ARTICLE IN PRESS

Ecotoxicology and Environmental Safety 65 (2006) 252–264

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
b
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

Received 8 September 2004; received in revised form 3 June 2005; accepted 9 July 2005
Available online 29 August 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-the-
year 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 used in commercial operations, produced waters gen-

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: vnero@sciborg.uwaterloo.ca (V. Nero).
erally 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 aro-
matic hydrocarbons (PAHs). The Microtox bacterial

0147-6513/$ - see front matter r 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2005.07.009
 ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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 chromato-
graphy–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 Formula Structure
O
C3H6O2
Z=0
(if x =1 methyl group) [ ]x
C8H14O2
Z=-2
(if x = 1 methyl group) [ ]x
C11H18O2
Z=-4
(if x = 1 methyl group)
C14H2202
Z=-6
(if x = 1 methyl group)
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.
253
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 parti-
cularly 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 process-
affected 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 degen-
eration) 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.
OH
The purpose of this study was to investigate the effects
of NAs alone and in conjunction with salinity on young-
O of-the-year (YOY) yellow perch gill and liver histo-
pathology under controlled laboratory conditions, to
OH
determine if salinity is an important modifying factor
for NA toxicity. Two NA mixtures (CNA; commercial
O
NA sodium salts; ENA, extracted NA from oil sands
[ ]x OH
process-affected water) with different distributions of
carbon numbers and Z families were tested to determine
O
if differences in NA composition affect gill and liver
[ ]x OH 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
254 V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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 hydrocar-
bons. 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 0.45-
mm 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
(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 recon-
stituted 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 commer-
cial standard has two absorbance peaks at 1743 and
1706 cm
1 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 corre-
sponded 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 pre-
pared (200 g/L) as required. Treatment solutions, pre-
pared 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
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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 cur-
rent) (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) 20.6
Potassium (K+, mg/L) BDL
Magnesium (Mg2+, mg/L) 10.2
Calcium (Ca2+, mg/L) 32
Anions Sulfate (SO2

4 , mg/L) 27.7
Chloride (Cl
, mg/L) 8.1
Carbonate (CO2
3 , mg/L) 2.2
Bicarbonate (HCO
3 , mg/L) 143.3
Salinity (ppt)a 0.24
Organics Naphthenic acid (NA, mg/L) 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

CO2
2


3 , SO4 , and Cl ) in mass per liter.
255
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 Scienti-
fic). 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 indepen-
dent 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 altera-
tion 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
3,
percentage of gill filaments or liver areas affected
determined the score value (0–6) for a particular
 ARTICLE IN PRESS
256 V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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 Description of general responsea
Proliferative Increase in the number of specific
cell types or structures
Degenerative Breakdown of tissue and/or cells
Inflammatory Increased presence of cells used in
tissue repair; response to damaged
tissue
Structural Changes in tissue architecture
Cytoplasmic Alterations in cellular volume, size
or storage products
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 impor-
tance 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 micro-
scope 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 thick-
ness (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
Examples of specific tissue alterations
Gills Liver
Chloride cell proliferation (2) Bile duct proliferation (2)
General necrosis (3) Hepatocellular necrosis (3)
Epithelial lifting (lamellar oedema) (1) Lymphocytic infiltration (2)
Lamellar tip fusion (2) Increased separation of hepatocytes (1)
Mucous cell hypertrophy (1) Hepatocyte hypertrophy (1)
filament appearing in the photomicrograph: two mea-
surements 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) rela-
tive 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
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264 257

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) 18.970.3 8.3370.08 30174 0.1 8.2770.1

NaOH (5 mg/L) Control 18.970.2 8.2170.05 666724 0.3 8.6570.07
Salt control (Na2SO4) (1 g/L) 18.470.2 8.5570.07 1672724 0.8 8.8970.06
CNA (0.9 mg/L) 18.570.2 8.3670.03 38072 0.2 8.9170.09
CNA (1.8 mg/L) 18.570.2 8.4070.04 46174 0.2 8.8970.08
CNA (3.6 mg/L) 18.470.2 (7) 8.3870.05 (7) 60874 (7) 0.3 (7) 8.9270.08 (7)
CNA (0.9 mg/L) +salt (1 g/L) 18.570.2 8.4670.03 176376 0.9 8.7770.11
CNA (1.8 mg/L) +salt (1 g/L) 18.570.2 8.3470.06 185178 0.9 8.8470.08
CNA (3.6 mg/L) +salt (1 g/L) 17.870.4 (3) 8.3070.10 (3) 197275 (3) 1.0 (3) 8.9770.28 (3)
ENA (1.7 mg/L) 18.770.2 8.2470.05 38773 0.2 8.7670.1
ENA (3.4 mg/L) 18.770.2 8.2670.06 46872 0.2 8.8170.11
ENA (6.8 mg/L) 17.670.3 (2) 8.1970.15 (2) 654716 (2) 0.3 (2) 8.9370.19 (2)
ENA (1.7 mg/L) +salt (1 g/L) 18.670.3 8.4070.09 1734720 0.9 8.8570.12
ENA (3.4 mg/L) +salt (1 g/L) 18.770.3 8.3470.07 1833716 0.9 8.7470.11
ENA (6.8 mg/L) +salt (1 g/L) 17.4 (2) 8.2970.15 (2) 1920762 (2) 1.0 (2) 8.90 (2)

increased based on additions of NaOH, salt (Na2SO4),

and/or NA solutions. 10

3.2. NA characterization 8
Percentage

6
The commercial and extracted NAs tested in the
Group % NAs
yellow perch exposures were characterized by GC–MS. 4 C5 to 13 28
Figs. 2a and b show the matrix of the possible NAs C14 to 21
C22 to 33
55
17
within the carbon number range 5–33, with 0–6 rings 2
Z0 to –4 61
(Z ¼ 0 to
12) for ENA and CNA, respectively. For 0 Z-6 to –12 39
comparison of NA mixtures, the matrix of possible NAs 5
7
was divided between carbon numbers 5–13, 14–21, and (a) 9
11
22–33 and Z families 0 to
4 and
6 to
12 as 13
15
described by Clemente et al. (2003a). The most Ca 17
rbo 19
nn 21 12
abundant NAs in the ENA had carbon numbers 14–21 um
ber 23 25
6
(55%) and Z families 0 to
4 (61%). The most 27

ily
fam Z
29 0

-
abundant NAs in the CNA had carbon numbers 5–13 10 31
33
(48%) and Z families 0 to
4 (84%). The ENA stock
8
had a significantly greater proportion of NAs with
carbon numbers 14–21 (P ¼ 0:040) and 22–33 6
Percentage

(Po0:001) and Z families
6 to
12 (X3 carbon rings) Group % NAs

(Po0:001) than the CNA stock. 4 C5 to 13 48
C14 to 21 40
C22 to 33 12
2
3.3. Mortality and organismal indices Z0 to –4 84
0 Z-6 to –12 16

5
Minimal mortality (10%) was observed for the 7
9
11
controls (Mildred Lake control, NaOH control) and (b) 13
15
salt (1 g/L) treatment. For both ENA and CNA Ca
17
19
rbo 21 12
treatments, there was 100% mortality at the highest nn
um 23
ber 25 6
test concentrations (ENA 6.8 mg/L; CNA 3.6 mg/L) 27
29 0
ily
fam Z

within 96 h of exposure. At lower concentrations (ENA 31

-

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 concentra-
tions 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.
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
258 V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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-
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 ex-
posed 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
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264 259

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 morpho-
metric 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 com-
pared to the ENA treatment.
3.7. Liver pathology
Yellow perch exposed to NA treatments had slightly
higher levels of degenerative, inflammatory, and struc-
tural 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
260 V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264

24
b
22
20
18 b No Salt
ab Salt (1g/l)
Pathological Index
16
b
14
*
12 a
10 * *
8 a

6
4
* *
2
0
Control

Control

Control

Control

Control

Control
CNA

CNA
Day 0

Day 0

Day 0

Day 0

Day 0

Day 0
CNA
ENA

CNA

ENA

CNA
ENA

ENA

ENA

CNA

ENA
Total Gill Proliferative Degenerative Inflammatory Structural 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 Anderson, 1995). Greater mortality was reported for
pathological index for NA treatments relative to naphthalene-exposed mummichog (Fundulus heterocli-
NA+salt treatments (Fig. 6). tus) (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
4. Discussion salinity compared to the intermediate salinities tested.
Although the salinity tested in this study was lower than
4.1. Lethal effects of NAs and NA+salt mixtures the isosmotic salinity reported for freshwater fish
(10 ppt; Craig, 1987), there may be some advantage in
Exposure of YOY yellow perch to NAs resulted in terms of reduced osmotic stress, which may in part
100% mortality in p96 h at the highest test NA contribute to the reduction in acute toxicity.
concentrations (CNA, 3.6 mg/L; ENA, 6.8 mg/L). The The acute toxicity of the CNA (80% mortality) to
response of YOY yellow perch was similar to that of yellow perch was higher than that of the ENA (0%
other fish species including rainbow trout (Oncorhynchus mortality) at lower concentrations (1.8 mg/L CNA,
mykiss) fingerlings (96 h LC50p10 mg/L extracted NA 1.7 mg/L ENA). The NAs tested in this study were
from MLSB, Syncrude Canada Ltd.) (Verbeek et al., found to have a significantly different composition,
1994), juvenile chum salmon (Oncorhynchus keta) (96 h which likely influenced the NA toxicity. Fig. 2 shows
LC50 ¼ 12 mg/L commercial NA, source unknown) and that the ENA had a greater proportion of C22–C33
juvenile kutum (Rutilus frisii) (LC100 ¼ 6.5 mg/L com- (17% vs. 12%) and less of the lowest carbon number
mercial NA, source unknown) (Dokholyan and Mago- group, C5–C13 (28% vs. 48%) compared to the CNA.
medov, 1984). Holowenko et al. (2002) also found that NAs composed
Similar to NA exposure, NA+salt mixtures resulted of a greater proportion of higher carbon numbers
in 100% mortality (p96 h) at 3.6 mg/L CNA and (‘‘+C22 group’’) were less toxic than NAs with a higher
6.8 mg/L ENA. However, at lower concentrations, proportion of the lower carbon numbers (‘‘
C22+
mortality was reduced by 40% for CNA (1.8 mg/L) group’’) using the Microtox bacterial assay. In field
and 50% for ENA (3.4 mg/L) with the addition of 1 g/L studies, Nero et al. (in press) and Siwik et al. (2000)
Na2SO4 (Fig. 3). Results from various studies suggest observed minimal or no mortality of caged goldfish and
that fish species are more resistant to acutely toxic larval fathead minnows, respectively, following expo-
conditions living in or exposed to isosmotic salinity (to sure to reclaimed environments containing NAs with a
fish blood) compared to both higher and lower salinity greater distribution of higher carbon numbers relative to
as a result of a minimization of osmotic stress (Hall and lower carbon numbers.
 ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264 261

No Salt Salt (1g/l)

18 a 18 No Salt Salt (1g/l)
a
b
15 ab 15 b ab
b ab
b a a
12 b 12

SLW (µm)
ID (µm)

9 9

6 6

3 3

0 0
Day 0 Control CNA (0.9 mg/l) ENA (1.7 mg/l) Day 0 Control CNA (0.9 mg/l) ENA (1.7 mg/l)

(a) Treatments (b) Treatments

120 No Salt Salt (1g/l) 35 b

No Salt Salt (1g/l)
b
100 30 ab a
a a
25 a a
80 b
SLL (µm)

BET (µm)
20
60
15
40
10
20
5

0 0
Day 0 Control CNA (0.9 mg/l) ENA (1.7 mg/l) Day 0 Control CNA (0.9 mg/l) ENA (1.7 mg/l)
(c) Treatments (d) Treatments

85
No Salt Salt (1g/l)
a a
80
a
b
b
75
PAGE (%)

b
70

65

60

55
Day 0 Control CNA (0.9 mg/l) ENA (1.7 mg/l)
(e) 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 words, the gill surface area decreased significantly
gill pathology (PAGE, 10% reduction compared to the controls)
which will inevitably impact the high efficiency of the
Although wide ranges of alterations were observed in fish gills. It is apparent from the gill sections (Fig. 4) that
this study, the predominant gill response of fish exposed the epithelial cell proliferation, epithelial lifting, and
to NAs and NA+salt mixtures (CNA and ENA) was a lamellar tip fusion of the ENA+salt exposed fish are a
proliferation of epithelial, chloride, and mucous cells. In protective response of the gill to reduce toxicant entry.
addition to proliferative alterations, the addition of low The elevated mucous cell production has also been
levels of salt (1 g/L sodium sulfate) to the ENA mixture implicated in reducing toxicant entry. Since mucus
caused a significant increase in inflammatory (epithelial contains elevated levels of sugars and proteins that
lifting) and structural (lamellar tip fusion) alterations carry an electrical potential capable of trapping tox-
compared to the control treatments. Based on gill icants (Perry and Laurent, 1993), it is possible that a
morphometrics fish gills had thicker (SLW) and shorter proliferative response could reduce the permeability of
(SLL) secondary lamellae, a reduced distance between the gill to NAs. This protective effect has been observed
secondary lamellae (ID) and a larger gill epithelium at in isolated rainbow trout (Partearroyo et al., 1992) and
the base of the secondary lamellae (BET). In other catfish (Rita rita) (Roy, 1988) gills exposed to surfac-
 ARTICLE IN PRESS
262 V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264

18

16

14 No Salt
Pathological Index Salt (1g/l)
12

10

0
Control

Control

Control

Control

Control

Control
CNA

CNA
Day 0

Day 0

Day 0

Day 0

Day 0

Day 0
CNA
ENA

CNA
ENA

CNA
ENA

ENA

ENA

CNA
ENA
Total Liver Proliferative Degenerative Inflammatory Structural 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 surfac- The differing sensitivity may be attributed to differences
tants (Brient et al., 1995) and therefore may respond in a in the chemical composition between naturally degraded
similar fashion. A proliferation of chloride cells is NAs (12 years) occurring in reclaimed environments
considered a general stress response in fish exposed to (Holowenko et al., 2002) and extracted NAs from
many different classes of toxicants such as metals relatively fresh oil sands tailings. This means that given
(Oronsaye and Brafield, 1984) and organics from a sufficient residence times in future aquatic reclamation
petrogenic source (van den Heuvel et al., 2000), but systems, the natural aging and biodegradation processes
since they are also key players in maintaining ionic that will proceed on the NA group of compounds will
balance in fish (Perry, 1997), it is not known whether the mitigate potentially toxic and sublethal effects to fish
NAs, the salt, or both are contributing to the prolifera- populations. However, the time required for this process
tion of chloride cells in this study. to occur is not known and there may be instances where
Although changes to gill structure following exposure fish will be exposed to varying compositions of NAs,
to NAs and salinity may be advantageous for fish by depending on the conditions and status of reclaimed
reducing toxicant entry and acute lethality compared to environments. This will result in different responses by
those for NAs alone, there is concern over the fish, not to mention potential changes in the modifying
consequences this may have for important physiological effects salinity contributes to water containing NAs of
functions of the gills, most notably gas exchange. As a varying composition as shown in this study (the addition
result of the reduced gill surface area, the efficiency of of 1 g/L salt increased gill toxicity to ENA, but had no
gas exchange will be severely impacted and may lead to effect on the CNA).
long-term issues in terms of fish health. Although this
was a short-term (3-week) exposure, there is evidence to 4.3. Sublethal effects of NAs and NA+salt mixtures on
suggest that gill cell proliferation is a long-term response liver pathology
based on the prevalence of gill epithelial cell proliferation
in yellow perch following 3- and 10-month exposures in There were no significant changes in categorical or
reclaimed environments with elevated levels of NAs total liver pathological indices for YOY yellow perch
(9 mg/L) and salinity (van den Heuvel et al., 2000). exposed to individual NAs or NA+salt mixtures at low
Mortality and histopathological results from this NA concentrations (1.7 mg/L ENA; 0.9 mg/L CNA).
current study indicate that YOY yellow perch exposed Similarly, no changes in liver pathology were found for
to the combination of ENA (1.7 mg/L) and salinity adult yellow perch exposed (3-week) to degraded NAs in
(0.9 ppt) under laboratory conditions were more sensi- oil sands reclaimed aquatic environments containing
tive than in field studies exposing adult yellow perch and 1.4–3.6 mg/L NAs and 0.2 ppt salinity; however, at
goldfish to reclaimed waters with elevated levels of NAs higher concentrations (24 mg/L NAs and 1.4 ppt sali-
(24 mg/L) and salinity (1.4 ppt) (Nero et al., in press). nity) yellow perch had significant degenerative and
 ARTICLE IN PRESS
V. Nero et al. / Ecotoxicology and Environmental Safety 65 (2006) 252–264
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 Z-
value) 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-
263
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).
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