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Marine and Freshwater Research
http://dx.doi.org/10.1071/MF14120
Sublethal effects of fluctuating hypoxia on juvenile
tropical Australian freshwater fish
Nicole Flint A,B,E, Michael R. Crossland C,D and Richard G. Pearson B,C
A
Central Queensland University, School of Business and Law and School of Medical and
Applied Sciences, North Rockhampton, Qld 4702, Australia.
B
College of Marine and Environmental Sciences, James Cook University, Townsville,
Qld 4811, Australia.
C
TropWater, James Cook University, Townsville, Qld 4811, Australia.
D
School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia.
E
Corresponding author. Email: [email protected]
Abstract. Hypoxia in freshwater ecosystems of the Australian wet tropics occurs naturally, but is increasing as a result of
anthropogenic influences. Diel cycling of dissolved oxygen (DO) concentration (fluctuating hypoxia) is common in the
region. Laboratory experiments sought to identify relationships between severity of fluctuating hypoxia and sublethal
effects on ventilation, feeding and growth for juvenile barramundi (Lates calcarifer), eastern rainbowfish (Melanotaenia
splendida splendida) and sooty grunter (Hephaestus fuliginosus). Fish continued to feed and grow under daily exposure to
severe fluctuating hypoxia treatments for several weeks. Ventilation rates increased in a significant direct quadratic
relationship with the severity of hypoxia treatments and increasing hypoxia caused ventilatory behaviour changes in all
species. Barramundi and rainbowfish attempted aquatic surface respiration and were more tolerant of severe hypoxia than
was sooty grunter; barramundi and rainbowfish are also more likely to experience hypoxia in the wild. There was a
significant quadratic relationship between growth and minimum DO saturation for barramundi. Although all three species
were tolerant of hypoxia, anthropogenic stressors on tropical Australian aquatic ecosystems may increase the frequency
and severity of hypoxic conditions causing a concomitant increase in fish kill events.
Additional keywords: agriculture, Hephaestus, Lates, Melanotaenia, oxygen, pollution.
Received 30 August 2013, accepted 21 July 2014, published online 19 November 2014
Introduction
Natural and human-enhanced hypoxia, or low concentration of
dissolved oxygen (DO), is a common cause of fish kills around
the world (e.g. Whitfield and Paterson 1995; Hamilton et al.
1997; Hernández-Miranda et al. 2010), and can cause severe
changes in behaviour, growth and reproduction of fish as a result
of metabolic, ventilatory and physiological effects (Ruggerone
2000; Richards 2011; Burt et al. 2013). In the long term, hypoxia
can alter community and food-web structure, contribute to
habitat loss, and reduce the overall health and sustainability of
populations (Eby et al. 2005) and ecosystems (Kramer 1987;
Zhang et al. 2010).
In Australia, hypoxia is one of the most common causes of
fish kills as a result of bacterial respiration following blackwater events and input of organic material of agricultural
origin, and high levels of plant respiration in eutrophic systems
(e.g. Townsend et al. 1992; Pearson et al. 2003a; King et al.
2012). However, few studies have focussed on the sublethal
effects of hypoxia on freshwater species in northern Australia.
High temperatures, nutrient enrichment and light availability,
especially in the absence of riparian shade, facilitate abundant
Journal compilation Ó CSIRO 2015
growth of aquatic plants and algae, which may contribute to
strong diel patterns of hypoxia cycling, in which DO concentrations are high in the late afternoon but drop during the night to a
minimum at dawn (Davis 1975; Brady et al. 2009; Bunch et al.
2010). For example, in Lagoon Creek in the Herbert catchment of
the wet tropics of northern Queensland, DO saturation can
fluctuate between 2 and 80% daily over several weeks (Pearson
et al. 2003a), and similar patterns have been reported in other
regions (e.g. Gulf of Mexico, Cheek et al. 2009; Florida, Bunch
et al. 2010). With increasing temperature, the solubility of oxygen
decreases and metabolic demands of aquatic organisms increase
(Cech et al. 1990; Smale and Rabeni 1995; Shimps et al. 2005), so
aquatic ecosystems in the tropics are under particular threat of
hypoxia, especially when low water levels and lack of water
movement reduce oxygen diffusion at the air–water interface.
As DO concentrations decline, many fish species attempt to
increase the rate of gas exchange at the gills to overcome the
reduced water-to-blood oxygen gradient (Moyle and Cech
2004). Mechanisms to achieve this include increasing the
volume of water pumped over the gills by increasing ventilation
rate (frequency of opercular pumping) or stroke volume
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(amount of water pumped with each stroke). For example,
channel catfish (Ictalurus punctatus) alter ventilation by changing stroke volume, whereas bluegill (Lepomis macrochirus) and
carp (Cyprinus carpio) increase stroke frequency in response to
hypoxia (Heath 1995). An increase in ventilation rate or volume
under mild hypoxia allows the oxygen pressure in arterial blood
to remain similar to that found under normoxic conditions
(Heath 1995). The level of hypoxia at which ventilation rate
increases differs among fish species, probably because of
different degrees of physiological adaptation to, or tolerance
of, hypoxia (Heath 1995).
Fish can also alter their behaviour to increase the availability
of DO. Aquatic surface respiration (ASR) is frequently used to
acquire oxygen by water-breathing fishes (e.g. Chapman et al.
1995; Richards 2011), including some south-eastern Australian
species that survive hypoxic events by increasing ventilation
rates and employing ASR (McNeil and Closs 2007). During
ASR, fish selectively utilise the thin layer of oxygenated water at
the air–water interface. Fish living in frequently hypoxic waters
are more likely to perform ASR than those from consistently
normoxic habitats (Kramer 1983). Guppies (Poecilia reticulata)
utilising ASR are able to survive for 10 h under hypoxic conditions that would otherwise be detrimental after 10 min (Kramer
and Mehegan 1981). This ability would be extremely useful
under fluctuating hypoxia, when dawn DO concentrations are
very low, but last only for a few hours.
DO is a limiting factor on growth, and sustained hypoxia can
cause a reduction in appetite (Pichavant et al. 2001; Braun et al.
2006; Chabot and Claireaux 2008; Burt et al. 2013) and an
increase in energetic costs (Brett 1979; Boeuf et al. 1999).
Additional growth depression when DO concentrations are
fluctuating rather than constant around a mean low DO concentration has also been suggested (Thetmeyer et al. 1999). However, some fish species have developed a high tolerance to
hypoxic conditions, reducing the effects of hypoxia on condition
and physical fitness. The ability to acclimatise to low DO tends
to be most prevalent in species that are commonly exposed to
hypoxia in their natural environment (Kramer 1987).
Intraspecific variability in hypoxia tolerance has been identified in the case of yellow perch (Perca flavescens), which is
more tolerant to hypoxia as a juvenile than as an adult (Robb and
Abrahams 2003); and hypoxia tolerance varies throughout the
larval and juvenile stages of red sea bream (Pagrus major;
Ishibashi et al. 2005) and Japanese flounder (Paralichthys
olivaceus; Ishibashi et al. 2007) in response to ontogenetic
changes in metabolic rates. Even within the juvenile phase,
hypoxia tolerance may vary with fish size, as identified for spot
(Leiostomus xanthurus) and Atlantic menhaden (Brevoortia
tyrannus) (Shimps et al. 2005).
The present study examined the responses of juvenile fish of
three common native species from tropical northern Queensland
to a range of conditions of fluctuating hypoxia, by examining the
relationship between the minimum level of hypoxia experienced
and effects on ventilation, feeding and growth. We concentrated
on juvenile fish because their smaller size makes them good
subjects for laboratory-based experiments, but we do not presume that juveniles are more susceptible to stressful conditions
than mature fish; we expect that different life stages will be
differentially affected by various stressors, and that tolerance to
N. Flint et al.
stressors may vary even within life-history stages. The three
species were exposed to various treatments of diel fluctuating
hypoxia in aquaria to test the hypothesis that the sublethal
responses of fish to fluctuating hypoxia will increase with
decreasing DO concentration.
Materials and methods
Test species
Juvenile barramundi, Lates calcarifer (Bloch), were purchased
from a commercial fish hatchery in northern Queensland.
Barramundi are an important commercial and recreational
fishing target and are widespread in coastal drainages across
northern Australia (Allen et al. 2003). Barramundi are important
predators, consuming microcrustaceans, fish and aquatic insects
(as a postlarval fish), macrocrustaceans, fish and aquatic insects
(as a juvenile fish), and fish and macrocrustaceans (as an adult
fish) (Pusey et al. 2004).
During the early juvenile stage, barramundi are regularly
exposed to harsh environmental conditions including high
salinities and high water temperatures (up to 388C) (Pusey
et al. 2004). Despite being occasionally affected by hypoxiarelated fish kills, barramundi are generally thought to be
moderately tolerant of hypoxia. They have been recorded living
in the wild in waters with surface DO concentrations down to
1.1 mg L1 (13–15% saturation at 25–308C; Pusey et al. 2004).
Sooty grunter, Hephaestus fuliginosus (Macleay), were provided by the Tablelands Fish Stocking Association, Atherton,
Queensland. The species occurs in tropical fresh waters of
Australia and New Guinea. In Australia, its range is fragmented,
and extends from the Northern Territory to central Queensland
(Pusey et al. 2004). Sooty grunter are a popular game fish for
recreational fishers and are successfully grown as an aquaculture species. They are voracious predators, and consume a
variety of invertebrates, fish and plant materials. Sooty grunter
are found in moderately to well-oxygenated running waters, and
have been recorded only from waters that are almost completely
fresh. The minimum DO concentration recorded for wild fish is
3.7 mg L1 (,45–50% DO saturation at 25–308C, Pusey et al.
2004).
Eastern rainbowfish, Melanotaenia splendida splendida
(Peters), were collected from a small tributary of the Ross River,
Townsville. Eastern rainbowfish are small schooling fish that
are widely distributed along the eastern coast of Queensland
(Pusey et al. 2004). Eastern rainbowfish are abundant wherever
they occur and tolerate a wide range of water-quality conditions
(Pusey et al. 2004). They are omnivorous feeders that consume
primarily small aquatic invertebrates and terrestrial insects from
the water surface. It is a popular aquarium species and is an
important test animal for biomonitoring and laboratory studies
(Humphrey et al. 2003).
Although eastern rainbowfish appear to prefer well oxygenated waters, they have been recorded in wetland habitats where
DO concentrations decreased to 0.2 mg L1 (2–3% saturation at
25–308C) in the bottom water layers (Hogan and Graham 1984;
cited in Pusey et al. 2004), although fish may have avoided these
anoxic conditions by remaining in the upper water column
(Pusey et al. 2004). Pusey et al. (2004) suggested that a DO
concentration of 4 mg L1 (50–55% DO saturation at 25–308C)
Sublethal effects of fluctuating hypoxia on fish
Experiments
All experiments were undertaken at James Cook University,
Townsville (JCU). Prior to experiments, fish were held in
1000-L tanks in a light- and temperature-controlled room. All
fish were housed with others of the same species and similar size
for at least 2 weeks before transfer to experimental aquaria.
Density in holding tanks was maintained at less than one fish
per 5 L. Holding tanks were fitted with canister filters, and water
quality in the holding tanks was monitored regularly. The
water used for holding tanks and experimental aquaria was tap
water filtered using a series of three filters (5-mm sediment,
1-mm sediment, 0.5-mm carbon). Testing by the TropWater
laboratory at JCU demonstrated that this filter series removes
chlorine, heavy metals, sediments and bacteria from the water
(M. Crossland, unpubl. data). Fish were randomly allocated to
experimental tanks.
Nitrogen gas was bubbled into the water in the experimental
aquaria to displace DO. This method has been extensively used
in previous studies (e.g. Cech and Massingill 1995; Taylor and
Miller 2001; Brady and Targett 2010) and is capable of producing extremely low DO concentrations. The advantages of using
nitrogen gas to deplete DO concentrations are that it is easy to
control, biologically inert and readily available. It does, however, have some drawbacks, including the presence of a ‘nitrogen
atmosphere’ above the water surface that prevents fish from
breathing air, or successfully performing ASR, and moderate
but unnatural cycling of pH in comparison to field situations
(Flint et al. 2012).
All experiments were carried out in glass aquaria with an
internal sealed lid, filled to the lid with 25 L of filtered water.
This design allowed tanks to be completely closed when necessary, blocking access of fish to the air–water interface. Industrial
nitrogen was delivered from cylinders fitted with a gas regulator
through a manifold, with plastic tubes running from the manifold and delivering equal pressures of nitrogen gas to each
experimental tank. A separate tube in each tank was used to
deliver air. Aquaria were kept on three levels of open shelving,
and allocation of DO treatments to aquaria was randomised and
maintained for all experiments. Water temperature was maintained at 288C (28C) to simulate summer conditions in the
field, when depleted DO concentrations are likely to be more
detrimental to fish (Martin and Saiki 1999).
Four aquaria were used as normoxic controls (.85% DO
saturation), and two aquaria were used for each of seven
treatments, which had minimum DO saturations of 5, 10, 20,
30, 40, 50 and 60%, with maximum DO concentration being
normoxic (.85% saturation) in all cases. DO concentrations
were cycled daily to create fluctuating hypoxic conditions and
were recorded half-hourly (Fig. 1 illustrates the typical DO
depletion and reoxygenation pattern experienced in all treatments). DO was depleted until the desired minimum concentration was reached, at which time the nitrogen was turned off and
aquaria were gradually aerated using compressed air.
During the 5-h DO depletion period, the experiment room
was lit with photographic-quality red globes to simulate nocturnal DO depletion, as would normally occur in the field.
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Fig. 1. Daily fluctuating hypoxia treatments as used in experiments.
The curves illustrate the typical depletion of dissolved oxygen (DO) and
reoxygenation pattern, including rate and severity, experienced in each
treatment on each day of each experiment.
A gradual DO depletion period was chosen over a sudden
reduction to simulate field situations. In the field, the time taken
for DO depletion may vary considerably depending on the rate
of deoxygenation and the duration of the period of darkness. The
5-h DO depletion period chosen here represents a compromise
between the usual period of darkness during summer in northern
Australia (,10 h) and the logistical constraints of maintaining
month-long laboratory experiments. Temperature and pH were
measured before DO reduction and at the time at which minimum DO concentration was reached for each aquarium. DO,
pH and temperature measurements were taken using a WTW
pH/Oxi 340i meter (Wissenschaftlich-Technische Werkstatten,
Weilheim, Germany), in combination with a WTW CellOx
325-3 DO probe and WTW SenTix pH probe. Both probes were
calibrated daily. Nitrogenous wastes (nitrate, nitrite and ammonia) were monitored using commercial test kits. Fifty percent
water changes were carried out daily.
Three separate experiments explored the effects of fluctuating hypoxia on ventilation rates, ventilatory behaviour, feeding
and growth on barramundi, eastern rainbowfish and sooty
grunter. So as to investigate the response of fishes to fluctuating
hypoxia in a predictive manner, the three experiments were
designed to produce data suitable for regression analysis, rather
than constraining comparisons between fixed treatments (cf.
Stergiou et al. 1997; Collins et al. 2012; Zhou et al. 2014).
Therefore, replicated treatments and controls were selected to
explore the shape of the relationship between ventilation,
feeding and growth of juvenile fishes and increasingly severe
fluctuating hypoxia. Quadratic models were used throughout
(using SigmaPlot 11) as they provided a good fit to the data,
indicated by significance of the fit, coefficients of determination
and the form of the curve. Other models considered included
cubic, sigmoidal and piecewise regression. Quadratic models
could be readily interpreted biologically, which is an important
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factor in selecting the most appropriate regression model
(McDonald 2009).
Experiment 1 – barramundi
Aquaria were divided into four equal compartments using
5.0-mm plastic mesh to prevent contact between individual fish,
because conspecifics may behave aggressively. Seventy-two
barramundi were used in the experiment, with four fish (in
separate compartments) in each of 18 tanks. Fish were weighed
and placed into the experimental aquaria where they were
acclimated for several days until feeding normally. After acclimation, DO cycling commenced and was carried out on a daily
basis for 21 days. Fish were fed with commercially prepared
aquaculture pellets. As barramundi grew quickly in the experimental tanks, the experiment was concluded after 21 days to
avoid confounding effects of high stocking density. After 21
days of cycling, fish were left for 24 h with no food, then
euthanased, weighed and measured.
Experiment 2 – eastern rainbowfish
The procedure was the same as for barramundi, except that
fish were fed with high-quality commercial flake food for
tropical fish, and fish were euthanased, weighed and measured
after 28 days of the experiment plus 24 h with no feeding.
Experiment 3 – sooty grunter
A pilot trial found that sooty grunter could not be kept within
aquaria with mesh barriers because these fish damaged themselves on the mesh. Therefore, in Experiment 3, four fish were
initially placed in each aquarium, with no barriers to divide them.
The largest of the four fish in each aquarium became dominant
and showed aggressive behaviour towards conspecifics. The
severity of the aggressive behaviour suggested that the data
gathered from the three subservient fish would be confounded.
Hence, all but the dominant (largest) fish were removed from each
aquarium. This reduced the information to be collected from the
experiment, so a second set of aquaria was established, with one
new (previously untested) sooty grunter in each aquarium. Aquarium set-up was identical in the two experiments. Thirty-six sooty
grunter individuals were used in 36 experimental aquaria and fed
with commercially prepared aquaculture pellets. Otherwise, the
experimental procedure was the same as for rainbowfish.
Measurement of ventilation
Ventilation rate of juvenile fish was measured using a stopwatch
to record the time for 50 ‘breaths’ (beats of the operculum),
which were then converted to ‘beats per minute’ (bpm). Ventilation rate was measured before commencement of DO depletion every morning of the experiment (baseline ventilation rate),
as well as at the minimum DO concentration for each aquarium
every day. On some occasions, it was not possible to measure
ventilation rate, such as when breathing was so slight it was not
observable. In these instances, no rate was recorded. Ventilation
rates of two fish in each aquarium were recorded in the barramundi and eastern rainbowfish experiments, and data for all
sooty grunter were recorded. Ventilation rates were averaged
across fish and days to provide one measurement per aquarium
for analysis.
N. Flint et al.
Ventilation behaviour was recorded at the same time as
ventilation rate, and was classified into the following five
stages: (0) no opercular movement detectable; (1) mouth closed,
opercular movements barely visible, no erratic movements,
easily disturbed by external stimuli; (2) mouth closed, opercular
movements obvious but not large, no erratic movements, easily
disturbed; (3) mouth open, large and fast opercular movements,
appears to gulp at water, circles aquarium sides, not easily
disturbed; and (4) ASR behaviour attempted, mouth open at
surface, very large and fast opercular movements, circles
aquarium sides and spends large amounts of time at the surface,
not easily disturbed. In these trials, ASR could be attempted,
but was not successful because fish did not have access to the
air : water interface.
Measurement of feeding
An individual daily index of appetite was recorded. Fish were
observed to be feeding (1) immediately on introduction of food
to the tank, (2) tentatively following introduction of food, or
(3) later or not at all. Because sooty grunter were housed individually and fed on pellet food it was possible to accurately
record food consumption of individual fish each day of the
experiment. The number of pellets placed into each aquarium
was counted, as was the number of pellets remaining after 1 h,
and the number of pellets consumed by each fish was then
calculated.
Measurement of growth
Fish were weighed to 0.001 g before commencement of each
experiment and following each experiment. Weight change was
calculated as a percentage of initial weight, and change in
weight of all fish in each aquarium was averaged to provide a
single value per aquarium. For sooty grunter, because there was
only one fish per aquarium, each data point represented an
individual fish rather than an average.
Results
The treatment of 5% DO saturation at minimum was found to be
sublethal for barramundi but lethal for rainbowfish and sooty
grunter. In addition, one rainbowfish in the 10% DO treatment
died during Experiment 2, and one sooty grunter in the control
treatment in Experiment 3 died. Fish that died during experiments were excluded from analyses.
Ventilation rates
The shape of the relationship between DO and ventilation rate for
barramundi demonstrated a rapidly increasing ventilation rate in
treatments with the minimum DO saturation between 50 and
20% (Fig. 2a). Fish in treatments with the minimum DO saturation of 20, 10 and 5% all demonstrated very high ventilation
rates. In treatments where DO saturations remained consistently
higher than 50%, ventilation rates did not increase. There was a
significant quadratic regression relationship between DO saturation and ventilation rate (R2 ¼ 0.823, P , 0.001). At normoxia
(measured each day before DO depletion), there was a significant quadratic regression relationship (R2 ¼ 0.671, P , 0.001)
between baseline ventilation rates of barramundi and minimum
DO saturation of treatments (Fig. 2b).
Sublethal effects of fluctuating hypoxia on fish
Ventilation under hypoxia
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(c)
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Fig. 2. Ventilation rates (opercular beats per minute, bpm) of (a) juvenile barramundi at minimum saturation of dissolved oxygen (DO) in each aquarium
averaged over 21 days; quadratic regression: y ¼ 175.1636 1.6278x þ 0.0093x2, R2 ¼ 0.823, P , 0.001; (b) juvenile barramundi under normoxic
conditions, averaged for each aquarium over 21 days; quadratic regression; y ¼ 70.1008 þ 0.1586x þ 0.0011x2, R2 ¼ 0.671, P , 0.001; (c) juvenile eastern
rainbowfish at minimum DO saturation in each aquarium, averaged for each aquarium over 28 days; quadratic regression: y ¼ 406.5385 6.9324x þ
0.0411x2, R2 ¼ 0.954, P , 0.001; (d ) juvenile eastern rainbowfish under normoxic conditions, averaged for each aquarium over 28 days; quadratic
regression: y ¼ 115.7985 þ 0.1830x 0.0008x2, R2 ¼ 0.117, P , 0.001; (e) juvenile sooty grunter at minimum DO saturation in each tank averaged for each
fish over 28 days; quadratic regression: y ¼ 326.7766 2.5404x þ 0.0101x2, R2 ¼ 0.892, P , 0.001; and ( f ) juvenile sooty grunter under normoxic
conditions, averaged for each aquarium over 28 days; quadratic regression: y ¼ 167.2398 0.0798x þ 0.0015x2, R2 ¼ 0.062, P , 0.001. Because only one
eastern rainbowfish and no sooty grunter survived at 5% DO, this treatment was excluded from analyses for these species.
Ventilation rates of eastern rainbowfish at minimum DO
saturation increased rapidly with decreasing DO saturation,
especially between 60 and 10% DO saturation (Fig. 2c). There
was a significant quadratic regression relationship between DO
saturation and ventilation rate (R2 ¼ 0.954, P , 0.001). Baseline
ventilation rate (rate at normoxia in each treatment) of rainbowfish was not affected by decreasing minimum DO saturation
(Fig. 2d ).
There was no significant difference between the two sets of
data for sooty grunter, except for the baseline ventilation rate
(Flint 2005), for which no treatment effect was identified in
either set. Therefore, the two sets of data were combined for
further analysis. There was a significant quadratic relationship
between ventilation rate at minimum DO saturation and DO
treatment (Fig. 2e, R2 ¼ 0.892, P , 0.001). There was no relationship between exposure to progressively lower minimum DO
saturation and ventilation rate under normoxic conditions
(Fig. 2f ).
Ventilation behaviour
Ventilation behaviour of all three species began to shift from
‘normal’ (Stages 0 and 1) at 60% DO saturation (Fig. 3a–c).
At DO saturations less than 30%, all three species frequently
exhibited an open-mouthed ventilatory behaviour (Stage 3).
Only at the 5% treatment did Stage 4 behaviour, including ASR,
become apparent for barramundi (Fig. 3a). Eastern rainbowfish
attempted to perform ASR in the 10% DO treatment (Fig. 3b).
Sooty grunter did not attempt ASR, except possibly for one
individual in the 20% DO treatment that moved to the surface,
but it was unclear whether this was a stress response rather than a
deliberate attempt to access DO at the water surface (Fig. 3c).
Feeding and growth
Barramundi showed consistent feeding behaviour throughout
the experiment. Fish in the control (normoxic) treatment fed
eagerly throughout the entire 21-day experiment (Fig. 4a).
Barramundi feeding actively decreased progressively with
decreasing minimum DO saturation, but the fish in the 5% DO
treatment still fed actively 70% of the time. Eastern rainbowfish
and sooty grunter did not often feed readily while being
observed (Fig. 4b, c), and no consistent relationship between
feeding behaviour and DO treatment was observed for either
species.
Juvenile barramundi experiencing minimum DO saturation
of 10% and below grew more slowly than in other treatments
(Fig. 5a; quadratic regression, R2 ¼ 0.377, P , 0.001). There
was no significant relationship between DO treatment and
growth of juvenile eastern rainbowfish (quadratic regression,
R2 ¼ 0.421, P ¼ 0.243), although it was notable that in the 10%
treatment, growth was negative (Fig. 5b); that is, there was a
reduction in average fish size over the course of the experiment
in that treatment. There was no significant relationship between
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N. Flint et al.
Ventilation stage 0
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Fig. 3. Behavioural ventilation stages recorded for (a) barramundi, (b)
eastern rainbowfish and (c) sooty grunter in each treatment as a percentage
of days that each behavioural stage was recorded. Ventilatory behaviour was
recorded daily when minimum dissolved oxygen (DO) was reached for
each treatment. Because only one eastern rainbowfish and no sooty grunter
survived at 5% DO, this treatment was excluded from analyses for these
species.
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Fig. 4. Feeding behaviour of (a) barramundi, (b) eastern rainbowfish
and (c) sooty grunter grouped by treatment, presented as the percentage
of days recorded at each index of feeding behaviour by all fish in a treatment.
Because only one eastern rainbowfish and no sooty grunter survived at 5%
dissolved oxygen (DO), this treatment was excluded from analyses for these
species.
Sublethal effects of fluctuating hypoxia on fish
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Fig. 6. Sooty grunter food consumption. Average number of pellets
consumed by each sooty grunter over 28 days; quadratic regression:
y ¼ 19.7003 þ 0.4865x 0.0032x2, R2 ¼ 0.206, P ¼ 0.002. Because no
sooty grunter survived at 5% dissolved oxygen (DO), this treatment was
excluded from the analysis.
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20
30
40
50
60
70
80
90
100
Treatment (% DO saturation at minimum)
Fig. 5. Growth of juvenile fishes in each experimental treatment, with
change in weight expressed as a percentage of initial weight. (a) Growth of
barramundi after 21 days; quadratic regression: y ¼ 316.2044 þ 3.6524x 0.0249x2, R2 ¼ 0.377, P , 0.001. (b) Growth of eastern rainbowfish
after 28 days; quadratic regression: y ¼ 11.3744 þ 1.1770x 0.0091x2,
R2 ¼ 0.421, P ¼ 0.243; and (c) growth of sooty grunter after 28 days;
quadratic regression: y ¼ 37.9986 þ 1.8460x 0.0115x2, R2 ¼ 0.206,
P ¼ 0.116. Because only one eastern rainbowfish and no sooty grunter
survived at 5% dissolved oxygen (DO), this treatment was excluded from
analyses for these species.
DO treatment and growth of sooty grunter (R2 ¼ 0.206,
P ¼ 0.116); however, all sooty grunter in the 10% treatment
grew by less than 60% of their initial weight, whereas most sooty
grunter in other treatments grew by more than 60% over 28 days,
and up to 177% in the control aquaria (Fig. 5c).
For sooty grunter, food consumption (number of pellets),
averaged for the duration of the experiment for each fish,
followed a pattern similar to that for growth (quadratic regression, R2 ¼ 0.206, P ¼ 0.002), with lowest feeding rates occurring at 10% minimum DO (Fig. 6).
Discussion
Ventilation
An inverse relationship between ventilation rate and DO saturation was identified for all three species of fish. This was
expected, because hyperventilation is a typical response of
water-breathing fishes to hypoxia (Perry et al. 2009); however,
the level of hypoxia at which ventilation rate begins to increase
and the maximum ventilation rate achieved differs among fish
species and with experimental conditions for a variety of reasons, including different tolerances to hypoxia. For example,
two morphologically similar species of Erythrinidae, Hoplias
malabaricus and H. lacerdae, have different gill ventilation
rates and oxygen extraction efficiencies under hypoxia;
H. malabaricus exhibits lower gill ventilation rates and higher
oxygen extraction efficiency when exposed to hypoxia, probably
because it inhabits stagnant hypoxic waters, whereas H. lacerdae
inhabits well oxygenated streams (Rantin et al. 1992). Prior
history of exposure to hypoxia is one factor affecting the hypoxic
ventilatory response (Perry et al. 2009). McNeil and Closs
(2007) demonstrated the ability of south-eastern Australian
floodplain fishes to survive hypoxic events by means of higher
ventilation rates and ASR, and concluded that the species studied
were generally tolerant to periodic hypoxia. In the present study,
average baseline ventilation rate (during periods of normoxia)
of barramundi was lower for fish experiencing the more severe
H
Marine and Freshwater Research
treatments. This may suggest that oxygen extraction efficiency
of barramundi improves with exposure to hypoxia.
ASR was attempted by fish in these experiments, but it was
unsuccessful, because the air–water interface was inaccessible
in the experimental aquaria. Nevertheless, under severe
hypoxia, barramundi and eastern rainbowfish showed the same
behaviour as is exhibited during successful ASR. This implies
that for eastern rainbowfish and barramundi, ASR is an automatic behavioural response to hypoxia that does not cease if
there is no increase in oxygen attainment, unlike for some other
fish species that alter their behaviour to limit metabolic energy
use if surface access is prevented (Richards 2011). Fish that
perform ASR under reduced DO conditions are typically
species that inhabit potentially hypoxic habitats (Kramer
1983; Verheyen et al. 1994), where 80% or more of the fish
species present may utilise the behaviour (Congleton 1980;
Kramer and McClure 1982; Mandic et al. 2009). Using ASR
makes it possible for fish to endure hypoxia for longer periods of
time, and is considered to be an important behavioural adaptation
in fish species that are unable to breathe air (Chippari-Gomes
et al. 2003; Stierhoff et al. 2003; Yang et al. 2013).
Specialised morphological characters often exist in species
adapted to perform ASR, including a fairly flat dorsal surface
and pointed head with upturned mouth (Jobling 1994; Chapman
and McKenzie 2009). Fishes with this shape, such as juvenile
eastern rainbowfish, are able to perform ASR without major
reorientation of their body within the water column, and to swim
along almost normally while skimming highly oxygenated
water from the surface. In contrast, sooty grunter, which did
not utilise ASR, have a curved dorsal surface and a ventrally
located jaw. Sooty grunter would have to adopt an almost
vertical position in the water column to perform ASR. Barramundi have an intermediate body shape between the other two
fish species tested here, and were found to be adept at performing ASR in the juvenile phase. Adult barramundi (30–45 cm
total length) have also been observed performing ASR, and have
survived subsurface (,0.3-m depth) DO saturations of between
2.4 and 6.0% over several days at Lagoon Creek in northern
Queensland (Pearson et al. 2003a, 2003b).
There are costs to performing ASR, including an increased
risk of aerial predation (Kramer et al. 1983; Riesch et al. 2010),
so it is beneficial to delay commencement of ASR for as long as
possible, until metabolic requirements force fish to the surface
(Yoshiyama et al. 1995; Watters and Cech 2003). A negative
correlation between hypoxia tolerance and the DO concentration at which a species commences ASR has been established for
sculpins (Mandic et al. 2009). A similar pattern was identified in
the present study, in which the less hypoxia-tolerant eastern
rainbowfish commenced ASR at a higher DO concentration than
did the more tolerant barramundi.
Comparison of the ventilatory capacities of the three
species presented here supports the notion that fish species
commonly exposed to hypoxia in their natural environment
are more able to tolerate low DO (e.g. Rantin et al. 1992;
McNeil and Closs 2007). Both barramundi and eastern rainbowfish, unlike sooty grunter, are found in habitats that may
naturally experience hypoxia. Sooty grunter prefer to inhabit
flowing streams and are unlikely to be found in hypoxic
waters (Pusey et al. 2004).
N. Flint et al.
Feeding and growth
Growth rates (percentage increase in weight) were not strongly
affected by fluctuating hypoxia, although there was a significant
quadratic regression relationship for barramundi and repressed
growth in the most severely hypoxic treatments (5 and 10%
DO saturation). A longer experimental period may have produced clearer results for this species. Growth in the lowest
surviving treatment (10% DO saturation) was repressed in sooty
grunter and negative in rainbowfish, although the regressions for
these species were not statistically significant.
The reduced growth of juvenile barramundi under fluctuating hypoxia was likely to be due to decreased food intake during
the experimental period. Barramundi showed reduced interest in
food in severely hypoxic treatments compared with normoxic
and intermediate treatments, with a 30% reduction in active
feeding time under the lowest DO treatment. Similar effects of
hypoxia on food consumption have been recorded for other
species, including Ictalurus punctatus and Perca flavescens
(Carlson et al. 1980), Pseudopleuronectes americanus (Bejda
et al. 1992), Gadus morhua (Chabot and Dutil 1999), Scophthalmus maximum and Dicentrarchus labrax (Pichavant et al. 2001),
and Salmo salar (Burt et al. 2013; Remen et al. 2012). There are
few studies on the effects on growth of diel fluctuations in DO.
Silurus meridionalis exposed to hypoxia (,36% DO) for ,10 h
each night for 15 days exhibited lower growth rates than did
fish in the control treatment maintained at ,85% DO or higher
day and night (Yang et al. 2013).
In comparisons of the effects of fluctuating hypoxia with
effects of chronic hypoxia, Carlson et al. (1980) found that
growth of Perca flavascens was significantly affected by
chronic hypoxia of ,25%, but not by fluctuating hypoxia where
the mean of fluctuations was the same as the tested level of
chronic hypoxia (cycling between ,17 and 46% DO saturation).
Bejda et al. (1992) found that Pseudopleuronectes americanus
grew more slowly under chronic hypoxia (,30% DO saturation)
than when exposed to diel fluctuations in hypoxia (cycling
between ,30 and 90% DO), but that recovery times for fish
that had been exposed to chronic hypoxia were faster than for
fish that had been exposed to fluctuating hypoxia. Conversely,
Paralichthys lethostigma showed lower growth under fluctuating hypoxia (cycling between ,34 and 75% DO) than under a
chronic treatment of approximately the same mean DO saturation (,60% DO); however, fish in the fluctuating treatment
grew more than fish in a chronic treatment that was equal to the
minimum DO saturation reached each day in the fluctuating
treatment (i.e. ,34% DO) (Taylor and Miller 2001). Thetmeyer
et al. (1999) also found that chronic hypoxia (40% DO)
depressed growth more than a fluctuating treatment (40–86%
DO saturation) in Dicentrarchus labrax. In each of these
examples, reduced food intake was the most likely cause of
growth suppression.
In comparison with other studies of the sublethal effects of
hypoxia on fish (e.g. Dicentrarchus labrax, Thetmeyer et al.
1999; Paralichthys lethostigma, Taylor and Miller 2001; Gadus
morhua, Chabot and Claireaux 2008), the species studied here
displayed few negative effects of exposure to fluctuating hypoxia, except in the most severe treatments. Importantly, the
present study focussed on juvenile fishes, and the effects of
Sublethal effects of fluctuating hypoxia on fish
hypoxia on other life-history stages of the same species may
differ. For example, a study on physiology of adult and juvenile
Perca flavescens exposed to hypoxia found that juveniles were
more tolerant than adults (Robb and Abrahams 2003). The
authors postulated that the higher tolerance of younger (and
smaller) fish may give them an advantage in seeking refuge from
predators by entering hypoxic waters. Conversely, Leiostomus
xanthurus becomes more tolerant to hypoxic conditions with
increasing size, indicating that differences in hypoxia tolerance
based on size and life-history stage are species-specific (Shimps
et al. 2005).
It is likely that long-term natural exposure to hypoxia has
conditioned juvenile barramundi and eastern rainbowfish to
survive and prosper under notionally challenging DO regimes.
In their natural environment, fish are usually able to access the
water surface (except in cases of high macrophyte cover), and in
this situation hypoxia tolerance may be increased by the ability to
utilise ASR (Flint et al. 2012). Eastern rainbowfish and barramundi are more likely than sooty grunter to experience hypoxia
in the field (Pusey et al. 2004) and, accordingly, juvenile sooty
grunter are less hypoxia-tolerant than the other two species.
Hypoxia occurs naturally in many areas and a recent review
focusing on chronically hypoxic East African papyrus swamps
suggested that some aquatic organisms, particularly small
organisms, may experience benefits to their fitness as a result
of living in habitats that we have traditionally considered to be
suboptimal (Joyner-Matos and Chapman 2013). It is interesting
to note that even anthropologically derived increases in the
incidence of hypoxia do not always have catastrophic effects on
individual fish species. Microevolutionary responses to hypoxic
stress have been identified in some fish species, in areas where
anthropogenic activities resulted in more frequent hypoxic
episodes. Haplochromis (Yssichromis) pyrrhocephalus, a cichlid of Lake Victoria, showed very high phenotypic plasticity in
response to a changing environment. Apparently in response
to increasing hypoxia, the average number of secondary gill
lamellae in fish from the lake increased by 25% between 1978
and 1999 (Witte et al. 2000). Gill surface area has increased in
Rastrineobola argentea (family Cyprinidae) between 1983 and
1988 (Wanink and Witte 2000). Similar increases in gill surface
area have been recorded from laboratory studies on cichlids
(Chapman et al. 2000) and sea bass (Saroglia et al. 2002).
A recent study demonstrated the ability of Sernotilus atromaculatus to adjust its physiological responses so as to function
more effectively in disturbed environments. When exposed to
low DO (,40% saturation for 4 h), fish from agricultural areas
showed a reduced short-term stress response in comparison to
those collected from streams in forested areas (Blevins et al.
2013). So as to better understand the ramifications of this type
of physiological plasticity for freshwater ecosystems, further
research on medium-term responses such as growth rates and
reproduction would also be useful.
The duration of the experiments in the present study was
chosen to reflect medium-term effects of fluctuating hypoxia.
Greater effects, or potentially a greater adaptive ability, may
accrue over a longer period. However, short-term sublethal
effects of chronic hypoxia have been observed in other local
studies (e.g. Pearson et al. 2003a), and thus a range of time
scales is appropriate for further investigation.
Marine and Freshwater Research
I
Determination of the synergistic effects of hypoxia on the
ability of fish to withstand disease, parasites and reduction in
water quality is important to understanding the true effect of
hypoxia on fish species and communities. When caused by
agricultural runoff, hypoxia often occurs in tandem with
increased concentrations of dissolved carbon dioxide, high
sediment loads, high concentrations of nitrogenous wastes,
including the extremely toxic form of ammonia (Økelsrud and
Pearson 2007), and pesticides (Pearson et al. 2003a). It is
possible that uptake rate of toxicants increases as a result of
higher respiration rates during periods of hypoxia. To mitigate
the effects of these activities on fish populations, it is necessary
to understand how combinations of changes to water chemistry
and pollutants interact with each other in each fish species, and
in life-history stages of each species.
Conclusions
The present study has demonstrated sublethal effects of fluctuating hypoxia on juveniles of three species of freshwater fishes
native to tropical Australia and provided results that can be
readily compared with field data to predict the likely effects of
minimum DO concentrations on these species. Despite the
natural tolerance of some tropical fish species to hypoxic stress
that has been identified in this and other studies, fish kills
attributed to hypoxic episodes are still known to occur regularly
(Bishop 1980; Townsend et al. 1992; Pearson et al. 2003a,
2003b). Nutrient-rich runoff from agriculture, aquaculture,
industry and urban development increases the frequency and
intensity of hypoxic episodes (e.g. Tucker and Burton 1999;
Collins et al. 2000; Chabot and Claireaux 2008) such that even
the most tolerant fish are affected. Therefore, sustained efforts to
ameliorate human impacts on fish habitats are warranted, and
this is no less important in the case of the wetlands of the wet
tropics in Australia, with their unique fauna and where there
remains only a small area of suitable habitat, much of which
suffers from the effects of agricultural and other contaminated
runoff (Januchowski-Hartley et al. 2011; Pearson et al. 2013).
Acknowledgements
This research was funded primarily by the Sugar Research and Development
Corporation, with additional funds provided by the Rainforest CRC’s
‘Catchment to Reef’ program and the Queensland Government’s ‘Growing
the Smart State’ Program. Thanks go to Tablelands Fish Stocking Association for sooty grunter juveniles; M. Sheaves for discussions and statistical
advice; A. Hogan for assistance and advice in relation to sooty grunter; P. Roy
for transporting fish to Townsville; R. Gegg for assisting with aquarium
construction; and B. Butler for early discussions. We thank two anonymous
reviewers and G. Closs for their helpful comments on the manuscript. Eastern
rainbowfish were collected under Queensland Fisheries Management
Authority General Fisheries Permit PRM00430H. Treatment of animals in
this study was approved by James Cook University’s Ethics Review Committee, Animal Ethics Sub-Committee, approval number A682_01.
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