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

iii

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

v

Dedication

To my parents, who have always supported me through

my many changes in life direction

vi

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.

vii

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

viii

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

ix

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

x

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

xi

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

xii

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

1

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

2

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.

4

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