Use of aquatic plants to create fluctuating hypoxia in an experimental environment

Abstract

In freshwater systems, dissolved oxygen (DO) saturation frequently fluctuates, falling at night and rising during the day in response to respiration and photosynthesis, respectively, of aquatic biota. Low DO (hypoxia) is a common cause of fish kills in freshwater systems around the world. Laboratory studies on responses of fish to fluctuating DO are currently limited, and require techniques that produce a realistic cycle of DO depletion and replacement. Artificial DO-depletion mechanisms frequently used for hypoxia studies may underestimate the field effects of hypoxia on fish because of the lack of the naturally occurring synergistic effect of lower pH, and seldom allow fish to employ behavioural adaptations to hypoxia, such as aquatic surface respiration. We demonstrate proof-of-principle for an alternative method of creating fluctuating hypoxia in an experimental environment, using the natural rhythms of photosynthesis and respiration of aquatic plants to create realistic conditions. A range of volumes of aquatic macrophytes were used alone and in combination with fish to lower DO saturation in sealed freshwater aquaria, and achieved DO saturations as low as 1.3%. This cost-effective method can be deployed over long periods with minimal effort in comparison to traditional methods of DO reduction.

Full text

Use of aquatic plants to create fluctuating hypoxia
in an experimental environment
Nicole Flint
A
,
D
,
E
,Richard G. Pearson
A
,
B
and Michael R. Crossland
B
,
C
A
School of Marine and Tropical Biology, James Cook University, Townsville, Qld 4811, Australia.
B
Australian Centre for Tropical Freshwater Research, James Cook University, Townsville,
Qld 4811, Australia.
C
School of Biological Sciences, University of Sydney, Sydney, NSW 2006, Australia.
D
Present address: Centre for Environmental Management, Central Queensland University,
Bruce Highway, Rockhampton, Qld 4702, Australia.
E
Corresponding author. Email: n.flint@cqu.edu.au
Abstract. In freshwater systems, dissolved oxygen (DO) saturation frequently fluctuates, falling at night and rising
during the day in response to respiration and photosynthesis, respectively, of aquatic biota. Low DO (hypoxia) is a
common cause of fish kills in freshwater systems around the world. Laboratory studies on responses of fish to fluctuating
DO are currently limited, and require techniques that produce a realistic cycle of DO depletion and replacement. Artificial
DO-depletion mechanisms frequently used for hypoxia studies may underestimate the field effects of hypoxia on fish
because of the lack of the naturally occurring synergistic effect of lower pH, and seldom allow fish to employ behavioural
adaptations to hypoxia, such as aquatic surface respiration. We demonstrate proof-of-principle for an alternative method of
creating fluctuating hypoxia in an experimental environment, using the natural rhythms of photosynthesis and respiration
of aquatic plants to create realistic conditions. A range of volumes of aquatic macrophytes were used alone and in
combination with fish to lower DO saturation in sealed freshwater aquaria, and achieved DO saturations as low as 1.3%.
This cost-effective method can be deployed over long periods with minimal effort in comparison to traditional methods of
DO reduction.
Additional keywords: barramundi, Ceratophyllum demersum,Lates calcarifer.
Received 26 August 2011, accepted 22 November 2011, published online 2 April 2012
Introduction
Hypoxia (low DO saturation) is a major cause of fish deaths and
reduced fish diversity worldwide (e.g. Townsend et al. 1992;
Hamilton et al. 1997; Herna´ndez-Miranda et al. 2010). Hypoxia
occurs naturally in marine, estuarine and freshwater environ-
ments and can be exacerbated by anthropogenic nutrient sources
such as agricultural runoff (e.g. Bonsdorff et al. 1997; Martin
and Saiki 1999; Collins et al. 2000), urban runoff (Tucker and
Burton 1999), industrial effluents (Winn and Knott 1992) or
wastes from aquaculture facilities (Hargrave et al. 1993;
Bonsdorff et al. 1997).
Hypoxia has been defined as DO less than 2 mg L
1
(,18%
in seawater), or below the point that sustains most animal life
(Diaz 2001; Rose et al. 2009), so its definition depends on the
context of the study (Farrell and Richards 2009). We understand
the lethal and sublethal effects of hypoxia largely from numer-
ous studies on marine and freshwater fish from cold and
temperate regions (e.g. Kramer 1987; Miller et al. 2002;
Richards 2011). Comparatively little information exists on the
sublethal effects of hypoxia in tropical freshwater systems, with
some notable exceptions from South America (e.g. Rantin et al.
1992; Fernandes et al. 1995), Africa (e.g. Chapman et al. 1995;
Chapman and Chapman 1998; Corrie et al. 2008) and northern
Australia (Pearson et al. 2003; Butler et al. 2007).
Moreover, studies of hypoxia rarely involve testing fluctuat-
ing hypoxia, whereby in the presence of high plant biomass, DO
saturation varies over a diel cycle, falling at night owing to the
respiration of aquatic organisms, and rising during the day,
through production of oxygen by photosynthesis (Brady et al.
2009). The upper and lower saturations reached during the cycle
vary with conditions that include nutrient status, plant and
microbe abundance, abundance of particulate and dissolved
organic material, temperature and flow. In agricultural regions
in tropical northern Queensland, fluctuating hypoxia is common
(Pearson et al. 2003). Studies on responses of fish to fluctuating
hypoxia require techniques that produce a cost-efficient, low-
maintenance system for DO depletion and replacement.
There are hundreds of published studies reporting the effects
of hypoxia on fish of various species, spanning at least 60 years.
Many studies have used laboratory experiments to identify these
effects, employing a variety of methods to achieve depleted DO
concentrations, including addition of nitrogen gas, vacuum
CSIRO PUBLISHING
Marine and Freshwater Research, 2012, 63, 351–360
http://dx.doi.org/10.1071/MF11190
Journal compilation ÓCSIRO 2012 www.publish.csiro.au/journals/mfr

Table 1. Experimental oxygen-depletion methods used in marine and freshwater studies
Dissolved oxygen (DO) depletion methods are recorded from a sample of 80 studies. All references were either technical methods papers, or examined the
effects of hypoxia on fish. Marine/freshwater: M ¼marine/estuarine, F ¼freshwater; type of hypoxia: C ¼chronic and/or acute hypoxia, F ¼fluctuating
hypoxia, G ¼DO saturation gradually altered to a maximum or minimum level (progressive hypoxia); lethal/sublethal: L ¼experiment to determine lethal
level, S ¼experiment on sublethal effects
Technique Studies using this technique Location of study Marine/
freshwater
Type of
hypoxia
Lethal/
sublethal
Addition of nitrogen gas (51 of 80) Fry 1951 Toronto – – –
Downing 1954 UK F G L
Downing and Merkens 1955 UK F G L
Whitmore et al. 1960 Oregon F C S
Davis et al. 1963 Oregon F C S
Dahlberg et al. 1968 Oregon F C S
Siefert and Spoor 1974 Minnesota F C L & S
Swift and Lloyd 1974 UK F C S
Johnston 1975 UK F C S
McDonald and McMahon 1977 Canada F C S
Kramer and Mehegan 1981
A
Trinidad F C L & S
Drewett and Abel 1983 UK F C L
Petersen and Petersen 1990 Denmark M C L & S
Pihl et al. 1991 Virginia M C L & S
Kaufmann and Wieser 1992 Austria F C S
Schurmann and Steffensen 1994 Denmark M G S
Cech and Massingill 1995 California F G S
Fernandes et al. 1995 Brazil F C S
Thomason et al. 1996 UK M C S
Crocker and Cech 1997 California F C S
Schurmann and Steffensen 1997 Denmark M G S
Dalla Via et al. 1998
A
Italy M G S
Plante et al. 1998 Quebec M C L
Chabot and Dutil 1999 Quebec M C S
Jones and Reynolds 1999a, 1999b, 1999cUK M C S
Renshaw and Dyson 1999 Australia M C S
Tallqvist et al. 1999 Finland M C L & S
Geiger et al. 2000 Florida M C S
Pichavant et al. 2000 France M C S
Pichavant et al. 2001 France M C S
Richardson et al. 2001 New Zealand F C S
Taylor and Miller 2001 North Carolina M C & F S
Pearson et al. 2003 Australia F C L & S
Ishibashi et al. 2005 Japan M G L
Shimps et al. 2005 North Carolina M C L
Ishibashi et al. 2007 Japan M G L
Landry et al. 2007 Mississippi M C S
Ripley and Foran 2007 Virginia M C S
Hassell et al. 2008 Australia M C L & S
Sloman et al. 2008 British Columbia M G S
Wang et al. 2008 Taiwan F C S
Brady et al. 2009 Delaware M F L & S
Lefranc¸ois et al. 2009 Italy M G S
Martı´nez et al. 2009 Uganda F C S
Brady and Targett 2010 Delaware M F S
Vanlandeghem et al. 2010 Illinois F C S
Cheek 2011 Texas M F S
Laursen et al. 2011
A
UK F G S
Tzaneva et al. 2011 Canada F G S
Fish respiration (11 of 80) Hunn 1969 Wisconsin F G S
Courtenay and Keenleyside 1983
A
Central America F G S
Vig and Nemcsok 1989 Hungary F G S
Kakuta and Murachi 1992 Japan F G S
Kakuta et al. 1992 Japan F G S
(Continued )
352 Marine and Freshwater Research N. Flint et al.

degassing, addition of sodium sulfite and sealing the experi-
mental containers so that the fish’s own respiration removes
oxygen from the water (Table 1). Other studies have aimed to
achieve realism by inserting cages or mesocosms into low-
oxygen environments in the field (Dunson and Dunson 1999;
Ruggerone 2000).
We assessed 80 published laboratory studies that examined
the effects of hypoxia on fish. The most common method of DO
depletion in these experiments was the displacement of oxygen
gas by bubbling water with nitrogen gas (64% of papers;
Table 1). This technique is very effective and has the advantage
that nitrogen gas is biologically inert. However, some aspects of
this method are unnatural, including the presence of a ‘nitrogen
atmosphere’ above the water’s surface, preventing fish from
effectively employing aquatic surface respiration (ASR; Kramer
and Mehegan 1981) or facultative air-breathing.
In natural waterways, fluctuating hypoxia is often caused by
abundant macrophyte growth, encouraged by high light condi-
tions or nutrient concentrations (Kaenel et al. 2000). During the
night, respiration by organisms in the water body removes
oxygen from the water and replaces it with carbon dioxide,
causing pH levels to drop (except in extremely hard water)
(Burnett 1997). Adding nitrogen gas to water causes the reverse
effect because nitrogen displaces both oxygen and carbon
dioxide from solution. This artificial situation is not ideal, given
that the low pH caused by high concentrations of dissolved
carbon dioxide in field situations can disrupt the acid–base
balance and gas transfer across fish gills (Cruz-Neto and
Steffensen 1997), thereby lowering the efficiency of oxygen
uptake (Dahlberg et al. 1968). This artificial effect may be
avoided by bubbling carbon dioxide gas into the water
(e.g. Pearson et al. 2003), or by adjusting pH using buffers.
However, the disadvantage is that such methods make experi-
ments more expensive, time-consuming and difficult to control,
particularly for long-running experiments.
Here, we demonstrate the use of aquatic plants to create
conditions of hypoxia, thereby exposing test organisms to diel
cycling of DO and pH, which replicates natural environments
and avoids some of the problems associated with other
methods. We present a cost-effective laboratory method,
which can be deployed over long periods of time with
minimal effort in comparison to some traditional methods
of DO reduction.
Materials and methods
We used Ceratophyllum demersum (commonly known as
hornwort), a cosmopolitan aquatic macrophyte, as our test
species. Hornwort is a non-rooted plant that absorbs nutrients
from the surrounding water, has high photosynthetic perfor-
mance (Blu
¨met al. 1997) and produces increasing DO satura-
tion with increasing light intensity (Pearson et al. 2003). It grows
quickly in high-nutrient waters, can tolerate a wide range of
water hardness, and can become a noxious weed in areas where
it is exotic (Global Invasive Species Database 2006). It is
commonly used in the aquarium trade, making it freely available
Table 1. (Continued)
Technique Studies using this technique Location of study Marine/
freshwater
Type of
hypoxia
Lethal/
sublethal
van Raaij et al. 1994 Netherlands F G S
Thetmeyer et al. 1999 Germany M C & F S
Waller et al. 2000
A
British Columbia M C S
Cerezo Valverde et al. 2006 Spain M G L & S
Lays et al. 2009 Norway M G S
Barnes et al. 2011 Australia F G L
Vacuum degassing (8 of 80) Mount 1961 Ohio F G L
Carlson and Herman 1978
A
Wisconsin F C & F S
Carlson et al. 1980 Minnesota F C & F L & S
Scott and Rogers 1980 Alabama F C S
Bejda et al. 1987 New Jersey M G S
Pouliot et al. 1988 Quebec F C S
Pouliot and de la Nou
¨e 1989 Quebec F C S
Miller et al. 2002 East USA M C L
Sodium sulfite – including when
used in conjunction with nitrogen
gas (7 of 80)
Chapman et al. 1995
A
Lake Victoria F G S
Gee and Gee 1995 Australia M C S
Chapman and Chapman 1998
A
Lake Nabugabo F G S
Schofield and Chapman 2000
A
Lake Nabugabo F G S
Melnychuk and Chapman 2002
A
Lake Kabaleka F G S
Schofield et al. 2007 Florida F G L
Corrie et al. 2008
A
Uganda F G S
Cages in the field (3 of 80) Moore 1942 Minnesota F C L
Dunson and Dunson 1999 Florida M C & F S
Ruggerone 2000 Alaska F C L
A
Location the fish were sourced from.
Using aquatic plants to create fluctuating hypoxia Marine and Freshwater Research 353

as a test subject. Plants were collected from weir pools in Ross
River, Townsville (198180S, 1468450E), and held in carbon-
filtered (0.5 mm) tap water in 500-L white plastic mesocosms in
open sunlight. Plants were maintained with a nutrient mix of
aquarium plant food, sodium nitrate and potassium dihydrogen
orthophosphate, and kept outside for 5 days before using in
experiments.
The trials were conducted in a light- and temperature-
controlled room at a constant water temperature of 298C, using
30-L glass aquaria filled with 25 L of carbon-filtered water. The
aquarium design left a 5-L pocket of air between the water
surface and the lid that was accessible to fish. Each aquarium
was sealed with a PVC plastic lid and silicon grease (Fig. 1).
Sealable ports in the lid allowed access for measuring DO. Plant
volume was measured as the amount of water displaced in a
graduated measuring cylinder. Treatments consisted of different
volumes of hornwort (50–300 mL) added to the aquaria, and
controls (0 mL plant material). Only healthy parts of the plants,
especially the dense green tips, were used in the experiments.
A submersible pump (Resun SP-600: 5 W, 220 V, 60/50 Hz,
250 L h
1
delivery, Shenzhen, China) in each aquarium
maintained water circulation. Trials with fish used juvenile
barramundi (Lates calcarifer) of 40–50-mm total length.
In all trials, DO, pH and temperature were regularly moni-
tored with 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. The readings were
taken by inserting the probes through a resealable opening in the
lid of each aquarium.
Presented here are the results of three experiments investi-
gating (1) the relationship between plant biomass and minimum
DO saturation achieved overnight, (2) the use of fish alone to
decrease DO saturation overnight and (3) the effectiveness of
the plants to reduce DO saturation overnight in the presence
of fish for prolonged time periods. Graphical illustration of data
and regression analyses were carried out in SigmaPlot 11.0
(Systat Software Inc. 2008, Chicago, IL).
Experiment with plants
Hornwort (0-mL plant material ¼control, and 50-, 100-, 150-,
200- and 300-mL treatments, n¼2 replicates per treatment) was
used to deplete DO saturation in the aquaria over 18 h, during
which time aquaria were kept in complete darkness. Minimum
DO saturation in each aquarium and pH data were analysed by
simple linear regression with 95% confidence intervals.
Experiment with fish
Reduction in DO saturation owing to fish respiration was tested
using sealed aquaria, each containing four barramundi (n¼2
replicates). This trial was carried out concurrently with the plant
experiment detailed above, and under the same conditions.
Experiment with fish and plants
This experiment aimed to create diel reductions in DO con-
centration that were as low as possible while remaining suble-
thal to fish, to demonstrate the concentrations to which DO
could be successfully and repeatedly lowered using a combi-
nation of fish (in this case barramundi) and plants (hornwort).
The best combination of plants and fish to achieve this aim was
found to be four barramundi and 275 mL of hornwort. Four
barramundi were placed into each of two experimental aquaria,
each of which also contained 275 mL of hornwort. Aquaria were
sealed and kept in the dark for 16 h, during which time one
aquarium was bubbled with compressed air, as a control,
whereas the other one was not. In initial trials, it was found that
18 h of darkness (as used in the tests with plants and fish alone,
above) resulted in excessive DO depletion, so 16-h dark periods
were employed. During the 8-h ‘day’, when overhead fluores-
cent lights were switched on in the experimental room, addi-
tional aquarium lamps (Hagen Aqua Glo – 55 lux, 18 000 K,
Montreal, Canada) were switched on behind the aquaria, closest
to the plants, to encourage photosynthesis, and compressed air
was provided to enhance DO renewal.
For 20 days, DO, pH and temperature were recorded every
morning (when lights were switched on at 0900 hours) and
evening (before lights were switched off at 1700 hours) in both
Removable PVC covers
for each access hole
Sealed PVC lid with access holes
Water level (25 L)
Plant material within meshed section
Pump
Air pocket (5 L) Mesh dividers
Fig. 1. Diagram of experimental aquaria. Polyvinyl chloride (PVC) lids were sealed to the tops of the aquaria by using high-
vacuum silicon grease. Plastic mesh (5 mm) was used to divide plant material and pump compartment from the large fish
compartment. Aquaria were of 30-L capacity, filled with 25 L of water, leaving a 5-L air pocket between the air–water interface
and the lid.
354 Marine and Freshwater Research N. Flint et al.

aquaria. Maintenance of fish during this time included daily
feeding as lights came on at 0900 hours, and a daily 50% water
change using carbon-filtered tap water (DO saturation .90%),
which was carried out 1 h before lights were switched off. By
this time, DO concentration had returned to normoxia. Hornwort
in experimental aquaria was replaced every 3 days with fresh
material that had been kept in large outdoor mesocosms (as
described above) for at least 5 days.
Results
Experiment with plants
Plant material alone substantially reduced the concentration of
DO in aquaria overnight, and percentage DO saturation recorded
in the morning decreased with increased volume of plant
material (Fig. 2a,R
2
¼0.970, P,0.001, 95% CI). Maximum
DO before depletion was constant across treatments (98.3% 
2.4% (s.d.)). The two control aquaria showed small decreases in
DO saturation overnight (Control 1: from 98% to 94%; Control
2: from 99% to 91%).
The pH levels recorded in the morning, when DO was at a
minimum, decreased with increased volume of plant material
(Fig. 2b,R
2
¼0.578, P,0.001, 95% CI). In the evening, when
DO was at a maximum, pH levels were relatively constant
across treatments (7.13 0.12). In the control aquaria, pH fell
slightly overnight (Control 1: from 7.2 to 6.8; Control 2: from
7.21 to 6.83).
Water circulation in the experimental aquaria was found be
to be effective, with variation within the water column of ,0.5%
DO saturation.
Experiment with fish
The respiration of four barramundi depleted DO saturation in the
sealed experimental aquaria by little more than for the control
aquaria (described above), to a maximum of 9% (Fish treatment
1: from 98% to 89%; Fish treatment 2: from 98% to 92%).
Concurrently, the change in pH in the treatment aquaria was
similar in magnitude to that of the control aquaria (Fish treat-
ment 1: from 7.98 to 7.45; Fish treatment 2: from 7.9 to 7.41).
Experiment with fish and plants
When 275 mL of hornwort were used to create fluctuating hypoxic
conditions in an aquarium containing four barramundi, the
resulting DO and pH reductions were highly repeatable over
20 days (Fig. 3). In the treatment aquarium, the mean minimum
(‘day’) DO was 4.8% 2.9% (s.d.) saturation, and the mean pH at
this time was 6.50 0.09. The mean maximum (‘night’) DO sat-
uration in the same aquarium was 89.4% 5.4%, and the mean pH
at this time was 7.36 0.16. The control aquarium during this test
had a ‘day’ DO sat uration of 97.5% 4.1% and pH of 7.50 0.11.
The ‘night’ DO saturation in the control tank was 94.1% 5.6%
and the pH averaged 7.51 0.14.
Discussion
In the present study, different volumes of aquatic plants (horn-
wort) in aquaria were used successfully to create diel cycles in
DO saturation, to various levels of hypoxia. Aquaria containing
barramundi alone achieved minor DO reduction overnight.
A higher ratio of fish to water volume would be required to sub-
stantially deplete DO using fish respiration alone (e.g. Lays et al.
2009), but for some species, this might cause additional stress to
fish through density effects on behaviour and physiology.
When plants were added to aquaria containing barramundi,
DO depletion was rapid and repeatable, and followed a natural
diel cycle. The DO cycling in the treatment aquarium containing
barramundi and hornwort was similar to that under field condi-
tions in areas where there is a high level of plant and algal
material in still water. For example, in a lentic habitat in northern
Queensland, Pearson et al. (2003) found that DO frequently
cycled between 2% and 85% saturation over 24 h, and similar
diel DO fluctuations have also been reported from other areas
(e.g. Gulf of Mexico, Cheek et al. 2009; Florida, Bunch et al.
2010).
The extremely low DO concentrations (as low as 1.3% and
frequently ,5% saturation) survived by juvenile barramundi in
the present study demonstrated an even greater capacity to
withstand hypoxic stress than has been shown in previous
studies. For example, Flint (2005) reported a lethal concentra-
tion of ,2% DO saturation for barramundi of 50–70-mm total
length when oxygen is gradually depleted (noting that study and
the present study were not designed to test lethal limits), and
120
(a)
(b)
100
80
60
40
20
0
Minimum DO saturation (%) pH at minimum DO saturation
0 50 100 150 200 250 300 350
0 50 100 150 200 250 300 350
Volume of plant material (mL)
7.0
6.9
6.8
6.7
6.6
6.5
6.4
6.3
Fig. 2. Magnitude of (a) dissolved oxygen and (b) pH depletion in
experimental aquaria containing varying amounts of plant material after
18 h of darkness. Each aquarium contained different volumes of hornwort, as
shown on the x-axes (two aquaria per treatment). There were no fish in any
aquaria.
Using aquatic plants to create fluctuating hypoxia Marine and Freshwater Research 355

120
Treatment tank Control tank
100
80
60
40
DO saturation (%)pH
20
0
8.0
(b)
(a)
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
Night 1
Night 2
Night 3
Night 4
Night 5
Night 6
Night 7
Night 8
Time of reading
Night 9
Night 10
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
Night 15
Night 16
Day 17
Day 18
Day 19
Day 20
Night 17
Night 18
Night 19
Night 20
Day 14
Day 15
Day 16
Night 11
Night 12
Night 13
Night 14
Night 1
Night 2
Night 3
Night 4
Night 5
Night 6
Night 7
Night 8
Night 9
Night 10
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
Night 15
Night 16
Day 17
Day 18
Day 19
Day 20
Night 17
Night 18
Night 19
Night 20
Day 14
Day 15
Day 16
Night 11
Night 12
Night 13
Night 14
Fig. 3. Dissolved oxygen (DO) cycling using fish and plants to reduce saturation overnight. (a)DO
saturation (%) and (b) pH in two aquaria each containing four barramundi fingerlings, and 275 mL hornwort.
The treatment aquarium was sealed and not aerated for 16 h overnight and then aerated for 8 h during the day.
The control aquarium was lightly sealed and aerated continuously. The absence of pH data at Day 6 was due
to equipment malfunction. Readings were taken in the morning (day) and evening (night) only, and were not
continuous. Continuous lines are for illustrative purposes only.
356 Marine and Freshwater Research N. Flint et al.

Pearson et al. (2003) identified a 24-h lethal concentration of
,11–15% saturation at 298C for fish of 85–105-mm total length.
In the present study, oxygen depletion was gradual, and animals
had access to the air–water interface, and so were likely to have
access to more highly oxygenated water than the minimum
concentrations measured within the water column, as would
often be possible in field situations (e.g. McNeil and Closs 2007;
Riesch et al. 2010).
Aquaria containing both plants and fish showed some
variability in the minimum DO saturation reached in a set time.
The use of larger aquaria would probably reduce this effect.
Conducting experiments under natural light may increase plant
productivity and reduce the amount of plant material required to
deplete DO to a required concentration, and reduce the need to
regularly replace used plants.
The use of plants as the primary oxygen consumer in
laboratory experiments creates natural fluctuations in dissolved
carbon dioxide, and hence pH, and allows fish access to the
higher oxygen concentrations in surface waters, such that
aquatic surface respiration (ASR) (Kramer and Mehegan
1981; Kramer and McClure 1982) and facultative air breathing
(e.g. Geiger et al. 2000) may be effectively utilised. This
advantage is in contrast with other methods commonly used to
create hypoxic conditions, such as vacuum degassers and nitro-
gen replacement, where access to higher oxygen saturations at
the air–water interface may be unavailable. In situations where
surface access is not desired for an experiment, a simple plastic
mesh divider could easily be incorporated into the aquarium
design to physically prevent fish from accessing the air–water
interface. The method described here produces DO cycling
regimes that are cost-effective and repeatable over several
weeks and that simulate field conditions.
Limitations of the method described here include the time
involved in changing water daily and plant material every 3 days,
and the necessity to maintain stands of plants for use in the
experiment. The regular water changes that were required may
also cause stress to fish. Despite these limitations, the technique
described here is easily implemented and could replace other
methods as a DO-depletion mechanism. It would be difficult to
use this method to maintain DO saturation at a minimum level
for a long period of time, as is necessary for experiments on the
effects of chronic hypoxia (e.g. Miller et al. 2002; Landry et al.
2007; Wang et al. 2008), but this method is particularly
appropriate to simulate diel DO cycling or progressive DO
depletion to a non-sustained minimum. Several factors may
influence variability in results, including aquarium size, the
amount of natural light available, temperature, water chemistry
and the health of plants used. However, this method offers an
alternative to traditional methods of DO depletion and should be
considered as a means of creating natural diel cycles in DO
saturation in experimental situations.
Acknowledgements
This research was funded primarily by the Sugar Research and Development
Corporation, through a PhD scholarship to NF at James Cook University
(JCU). The authors thank two anonymous reviewers and A. J. Boulton for
their helpful comments on the text, B. Butler for valuable discussions, and
R. Gegg for assistance in building aquaria. Treatment of all animals in this
study was approved by JCU’s Ethics Review Committee, approval number
A682_01. Aquatic plants were collected under Queensland Government
Environmental Protection Agency Permit WISP00739802.
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