Article
pubs.acs.org/est
In Vitro Assessment of Endocrine Disrupting Potential of Naphthenic
Acid Fractions Derived from Oil Sands-Influenced Water
Liane A. Leclair,† Lani Pohler,‡ Steve B. Wiseman,‡ Yuhe He,‡ Collin J. Arens,† John P. Giesy,‡,§
Stephen Scully,⊥ Brian D. Wagner,⊥ Michael R. van den Heuvel,† and Natacha S. Hogan*,‡,∥
†
Canadian Rivers Institute, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada
Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B3, Canada
§
Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5B4, Canada
∥
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A8, Canada
⊥
Department of Chemistry, University of Prince Edward Island, Charlottetown, Prince Edward Island C1A 4P3, Canada
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Publication Date (Web): April 22, 2015 | doi: 10.1021/acs.est.5b00077
‡
ABSTRACT: Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A
activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the
predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to
examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms
to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive
processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced
waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions
were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by
use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast
androgen screen. The mixtures were characterized by high resolution mass spectrometry, 1H nuclear magnetic resonance, and
attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not
after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause
reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and
main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed.
Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent
antiandrogen or antiestrogens.
■
INTRODUCTION
Exposure to oil sands-influenced waters has been reported to
induce a range of toxicological effects in fishes including
endocrine disruption. Studies have demonstrated decreased
plasma sex steroid concentrations and reduced gonad development associated with elevated CYP1A activity in yellow perch
(Perca f lavescens).2,3 Reduced sex steroids have also been
In 2010, the Athabasca oil sands industry in northern Alberta
had accumulated just under 840 million m3 of tailings and
process-affected water.1 Under a zero-discharge policy, tailings
and affected water must be stored on site within tailings ponds
or incorporated back into a reclaimed landscape. This waste
material contains particulate matter (sand and silt) as well as
inorganic and organic compounds such as metals, ions,
naphthenic acids (NAs), and polycyclic aromatic hydrocarbons
(PAHs).
© 2015 American Chemical Society
Received:
Revised:
Accepted:
Published:
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January 6, 2015
March 31, 2015
April 2, 2015
April 2, 2015
DOI: 10.1021/acs.est.5b00077
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observed in goldfish (Carassius auratus)4 and fathead minnows
(Pimephales promelas)5 exposed to aged oil sands-affected
process waters (OSPW), as well as decreased sex steroid
production in gonadal tissue from slimy sculpin (Cottus
cognatus) collected within tributaries of the Athabasca river in
the Athabasca oil sands area relative to fish from a reference
site.6 Explants of ovarian and testicular tissue from goldfish
exposed to OSPW also had decreased synthesis of testosterone
(T) and 17β-estradiol (E2).4 In vitro studies have shown that
OSPW from an active settling basin (receiving raw fresh
OSPW) can decrease production of T and increase E2 in a
steroidogenesis assay using H295R cells7 as well as affect
androgen and estrogen receptor signaling.8
The constituents or mechanisms of oil sands-influenced
waters responsible for the endocrine disrupting effects are
largely unknown. NAs have been suspected of being involved in
reproductive responses largely because they are the dominant
class of organic compounds present at mg/L concentrations,3,5
not because of any mechanistic evidence. NAs are composed of
acyclic, monocyclic, and polycyclic carboxylic acids, with the
general formula of CnH2n+ZO2, where n represents the carbon
number and Z is zero or a negative, that specifies the hydrogen
deficiency resulting from ring formation or double bonds.9
Fresh (subject to little environmental weathering or biodegradation) NA mixtures have been shown to be responsible for
acute lethality in aquatic biota in oil sands-influenced water.9,10
Fathead minnows exposed to NAs extracted from oil sandsinfluenced waters (which we define as any water being
influenced by either anthropogenic or natural chemical
influence from oil sands bitumen) spawned fewer eggs, and
males had fewer secondary sexual traits as well as lower levels of
T and 11-ketotestosterone.11 Recently, the aromatic components of oil sands derived NAs have been shown to possess
weak estrogen agonist activity.12 There is a need for further
research on mechanisms of toxicity to determine whether NAs
derived directly from oil sands sources are able to influence
reproductive pathways directly. In vitro tools are best suited for
this approach.
The objective of this study was to assess the potential for
NAs purified from OSPW to interact with receptor-mediated
and steroidogenic reproductive pathways. Given the consistently observed CYP1A activity in fishes in vitro and the
possible involvement of Ah receptor-active compounds in
reproductive toxicity, the ability of NAs to induce CYP1A is
also important. NAs were extracted using an acid precipitation
method, separated from humic material, and further purified
into four fractions. Four well-established in vitro assays were
used to screen for compounds that (1) bind the arylhydrocarbon receptor (AhR) using H4IIE-luc cell line, (2)
interfere with steroid biosynthesis using the H295R steroidogenesis cell line, and (3) exhibit estrogenicity, androgenicity,
antiestrogenicity, and antiandrogenicity using the using the
yeast androgen screen (YAS) and the yeast estrogen screen
(YES). Fractions were chemically characterized by high
resolution mass spectrometry (HRMS), 1H nuclear magnetic
resonance (NMR), and attenuated total reflectance (ATR)
infrared spectroscopy to relate their chemical nature to activity
observed in the chosen bioassays.
Syncrude Canada, Fort McMurray, Canada. This water was
chosen as it represented tailings water that had been aged
approximately 17 years at the time of study in a storage pond
open to the elements. Pond 10 water has also been observed to
contain 40 mg/L of naphthenic acids, one of the higher levels at
experimental ponds in the area, providing for the greatest
possible yield of naphthenic acids for the effort. Furthermore,
waters in the Syncrude experimental pond complex from the
exact same tailings source have been found to show
reproductive effects in fishes.3,5 Extraction methods were
based on those developed by Frank et al.13 and modifications
thereof detailed by MacDonald et al.14 (Figure 1). Further
modifications described here were developed in order to
capture all components of the mixture in our fraction,
particularly that part of the mixture that did not reprecipitate
in the last step and to characterize the neutral extract. The
result of this was four fractions that captured the maximum
possible amount and diversity of the NAs extracted. Briefly,
4000 L of water was acidified to pH 2 ± 0.2 with H2SO4
(Sigma, Oakville, Canada), and the precipitate was removed
and redissolved in pH 10 ± 0.2 with 0.1 M NaOH (Sigma).
Particulate matter was removed via centrifugation of the basic
solution, and humic material was removed via DEAE cellulose
filtration. Liquid−liquid extraction with dichloromethane
(DCM) was performed to remove neutrals, and the DCM
was removed by nitrogen evaporation (henceforth called the
“DCM fraction”). The NAs were reprecipitated after adjustment to pH 2.0 and spun at 17 000g for 15 min. The pellet was
washed with distilled water and freeze-dried to produce a solid
material (henceforth called the “main fraction”). The supernatant was passed through a C18 cartridge and eluted with
100% MeOH (called “C18 MeOH” fraction) followed by 1:1
MeOH/0.1 M NaOH (“C18 NaOH” fraction). Those four
fractions were (1) the DCM fraction, (2) the main fraction, (3)
the C18 MeOH fraction, and (4) the C18 NaOH fraction were
subsequently used for chemical analyses and in vitro bioassays.
In Vitro Bioassay Justification. Hormone mimics or
antagonists of estrogen or androgen receptor-mediated
processes have been shown to play major roles in endocrine
disruption. While receptor binding can be conducted for fishes,
the inability of receptor binding approaches to differentiate
agonism from antagonism led to the choice of a reporter gene
assay that has the potential to determine both mechanisms. As
there are currently no commonly available fish-based bioassays
to suit this purpose, the well-established YES and YAS bioassays
were chosen. While these are based on the mammalian
(human) androgen and estrogen receptors, studies have
shown that receptor binding affinity between mammals can
vary as much as between mammals and fishes.15 Steroidogenesis is commonly disturbed in organisms exposed to
endocrine disrupting substances, and there is only one
established bioassay for steroidogenesis that is based on an
immortal cell line, the H295R human cell line. Short of
steroidogenesis in fish explants, which can have high variability
and can usually only be conducted at particular times of year,
this bioassay represents one of the only tools available for this
end point. While there are multiple bioassays for Ah-receptor
active substances, most depend on measurement of catalytic
activity of induced CYP1A enzymes that can be inhibited by
other compounds in complex mixtures such as those used here.
The H4IIE-luc reporter gene assay was chosen as it measures
Ah-receptor binding without the use of a catalytic end point
■
METHODS
Naphthenic Acid Extraction. NAs used in this study were
extracted by acid precipitation from 17 year old tailings pond
water from a small tailings storage pond (Pond 10) located at
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Figure 1. Procedure for bulk extraction and fractionation of NA extracted from oil sands-influenced waters.
incubated for 24 h. Media was removed and fractions dissolved
(in 0.05% v/v, 1:1 NaOH/DMSO) were diluted in media and
added to the plates in triplicate wells. Final NA concentrations
ranged from 0.005 to 5 mg/L with time points at 24, 48, and 72
h conducted in two separate trials. Cells were harvested at each
time point, and luciferase activity was measured by use of the
SteadylitePlus Kit (PerkinElmer, MA, USA). Dose−response
curves for the NA fractions were compared with that obtained
using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; Wellington
Laboratory, Guelph, ON) to determine TCDD equivalent
concentration.
H295R Steroidogenesis Assay. The H295R steroidogenesis assay was performed according to established
protocols.18,19 H295R adrenal cells were obtained from
ATCC (CRL-2128) and propagated at 37 °C and 5% CO2 in
a 1:1 mixture of DMEM/Hams F-12 medium supplemented
with 2.5% Nu-Serum (BD Biosciences, San Jose, CA, USA), 1%
ITS + Premix (BD Biosciences), and 1.2 g/L Na2CO3. Cells
were plated in 24 well plates at a density of 3 × 105 cells/mL
and incubated for 24 h. Media was removed, and media
containing final concentrations for each fraction from 0.005 to
5 mg/L (four concentrations total) dissolved in 1:1
NaOH:DMSO (0.05% v/v final carrier volume) was added
and incubated for 48 h. Forskolin (0.1 and 10 μM) and
prochloraz (0.3 and 3 μM) were used as positive controls.
Media was collected and measured for testosterone, progesterone, corticosterone, and androstenedione by high-performance
liquid chromatography (HPLC)/mass spectrometry (MS)
using an aqueous 0.1% formic acid in a nanopure and methanol
gradient delivered at a flow rate of 250 μL/min. Samples were
injected onto a Betasil C18 column (Thermo Electron
Corporation, Waltham, MA) connected to a C18 guard
column. A triple quadruple mass spectrometer, SpectraMax
CYP1A determination and has been shown to be more sensitive
than other forms of the H4IIE bioassay.16
Cytotoxicity. Cytotoxicity was conducted only using the
H4IIE-luc cells since, based upon experience in the laboratory,
cytotoxicity between H295R and H4IIE cells varies by only
20%. The YES and the YAS bioassays incorporate an
absorbance measure of cell density/proliferation during the
bioassay, so cytotoxicity is incorporated into every bioassay.
The WST-1 bioassay was performed with cells to determine
cytotoxic concentrations of each fraction using the H4IIE-luc
cells. The principle is based on cleavage of the tetrazolium salt
WST-1 by mitochondrial dehydrogenases in viable cells to form
a yellow formazan dye. A lesser absorbance is therefore
interpreted as a decrease in cell viability. H4IIE-luc rat
hepatoma cells were used,17 and cells were propagated in
DMEM/F-12 media containing 10% fetal bovine serum (FBS)
at 37 °C, 5% CO2. Cytotoxicity of fractions were evaluated by
exposing 8 × 104 H4IIE-luc cells to concentrations of fractions
ranging from 0.05 to 50 mg/L (4 concentrations plus controls)
for 24 h with 3 replicates. A 30 min treatment with the
tetrazolium salt WST-1 (Roche Applied Science, Indianapolis,
IN) was performed, and the absorbance was measured at 450
nm using a POLARStar OPTIMA microplate reader (BMG
Labtech).
Aryl Hydrocarbon Receptor Transactivation Assay.
The assay to determine the potential for each fraction to
activate a reporter gene through binding to the AhR was
conducted as previously described17 with modifications. The
amount of AhR-induced luciferase was quantified using the
LucLite(R) Reporter Gene Assay System (PerkinElmer,
Netherlands). H4IIE-luc cells were propagated in DMEM
containing 10% FBS at 37 °C, 5% CO2. Cells were plated to a
concentration of 8 × 104 cells/mL in 96-well plates and
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Nuclear Magnetic Resonance. Proton (1H) nuclear
magnetic resonance (NMR) characterization data were
collected at 298 K on a Bruker AV-300 spectrometer operating
at 300.1 MHz with chemical shifts reported in parts per million
(ppm) downfield of SiMe4. Each fraction was dissolved in
dDMSO (Cambridge Isotope, St Léonard, QC, Canada) and
held in NMR tubes (5 mm, Sigma). Spectra data was analyzed
for peak intensities and chemical shifts.
Statistics. NA mixtures in the H4IIE-luc assay were
quantified as their TCDD equivalent factor; YES were
evaluated as 17β-estradiol (agonist) and 4-hydroxytamoxifen
(antagonist) equivalent factors, and YAS cell assays were
evaluated for DHT (agonist) and flutamide antagonist
equivalent factors. Nonlinear logistic dose vs effect best fit
curves, Y = Xmin + ((Xmax − Xmin)/(1 + ((X/EC50)
^Hillslope))), were used to determine the EC50 (n = 3) for
each treatment on a mass basis since no molecular weight can
be attributed to the NA mixture. GraphPad Prism 5.0
(GraphPad Software, San Diego, CA) was used for these
analyses.
H295R cell line assay data were analyzed by testing for
normality and homogeneity of variance (Levene’s and BrownForsythe tests, respectively) with appropriate transformations
where those assumptions were not met. A full factorial two-way
ANOVA comparing both trials and dose effect was performed
for the H295R cell line assay followed by a posthoc test of
treatments against solvent controls by the use of Dunnett’s test.
Statistical analyses were conducted using STATISTICA (v8.0,
Statsoft Corporation, Tulsa, OK) with an experiment-wise
alpha of 0.05.
190 (Molecular Devices Corp., Sunnyvale, CA, USA), operating
in positive electrospray ionization multiple reaction monitoring
(MRM) mode was used to measure hormone concentrations.
Yeast-Based Screening Assays. The yeast estrogen
screen (YES) and yeast androgen screen (YAS) were used to
quantify the estrogenic, antiestrogenic, androgenic, and
antiandrogenic activity of the NA fractions. Genetically
modified (recombinant) yeast (Saccharomyces cerevisiae) cells
were cultured, and assays were performed as per Routledge and
Sumpter20 with some modifications. Yeast cells were cultured in
a 250 mL conical flask in media for 24 h at 28 °C. The fractions
dissolved in 5% NaOH in DMSO were serially diluted in 2-fold
steps, and 4 μL of each concentration was transferred to a 96well flat-bottom microtiter plate in triplicate to give final
concentrations in media of 0.019−9.9 mg/L (10 concentrations). YES standard and fraction plates tested were
incubated at 38 °C for 72 h, and YAS standard and fractions
plates were incubated for 24 h at 38 °C and 48 h at room
temperature. Plates were subsequently read on a microplate
reader using a Bio-Tek ELx800UV plate reader (USA) at 550
and 630 nm. The 630 nm optical density reflects the yeast cell
number; thus, cytotoxicity is incorporated into the bioassay.
While the H4IIE cytotoxicity bioassay showed effects at greater
concentrations used for YES and YES, no effects on cell density
were observed at any of the concentrations used here.
Absorbance derived from the exposure to fractions was
compared with a standard curve for E2 and dihydroxytestosterone (DHT) for estrogenicity and androgenicity, respectively,
and curves of 4-hydroxytamoxifen and flutamide for antiestrogenicity and antiandrogenicity, respectively, to determine
equivalents of the respective standards.
High Resolution Mass Spectrometry. Each fraction was
examined by high resolution mass spectrometry (HRMS) using
a Thermo Scientific Velos Orbitrap mass spectrometer
equipped with an electrospray ionization interface. The sample
was infused into the mass spectrometer at a rate of 2.5 μL
min−1. The mass spectrometer was equipped with an
electrospray ionization (ESI) source operated in negative
mode. Analyses were recorded with the highest mass resolution
mode (100 000), and the observed ions [M − H]− from m/z
90 to 400 were used to determine the relative concentration of
the NAs that fit the formula CnH2n+ZO2, where Z is the
hydrogen deficiency (an indication of the number of rings and/
or double bonds). Acid extractable structures not corresponding to the CnH2n+ZO2 formula are known to be present in
weathered oil sands material, particularly O3 and O4 NAs,
presumed to be alcohols of the primary NAs structures. In
addition to the O2 structures described above, O3 and O4 NAs
were evaluated using their predicted molecular mass. Ratios of
O2, O3, and O4 NAs were derived by summing the total ion
intensity of all ions within those three groups corresponding to
m/z ratios of possible structures from to 5 to 30 carbons and
from Z = 0 to Z = −30.
Attenuated Total Resonance Infrared Spectroscopy.
Fractions were characterized using attenuated total resonance
(ATR) infrared spectroscopy to determine the functional
groups found in each fraction. The data were collected at 293 K
on a Bruker Alpha spectrometer with an optical resolution of
0.9 cm−1 equipped with an Alpha-P ATR accessory with a
diamond crystal. Dry samples (C18 NaOH, C18 MeOH, main)
were grounded to a fine powder before applying them directly
to the plate. The spectra were then analyzed and compared for
peak location and intensity.
■
RESULTS
Toxicity, Endocrine Disruption, and Ah-Receptor
Binding of Fractions. The WST-1 assay showed cytotoxicity
at 50 mg/L of each fraction tested. On the basis of these results,
final concentrations in media used for the H4IIE-luc and
H295R cell assays ranged from 0.005 to 5 mg/L. The DCM
fraction exhibited AhR agonist activity at 5 mg/L after an
incubation period of 24 h (Figure 2). This corresponded to a
TCDD equivalent concentration of 1.99 × 10−6 mg/L after 24
h, and activity dissipated at 48 and 72 h. The other three
fractions tested did not cause transactivation after 24, 48, or 72
h.
Figure 2. Dose−response curve of TCDD and NA fractions using
H4IIE-luc cells after 24 h. Samples were assayed in triplicate, and two
trials were conducted. Average, SEM, and nonlinear fit curve are
shown (n = 2).
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Environmental Science & Technology
Figure 3. Concentration (ng/mL) of androstenedione (A), testosterone (B), progesterone (C), and corticosterone (D) in media of H295R cells
exposed to NA fractions for 48 h. Incubation with the carrier NaOH/DMSO (0.05% v/v) was included as a control (C) as well as prochloraz (P) or
forkoslin (F) as modulators of steroidogenesis. Samples were assayed in triplicate, and two trials were conducted. Average and SEM are shown (n =
2/treatment). Asterisk indicates a significant difference between the exposed and control group within each experiment by two-way ANOVA with
Dunnett’s test.
also suggested by ATR infrared spectrometry by the broad
shoulder peak around 3500−3200 cm−1 (Figure 5). Each
fraction demonstrated a carboxylic acid dimer and a broad
carboxylic acid OH stretch signature underlying the CH bend
at 1700 and 3200−3000 cm−1, respectively. 1H NMR analysis
of each fraction demonstrated that there was aromatic content
in all fractions (Figure 6A). Unresolved peaks in the 6.8−7.4
ppm region suggested monocyclic aromatic moieties in all
fractions (Figure 6B). A more complex pattern of conjugated
aromatic rings is suggested by the peaks at 7.7 ppm in the
DCM and main fraction only. However, on the basis of
aromatic/aliphatic proton ratios from the proton integrations,
the DCM fraction had a lesser proportion of aromatic protons
than the other fractions (Table 2). The two C18 fractions
contained the greatest relative quantity of aromatic protons.
The DCM fraction was the only fraction that had the clear
presence of olefinic protons in the 5−6 ppm region.
The H295R steroidogenesis assay was used to determine
whether fractions would affect production of steroid hormones
(androstenedione, testosterone, progesterone, and corticosterone) (Figure 3). Exposure to the main fraction at 5 mg/L
resulted in a significant increase in corticosterone production
compared to media collected from cells exposed to solvent
control. Exposure to 0.05−0.5 mg/L of the C18 MeOH
fraction resulted in greater production of progesterone when
compared to the solvent control although no dose−response
relationship was observed. The DCM and C18 NaOH fractions
had no effect on hormone production at any concentration
tested.
Neither of the fractions of NAs had estrogenic effects in the
YES assay. The DCM, main, and C18 MeOH fractions of NAs
all showed antagonized effects of 17β-estradiol (Figure 4A).
The 4-hydroxytamoxifen equivalent factor for those fractions
was 3.7 × 10−3, 1.2 × 10−3, and 2.8 × 10−4 for the DCM, main,
and C18 MeOH fractions, respectively (Table 1). There was no
androgenicity present in any of the fractions at the
concentrations tested. The DCM and main NA fractions
showed an antagonistic effect in the YAS (Figure 4B). The
flutamide equivalent factor for those fractions was 5.9 and 4.72
for DCM and main NA fractions, respectively (Table 1).
Spectroscopic Analyses. HRMS demonstrated that all
fractions contained a suite of compounds with m/z ratios
consistent with NAs. The C18 MeOH and C18 NaOH fraction
were generally enriched in NAs with 14 or less carbons and
depleted in NAs with 15 or more carbons when compared to
the main fraction (Table 2). In addition, the C18 MeOH
fraction had a greater proportion of NAO3 and NAO4
presumably caused by one or two additional OH groups
(Table 2) as HRMS analysis did not show −2 charge with any
of the NAO3 or NAO4 ions. Additional hydroxyl groups were
■
DISCUSSION
There were stimulatory effects of NA fractions on steroid
hormone production. There was no measurable estrogenic or
androgenic activity in any of the fractions as assessed by YES
and YAS. Antiestrogenic and antiandrogenic activity was
detected in most of the fractions and was greatest in the
DCM fraction. Only the DCM fraction was found to contain
AhR agonists. The results for antiestrogenicity, antiandrogenicity, and AhR agonism were not consistent with increased
aromaticity of extracted fractions according to 1H NMR.
It was likely that neutral compounds present in the DCM
fraction caused the AhR potency observed in H4IIE-luc cells
after 24 h exposure. Planar compounds such as polycyclic
aromatic hydrocarbons (PAHs), biphenyls (PCBs), and 2,3,7,8tetrachlorodibenzodioxin (TCDD) induce CYP1A activity.21,22
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Environmental Science & Technology
properties.23,24 However, the waters used in that study were
relatively fresh as opposed to the aged waters used in the
present study. Furthermore, the AhR agonist compounds found
in the DCM fraction are concentrated from 4000 L of aged
tailings water, and enriched in that fraction, extrapolating to the
original tailings water, the concentration would have been
approximately 0.2 ng/L as 2,3,7,8-TCDD. The acid precipitation technique was not designed for the extraction of
neutrals, and no information is available as to the efficiency of
the coextraction of neutrals; so, this is likely an underestimate
of what might be present. The environmental relevance of the
H4IIE-luc bioassay results were supported by the observations
that tailings waters of the same origin consistently showed
CYP1A induction in fishes exposed to tailings-influenced waters
of the same origin.2,3,25,26 Also consistent with previous results
was that the main NA fraction used here also failed to induce
CYP1A in rainbow trout as measured by EROD activity in
previous studies.14,27
Aromatic compounds found in the DCM fraction are
suggestive of a structure containing at least two benzene
rings. On the basis of the 1H NMR spectra, the two benzene
rings are likely not conjugated, as such conjugated rings would
show peaks in the spectrum at higher than 8 ppm. The peak
present around 7.7 ppm is consistent with compounds such as
dibenzothiophenes or other heterocycles. Dibenzothiophenes
and heterocyclic aromatic compounds have been used in
previous investigations as simple analogs of organic S found in
petroleum materials.28 Dibenzothiophenes, heterocycles, and
PAHs have previously been found in oil sands tailings pond in
sediments29 as well as in insect larvae and adult insects.30 The
presence of hydroxy acids (O3 compounds) has been reported
in OSPW in northern Alberta.31 The present study also
identified oxidized NAs and showed that they are removed
from the main NA fraction by the extraction process. Despite
rapid degradation, other NA compounds remain recalcitrant for
decades and we would speculate that diamondoid-type
structures could be resistant to bacterial degradation.
The lesser molecular weight and more water-soluble C18
fractions showed little potency in the assays conducted here,
with the exception of some weak antiestrogenicity. These
fractions contained a larger proportion of NAO3 and NAO4
when compared to the DCM and main fraction. The NAO3 and
NAO4 compounds would be less likely to precipitate with the
addition of H2SO4 due to greater polarity imparted by one or
two hydroxyl or ether groups. The smaller average molecular
weight of the C18 fractions would also have contributed to
Figure 4. Dose−response curves for estrogen receptor antagonist
(YES) activity (A) and androgen receptor antagonist (YAS) activity
(B) of each NA fraction determined using the yeast screening assays.
NaOH/DMSO (5% NaOH) was used as a solvent control. Average,
SEM, and nonlinear best fit curve are shown (n = 3).
Furthermore, the CYP1A induction observed was attenuated
after 48 and 72 h, which suggested that compounds found in
this fraction are labile and biotransformed within a day,
consistent with PAHs. However, the results of 1H NMR
analyses suggested that the AhR-active fraction was not more
enriched in aromatic moieties, and IR showed that this fraction
was still dominated by NAs. Neutral chemicals, such as PAHs,
may have comprised only a small portion of the mixture. In
previous studies employing the same assay, OSPW from WestIn-Pit, an active settling basin, did not show AhR-active
Table 1. EC50 Concentrations and Toxicity Equivalency Factors (TEF) for Each Fraction and Standards for the YES, YAS, and
H4IIE-luc Assaya
EC50 (mg/L)
YES
a
YAS
DCM
C18 MeOH
C18 NaOH
main
0.45
5.88
9.57
1.43
1.49
11.34
16.37
1.87
4-hydroxyTamoxifen
flutamide
TCDD
0.0017
−
−
−
8.81
−
TEF
H4IIE-luc
Fractions
9.82
−
−
−
standards
−
−
1.96 × 10−5
YES
YAS
H4IIE-luc
0.0037
0.00028
0.00017
0.0012
5.90
0.78
0.52
4.72
1.99 × 10−6
−
−
−
−
−
−
−
−
−
−
−
−
Hyphens indicate either no significant activity in the respective bioassay or an inability to calculate an EC50 (less than 50% of maximal response).
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Environmental Science & Technology
Table 2. Average (SEM) and Most Abundant Carbon and Z Number for Each Fractiona
average
fractions
C18 MeOH
C18 NaOH
DCM
main
carbon
12.62
13.08
15.28
15.35
(0.23)
(0.27)
(0.27)
(0.34)
most abundant
Z number
−3.75
−4.08
−4.38
−5.33
(0.20)
(0.07)
(0.06)
(0.24)
NA (%)
carbon
Z number
O2
O3
O4
aromatic/aliphatic ratio
14
14
16
15
−4
−4
−4
−4
88.08
96.06
96.42
99.82
9.79
3.37
2.97
0.15
2.14
0.57
0.61
0.03
1:5.7
1:2.7
1:35.4
1:26.3
a
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Publication Date (Web): April 22, 2015 | doi: 10.1021/acs.est.5b00077
NA percentages demonstrate the relative concentration of each compound family, the NA O2, NA with one additional oxygen (O3), and NA with
two additional oxygens (O4) (charge of −1) based on parent ion intensity.
Figure 5. ATR infrared spectroscopy of four NA fractions extracted from oil sands process-affected waters. (A) hydroxyl groups, (B) CH bend, and
(C) carboxylic acid dimer.
their greater water solubility. The higher relative aromatic
content of these fractions might also suggest that they were
contaminated with some residual humic material not captured
by the DEAE cellulose.
The antiestrogenic and antiandrogenic activity demonstrated
herein is in accordance with previously observed effects of NAs
or oil sands-influenced waters. In the only other similar study,
NAs present in North Sea offshore produced water were weak
estrogen receptor (ER) agonists and androgen receptor (AR)
antagonists.32 This differs from the results of the present study;
however, the chemical nature of the material used in the
respective studies could vary significantly. It should also be
cautioned that antiandrogenicity and antiestrogenicity found in
the present study does not demonstrate that this effect occurs
due to competitive binding at the receptor level. As one
possible example, the fractions may influence the potency or
bioavailabilty of T or E2 by altering bioavailability as has been
observed for other hydrophobic organic compounds.33
Reproductive-endocrine disruption has been reported in
several fishes exposed to OSPW and extracted constituents with
suppression of steroidogenesis being proposed as a predominant mechanism of action.7,34 The extracted fractions in the
present study did not alter steroid hormone production in
H295R cells. The difference in effects observed in this study,
versus the results of previous in vitro studies, may be due to
several factors. For example, in the in vitro study by He et al.34
where steroidogenesis was affected, whole fresh OSPW was
applied to the cells. This would contain not only unweathered
NAs but also potentially salts and ammonia not contained in
our extracts that sought to isolate the effects of naphthenic
acids. Aging of OSPW has also been shown to change the NA
profile; therefore, the compounds responsible for the effects on
steroidogenesis may have undergone degradation in aged
OSPW used in this study.
The most important conclusion of this study was that
naphthenic acids derived from oil sands-affected waters can act
through a mechanism of steroid antagonism at environmentally
relevant concentrations. The novel aim of this study was to
separate out NAs from other components of the mixture such
as neutrals, salts, and ammonia so that conclusions could be
definitely made regarding the endocrine disrupting effects of
this family of compounds. Antiandrogenic/antiestrogenic
potency of NAs demonstrated here was found at concentrations
that would be found in oil sands-influenced waters.
Reproductive impacts in yellow perch were not obvious at
concentrations less than 7 mg/L of NAs35 but did become
apparent at concentrations of 13 mg/L NA.3 A number of other
studies using fathead minnow have found reproductive effects,
in some cases in the same pond water, at NA concentrations
within this same range,5,11,36 and thus steroid antagonism by
NAs could be a potential mechanism for reproductive effects
observed in vivo. The YES and YAS EC50s ranged from
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DOI: 10.1021/acs.est.5b00077
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Environmental Science & Technology
Figure 6. 1H NMR of the main fraction (A) and a comparison of four NA fractions (B) extracted from oil sands process-affected waters. Spectra
were centered on the aromatic region for comparison.
disrupters at concentrations found in tailings waters and in a
number of reclamation scenarios. While this may have been
suspected previously, this is one of the only studies to separate
these components from the rest of the oil sands mixture.
Second, neutral compounds, such as PAHs, may also be
relevant as these have an alternative mechanisms of action in
this mixture (that can also influence reproduction), though the
precise identity of these compounds has yet to be determined.
approximately 0.5 to 16 mg/L NA, well within the range
observed to cause effects on fishes. While the compounds
responsible for the antiandrogenic and antiestrogenic effects
seen here are not known, steroidal aromatic NAs37 as well as
alkylphenols35,38 have been previously reported in the
Athabasca OSPW. Recently, steroidal NAs have been shown
to be weakly estrogenic to zebrafish.12
The research conducted has important implications for the
management of tailings water in oil sands reclamation. As a zero
discharge industry with nearly a billion cubic meters of tailings
and tailings-contaminated waters stored, the need to eventually
release these wastewaters is inevitable. This study and others
have shown that oil sands-influenced waters can be aged nearly
two decades and still disrupt endocrine function, which can
result in adverse effects on reproduction of fish. When those
waters are released, they must be treated in such a way that they
pose little environmental impacts. However, this is very
challenging when the environmental impacts of the mixture
are uncertain and there are no guidelines for what constitutes
safe release. This research contributes to that challenge in two
ways. First, NAs are of concern as potential endocrine
■
AUTHOR INFORMATION
Corresponding Author
*Phone: 306-966-6862; fax: 306-966-4151; e-mail: natacha.
hogan@usask.ca.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by Syncrude Canada, Suncor Energy,
Shell Albian Sands, Total E&P Canada, and Canadian Natural
Resources Limited under the auspices of Canadian Oil Sands
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