ARTICLE IN PRESS
Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
www.elsevier.com/locate/ecoenv
The effects of salinity on naphthenic acid toxicity to yellow perch:
Gill and liver histopathology
V. Neroa,, A. Farwella, L.E.J. Leeb, T. Van Meerc, M.D. MacKinnond, D.G. Dixona
a
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
Department of Biology, Wilfrid Laurier University, Waterloo, Ont., Canada N2L 3C5
c
Syncrude Canada Ltd., Environment Department, Fort McMurray, Alta., Canada T9H 3L1
d
Syncrude Canada Ltd., Edmonton Research Center, Edmonton, Alta., Canada T6N 1H4
b
Received 8 September 2004; received in revised form 3 June 2005; accepted 9 July 2005
Abstract
Naphthenic acids (NAs) are naturally occurring saturated linear and cyclic carboxylic acids found in petroleum, including the
bitumen contained in the Athabasca Oil Sands deposit in Alberta, Canada. The processing of these oil sands leads to elevated
concentrations of NAs, as well as increased salinity from produced waters as a result of ions leaching from the ores, the process aids,
and the water associated with the deeper aquifers. These changes can result in waters that challenge reclamation of impacted waters
associated with oil sands development. Laboratory tests examined the effects of salinity on NA toxicity using local young-of-theyear yellow perch exposed to a commercially available mixture of NAs (CNA) and an NA mixture that was extracted from oil sands
process-affected water (ENA), with and without the addition of sodium sulfate (Na2SO4). Gill and liver histopathological changes
were evaluated in the surviving fish after 3 weeks of exposure. At 6.8 mg/L ENA and 3.6 mg/L CNA, 100% mortality was observed,
both with and without the addition of salt. Exposure of yellow perch to 25% of the NA required to give an LC100 (0.9 mg/L CNA;
1.7 mg/L ENA) resulted in high levels of gill proliferative (epithelial, mucous, and chloride cell) changes, a response that was
increased with the addition of 1 g/L salt (Na2SO4) for the ENA. The significance of these changes was a reduced gill surface area,
which likely caused a reduction in both the transport of NAs within the fish and the exchange of vital respiratory gases. While the
gills were affected, no liver alterations were identified following NA or NA+salt exposures. Differences in the chemical composition
of the NAs tested may explain the differences in the lethality and histopathology of yellow perch.
r 2005 Elsevier Inc. All rights reserved.
Keywords: Naphthenic acids; Salinity; Gill and liver histopathology; Yellow perch
1. Introduction
Naphthenic acids (NAs) are naturally occurring
organic acids in most sources of petroleum. Recent work
of direct extraction, using hot water and caustic soda
(1 M sodium hydroxide), of the various oil sands ores
from the surface mining deposits in the Athabasca Oil
Sands showed that the organic acids ranged from less
than 0.05–0.5% of the bitumen by weight (Clemente,
2004). Based on the caustic hot water extraction method
Corresponding author. Fax: +1 519 746 0614.
E-mail address: [email protected] (V. Nero).
0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2005.07.009
used in commercial operations, produced waters generally contain less than 100 mg/L of NAs (Schramm
et al., 2000). In addition, the salts associated with the ore
itself plus those utilized during the processing of oil
sands (NaOH) add ions to the water inventories. Further
enhancement of ionic concentrations results from
recycling of water in the operating plants. Requirements
for water for the separation of bitumen from oil sands
produce large volumes of process-affected water and
tailings containing high concentrations of NAs and
inorganic ions (sodium, chloride, and sulfate), as well as
saturated concentrations of alkylated polycyclic aromatic hydrocarbons (PAHs). The Microtox bacterial
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
2
assay (EC50) and routine rainbow trout (Oncorhynchus
mykiss) (LC50) and Daphnia (EC50) toxicological testing
have shown that the majority of the acute toxicity
associated with oil sands process-affected water can be
attributed to NAs (MacKinnon and Boerger, 1986;
Madill et al., 2001; Schramm et al., 2000).
NAs are a complex mixture of hundreds of relatively
low molecular weight (o500 amu) linear and cyclic
saturated carboxylic acids (Alberta Environmental
Protection (AEP), 1996). The typical structures are
represented by the general formula CnH2n+ZO2, where n
indicates the carbon number and Z (0 to 12) specifies
the number of hydrogen ions removed to accommodate
a cycloalkane ring structure (Brient et al., 1995).
Therefore, Z ¼ 0 represents a linear NA, Z ¼ 2
represents a 1-ring NA, Z ¼ 4 represents a 2-ring
NA, Z ¼ 6 represents a 3-ring NA, and so forth
(Fig. 1). Recent techniques have used gas chromatography–mass spectrometry (GC–MS) to characterize the
NA chemical profile (Clemente et al., 2003a; Holowenko
et al., 2002), which yields partial information on the
mass and relative distribution of NAs based on carbon
numbers and Z families. Studies of NAs from various
sources have found a reduction in the concentration and
altered carbon numbers and Z families as a result of
microbial activity (Clemente et al., 2003b, 2004; Herman
et al., 1994; Holowenko et al., 2002). These changes
have been associated with a reduction in NA toxicity
(Herman et al., 1994; Lai et al., 1996; Schramm et al.,
2000).
Methods such as water capping of soft tailings
(mature fine tailings, MFT) appear to minimize adverse
conditions to viable aquatic ecosystems (Harris, 2001).
Z Family
Z=0
Z=-2
Z=-4
Z=-6
Formula
C3H6O2
(if x =1 methyl group)
C8H14O2
(if x = 1 methyl group)
C11H18O2
(if x = 1 methyl group)
C14H2202
(if x = 1 methyl group)
Structure
O
[ ]x
OH
O
[ ]x
OH
O
[ ]x
OH
O
[ ]x
OH
Fig. 1. Typical structures and formulas of linear (Z ¼ 0) and cyclic
(Z ¼ 2 to 6) NAs including the chemical formula. x (X1) is a
methyl group. The general formula is CnH2n+ZO2, where n is the
carbon number and Z is the number of hydrogen ions lost due to
cyclization.
In these experimental aquatic test systems, waters
containing various levels of NAs and salinity are
available. Although the water capping method has been
shown to remove the acutely toxic effects of NAs, likely
due to the biodegradation of NAs over time, this wet
landscape option raises the question of sublethal or
chronic effects of NAs to aquatic organisms (Nelson
et al., 1995). Furthermore, fish are known to be particularly sensitive to the toxic effects of NAs in
comparison to other aquatic organisms (diatoms and
snails, Patrick et al., 1968; Microtox and Daphnia,
Verbeek et al., 1994). However, our understanding of
the sublethal effects of NAs on fish is limited.
Based on field studies, NAs in oil sands processaffected water are thought to be the major cause of
histopathological alterations in the gills and liver of fish
(Nero et al., in press). Significant alterations of the gills
(cellular proliferation) and liver (hepatocellular degeneration) were found in yellow perch and goldfish
exposed to oil sands process-affected water after 3
weeks (Nero et al., in press). Gill cell proliferation was
also evident in yellow perch following long-term (3 and
10 months) exposure to process-affected water (van den
Heuvel et al., 2000). Interpretation of cause and effect in
field studies is complicated due to the complexity of oil
sands constituents found in reclaimed environments.
Few studies have reported the modifying effects of
salinity on the toxicity of organic chemicals to fish, of
which only a few studies have examined the interactive
toxicity of NAs and salinity. In vitro studies using
rainbow trout cell lines (gill, liver, spleen) and 7-day
fathead minnow larval bioassays showed some trends
toward enhanced NA toxicity at low levels of salinity
(Farwell and Dixon, 2001; Lee et al., 2000). Since
elevated ion levels are known to increase gill cell
proliferation (Perry, 1997), the addition of salinity could
induce even greater structural changes to fish gills, than
NAs alone, which would have negative consequences on
gill function.
The purpose of this study was to investigate the effects
of NAs alone and in conjunction with salinity on youngof-the-year (YOY) yellow perch gill and liver histopathology under controlled laboratory conditions, to
determine if salinity is an important modifying factor
for NA toxicity. Two NA mixtures (CNA; commercial
NA sodium salts; ENA, extracted NA from oil sands
process-affected water) with different distributions of
carbon numbers and Z families were tested to determine
if differences in NA composition affect gill and liver
histopathological endpoints. Sodium sulfate (Na2SO4)
was used to examine the interactive effects with salt,
since both sodium (Na+) and sulfate (SO2
4 ) ion levels
were found to be elevated in reclaimed systems
constructed with oil sands process-affected materials
(74–611 mg/L for Na+ and 26–717 mg/L for SO2
4 ; Nero
et al., in press). The histological information generated
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
by this work will improve our understanding of the
toxicity of NAs and the influence of salinity on NA
toxicity to fish. Such information will contribute to
improving future risk assessment and management
strategies related to oil sands reclamation.
2. Materials and methods
2.1. Sources of NA mixtures
A commercial (CNA) and an extracted (ENA) NA
mixture were examined in this study. The CNA was a
dense, amber-colored mass of NA–sodium salts (8–10%
sodium) purchased from Acros Organics. The ENA was
isolated from surface waters of the West InPit (WIP)
settling basin (Syncrude Canada Ltd.) in Alberta,
Canada. Stock solutions for each NA mixture were
prepared by dissolving the NAs into 0.1 N sodium
hydroxide (NaOH).
2.2. Extraction of NAs from oil sands process-affected
water
Oil sands process-affected water was collected in 20-L
high-density polyethylene containers. The whole water
was acidified to pH 2 with concentrated sulfuric acid
(H2SO4) to allow the precipitation of the NAs with the
suspended solids. After 24 h, the clarified overlaying
water was siphoned off and the precipitant was
centrifuged (3000 rpm for 8 min) to remove excess water.
The precipitant was washed three times with a 0.1 N
NaOH solution to dissolve the NAs into the water phase
that was produced by subsequent centrifugation of the
caustic mix. The resulting alkaline supernatant was
acidified with sulfuric acid to pH2 and extracted twice
with high-quality dichloromethane (DCM) (CH2Cl2;
X99.9% purity) in a 5:1 water:DCM volume. The DCM
containing NAs (some soluble hydrocarbons, including
PAHs, could be present) was then back-extracted with
0.1 N NaOH in separatory funnels to get the NAs in the
aqueous phase, free of the more lipophilic hydrocarbons. The 0.1 N NaOH solution (pH 10–12) containing
the NAs was stored in 2–50 L high-density polyethylene
containers before experimentation.
Samples of commercial and extracted NAs in 0.1 N
NaOH (stock solutions) were preserved for chemical
analyses by filtering the NaOH solutions through 0.45mm Millipore membrane filters and then acidifying with
sulfuric acid to pH 2–2.5.
2.3. Analysis of total NA concentration
Preserved stock solutions of NAs were analyzed for
total NA concentration (mg/L) using a Perkin Elmer
Spectrum RX 1 Fourier transform infrared spectroscopy
3
(FT-IR) system at the Department of Earth Sciences,
University of Waterloo, using the method developed by
Jivraj et al. (1996). Briefly, preserved samples were
extracted twice with DCM in a 1:1 sample:DCM
volume. The pooled volumes of DCM, containing the
NA fraction, were evaporated to dryness and reconstituted back into DCM for a 6 final concentration
factor and analysis by FT-IR.
The amounts of NAs in the CNA and ENA tested in
this study were determined by the use of a standard
calibration curve based on a commercially available NA
(Merichem Refined NA, 170–260 amu). This commercial standard has two absorbance peaks at 1743 and
1706 cm1 that are identical to the NAs extracted from
oil sands process-affected water. As a result, the
standard curve generated (standard calibration curve
of known concentrations vs. combined peak heights) for
the commercial standard and linear regression analysis
can be used to calculate the concentration of NAs in the
extracted sample taking into account the dilution factor
of the original sample.
2.4. Characterization of NAs
The characterization of NAs was determined using a
Varian CP-3800 GC/Saturn 2000 MS system at the
Department of Earth Sciences, University of Waterloo
using a modified derivatization protocol of St. John et
al. (1998), as described in Holowenko et al. (2002).
Samples were injected into the GC system at a
concentration of 2 mg/mL. Peak ion intensity values
were averaged over the elution of the NAs peak from
retention times between 10 and 30 min. Spectral data
was acquired using Varian Saturn GC/MS Workstation
software and entered in a Microsoft Excel spreadsheet
program, which selected only those masses that corresponded to derivatized NAs with carbon numbers 5–33
and Z-values of 0 to 12 (Holowenko et al., 2002). The
intensity value for each peak was divided by the sum of
all the peak intensities to produce a normalized value for
each carbon and Z number. This normalized data was
used to plot three-dimensional graphs.
2.5. Experimental design
2.5.1. Stock solutions and treatment preparation
Stock solutions of NAs in 0.1 N NaOH were prepared
before the start of yellow perch exposures in sufficient
volumes for the duration of the 21-day exposures,
whereas the sodium sulfate (Na2SO4) stock was prepared (200 g/L) as required. Treatment solutions, prepared in 60-L bins, included the control, NaOH control
(5 mg/L), 1 g/L salt (Na2SO4), and 3 concentrations of
NA and NA+ 1 g/L Na2SO4. The NaOH control had a
NaOH concentration equivalent to the highest NA
treatments. Stock solutions of ENA were diluted to
ARTICLE IN PRESS
4
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
prepare test treatments of 6.8, 3.4, and 1.7 mg/L.
Treatments of 3.6, 1.8, and 0.9 mg/L CNA were
prepared from the concentrated stock solution. The
pH was adjusted to levels comparable to Mildred Lake
control water (8.0–8.3) for all NaOH treatment
solutions using concentrated hydrochloric acid (HCl)
(36.5–38.0%).
2.6. Test organisms, experiments, and sampling
YOY yellow perch (length, 4.8–5.5 cm; weight,
1.1–1.9 g) were captured by electrofishing (direct current) (Boat type; Smith-Root SR-18, 3.4 V, 500 A,
containing a 74-gall live well) from Mildred Lake
Reservoir (MLR), located on Syncrude’s Lease 17/22
on August 22, 2003. Fish were held in aerated 60-L bins
containing MLR water for a short period (2 days) before
the start of exposures. Experiments were conducted in a
trailer at Syncrude’s Environmental Complex, located
adjacent to the MLR. Water from the MLR was used as
the sole source of control/dilution water for the
experiments. Detailed water chemistry, including major
ion and NA concentrations for MLR, has been included
in Table 1.
Yellow perch 3-week static renewal tests (100%
renewal every 3 days, loading density of 1.5 g fish/L/
day) were conducted in 35-L glass aquaria (containing
30 L of aerated test solution) under a 16:8 h light:dark
regime. Tanks with 10 fish/tank per treatment were
positioned in two large reservoirs (2000 L) to reduce
temperature fluctuations. Initially, yellow perch were fed
cultured brine shrimp nauplii; however, their feeding
performance was not satisfactory, so the feed was
replaced with frozen bloodworms. Yellow perch were
fed twice daily (morning and evening) a ration of about
9% wet body weight of bloodworms per tank. On days
Table 1
Salinity and concentrations of major ions (cations and anions) and
naphthenic acids for Mildred Lake Reservoir (MLR)
Water quality parameter
MLR
Cations
Sodium (Na+, mg/L)
Potassium (K+, mg/L)
Magnesium (Mg2+, mg/L)
Calcium (Ca2+, mg/L)
20.6
BDL
10.2
32
Anions
Sulfate (SO2
4 , mg/L)
Chloride (Cl, mg/L)
Carbonate (CO2
3 , mg/L)
Bicarbonate (HCO
3 , mg/L)
27.7
8.1
2.2
143.3
Salinity (ppt)a
Organics
Naphthenic acid (NA, mg/L)
0.24
0.7
Note: BDL refers to measurements below detection limit. Water
chemical data courtesy of Tara Hayes (University of Waterloo).
a
Salinity is expressed as the sum of the ionic composition of the
major cations (Ca2+, Mg2+, Na+, and K+) and anions (HCO
3,
2
CO2
3 , SO4 , and Cl ) in mass per liter.
of water renewal, fish were removed by net and placed in
clean tanks with fresh solutions. All tanks were
monitored daily for fish survival. Water characteristics
(temperature, pH, conductivity, salinity, and dissolved
oxygen) were measured using an Orion Model 1230 field
meter at the beginning and end of each renewal time
period. Ammonia (NH3) and nitrate levels (NO
3 ) were
monitored using Hagen water-testing kits and remained
constant between renewals.
Following the 3-week period, yellow perch were
sacrificed by a sharp blow to the head and severing of
the spinal column behind the skull. Fork length (l), body
weight (bw), and liver weight (lw) were recorded.
Portions of the liver and the right gill arches of each
fish were removed and immediately placed in 10%
neutral buffered (phosphate) formalin (Fisher Scientific). Condition factor, K ¼ 100 ðbw=l3 Þ, and liver
somatic index, LSI ¼ 100 (lw/bw), were calculated
and recorded for each fish.
2.7. Histological procedures and assessment
For histological examination, slides of gill (second gill
arch) and liver tissue (8 mm2) of fish were prepared and
stained with hematoxylin and eosin at the University of
Guelph Animal Health Laboratory. The slides were
coded and the tissues assessed blindly by two independent observers (V.N. and L.L.) without prior knowledge
of the sample identity. Histopathological alterations
were evaluated using a modified version of the protocol
described by Bernet et al. (1999). Each morphological
alteration was classified into one of five categories,
proliferative, degenerative, inflammatory, structural,
and cytoplasmic, each representing a general tissue
response by an organism to a particular stressor.
Morphological alterations do not overlap into other
categories, nor are they counted more than once per
tissue examined. Table 2 lists the category of alterations
examined in this study, accompanied by a description of
the general response of the fish tissue and examples of
specific tissue alterations assigned to each category. The
entire gill arch of each fish and a portion (8 mm2) of the
middlemost section of the entire liver sample were
examined under the light microscope. For gills, the
number of gill filaments possessing a particular alteration was recorded and divided by the total number of
gill filaments examined (dependent on the individual
fish) to give a percentage of filaments affected. The liver
tissue (8 mm2) was divided into 10 equal areas
(0.4 2 mm) to ensure that there was no examination
of overlapping areas. The number of areas containing a
particular alteration was recorded and divided by the
total number of areas examined (10) to give a percentage
of liver areas affected. For both gill and liver, the
percentage of gill filaments or liver areas affected
determined the score value (0–6) for a particular
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
5
Table 2
Descriptions of the category of alterations (proliferative, degenerative, etc.) and examples of specific alterations assigned to each category for gill and
liver tissue
Category of alterationa
Proliferative
Degenerative
Inflammatory
Structural
Cytoplasmic
Description of general responsea
Increase in the number of specific
cell types or structures
Breakdown of tissue and/or cells
Increased presence of cells used in
tissue repair; response to damaged
tissue
Changes in tissue architecture
Alterations in cellular volume, size
or storage products
Examples of specific tissue alterations
Gills
Liver
Chloride cell proliferation (2)
Bile duct proliferation (2)
General necrosis (3)
Epithelial lifting (lamellar oedema) (1)
Hepatocellular necrosis (3)
Lymphocytic infiltration (2)
Lamellar tip fusion (2)
Mucous cell hypertrophy (1)
Increased separation of hepatocytes (1)
Hepatocyte hypertrophy (1)
Note: Also included in parentheses are the importance factors given to the specific gill and liver alterations listed.
a
Information provided by Bernet et al. (1999).
alteration (0, none observed; 1, 10–20%; 2,
21–30%,y,6, 60% or more). In addition, importance
factors (ranging from 1 to 3) are assigned to each
alteration (Table 2) as a measure of the potential for a
particular alteration to impact fish health (alterations
posing a greater risk to fish health are given higher
importance factors) (Bernet et al., 1999). The importance factors and the score values were multiplied to
give an index for a particular alteration. The indices for
each alteration within a category of alterations were
summed to give an index for each category of alterations
(categorical alteration index). Finally, the indices for
each category of alterations were summed to give an
overall organ (gill and liver) pathological index (total
pathological index) (Bernet et al., 1999). Data for
categorical and total gill and liver pathological indices
are presented as the mean7standard error (SEM) for a
specified sample size.
2.8. Gill morphometric analysis
Photomicrographs of gill tissue were taken using a
digital camera (Nikon 9000) under medium power
magnification (100 ) in a Nikon TE3000 light microscope for six individuals per treatment. All filaments
occurring in photomicrographs were measured for
secondary lamellar length (SLL) and width (SLW),
interlamellar distance (ID), and basal epithelial thickness (BET), which represent the major dimensions of gill
tissue influencing the diffusion distance (gas exchange)
in fish (Hughes and Perry, 1976). For SLW and ID,
measurements were made on every second secondary
lamella appearing on the filament. Three measurements
were made for each secondary lamella: the lamellar tip,
the center of the lamellae, and the base of the lamellae,
to give an average SLW and ID for each measured
lamella. For BET, six measurements were made on each
filament appearing in the photomicrograph: two measurements at the top, two at the bottom and two in the
center of each filament to give an average BET for each
filament. The proportion of the secondary lamellae
available for gas exchange (PAGE) was averaged for
each filament of an individual and calculated using the
equation PAGE (%) ¼ 100 (mean SLL/(mean
BET+mean SLL)). All gill morphometric indices are
presented as mean7standard error (SEM) for six
individuals per treatment.
2.9. Statistical analysis
Comparison of the chemical composition (carbon
numbers and Z families) between ENA and CNA was
completed using a pairwise t-test as described in
Clemente et al. (2003a). Analysis of variance (ANOVA)
and Tukey pairwise tests were used to compare the
effects of individual chemicals (salt, CNA, and ENA)
and mixture treatments (CNA+salt, ENA+salt) relative to the control, as well as comparisons between CNA
and ENA treatments. Data were checked for normality
using normal probability plot. All statistical analyses
were conducted at a ¼ 0:05 using SYSTAT 10 (SPSS
Inc., 2000).
3. Results
3.1. Water chemistry
Measured water parameters included temperature, pH,
conductivity, salinity, and dissolved oxygen (Table 3).
Water temperatures ranged between 17.4 and 19.9 1C
over the 3-week exposure period. Measurements of pH
for all treatments were within the pH range of control
water (Mildred Lake). Conductivity and salinity levels
ARTICLE IN PRESS
6
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
Table 3
Measured water chemistry parameters (mean7SEM, n ¼ 14 unless otherwise stated in parentheses) for yellow perch exposures
Treatment
Temperature (1C)
pH
Conductivity (mS/cm)
Salinity (ppt)
Dissolved oxygen (mg/L)
Control (Mildred Lake)
NaOH (5 mg/L) Control
Salt control (Na2SO4) (1 g/L)
CNA (0.9 mg/L)
CNA (1.8 mg/L)
CNA (3.6 mg/L)
CNA (0.9 mg/L) +salt (1 g/L)
CNA (1.8 mg/L) +salt (1 g/L)
CNA (3.6 mg/L) +salt (1 g/L)
ENA (1.7 mg/L)
ENA (3.4 mg/L)
ENA (6.8 mg/L)
ENA (1.7 mg/L) +salt (1 g/L)
ENA (3.4 mg/L) +salt (1 g/L)
ENA (6.8 mg/L) +salt (1 g/L)
18.970.3
18.970.2
18.470.2
18.570.2
18.570.2
18.470.2 (7)
18.570.2
18.570.2
17.870.4 (3)
18.770.2
18.770.2
17.670.3 (2)
18.670.3
18.770.3
17.4 (2)
8.3370.08
8.2170.05
8.5570.07
8.3670.03
8.4070.04
8.3870.05
8.4670.03
8.3470.06
8.3070.10
8.2470.05
8.2670.06
8.1970.15
8.4070.09
8.3470.07
8.2970.15
30174
666724
1672724
38072
46174
60874 (7)
176376
185178
197275 (3)
38773
46872
654716 (2)
1734720
1833716
1920762 (2)
0.1
0.3
0.8
0.2
0.2
0.3
0.9
0.9
1.0
0.2
0.2
0.3
0.9
0.9
1.0
8.2770.1
8.6570.07
8.8970.06
8.9170.09
8.8970.08
8.9270.08 (7)
8.7770.11
8.8470.08
8.9770.28 (3)
8.7670.1
8.8170.11
8.9370.19 (2)
8.8570.12
8.7470.11
8.90 (2)
increased based on additions of NaOH, salt (Na2SO4),
and/or NA solutions.
10
3.2. NA characterization
Percentage
8
6
4
2
0
5
7
9
(a)
11
% NAs
28
55
17
Z0 to –4
Z-6 to –12
61
39
15
17
19
rbo
21
nn
um
ber 23 25
12
27
10
6
29
0
31
33
8
6
Group
4
2
3.3. Mortality and organismal indices
0
Minimal mortality (10%) was observed for the
controls (Mildred Lake control, NaOH control) and
salt (1 g/L) treatment. For both ENA and CNA
treatments, there was 100% mortality at the highest
test concentrations (ENA 6.8 mg/L; CNA 3.6 mg/L)
within 96 h of exposure. At lower concentrations (ENA
3.4 mg/L; CNA 1.8 mg/L), the addition of salt resulted
in a 40% (CNA) and 50% (ENA) reduction in mortality
compared to the chemicals alone. At similar concentrations of NAs (1.8 and 3.6 mg/L), CNA treatments had
between 20% and 80% higher mortality compared to
ENA treatments, with or without the addition of salt.
Group
C5 to 13
C14 to 21
C22 to 33
13
Ca
Percentage
The commercial and extracted NAs tested in the
yellow perch exposures were characterized by GC–MS.
Figs. 2a and b show the matrix of the possible NAs
within the carbon number range 5–33, with 0–6 rings
(Z ¼ 0 to 12) for ENA and CNA, respectively. For
comparison of NA mixtures, the matrix of possible NAs
was divided between carbon numbers 5–13, 14–21, and
22–33 and Z families 0 to 4 and 6 to 12 as
described by Clemente et al. (2003a). The most
abundant NAs in the ENA had carbon numbers 14–21
(55%) and Z families 0 to 4 (61%). The most
abundant NAs in the CNA had carbon numbers 5–13
(48%) and Z families 0 to 4 (84%). The ENA stock
had a significantly greater proportion of NAs with
carbon numbers 14–21 (P ¼ 0:040) and 22–33
(Po0:001) and Z families 6 to 12 (X3 carbon rings)
(Po0:001) than the CNA stock.
(2)
-
(2)
(2)
fam Z
ily
(2)
(3)
5
(b)
7
9
11
C5 to 13
C14 to 21
C22 to 33
% NAs
48
40
12
Z0 to –4
Z-6 to –12
84
16
13
15
17
19
Ca
rbo
21
nn
23
um
25
ber
27
12
6
29
31
0
-
(3)
(7)
fam Z
ily
(7)
33
Fig. 2. Three-dimensional plots (n ¼ 3) of the distribution of carbon
numbers and Z families of (a) extracted naphthenic acid (ENA) (WIP;
Syncrude Canada Ltd.) and (b) commercial naphthenic acid (CNA)
(Acros Organics). Included within the plots are tables providing the %
of NAs based on carbon numbers 5–13, 14–21, and 22–33 and Z
families 0 to 4 and 6 to 12.
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
Following the termination of the 3-week exposures,
organismal indices (l, bw, K, and LSI) were determined
for control and exposed fish and compared to baseline
values for yellow perch (day 0, a subset of fish taken
from the same stock of fish used for the exposures and
sampled at day 0). Compared to the day 0 baseline
values for yellow perch, there were reductions in yellow
perch body weight observed in all treatments following
the 3-week exposure, where day 0 weights ranged from
1.16 to 1.9 g. However, there were no statistically
significant differences (P40:05) between day 0, control
(0.99–1.98 g), salt (1.05–1.65 g), and NA treatments
(0.8–2.82 g). Day 0 yellow perch had a significantly
greater K (0.95–1.19) than fish from the controls
(0.75–0.97; Po0:001), salt (0.79–0.95; Po0:001) and
NA treatments (0.69–1.02; Po0:001). The reduced
condition factors observed in the exposure fish (control
and NA exposed) compared to day 0 values suggest that
the yellow perch were sensitive to our laboratory
conditions. Other studies have observed similar growth
reductions and decreased condition factors in juvenile
yellow perch held under intensive culture conditions for
up to 8 weeks (Malison et al., 1998; Head and Malison,
2000). There were no significant trends in LSI, which
ranged from 1.09 to 1.67 for day 0 and treatment fish.
3.4. Histopathological assessment
Due to mortality at the higher concentrations,
histopathological and morphometrical analyses were
limited to the lowest NA concentrations for each of the
chemical mixtures tested (CNA and CNA+salt, 0.9 mg/
L; ENA and ENA+salt, 1.7 mg/L).
3.5. Gill pathology
3.5.1. Effects of NAs
Yellow perch from the control treatment had normal
gill histology (Fig. 3a) relative to the proliferative
alterations of fish exposed to NAs (Figs. 3b and c).
Yellow perch from the NA treatments had greater
alterations for all categories of alterations compared to
the controls (Fig. 4); however, only proliferative
alterations were significantly greater for CNA
(P ¼ 0:001) and ENA (P ¼ 0:001) exposed fish (Fig. 4).
Greater proliferative alterations were attributed to
increased numbers of epithelial (complete and partial
fusion of lamellae) (Fig. 3b), chloride (Fig. 3b), and
mucous cells (Fig. 3c). Yellow perch from both CNA
and ENA treatments had significantly higher total gill
pathological indices than the control fish (CNA,
P ¼ 0:005; ENA, Po0:001) (Fig. 4).
3.5.2. Effects of NA+salt mixtures
Categorical and total gill pathological indices for
NA+salt treatments were compared to the salt treat-
7
ment (control; 1 g/L Na2SO4). There were no statistical
differences in categorical or total gill pathological
indices (Fig. 4) between the controls (no salt; salt).
Yellow perch from the NA+salt treatments had
increased indices for all categories, with the greatest
increase in the proliferative index compared to the
salt treatment (Fig. 4). Only fish from the ENA+salt
treatments had a significantly higher proliferative
index compared to the salt treatment (P ¼ 0:014).
Similarly to NA treatments without salt, epithelial,
mucous, and chloride cell proliferation increased. In
some cases, the high severity of gill proliferative
alterations resulted in fused secondary lamellae forming
a membrane-like structure covering the tips of the gills
and enclosing the filament (Fig. 3d). In addition,
ENA+salt exposed fish had significantly greater
structural (P ¼ 0:002) and inflammatory (P ¼ 0:001)
alterations compared to the salt treatment (Fig. 4).
Inflammatory alterations consisted mainly of a lifting of
the gill epithelium (epithelial lifting) and an infiltration
of lymphocytes into the secondary lamellae Fig. 3e),
while the main structural alteration was lamellar
synechiae (fusion of the secondary lamellar tips), which
was also evident in fish exposed to NAs only (Fig. 3c),
but found to be more prevalent in NA+salt treated
fish (Fig. 3f).
The total gill pathological index was elevated for
NA+salt treatments (CNA and ENA) compared to
salt-exposed fish (Fig. 4); however, only yellow perch
from the ENA+salt treatment had a significantly
higher total gill pathological index compared to fish
from the salt treatment (P ¼ 0:001) (Fig. 4). There
were no statistical differences between fish exposed to NAs alone (CNA and ENA) and those
exposed to NA+salt mixtures (CNA and ENA)
(Fig. 4).
3.6. Gill morphometrics
Measurements of yellow perch gills included SLW,
ID, SLL, and BET (Fig. 3a). Values for the SLL and
BET were used to calculate the PAGE index, which
represents the gill surface area. The gill alterations that
were found to have a significant impact on the changes
in gill dimensions were the proliferative (chloride,
mucous, and epithelial cell proliferation), inflammatory
(epithelial lifting), and structural changes (lamellar tip
fusion). Generally, an increased severity of these
particular alterations caused reductions in the ID,
SLL, and PAGE indices and increases in the SLW and
BET indices for NA and NA+salt treatments compared
to the control and salt treatments (Fig. 5a–e). The
greatest change was associated with exposure of
yellow perch to the ENA+salt mixture. While there
was no significant difference between yellow perch
exposed to ENA alone and those exposed to ENA+salt
ARTICLE IN PRESS
8
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
Fig. 3. Gill histological sections of yellow perch from control (a), NA (b, c), and NA+salt treatments (d–f): (a) Normal gill structure from the
control treatment showing a gill filament (f) and secondary lamellae (sl). Also shown are the four gill measurements (secondary lamellar width, SLW;
secondary lamellar length, SLL; Basal epithelial thickness, BET; Interlamellar distance, ID) examined for morphometric analysis. (b) Proliferation of
epithelial cells at the base (circles) and a proliferation of chloride cells (arrows) at the base and along the edges of the secondary lamellae. (c)
Proliferation of mucous cells at the base (arrows) of the secondary lamellae, areas of lamellar tip fusion (oval) and epithelial lifting (open arrows). (d)
Cellular proliferation between secondary lamellae leading to a complete fusion of the secondary lamellae and a membranous structure enclosing the
filament (arrows). (e) Mucous cell proliferation (open arrows), and fusion and infiltration of inflammatory cells of the lamellar tips (oval). (f) Epithelial
cell proliferation (open arrows) and chloride cell proliferation (arrows) causing lamellar synechiae (ovals).
using the more qualitative scoring system (categorical
and total gill pathological indices, Fig. 4), the morphometric analysis of the gills was found to be a more
sensitive indicator of subtle changes in gill structure
(Fig. 5a–e) with the simultaneous exposure of salt
and NAs.
Yellow perch from the ENA+salt mixture had gills
with significantly higher indices for SLW (Po0:001) and
BET (Po0:001) and lower indices for ID (Po0:001),
SLL (P ¼ 0:027), and PAGE (Po0:001) compared to
the salt treatment (Fig. 5a–e) and significantly greater
SLW (P ¼ 0:04) and BET (P ¼ 0:047) indices and lower
ID (P ¼ 0:008) and PAGE (P ¼ 0:018) indices compared to the ENA treatment.
3.7. Liver pathology
Yellow perch exposed to NA treatments had slightly
higher levels of degenerative, inflammatory, and structural alterations and lower levels of proliferative
alterations compared to the controls, but none were
significantly different (P40:05) (Fig. 6). Yellow perch
exposed to mixtures of NA+salt had trends for
proliferative, degenerative, and structural alterations
similar to those for NA treatments, with the exception
of the inflammatory index, which was lowered by the
addition of salt, but none were significantly different.
Although there were no significant differences in total
liver pathological indices between controls and treat-
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
24
9
b
22
20
b
Pathological Index
18
No Salt
Salt (1g/l)
ab
b
16
14
*
12
a
*
10
*
a
8
6
4
*
*
2
Total Gill
ENA
CNA
Day 0
Control
CNA
Structural
ENA
Control
ENA
Inflammatory
Day 0
CNA
Control
ENA
Degenerative
Day 0
CNA
Control
ENA
Proliferative
Day 0
CNA
Control
Day 0
ENA
CNA
Day 0
Control
0
Cytoplasmic
Pathological Alteration
Fig. 4. Total (total gill) and categorical (proliferative, degenerative, inflammatory, structural, cytoplasmic) gill pathological indices (mean7SEM) of
yellow perch exposed to control and NA treatments, with (1 g/L) and without salt (n ¼ 9, except for the ENA+salt treatment, where n ¼ 6). Also
included are the baseline level (day 0) of gill alterations of yellow perch from Mildred Lake. Different letters represent significant differences in total
gill pathological indices between treatments within the test groups (no salt, salt) and asterisks represent significant differences in gill categorical
pathological indices between control and treated fish.
ments, there were trends toward an elevated total liver
pathological index for NA treatments relative to
NA+salt treatments (Fig. 6).
4. Discussion
4.1. Lethal effects of NAs and NA+salt mixtures
Exposure of YOY yellow perch to NAs resulted in
100% mortality in p96 h at the highest test NA
concentrations (CNA, 3.6 mg/L; ENA, 6.8 mg/L). The
response of YOY yellow perch was similar to that of
other fish species including rainbow trout (Oncorhynchus
mykiss) fingerlings (96 h LC50p10 mg/L extracted NA
from MLSB, Syncrude Canada Ltd.) (Verbeek et al.,
1994), juvenile chum salmon (Oncorhynchus keta) (96 h
LC50 ¼ 12 mg/L commercial NA, source unknown) and
juvenile kutum (Rutilus frisii) (LC100 ¼ 6.5 mg/L commercial NA, source unknown) (Dokholyan and Magomedov, 1984).
Similar to NA exposure, NA+salt mixtures resulted
in 100% mortality (p96 h) at 3.6 mg/L CNA and
6.8 mg/L ENA. However, at lower concentrations,
mortality was reduced by 40% for CNA (1.8 mg/L)
and 50% for ENA (3.4 mg/L) with the addition of 1 g/L
Na2SO4 (Fig. 3). Results from various studies suggest
that fish species are more resistant to acutely toxic
conditions living in or exposed to isosmotic salinity (to
fish blood) compared to both higher and lower salinity
as a result of a minimization of osmotic stress (Hall and
Anderson, 1995). Greater mortality was reported for
naphthalene-exposed mummichog (Fundulus heteroclitus) (Levitan and Taylor, 1979) and surfactant-exposed
mummichog and eel (Anguilla rostrata) (Eisler, 1965) in
the lower (o10 ppt) and higher (420 ppt) ranges of
salinity compared to the intermediate salinities tested.
Although the salinity tested in this study was lower than
the isosmotic salinity reported for freshwater fish
(10 ppt; Craig, 1987), there may be some advantage in
terms of reduced osmotic stress, which may in part
contribute to the reduction in acute toxicity.
The acute toxicity of the CNA (80% mortality) to
yellow perch was higher than that of the ENA (0%
mortality) at lower concentrations (1.8 mg/L CNA,
1.7 mg/L ENA). The NAs tested in this study were
found to have a significantly different composition,
which likely influenced the NA toxicity. Fig. 2 shows
that the ENA had a greater proportion of C22–C33
(17% vs. 12%) and less of the lowest carbon number
group, C5–C13 (28% vs. 48%) compared to the CNA.
Holowenko et al. (2002) also found that NAs composed
of a greater proportion of higher carbon numbers
(‘‘+C22 group’’) were less toxic than NAs with a higher
proportion of the lower carbon numbers (‘‘C22+
group’’) using the Microtox bacterial assay. In field
studies, Nero et al. (in press) and Siwik et al. (2000)
observed minimal or no mortality of caged goldfish and
larval fathead minnows, respectively, following exposure to reclaimed environments containing NAs with a
greater distribution of higher carbon numbers relative to
lower carbon numbers.
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
10
No Salt
18
a
15
b
b
a
b
b
SLW (µm)
9
ab
a
b
ab
12
9
6
3
3
0
0
Day 0
Control
(a)
CNA (0.9 mg/l)
Day 0
ENA (1.7 mg/l)
No Salt
Control
(b)
Treatments
120
CNA (0.9 mg/l)
b
No Salt
Salt (1g/l)
ab
30
a
a
80
ENA (1.7 mg/l)
Treatments
35
Salt (1g/l)
100
a
25
b
BET (µm)
SLL (µm)
Salt (1g/l)
15
6
60
40
a
b
a
20
15
10
20
5
0
0
Day 0
(c)
No Salt
18
ab
12
ID (µm)
Salt (1g/l)
a
Control
CNA (0.9 mg/l)
ENA (1.7 mg/l)
Day 0
Control
(d)
Treatments
CNA (0.9 mg/l)
ENA (1.7 mg/l)
Treatments
85
No Salt
Salt (1g/l)
a
80
a
PAGE (%)
b
a
75
b
b
70
65
60
55
Day 0
Control
(e)
CNA (0.9 mg/l)
ENA (1.7 mg/l)
Treatments
Fig. 5. Gill morphometric indices (mean7SEM) of yellow perch exposed to control and NA treatments, with (1 g/L) and without salt (n ¼ 6):
(a) Interlamellar distance (ID). (b) Secondary lamellar width (SLW). (c) Secondary lamellar length (SLL). (d) Basal epithelium thickness (BET). (e)
Proportion of area of the secondary lamellae available for gas exchange (PAGE). Different letters represent significant differences between controls
and NAs in treatments with and without salt additions. Also included are the baseline levels (day 0) of gill measurements for yellow perch from MLR.
4.2. Sublethal effects of NAs and NA+salt mixtures on
gill pathology
Although wide ranges of alterations were observed in
this study, the predominant gill response of fish exposed
to NAs and NA+salt mixtures (CNA and ENA) was a
proliferation of epithelial, chloride, and mucous cells. In
addition to proliferative alterations, the addition of low
levels of salt (1 g/L sodium sulfate) to the ENA mixture
caused a significant increase in inflammatory (epithelial
lifting) and structural (lamellar tip fusion) alterations
compared to the control treatments. Based on gill
morphometrics fish gills had thicker (SLW) and shorter
(SLL) secondary lamellae, a reduced distance between
secondary lamellae (ID) and a larger gill epithelium at
the base of the secondary lamellae (BET). In other
words, the gill surface area decreased significantly
(PAGE, 10% reduction compared to the controls)
which will inevitably impact the high efficiency of the
fish gills. It is apparent from the gill sections (Fig. 4) that
the epithelial cell proliferation, epithelial lifting, and
lamellar tip fusion of the ENA+salt exposed fish are a
protective response of the gill to reduce toxicant entry.
The elevated mucous cell production has also been
implicated in reducing toxicant entry. Since mucus
contains elevated levels of sugars and proteins that
carry an electrical potential capable of trapping toxicants (Perry and Laurent, 1993), it is possible that a
proliferative response could reduce the permeability of
the gill to NAs. This protective effect has been observed
in isolated rainbow trout (Partearroyo et al., 1992) and
catfish (Rita rita) (Roy, 1988) gills exposed to surfac-
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
11
18
16
No Salt
Pathological Index
14
Salt (1g/l)
12
10
8
6
4
2
Total Liver
Proliferative
Structural
ENA
CNA
Control
ENA
Day 0
CNA
Control
ENA
Inflammatory
Day 0
CNA
Control
ENA
Degenerative
Day 0
CNA
Control
ENA
Day 0
CNA
Control
ENA
Day 0
CNA
Control
Day 0
0
Cytoplasmic
Pathological Alteration
Fig. 6. Total (total liver) and categorical (proliferative, degenerative, inflammatory, structural, cytoplasmic) liver pathological indices (mean7SEM)
of yellow perch exposed to control and NA treatments, with (1 g/L) and without salt (n ¼ 9, except for the ENA+salt treatment, where n ¼ 6). Also
included are the baseline levels (day 0) of liver alterations of yellow perch from MLR.
tants. NAs have properties similar to those of surfactants (Brient et al., 1995) and therefore may respond in a
similar fashion. A proliferation of chloride cells is
considered a general stress response in fish exposed to
many different classes of toxicants such as metals
(Oronsaye and Brafield, 1984) and organics from a
petrogenic source (van den Heuvel et al., 2000), but
since they are also key players in maintaining ionic
balance in fish (Perry, 1997), it is not known whether the
NAs, the salt, or both are contributing to the proliferation of chloride cells in this study.
Although changes to gill structure following exposure
to NAs and salinity may be advantageous for fish by
reducing toxicant entry and acute lethality compared to
those for NAs alone, there is concern over the
consequences this may have for important physiological
functions of the gills, most notably gas exchange. As a
result of the reduced gill surface area, the efficiency of
gas exchange will be severely impacted and may lead to
long-term issues in terms of fish health. Although this
was a short-term (3-week) exposure, there is evidence to
suggest that gill cell proliferation is a long-term response
based on the prevalence of gill epithelial cell proliferation
in yellow perch following 3- and 10-month exposures in
reclaimed environments with elevated levels of NAs
(9 mg/L) and salinity (van den Heuvel et al., 2000).
Mortality and histopathological results from this
current study indicate that YOY yellow perch exposed
to the combination of ENA (1.7 mg/L) and salinity
(0.9 ppt) under laboratory conditions were more sensitive than in field studies exposing adult yellow perch and
goldfish to reclaimed waters with elevated levels of NAs
(24 mg/L) and salinity (1.4 ppt) (Nero et al., in press).
The differing sensitivity may be attributed to differences
in the chemical composition between naturally degraded
NAs (12 years) occurring in reclaimed environments
(Holowenko et al., 2002) and extracted NAs from
relatively fresh oil sands tailings. This means that given
sufficient residence times in future aquatic reclamation
systems, the natural aging and biodegradation processes
that will proceed on the NA group of compounds will
mitigate potentially toxic and sublethal effects to fish
populations. However, the time required for this process
to occur is not known and there may be instances where
fish will be exposed to varying compositions of NAs,
depending on the conditions and status of reclaimed
environments. This will result in different responses by
fish, not to mention potential changes in the modifying
effects salinity contributes to water containing NAs of
varying composition as shown in this study (the addition
of 1 g/L salt increased gill toxicity to ENA, but had no
effect on the CNA).
4.3. Sublethal effects of NAs and NA+salt mixtures on
liver pathology
There were no significant changes in categorical or
total liver pathological indices for YOY yellow perch
exposed to individual NAs or NA+salt mixtures at low
NA concentrations (1.7 mg/L ENA; 0.9 mg/L CNA).
Similarly, no changes in liver pathology were found for
adult yellow perch exposed (3-week) to degraded NAs in
oil sands reclaimed aquatic environments containing
1.4–3.6 mg/L NAs and 0.2 ppt salinity; however, at
higher concentrations (24 mg/L NAs and 1.4 ppt salinity) yellow perch had significant degenerative and
ARTICLE IN PRESS
12
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
inflammatory alterations which led to a significant
increase in liver pathology (Nero et al., in press).
The fact that there were no significant liver alterations
in NA+salt treatments may be a function of reduced
NA uptake due to the increased sensitivity of the gills of
YOY yellow perch to NAs and salinity. The observed
changes to the gill surface area using gill morphometric
analysis (PAGE, SLW, ID) in NA and NA+salt
exposed fish may potentially reduce water flow over
the gills and gill perfusion. This reduction has been
implicated with limiting gill uptake of organic chemicals
(Schmieder and Weber, 1992), which may lead to a
decreased binding and entry of NAs into the vascular
system and transport to the liver.
5. Conclusions
YOY yellow perch were highly sensitive to NA
exposure, with 100% mortality at 3.6 mg/L CNA and
6.8 mg/L for the ENA. At lower NA concentrations
(CNA, 1.8 mg/L; ENA, 3.4 mg/L), the addition of 1 g/L
of salt reduced the NA toxicity by 40–50% from levels
seen with no salt addition. CNA was shown to be more
acutely toxic compared to ENA, which may be a
function of the differences in the relative composition
of the NA group of compounds (C-number and Zvalue) in the CNA and ENA. Yellow perch exposed to
NAs and mixtures of NAs+salt showed a high level of
gill proliferative alterations, in the form of epithelial,
chloride and mucous cell proliferations. Quantifying
these proliferative alterations using gill morphometrics
revealed significantly greater changes to the secondary
lamellar morphology of yellow perch exposed to the
NA+salt mixture as opposed to NA alone. These
changes had significant impacts on the gill surface area,
which may have consequences for fish health (gas
exchange) and NA uptake. Although there were signs
of degenerative and inflammatory alterations of the
liver, there were no significant differences in liver
pathology of NA or NA+salt exposed fish. There is
some indication that the addition of salt may have led to
differing levels of response, depending on the chemical
composition of NAs. This needs to be taken into
consideration in assessing the effects of oil sands
reclaimed environments of different ages.
Acknowledgments
Funding for this research was provided by the
Canadian Water Network, an NSERC Discovery Grant
to D.G.D and Syncrude Canada Ltd. Special thanks to
Neil Rutley, Joanne Hogg and others from the
Environmental Complex, Syncrude Canada Ltd. for
electrofishing support. Thanks to Phil Fedorak (Uni-
versity of Alberta) for use of software for NA analyses
and Shirley Chatten (University of Waterloo) for NA
analyses. Thanks to Tara Hayes and Dr. Ralph Smith
(University of Waterloo) for water chemistry data.
Thanks to Monalisa Elshayeb, Karla Spence and Chris
Bierling for additional laboratory assistance. Gill and
liver tissue slides were prepared at the Animal Health
Laboratory (University of Guelph).
References
Alberta Environmental Protection (AEP), 1996. Naphthenic Acids
Background Information, Discussion Report. Environmental
Criteria Branch, Environmental Assessment Branch, Environmental Regulatory Services, Alberta Environmental Protection.
Bernet, D., Schmidt, H., Meier, W., Burkhardt-Holm, P., Wahli, T.,
1999. Histopathology in fish: proposal for a protocol to assess
aquatic pollution. J. Fish. Dis. 22, 25–34.
Brient, J.A., Wessner, P.J., Doly, M.N., 1995. Naphthenic acids. In:
Kroschwitz, J.I. (Ed.), Encyclopedia of Chemical Technology,
fourth ed., vol. 16. Wiley, New York, pp. 1017–1029.
Clemente, J.S., 2004. The characterization, analyses and biodegradation of naphthenic acids. M.Sc. Thesis, University of Alberta,
Edmonton.
Clemente, J.S., Prasad, N.G.N., MacKinnon, M.D., Fedorak, P.M.,
2003a. A statistical comparison of naphthenic acids characterized
by gas chromatography-mass spectrometry. Chemosphere 50,
1265–1274.
Clemente, J.S., Tin-Wing, Y., Fedorak, P.M., 2003b. Development of a
high performance liquid chromatography method to monitor the
biodegradation of naphthenic acids. J. Environ. Eng. Sci. 2, 177–186.
Clemente, J.S., MacKinnon, M.D., Fedorak, P.M., 2004. Aerobic
biodegradation of two commercial naphthenic acids preparations.
Environ. Sci. Technol. 38, 1009–1016.
Craig, J.G., 1987. The Biology of Perch and Related Fish. Timber
Press, Portland, OR.
Dokholyan, B.K., Magomedov, A.K., 1984. Effect of sodium
naphthenate on survival and some physiological–biochemical
parameters of some fishes. J. Ichthyol. 23, 125–132.
Eisler, R., 1965. Some effects of synthetic detergent on estuarine fish.
Trans. Am. Fish. Soc. 94, 26–31.
Farwell, A., Dixon, D.G., 2001. Effects of salinity and water hardness
on naphthenic acid toxicity. SETAC 2001: Society of Environmental Toxicology and Chemistry, Baltimore, MD.
Hall, L.W., Anderson, R.D., 1995. The influence of salinity on the
toxicity of various classes of chemicals to aquatic biota. Crit. Rev.
Toxicol. 25, 281–346.
Harris, M., 2001. Aquatic Ecosystems Associated with Oil Sands
Development: Syncrude Canada’s Progress in Optimizing Freshwater Environments. Summary of the University of WaterlooSyncrude Canada Partnership, 1995–1998. University of Waterloo,
Waterloo, Ontario.
Head, A.B., Malison, J.A., 2000. Effects of lighting spectrum and
disturbance level on the growth and stress responses of yellow
perch, Perca flavescens. J. World. Aquacult. Soc. 31, 73–80.
Herman, D.C., Fedorak, P.M., MacKinnon, M.D., Costerton, J.W.,
1994. Biodegradation of naphthenic acids by microbial populations
indigenous to oil sands tailings. Can. J. Microbiol. 40, 467–477.
Holowenko, F.M., MacKinnon, M.D., Fedorak, P.M., 2002. Characterization of naphthenic acids in oil sands wastewaters by gas
chromatography-mass spectrometry. Water Res. 36, 1–13.
Hughes, G.M., Perry, S.F., 1976. Morphometric study of trout gills: a
light-microscope method suitable for the evaluation of pollutant
action. J. Exp. Biol. 64, 447–460.
ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]–]]]
Jivraj, M.N., MacKinnon, M., Fung, B., 1996. Naphthenic Acids
Extraction and Quantitative Analyses with FT-IR Spectroscopy.
Syncrude Analytical Methods Manual, fourth ed. Syncrude
Canada Ltd., Research Department, Edmonton.
Lai, J.W.S., Pinto, L.J., Kiehlmann, E., Bendell-Young, L.I., Moore,
M.M., 1996. Factors that affect the degradation of naphthenic
acids in oil sands wastewater by indigenous microbial communities.
Environ. Toxicol. Chem. 15, 1482–1491.
Lee, L.E.J., Haberstroh, K., Dixon, D.G., Bols, N.C., 2000. Salinity
effects on the toxicity of naphthenic acids to rainbow trout cell
lines. Canadian Technology Report of Fisheries and Aquatic
Science 2331:91, 27th Annual Aquatic Toxicology Workshop,
St. John’s, Newfoundland.
Levitan, W.M., Taylor, M.H., 1979. Physiology of salinity-dependent
naphthalene toxicity in Fundulus heteroclitus. J. Fish. Res. Board
Can. 36, 615–620.
MacKinnon, M.D., Boerger, H., 1986. Description of two treatment
methods for detoxifying oil sands tailings pond water. Water
Pollut. Res. J. Can. 21, 496–512.
Madill, R.E.A., Orzechowski, M.T., Chen, G., Brownlee, B.G., Bunce,
N.J., 2001. Preliminary risk assessment of the wet landscape option
for reclamation of oil sands mine tailings: bioassays with mature
fine tailings pore water. Environ. Toxicol. 16, 197–208.
Malison, J.A., Mellenthin, J., Head, A.B., Procarione, L.S., Barry,
T.P., Held, J.A., 1998. Cortisol stress responses and growth of
yellow perch (Perca flavescens) reared under selected intensive
culture conditions. Aquaculture Book of Abstracts, p. 344.
Nelson, L.R., Gulley, J.R., MacKinnon, M., 1995. Environmental
issues on reclamation of oil sands fine tails. In: Heavy Crude and
Tar Sands—Fueling for a Clean and Safe Environment. Alberta
Department of Energy, Oil Sands Research Division, pp. 705–718.
Nero, V., Farwell, A., Lister, A., Van Der Kraak, G., Lee, L.E.J., Van
Meer, T., MacKinnon, M.D., Dixon, D.G. Gill and liver
histopathological changes in yellow perch and goldfish exposed
to oil sands process-affected water. Ecotoxicol. Environ. Saf., in
press, doi:10.1016/j.ecoenv.2005.04.014.
Oronsaye, J.A.O., Brafield, A.E., 1984. The effect of dissolved
cadmium on the chloride cells of the gills of the stickleback,
Gasterosteus aculeatus. J. Fish. Biol. 25, 253–258.
13
Partearroyo, M.A., Pilling, S.J., Jones, M.N., 1992. The effects of
surfactants on the permeability of isolated perfused fish gills to
urea. Comp. Biochem. Physiol. A 101, 653–659.
Patrick, R., Cairns, J., Scheier, A., 1968. A comparison of the toxicity
of some common industrial waste components tested individually
and combined. Prog. Fish. Cult. 30, 3–8.
Perry, S.F., 1997. The chloride cell: structure and function in the gills
of freshwater fishes. Ann. Rev. Physiol. 59, 325–347.
Perry, S.F., Laurent, P., 1993. Environmental effects on fish gill
structure and function: recent advances and future directions. In:
Jensen, F., Rankin, C. (Eds.), Fish Ecophysiology. Chapman &
Hall, London, pp. 231–264.
Roy, D., 1988. Statistical analysis of anionic detergent-induced
changes in the goblet mucous cells of opercular epidermis and gill
epithelium of Rita rita (Ham.) (Bagridae: Pisces). Ecotoxicol.
Environ. Saf. 15, 260–271.
Schmieder, P.K., Weber, L.J., 1992. Blood and water flow limitations
on gill uptake of organic chemicals in the rainbow trout
(Onchorynchus mykiss). Aquat. Toxicol. 24, 103–122.
Schramm, L.L., Stasiuk, E.N., MacKinnon, M., 2000. Surfactants in
Athabasca oil sands slurry conditioning, flotation recovery, and
tailings processes. In: Schramm, L.L. (Ed.), Surfactants: Fundamentals and Applications in the Petroleum Industry. Cambridge
University Press, Cambridge, UK, pp. 365–430.
Siwik, P.L., Van Meer, T., MacKinnon, M.D., Paskowski, C.A., 2000.
Growth of fathead minnow in oilsands processed wastewater in
laboratory and field. Environ. Toxicol. Chem. 19, 1837–1845.
St. John, W.P., Rughani, J., Green, S.A., McGinnis, G.D., 1998.
Analysis and characterization of naphthenic acids by gas chromatography-electron massspectrmetry of tert.-butyldimethylsilyl derivatives. J. Chromatogr. A 807, 241–251.
van den Heuvel, M.R., Power, M., Richards, J., MacKinnon, M.D.,
Dixon, D.G., 2000. Disease and gill lesions in yellow perch (Perca
flavescens) exposed to oil sands mining-associated waters. Ecotoxicol. Environ. Saf. 46, 334–341.
Verbeek, A.G., Mackay, W.C., MacKinnon, M.D., 1994. A toxicity
assessment of oil sands wastewater: a toxic balance. Canadian
Technical Report of Fisheries and Aquatic Sciences, No. 1989,
pp. 196–207.