occur when an organic compound causes
5
a disturbance of phospholipids in biological membranes (Tollefsen et al., 2012). Exposure
to NAs has associated with toxicity to microorganisms, algae, fish and invertebrates
(Headley and McMartin, 2004; Miskimmin et al., 2010; Kindzierski et al., 2012). Exposure
to NAs has been correlated with embryo deformities and reduced hatch length in yellow
perch (Perca flavescens) and Japanese medaka (Oryzias latipes) (Peters et al., 2007).
Petrogenic NAs can also mimic the hormone estrogen, acting as weak estrogen receptor
(ER) agonists and potentially disrupting sexual differentiation in aquatic species (Thomas
et al., 2009). Kavanagh et al. (2012) found that fathead minnows (Pimephales promelas)
exposed to NAs extracted from oil sands process waters showed reproductive
dysfunction and reduced female fecundity. Exposed fish also exhibited reduced
concentrations of reproductive hormones and secondary sexual characteristics in males.
However, NAs are difficult to analyze separately from oil sands mixtures, and commercial
mixtures of NAs behave differently than oil sands NAs during toxicity testing, therefore
the environmental relevance of laboratory testing results is unclear.
Aromatic Hydrocarbons
Aromatic hydrocarbons do not have one single toxic mechanism of action, and
instead exert their effects through multiple mechanisms that affect multiple physiological
systems. The PAHs are considered to be the main toxic components of dilbit due to their
high hydrophobicity, persistence in the environment, and bioavailability to aquatic
organisms (Incardona et al., 2005; Dew et al., 2015; Finch et al., 2017). These PAHs
commonly, but not always, activate the aryl hydrocarbon receptor (AhR), which through
a series of reactions initiates upregulation of metabolic enzymes like CYP1a (Madison et
al., 2015, 2017; Alderman et al., 2017a). Although this upregulation can result in the
biotransformation of contaminants, some of the reactive metabolites formed through this
process can cause damage to DNA, lipids and proteins (Akcha et al., 2003; Madison et
al., 2015). DNA adducts can form when PAH metabolites bind to DNA and may result in
mutagenicity which can lead to tumor formation (Akcha et al., 2003). Additionally, PAH
metabolites can cause lipid peroxidation and result in membrane damage followed by cell
death (Barron et al., 2005).
There is a delicate balance between the detoxification and excretion of PAHs and
the formation of toxic metabolites like alkyl PAHs (Hodson, 2017). Alkyl PAHs often have
6
a greater binding affinity to AhR, particularly alkylated congeners like phenanthrenes
(Billiard et al., 2002), and are often more bioavailable and have higher partition
coefficients than their parent compounds (King et al., 2017b). For example, the alkyl PAH
retene has been shown to be over 100 times more toxic than the parent compound,
phenanthrene (Hawkins et al., 2002). Barata et al. (2005) have also showed that alkylated
naphthalenes are much more toxic than the parent compound to marine copepods
(Oithona davisae). The toxicity of alkyl PAHs increases with increasing number of rings,
alkyl carbons and the octanol-water partition coefficient, KOW (Rice et al., 2001). Most
studies agree that it is 3- to 5-ringed alkyl PAHs, particularly alkyl phenanthrenes,
naphthalenes, fluorenes, naphthobenzothiophenes, chrysenes and dibenzothiophenes
that are highly toxic to aquatic species (Wu et al. 2012; King et al., 2017b).
Specific toxic mechanisms of action depend on the compound, exposure duration,
species, and environmental conditions (Bejarano et al., 2014; Alderman et al., 2017a).
Previous literature suggests that the lighter oil components, particularly MAHs like BTEX
and low molecular weight (LMW) PAHs are responsible for acute toxicity (Lee et al., 2015;
Philibert et al., 2016). Although they tend to readily evaporate after a spill, exposure to
these compounds has been shown to cause nonpolar narcosis in biological membranes
(Almeda et al., 2013; Lee et al., 2015). Chronic toxicity is believed to be caused by HMW
PAHs (3 rings or greater) which are more persistent in the environment (Couillard et al.,
2005; Turcotte et al., 2011; Fallahtafti et al., 2012; Lin et al., 2015). Analyzing PAH toxicity
in dilbit is complicated by the fact that multiple mechanisms of toxicity can occur
simultaneously. Because dilbit contains such a wide variety of AhR-inducing and non-
AHR-inducing PAHs, each with a different binding affinity, it is difficult to assert which
components cause the toxicity shown in laboratory experiments.
Previous literature suggests aquatic organisms exposed to dilbit show similar
signs of toxicity compared to conventional crude oils, although most studies to date have
assessed freshwater species (Colavecchia et al., 2004, 2006; van den Heuvel et al.,
2012; Yergeau et al., 2013; Winter, 2013; Philibert et al., 2016). The water-
accommodated fraction (WAF) of two types of Canadian dilbit showed toxicities to
fathead minnows (P. promelas) and silverside minnows (Menidia beryllina) within the
same range as conventional crude oils (Barron et al., 2018). General trends have been
7
found for greater dilbit toxicity in estuarine species compared to freshwater species, but
additional research is needed to investigate this further (Madison et al., 2015, 2017;
Bejarano et al., 2017; Barron et al., 2018)
Exposure to mixtures of PAHs in crude oil have also resulted in many categories
of sublethal toxic effects including reproductive and developmental toxicity,
teratogenicity, cardiotoxicity, neurotoxicity, immunotoxicity and changes in behaviour
(plus others) in a variety of different aquatic species. Exposure to PAHs has been shown
to suppress growth in juvenile Chinook salmon (Oncorhynchus tshawytscha) (Meader et
al., 2006). Barron et al. (2018) found that concentrations of two weathered dilbits
decreased growth in mysids (M. bahia), similar to crude oil. Colavecchia et al. (2006)
found that white suckers (Catostomus commersoni) exposed to sediment from naturally-
occurring bitumen deposit sites showed decreased growth and weight in embryos.
Exposure to PAHs can also affect the endocrine system, and correspondingly,
reproduction. Barron et al. (2018) found that weathered dilbit impaired reproduction in
invertebrates (Ceriodaphnia dubia) at similar PAH concentrations as crude oil. Exposure
can affect vitellogenesis, and rainbow trout (Oncorhynchus mykiss) liver cells exposed to
PAH mixtures in vitro in laboratory studies have shown decreases in plasma vitellogenin
(Anderson et al., 1996). Similar impacts on vitellogenin have been shown in the field, with
killfish (Fundulus heteroclitus) showing reduced blood vitellogenin associated with PAH
concentrations in sediment (Pait and Nelson, 2009). Additionally, PAHs impact hormonal
regulation and may potentially reduce ovarian responsiveness to hormones by interfering
with hormone membrane receptors (Thomas and Budiantara, 1995). Exposure to PAHs
has also been found to decrease concentrations of 17β-estradiol in exposed females
(Stein et al., 1991; Johnson et al., 1995). Interestingly, van den Heuvel et al. (2012) found
that female yellow perch (Perca flavescens) transported to a bitumen-containing lake
showed a significant increase in testosterone levels in females compared to control fish,
although no differences were found in males. Other reproductive effects include delay in
oocyte maturation shown in female Atlantic croaker (Micropogonias undulatus) and
Atlantic Cod (Gadus morhua) exposed to PAHs (Thomas and Budiantara, 1995; Khan,
2013).
8
Dilbit and its components have also been associated with developmental and
teratogenic effects. Blue sac disease (BSD), and toxicity closely resembling BSD, has
been observed in fish embryos in multiple freshwater species exposed to crude oil, or to
individual PAHs (Ramachandran et al., 2004; Hodson, 2017). Symptoms of BSD include
pericardial and yolk sac edema, craniofacial malformations and spinal curvatures
(Colavecchia et al. 2004, 2006). BSD symptoms have been shown in fathead minnows
(P. promelas) and common white sucker (C. commersonii) embryos exposed to dilbit
from the Alberta oil sands (Colavecchia et al. 2004, 2006). Dilbit exposure leading to
increased CYP1a expression was shown to have a strong correlation to blue sac disease
(BSD) presence in Japanese medaka (O. latipes) embryos, which showed increased yolk
sac and pericardial edemas, as well as craniofacial malformations (Madison et al., 2015,
2017). Although not directly acutely lethal, BSD can lead to harmful effects on growth and
development, lower percentage survival to adulthood and increased predator
susceptibility (Carls and Thedinga, 2010). Evidence of BSD was found in zebrafish (Danio
rerio) in response to dilbit exposure, with pericardial edema being the most common
response (Philibert et al., 2016). Dilbit in sediment decreased hatching success, size of
hatched larvae and steroid production in fathead minnows (P. promelas), and increased
larval mortality (Colavecchia et al., 2004). A follow-up study reported increased mortality,
teratogenesis, and decreased growth and weight in white sucker (C. commersoni)
embryos exposed to bitumen-deposit sediment (Colavecchia et al., 2006).
Dilbit has been shown to have potential cardiotoxic effects similar to crude oil,
which can result in changes in swimming behaviour. Cardiotoxic effects such as the
blockage of potassium and calcium channels have been shown to precede other
deformities like pericardial and yolk sac edemas found in BSD (Incardona et al., 2014).
Juvenile sockeye salmon (Oncorhynchus nerka) exposed to the dissolved fraction of Cold
Lake Blend (CLB) dilbit for 1 and 4 weeks showed concentration-dependent alterations
in cardiac morphology (Alderman et al., 2017a). These alterations are correlated with
impairments in swimming performance that may result in reduced migratory success
(Alderman et al. 2017a). Other changes in behaviour were found by Philibert et al. (2016)
who showed that zebrafish exposed to dilbit showed a reduction in shelter-seeking
behavior, which could increase predator susceptibility and affect their survival in the
natural environment.
9
Other potential effects of dilbit on aquatic species include neurotoxicity and
immunotoxicity. Although not previously investigated in dilbit, evidence of neurotoxicity
has been shown in fish exposed to crude oils (Irie et al., 2011; Almeida et al., 2012). For
example, Japanese medaka (O. latipes) exposed to dilbit commonly exhibited non-
inflated swim bladders, indicating an effect on the autonomic nervous system (Madison
et al., 2017). Kawaguchi et al. (2012) found that pufferfish larvae exposed to crude heavy
oil showed an abnormal swimming pattern which suggested a developmental disorder of
the brain. Previous studies also suggest that dilbit impairs the immune response in fish
(Kennedy and Farrell, 2008). Sockeye salmon (O. nerka) exposed to the dissolved
fraction of dilbit experienced inflammatory responses and protein leakage when forced to
exercise, which is evidence of cell damage (Alderman et al., 2017b). Additionally, PAH
exposure has shown impacts on adaptive immunity via alterations in B and T cell
functioning in sheepshead minnows (Cyprinodon variegatus) (Jones et al., 2017). It is
evident that PAH exposure can affect multiple systems via multiple mechanisms of action,
and further research on the specific effects PAH toxicity in dilbit, particularly to saltwater
species, is warranted.
1.1.5. Previous Dilbit Spills in the Environment
Spills of bitumen-containing products into the environment have been few. In
2007, a Kinder Morgan pipeline carrying a dilsynbit blend, which contained bitumen with
a condensate and synthetic light crude, ruptured in Burnaby, BC (Environment Canada,
2013). This spill resulted in 100,000 L (224 m3) of Albian heavy synthetic crude oil
entering Burrard Inlet and Kask Creek in BC, Canada (Stantec, 2012), with 15 km of
shoreline being affected (Environment Canada, 2013). Remediation efforts included
skimming and booming in addition to flushing. Surface water quality guidelines were met
in 2007 for both extractable hydrocarbons and PAHs (Environment Canada, 2013).
Following the spill, brown algae (Fucus spp.) populations declined, which subsequently
caused mortalities in fauna that use algae as a habitat, although it is unclear whether
algal death was related to oil exposure or to remediation techniques (Dew et al., 2015).
In the 1 to 2 months following the spill, 10 out of 78 monitored sites for sediment quality
showed measured PAH concentrations exceeding guidelines that could be directly
attributed to the spill (Stantec, 2012). Elevated PAH tissue concentrations were
10
measured in mussels (Mytilus spp.) and red rock crabs (Cancer productus) (Environment
Canada, 2013). As of 2011, the only endpoint that had not met guideline requirements
was TPAH concentration in blue mussels (Mytilus edulis) (Environment Canada, 2013).
Fortunately, this spill occurred during ideal environmental conditions, which included a
slack tide, no rainfall and outside the salmon migration period (Environment Canada,
2013). Several of these conditions lessened the potential environmental damage and
allowed most of the dilbit to remain floating (Environment Canada, 2013).
The Kalamazoo River spill in Michigan in 2010 released 3.2 million L (3190 m3)
of dilbit into the environment from an Enbridge pipeline (Environment Canada, 2013).
These conditions represented a worst-case scenario, as the dilbit had time to weather on
land before entering the fast-flowing river, where it was then mixed with suspended solids
(Dew et al., 2015). Between 10 and 20% of the oil mixed with particulate matter and sank
to the bottom of the river (USEPA, 2013). Papoulias et al. (2014) found that 3 weeks after
the spill, smallmouth bass (Micropterus dolomieu) and golden redhorse (Moxostoma
erthrurum) showed biomarkers indicative of PAH exposure, including a higher frequency
of gill and spleen lesions, macrophage aggregates and higher CYP1a expression.
Additionally, mussel mortality in the river was higher in contaminated areas than
uncontaminated regions (Winter, 2013). Lee et al. (2012) determined that the dilbit
formed stable oil particulate aggregates within the river sediments. Years later, and after
extensive dredging, the USEPA estimated about 680 m3 oil remained submerged within
river sediments (USEPA, 2013). Dredging continues today, continuing to affect the
ecology of the river ecosystem years after the spill.
1.2. Use of Chemical Dispersants
1.2.1. Purpose and Effectiveness of Dispersants
One remediation technique used to treat oil spills involves the use of chemical
dispersants. Dispersants are mixtures of surfactants in solvent that contain anionic soaps
and non-ionic detergents which can orient to the oil-water interface (George-Ares and
Clark, 2000). Dispersants reduce surface tension in order to break down the oil into
smaller oil-surfactant droplets which are more easily biodegraded (Adams et al., 2014).
11
Dispersants increase the surface area-to-volume ratio of oil, as well as the partitioning
rate into aqueous solution (Adams et al., 2014). This produces a chemically-enhanced
water-accommodated fraction (CEWAF) which disperses into the water column (Adams
et al., 2014).
Many factors impact the effectiveness of dispersants, including temperature,
salinity, amount of oil weathering and wave energy (Moles et al., 2002). Dispersants are
only recommended under certain high-energy state conditions, where wind or wave
intensities are high (Environment Canada, 2013). However, when high wave energy is
present, oil-water emulsions are likely to form quickly, reducing the window of opportunity
for effective dispersant use (Lunel and Davies, 2001). Dispersant effectiveness also
decreases as oil viscosity increases, which happens at colder temperatures and as the
oil weathering process progresses (Zhao et al., 2014; King et al., 2017a). Dilbit is
classified as a high-viscosity oil, therefore reaching the oil-water interface may be more
difficult for a dispersant than with traditional crude oils (Environment Canada, 2013).
Environment Canada (2013) suggest that due to its density, viscosity and adhesiveness,
the effectiveness of chemical dispersants on dilbit may be limited, especially in marine
environments where seawater temperatures are less than 8°C.
1.2.2. Corexit 9500A Composition
Corexit 9500A (referred to as Corexit from this point forward) has been recently
approved for use in Canada in 2016 (Canada Gazette, 2016). Corexit has a density of
0.95 g/cm3, a pH of 6.2, and boiling point of 147°C (Nalco, 2014). The dispersant mixture
contains amphoteric ingredients, containing both hydrophilic and hydrophobic regions,
the most noteworthy being dioctyl sodium sulfosuccinate (DOSS), an anionic surfactant,
at between 10-30% (Adams et al., 2014; Anderson et al., 2011). Also present in Corexit
is propylene glycol and a group of non-ionic sorbitan ester and ethoxylate-based
surfactants (Span 80, Tween 80, Tween 85), as well as ether and hydrocarbon-based
solvents (Dasgupta and McElroy, 2017). Other than DOSS, these specific surfactants
have not been well studied. A CDC report stated that the “ingredients are not considered
to cause chemical sensitization; the dispersants contain proven, biodegradable and low
toxicity surfactants” (CDC, 2011).
12
1.2.3. Use of Corexit in Spill Scenarios
To date, there is no record of Corexit being used in response to a dilbit spill in the
marine environment. Corexit is up to 99% effective at dispersing low-viscosity crude oils
(Belore et al., 2009), but as viscosity increases, its effectiveness decreases (Moles et al.,
2002). Li et al. (2010) showed that as water temperature decreased from temperate
(15.6°C) to colder (9.8°C) water, the effectiveness of Corexit with Intermediate Fuel Oil
(IFO), a heavy oil, decreased heavily from 95% to 12.9%. Pan et al. (2017) showed that
Corexit increased dispersion from 10% to 60% in Cold Lake Blend (CLB) dilbit in
temperate waters (15°C) experimentally using EPA baffled flask test, but this was within
the 120-min window after the spill, which may not be realistic in a real-world scenario.
Environment Canada (2013) found that Corexit can cause partial dispersion (45%) of
dilbit in a wave tank in the presence of high-energy breaking waves at an average
seawater temperature of 8°C, which falls within the temperature range along Pacific coast
of Canada. However, this dispersion was tested within the first 60-min window after the
spill, which again may not be realistic in a post-spill environment. Experiments conducted
by King et al. (2017a) suggest that dispersants would be almost completely ineffective
on dilbit past 3-h post-spill because oil viscosity would increase significantly within that
time period (Pan et. Al., 2017). King et al. (2017a) created an oil spill response decision-
making matrix and concluded that chemical dispersion methods would be ineffective after
a dilbit spill.
1.2.4. Environmental Fate
In the environment, Corexit partitions at the highest percentage into the
soil/sediment (50 – 70%), but the proportion that partitions into water (10-30%) is water-
soluble (Nalco, 2014). The ‘organic’ portion of the Corexit mixture is expected to be
readily biodegradable (Nalco, 2014). The measurements of DOSS taken during the
application of Corexit during the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico
in 2010 suggest that this important component readily photodegrades, as beyond the
immediate area of continuous application, concentrations did not exceed USEPA
guidelines (Gray et al., 2014). However, Kover et al. (2014) suggest that two solvents in
Corexit, propylene glycol and 2-butoxyethanol, are not expected to readily degrade in
13
natural environments through direct or indirect photolysis. Since measurements of these
components have not been taken in post-spill environments, it is possible that these may
persist for longer periods of time.
1.2.5. Proposed Mechanisms of Action of Corexit
Corexit likely exerts its toxic effects by affecting oxidative balance and inducing
cytotoxicity. Zheng et al. (2014) found that Corexit can induce cytotoxicity by altering
intracellular oxidative balance and causing lipid peroxidation in mammalian cell lines.
They found that Corexit increased reactive oxygen species (ROS) that can cause
damage to DNA, proteins and cell membranes. Li et al. (2015) found that Corexit
activates C-reactive protein and NADPH oxidase 4, which are associated with ROS
production. Additionally, Zheng et al. (2014) found that Corexit depleted glutathione
levels, an antioxidant that works to prevent ROS damage. Catalase activity was also
altered by Corexit exposure, which is an enzyme that protects cells from oxidative
damage by ROS. This is further supported by the findings of Dussauze et al. (2015) that
antioxidant enzyme superoxide dismutase (SOD), responsible for transforming ROS and
preventing oxidative stress, significantly decreased in the intestine and brain of European
sea bass (Dicentrarchus labrax) exposed to Corexit.
Li et al. (2015) found that Corexit also inhibits junctional proteins, leading to an
increase in cell permeability and eventually to apoptosis. They also found that Corexit
induced caspase-3 activation, resulting in apoptosis of epithelial cells. This was
supported by Zheng et al. (2014) who showed that Corexit increases caspase-3, followed
by subsequent apoptosis. Zheng et al. (2014) showed that Corexit can alter mitochondrial
function by inhibiting mitochondrial complex-I and increasing BAX expression, which also
promotes cellular apoptosis. Chen and Reese (2016) found that Corexit 9527, a similar
dispersant to Corexit 9500, inhibited retinoic acid biosynthesis from retinal, which
inhibited retinol-induced expression of the Hoxa1 gene. This suppressed P19 cell
differentiation into neuronal cells and suggested that Corexit can also be neurotoxic. This
is supported by Sriram et al. (2011) who found that inhalation exposure to Corexit causes
disruption in olfactory signal transduction, axonal function and synaptic vesicle fusion in
a rat model, which may impact proper neurotransmitter signaling.
14
Multiple studies have suggested that DOSS is the most toxic surfactant
component in the Corexit mixture (Chen and Reese, 2016; Dasgupta and McElroy, 2017).
Bandele et al. (2012) found that both Corexit and its main ingredient, DOSS, induced
cytotoxicity and were equally toxic in human hepatocyte (HepG2/C3A) cell lines after 24h,
suggesting that DOSS is the main toxic component which causes cytotoxicity. This
observation was supported by in vivo data using sheepshead minnow (C. variegatus)
embryos, showing that DOSS affects physiology at multiple levels and induces
genotoxicity and reduces survival (Dasgupta, 2016). Interestingly, in a follow-up study,
Corexit showed more cytotoxicity than DOSS after 72-h, suggesting that over time, the
other surfactants in Corexit can also contribute to cytotoxicity (Dasgupta and McElroy,
2017). Dasgupta and McElroy (2017) showed that other anionic surfactants in Corexit,
Tween 80 and 85, inhibited CYP1a activity induced by a model agonist, which could
disrupt the metabolism of toxic hydrocarbon components in oil (Dasgupta and McElroy,
2017). Tween 80 has also been reported to almost completely inhibit CYP1a activity in
fish (Prochilodus scrofa) hepatic cells in vitro, possibly due to membrane disruption (da
Silva and Meirelles, 2004). More toxicity information on the individual toxicities of the
components in Corexit would be beneficial when evaluating exactly how toxicity may
occur.
1.2.6. Corexit Toxicity
Many studies have shown that chemical dispersant toxicities are both species-
and compound-specific, yet few studies to date have evaluated Corexit toxicity in cold
temperatures and using coldwater species. Hemmer et al. (2011) reported that Corexit
was only slightly toxic to mysids (A. bahia) and practically non-toxic to inland silversides
(Menidia beryllina). This was supported by Word et al. (2014) who conducted a literature
review of published data and determined that Corexit would be considered slightly toxic
to probably not toxic to aquatic species, according to USEPA criteria. However, the
MSDS says that Corexit is harmful to aquatic life (Nalco, 2014), particularly to small
invertebrates like copepods and bivalves. Cohen et al. (2014) found that Corexit caused
acute mortality in copepods (Labidocera aestiva), and Salehi et al. (2017) found that
Corexit alone is very toxic to oysters (Crassostrea virginica), particularly in early life
stages. Almeda et al. (2014) found that Corexit was highly toxic to marine
15
microzooplankton, including oligotrich ciliates, tintinnids and heterotrophic
dinoflagellates.
Corexit exposure has been associated with many sublethal toxic effects including
developmental toxicity, teratogenicity, immunotoxicity, neurotoxicity and cardiotoxicity. In
mallard ducks (Anas platyrhynchos), embryo hatching success was significantly reduced
by Corexit exposure (Wooten et al. 2011). DeLorenzo et al. (2017) found that in grass
shrimp (Palaemonetes pugio) exposed to Corexit, embryo hatching success was
significantly reduced and lipid peroxidation activity was increased. Adams et al. (2014)
found that Corexit exposure caused gill disruption, opaque yolk sacs and spinal curvature
in rainbow trout (Oncorhynchus mykiss). As mentioned previously, studies by Chen and
Reese (2016) and Sriram et al. (2011) showed that Corexit can impact neurotransmitter
signaling and cause neurotoxicity. Jones et al. (2017) showed that Corexit affects gene
expression in both immunity pathways and blood and circulation processes. Krajnak et
al. (2011) found that after 5 h of Corexit inhalation exposure to male Sprague-Dawley
rats, increased heart rate and blood pressure was shown at 1 d post-exposure, but not
at 7 d, suggesting that acute exposure exerts transient cardiovascular effects. Anderson
et al. (2011) looked at the irritancy and immunotoxicity of Corexit and DOSS and found
that both caused increased dermal irritation and lymphocyte proliferation in a dose-
dependent manner.
Corexit can also cause indirect impacts on the survival of aquatic organisms. Chiu
et al. (2017) showed that Corexit can inhibit spontaneous DOM (dissolved organic matter)
assembly to form microgels (POM, particulate organic matter), an important natural
process in surface ocean waters. POM formation impacts microbial loops and nutrition
availability in the ocean, and interfering with these biological loops could result in entire
ecosystem effects. Additionally, Hamdan and Fulmer (2011) found that Corexit caused
almost 100% reduction in hydrocarbon-degrading Marinobacter viability and production,
suggesting that organisms capable of bioremediating after the spill may be instead killed
off by dispersant. This was supported by Kleindienst et al. (2015) who reported that
Corexit decreased microbial degradation of hydrocarbons through selection for
dispersant-degrading bacteria (Colwellia) instead of hydrocarbon-degrading
Marinobacter. Overholt et al. (2016) investigated this further, finding that Corexit inhibited
16
the growth and crude oil degradation potential of Acinetobacter by 34 and 40%,
respectively. These observations should all be taken into account when determining
whether use of Corexit would be appropriate in a marine environment.
1.2.7. Oil Dispersed by Corexit
Proposed Mechanisms of Action
Similar to dilbit, chemically-dispersed dilbit toxicity is likely due to the presence of
toxic PAHs. Dispersant breaks the oil down into particles that are more digestible by
microorganisms but also increases the potential bioavailability of the toxic soluble
components in oil (Adams et al., 2014). For this reason, CEWAF toxicity is often greater
than WAF toxicity at the same nominal oil loading (Greer et al., 2012; Wu et al., 2012;
Adams et al., 2014). The CEWAF contains a higher percentage of alkyl PAHs than WAF
(Madison et al., 2015; Peiffer and Cohen, 2015; Wu et al., 2012; Gardiner et al., 2013).
In particular, CEWAF toxicity has been attributed to a higher percentage of naphthalenes,
dibenzothiophenes, phenanthrenes, anthracenes and HMW PAHs dispersed in water
(Dasgupta et al., 2015). These HMW PAHs with three or more benzene rings are more
toxic than low molecular weight (LMW) PAHs (Couillard et al., 2005). Similar to dilbit
alone, PAHs in CEWAF can activate the AhR to initiate upregulation of metabolic
enzymes like CYP1a, forming reactive metabolites which can damage DNA, lipids and
proteins (Akcha et al., 2003; Madison et al., 2015, 2017; Alderman et al., 2017a).
Dussauze et al. (2015) found that oil CEWAF reduced antioxidants superoxide dismutase
(SOD) and glutathione peroxidase (GPX) in the intestine and brain of European sea bass
(Dicentrarchus labrax), which are responsible for transforming ROS and preventing
oxidative stress. However, they also found that CEWAF activated these antioxidants in
the liver, suggesting that toxic effects are organ-specific.
Oil dispersed by Corexit has been shown to have endocrine-disrupting effects in
reptiles. Williams et al. (2017) showed that CEWAF induced estrogen receptor
transcriptional activation in vitro and also increased female to male ratios in the American
alligator (Alligator mississippiensis). The same study found that CYP19A1, an enzyme
involved in ovarian development and converting androgens to estrogens, was reduced
after CEWAF exposure. This reduction of gonadal CYP19A1 was consistent with results
17
found in Lake Apopka, a well-known example of the endocrine-disrupting effects of
anthropogenic chemicals, and suggests oil dispersed by Corexit may affect estrogen
synthesis (Kohno et al., 2008). The CEWAF has also been shown to suppress growth
factor anti-Müllerian hormone (AMH) and transcription factor sex determining region Y-
box 9 (SOX9), both involved in sex determination, but not mediated by estrogen receptor
activation (Williams et al., 2017). This suggests that the endocrine-disrupting effects of
Corexit are not mediated solely by estrogen receptor activation. While CEWAF has been
shown to induce ER activity, Judson et al. (2010) found that Corexit alone did not induce
ER activity in vitro, so further research is necessary regarding the potential for endocrine
disruption in organisms exposed to Corexit.
An area of debate has been whether oil and oil dispersants exhibit synergistic
toxicity to marine organisms. The purpose of dispersants is to reduce oil droplet size to
enhance oil-water partitioning of water-soluble fractions (Madison et al., 2017). In
CEWAF, there is a greater exposure to dissolved hydrocarbons that partition into the
water from oil droplets (Adams et al., 2014). One compelling study conducted by Rico-
Martínez et al. (2013) found that CEWAF toxicity in rotifers (Brachionus plicatilis) was 47
to 52 times higher than the WAF of crude oil alone. However, they evaluated this using
nominal concentrations of oil and dispersant and did not measure the concentration of
hydrocarbons in their solutions. Bejarano et al. (2014) found that when studies reported
toxicity values based on nominal loading rates, 93% of the CEWAF toxicity values
(LC50/EC50) were smaller than toxicity values for WAF, indicating a much greater toxicity
of CEWAF. In contrast, when studies reported toxicity based on measured concentrations
of hydrocarbons (TPAH/TPH [total petroleum hydrocarbons]), 78% of oil CEWAF toxicity
values (LC50/EC50) were greater than or equal to toxicity values for WAF, indicating
lower or equal toxicity. They suggest that toxicity values based on nominal concentrations
greatly overestimate the CEWAF toxicity in comparison to the WAF. This has been
supported by studies evaluating a wide variety of marine species (Wu et al., 2012; Greer
et al., 2012; Adams et al., 2014). When expressed as measured TPAH concentrations,
most of these studies conclude that WAF and CEWAF have similar toxicities.
18
Toxicity of Dispersed Oil
Most studies to date have evaluated crude oil (not dilbit) toxicity when dispersed
with Corexit. Results from studies that have evaluated the toxic effects of crude oil and
Corexit in combination may not be directly extrapolated to dilbit, due to its unique physical
and chemical properties. The CEWAF of crude oil dispersed by Corexit has been shown
to be acutely toxic to many marine species, including tropical coral, ctenophores,
microalgae and microzooplankton (Goodbody-Gringley et al., 2013; Peiffer and Cohen,
2015; Garr et al., 2014; Almeda et al., 2014). Cohen et al. (2014) found that Corexit alone
caused acute mortality in copepods (L. aestiva), whereas CEWAF exposure caused
chronic effects, including impaired swimming ability. This was also shown by Adams et
al. (2014) who reported that Corexit alone caused acute embryo mortality in rainbow trout,
whereas CEWAF was chronically toxic and caused embryo mortality near the end of the
experiment. This suggests that CEWAF toxicity may contribute to chronic toxicity in
aquatic species, whereas Corexit alone may be more acutely toxic.
Similar to WAF of oil, CEWAF has been associated with reproductive and
developmental toxicity, teratogenicity, cardiotoxicity and immunotoxicity in aquatic
species. BSD symptoms have been shown in CEWAF exposure to rainbow trout
(Oncorhynchus mykiss) embryos (Wu et al., 2012). DeLorenzo et al. (2017) found a
significant reduction in hatching success and an increase in mean time-to-hatch in
sheepshead minnows (C. variegatus) exposed to CEWAF. Embryo hatching significantly
decreased in grass shrimp (Palaemonetes pugio), and no embryos at the highest CEWAF
concentrations hatched at all (DeLorenzo et al., 2017). Atlantic herring (Clupea harengus)
embryos exposed to Medium South American (MESA) crude oil CEWAF showed
premature hatching and did not physically appear normal (Adams et al., 2014). Olsen et
al. (2013) found that reproduction was impaired in copepods (Calanus finmarchicus) after
exposure to dispersed crude oil, although after 13 d of recovery, no significant differences
in hatching success or egg production rates were found, suggesting species may be able
to recover after CEWAF exposure.
Evidence of immunotoxicity and cardiotoxicity was shown by Jones et al. (2017)
who found that CEWAF affects gene expression in both immunity pathways and blood
and circulation processes, altering 109 genes in total. A decrease in macrophage function
19
was shown in all treatment groups, which suggests innate immunity suppression was
occurring (Jones et al., 2017). However, studies evaluating the adaptive immune
responses after the DWH oil spill, remediated with Corexit, have shown conflicting results.
One study showed that the gene for immunoglobulin mu (IgM) was up-regulated in gulf
killfish (Fundulus grandis) liver at oil-impacted sites, indicating a B-cell-mediated immune
response (Garcia et al., 2012). Other studies have shown that exposed fish showed
down-regulation of IgM transcripts (Bayha et al., 2017; Song et al., 2012). Other effects
that include decreases in blood clotting, erythrocyte damage and hepatic vascular
congestion have been shown in DWH oil- and sediment-exposed fish (Incardona et al.,
2013, 2014; Jones et al., 2017). Additionally, laboratory experiments with Atlantic herring
(Clupea harengus) embryos exposed to Medium South American (MESA) crude oil
CEWAF showed significantly decreased heart rates (Adams et al., 2014).
There have only been a few studies to date that have directly assessed the toxicity
of dilbit dispersed by Corexit. Madison et al. (2015) showed developmental
malformations, including non-inflated swim bladders and BSD, in Japanese medaka (O.
latipes) embryos exposed to CEWAF, although fish responded similarly to both WAF and
CEWAF. They showed similar results in a follow-up study with Japanese medaka
(Madison et al., 2017). Further evaluation of the toxicity of a dilbit-Corexit mixture to
Pacific marine species is essential to understand the impact of using Corexit as a
remediation technique.
1.2.8. Previous Spills Remediated Using Corexit
Corexit was applied in large quantities during the DWH crude oil spill in the Gulf
of Mexico in 2010. This spill is still considered to be the largest marine oil spill in history,
and required 7.9 million litres of chemical dispersants, primarily Corexit, to be applied at
the water’s surface and subsurface at the wellhead (Kujawinski et al., 2011).
Approximately 16% of the oil was dispersed using chemical dispersants (Gray et al.,
2014). Concentrations of dispersant ranged from 10 to 100 µg/L during and after the spill
(Kujawinski et al., 2011). The measurements of DOSS taken beyond the immediate area
where Corexit was being continuously applied did not exceed USEPA guidelines (Gray
et al., 2014). About 1 month after the DWH wellhead was capped, the total concentration
20
of 70 PAH analytes was 0.49 µg/L in water from an oiled site in Louisiana (Whitehead et
al., 2012). Evidence in the field suggests that low PAH concentrations may persist for
long periods of time due to sediment re-suspension (Jones et al., 2017). Allan et al.
(2012) found that about a year after the spill, PAH concentrations in the water had
returned to pre-spill concentrations, but when measured two months after that, levels had
increased to peak-oil levels, likely due to sediment perturbation. Three years after the
spill, measured PAH concentrations in sediment still remained high, indicating that the
risk of re-suspension remains long after use (Turner et al., 2014).
1.3. Juan de Fuca and Strait of Georgia Ecosystem
1.3.1. Burrard Inlet Representation
Burrard Inlet is one of Canada’s most productive aquatic ecosystems and is home
to a variety of species, including echinoids, crustaceans, and many fish species.
Representative species from each of these categories were chosen according to their
prevalence along the coastal areas of British Columbia. These species include the purple
sea urchin (Strongylocentrous purpuratus), spot prawn (Pandalus platyceros) and
topsmelt (Atherinops affinis). Additionally, a tropical shrimp species, mysid shrimp
(Mysidopsis bahia), was also chosen due to their easy accessibility, relative sensitivity to
pollutants, and the prevalence of toxicity data available for this test species using crude
oil (Barron et al., 2018; DeLorenzo et al., 2017).
1.3.2. Purple Sea Urchins (Strongylocentrous purpuratus)
Purple sea urchins were chosen to represent phylum Echinodermata due to their
prevalence in the Juan de Fuca area and the relative ease of obtaining them for
laboratory toxicity testing. Previous toxicity studies have evaluated the toxicity of crude
oil and Corexit to sea urchins but not in combination. After the Prestige tanker spill, and
even after the oil had visibly disappeared, embryogenesis was completely inhibited in
sea urchins exposed to samples from the spill site (Beiras and Saco-Álvarez, 2006). This
water also caused growth impairment in sea urchins, oyster larvae and mussel embryos.
Vashchenko (1980) found that hydrocarbon exposure caused prominent delay,
21
asynchronism and abnormal non-viable larvae in artificially-fertilized sea urchins. The
long-term effects of this sublethal exposure caused deformed sex cells and high mortality
of larvae. Corexit 9527, a similar dispersant to Corexit 9500, caused significant delay in
fertilization rates in several species of sea urchins and fish (Lonning and Hagstrom,
1976). A follow-up study showed that 10 min of sperm exposure to Corexit 9527 reduced
fertilization from 55% to 8% (Hagstrom and Lonning, 1977). Both studies also showed
that polyspermy occurred after Corexit exposure, where the hyaline layer of the egg does
not seal off to prevent additional fertilization, allowing multiple fertilizations to occur. The
lack of toxicity data on sea urchins specifically for dilbit, and dilbit and Corexit in
combination, highlights the need for the current research.
1.3.3. Mysid Shrimp (Mysidopsis bahia)
Mysid shrimp were chosen due to their ease of access, relative sensitivity to
pollutants, and the prevalence of toxicity data available for this species using crude oil
(Barron et al., 2018; DeLorenzo et al., 2017). Previous studies have shown that crude oil
is slightly toxic to mysids (DeLorenzo et al., 2017; Barron et al., 2018). Barron et al. (2018)
also evaluated growth in mysids for 7 d and found that growth was reduced in groups
exposed to dilbit. Detoxification is an energy-expending process, suggesting that this
energy expended in detoxification may come at a cost to growth or fecundity. DeLorenzo
et al. (2017) found that crude oil dispersed by Corexit showed slight toxicity to mysids,
and LC50 values fell within the same range of measured TPAH concentrations as dilbit
alone. Previous literature evaluating the toxicity of Corexit alone has shown that it is
slightly toxic to mysids (Hemmer et al., 2011; Word et al., 2014). The current study will
assess the impact of a dilbit-Corexit mixture on mysids to determine whether the
response is similar to that of crude oil.
1.3.4. Topsmelt (Atherinops affinis)
Topsmelt are abundant in nearshore waters along the southern coast of BC
(Allen, 1982). Since topsmelt embryos develop in benthic habitats like bays and estuaries
that may be affected by oil spills, they are an important species to evaluate for potential
toxicity (Incardona et al., 2005; Anderson et al., 2009). Spawning occurs between April
22
and October, leaving embryos in particular susceptible to oil exposure in the event of a
spill (Incardona et al., 2005). Previous studies have shown that topsmelt larvae are
sensitive to WAF of crude oil, as well as crude oil dispersed by Corexit (Singer et al.,
1998; Anderson et al., 2009). Effects included cardiovascular abnormalities, as well as
significant inhibition of development and survival to hatching in embryos (Anderson et al.,
2009). Cardiovascular abnormalities were also shown in a study by Van Scoy et al. (2012)
which found that crude oil dispersed by Corexit caused a reduction in egg production in
adults, not only immediately following the exposure, but also after a 5 month recovery
period, suggesting lasting effects on reproduction. The same study also found that the
highest concentrations of crude oil dispersed by Corexit caused nearly 100% fish
mortality. However, these studies did not assess the potential effects of Corexit-only
exposure to topsmelt. The current study will evaluate the toxicity of Corexit alone, as well
as dilbit toxicity with and without dispersant.
1.3.5. Spot Prawn (Pandalus platyceros)
Spot prawns are a benthic species found along the Pacific coast of BC (DFO,
2018). They are an economically important species to the Pacific region, with $33.5 - $39
million of landed value in 2013-2015 (DFO, 2018). Approximately 1600 to 1850 tonnes
of prawns are landed each year, with about 60% of harvesting coming from the Strait of
Georgia and inside of Vancouver Island (DFO, 2018). This highlights the potential impact
of a dilbit spill in the Juan de Fuca or Strait of Georgia to spot prawns. This limited entry,
competitive fishery has 246 license eligibilities, with 60 of these licenses designated to
First Nations communities (DFO, 2018). One report suggests that over half of commercial
license-holders fish exclusively for spot prawns and are highly dependent on this industry
(Mormorunni, 2001). Not only are they economically viable, adults are an important food
source for other fish species like rockfish and octopus, while larvae are important prey
for other pelagic marine species (Bergstrom, 2000).
Spot prawns are the largest species of local shrimp, growing to an average of 20
cm and living up to four years (DFO, 2018). Spot prawns spawning period is from August
to October, and each female carries between 2,000 and 4,000 eggs for five months
before releasing their hatched larvae in the spring (DFO, 2018). Adult prawns are
23
normally found in rocky crevices and under boulders, but juveniles can often be found on
muddy bottoms or feeding in shallow water (DFO, 2018). Exposure to dilbit and Corexit
could potentially affect multiple life stages of the spot prawn, depending on the time of
year and location of the spill.
Chemical stimuli are used by aquatic species to identify potential feeding sources,
as well as escape predators and locate mates (Rittschof, 1992). Previous studies have
shown that pollutants can affect the ability of crustaceans like spot prawns to detect
chemical stimuli (Blinova and Cherkashin, 2012). A hierarchical sequence of feeding
behaviours was first described by Dethier et al. (1960) and has been since modified by
Lindstedt (1971) and Lee and Meyers (1996). It proposes that the response of decapod
crustaceans to food stimuli occurs in five phases: (1) detection of chemical stimulus; (2)
orientation toward the stimuli; (3) locomotion toward or away from the stimuli; (4) initiation
of feeding; and (5) continuation or termination of feeding. The antennules (first antennae)
contain primary chemoreceptors which can sense when a chemical stimulus is
introduced. By flicking the antennules, crustaceans are able to detect odorants (Lee and
Meyers, 1996). Antennular flicking appears to be the most sensitive and common
behaviour associated with sensing a chemical stimulus at a distance (Lee and Meyers,
1996). Additionally, by wiping antennules with the third maxilliped, crustaceans can
remove debris from the receptors to obtain a better signal (Barbato and Daniel, 1997;
Daniel et al., 2008). During orientation, crustaceans will use their dactyl probe to rake,
probe or dig at the chemical stimulus, and then turn toward or away (Lee and Meyers,
1996). Locomotion consists of moving toward the food, either in a calm or frantic manner
(Lee and Meyers, 1996). The crustacean will then initiate feeding, whereby they will grab,
lunge, pounce, hold or taste the food, followed by either the continuation or cessation of
eating (Lee and Meyers, 1996). The importance of spot prawns economically, as well as
ecologically, supports the need for the current research on the potential toxicity of dilbit
and Corexit to sensory reception.
1.4. Objectives of Study
Currently, very little information exists on the toxicity of dilbit and Corexit,
particularly in combination, to marine organisms, especially those native to the Pacific
24
coast of Canada. To date, most studies assessing the toxicity of oil dispersed by Corexit
have evaluated either tropical marine species or freshwater species. The research goal
of this study was to generate new empirical data that directly assesses the effects of
environmentally realistic concentrations of dilbit and Corexit on aquatic biota from the
Strait of Georgia and Juan de Fuca, BC. Furthermore, this research addresses the
potential for synergistic toxicity between dilbit and Corexit. The assessment of toxic
effects on multiple marine species will address Department of Fisheries and Oceans
(DFO) data gaps on the environmental impacts of dilbit and Corexit. The overarching
hypothesis is that acute exposures of dilbit, Corexit and a dispersant-oil mixture will cause
mortality and adverse behavioral effects in Pacific marine organisms at increasing
concentrations. Additionally, that the addition of Corexit to dilbit will render it more toxic
than dilbit WAF alone.
25
Chapter 2. The Effects of Diluted Bitumen and the Dispersant Corexit 9500A on Pacific Marine Organisms
Kassondra N. Rhodenizera, Christopher J. Kennedya
aDepartment of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada
2.1. Introduction
Canada is one of the world’s largest oil-producing countries, with the majority of
production coming from the oil sands in Northern Alberta (Alsaadi et al., 2017). This area
is also the largest producer of oil sands bitumen in the world, containing over 169 billion
barrels of recoverable bitumen using current extraction methods (ERCB, 2012). Bitumen
extraction, production and exportation levels are expected to triple within the next decade
(Alsaadi et al., 2017). Due to its high viscosity, raw bitumen is combined with natural gas
condensate or synthetic oils before transport, producing diluted bitumen (dilbit) and
facilitating its efficient flow through pipelines (Barron et al., 2018). Major new pipelines
for the transmission of dilbit have been proposed to accommodate a rise in production
levels. Although the recent proposals for the Northern Gateway and Energy East pipeline
were rejected, other proposed pipelines destined to carry dilbit have been approved, with
the Kinder Morgan pipeline expansion attracting the most attention. In BC, the existing
and proposed pipelines will transport dilbit to coastal ports, then be shipped by marine
tankers to overseas markets, greatly enhancing the potential risks of a spill of dilbit into
the marine environment (Environment Canada, 2013).
Dilbit entering the aquatic environment is very difficult to remediate, as it can
disperse quickly from the release site, as well as partition into different compartments of
the environment (Environment Canada, 2013; Hua et al., 2018). Dilbit exposure has
resulted in similar types of adverse effects as seen with conventional crude oils, however
most studies to date have assessed effects on freshwater species, highlighting the need
for the current research on marine organisms. Similar to crude oil, dilbit contains a wide
variety of polycyclic aromatic hydrocarbons (PAHs) which can exert their toxic effects
26
through multiple mechanisms that affect multiple physiological systems. Specific toxic
mechanisms of action depend on the compound, exposure duration, species, and
environmental conditions (Bejarano et al., 2014; Alderman et al., 2017a). Understanding
the potential environmental impacts of dilbit to Canada’s Pacific organisms is necessary
to ensure that adequate regulation and effective mitigation procedures are in place,
should a spill into the marine environment occur.
One remediation technique for oil spills involves the use of chemical dispersants
which break down oil into smaller oil-surfactant droplets to facilitate biodegradation
(Adams et al., 2014). Although these smaller droplets allow increased biodegradation
and dilution of the oil, they can increase the concentrations of toxic hydrocarbons in the
water column, increasing bioavailability and the potential for adverse effects to aquatic
organisms. Corexit 9500A (Corexit), was recently approved for use in Canada in 2016
(Canada Gazette, 2016). Corexit alone (not in combination with oil) has been shown to
be toxic to many marine species, although most of the reported literature has evaluated
tropical marine species.
Crude oil chemically-dispersed by Corexit has been associated with reproductive
and developmental toxicity, teratogenicity, cardiotoxicity and immunotoxicity in aquatic
species (Wu et al., 2012; Olsen et al., 2013; Adams et al., 2014; DeLorenzo et al., 2017;
Jones et al., 2017). However, there have only been a few studies to date that have directly
assessed the toxicity of dilbit dispersed by Corexit. Madison et al. (2015, 2017) showed
evidence of developmental toxicity in Japanese Medaka (O. latipes) embryos exposed to
chemically-dispersed dilbit, although fish responded similarly to both oil-only and
chemically-dispersed oil treatments. The lack of data to cold water species is an
information gap that needs to be filled before proceeding to using Corexit in Pacific
coastal waters.
An area of debate has been whether oil and oil dispersants exhibit synergistic
toxicity to marine organisms. Studies that have evaluated the effects of crude oil and
Corexit in combination may not be directly extrapolated to those which may occur with
dilbit, due to its unique physical and chemical properties. Rico-Martínez et al. (2013)
evaluated toxicity in rotifers (B. plicatilis) and found that the synergistic acute toxicities of
27
crude oil and dispersant were 47- to 52-times higher than oil alone, although hydrocarbon
concentrations were not measured. When evaluating toxicity based on measured TPAH
concentrations, most studies suggest that oil-only and chemically-dispersed oil
treatments are similar in toxicity. Determining whether dilbit and Corexit show a
synergistic toxic effect is important in determining whether Corexit would be appropriate
in a dilbit spill.
Georgia Straight is one of Canada’s most productive marine ecosystems and is
home to a variety of species, including echinoids, crustaceans, and many fish species.
Representatives from echinoderm, crustacean, and teleost species were chosen
according to their prevalence along the coastal areas of British Columbia. These species
included the purple sea urchin (Strongylocentrous purpuratus), spot prawn (Pandalus
platyceros) and topsmelt (Atherinops affinis). In addition, the tropical mysid shrimp
(Mysidopsis bahia) was also used due to their ease of access, relative sensitivity to
pollutants, and the prevalence of toxicity data available for this species using crude oil
(Barron et al., 2018; DeLorenzo et al., 2017). This study directly assesses the effects of
environmentally realistic concentrations of dilbit, Corexit, and dilbit and Corexit in
combination on aquatic biota from the Straights of Georgia and Juan de Fuca, BC. The
overarching hypothesis is that acute exposures of dilbit, Corexit and a dispersant-oil
mixture will cause mortality and adverse behavioral effects in Pacific marine organisms
at increasing concentrations. The assessment of effects on multiple marine species will
address Department of Fisheries and Oceans (DFO) data gaps on the environmental
impacts of dilbit and Corexit.
2.2. Materials and Methods
2.2.1. Organisms
Representative marine species were chosen according to their prevalence and
ecological importance along the coastal areas of British Columbia. These species
included the purple sea urchin (Strongylocentrous purpuratus), spot prawn (Pandalus
platyceros) and topsmelt (Atherinops affinis). In addition, the tropical mysid shrimp
(Mysidopsis bahia) was used due to their accessibility, relative sensitivity to
28
contaminants, and the prevalence of toxicity data available for this species using crude
oil (Barron et al., 2018; DeLorenzo et al., 2017). Juvenile mysids and juvenile topsmelt
were purchased and shipped from Aquatic BioSystems Inc. (Fort Collins, CO). Purple
sea urchins were purchased and shipped from Nautilus Environmental (San Diego, CA).
Adult spot prawn were purchased from T&T Supermarket (Richmond, BC).
2.2.2. Test Chemicals
Chemicals
Summer blend dilbit from the Cold Lake region (Canada’s second largest oil
sands deposit) was obtained from the Centre for Offshore Oil, Gas, and Energy Research
(Fisheries and Oceans Canada). Summer Cold Lake blend (CLB) is a crude bitumen
blended with 20% condensate (King et al., 2017b). Corexit was obtained from Nalco
Environmental Solutions LLC (Sugar Land, TX). Life brand mineral oil was purchased
locally. Seawater for all tests was provided by the Vancouver Aquarium, and was filtered
and UV sterilized before use.
WAF and CEWAF
WAF and CEWAF solutions were prepared according to methods previously
described by Madison et al. (2017), with a few modifications. Dilbit WAF solutions (100%
v/v) were generated fresh daily. The WAF solutions were stirred in 23 L glass carboys
using stainless steel stirring rods attached to a Teflon tube, with a 12 V DC gearmotor
which rotated at 168 rpm. Briefly, 5 mL of CLB summer blend dilbit was added to 23 L of
seawater and stirred for 18 h to create the maximum WAF (100% v/v), creating a
maximum oil loading of 0.217 mL/L. Mixtures were left to settle for 1 h, after which WAF
was extracted by siphoning off of the bottom layer. The CEWAF was generated the same
way, although after 18 h of spinning, the WAF was settled and siphoned and Corexit was
added to the surface at a dispersant-oil ratio (DOR) of 1:10. For the spot prawn
experiment, a DOR of 1:20 was used in order to assess sublethal behavioural effects. A
DOR of 1:5 was used for the topsmelt test to evaluate the potential impact of a higher
DOR. Nominal loadings of Corexit ranged from 10.33 to 41.31 mg/L. The CEWAF was
29
stirred for an additional 1 h, allowed to settle for 1 h, and the CEWAF was siphoned and
removed.
An oil control WAF was also prepared by the same method as dilbit WAF, using
a non-toxic mineral oil alone, as a control that contained no PAHs (Adams et al., 2014).
A dispersant control CEWAF solution was also prepared by the same method as dilbit
CEWAF, using non-toxic mineral oil instead of dilbit, as a control that contained no PAHs
(Madison et al., 2017; Adams et al., 2014). Corexit-only treatments were made using the
same nominal loading (mg/L) of Corexit stock solution as was used in the CEWAF to
create the 100% Corexit solution which was then diluted. The total range of Corexit
concentrations used in each respective test can be found in Table A1. Each experiment
included dilbit WAF, mineral oil WAF, dilbit CEWAF, mineral oil CEWAF, Corexit alone,
and a saltwater control. Serial dilutions were used as recommended by Barron and
Ka'aihue (2003) to allow water chemistry analysis extrapolation from 100% WAF to lower
dilutions. Dilutions of 100% v/v solutions depended on the specific test and ranged from
1.0 to 50.0% v/v. Water quality was monitored for dissolved oxygen, temperature and pH
throughout the exposures.
Water Analyses
Individual PAH concentrations were measured by Axys Analytical Services
following standard procedure as described in Alderman et al. (2017a). Briefly, 1L of each
water sample was sent to Axys Analytical (Sidney, BC) where concentrations of individual
PAHs were identified using gas chromatography-mass spectrometry. Samples were first
spiked with deuterated surrogate standards, followed by extraction with dichloromethane
and were cleaned up with column chromatography on silica. Instrumental analysis was
conducted using low-resolution mass spectrometry with an RTX-5 capillary gas
chromatography column. This was operated in the electron impact ionization mode using
multiple ion detection. At least 1 characteristic ion for each target analyte and surrogate
standard was acquired. Concentrations of PAHs were then calculated using the isotope
dilution method of quantification. Reporting limits for individual compounds ranged from
0.055 to 10800 ng/L. Average percentage of recovery was 100.1%. The measured PAHs
consisted of high molecular weight (HMW) PAHs including C3-naphthalene,
fluoranthrenes and phenanthrenes as these have been shown to be higher in CEWAF
30
treatments than WAF treatments (Couillard et al., 2005; Peiffer and Cohen, 2015). All
test chemicals at 100% v/v were tested using this method, with the exception of the
mineral oil CEWAF at a DOR of 1:5. Solutions were not analyzed for concentrations of
Corexit, so the estimated concentrations were calculated based on nominal loadings of
Corexit and assuming no dispersant was lost in solution and dilution preparation.
2.2.3. Standardized Toxicity Tests
Standardized toxicity tests included a 20-min purple sea urchin (S. purpuratus)
fertilization assay, a 48-h static-renewal test for mortality using juvenile mysids (M. bahia)
and a 96-h static-renewal test for mortality using juvenile topsmelt (A. affinis) as
summarized in Table 1. All experiments were approved by the University Animal Care
Committee of Simon Fraser University in accordance with Canadian Council on Animal
Care guidelines. All tests were conducted at Nautilus Environmental laboratory in
Burnaby, BC between May 2016 and July 2017. All toxicity tests met QA/QC
requirements, which includes control survival and water quality criteria.
Purple sea urchin (Strongylocentrous purpuratus) 20-min fertilization assay
The 20-min purple sea urchin (Strongylocentrous purpuratus) fertilization assay
followed the protocol of Environment Canada (2011). Chemicals tested included dilbit
WAF, mineral oil WAF, dilbit CEWAF (DOR 1:10), mineral oil CEWAF (DOR 1:10),
Corexit alone, and a saltwater control. Seven test concentrations were created using a
geometric dilution series (100, 50, 25, 12.5, 6.25, 3.12, 1.56 %v/v) created from the stock
(100% v/v) of each chemical. There were 3 replicates for each treatment. Urchins were
held at 10°C prior to test commencement in which gametes were isolated from adult
urchins and assessed for viability. Urchins were placed at 15 °C to spawn. Urchins were
induced to spawn using 0.1M KCl; gloves were changed frequently to avoid pre-fertilizing
eggs. Semen from each male was stored separately on ice. Sperm of high quality were
determined by activating a small portion of each male's sperm by diluting with control
water and placing on a microscope slide so motility could be judged. Sperm quality was
assessed by looking at shape, color and size. High-quality sperm were then pooled and
the appropriate sperm:egg ratio was determined based on which testing ratio gave 60-
31
98% fertilization, which was 300:1. Finally, 0.1 mL of pooled sperm were added to each
30 mL vial which contained 10 mL of the respective treatment.
High-quality eggs were determined by pipetting each egg sample onto a
microscope slide and analyzing at 40x magnification. Eggs of poor quality are small in
size, irregular in shape and display vacuolization (Environment Canada, 2011). Eggs
were also evaluated to ensure they were not previously fertilized before commencing the
test, which would appear as a halo surrounding the circular egg (Environment Canada,
2011). Eggs determined to be of high-quality were then pooled. After sperm had been
added to the treatments for 10 min, 1 mL of the pooled eggs were added to each vial for
an additional 10 min prior to test termination. The test was terminated at 20 min using 10
drops of 10% neutral buffered formalin. After exposure, the number of eggs fertilized was
counted using a Sedgwick-Rafter chamber at 100x magnification using phase-contrast
microscopy, to determine percent fertilization for each treatment. Fertilized eggs were
identified by the presence of a halo close to the circular egg that appeared to engulf it
(Environment Canada, 2011).
Mysid (Mysidopsis bahia) 48-h static renewal test
The 48-h static-renewal test for mortality used juvenile mysids (Mysidopsis bahia)
aged 1 to 5 d old and was conducted according to USEPA protocol (2002). Briefly, 10
mysids were placed randomly in each test chamber containing 200 mL of its respective
treatment solution at 25°C. Mysids were fed Artemia 2 h prior to commencing test and 2
h prior to solution renewal at 24 h. Chemicals tested included dilbit WAF, mineral oil WAF,
dilbit CEWAF (DOR 1:10), mineral oil CEWAF (DOR 1:10), Corexit alone, and a saltwater
control. Six test concentrations were created using a geometric dilution series (100, 50,
25, 12.5, 6.25, 3.12 %v/v) created from the stock (100% v/v) of each chemical. There
were 4 replicates of each treatment. At 24 and 48 h, mortality was determined visually by
counting the number of mysids in each test chamber. Those who showed any sign of
movement (swimming or trying to swim) during 15 seconds of visual inspection were
classified as alive, while those showing no signs of movement were classified as dead.
Qualitative observations on the general movement and swimming speed in each
treatment group were also noted.
32
Topsmelt (Atherinops affinis) 96-h static renewal test
The 96-h static-renewal test for mortality used juvenile topsmelt (Atherinops
affinis) and was conducted according to Washington State Department of Ecology
protocol (WSDoE, 2008). Briefly, 5 topsmelt were placed in each test chamber containing
200 mL of a respective treatment at 20°C. Chemicals tested included dilbit WAF, mineral
oil WAF, dilbit CEWAF (DOR 1:5), mineral oil CEWAF (DOR 1:5), Corexit alone, and a
saltwater control. Five test concentrations were created using a geometric dilution series
(100, 50, 25, 12.5, 6.25 %v/v) created from the stock (100% v/v) of each chemical. There
were 4 replicates of for each treatment. Mortality was determined visually at each 24 h
interval by counting the number of topsmelt in each test chamber. Those who showed
any sign of movement (swimming or trying to swim) during 15 seconds of visual
inspection were classified as alive, while those showing no signs of movement were
classified as dead. Qualitative observations on the general movement and swimming
speed in each treatment group were noted.
33
Table 1 Bioassay parameters for each species tested, including life stage, test duration, concentrations, replicates, individuals per replicate, volume used, temperature, photoperiod, salinity, feeding regimen, protocol and endpoints.
Sea urchin (Strongylocentrous
purpuratus)
Mysid (Mysidopsis
bahia)
Topsmelt (Atherinops
affinis)
Spot prawn (Pandalus
platyceros)
Life stage Gametes Juveniles Juveniles Adults
Test duration 20 min 48 h 96 h 7 d
# Concentrations
7 6 5 5
Definitive test concentrations
(%v/v WAF and CEWAF)
100, 50, 25, 12.5, 6.25, 3.12, 1.56
100, 50, 25, 12.5, 6.25,
3.12
100, 50, 25, 12.5, 6.25
100, 32, 10, 3.2, 1.0
# Replicates per
concentration
3 4 4 2
# Individuals per test
chamber
0.1 mL pooled sperm, 1 mL pooled
eggs
10 5 3
Volume in test
chamber (mL)
10 200 200 6,000
Temperature
(°C)
15 25 20 12
Photoperiod Regular light 16h light : 8h
dark
16h light : 8h
dark
16h light : 8h dark
Salinity (ppt) 28 +/- 2 30 +/- 2 28 +/- 2 28 +/- 2
Feeding n/a 2h before test, 2h prior
to 24h
renewal
n/a Day 4
Endpoint Egg Fertilization Mortality Mortality a) Mortality b) Antennule
Flicking
c) Pre-Feeding
and
Feeding Behaviours
Protocol Environment Canada (2011)
USEPA (2002)
WSDoE (2008)
n/a
34
2.2.4. Spot Prawn (Pandalus platyceros) Experiments
Exposure
Spot prawns (P. platyceros) were acclimated for at least 2 weeks in laboratory
conditions in large fibreglass tanks before use in any assay. Prawns were randomly
sorted and not weighed prior to placement in 11 L (W 36 cm x H 26 cm x D 12.5 cm)
fiberglass tanks (n = 3 per tank), to minimize stress and handling. Prawns were exposed
statically to 5 concentrations (100.0, 32.0, 10.0, 3.2 and 1.0% v/v) of each treatment listed
in Section 3.2.2. for 7 d; solutions were prepared with a DOR of 1:20 (Table 1). Each
treatment had a total of 3 prawns per tank, 2 tanks per treatment (n = 2). Exposures were
staggered in two groups so that one tank per treatment (n = 3) was done in the first group
and the other tank for the same treatment (n = 3) was done in the second group. Prawns
were fed thawed frozen mysids 2 h before exposure and 2 h before water change at 4 d.
Mortality was recorded daily. To determine mortality, prawns were observed for 5 min for
signs of active swimming and were prodded with a net once 5 min had passed without
movement. Those lacking any movements were considered dead and were removed
from the exposure tank.
Behavioural Tests
Antennule Flicking
After the 7-d exposure period, prawns were moved individually to 11 L plexiglass
tanks containing fresh seawater. Tanks were surrounded by black plexiglass on all sides
to ensure a minimal observer effect. After 10 min of acclimation, the tank was covered
with a black plexiglass cover, so prawns were in the dark, with a 3x3 cm hole through
which a camera was inserted and recording began. At 5 min after recording started, 1 mL
of a mysid broth (a liquid food stimulus) was injected gently and directly into the tank by
a syringe and connecting tube. Prawns were video taped for 8 min in total. Videos were
analyzed for every prawn by quantitative evaluation for the number of antennular flicks
(both left and right). This behaviour was counted for a period of 2 min prior to the
introduction of the food stimulus (between minutes 3 to 5 of the recording). At 30 sec after
the introduction of the food stimulus (between minutes 5:30 to 7:30 of the recording) these
behaviors were once again counted. The 30-sec delay was to ensure that the prawn’s
35
reactions were not due to being startled but were due to the detection of the food stimulus.
Also noted was whether the prawn showed apparent signs of stress by swimming
erratically back and forth.
Pre-Feeding and Feeding Behaviours
Following the antennule-flicking evaluation, individual prawns were moved to
clean 11 L tanks and acclimated for 10 min. A stainless-steel scoop was used to introduce
0.75 to 1.00 g of sardines in spring water (Brunswick Canadian) as solid food on one side
of the aquarium. Prawns were then observed for 5 min and evaluated for the following
hierarchical sequence of pre-feeding behaviors: (1) antennule wiping by the third
maxilliped; (2) dactyl probing where dactyls of pereiopods are used to probe the food; (3)
orientation toward the chemical stimulus; and (4) eating solid food. Each behaviour was
scored as a +ve/-ve response. Other qualitative observations were taken, including
whether prawns movements appeared agitated or lethargic.
2.2.5. Statistical Analyses
Comparisons between WAF and CEWAF were expressed based on both nominal
loading of oil (% v/v) and measured TPAH concentrations (µg/L). Comparisons between
CEWAF treatments and Corexit-only treatments were expressed based on nominal
loading of Corexit (mg/L). For the topsmelt and mysid tests, LC50 (Lethal Concentration
to 50 percent of the population) values were calculated using the Comprehensive
Environmental Toxicity Information System (CETIS) from Tidepool Scientific Software
(McKinleyville, CA). The LC10/LC20 values were also calculated when an LC50 could
not be generated. For the echinoderm fertilization test, the concentration inhibiting 50%
of fertilization (IC50) and 20% of fertilization (IC20) and 95% confidence intervals (CI) for
each chemical and chemical combination were estimated. Data was checked for
normality and parametric assumptions were checked before the use of non-parametric
tests. The IC20 and IC50 values were calculated using linear interpolation, as data did
not meet assumptions of normality and homoscedasticity (Environment Canada, 2011).
For the topsmelt and mysid toxicity tests, parametric assumptions were checked before
the use of non-parametric tests. Probit and Spearman-Karber method were not
appropriate given the data, and instead a trimmed Spearman-Karber model was fit to the
36
data. Linear interpolation was used where mortalities were insufficient to calculate LC50
values, to instead calculate LC10 or LC20 values. All concentration-response graphs
were created using PRISM8 by Graphpad Software (San Diego, CA). A non-linear
regression curve fit was used for each graph with hillslope constrained to 1.
A one-way Analysis Of Variance (ANOVA) was used to evaluate the difference in
the mean number of antennule flicks before and following liquid food exposure (p < 0.05).
Each chemical-concentration combination was grouped as one factor (“treatment” factor)
to evaluate whether treatment had an effect on the change in number of flicks. A general
linear model (two-factor completely randomized design [CRD] ANOVA; chemical x
concentration) was used to compare the mean number of antennule flicks between
treatment groups in the 2-min period preceding the food introduction, after the prawns
had been acclimated to clean water. In this model, seawater control data had to be
dropped in order to run a full factorial analysis, since the control data had only one factor
in a two-factor analysis. A post-hoc Tukey’s multiple comparison (p < 0.05) procedure
was used to determine which groups showed evidence of a difference between their
means. All statistical analysis done using JMP (SAS Institute, 2012) and graphing was
done using PRISM8 by Graphpad Software.
For each of the pre-feeding behaviours that were evaluated as a +ve/-ve
response, the proportion of prawns per tank (n = 3 per tank, 2 tanks per treatment) that
exhibited this particular pre-feeding behaviour was calculated and used in a one-factor
SAS binomial logistic model (p < 0.05) (SAS Institute, 2012). Each chemical-
concentration combination was grouped as one factor (“treatment” factor) and all
treatments were analyzed using a Firth bias adjustment to account for value “0” in multiple
responses. In addition, a two-factor SAS logistic model was also run to evaluate the data
as a full factorial analysis (chemical x concentration). A post-hoc Tukey-Kramer
procedure was used to determine differences between means (p < 0.05). In these
models, seawater control data had to be dropped in order to run a full factorial analysis,
since the control data had only one factor in a two-factor analysis. Since many of the data
37
values were zeroes, a Firth bias adjustment was used in all models. All graphing was
done using PRISM8 by Graphpad Software (San Diego, CA).
2.3. Results
2.3.1. Chemical Analyses
Mineral oil WAF had an extremely low measured TPAH concentration (0.056
µg/L; Table 2). The TPAH concentration in 100% dilbit WAF was 15.48 µg/L; the highest
measured TPAH concentrations were in the 100% dilbit CEWAF (DOR 1:5) at 25.22 µg/L
TPAH. There was no significant increase in concentrations of some high molecular weight
(HMW) PAHs in CEWAF compared to WAF including C3-naphthalene, fluoranthrenes
and phenanthrenes, as seen in previous studies (Couillard et al., 2005; Peiffer and
Cohen, 2015), likely due to the slightly different CEWAF preparation method used in the
current study. There was, however, a significant increase in the concentration of C4-
dibenzothiophenes in CEWAF compared to WAF (Table A2). The C4-dibenzothiophenes
concentrations increased by more than 166-fold in the dilbit CEWAF (DOR 1:5) compared
to dilbit alone. Even more interesting was the fact that measured CEWAFs of both mineral
oil and dilbit showed a similar increase in C4-dibenzothiophenes.
38
Table 2 Nominal loadings of oil (mL/L) and Corexit (mg/L) with measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) in 100%v/v water-accommodated fraction (WAF) and chemically-enhanced water-accommodated fraction (CEWAF) solutions of diluted bitumen (dilbit) and mineral oil at various dispersant-oil ratios (DORs).
Oil WAF or Dispersant-Oil
CEWAF DOR Nominal loading
of oil (mL/L) Nominal loading of Corexit (mg/L)
Measured TPAH (µg/L)
Dilbit WAF 0.217 15.48
Dilbit CEWAF 1:20 0.217 10.32 15.56
Dilbit CEWAF 1:10 0.217 20.65 17.90
Dilbit CEWAF 1:05 0.217 41.31 25.22
Mineral oil WAF 0.217 0.06
Mineral oil CEWAF 1:20 0.217 10.33 6.21
Mineral oil CEWAF 1:10 0.217 20.65 8.47
Mineral oil CEWAF 1:05 0.217 41.31 n/a
2.3.2. Effects of Dilbit and Corexit on Juvenile Topsmelt
The mineral oil CEWAF treatment (DOR 1:5) was the most acutely toxic to
topsmelt juveniles, and all fish in the 100% CEWAF treatment group died by 24 h (not
shown). Based on the nominal loading of Corexit, the mean percent survival of mineral
oil CEWAF is very similar to Corexit alone, with 0% survival in the top nominal loading
treatments (Table 3). The concentration-response curves for dilbit WAF and dilbit
CEWAF, expressed as measured TPAH concentrations can be found in Figure 1A and
1B, respectively. The concentration-response curves for Corexit alone and mineral oil
CEWAF, expressed as nominal loading of Corexit (mg/L), can be found in Figure 1C and
1D, respectively.
39
Mineral oil CEWAF and Corexit-only treatments also had similar toxicity values
based on nominal loadings of Corexit, with 96-h LC50 values of 24.3 (21.1 – 28.0) and
25.6 (22.6 – 29.0) mg/L, respectively (Table 4). Dilbit CEWAF toxicity was similar based
on nominal loading of Corexit, with a 96-h LC50 value of Corexit of 29.5 (28.2 – 30.9)
mg/L. Dilbit WAF toxicity, based on an LC50 value as measured TPAH concentration,
was similar at 24 h and 96 h, indicating that the most toxicity occurred within the first 24
h of exposure (Table 4). In contrast, the dilbit CEWAF LC50 values expressed as both
measured TPAH concentration and nominal loading of Corexit, were lower at 96 h
compared to 24 h, suggesting that toxicity increased over time. This was the same for
the Corexit-only treatment group, which showed an LC50 value lower at 96 h compared
to 24 h. When using measured TPAH concentrations, 96-h LC50 values for dilbit WAF
and dilbit CEWAF were similar at 14.9 and 18.0 µg/L TPAH, respectively (with
overlapping 95% CIs). For LC50s based on nominal loadings, dilbit CEWAF, mineral oil
CEWAF and Corexit alone were more toxic to topsmelt than dilbit alone, with mineral oil
CEWAF being the most toxic.
40
Table 3 Mean percent survival of juvenile topsmelt (Atherinops affinis) based on nominal oil loading (%v/v) and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) following a 96-h exposure to dilbit water-accommodated fraction (WAF) and chemically enhanced water-accommodated fraction (CEWAF), mineral oil CEWAF, and Corexit.
Dilbit WAF Dilbit CEWAF Mineral oil CEWAF Corexit
Nominal Loading
(%v/v)
TPAH (µg/L)
Mean Survival (%)
TPAH (µg/L)
Mean Survival (%)
TPAH (µg/L)
Mean Survival
(%)
Mean Survival
(%)
100 15.48 45 25.22 5 n/a 0 0
50 7.74 100 12.61 100 n/a 75 80
25 3.87 95 6.30 100 n/a 95 95
12.5 1.93 95 3.15 90 n/a 95 100
6.25 0.97 95 1.58 80 n/a 95 95
Note: Dilbit CEWAF and mineral oil CEWAF were created with Corexit at a DOR of 1:5. Corexit 100% solution was created using the same nominal loading of Corexit as in the 100% CEWAF solutions. Concentrations of TPAH (µg/L) were measured for the 100% v/v solution for each treatment (except mineral oil CEWAF and Corexit), and concentrations in dilutions estimated from this measured value. Mineral oil WAF solutions were not shown because no significant mortalities were found in this treatment group.
41
Figure 1 Concentration-response relationship for juvenile topsmelt mortality after 96-h exposure to: a) diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent mortality of topsmelt vs. measured total polycyclic aromatic hydrocarbon (TPAH) concentrations [µg/L, log-scale]); b) dilbit chemically-enhanced water-accommodated fraction (CEWAF) (plotted as percent mortality of topsmelt vs. measured TPAH concentrations [µg/L, log-scale]); c) Corexit (plotted as percent mortality of topsmelt vs. nominal loading of Corexit [mg/L, log-scale]), and d) mineral oil CEWAF (plotted as percent mortality of topsmelt vs. nominal loading of Corexit [mg/L, log-scale]).
Qualitative, sublethal effects were also evident throughout the exposure. Many of
the fish in the dilbit WAF and dilbit CEWAF treatment groups exhibited darting and erratic
swimming behaviours at 24 and 96 h; all fish which remained alive in the dilbit WAF, dilbit
CEWAF and mineral oil CEWAF treatment groups exhibited this agitation. Interestingly,
in the Corexit-only treatments at 96 h, at the higher concentrations, fish swam extremely
slow and appeared lethargic.
42
Table 4 24-h and 96-h LC50 (Lethal Concentration to 50 percent of the population) values for diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) by Corexit, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L), and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) for topsmelt (Atherinops affinis) juveniles.
24-h LC50 (95% CI) 96-h LC50 (95% CI)
WAF or
CEWAF Treatment
Oil (% v/v)
Corexit (mg/L)
Measured
TPAH (µg/L)
Oil (%v/v)
Corexit (mg/L)
Measured
TPAH (µg/L)
Dilbit WAF
93.7 (72.1 -
121.8)a
14.5 (11.2 -
18.8)a
96.0 (73.0 -
126.3)a
14.9 (11.3 -
19.6)a
Dilbit CEWAF
93.9 (72.8 -
121.2)a
43.2 (30.1 –
50.1)
23.7 (18.3 -
30.6)a
71.4 (68.2 -
74.8)a
29.5 (28.2 –
30.9)
18.0 (17.2 -
18.9)a
Mineral oil
WAF > 100%b > 0.056 > 100%b > 0.056
Mineral oil CEWAF
66.5 (59.9 - 73.8)a
27.5 (24.8 – 30.5) n/a
58.9 (51.0 - 68.0)a
24.3 (21.1 – 28.0) n/a
Corexit > 41.3b n/a
25.6 (22.6 – 29.0)a n/a
Note: Oil refers to either dilbit or mineral oil, as listed in Treatment column. Oil WAF and dispersant-oil
CEWAF nominal loadings are expressed as %v/v oil loading, with a dispersant-oil ratio (DOR) of 1:5 in CEWAF solutions. Corexit nominal loadings are expressed as mg/L of original loading in solution. Measured TPAH concentrations are expressed as µg/L and are based on measurements taken at t = 0. a Estimated by Trimmed Spearman-Karber method b Estimated by Linear Interpolation
2.3.3. Effects of Dilbit and Corexit on Juvenile Mysids
Over the 48-h exposure period, dilbit CEWAF was the most acutely toxic to mysid
shrimp, with most shrimp in the 100% treatment group dying by 48 h (Table 5). The
concentration-response curves for dilbit WAF and dilbit CEWAF, expressed as measured
43
TPAH concentrations, can be found in Figures 2A and 2B, respectively. The LC50 based
on measured TPAH was 9.2 (7.8 - 10.8) µg/L TPAH for dilbit CEWAF, while for dilbit WAF
it was higher, at 15.6 (10.9 -17.0) µg/L TPAH (Table 6). Insufficient mortality occurred in
other treatment groups for LC50 value determinations, so LC10 values are given. Based
on nominal loadings of Corexit, Corexit was much more toxic in the dilbit CEWAF than
the mineral oil CEWAF or Corexit alone (Table 6). The 24-h LC50 values for dilbit WAF
and CEWAF are shown in Table A3.
Table 5 Mean percent survival of juvenile mysids (Mysidopsis bahia) based on nominal oil loading (%v/v) and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) following exposures to the water-accommodated fractions (WAF) and chemically-enhanced water-accommodated fractions (CEWAF) of dilbit and mineral oil, and Corexit alone for 48 h.
Dilbit WAF Dilbit CEWAF Mineral oil WAF Mineral oil
CEWAF Corexit
Nominal Oil
Loading (%v/v)
TPAH
(µg/L)
Mean Survival
(%)
TPAH
(µg/L)
Mean Survival
(%)
TPAH
(µg/L)
Mean Survival
(%)
TPAH
(µg/L)
Mean Survival
(%)
Mean Survival
(%v/v)
100 15.48 43 17.91 3 0.056 88 8.47 68 90
50 7.74 83 8.95 75 0.028 98 4.24 90 100
25 3.87 100 4.48 73 0.014 100 2.12 98 98
12.5 1.93 100 2.24 98 0.007 88 1.06 88 95
6.25 0.97 95 1.12 93 0.004 98 0.53 98 100
3.12 0.48 93 0.56 88 0.002 98 0.26 98 100
Note: Dilbit CEWAF and mineral oil CEWAF were created with Corexit at a DOR of 1:10. Corexit 100%
solution was created using the same nominal loading of Corexit as in the 100% CEWAF solutions, to allow comparison. Concentrations of TPAH (µg/L) were measured for the 100% v/v solution for each treatment, and dilutions were estimated from this measured value.
44
Figure 2 Concentration-response relationship for juvenile mysid mortality after 48 h of exposure to: a) diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent mortality of mysids vs. measured total polycyclic aromatic hydrocarbon (TPAH) concentrations [µg/L, log-scale]), and b) dilbit chemically-enhanced water-accommodated fraction (CEWAF) (plotted as percent mortality of mysids vs. measured TPAH concentrations [µg/L, log-scale]).
Qualitative observation of sublethal effects of these treatments to mysids were
also evident. The most common effect was lethargy and slow swimming speeds, which
was seen in the dilbit WAF, dilbit CEWAF, mineral oil CEWAF and Corexit treatments
compared to controls. Although mysids were moving extremely slowly at the two highest
concentrations of Corexit at 24 h and 48 h, almost no mortalities were observed in these
treatment groups (Table 5). For LC50s values based on both nominal loadings and
measured TPAHs, dilbit CEWAF was most toxic, followed by dilbit WAF.
45
Table 6 48-h LC10 and LC50 values for diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) by Corexit, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L), and measured total polycyclic aromatic hydrocarbon (TPAH) concentrations (µg/L) for mysid (Mysidopsis bahia) juveniles.
48-h LC10 (95% CI) 48-h LC50 (95% CI)
Oil WAF or Dispersant-Oil
CEWAF
Oil (%v/v)
Corexit (mg/L)
Measured TPAH (µg/L)
Oil (%v/v) Corexit (mg/L)
Measured TPAH (µg/L)
Dilbit WAF
87.8 (70.2
- 109.8)a
15.6 (10.9 -
17.0)a
Dilbit CEWAF 51.1 (43.3
- 60.2)a 10.6 (8.9 – 12.4)
9.2 (7.8 - 10.8)a
Mineral oil WAF
79.4 (42.2 - n/a)b
0.044 (0.024 – n/a)b
Mineral oil
CEWAF
50.0 (n/a –
62.0)b
10.3 (n/a –
12.8)
4.24 (n/a –
5.25)b
Corexit 20.6b n/a
Note: Oil refers to either dilbit or mineral oil, as listed in Treatment column. Oil WAF and dispersant-oil CEWAF nominal loadings are expressed as %v/v oil loading, with a dispersant-oil ratio (DOR) of 1:10 in CEWAF solutions. Corexit nominal loadings are expressed as mg/L of original loading in solution. Measured TPAH concentrations are expressed as µg/L and are based on measurements taken at t = 0. a Estimated by Trimmed Spearman-Karber method b Estimated by Linear Interpolation
2.3.4. Effects of Dilbit and Corexit on Echinoderm Fertilization
Nearly 100% of urchin eggs remained unfertilized in all treatments which
contained Corexit, including dilbit CEWAF, mineral oil CEWAF and Corexit alone (Table
A4). In each of these treatments, the lowest nominal loading of Corexit used was 0.322
mg/L (Table 7), indicating fertilization would be completely inhibited at this concentration
and above. The IC50 for dilbit WAF was 0.80 (0.52 – 1.00) µg/L TPAH, and a nominal oil
loading of 5.14 (3.36 – 6.48) %v/v oil. The concentration-response curve for dilbit WAF
in measured TPAH can be found in Figure 3. Interestingly, the mineral oil WAF solution
46
had similar IC50 based on nominal oil loading of 2.90 (2.33 - 24.74) % v/v oil. When
based on measured TPAH concentrations, the IC50 for mineral oil WAF was only 0.002
(0.001 – 0.015) µg/L TPAH, suggesting that a mechanism other than TPAH toxicity was
likely occurring.
Table 7 Concentrations inhibiting 20% fertilization (IC20) and 50% fertilization (IC50) after 20-min purple sea urchin (Strongylocentrous purpuratus) fertilization assay using diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically-enhanced water-accommodated fractions (CEWAF), and Corexit alone, expressed as nominal oil loading (%v/v), nominal loading of Corexit (mg/L) and measured total polycyclic aromatic hydrocarbon (TPAH) concentration (µg/L).
IC20 (95% CI) IC50 (95% CI)
Oil WAF or
Dispersant-Oil
CEWAF Oil (%v/v) Corexit (mg/L)
Measured TPAH (µg/L) Oil (%v/v)
Corexit (mg/L)
Measured TPAH (µg/L)
Dilbit WAF 0.93 (0.26 – 5.91)a
0.144
(0.040 – 9.146)a
5.14 (3.36 – 6.48)a
0.80 (0.52 – 1.00)a
Dilbit
CEWAF < 1.56% < 0.322 < 0.279 < 1.56 < 0.322 < 0.279
Mineral oil
WAF
1.64 (n/a –
2.55)a
0.009 (n/a
– 0.014)a
2.90 (2.33
- 24.74)a
0.002 (0.001 –
0.015)a
Mineral oil CEWAF < 1.56% < 0.322 < 1.32 < 1.56 < 0.322 < 1.32
Corexit < 0.322 n/a < 0.322 n/a
Note: Oil refers to either dilbit or mineral oil, as listed in Treatment column. Oil WAF and dispersant-oil
CEWAF nominal loadings are expressed as %v/v oil loading, with a dispersant-oil ratio (DOR) of 1:10 in CEWAF solutions. Corexit nominal loadings are expressed as mg/L of original loading in solution. Measured TPAH concentrations are expressed as µg/L and are based on measurements taken at t = 0. a Estimated by Linear Interpolation
47
Figure 3 Concentration-response relationship for percentage of unfertilized echinoderm eggs after 20-min fertilization assay with exposure to diluted bitumen (dilbit) water-accommodated fraction (WAF) (plotted as percent unfertilized vs. measured total polycyclic aromatic hydrocarbon [TPAH] concentrations [µg/L]).
2.3.5. Effects of Dilbit and Corexit on Behaviour in Spot Prawns
Mortality During the 7-d Exposure
Spot prawn mortality did not occur in numbers high enough in any treatment that
would allow toxicity parameters to be calculated (Table A5). In general, the highest
number of mortalities occurred in the dilbit WAF treatment group, but they appeared to
be random and across all concentrations.
Behavioural Tests
Antennule Flicking
There was no evidence of a difference in the mean number of antennule flicks
before and after liquid food exposure between any treatment group (p = 0.78), including
the control group. Flicking appeared to occur regardless of the introduction of the food
stimulus. In the 2-min period preceding the food introduction, just after the prawns had
been acclimated to clean water, there was a significant difference in the mean number of
antennule flicks between chemical groups (p = 0.011; Figure 4), but not between
concentrations (p = 0.066). The mean number of flicks in the control group was 148.3
48
(SE 22.0), and was similar to dilbit WAF, with a mean number of flicks of 151.7 (SE 12.8).
The number of mean antennule flicks was significantly lower in the Corexit (p = 0.0028)
and mineral oil CEWAF treatment groups (p = 0.0019) compared to dilbit WAF (Table
A6). The Corexit and mineral oil CEWAF treatment groups showed much less flicking
overall, regardless of concentration, with a mean number of flicks in Corexit treatments
of 85.3 (SE 13.8) and in the mineral oil CEWAF of 79.5 (SE 14.8), almost half of the
control group.
Figure 4 Graphical representation of mean antennule flicks (least squares mean) counted in the 2-min period after acclimatization to clean water, before the addition of the liquid food stimulus, between chemical groups including control, diluted bitumen (dilbit) water-accommodated fraction (WAF), dilbit chemically-enhanced water-accommodated fraction (CEWAF), Corexit, mineral oil WAF and mineral oil CEWAF calculated using a 2-factor completely randomized design (CRD) Analysis Of Variance (ANOVA). Error bars express standard error. Control data was not run in the 2-factor model but is shown here for comparison.
Pre-feeding and Feeding Behaviours
When grouping each chemical-concentration combination as one “treatment”
factor, the one-factor SAS binomial logistic model showed that there was no significant
effect of treatment on any of the pre-feeding or feeding behaviours (wiping [p = 0.952];
49
probing [p = 0.914], orienting [p = 0.862] and eating solid food [p = 0.860]). This means
that this model did not show evidence that any chemical or concentration was significantly
different from the control, and no concentration-response relationship could be detected.
Although a few of the unadjusted p-values were close to significant, there was no
statistically significant differences between treatments when p-values were adjusted for
multiple comparisons.
The two-factor SAS logistic model showed that there was no significant effect of
chemical, concentration, or any chemical-concentration interaction seen in antennule
wiping and dactyl probing (Table A7 and A8). For the third pre-feeding behaviour,
orienting toward the food stimulus, control prawns oriented towards food 60% of the time
(proportion 0.6). This behaviour showed evidence of a chemical effect (p = 0.056),
indicating indicated that one or more chemical groups showed an altered response of
fewer prawns orienting toward the food stimulus (Table A9). Table A10 shows the
differences in orienting between chemicals. Although a significant chemical effect was
found overall (p = 0.056), when the p-values were adjusted and corrected for multiple
comparisons, there was no statistically significant difference shown between chemical
groups.
The two-factor SAS logistic model showed that there was a chemical effect on the
final response, eating solid food (p = 0.037), indicating exposure to chemical, and not
concentration affects eating (Table A11). Table A12 shows the differences in eating
between chemical groups. Although a significant chemical effect was found (p = 0.037),
and the unadjusted p-values showed a significant difference between certain chemical
groups, when the p-values were adjusted and corrected for multiple comparisons, there
was no statistical significance shown between chemical groups. Figure 5 shows the
proportion of prawns for each chemical and concentration that ate solid food. Prawns at
nearly every concentration of Corexit (ranging from 0.103 to 10.3 mg/L; Figure 5c) did
not eat, with 26 out of 29 exposed prawns giving a “no” response to eating, while control
prawns ate 50% of the time. Prawns exposed to Corexit were generally more lethargic
than the other treatment groups.
50
Figure 5 Mean proportion of prawns at each chemical and concentration that ate solid food after the 7-d exposure, expressed as %v/v (ranging from 1.0%v/v to 100%v/v), for: a) diluted bitumen (dilbit) water-accommodated fraction (WAF); b) dilbit chemically-enhanced water-accommodated fraction (CEWAF); c) Corexit; d) mineral oil WAF, and e) mineral oil CEWAF. Error bars express standard error. Data for control prawns are expressed as 0.0 %v/v to allow visual expression on the log scale. Total number of prawns shown is N = 144.
51
2.4. Discussion
The current study demonstrates that exposing Pacific marine species to
environmentally realistic concentrations of dilbit and Corexit can cause acutely toxic
effects. It does not appear that dilbit and Corexit act synergistically, although dispersant
increases the concentration of potentially toxic hydrocarbons that are bioavailable to
marine organisms. Corexit in all treatments was extremely toxic to echinoderm
fertilization, causing nearly 100% of all eggs to remain unfertilized at concentrations as
low as 0.322 mg/L. Concentrations of dispersant measured after the DWH spill ranged
between 10 to 100 µg/L (Kujawinski et al., 2011), showing that the lowest concentrations
used here are environmentally relevant, especially during the initial application of
dispersant. The finding of Corexit causing complete inhibition of echinoderm fertilization
carries important implications because it demonstrates that the timing and location of a
dilbit release into the environment should determine whether the use of Corexit is
appropriate (Environment Canada, 2011). If a spill were to occur during spawning season
between January and May (Environment Canada, 2011), the application of Corexit could
cause complete inhibition of fertilization and impact future generations of echinoderms.
The reduction in antennule flicking for prawns exposed to Corexit and mineral oil
CEWAF suggest that Corexit may reduce the ability for olfactory perception. Previous
studies by Chen and Reese (2016) and Sriram et al. (2011) showed that Corexit can
impact neurotransmitter signaling and cause neurotoxicity. Sriram et al. (2011) found that
rats exposed to Corexit via whole-body inhalation exposure experienced disruptions in
olfactory signal transduction, axonal function and synaptic vesicle fusion and suggested
that Corexit may impact proper neurotransmitter signaling. Since the detection of
chemical stimuli is used by aquatic species to identify potential feeding sources, as well
as escape predators and locate mates (Rittschof, 1992), impacts on the olfactory
behaviours could severely impact crustacean populations. It is also possible that Corexit
reduced movement capabilities in prawns, as they also generally appeared more
lethargic than other treatment groups. Reducing the capability of prawns to detect
chemical stimuli, or move toward or away from stimuli, could adversely impact their ability
to survive in the natural environment.
52
The presence of PAHs in our mineral oil CEWAF solutions was surprising (6.21
and 8.47 µg/L TPAH in DORs of 1:20 and 1:10, respectively). This suggests that some
component of Corexit is contributing to the concentration of PAHs, particularly since
TPAH concentrations in mineral oil WAF were extremely low (0.056 µg/L). Even more
interesting was the fact that mineral oil CEWAFs showed an increase in C4-
dibenzothiophenes similar to dilbit CEWAF, with concentrations of 5440 and 7480 ng/L
for DOR 1:20 and 1:10, respectively. This suggests that perhaps Corexit itself is
composed of C4-dibenzothiophenes that remained unbound in the mineral oil CEWAF
solution. It is also a possibility that Corexit increases the solubility of C4-
dibenzothiophenes in the mineral oil itself, as mineral oil is a distilled petroleum base,
and although it is very refined, does contain a complex mixture of hydrocarbons such as
alkanes and cycloalkanes (Marinescu et al., 2004). This is an interesting observation,
and future research should include measured TPAH concentrations for all Corexit
solutions, as well as measurements of the individual components of Corexit itself.
Dilbit WAF toxicity was relatively low compared to toxicity found in Corexit
exposures. Dilbit WAF was only slightly toxic to mysid shrimp and topsmelt juveniles,
results similar to those found with crude oil. Dilbit WAF also showed no significant effect
on any behaviour in spot prawns. Although dilbit WAF was toxic to echinoderm
fertilization, mineral oil WAF was just as toxic at similar nominal oil loadings. This
suggests that the toxic effects of both dilbit and mineral oil may both be due to physical
interference, as opposed to chemically-induced toxicity from PAHs. This is very possible,
due to the inherently small size of echinoderm gametes that must undergo fertilization
and suggests that any amount of oil can inhibit echinoderm fertilization, even when toxic
PAHs are not present. Previous studies have showed that when preparing oil CEWAFs,
some oil remains in the water as particulate oil, especially when higher mixing energy is
used in CEWAF preparation (Singer et al., 2000; Adams et al., 2011). Although the mixing
energy was low in the current study, it is possible that some oil droplets may have
remained in the WAF and physically impaired the ability of the sperm to penetrate the
eggs. This suggests that both undispersed and dispersed dilbit in the environment could
have a significant impact on the ability of urchins to reproduce.
53
Most previous studies have expressed WAF and CEWAF toxicity by measured
TPAH concentrations. Since CEWAFs commonly have higher TPAH concentrations at
the same oil loading as WAFs, these studies conclude that synergistic toxicity is not
occurring, even though CEWAF may show toxicity at lower nominal oil loadings. Bejarano
et al. (2014) summarized the literature and reported that when toxicity values based on
nominal loading rates, 93% of the CEWAF toxicity values were lower than WAF values,
indicating a much greater toxicity of CEWAF. Although it is useful to compare toxicities
using measured TPAH concentrations, if dilbit is much more toxic with the addition of
Corexit, at the same nominal loadings, this is compelling evidence against the use of
Corexit as a remediation technique.
The toxicity of the mineral oil CEWAF treatment, particularly in topsmelt, suggests
that some Corexit remained bioavailable in solution and was not sequestered by oil
(Adams et al., 2014; Madison et al., 2017). Since a higher DOR (1:5) than tests with other
DORs (1:10 and 1:20) was used in this exposure, it is possible that there was insufficient
mineral oil to sequester the Corexit. Based on the nominal loading of Corexit, the mean
percent survival in both groups (mineral oil CEWAF and Corexit alone) were very similar,
supporting this hypothesis. Adams et al. (2014) found that mineral oil Nujol sequestered
Corexit at a DOR of 1:20 and rendered it non-toxic, but at higher DORs (1:10, 1:5 and
1:2.5) there was evidence of toxicity. Madison et al. (2015) also found that at a higher
DOR (1:10), mineral oil CEWAF showed evidence of toxic effects, suggesting that there
was insufficient oil to sequester the Corexit in solution. No studies to date that have
evaluated mineral oil CEWAF toxicity have measured total hydrocarbon or Corexit
concentrations in these solutions, so this hypothesis regarding unbound Corexit in
solution is untested (Adams et al., 2014; Madison et al., 2017). The results of the current
study clearly show some form of toxicity in the mineral oil CEWAF treatment, and future
experiments should further investigate this interaction by measuring PAH concentrations
in all treatments (including Corexit) as well as measuring concentrations of the individual
components of Corexit.
Corexit, both in combination with dilbit and alone, showed more evidence of
toxicity than dilbit WAF alone. This demonstrates that further evaluation should be done
before using the dispersant in marine waters along the Pacific coast of BC. Environment
54
Canada (2013) suggests that in all situations where Corexit may be applied, it is important
to ensure that a “net environmental benefit” will be achieved. A net environmental benefit
occurs when the increase in value of environmental or ecological services is gained
through remediation, minus the adverse environmental effects due to the action of
remediation (Efroymson et al., 2004). The toxicity of Corexit and dispersed dilbit should
be contrasted with the potential increase in biodegradation rates by microorganisms, as
well as the direct impact undispersed dilbit could have on the surrounding ecosystem.
The results from this research directly address a data gap in spill mitigation procedures
on the west coast of Canada, and it is suggested that toxicity testing be conducted with
additional Pacific marine species, and that measures of toxicity be expressed not only
just as TPAH, but as measured concentrations of the individual components of Corexit.
55
Chapter 3. Extended Discussion
3.1. Chemical Analyses
In the present study, measured TPAH concentration in the 100% dilbit WAF of
15.48 µg/L was similar to previous data from Madison et al. (2017) showing 20.3 µg/L
TPAH in 100% dilbit WAF. Our highest measured TPAH concentration in the 100% dilbit
CEWAF (DOR 1:5) at 25.22 µg/L TPAH is lower than shown in previous studies, as
Madison et al. (2017) measured 62.8 µg/L TPAH in their 100% dilbit CEWAF (DOR 1:10).
This is likely due to the fact that in the present study, Corexit dispersed the WAF only,
and not residual oil left on the surface. Our results simulate the potential effects of Corexit
as it mixes with oil that has already dispersed into the water column, which can occur in
the presence of high wave energy.
3.2. Toxicity Tests
3.2.1. Effects on Juvenile Topsmelt
Sublethal effects of oil and dispersant on topsmelt have been shown in previous
studies. However, these studies have expressed toxicity as a measure of total
hydrocarbon content (THC) so direct comparison to the current study cannot be made.
Anderson et al. (2009) found that crude oil dispersed by Corexit caused significant
inhibition of development and survival to hatching in topsmelt embryos, which they did
not see in WAF-only exposures, at their lowest tested CEWAF concentrations (23 and
25 mg/L). They also found cardiovascular and other abnormalities at all CEWAF
concentrations, with pericardial and yolk-sac edemas, and tube hearts with incomplete
circulation. Cardiovascular abnormalities were also shown in a study by Van Scoy et al.
(2012) which also found that crude oil dispersed by Corexit caused a reduction in egg
production in adult topsmelt (LC50 63.1 mg/L THC), not only immediately following the
exposure, but also after a recovery period of 5 months. This suggests significant impacts
56
on reproduction, which could have detrimental effects on populations for many
generations.
Previous studies have suggested that oil toxicity in other small aquatic species
like zooplankton occurs due to concentration-dependent narcosis (Barata et al., 2005;
Almeda et al., 2013). Nonpolar narcosis is a non-specific form of toxicity that occurs when
an organic compound causes a disturbance of phospholipids in biological membranes
(Tollefsen et al., 2012). Cohen et al. (2014) also found that incapacitation occurred in
copepods (Labidocera aestiva) in their oil CEWAF treatments occurred by concentration-
dependent narcosis. Aromatic hydrocarbons, NAs and other individual components of oil
have been shown to cause nonpolar narcosis (Headley and McMartin, 2004; Tollefsen et
al., 2012; Almeda et al., 2013; Lee et al., 2015). All potential toxic effects, both lethal and
sublethal, should be taken into affect in the decision of whether the use of chemical
dispersants is appropriate.
3.2.2. Effects on Juvenile Mysids
Barron et al. (2018) found that the 48-h LC50s for fresh CLB and Western
Canadian Select (WCS) dilbit were 14.6 and 23.0 µg/L TPAH, respectively, in juvenile
mysids. The LC50 value calculated for mysids for CLB dilbit in the present study was 15.6
(10.9 -17.0) µg/L TPAH, falling well within this range. In the current experiment, the
qualitative observation of reduced swimming speed of the mysids suggests some form of
sublethal toxicity. It is possible that the effects on swimming speed were caused by
cellular narcosis, as similar reductions of swimming speed in copepods have been
previously shown in both oil and dispersant exposure (Gardiner et al., 2013; Cohen et al.,
2014). Cohen et al. (2014) found that incapacitation occurred in copepods (Labidocera
aestiva) in their CEWAF treatments by concentration-dependent narcosis.
Concentration-dependent narcosis has also been reported in zooplankton after exposure
to crude oil (Barata et al., 2005; Almeda et al., 2013). Barron et al. (2018) also evaluated
growth in mysids for 7 d and found IC25 values of 5.72 and 7.82 µg/L TPAH for CLB and
WCS, respectively, which fall within the currently tested range of concentrations. Energy
for growth may be reallocated to detoxification or reparation of damage in the presence
of toxic PAHs.
57
Previous work by DeLorenzo et al. (2017) showed a range of LC50 values for
Corexit CEWAF (DOR of 1:20) for 3 different crude oils in the range of 1.16 – 17.6 µg/L
TPAH. The LC50 value in this study falls within this range found for traditional crude oils,
suggesting that dilbit CEWAF toxicity behaves similarly to other crude oil CEWAF toxicity,
and that previous literature for oil CEWAF toxicity may be applicable to dilbit. Since the
mysid is an EPA reference species, it also suggests our results can be cross-compared
with confidence. Bejarano et al. (2017) analyzed toxicity data for Arctic and non-Arctic
species and found that the mysid was generally more sensitive than the most sensitive
Arctic species to WAF of crude oil and CEWAF of crude oil and Corexit. They suggest
that species sensitivity distributions (SSDs) that include data from mysid toxicity testing
may therefore also be protective of most temperate species.
Literature toxicity values for Corexit fall between 32.2 and 42 mg/L for this species
(48-h LC50) (Hemmer et al., 2011; Word et al., 2014). In the current experiment, it was
beneficial to use a lower loading rate of Corexit, to allow for direct comparison between
the mineral oil CEWAF treatment and Corexit-alone treatment, as expressing toxicity as
a measure of TPAH concentration was not appropriate. Corexit was much more toxic
based on nominal loading in the dilbit CEWAF than the mineral oil CEWAF or Corexit
alone in mysids, which was likely due to its interaction with dilbit increasing soluble PAH
concentrations.
3.2.3. Effects on Echinoderm Fertilization
The observation that nearly 100% of urchin eggs remained unfertilized at all
concentrations of Corexit, dilbit CEWAF and mineral oil CEWAF, show that Corexit is
extremely harmful to fertilization success. It was not possible to determine whether
synergistic toxicity occurred in the echinoderm fertilization since Corexit was extremely
toxic at very low concentrations. Both dilbit WAF and mineral oil WAF were also toxic to
fertilization at low nominal oil loadings, suggesting that physical interference may be
preventing fertilization. The present results support the observations made following real-
world spill scenarios that oil is extremely harmful to echinoderms. After the Prestige
tanker spill, and even after the oil had visibly disappeared, embryogenesis was
completely inhibited in sea urchins exposed to samples from the spill site (Beiras and
58
Saco-Álvarez, 2006). This water also caused growth impairment in sea urchins, oyster
larvae and mussel embryos. Vashchenko (1980) found that hydrocarbon exposure
caused prominent delay, asynchronism and abnormal non-viable larvae in artificially
fertilized sea urchins, although the concentrations used were much higher and ranged
from 10-30 mg/L. The long-term effects of this sublethal exposure caused deformed sex
cells and high mortality of larvae. The present results also show that the addition of
Corexit in a spill scenario amplifies the adverse effects of oil. Corexit 9527, a similar
dispersant to Corexit 9500, caused significant delay in fertilization rates in several species
of sea urchins and fish, although concentrations used (1-10,000 mg/L) were much higher
than in the current experiment (Lonning and Hagstrom, 1976). A follow-up study by
Hagstrom and Lonning (1977) showed that 10 min of sperm exposure to Corexit 9527
reduced fertilization from 55% to 8% at much more environmentally-realistic
concentrations (up to 10 mg/L), similar to the highest nominal loading of Corexit in the
current study (20.65 mg/L). These results suggest that Corexit should not be used during
spawning season of echinoderms, as fertilization and survival in early life stages are
critical to the long-term survival of adult populations.
3.2.4. Effects on Spot Prawns
Antennule flicking appeared to occur regardless of the introduction of the food
stimulus, which is not surprising as flicking is used to detect all odorants present in the
water column, not just in the event of a new stimulus introduction (Lee and Meyers, 1996).
It is possible that prawns were already on high alert after being placed in a new, clean
tank after their 7-d exposure period, and that the introduction of the food stimulus was
irrelevant. When evaluating the mean number of antennule flicks between treatment
groups, it is interesting to note that the dilbit WAF treatment group showed a similar
number of mean antennule flicks as the control group, suggesting that the prawns were
not affected by dilbit exposure. In contrast, in both the Corexit and mineral oil CEWAF
treatment groups, the mean number of antennule flicks was almost half of the flicks
measured in the control group. This suggests that Corexit does have some effect on
prawn physiology, although exact mechanisms cannot be determined. Overall, the
behavioural tests employed here showed that Corexit likely exerted some toxic effect on
exposed prawns, and dilbit alone appeared to have no effect.
59
3.3. PAH Toxicity at Low Concentrations
Although measured TPAH concentrations of the 100%v/v dilbit WAF in the current
study was relatively low (15.48 µg/L), dilbit has been linked to changes in gene
expression and changes in morphology at much lower concentrations. Madison et al.
(2017) found that CLB dilbit increased CYP1a expression in Japanese medaka (O.
latipes) embryos exposed to concentrations as low as 0.4 µg/L TPAH. Alderman et al.
(2017a) also found that dilbit concentrations as low as 3.5 µg/L TPAH caused alterations
in cardiac morphology. Jones et al. (2017) found that crude oil CEWAF concentrations of
0.35 to 1.10 µg/L TPAH caused significant changes in gene expression in sheepshead
minnows (C. variegatus). Both undispersed and chemically-dispersed dilbit introduce
PAHs into the water, and both cases can have potentially detrimental effects on species
living in close proximity, even with only small amounts of PAHs.
3.4. Limitations
3.4.1. Measured Concentrations
Expressing toxicity as a measure of TPAH concentrations can be problematic, as
demonstrated when the mineral oil CEWAF was found to be toxic to topsmelt at much
lower TPAH concentrations than in dilbit CEWAF. Furthermore, there are some PAHs
included in TPAH measurements that are acutely, but not chronically, toxic, such as
naphthalenes (Adams et al., 2014). The opposite is also true; compounds like chrysenes
are more chronically toxic (Lin et al., 2015). There are also conflicting literature reports of
which hydrocarbon measurement best correlates with toxicity. Barron et al. (2018) found
that dilbit lethality correlated well with TPH concentrations, but not TPAH. Couillard et al.
(2005) found that CEWAF toxicity strongly correlated with TPAH concentrations in
mummichog embryos, while whole-body EROD activity correlated with only HMW PAH
concentrations. Additionally, each study typically chooses a different set of hydrocarbons
to analyze, which can make it difficult to accurately compare toxicity data between studies
(Couillard et al., 2005; Adams et al., 2014; Barron et al., 2018). Other measures of toxicity
should therefore be used to evaluate WAF and CEWAF toxicity, like naphthenic acid (NA)
measurements. The concentrations of NAs were not measured in the current experiment;
60
however, a recent study by Alderman et al. (2017a) measured about 15 µg/L NAs in their
dilbit WAF solutions that contained about 15 µg/L TPAH, which is likely similar to the
concentrations in solutions in the present study.
Corexit toxicity in previous studies has been expressed as measured DOSS
concentrations (Dasgupta and McElroy, 2017; Jones et al., 2017). As DOSS
concentrations were not measured in the present study, this presents a data gap which
may not allow accurate comparisons between CEWAF and Corexit only treatments.
Measuring the concentration of DOSS in Corexit treatments may have given more insight
into the high toxicity shown in the mineral oil CEWAF treatments, and also allowed for
the determination of which components of Corexit remain bioavailable in solution.
Furthermore, since the mineral oil CEWAF treatments in the current study showed
measured TPAH concentrations higher than expected, it would be beneficial to analyze
Corexit alone for the potential presence of TPAHs.
Additionally, calculating LC50 values from initial concentrations may
underestimate toxicity (Clark et al., 2001; DeLorenzo et al., 2017). DeLorenzo et al.
(2017) showed that TPAH concentrations in Corexit-dispersed crude oil were 43% and
16% of the initial concentrations after 24 and 96 h, respectively. Measurements should
therefore be taken at regular intervals over the exposure period. In addition, in real-world
spill scenarios, oil becomes diluted and also disperses from the spill site, suggesting that
static exposures may not accurately simulate a real-world spill (Environment Canada,
2013; King et al., 2017b; Madison et al., 2017).
3.4.2. Spot Prawn Behavioural Tests
Although the liquid food was injected at this same location for every prawn, each
prawn was not in the same location within the tank at the time the food was injected. This
resulted in some prawns being closer to the food stimulus than others, and some being
oriented in the opposite direction. In future research, it is suggested to begin the test with
some sort of barrier between the prawn and the location of the injected food, to ensure
all prawns begin at the same distance away from the food. Additionally, it would have
been beneficial to monitor the spot prawns post-exposure for a longer period of time to
61
determine any additional sublethal effects. Adams et al. (2014) found that oil CEWAF
mortalities in rainbow trout embryos occurred in the latter half of their 24-d exposure
period and showed BSD signs before death, while Corexit mortalities occurred within the
first 4 d of exposure. This suggests that oil CEWAF toxicity may take longer to occur, and
shorter monitoring periods may not show indication of all toxicity.
3.5. Applied Aspects of the Study
The current study demonstrates that exposing Pacific marine species to
environmentally realistic concentrations of dilbit and Corexit causes acutely toxic effects.
It does not appear that dilbit and Corexit act synergistically, although dispersant
increases the concentration of potentially toxic hydrocarbons that are bioavailable to
marine organisms. This was shown by the similarities between WAF and CEWAF LC50
values based on measured TPAHs. Although it is useful to compare toxicities using
measured TPAH concentrations, toxicities based on nominal loadings of oil and
dispersant may be more relevant to real-world spill scenarios. If dilbit is much more toxic
with the addition of Corexit, at the same nominal loadings, this is compelling evidence
against the use of Corexit as a remediation technique. Corexit in all treatments was
extremely toxic to echinoderm fertilization, causing nearly 100% of all eggs to remain
unfertilized in these treatments. The finding of Corexit causing complete inhibition of
echinoderm fertilization, even at the lowest concentrations, carries important implications
because it demonstrates that the timing and location of a dilbit release into the
environment should determine whether the use of Corexit is appropriate (Environment
Canada, 2011). The data suggest that Corexit, both alone and in combination with dilbit,
can be extremely toxic to Pacific marine organisms, particularly echinoderms.
Environment Canada (2013) suggests that in all situations where Corexit may be applied,
it is important to ensure that a “net environmental benefit” will be achieved. The toxicity
of Corexit and dispersed oil should be contrasted with the potential increase in
biodegradation rates by microorganisms, and also the direct impact undispersed dilbit
would have on the surrounding ecosystem.
This study reinforces the importance of timing of a spill into a marine environment.
As previously indicated, once dilbit forms oil particulate aggregates and sinks to the
62
bottom, remediation is extremely difficult. Dilbit within the sediments can cause lower
fitness in benthic fish species and cause subsequent decreases in fish populations (Dew
et al., 2015). It is possible that sediment resuspension may cause PAH concentrations to
fluctuate over time, as was exhibited when PAH increases in coastal waters were found
one year after the DWH oil spill (Allan et al., 2012). If a spill occurs during echinoderm
spawning, the effects could cause a significant impact on population dynamics.
Furthermore, the effects on spot prawns feeding behaviour and olfactory sense
perception could adversely impact their fitness and populations which has both
economical and ecological relevance. The results from this research directly address a
data gap in spill mitigation procedures on the west coast of Canada. Responders will be
better informed on appropriate spill responses depending on location and time of year.
3.6. Future Research
Alternatives to chemical dispersants should continue to be investigated. A recent
study by Salehi et al. (2017) suggests that a new hyperbranched polyethylenimine (HPEI)
dispersant‐like compound is equally as effective at dispersing crude oil as Corexit and
was much less acutely toxic to Daphnia magna and the Eastern oyster (Crassostrea
virginica). If products like this could be approved in Canada, this would eliminate the need
to use toxic chemical dispersants in the event of a spill.
Additionally, the impact of weathering and oil-sediment interactions on dilbit
dispersion and toxicity in the field requires further evaluation, particularly in real-world
coldwater scenarios. Future research should also be conducted using additional
coldwater species to understand how factors such as their metabolic rates, resilience and
trophic level interactions are impacted by dilbit and Corexit (Bejarano et al., 2017).
Assessing multiple species at different life stages would be important to classify risk at
different times of the year in which a spill may occur. One area where data is lacking is
on dilbit and Corexit toxicity to bivalves, particularly with the knowledge that TPAHs
accumulate in mussels (Environment Canada, 2013). It is suggested that toxicity testing
be conducted with additional Pacific marine species, and that measures of toxicity be
expressed not only just as TPAH, but as measured concentrations of the individual
components of Corexit.
63
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Appendix
Supplementary Tables
Table A1 Range of Corexit used for each test species, based on nominal loadings in µL/L and mg/L.
Test Species Range in µL/L Range in mg/L
Mysid 0.678 to 21.74 0.644 to 20.653
Echinoderm 0.339 to 21.74 0.322 to 20.653
Topsmelt 2.717 to 43.48 2.582 to 41.306
Spot Prawns 0.1087 to 10.87 0.1033 to 10.326
Table A2 Measured concentrations of C4-dibenzothiophenes in 100% water-accommodated fraction (WAF) and 100% chemically-enhanced water-accommodated fraction (CEWAF) solutions of diluted bitumen (dilbit) and mineral oil used in toxicity tests, at various dispersant-oil ratios (DOR).
Oil WAF or Dispersant-Oil CEWAF DOR
Measured concentrations of C4-dibenzothiophenes (ng/L)
Dilbit WAF 64.3
Dilbit CEWAF 1:20 2010
Dilbit CEWAF 1:10 4860
Dilbit CEWAF 1:05 10,800
Mineral oil 3.65
Mineral oil CEWAF 1:20 5440
Mineral oil CEWAF 1:10 7480
Mineral oil CEWAF 1:05 n/a
Note: Concentrations were not measured in mineral oil CEWAF solution at a DOR of 1:5.
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Table A3 LC50 values (24-h and 48-h) for diluted bitumen (dilbit) water-accommodated fraction (WAF) and chemically enhanced water-accommodated fraction (CEWAF) by Corexit, expressed as % v/v nominal oil loading, nominal loading of Corexit, and total polycyclic aromatic hydrocarbons (TPAH) for mysid (Mysidopsis bahia) juveniles.
24-h LC50 48-h LC50
Oil WAF or
Dispersant-Oil CEWAF Oil (%v/v)
Corexit (mg/L)
TPAH (µg/L) Oil (%v/v)
Corexit (mg/L) TPAH (µg/L)
Dilbit WAF > 100b > 15.476b 87.8 (70.2 -
109.8)a 15.59 (10.86 -
16.99)
Dilbit CEWAF 88.9 (76.3 -
103.4)a
18.36 (15.76 – 21.36)a
15.92 (13.66 - 18.52)a
51.1 (43.3 - 60.2)a
10.55 (8.94 – 12.43)a
9.15 (7.75 - 10.78)
Note: Oil WAF and dispersant-oil CEWAF nominal loadings are expressed as %v/v oil loading, with a
dispersant-oil ratio (DOR) of 1:10 in CEWAF solutions. Corexit nominal loadings are expressed as mg/L of original loading in solution. Measured TPAH concentrations are expressed as µg/L and are based on measurements taken at t = 0. a Estimated by Trimmed Spearman-Karber method b Estimated by Linear Interpolation
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Table A4 Mean percent fertilization of purple sea urchins (Strongylocentrous purpuratus) following 20-min exposure to diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) and Corexit.
Oil WAF or Dispersant-Oil CEWAF
Nominal Loading (%v/v) Mean % Fertilized Mean % Unfertilized
Dilbit WAF 100 0 100
50 0 100
25 6 94
12.5 21 79
6.25 33 67
3.12 75 25
1.56 41 59
Dilbit CEWAF 100 0 100
50 0 100
25 0 100
12.5 0 100
6.25 0 100
3.12 0 100
1.56 1 99
Mineral oil WAF 100 0 100
50 9 91
25 14 86
12.5 37 63
6.25 37 63
3.12 37 63
1.56 67 33
Mineral Oil CEWAF 100 0 100
50 0 100
25 0 100
12.5 0 100
6.25 0 100
3.12 0 100
1.56 0 100
Corexit 100 0 100
50 0 100
25 0 100
12.5 0 100
6.25 0 100
3.12 0 100
1.56 7 93
81
Table A5 Number of spot prawn (Pandalus platyceros) deaths that occurred in each 7-d chemical treatment.
Oil WAF or Dispersant-Oil CEWAF Number of Deaths Concentrations
Control 0 n/a
Dilbit WAF 8 1%, 10%, 32%, 32%,
32%, 32%, 100%, 100%
Dilbit CEWAF 3 1%, 10%, 100%
Mineral oil WAF 0 n/a
Mineral oil CEWAF 3 10%, 10%, 10%, 10%
Corexit 4 3.2%, 10%, 10%
Table A6 Tukey’s Multiple Comparisons procedure for mean flicks counted in the 2-min period after acclimatization to clean water between treatment groups using a 2-factor completely randomized design (CRD) Analysis Of Variance (ANOVA).
Level Least Sq Mean Std Error
DILBIT A 151.7 12.8
DIL+COR A B 117.6 11.7 MINOIL A B 109.0 14.8 COR B 85.3 13.8
MIN+COR B 79.5 14.8
Note: Levels not connected by same letter are significantly different.
Table A7 Analysis of effects for chemical, concentration, and each chemical x concentration combination for shrimp wiping antennules after the 7-d exposure.
Type 3 Analysis of Effects
Effect DF Wald Chi-Square Pr > ChiSq (P-value)
Chemical 4 4.9382 0.2937
Concentration 4 4.3318 0.3630
Chem*Concentration 16 5.2582 0.9943
82
Table A8 Analysis of effects for chemical, concentration, and each chemical x concentration combination for shrimp dactyl probing after the 7-d exposure.
Type 3 Analysis of Effects
Effect DF Wald Chi-Square Pr > ChiSq (P-value)
Chemical 4 6.7599 0.1491
Concentration 4 3.1009 0.5411
Chem*Concentration 16 8.7251 0.9243
Table A9 Analysis of effects for chemical, concentration, and each chemical x concentration combination for shrimp orienting toward food after the 7-d exposure.
Type 3 Analysis of Effects
Effect DF Wald Chi-Square Pr > ChiSq (P-value)
Chemical 4 9.1929 0.0565
Concentration 4 2.0286 0.7305
Chem*Concentration 16 8.3774 0.9368
Table A10 SAS Logistic Model output for prawns orienting toward food after 7-d exposure to chemicals, with standard error, unadjusted p-values and p-values adjusted for multiple comparisons.
Chemical Chemical Standard Error Unadjusted P-
values Adjusted P-
values
DILBIT WAF DILBIT CEWAF 0.6925 0.1069 0.4893
DILBIT WAF COREXIT 0.7695 0.0381 0.2314
DILBIT WAF MINERAL OIL 0.6024 0.7783 0.9986
DILBIT WAF MINERAL OIL CEWAF
0.6275 0.8516 0.9997
DILBIT CEWAF COREXIT 0.7947 0.5466 0.9747
DILBIT CEWAF MINERAL OIL 0.6343 0.0426 0.2525
DILBIT CEWAF MINERAL OIL
CEWAF
0.6581 0.1289 0.5504
COREXIT MINERAL OIL 0.7175 0.0139 0.0998
COREXIT MINERAL OIL CEWAF
0.7387 0.0454 0.2652
MINERAL OIL MINERAL OIL CEWAF
0.5626 0.6099 0.9864
83
Table A11 Analysis of effects for chemical, concentration, and each chemical x concentration combination for shrimp eating solid food after the 7-d exposure.
Type 3 Analysis of Effects
Effect DF Wald Chi-Square Pr > ChiSq (P-value)
Chemical 4 10.21 0.037
Concentration 4 2.20 0.700
Chem*Concentration 16 8.72 0.925
Table A12 SAS Logistic Model output for prawns eating solid food after 7-d exposure to chemicals diluted bitumen (dilbit) and mineral oil water-accommodated fractions (WAF) and chemically enhanced water-accommodated fractions (CEWAF) and Corexit, with standard error, unadjusted p-values and p-values adjusted for multiple comparisons.
Chemical Chemical Standard Error Unadjusted P-
values Adjusted P-
values
DILBIT WAF DILBIT CEWAF 0.692 0.107 0.489
DILBIT WAF COREXIT 0.808 0.022 0.149
DILBIT WAF MINERAL OIL 0.628 0.852 1.000
DILBIT WAF MINERAL OIL CEWAF
0.602 0.778 0.999
DILBIT CEWAF COREXIT 0.832 0.379 0.904
DILBIT CEWAF MINERAL OIL 0.658 0.129 0.550
DILBIT CEWAF MINERAL OIL
CEWAF
0.634 0.043 0.252
COREXIT MINERAL OIL 0.779 0.026 0.171
COREXIT MINERAL OIL CEWAF
0.759 0.008 0.060
MINERAL OIL MINERAL OIL CEWAF
0.563 0.610 0.986