Isotopic and microbial evidence for biodegradation of diluted bitumen in the unsaturated zone.

The oil sands region in Western Canada is one of the world's largest proven oil reserves. To facilitate pipeline transport, highly viscous oil sands bitumen is blended with lighter hydrocarbon fractions to produce diluted bitumen (dilbit). Anticipated increases in dilbit production and transport raise the risk of inland spills. To understand the behaviour of dilbit in the unsaturated or vadose zone following a surface spill, we ran parallel dilbit and conventional heavy crude exposures, along with an untreated control, using large soil-filled columns over 104 days. Phospholipid fatty acids (PLFAs), biomarkers for the active microbial population, were extracted from column soil cores. Stable carbon isotope contents (δ13C) of individual PLFAs and radiocarbon contents (Δ14C) of bulk PLFAs were characterized over the course of the experiment. The Δ14CPLFA values in soils impacted by dilbit (-221.1 to -54.7‰) and conventional heavy crude (-259.4 to -97.9‰) indicated similar levels of microbial uptake of fossil carbon. In contrast, Δ14CPLFA values in the control column (-46.1 to +53.7‰) reflected assimilation of more recently fixed organic carbon. Sequencing of 16S ribosomal RNA genes extracted from soil cores revealed a significant increase in the relative abundance of Polaromonas, a known hydrocarbon-degrader, following exposure to both types of oil. This study demonstrates that in the first several months following a surface spill, dilbit has a similar potential for biodegradation by a native shallow subsurface microbial community as conventional heavy crude oil.

Early developmental toxicity of Atlantic salmon exposed to conventional and unconventional oils.

Atlantic salmon is an important species for Canadian culture and economy and its importance extends beyond Canada to Scandinavia and Western Europe. However, it is a vulnerable species facing decline due to habitat contamination and destruction. Existing and new Canadian pipeline projects pose a threat to salmonid habitat. The effects of diluted bitumen (dilbit), the main oil circulating in pipelines, are less studied than those of conventional oils, especially during the critical early embryonic developmental stage occurring in freshwater ecosystems. Therefore, this study aimed to compare the effects of water-accommodated fractions (WAF) of the Clearwater McMurray dilbit and the Lloydminster Heavy conventional oil on Atlantic salmon embryos exposed either from fertilization or from eyed stage. The dilbit contained the highest concentrations of low molecular weight (LMW) compounds (including BTEX and C6-C10), while the conventional oil contained the highest concentrations of PAHs. The Clearwater dilbit caused a higher percentage of mortality and malformations than the conventional oil at similar WAF concentrations. In addition, the embryos exposed from fertilization suffered a higher mortality rate, more developmental delays, and malformations than embryos exposed from the eyed stage, suggesting that early development is the most sensitive developmental stage to oil exposure. Gene expression and enzymatic activity of the detoxification phase I and II enzymes (CYP1A and GST) were measured. Data showed increases in both cyp1a expression and GST activity with increasing WAF concentrations, while gst expression was not affected by the exposures. Also, gene expression of proteins involved in the biotransformation of vitamin A and DNA damage repair were modified by the oil exposures. Overall, this study indicates that Atlantic salmon is mostly affected by oil exposure at the beginning of its development, during which embryos accumulate deformities that may impact their survival at later life stages.

Comparative developmental toxicity of conventional oils and diluted bitumen on early life stages of the rainbow trout (Oncorhynchus mykiss).

Petroleum hydrocarbons are widely used and transported, increasing the risks of spills to the environment. Although conventional oils are the most commonly produced, the production of unconventional oils (i.e. diluted bitumen or dilbit) is increasing. In this study, we compared the effects of conventional oils (Arabian Light and Lloydminster) and dilbits (Bluesky and Clearwater) on early life stages of a salmonid. To this end, aqueous fractions (WAF: water accommodated fraction) of these oils were extracted using mountain spring water. Rainbow trout (Oncorhynchus mykiss) larvae were exposed to 10 and 50% dilutions of these WAFs from hatching (340 DD; degree days) until yolk sac resorption (541 DD). Exposure to WAFs increased skeletal malformations (both dilbits) and hemorrhage (both conventional oils and Bluesky) and decreased head growth (Arabian Light). In addition, increases in EROD activity and DNA damage were measured for all oils and an increase in cyp1a gene expression was measured for Arabian Light, Bluesky and Clearwater. The PAH and C10C50 concentrations were positively correlated to total larval EROD activity, whereas concentrations of total hydrocarbons, VOCs, PAHs, and C10C50 were positively correlated to cyp1a expression. Total hydrocarbon, VOC, and C10C50 concentrations were also negatively correlated to larval growth. This study supports that petroleum hydrocarbons are toxic to early developmental stages of rainbow trout and show that their degree and spectrum of toxicity depends on their chemical composition.

Lethal and sublethal effects of diluted bitumen and conventional oil on fathead minnow (Pimephales promelas) larvae exposed during their early development.

The increasing extraction of bitumen from the oil sands region in Canada is creating a need for transport. Spills from current and projected pipelines represent a significant environmental risk, especially for freshwater ecosystems. The toxicity of diluted bitumen (dilbit) on freshwater fish is largely unknown. This study assessed the toxicity of two dilbits (Clearwater McMurray and Bluesky) and compared their toxicity to a conventional oil (Lloydminster Heavy) on fathead minnow (Pimephales promelas) larvae. Larvae were exposed to various concentrations of the water-accommodated fraction (WAF) of the oils during 7 days from hatching. In the WAF treatments, the concentrations of volatile organic compounds (VOCs), including benzene, toluene, ethylbenzene, xylene (BTEX), hydrocarbons containing 6 to 10 carbon atoms (C6-10), and polycyclic aromatic hydrocarbons (PAHs) and their alkylated forms were measured. Both dilbits contained higher concentrations of light components, while the conventional oil contained the highest concentrations of PAHs and alkylated PAHs. The Clearwater McMurray dilbit induced a higher mortality, with a maximum of 65.3%, while the other oils induced a similar mortality up to 16.5% and 18.6% for Lloydminster and for Bluesky, respectively. All three oils induced an increase in gene expression of the phase I detoxification enzyme (cyp1a) with increasing total hydrocarbon concentrations. All three exposures induced a similar increase in glutathione S-transferase (GST) activity, but no change in gst gene expression. For the Bluesky and Lloydminster exposures, an increase in malondialdehyde concentration was also observed, suggesting a rate limiting capacity of GST and phase II enzymes to perform the biotransformation of the PAH metabolites. Overall, this study brings new insights on the toxicity of dilbits in comparison to conventional oils on early life stages of North American freshwater fish and demonstrated that dilbits can be more toxic than conventional oils, depending on their composition and diluent proportions.

Fate and Fathead Minnow Embryotoxicity of Weathering Crude Oil in a Pilot-Scale Spill Tank.

For several years now, the Natural Resources Canada research facility at CanmetENERGY Devon (AB, Canada) has been performing experiments in a pilot-scale spill tank using 1200 L of river water to examine the physical and chemical behaviors of various crude oil/water mixtures under varying water temperature regimes. Because oil toxicity can be modulated by weathering of the petroleum products, the present study aimed to assess changes in fish embryotoxicity to mixed sweet blend crude oil as it weathered at air and water temperatures of 14 °C and 15 °C, respectively, for 28 d. The physicochemical behavior of the oil was also monitored. Water samples were taken from the spill tank 5 times during the 28-d experiment on days 1, 6, 14, 21, and 28 and were used to perform toxicity exposures using fathead minnow embryos (Pimephales promelas). For each water sampling day, newly fertilized embryos were exposed to a serial dilution of the spill tank water, noncontaminated river water (used in the spill tank), and a reconstituted water laboratory control. Embryos were raised until hatching. Although mortality was not significantly altered by the oil contamination over the time period, malformation occurrence and severity showed concentration-dependent responses to all contaminated water collected. The results suggest that days 14, 21, and 28 were the most toxic time periods for the fish embryos, which corresponded to increasing concentrations of unidentified oxidized organic compounds detected by a quadropole-time-of-flight system. The present study highlights a novel area for oil research, which could help us to better understand the toxicity associated with oil weathering for aquatic species. Environ Toxicol Chem 2021;40:127-138. © 2020 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Natural Resources Canada.

Effects of Environmentally Relevant Residual Levels of Diluted Bitumen on Wild Fathead Minnows (Pimephales promelas).

Transportation of crude oil across North America's boreal ecozone creates the potential for spills in freshwater where less is known about the sensitivity of resident fish than for marine systems. The sensitivity of wild fathead minnows (FHM) to residual concentrations (ppb range) of the water accommodated fraction (WAF) of diluted bitumen (dilbit) was assessed by exposing them for 21 days followed by a 14 days depuration. Target concentrations were well below detection limits for GC-MS, but were estimated by dilution factor (1:100,000 and 1:1,000,000 WAF:water) to contain less than 0.0003 µg/L of polycyclic aromatic compounds. Confinement and handling stress caused by transfer of wild fish into tanks much smaller than their natural range resulted in mortality and lower body condition among all groups, but interactive effects of oil exposures still resulted in females with smaller cortical alveolar oocytes, and males with larger testicular lobe lumen sizes. Additional studies examining the compounded effects of stress and environmentally relevant oil exposures in wild fishes are needed.

Effects on Trout Alevins of Chronic Exposures to Chemically Dispersed Access Western Blend and Cold Lake Blend Diluted Bitumens.

The present study assessed the chronic toxicity of 2 chemically enhanced water accommodated fractions (CEWAFs) of diluted bitumens (dilbits), Access Western Blend (AWB) and Cold Lake Blend (CLB), to rainbow trout alevins. Chemical dispersion was used to overcome the resistance to dispersion of dilbits and to generate test solutions that contained more and smaller oil droplets for increased partitioning of petroleum hydrocarbons into water. Test solutions were characterized by fluorescence spectroscopy, a rapid and inexpensive analytical tool to compare toxicity endpoints measured by fluorescence (total petroleum hydrocarbons measured by fluorescence [TPH-F]). Cumulative mortality and the prevalence and severity of malformations increased following exposure of alevins to dispersed dilbits. Toxicity curves overlapped for AWB and CLB when expressed as TPH-F and 22- to 24-d median lethal and effect concentrations ranged from 0.36 to 1.5 mg/L. Gene expression in alevins was also altered following exposure to dispersed dilbit, with relative cytochrome P450-1A mRNA levels increasing up to 170-fold for AWB and up to 240-fold for CLB. Access Western Blend and CLB caused similar toxicity to rainbow trout alevins as light to medium conventional crude oils, and rainbow trout alevins were more sensitive than yellow perch, Japanese medaka, and fathead minnow embryos exposed to dispersed AWB and CLB. The present study is the first to assess the embryotoxicity of dilbits to a Canadian freshwater salmonid species. Environ Toxicol Chem 2020;39:1620-1633. © 2020 SETAC.

Impact of crude oil and the dispersant Corexit™ EC9500A on capelin (Mallotus villosus) embryo development.

Marine food webs are particularly vulnerable to oil spills if keystone species are impacted. To quantify lethal and sublethal toxicity in a key Holarctic forage fish, capelin embryos were exposed to Hibernia crude oil water accommodated fraction (WAF) produced at an oil-to-water ratio of 1:9 (v:v) and chemically-enhanced WAF (CEWAF) produced with the dispersant Corexit™ EC9500A at a dispersant-to-oil ratio of 1:10 (CEWAF H) or 1:50 (CEWAF L). Corexit alone yielded similar embryotoxicity to CEWAF. 10% CEWAF H, with total polycyclic aromatic hydrocarbons of 99.2 µg/L, decreased embryo survival following 10 h of exposure, while continual exposed to 1% CEWAF L decreased hatching and heart rates. Concentrations down to 0.1% CEWAF L increased in a dose-dependent manner the transcript level of cytochrome P4501a1 (cyp1a1) in hatched larvae. These data indicate that embryo-larval survival of capelin is likely at risk if an oil spill coincides in space and time with spawning.

An Embryonic Field of Study: The Aquatic Fate and Toxicity of Diluted Bitumen.

Canada has experienced a significant increase in the transport of diluted bitumen (dilbit), a predominant oil sands product that combines bitumen with diluents derived from oil-gas condensates and other proprietary compounds. The proportion of diluent and the chemical composition of dilbit vary to meet seasonal transport requirements. While the toxic effects of a variety of crude and refined oils are well-studied, the toxicity of dilbit to aquatic species is less well known. This focused review summarizes dilbit production, chemistry, and the few data on toxicity to aquatic species. These data suggest that un-weathered dilbit would cause effects on fish equivalent to those of conventional oils, but its toxicity may be lower, depending on interactions among test conditions, the behavior of dilbit added to water and the species tested.

Cold Lake Blend diluted bitumen toxicity to the early development of Japanese medaka.

Diluted bitumen (dilbit) from Alberta oil sands (Canada) is transported across major continental watersheds, yet little is known about its toxicity to fish if spilled into aquatic environments. The toxicity of Cold Lake (CLB) dilbit was assessed for medaka embryos (Oryzias latipes) exposed to water accommodated fractions (WAF) and chemically-enhanced WAF (CEWAF) using Corexit®EC9500A as dispersant. The effects of CLB toxicity were similar to conventional crude oils and Access Western Blend (AWB) dilbit. The prevalence of malformations and cyp1a mRNA synthesis in hatched fish increased monotonically with concentration during WAF and CEWAF treatments and provided a novel indicator of dilbit PAH toxicity. Apart from nfe2 (an antioxidant transcription factor), there were no statistically significant monotonic exposure-responses of ahr, arnt2, cat, sod, gpx, gst, gsr, g6pdh, p53, and hsp70 transcripts at total polycyclic aromatic hydrocarbons (TPAH) concentrations bracketing EC50s for embryotoxicity (WAF ≅ 3 µg/L; CEWAF ≅ 0.1 µg/L TPAH). Based on measured TPAH concentrations in exposure test solutions, CLB dilbit was 6-10 fold more toxic to medaka than AWB during chronic exposures. Lack of direct monotonic gene transcription responses to increasing oil concentrations during exposures that were embryotoxic suggests that the capacity of the oxidative stress response is limited in earlier lifestages or that differences exist among species in mechanisms of toxicity. This study provides a comparative framework for identifying suitable biomarkers and toxicity methods for those fish species in sensitive lifestages at highest risk of Canadian oil sands dilbit exposure following a spill in the freshwater environment.