Aquatic Toxicology 165 (2015) 222–230
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Diluted bitumen causes deformities and molecular responses indicative of oxidative stress in Japanese medaka embryos Barry N. Madison a , P.V. Hodson b , V.S. Langlois a,b,∗ a b
Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Canada School of Environmental Studies, Queen’s University, Kingston, ON, Canada
a r t i c l e
i n f o
Article history: Received 4 May 2015 Received in revised form 8 June 2015 Accepted 10 June 2015 Available online 17 June 2015 Keywords: Diluted bitumen Dilbit Water accommodated fraction Polycyclic aromatic hydrocarbons PAHs Blue sac disease Oxidative stress Embryotoxicity Fish
a b s t r a c t This study characterized the toxicity and physiological effects of unweathered diluted bitumen (Access Western Blend dilbit; AWB) to fish. Embryos of Japanese medaka (Oryzias latipes) were exposed for 17 days to dilutions of physically-dispersed (water accommodated fraction; WAF) and chemically-dispersed (chemically-enhanced WAF; CEWAF) dilbit. AWB dilbit exposure was not lethal to medaka, but resulted in a high prevalence of blue sac disease (BSD), impaired development, and abnormal or un-inflated swim bladders at hatch. Physiological effects were indicated by the relative mRNA levels of key genes associated with, among others, cell cycling and the response to mutations (p53), xenobiotic metabolism (ahr, arnt2), phase I (cyp1a) and II processes associated with oxidative stress (cat, g6pdh, hsp70, gst, gpx, gsr, nfe2, and sod). AWB dilbit treatment increased p53 and cyp1a transcript levels (1.5-fold and >15-fold, respectively), with significant, but less pronounced changes in indicators of oxidative stress and metabolism. The exposure-related changes in embryotoxicity and mRNA synthesis were consistent with metabolism of polycyclic aromatic hydrocarbons (PAHs) to reactive and toxic metabolites. Medaka embryos responded similarly to WAF and CEWAF treatments, but CEWAF was about 100 times more efficient in delivering toxic concentrations of PAHs. The toxicity of chemically-dispersed nujol, a non-toxic mineral oil used as an experimental control, suggested that a portion of the observed effects of AWB could be attributed to excess dispersant in solution. This first study of the physiological effects of dilbit toxicity to fish embryos provides a baseline to compare toxicity between dilbit and conventional crude oils, and the groundwork for the development of molecular biomarkers of the sensitivity and level of risk of native Canadian fish species to dilbit exposure. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The growing pipeline and railway shipment of diluted bitumen (dilbit) products from Canada’s oil sands industries has generated public concern about the effects of potential spills on aquatic ecosystems (Dupuis and Ucan-Marin, 2014). Of particular importance are the effects on the early developmental stages of fish species, which seem particularly vulnerable due to their inability to avoid oil exposure, and their high sensitivity to toxicity (Carls et al., 2000). The embryo toxicity of crude and refined oils is well established (e.g., Martin et al., 2014; Schein et al., 2009; Carls et al.,
∗ Corresponding author at: Canada Research Chair (CRC) in Ecotoxicogenomics and Endocrine Disruption, Environmental Toxicology & Endocrinology (ETE) Laboratory, Royal Military College of Canada (RMCC), Department of Chemistry & Chemical Engineering, 2526 Sawyer (Bldg 69), 11 General Crerar Cr. Kingston, ON K7K 7B4, Canada. Fax: 1 613 542 9489. E-mail address: [email protected] (V.S. Langlois). http://dx.doi.org/10.1016/j.aquatox.2015.06.006 0166-445X/© 2015 Elsevier B.V. All rights reserved.
2000) and has been linked to the concentrations of 3- to 5-ringed alkyl polycyclic aromatic hydrocarbons (alkyl PAHs) that can partition from oil droplets into water at toxic concentrations (Adams et al., 2014b; Bornstein et al., 2014; Hodson et al., 2007). Phenotypic responses include an exposure-dependent increase in the prevalence of signs of blue-sac disease (BSD; including edemas, heart malformations, craniofacial malformations, and fin erosion) and impairment of development (He et al., 2012; Farwell et al., 2006). Physiologically, oil exposure elevates the synthesis and total activity of cytochrome P450 enzymes, specifically CYP1A (Kim et al., 2014, 2013; Gagné et al., 2012; He et al., 2012; Mu et al., 2012; Carney et al., 2008), the synthesis of the aryl-hydrocarbon receptor protein (AHR) (Incardona et al., 2011a, 2006; Scott et al., 2011; Hanno et al., 2010), and the synthesis and activity of phase II biotransformation enzymes regulating oxidative stress (Olsvik et al., 2011). In contrast, the toxicity of dilbit to fish embryos has not been previously reported, although bituminous oil sands were quite toxic to embryos of common white sucker (Catostomus commer-
B.N. Madison et al. / Aquatic Toxicology 165 (2015) 222–230
soni; Colavecchia et al., 2006). If dilbit embryo toxicity is due solely to its PAH constituents, the toxic effects and overall toxicity should correspond to those of conventional oils. While the concentrations of total PAH in dilbit are lower than in conventional crudes, they include 3- to 5-ringed alkyl PAH at similar concentrations. The 2ringed naphthalenes that comprise the most abundant PAH in crude oil are at substantially lower concentrations in dilbit because the parent bitumen represents highly weathered oil (GOC, 2013). Raw bitumen is too viscous to flow at room temperature in a pipeline and will sink in water because its density may exceed 1.0. However, when mixing raw bitumen with diluents, such as oil–gas condensate, dilbit becomes highly volatile and its viscosity and density are closer to those of conventional medium crude oils. Because many components of oil–gas condensates can evaporate quickly from dilbit, the physical properties, environmental fate and behavior, and susceptibility to chemical dispersion and sinking of dilbit change rapidly after a spill (King et al., 2014; Fingas, 2012). While the relative embryo toxicity of dilbit might be inferred from its PAH content, predicting toxicity is also complicated by variations in the composition of the raw bitumen, depending on its source and method of production. There are also variations in the composition of oil–gas condensates, depending on their sources, and in the proportions added seasonally to dilbit to ensure viscosities suitable for transport (Crosby et al., 2013). Because of the marked uncertainties in the predicted environmental fate, behavior, and chemical constituents of spilled dilbit, we initiated a program of research to measure the embryo toxicity of dilbit to a variety of marine and freshwater fish species. In addition to comparing relative toxicity among dilbits and between dilbit and conventional crude oils, our objectives include: identifying molecular responses of embryos unique to dilbit exposure; the characterization of genes that could be used as biomarkers of dilbit toxicity in assessments of spill impacts; associating toxicity with the chemical composition of dilbit; and identifying any properties of dilbit that could bias measurements of toxicity. This is the first report of dilbit chronic toxicity to fish embryos and the first to describe changes to gene regulation of embryos in response to dilbit exposure.
223
2.3. Embryo collection Embryo clutches were stripped twice from a population of sexually mature females (n = 60) over four days, totaling 520 embryos. The embryos were held in plastic Petri dishes immersed in an embryo rearing solution (ERS), containing: 1000 mg/L NaCl, 40 mg/L CaCl2 , 30 mg/L KCl, and 163 mg/L MgSO4 . Healthy, fertilized embryos (< 24 h post fertilization) were randomly assigned to treatment groups. 2.4. Preparation of dilbit exposure solutions We assessed the toxicity of the water accommodated fraction (WAF) and chemically-enhanced water accommodated fraction (CEWAF) of unweathered AWB on embryos. WAF and CEWAF were prepared following the methods of Adams et al. (2014a,b) and Martin et al. (2014) as modified from Singer et al. (2000). Briefly, WAF was prepared by adding dilbit to water at a ratio of 1:9, stirring gently for 18 h and allowing the solution to settle for 1 h. CEWAF was prepared by the same procedure, but after 18 h, Corexit® 9500A (oil dispersant; ECOLAB/NALCO, Illinois, USA) was added to the surface layer of residual oil at a dispersant-to-oil ratio of 1:10. The CEWAF was stirred for an additional hour and allowed to settle for 1 h, after which surface oil layers were removed and stock solutions of WAF and CEWAF without visible surface oil were transferred to 20 mL vials. The effectiveness of dilbit dispersion seemed more variable than in previous experiments with conventional crude oil, based on the apparent turbidity of the final CEWAF stock and the behavior of the floating dilbit when the dispersant was added after 18 h of stirring. Stock solutions were prepared fresh daily and diluted for static renewal exposures in clean exposure jars. Used exposure jars were cleaned with methanol-soaked wipes to remove residual oil, rinsed with de-chlorinated water, and dried for re-use the following day. Nominal WAF and CEWAF concentrations were 0.32, 1.0, 3.2, 10 and 32% v/v, and 0.0001, 0.001, 0.01, 0.1, and 1% v/v, respectively. Methylene blue (0.0001% v/v) was added to all treatments to inhibit fungal growths and to aid the recognition of unfertilized or dead embryos.
2. Materials & methods 2.5. Chemical analyses of exposure solutions 2.1. Oils and chemicals AWB dilbit was identified as a dominant blend transported in high volumes across Canada (GOC, 2013) and chosen as the focus of our study. Unweathered AWB winter blend dilbit and Corexit® 9500A dispersant were supplied by the Centre for Offshore Oil, Gas and Energy Research, Department of Fisheries and Oceans (DFO, Dartmouth, NS). Retene (7-isopropyl1-methylphenanthrene), mineral oil, and methylene blue were purchased from MP Biomedicals (Montréal, QC) or Sigma–Aldrich (St. Louis, MO).
2.2. Experimental animals Japanese medaka (Oryzias latipes) embryos were collected from our colony held at Queen’s University, Kingston, ON, Canada. All fish were held in accordance with approved protocols of Queen’s University’s Animal Care Committee and followed the Guidelines of the Canadian Council on Animal Care. Both parental fish and embryos were maintained throughout the experiment in de-chlorinated water from the City of Kingston’s municipal freshwater supply, originating from Lake Ontario, ON, Canada at 27 ± 1 ◦ C, pH 7.9 ± 0.2, and hardness 131 ± 3 mg/L (CaCO3 ).
The hydrocarbon composition of test solutions was characterized by measurement of 55 PAHs (Supplementary material – Table S1; Maxxam analytics, Mississauga, ON, Canada) by gas chromatography–mass spectrometry (GC–MS), following a standard Environment Canada test method modified from a USEPA method (ESTD/OR/20 / EPA 8270D). Samples for hydrocarbon analysis were collected on days 3, 5, and 14, at the same time as the daily renewal of test solutions. One-liter water samples were collected from the control and highest concentrations of WAF (1, 3.2, 10, and 32% v/v) and CEWAF (0.01, 0.1, and 1% v/v), and from the negative control (water), positive control (retene), and dispersant control (chemically-dispersed nujol (mineral oil) CEWAF) solutions. All samples were kept on ice in coolers and immediately sent to Maxxam for GC–MS analysis (
Contents lists available at ScienceDirect
Aquatic Toxicology journal homepage: www.elsevier.com/locate/aquatox
Diluted bitumen causes deformities and molecular responses indicative of oxidative stress in Japanese medaka embryos Barry N. Madison a , P.V. Hodson b , V.S. Langlois a,b,∗ a b
Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Canada School of Environmental Studies, Queen’s University, Kingston, ON, Canada
a r t i c l e
i n f o
Article history: Received 4 May 2015 Received in revised form 8 June 2015 Accepted 10 June 2015 Available online 17 June 2015 Keywords: Diluted bitumen Dilbit Water accommodated fraction Polycyclic aromatic hydrocarbons PAHs Blue sac disease Oxidative stress Embryotoxicity Fish
a b s t r a c t This study characterized the toxicity and physiological effects of unweathered diluted bitumen (Access Western Blend dilbit; AWB) to fish. Embryos of Japanese medaka (Oryzias latipes) were exposed for 17 days to dilutions of physically-dispersed (water accommodated fraction; WAF) and chemically-dispersed (chemically-enhanced WAF; CEWAF) dilbit. AWB dilbit exposure was not lethal to medaka, but resulted in a high prevalence of blue sac disease (BSD), impaired development, and abnormal or un-inflated swim bladders at hatch. Physiological effects were indicated by the relative mRNA levels of key genes associated with, among others, cell cycling and the response to mutations (p53), xenobiotic metabolism (ahr, arnt2), phase I (cyp1a) and II processes associated with oxidative stress (cat, g6pdh, hsp70, gst, gpx, gsr, nfe2, and sod). AWB dilbit treatment increased p53 and cyp1a transcript levels (1.5-fold and >15-fold, respectively), with significant, but less pronounced changes in indicators of oxidative stress and metabolism. The exposure-related changes in embryotoxicity and mRNA synthesis were consistent with metabolism of polycyclic aromatic hydrocarbons (PAHs) to reactive and toxic metabolites. Medaka embryos responded similarly to WAF and CEWAF treatments, but CEWAF was about 100 times more efficient in delivering toxic concentrations of PAHs. The toxicity of chemically-dispersed nujol, a non-toxic mineral oil used as an experimental control, suggested that a portion of the observed effects of AWB could be attributed to excess dispersant in solution. This first study of the physiological effects of dilbit toxicity to fish embryos provides a baseline to compare toxicity between dilbit and conventional crude oils, and the groundwork for the development of molecular biomarkers of the sensitivity and level of risk of native Canadian fish species to dilbit exposure. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The growing pipeline and railway shipment of diluted bitumen (dilbit) products from Canada’s oil sands industries has generated public concern about the effects of potential spills on aquatic ecosystems (Dupuis and Ucan-Marin, 2014). Of particular importance are the effects on the early developmental stages of fish species, which seem particularly vulnerable due to their inability to avoid oil exposure, and their high sensitivity to toxicity (Carls et al., 2000). The embryo toxicity of crude and refined oils is well established (e.g., Martin et al., 2014; Schein et al., 2009; Carls et al.,
∗ Corresponding author at: Canada Research Chair (CRC) in Ecotoxicogenomics and Endocrine Disruption, Environmental Toxicology & Endocrinology (ETE) Laboratory, Royal Military College of Canada (RMCC), Department of Chemistry & Chemical Engineering, 2526 Sawyer (Bldg 69), 11 General Crerar Cr. Kingston, ON K7K 7B4, Canada. Fax: 1 613 542 9489. E-mail address: [email protected] (V.S. Langlois). http://dx.doi.org/10.1016/j.aquatox.2015.06.006 0166-445X/© 2015 Elsevier B.V. All rights reserved.
2000) and has been linked to the concentrations of 3- to 5-ringed alkyl polycyclic aromatic hydrocarbons (alkyl PAHs) that can partition from oil droplets into water at toxic concentrations (Adams et al., 2014b; Bornstein et al., 2014; Hodson et al., 2007). Phenotypic responses include an exposure-dependent increase in the prevalence of signs of blue-sac disease (BSD; including edemas, heart malformations, craniofacial malformations, and fin erosion) and impairment of development (He et al., 2012; Farwell et al., 2006). Physiologically, oil exposure elevates the synthesis and total activity of cytochrome P450 enzymes, specifically CYP1A (Kim et al., 2014, 2013; Gagné et al., 2012; He et al., 2012; Mu et al., 2012; Carney et al., 2008), the synthesis of the aryl-hydrocarbon receptor protein (AHR) (Incardona et al., 2011a, 2006; Scott et al., 2011; Hanno et al., 2010), and the synthesis and activity of phase II biotransformation enzymes regulating oxidative stress (Olsvik et al., 2011). In contrast, the toxicity of dilbit to fish embryos has not been previously reported, although bituminous oil sands were quite toxic to embryos of common white sucker (Catostomus commer-
B.N. Madison et al. / Aquatic Toxicology 165 (2015) 222–230
soni; Colavecchia et al., 2006). If dilbit embryo toxicity is due solely to its PAH constituents, the toxic effects and overall toxicity should correspond to those of conventional oils. While the concentrations of total PAH in dilbit are lower than in conventional crudes, they include 3- to 5-ringed alkyl PAH at similar concentrations. The 2ringed naphthalenes that comprise the most abundant PAH in crude oil are at substantially lower concentrations in dilbit because the parent bitumen represents highly weathered oil (GOC, 2013). Raw bitumen is too viscous to flow at room temperature in a pipeline and will sink in water because its density may exceed 1.0. However, when mixing raw bitumen with diluents, such as oil–gas condensate, dilbit becomes highly volatile and its viscosity and density are closer to those of conventional medium crude oils. Because many components of oil–gas condensates can evaporate quickly from dilbit, the physical properties, environmental fate and behavior, and susceptibility to chemical dispersion and sinking of dilbit change rapidly after a spill (King et al., 2014; Fingas, 2012). While the relative embryo toxicity of dilbit might be inferred from its PAH content, predicting toxicity is also complicated by variations in the composition of the raw bitumen, depending on its source and method of production. There are also variations in the composition of oil–gas condensates, depending on their sources, and in the proportions added seasonally to dilbit to ensure viscosities suitable for transport (Crosby et al., 2013). Because of the marked uncertainties in the predicted environmental fate, behavior, and chemical constituents of spilled dilbit, we initiated a program of research to measure the embryo toxicity of dilbit to a variety of marine and freshwater fish species. In addition to comparing relative toxicity among dilbits and between dilbit and conventional crude oils, our objectives include: identifying molecular responses of embryos unique to dilbit exposure; the characterization of genes that could be used as biomarkers of dilbit toxicity in assessments of spill impacts; associating toxicity with the chemical composition of dilbit; and identifying any properties of dilbit that could bias measurements of toxicity. This is the first report of dilbit chronic toxicity to fish embryos and the first to describe changes to gene regulation of embryos in response to dilbit exposure.
223
2.3. Embryo collection Embryo clutches were stripped twice from a population of sexually mature females (n = 60) over four days, totaling 520 embryos. The embryos were held in plastic Petri dishes immersed in an embryo rearing solution (ERS), containing: 1000 mg/L NaCl, 40 mg/L CaCl2 , 30 mg/L KCl, and 163 mg/L MgSO4 . Healthy, fertilized embryos (< 24 h post fertilization) were randomly assigned to treatment groups. 2.4. Preparation of dilbit exposure solutions We assessed the toxicity of the water accommodated fraction (WAF) and chemically-enhanced water accommodated fraction (CEWAF) of unweathered AWB on embryos. WAF and CEWAF were prepared following the methods of Adams et al. (2014a,b) and Martin et al. (2014) as modified from Singer et al. (2000). Briefly, WAF was prepared by adding dilbit to water at a ratio of 1:9, stirring gently for 18 h and allowing the solution to settle for 1 h. CEWAF was prepared by the same procedure, but after 18 h, Corexit® 9500A (oil dispersant; ECOLAB/NALCO, Illinois, USA) was added to the surface layer of residual oil at a dispersant-to-oil ratio of 1:10. The CEWAF was stirred for an additional hour and allowed to settle for 1 h, after which surface oil layers were removed and stock solutions of WAF and CEWAF without visible surface oil were transferred to 20 mL vials. The effectiveness of dilbit dispersion seemed more variable than in previous experiments with conventional crude oil, based on the apparent turbidity of the final CEWAF stock and the behavior of the floating dilbit when the dispersant was added after 18 h of stirring. Stock solutions were prepared fresh daily and diluted for static renewal exposures in clean exposure jars. Used exposure jars were cleaned with methanol-soaked wipes to remove residual oil, rinsed with de-chlorinated water, and dried for re-use the following day. Nominal WAF and CEWAF concentrations were 0.32, 1.0, 3.2, 10 and 32% v/v, and 0.0001, 0.001, 0.01, 0.1, and 1% v/v, respectively. Methylene blue (0.0001% v/v) was added to all treatments to inhibit fungal growths and to aid the recognition of unfertilized or dead embryos.
2. Materials & methods 2.5. Chemical analyses of exposure solutions 2.1. Oils and chemicals AWB dilbit was identified as a dominant blend transported in high volumes across Canada (GOC, 2013) and chosen as the focus of our study. Unweathered AWB winter blend dilbit and Corexit® 9500A dispersant were supplied by the Centre for Offshore Oil, Gas and Energy Research, Department of Fisheries and Oceans (DFO, Dartmouth, NS). Retene (7-isopropyl1-methylphenanthrene), mineral oil, and methylene blue were purchased from MP Biomedicals (Montréal, QC) or Sigma–Aldrich (St. Louis, MO).
2.2. Experimental animals Japanese medaka (Oryzias latipes) embryos were collected from our colony held at Queen’s University, Kingston, ON, Canada. All fish were held in accordance with approved protocols of Queen’s University’s Animal Care Committee and followed the Guidelines of the Canadian Council on Animal Care. Both parental fish and embryos were maintained throughout the experiment in de-chlorinated water from the City of Kingston’s municipal freshwater supply, originating from Lake Ontario, ON, Canada at 27 ± 1 ◦ C, pH 7.9 ± 0.2, and hardness 131 ± 3 mg/L (CaCO3 ).
The hydrocarbon composition of test solutions was characterized by measurement of 55 PAHs (Supplementary material – Table S1; Maxxam analytics, Mississauga, ON, Canada) by gas chromatography–mass spectrometry (GC–MS), following a standard Environment Canada test method modified from a USEPA method (ESTD/OR/20 / EPA 8270D). Samples for hydrocarbon analysis were collected on days 3, 5, and 14, at the same time as the daily renewal of test solutions. One-liter water samples were collected from the control and highest concentrations of WAF (1, 3.2, 10, and 32% v/v) and CEWAF (0.01, 0.1, and 1% v/v), and from the negative control (water), positive control (retene), and dispersant control (chemically-dispersed nujol (mineral oil) CEWAF) solutions. All samples were kept on ice in coolers and immediately sent to Maxxam for GC–MS analysis (
