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First published online February 20, 2004
Journal of Experimental Biology 207, 1249-1261 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.00871
The physiological consequences of exposure to chronic, sublethal waterborne nickel in rainbow trout (Oncorhynchus mykiss): exercise vs resting physiology
1 Department of Biology, McMaster University, Hamilton, Ontario, Canada, L8S
4K1
2 Department of Biological Sciences, University of Alberta, Edmonton,
Alberta, Canada, T6G 2E9
* Author for correspondence (e-mail: michanderic{at}yahoo.com)
Accepted 9 January 2004
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We present evidence for a clear, persistent cost of acclimation to chronic,
sublethal Ni exposure. Chronic (40–99 day) exposure to sublethal
waterborne Ni (243–394 µg Ni l–1; 
1% of the 96
h LC50) impaired the exercise physiology, but not the resting
physiology, of rainbow trout. Ni acted as a limiting stressor, decreasing
maximal rates of oxygen consumption
(
)
during strenuous exercise in trout exposed for 34 days to sublethal Ni. This
drop in high-performance gas exchange was attributed mainly to a reduction in
relative branchial diffusing capacity (Drel) caused by
thickening of secondary lamellae. Morphometric analysis of the gills of
chronically exposed fish revealed overall swelling of secondary lamellae, as
well as hypertrophic respiratory epithelia within secondary lamellae.
Additionally, contraction of the lamellar blood pillar system and narrowing of
interlamellar water channels occurred, possibly contributing to decreased
high-performance gas exchange. Decreased aerobic capacity persisted in fish
previously exposed to nickel despite a clean-water exposure period of 38 days
and an almost complete depuration of gill Ni, suggesting that extrabranchial
mechanisms of chronic Ni toxicity may also be important.
Chronic impairment of such a dynamically active and critical organ as the gill may depress the overall fitness of a fish by impairing predator avoidance, prey capture and migration success with obvious environmental implications.
Key words: nickel toxicity, acclimation, exercise, gill, Oncorhynchus mykiss
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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), Al (Playle et al.,
1989), Ag (Wood et al.,
1996), Mo (Reid,
2002) and Ni (Pane et al.,
2003). These studies provide valuable information for modeling the
acute toxicity of trace metals by incorporating a component of the unique
physiological impact of each toxicant (e.g. the biotic ligand model, or BLM;
see Di Toro et al., 2001 and
Paquin et al., 2002 for
detailed explanations of this model).
An obvious limitation of this acute diagnostic approach, however, is that
the concentrations used in these studies are often environmentally
unrealistic. In the case of acute waterborne Ni exposure, adverse effects on
gill ultrastructure and respiration in freshwater fish have been investigated
using concentrations of 3200 µg Ni l–1
(Hughes and Perry, 1976;
Hughes et al., 1979; rainbow
trout), 11 700 µg Ni l–1 (Pane et al., 2003a; rainbow
trout), 10 700 µg Ni l–1 (E. F. Pane, A. Haque and C. M.
Wood, submitted; rainbow trout) and 14 000 µg Ni l–1
(Nath and Kumar, 1989;
Colisa fasciatus). These acute concentrations are far higher than
concentrations of Ni found in contaminated freshwaters (typically <500
µg l–1; Chau and
Kulikovsky-Cordeiro, 1995;
Eisler, 1998). Additionally,
the information gained typically comes from animals completely out of balance
with their environment and fighting a losing battle with a toxicant.
Chronic, sublethal exposure, however, allows one to investigate
steady-state conditions that exist between an aquatic organism and a toxicant
and the processes of acclimation leading to this steady state. When applied to
a general target tissue, the acclimation phenomenon can be divided temporally
into three phases: (1) the `shock', or damage, phase, during which the
morphology and physiology of the target tissue are disturbed, (2) a defense
phase, during which tissue-specific responses are mounted in an attempt to
decrease the rate of influx or accumulation of the toxicant and (3) a recovery
phase, during which compensation and repair occur to restore perturbed
physiological processes and increase resistance to the toxicant
(McDonald and Wood, 1993).
In the present study, we concentrated on the third phase and examined the
physiology of juvenile and adult rainbow trout following 40–99 days of
exposure to sublethal concentrations of waterborne Ni. Initially, a
concentration of 2034 µg Ni l–1 was used as a screening
tool to gauge the impact of a relatively high (partially lethal) chronic
concentration. This concentration is 13% of the 96-h LC50 for
juvenile trout in the same Hamilton city tap water
(Pane et al., 2003) and 6% of
the 96-h LC50 for adult trout
(Segner et al., 1994). While
such a concentration is probably greater than Ni concentrations measured in
the most heavily contaminated industrial sites
(Chau and Kulikovsky-Cordeiro,
1995; Eisler,
1998), it served as a reference point, as we know of only two
other studies that have examined the effects of chronic waterborne Ni exposure
on freshwater fish. Pickering
(1974) assessed the
reproductive effects of chronic Ni exposure, while Calamari et al.
(1982) examined the kinetics of
Ni accumulation. Neither study investigated physiological mechanisms.
Most of the experiments detailed herein were conducted at concentrations
between 243 µg Ni l–1 and 394 µg Ni
l–1. These concentrations fall within the range of Ni
concentrations found in watersheds heavily impacted by mining and industrial
activity (Chau and Kulikovsky-Cordeiro,
1995; Eisler,
1998), and the values are only approximately 2% and 1% of the 96-h
LC50 values for juvenile and adult trout, respectively. These
concentrations are entirely sublethal, and our goal was to extensively
characterize the physiology of rainbow trout chronically acclimated to this
range of waterborne Ni concentrations. Wilson et al.
(1994) conducted similar
chronic, sublethal exposures with rainbow trout and Al and showed increased
energy expenditures associated with exposure. Therefore, we also set out to
document whether similar costs of acclimation occurred during chronic,
sublethal Ni exposure.
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1 mmol
l–1, Mg2+ 0.2 mmol l–1,
Na+ 0.6 mmol l–1, Cl–
0.8 mmol l–1, SO42–
0.25 mmol l–1, titratable alkalinity to pH 4.0 1.9
mmol l–1, background Ni 4 µg l–1,
dissolved organic carbon (DOC) 3 mg l–1, total hardness
(as CaCO3) 140 mg l–1 and pH
7.9–8.0. Fish were starved at least 24 h prior to and throughout all
experiments.
Chronic exposure conditions
In all exposures, Ni was delivered as NiSO4.6H2O by
gravity feed from a concentrated stock solution in a flowthrough set-up with
dechlorinated Hamilton tap water. Three exposure regimes were used: (1)
juvenile trout (20–50 g) were exposed to either control, 384 µg Ni
l–1 or 2034 µg Ni l–1 for 42 days, (2)
adult trout (200–350 g) were exposed to either control or 243 µg Ni
l–1 for 40 days and (3) juvenile trout (10–20 g) were
exposed initially to either control or 394 µg Ni l–1 for
99 days, followed by 38 days of exposure (of both groups of fish) to clean
water. In all experiments, fish were fed 1% of their body mass daily. The
composition of the food was: crude protein 40%, crude fat 11%, crude
fiber 3.5%, Ca 1.0%, P 0.85%, Na 0.45% and Ni 3.86 mg
kg–1 dry mass. Water samples for analysis of dissolved Ni
were taken every other day, 0.45-µm filtered, acidified with trace metal
grade HNO3 (Fisher Scientific, Nepean, ON, Canada) and analyzed for
dissolved Ni by graphite furnace atomic absorption spectrophotometry (GFAAS;
220 SpectrAA; Varian, Palo Alto, CA, USA) against certified atomic absorption
standards (Fisher Scientific). Atomic absorption values were normalized to an
independent reference standard (Fisher Scientific) interspersed at every 10
samples. The accepted recovery limits of this reference standard were
90–110%.
Sampling protocols–Experiment 1
On days 12 and 24 of the exposure, critical swimming speed
(Ucrit) was determined in fish exposed to either control,
384 µg Ni l–1 or 2034 µg Ni l–1. The
evening before the swim trial, fish were transferred in groups of five (all
from the same exposure tank) to a large (150 liter) swim respirometer and
left overnight in clean, flowing dechlorinated Hamilton city tap water. Fish
were then swum in clean water at 1-h intervals, increasing water speed by 7 cm
s–1 during each interval, until exhaustion (as determined by
each fish being impinged on the rear screen of the respirometer and refractive
to physical stimulation). Fork length was measured to the nearest 0.1 cm, and
individual Ucrit values were calculated according to the
formula of Brett (1964):
 | (1) |
V is
the speed increment in cm s–1. Individual
Ucrit values were then converted to body lengths per
second (BL s–1). On day 42, fish in all three treatments were euthanized by an overdose of MS-222 and placed on ice. A blood sample was then taken by caudal puncture and, following brief centrifugation (14 000 g for 1 min), the plasma was frozen in liquid nitrogen and stored at –80°C for later analysis. Tissues surgically removed to measure Ni concentration included the gills, heart, liver, stomach, intestine, kidney and white muscle. After tissues were digested at 60°C for 48 h in trace metal grade 1 mol l–1 HNO3, the digest was homogenized by vortexing, centrifuged at 14 000 g for 10 min and the supernatant diluted with double-distilled water for Ni analysis by GFAAS as described above.
Prior to all analyses, plasma samples were sonicated on ice for 5 s at 5 W
(Microson; Misonix Inc, Farmingdale, NY, USA) to ensure homogeneity. Plasma
[Na+], [Ca2+] and [Mg2+] were determined by
flame atomic absorption spectrophotometry (FAAS; 220FS SpectrAA; Varian),
while plasma [Cl–] was measured by the mercuric thiocyanate
method (Zall et al., 1956).
Plasma [Ni] was determined by GFAAS as described above. Plasma protein was
determined using Bradford reagent
(Bradford, 1976) and bovine
serum albumin standards (Sigma-Aldrich, St Louis, MO, USA). Plasma total
ammonia and lactate concentrations were determined enzymatically (glutamate
dehydrogenase/NADP and L-lactate dehydrogenase/NADH, respectively;
Sigma-Aldrich). Prior to analysis, plasma for lactate was deproteinized in two
volumes of 6% perchloric acid (Milligan
and Wood, 1986). Plasma cortisol was determined using an
125I radioimmunoassay (ICN Biomedicals, Montreal, QC, Canada) with
radioactivity measured by 
counting (Minaxi ; Canberra-Packard,
Meriden, CT, USA).
Sampling protocols–Experiment 2
On day 40, adult trout (200–350 g) chronically exposed to either
control or 243 µg Ni l–1 were anesthetized with 0.075 g
l–1 of MS-222 (neutralized with NaOH; pH 8.0) and fitted with
indwelling dorsal aortic catheters (Soivio
et al., 1972). During surgery, the anesthetic solution irrigating
the gills of chronically Ni-exposed fish was spiked with
NiSO4.6H2O to yield an Ni concentration comparable to
that to which these fish had been chronically exposed. Post surgery, fish were
transferred to individual darkened Plexiglas chambers (3 liter) served with a
water flow of 100 ml min–1 and continuous aeration and
allowed to recover for 48 h prior to sampling on day 42. Boxes housing
chronically Ni-exposed fish received a comparable Ni solution delivered from a
stock solution by gravity flow as described above.
After recovery, control and experimental fish (N=9; both
treatments) were sampled once for the various parameters shown in
Table 1. The sampling protocol
closely followed that of Wood et al.
(1996), as described in Pane
et al. (2003a). Each fish was sampled as follows: ventilation rate was counted
visually and then water samples from in front of the mouth of each fish were
filtered (0.45 µm) and analyzed for dissolved Ni by GFAAS as described
above. Unfiltered water samples were then taken for inspired O2
tension (PIO2) and inspired pH
(pHI). Blood (1 ml) was drawn anaerobically via the
arterial catheter into an ice-cold, Li-heparinized (50 i.u.
ml–1; Sigma-Aldrich), gas-tight Hamilton syringe for analysis
of arterial blood pH (pHa), O2 tension (PaO2),
plasma total CO2 (CaCO2), hematocrit (Ht),
blood hemoglobin (Hb) and plasma concentrations of lactate, protein, cortisol,
total ammonia and water content. Plasma was separated by centrifugation at 14
000 g for 1 min, and erythrocytes were reserved for
determination of water content.
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At the end of the experiment, fish were euthanized with an overdose of MS-222, and a piece of gill tissue (approximately 50 filaments) was trimmed off the central portion of the second gill arch on the left side of the fish, wrapped in foil, frozen in liquid nitrogen and stored at –20°C for later analysis of gill [Ni]. Additionally, a sample of white muscle was taken for determination of water content.
Analytical methods–Experiment 2
For the analyses of pHa, PaO2, water pHI and
PIO2, we used Radiometer electrodes and meters,
similar to those used by Wood et al.
(1988), thermostatically set
to the experimental temperature. Hb was determined by the colorimetric
cyanmethemoglobin method (Sigma-Aldrich reagents). Plasma for
CaCO2 was obtained by centrifuging whole blood (5000
g for 30 s) in ammonium-heparinized microhematocrit tubes in
duplicate. Ht was measured directly from the tubes, while
CaCO2 was analyzed on true plasma using a Corning 965
CO2 analyzer (Corning Life Sciences, Acton, MA, USA). Plasma
protein, total ammonia, lactate and cortisol concentrations were determined as
described above. Water content of plasma, erythrocytes and white muscle was
determined by pre- and post-weighing samples after drying to a constant mass
in a 70°C oven.
Calculations–Experiment 2
Calculations of PaCO2 and plasma
HCO3– based on measured pHa and
CaCO2 were identical to those described in
Playle et al. (1989) using the
Henderson–Hasselbach equation and values for CO2 solubility
(
CO2) and apparent pK (pK') at the appropriate
temperature from Boutilier et al.
(1984). Mean cellular
hemoglobin concentration (MCHC) was calculated as the ratio of simultaneous
measurements of Hb to Ht in whole blood samples and is expressed as g Hb
ml–1 of red blood cells (RBC).
Sampling protocols–Experiment 3
On days 0 (initial control), 9 and 34 of chronic Ni exposure (394 µg Ni
l–1) and on day 38 of subsequent exposure to clean water,
oxygen consumption of swimming fish was measured using a variation of a
technique described in Wilson et al.
(1994). Briefly, fish
(N=9; both treatments) were transferred the night before an
experiment to small Blazka-type swim respirometers (3.2 liter) served
overnight with a water flow of 300 ml min–1 and an
orientation velocity of 15 cm s–1 (approximately 1
BL s–1). Temperature control was achieved throughout
the experiment by submersing the respirometers in a wet table receiving a
constant flow of water. The overnight acclimation temperature was 15°C
and, over the 5 h needed to complete the respirometry experiment, the
temperature rose to 16.5°C due to increased thermal output by the
respirometers at greater r.p.m. This temperature increase, however, was
consistent across all respirometry trials involving both control and treated
fish and therefore should not have contributed greatly to differences in
oxygen consumption between treatments. On days 9 and 34, respirometers housing
experimental fish were served with a comparable Ni concentration delivered
from a stock solution by gravity flow as described above.
At each water velocity (increments of 5 cm s–1;
Wilson et al., 1994), oxygen
consumption was determined using a variation on a closed respirometry
technique. At the start of each hour, immediately following a water velocity
increase, the respirometers were opened to flowing water for 20 min toallow
for near saturation of water with oxygen. After 20 min, the respirometers were
sealed and an initial water sample was taken for partial pressure of oxygen
(PO2) followed 40 min later by a final water sample. The
process was continued until each fish was exhausted.
Immediately following exhaustion, fish were killed with an overdose of MS-222, weighed to the nearest 0.01 g, and fork length measured to the nearest 0.1 cm for calculation of individual Ucrit values as described above. On day 34 of Ni exposure and day 38 of clean water exposure, a gill sample was quickly removed and analyzed for Ni as described above.
At all times, oxygen consumption
()
was calculated according to the formula:
 | (2) |
O2 is the solubility co-efficient of oxygen in water
at the experimental temperature (Boutilier
et al., 1984), P is the difference in partial
pressures between initial and final water samples, V is the volume of
water, M is the mass of the fish and t is the time interval
in hours. The periodic opening of the system to flowing water kept the partial
pressure of oxygen in the water above 100 torr (13.3 kPa) at all times. Oxygen
consumption rates were corrected for `blank' oxygen consumption by the
experimental apparatus in the absence of fish. Oxygen was typically consumed
by the apparatus at a rate of 1.7–3.2 torr h–1,
depending on the individual respirometer. These values were approximately
15–30% of the lowest rates of oxygen consumption (11 torr
h–1) measured at the lowest swimming speed tested (15 cm
s–1).
For each fish, the log of oxygen consumption was plotted against swimming
speed (in BL s–1; see
Fig. 1). The regression line of
each fish was extrapolated back to 0 BL s–1 to yield
basal oxygen consumption
(
)
and out to the individual Ucrit value of each fish (see
Fig. 1), at which point oxygen
consumption was taken to be
(Wilson et al., 1994). Aerobic
scope for activity was calculated as the difference between
and
.
Only fish yielding a significant linear regression (P<0.05) of the
log of oxygen consumption vs swimming speed were included in the
analysis.
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Gill morphometric analysis–Experiment 3
After 69 days of Ni exposure, gills from control and experimental fish
(N=5; both treatments) were fixed for light microscopic examination
of morphometrics. Fish were netted and immediately euthanized by a blow to the
head. A large section of filaments (approximately 50) was cut away from the
second gill arch on the left side of the head, rinsed quickly with 0.1 mmol
l–1 Sorenson phosphate buffer
(Hayat, 1981) and placed in
neutral buffered formalin (NBF) for 1 h. The NBF was adjusted to pH 7.5 with
NaOH and vacuum filtered (0.45 µm). After 1 h, individual filaments were
placed in fresh NBF for 24 h at 4°C and then placed in tap water overnight
at 4°C. Filaments were then dehydrated in a graded alcohol series and
embedded (one filament per block) in Spurrs resin
(Hayat, 1981).
Tissue blocks were oriented along the axis of the gill filament to allow
for longitudinal (saggital) sectioning of the filament. Thick sections (1
µm) were cut with a Reichert Jung Ultracut microtome (Vienna, Austria) and
stained with Richardson's stain
(Richardson et al., 1960).
Sections were examined and digitally captured with a Leica DM IRBE inverted
microscope. Digitally captured images were adjusted for contrast only using
Adobe Photoshop 6.0 software.
Determination of the various volume ratios and blood–water diffusion
distances (BWDD: see Table 2)
closely followed techniques outlined by Hughes and Perry
(1976) and Hughes et al.
(1979). Briefly, a six-lined
anisotropic Merz grid was laid over images of sections magnified 715x,
and point counts were used to estimate relative volumes within secondary
lamellae (for details, see Hughes et al.,
1979). A typical section included a portion of an individual
filament body with 30–40 secondary lamellae. Measurements included the
volume of the lamellar region (VLR), as defined by the
area between the body of the filament and the distal tips of the lamellae, the
volume of the secondary lamellar tissue (VSL), the volume
of epithelial tissue lying outside the blood pillar system
(VOPS) and the volume of the pillar system
(VPS). The ratios tabulated were the portion of the
lamellar region occupied by secondary lamellae
(VSL/VLR) and the portion of the
secondary lamellae occupied by tissue outside the pillar system (blood
channels) (VOPS/VSL). The portion of
the lamellar region occupied by the pillar system
(VPS/VLR) was then calculated by:
 | (3) |
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BWDD were determined at the same magnification using the Merz grid to
randomize the measurement points (Wilson
et al., 1994). Distances were measured from the intersection of
the grid with the lamellar epithelium to the nearest erythrocytic surface. If
the path between the epithelial intersection and the nearest erythrocyte
crossed an empty blood channel, that measurement was discarded
(Hughes et al., 1979).
Each fish (N=5 per treatment) was assigned a mean value for each
parameter based on a total of approximately 200 point counts per individual
using three or four fields of view per section on two or three sections per
fish (Hughes et al.,
1979).
Additionally, a relative diffusing capacity (Drel;
Hughes et al., 1979) was
calculated as:
 | (4) |
). Because the reference area used (a Merz grid) was the same
for both control and experimental fish, a direct comparison can be made
between the two treatments, yielding relative diffusing capacities and a
comparison of relative surface areas available for gas exchange.
Statistical analyses
Data are presented as means ±
S.E.M. (N=number of fish). Where
appropriate (Figs 2,
3,
4,
5), experimental means (at two
Ni concentrations) were compared with control means using a one-way analysis
of variance (ANOVA) with a two-sided Dunnett's post-hoc multiple
comparison test. When only one Ni concentration was used (Figs
6,
8), experimental means were
compared with control means by an unpaired two-tailed Student's
t-test. Additionally, where appropriate
(Fig. 6), time-dependent
responses of both control and experimental fish were tested against respective
time 0 values by a one-way ANOVA with a two-sided Dunnett's post-hoc
multiple comparison test. The slopes and intercepts of group regression
equations (Fig. 7) were
compared as described by Zar
(1984). Statistical
significance in all cases was accepted at P<0.05.
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Chronic exposure to 384 µg Ni l–1 had no impact on plasma ion concentrations (Fig. 3). This low concentration of Ni did not appear to induce any markedly deleterious effects on any measured parameter in resting fish (cf. Figs 4, 5; Table 1).Table 1 presents the results of a more detailed analysis of the effects of chronic, low-level (243 µg Ni l–1) Ni exposure on resting, cannulated rainbow trout. There were no significant differences between control and Ni-exposed fish with respect to 15 different blood gas, acid–base, hematological, stress, water balance and ventilatory parameters. In this experiment, the gill Ni burden of chronically Ni-exposed fish was increased by approximately 3.2-fold over that of control fish (2035±527 vs 642±26 µg kg–1 wet mass).
Plasma concentrations of Na+, Cl–, Ca2+ and Mg2+ in juvenile rainbow trout (30–50 g) exposed for 42 days to 2034 µg Ni l–1 were only slightly affected (Fig. 3) despite the marked effects of exposure to this concentration on certain hematological parameters and swimming performance (Figs 4, 5) and the fact that this Ni concentration produced 33% mortality over 42 days. Although the reductions in plasma Na+ and Cl– were statistically significant in fish exposed to 2034 µg Ni l–1 (Fig. 3), losses of these two ions from the plasma were only 4% and 5%, respectively. Plasma [Ca2+] was well conserved, and plasma [Mg2+] was actually elevated.
While plasma protein, total ammonia, cortisol and lactate concentrations were very similar in control fish and fish exposed to 384 µg Ni l–1, exposure to 2034 µg Ni l–1 had a marked impact on both plasma protein and total ammonia concentration, with these two parameters being significantly increased by 29% and 200%, respectively (Fig. 4A,B). Additionally, although the changes were not statistically significant, plasma lactate concentration was elevated at this higher Ni concentration and plasma cortisol was suppressed (Fig. 4C,D).
The discrepancy between the effects of these two chronic Ni concentrations
(384 µg Ni l–1 vs 2034 µg Ni
l–1) was further evidenced by measurements of
Ucrit. Ucrit in fish exposed to the
higher Ni concentration (2034 µg Ni l–1) was markedly
reduced by 42% and 35% after 12 days and 24 days ofexposure, respectively
(Fig. 5). Exposure to the lower
Ni concentration (384 µg Ni l–1) resulted in slight
(7%), but not statistically significant, decreases of
Ucrit on both sampling days
(Fig. 5).
In contrast to the lack of effects found in resting fish, chronic Ni
exposure had a significant impact on oxygen consumption patterns when fish
were exercised. After 34 days of exposure to 394 µg Ni
l–1, treated fish exhibited a significantly lower maximal
oxygen consumption rate
(;
33%) and aerobic scope for activity (38%;
Fig. 6B,C). These trends
persisted even after 38 days of exposure of both groups of fish to clean
water, with the reduction in aerobic scope remaining statistically significant
(Fig. 6C). Basal oxygen
consumption rates
()
were not changed throughout the exposure regime
(Fig. 6A). Additionally,
Ucrit values were not significantly changed, despite a
tendency towards reduced values (6–10% lower) in the Ni group at
all times after the initial control (Fig.
6D; cf. Fig.
5).
Group regressions of the log of oxygen consumption rate vs
swimming speed are shown for both groups of fish swum on days 0 and 34 in
Fig. 7A and 7B, respectively.
The slopes and intercepts of the two regressions on day 0 were essentially
identical (Fig. 7A), while the
slope of the regression line for Ni-exposed fish on day 34 was significantly
lower than its control counterpart (Fig.
7B; P<0.05). The group regression lines showed little
change in
(note the log scale on the y-axis), and the intercepts in
Fig. 7B were not significantly
different from one another.
,
however, was noticeably reduced and, correspondingly, so was aerobic scope for
activity (cf. Fig.
6A–C).
Morphometric analysis of gills from control and experimental fish after 69 days of exposure revealed significant Ni-induced changes in the ultrastructure of secondary lamellae (Table 2). The percentage of the lamellar region occupied by secondary lamellae (VSL/VLR), and the percentage of secondary lamellae occupied by tissue outside the pillar system (blood channels; VOPS/VSL) increased significantly in Ni-exposed fish by 15.4% and 30.9%, respectively (Table 2). Additionally, the percentage of the lamellar region occupied by the pillar system (VPS/VLR) decreased significantly by 19.4%. Although elevated by slightly more than 10%, blood–water diffusion distance (BWDD) in experimental fish was not significantly different from that of control fish. These Ni-induced changes to the lamellar ultrastructure are illustrated by the light micrographs in Fig. 8.
The persistence of significantly reduced scope for aerobic activity in Ni-exposed fish following exposure to clean water (Fig. 6C) can be contrasted with the almost complete depuration of gill Ni burden in these fish. Fig. 9 shows the near return of gill Ni to control levels in fish previously exposed to Ni (99 days) followed by 38 days of exposure to clean water. Although the gill burden of fish previously exposed to Ni was still significantly elevated, the Ni burden of these fish was only 36% higher than that of control fish (593±39 µg kg–1 vs 436±9 µg kg–1). The gill burden of fish previously exposed to Ni after exposure to clean water falls towards the higher end of typical background gill Ni concentrations and was similar to that of control trout from both experiment 1 (560±10 µg kg–1; see Fig. 2) and experiment 2 (642±26 µg kg–1; see Results above). In comparison, the gill burden in Ni-exposed fish after 34 days (3434±530 µg kg–1) was approximately seven times higher than that of control fish (Fig. 9).
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) Ni exposure, the plasma was the main sink for Ni. Using
tissue Ni accumulation data (Fig.
2A) and the relative proportion of total body mass represented by
each tissue or body fluid (using values for rainbow trout from
Hogstrand et al., 2003),
tissue-specific Ni burdens were calculated to estimate internal distribution
of accumulated Ni within certain tissues of a hypothetical 1-kg rainbow trout
following chronic Ni exposure to 384 µg Ni l–1 and 2034
µg Ni l–1 (Fig.
2B). The plasma accumulated more than three times as much Ni as
any other tissue at both exposure concentrations, followed by the kidney,
white muscle, intestine and gill. Note that although white muscle was the
third largest Ni sink, this poorly vascularized tissue did not significantly
accumulate Ni (Fig. 2A).
Given such high amounts of plasma Ni, one might speculate that a large
portion of Ni accumulated in tissues may be a function of vascularization,
especially in tissues that are highly vascularized.
Fig. 2C plots the percentage of
accumulated Ni in each tissue that can be explained simply by accounting for
the degree of vascularization, using estimates of salmonid 125I
plasma space values for each tissue from Olson
(1992). At both exposure
concentrations, all of the Ni accumulated by the liver and heart was present
in the blood perfusing these tissues, as was a substantial portion of Ni in
the white muscle (31–40%). Interestingly, the two tissues with markedly
higher overall Ni burdens (kidney and gill) had the lowest percentage of Ni
burden that could be explained by vascularization and are the tissues in most
intimate contact with either the exposure water (gill) or the urine (kidney).
While the exposure water is obviously high in Ni, it is also assumed that the
Ni concentration of the urine is elevated during waterborne Ni exposure, given
that renal clearance is the primary excretory mechanism of bloodborne Ni
(Eisler, 1998;
USEPA, 1986). Although the
distribution of Ni among various ligands within the blood plasma of fish is
poorly understood, the relative affinity of mammalian serum albumin for Ni
determines the extent of Ni capable of crossing biological membranes bound to
low-molecular-mass ligands (USEPA,
1986; Kasprzak,
1987). The present data suggest that Ni is not easily accessing
the interstitial space (and the intracellular compartment) and is primarily
being retained in the blood plasma, perhaps bound to either serum albumin or
as another protein complex.
After 42 days of exposure, Ni concentrations in the gill and kidney were
approximately equilibrated with plasma Ni at both exposure concentrations (384
µg Ni l–1 and 2034 µg Ni l–1;
Fig. 2A). A similar phenomenon
occurred following 120 h of acute, high-concentration Ni exposure
(Pane et al., 2003), although
in that case Ni concentrations in the gill and kidney were equilibrated with
those in both the plasma and the exposure water. During chronic Ni exposure,
however, plasma concentrations exceeded Ni concentrations in the exposure
water (see Results). Additionally, during acute Ni exposure, plasma Ni
concentrations increased linearly with time over 120 h of exposure
(Pane et al., 2003). What is
not known during chronic exposure, however, is whether the high plasma Ni seen
after 42 days represents a plateau (homeostatically regulated level) or simply
a point during a time course of slowly but continually increasing plasma Ni
concentrations.
Exposing rainbow trout chronically to a relatively high Ni concentration
(2034 µg Ni l–1) provided insight into the mode of chronic
toxicity. Clearly, ionoregulatory disruption is far less important than
respiratory toxicity under these conditions (Figs
3,
4). Despite substantial
mortality and signs of respiratory distress in resting fish exposed to 2034
µg Ni l–1 (Fig.
4), plasma ion disturbances were minimal
(Fig. 3). These results agree
well with those of acute Ni studies in which respiratory toxicity is very
pronounced while ionoregulatory disturbance is not substantial
(Pane et al., 2003).
The respiratory effects of chronic, very low-level Ni exposure were quite subtle and were only unmasked by strenuous aerobic exercise (Figs 6, 7). The following discussion of Ni-induced limitation of aerobic swimming performance focuses on the gill as a key site of toxic action underlying the observed reductions in maximal oxygen utilization rates. In support of this specific focus on the gill during chronic Ni exposure are three pieces of evidence: (1) acute respiratory toxicity occurs exclusively at the gill and involves no bloodborne or systemic component (E. F. Pane, A. Haque and C. M. Wood, submitted); (2) chronically, white muscle, which contributes to aerobic swimming at speeds close to Ucrit, did not significantly accumulate Ni at either chronic concentration used (Fig. 2A); and (3) significant ultrastructural alterations to the branchial epithelium were observed, consistent with diffusive limitations of high-performance gas exchange (Table 2; Fig. 8).
Within the context of the rainbow trout gill, the connection between
decreased available surface area for diffusion and decreased maximal oxygen
exchange capacity has been well established. It is thought that at times of
maximal oxygen usage, the gills are fully perfused with blood and the system
is diffusion limited rather than perfusion limited
(Daxboeck et al., 1982;
Duthie and Hughes, 1987).
Accordingly, small decreases in gas exchange capacity may not be detected at
rest or at lower swimming speeds (Duthie
and Hughes, 1987) but may become important as the intensity of
exercise increases (Nikl and Farrell,
1993). Indeed, this phenomenon clearly applies during strenuous
exercise following chronic Ni exposure. At or near Ucrit,
maximal oxygen consumption rates
()
in the present study decreased by 33.0% (Figs
6B,
7). An alternative explanation
of the data in Figs 6B,
7, that swimming became more
efficient in treated fish at higher swimming speeds, is inconsistent with our
unquantified observations that treated fish consistently relied more
frequently on erratic burst swimming near Ucrit.
The 33% decrease in
was consistent with a 10.3% decrease in the relative diffusing capacity
(Drel) of experimental fish
(Table 2). This decrease in
Drel was driven more by increased blood water diffusion
distance (BWDD) in experimental fish (Table
2) rather than a distinct decrease in available lamellar surface
area (S) (from equation 4, Drel is directly
proportional to S and inversely proportional to BWDD). Because it
incorporates both S and BWDD, Drel is a more
comprehensive parameter than either S or BWDD taken alone and is a
better morphometrically determined approximation of the efficiency of oxygen
transfer from the water to the blood across the branchial epithelium
(Hughes and Perry, 1976;
Hughes et al., 1979).
The observed decrease in Drel corresponded well with a
slight swelling of the secondary lamellae in chronically Ni-exposed fish, as
evidenced by significantly increased
VSL/VLR (15.4%;
Table 2; Fig. 8). The most prominent
Ni-induced change in branchial ultrastructure was swelling of the lamellar
epithelial layer, as indicated by a 30.9% increase in
VOPS/VSL
(Table 2;
Fig. 8). In the gills of
Ni-exposed fish, lifting of the lamellar epithelium from the blood channel
system appeared more frequently than in the lamellae of control fish (E. F.
Pane, personal observation), and the increases in
VSL/VLR and
VOPS/VSL appeared to be driven more by
hypertrophy, or cell swelling, than a hyperplastic increase in cell number
(Fig. 8). Mallat
(1985) cited both epithelial
lifting and hypertrophy among common lesions associated with metal exposure,
with epithelial lifting being the most common response. Additionally,
hypersecretion of mucus and hyperplasia were identified as common defense
mechanisms. In the present study, hyperplasia was not evident, while
hypertrophic pavement cells were commonly observed in fish subjected to
chronic low-level Ni exposure (Fig.
8).
Ni-induced edema in the lamellar epithelium of the gill has been documented
during acute exposure to high concentrations of Ni in several earlier studies.
Nath and Kumar (1989) reported
extensive hypertrophy of the respiratory epithelium leading to separation from
the pillar system in the gills of Colisa fasciatus acutely exposed
(96 h) to approximately 14 000 µg Ni l–1. Additionally,
marked increases were observed in
VSL/VLR and
VOPS/VSL (57% and 49%, respectively)
following only 3 days of exposure of rainbow trout to 3200 µg Ni
l–1 (Hughes and Perry,
1976; Hughes et al.,
1979). Such profound acute swelling within the delicate
respiratory surface is presumably the cause of marked Ni-induced disturbances
in blood gases and acid–base balance, such as those observed by Pane et
al. (2003) in trout acutely
exposed to 11 700 µg Ni l–1. These authors observed a
linear decrease in arterial oxygen tension with time (96 h) to less than 35%
of control values, with a twofold increase in carbon dioxide tension and a
concomitant respiratory acidosis, suggestive of a substantial limitation of
branchial diffusive capacity. Indeed, in a separate study by Pane et al. (E.
F. Pane, A. Haque and C. M. Wood, submitted), rainbow trout acutely exposed to
10 700 µg Ni l–1 experienced a 46.4% increase in
VSL/VLR as well as significantly
increased VOPS/VSL, BWDD and lamellar
width. Additionally, Hughes and Perry
(1976) examined the relative
contributions to Ni-induced edema of both tissue and non-tissue (lymphoid)
spaces, concluding that overall lamellar swelling was due to swelling in both
epithelial components.
In mammals, Ni is considered a moderate contact allergen
(Kligman, 1966), and it is
possible that lamellar swelling in fish may be an inflammatory response.
Although leukocyte infiltration of lamellar blood channels and vasodilation
have occasionally been observed in acutely exposed fish (E. F. Pane, A. Haque
and C. M. Wood, submitted), the contraction of the pillar system seen during
chronic Ni exposure (decreased
VPS/VLR;
Table 2) argues against a
chronic inflammatory response. An alternative suggestion proposed by Mallat
(1985) is that intraepithelial
fluid may result not from blood exudates but from the overlying freshwater
medium; this is particularly relevant in the case of freshwater fish that are
continually faced with the osmotic challenge of water absorption from a very
hypoosmotic medium.
The decreases in
and Drel observed during Ni exposure are qualitatively
similar to the findings of Duthie and Hughes
(1987), who surgically reduced
the available gill surface area in trout and reported decreases in
that almost exactly corresponded (i.e. a 1:1 ratio) to the decreases in
available surface area. Additionally, following 34 days of exposure to
sublethal Al at moderately low pH, an approximate 1.2:1 ratio was found
between these two variables in rainbow trout
(Wilson et al., 1994).
According to Duthie and Hughes
(1987), however, percent
decreases in
greater than percent decreases in available lamellar surface area suggest some
extra-diffusional limitations, such as perfusion or convection limitations. In
the present study, the ratio of decreased
(33%) to Drel (10.3%) was approximately 3.2:1. Despite the
conventional belief that the branchial vascular network is relatively
resistant to waterborne irritants, a significant contraction of the lamellar
vasculature was observed following chronic Ni exposure
(Table 2) and may have
contributed to the increased negative impact on maximal oxygen consumption
rates. Additionally, the decreased interlamellar water space in chronically
exposed fish (see Fig. 8B)
would decrease the volume of water flowing over the respiratory surface and
impose a ventilatory convective limitation also capable of impairing
high-performance oxygen uptake at the gill.
Furthermore, the 3.2:1 ratio of the present study suggests that some
extra-branchial mechanisms such as muscle, erythrocytic or renal impairment
may be responsible for some portion of decreased aerobic capacity in
Ni-exposed fish (see above). Although Ni did not accumulate significantly in
white muscle, muscle ammonia concentrations, for example, were not measured.
Therefore, we cannot entirely exclude the possibility that Ni-induced
perturbation of swimming performance was mediated through elevated muscle
ammonia concentrations, as is the case with acute exposure of brown trout to
sublethal copper and low pH (Beaumont et
al., 2003). The latter effect is caused by a significant
ammonia-induced depolarization of the resting membrane potential of muscle
fibers (Beaumont et al., 2000).
Plasma ammonia in rainbow trout chronically exposed to 384 µg Ni
l–1, however, was not significantly elevated
(Fig. 4), suggesting that
ammonia is not a mediating factor of reduced swimming performance at this low
concentration. Plasma ammonia was substantially elevated, however, following
chronic exposure to 2034 µg Ni l–1. Unfortunately, we did
not measure Ni in the red muscle.
Given such high (comparable to gill) concentrations of Ni in the plasma
(blood) and kidney (Fig. 2)
following chronic exposure, we also cannot entirely dismiss the possibility
that these two tissues may contribute to the limitation of high-performance
aerobic function in Ni-exposed fish. Chronically impaired hemoglobin would
cause such a decline, as might chronic renal damage, due to the importance of
renal handling of water and electrolytes during exercise
(Wood and Randall, 1973).
It must also be considered, given the persistence of impaired aerobic
capacity several weeks after the removal of Ni from the exposure water, that
some degree of extrabranchial limitation of exercise performance may have been
due either to the persistence of extrabranchial accumulated tissue Ni, to
specific organ damage that persisted after the Ni exposure period, or to some
combination of both. Unfortunately, we can only speculate at this point in
time, as the kinetics of tissue Ni handling and specific organ function during
Ni exposure and depuration remain to be tested. Additionally, there is the
possibility that decreased aerobic performance in both the presence and
absence of direct toxicant insult may be secondary to Ni-induced impairment of
physiological function at a higher level of organization than that of an
individual organ. Although Ni is both immunogenic
(Barchowsky et al., 2002) and
carcinogenic (Costa, 1991) in
mammalian systems, such effects of Ni in fish are currently unknown.
In summary, we present evidence of a clear cost of acclimation to
chronically sublethal Ni exposure in terms of subtle alterations to the
branchial ultrastructure and reduced aerobic swimming performance. In the
classic terms of Brett (1958),
regarding environmental contaminants and aerobic metabolism, Ni acted as a
`limiting stressor' at 34 days of exposure by limiting oxygen exchange with
the environment during high demand, thereby reducing
(Figs 6B,
7B). `Loading stress', or
increased costs of day-to-day living, was not seen, as
remained unchanged throughout Ni exposure and subsequent exposure to clean
water (Figs 6A,
7A,B). It is also evident that
the cost of acclimation to Ni is not a transient phenomenon. To some degree,
chronically depressed aerobic capacity persisted for 38 days post Ni-exposure
(Fig. 6C), despite an almost
complete depuration of gill Ni burden (Fig.
9). Because we only measured oxygen consumption rates and gill Ni
burdens after the extended depuration period, we can only speculate as to the
possible causes of long-lasting effects of Ni even after fish were returned to
clean water.
Chronic impairment of such a dynamically active and critical organ is
likely to depress the overall fitness of a fish
(Wood, 2001) with obvious
environmental implications. In the context of chronic sublethal Ni exposure
(394 µg Ni l–1), reduced maximal oxygen consumption could
compromise fitness by possibly impairing both predator avoidance and prey
capture. Additionally, possible impairment of migratory success is
particularly relevant to salmonids returning to freshwater spawning
streams.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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