Ảnh hưởng của nitrite, nhiệt độ và CO2 lên quá trình sinh lý và tăng trưởng của cá thát lát còm (chitala ornata, gray, 1831) anh

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

DOCTORAL DISSERTATION SUMMARY
Major: Aquaculture
Major code: 9 62 62 03 01

LE THI HONG GAM

EFFECTS OF NITRITE,
TEMPERATURE AND HYPERCAPNIA
ON PHYSIOLOGICAL PROCESSES AND
GROWTH IN CLOWN KNIFEFISH
(Chitala ornata, Gray 1831)

Can Tho, 2018


THE RESEARCH WAS PERFORMED AND
COMPLETED AT CAN THO UNIVERSITY

Supervisor: Prof. Dr. Nguyen Thanh Phuong
Co-supervisor: Assoc. Prof. Dr. Mark Bayley

The dissertation will be defended at the Doctoral Dissertation
Assessment Committee at the University Level
At: ……………………………………….……………...
Time & Date:……………………………………………

Reviewer 1: …………………………………………….
Reviewer 2: …………………………………………….

The dissertation can be found at:
The Learning Resource Center, Can Tho University
The National Library of Vietnam


THE LIST OF PUBLISHED PAPERS
1. Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong,
D.T.T., Phuong, N.T. and Bayley, M., 2017. Extreme
nitrite tolerance in the clown knifefish Chitala ornata is
linked to up-regulation of methaemoglobin reductase
activity. Aquatic Toxicology. 187: 9–17.
2. Gam, L.T.H., Jensen, F.B., Huong, D.T.T., Phuong,
N.T., and Bayley, M., 2018. The effects of elevated
environmental CO2 on nitrite uptake in the air-breathing
clown knifefish, Chitala ornata. Aquatic Toxicology.
196: 124-131.
3. Gam, L.T.H., Vu, N.T.T., Nhu, P.N., Phuong, N.T. and
Huong, D.T.T., 2018. Effects of nitrite exposure on
haematological parameters and growth in clown knifefish
(Chitala ornata, Gray1831). Can Tho University Journal
of Science. 54(2): 1-8.


CHAPTER 1
INTRODUCTION
1.1 Background
Climate change is defined as a change of climate that affected directly
or indirectly human activity, replacing the composition of the global
atmosphere, and natural climate change recorded over long-term

comparable periods of time (UNFCCC, 1992). This change has been
caused by the increases of toxic gases such as CO2, N20, CH4 and green
house gas concentrations as well as a temperature rise of 2.5 degrees
Fahrenheit (1-4 degrees Celsius) over the next century (IPCC, 2013).
According to the evaluation of vulnerability, Vietnam had the 27 th rank
among 132 countries over the world, which is under the impacts of
climate change.
With topographic characteristics and natural geographical conditions,
the Mekong Delta (MD) becomes one of the areas having the most
impacts over the world. The increases of temperature induce the rise of
metabolism of organism and aquatic animals as well as decomposition
of toxic compounds. In the other hand, with the abundance of intensive
culture system, overfeeding with waste products from excretion of
aquatic animals has caused toxic gases such as: nitrite, carbon dioxide,
ammonia, hydro sulfur…Especially, nitrite which is a product of
nitrogen cycle, formed from ammonia in the condition of low dissolved
oxygen level is well-documented toxin in aquatic system because it
causes a lowering of blood oxygen with methaemoglobin formation
(brown blood phenomenon), then leading a disturbance of respiration,
physiological processes and growth (Kroupova et al., 2005). However,
there have been a limited number of studies about effects of these
environmental parameters to biological features, physiological
processes in air-breathers, which may be seriously influenced by global
climate change with their air-breathing activity. To date only two
studies exist in air-breathers in the striped catfish (Pangasionodon
hypophthalmus) reported by Lefevre et al., 2011 and the snakehead
(Channa striata) also reported by Lefevre et al., 2012 with typical
results driven by high tolerance of nitrite in reducing nitrite uptake via
gills and efficient denitrification mechanisms. Besides, there have
recently been several studies about effects of other environmental

factors in air-breathing fish such as Damsgaard et al. (2015) about
effects of carbon dioxide on acid-base regulation in P. hypophthalmus
with high capacity of acid-base regulation compared to other airbreathing species. Moreover, there is obviously not only one toxin
existing in aquatic environment; the best assumption is that the

1


combination of a variety of toxin may cause more bad effects by
competition to uptake into fish blood.
The facultative air-breathing C. ornata is an important species in
aquaculture throughout South East Asia. C. ornata is not only of high
commercial value as a source of protein for human consumption, but it
is also a costly ornamental fish species in tropical aquaria. Therefore,
the present dissertation about “Effects of nitrite, temperature and
hypercapnia on physiological processes and growth in clown knifefish
(Chitala ornata, Gray 1831)” was necessarily conducted to have an
understanding about effects and adaption mechanisms of this airbreathing fish under climate change.
1.2 The objectives of dissertation
The objectives of this dissertation were to investigate the effects of
nitrite, high concentrations of carbon dioxide and elevated temperatures
to physiological parameters and growth of the air-breathing C. ornata
during sub-lethal and chronic exposures of these factors in isolation and
combination in order to provide a better physiological understanding,
particularly recommendations and solutions for minimizing impacts of
nitrite toxicity and its combination with other environmental elements
in aquaculture ponds under global climate change.
1.3 The main projects of dissertation
a) Conducting a survey on some selected environmental parameters
in C. ornata ponds

b) Determining the 96 h LC50 of nitrite and examining the effect of
nitrite on haematological parameters and growth in C. ornata
c) Determining the activity of metHb reductase in metHb reduction in
sub-lethal nitrite exposures in C. ornata
d) Investigating the combined effect of nitrite and hypercapnia (high
concentration of carbon dioxide in the water) on haematological
parameters in small-sized and large sized C. ornata
e) Determining the temperature tolerance and the effect of various
levels of temperature on haematological parameters in small-sized
and large sized C. ornata
f) Determining 96 h LC50 of nitrite at elevated temperatures and
investigating the effects of nitrite at different temperature on
haematological paramters in C. ornata
g) Examining the effects of nitrite at different temperatures on
haematological parameters, growth and digestive enzyme activity
in C. ornata
1.4 The hypotheses of dissertation

2


During nitrite exposure, C. ornata reduce their branchial HCO3-/Clexchanging rate and/or increase the activity of erythrocyte NADH
metHb reductase for metHb reduction and experience significant
changes in exchanging rate of branchial ions for recovery.
b) pH regulation under a respiratory acidosis stimulate a reduction in
branchial HCO3-/Cl- exchanger and thereby protect against nitrite
toxicity in C. ornata
c) Chronic exposures of nitrite cause negative impacts to growth
parameters such as low weight gain, low survival rate and high
FCR in C. ornata

d) Elevated temperatures cause imbalance of acid-base status such as a
reduction in pH and a rise of PCO2 , leading negative disturbances
to blood cells, Hb and plasma ions in C. ornata
e) C. ornata has low tolerance of nitrite in the elevation of
temperature, leading to more significant effects to physiological
parameters and growth compared to those in isolated exposure of
nitrite or isolated elevated temperatures
1.5 Significant contributions and applicability of the dissertation
The dissertation provides a better understanding about physiological
knowledge of the air-breathing clown knifefish C. ornata including
recommendations and solutions for minimizing nitrite toxicity as well as
its combination with other environmental elements in aquaculture ponds
under global climate change.
With high tolerances of nitrite, temperature and hypercapnia in both
sub-lethal and chronic levels, C. ornata can properly adapt with
extreme environmental changes such as temperature (24-33ºC), partial
pressure of carbon dioxide (below 21 mmHg) and nitrite concentration
(below 2.5 mM) contributing to the sustainable development of aquatic
animals in the increases of temperature (1- 4ºC) in the next century and
accumulation of toxic gases such as nitrite, carbon dioxide in intensive
farming systems.
The results of dissertation will be reliable background for conducting
deeper further studies about physiology in C. ornata, other airbreathing species or comparing with physiological responses of this
species to those in other aquatic animals under extreme environmental
changes.
CHAPTER 2
METHODOLOGY
a)

3

2.1 Project 1: Extreme nitrite tolerance in the clown knifefish
Chitala ornata is linked to up-regulation of methaemoglobin
reductase activity
Fish: C. ornata (8-10 g; 28-31 ± 0.05 g); Chemical: NaNO2 (Merck)
Determination of acute nitrite toxicity (96 h LC50): Fish (8-10g, n=576)
were randomly distributed to 48 tanks (36-L water each) and 12 fish per
tank. The fish were fasted for 2 days before exposure to 0, 2.6, 3.7, 4.8,
5.9; 7.0, 8.0, 9.1, 10.2, 11.3, 12.4 or 13.5 mM nitrite, with four replicate
tanks for each concentration.
Sub-lethal exposures: Fish (n=300, body mass 31.8 ± 1.8g) were
randomly taken from the 1m3 holding tank in a recirculation system
with optimal water and placed in 200L experimental tanks with aerated
water two days before experimentation. Fish were fasted from this time
until experiment termination. Sub-lethal nitrite exposure concentrations
were control, 1 mM and 2.5 mM, with one tank per treatment (100 fish
per tank). Extra nitrite was added during the experiment to maintain the
chosen concentration Ten fish were sampled from each tank at days 0,
1, 2, 3, 4, 5, 6 and 7.
- Blood sampling: Blood was sampled by caudal puncture. The fish
were placed on ice (which causes gentle initiation of a comatose state in
this species) and 1mL of blood was withdrawn by from the caudal vein,
using a heparinized syringe. The fish were subsequently euthanized by
severing the spine. The blood was divided into two parts. Half was used
immediately for measurements of haemoglobin derivatives, haematocrit
(Hct, ratio between volume of red blood cells), mean corpuscular
haemoglobin concentration (MCHC, Huong and Tu, 2010) and
extracellular pH (pHe), carbon dioxide tension (pCO2) and lactate, using
the iSTAT analyzer (i-STAT Corporation, Princeton, USA) with CG4+

cartridges. The remainder of the blood was centrifuged, and the plasma
was stored at -80°C for subsequent analysis of ions and osmolality. The
values for pH and PCO2, HCO3- in the blood (Boutilier et al., 1985;
Cameron, 1971).
- Analysis procedures
+ Haemoglobin derivatives: The concentrations of oxyHb, deoxyHb,
metHb, HbNO were calculated by spectral deconvolution, following the
procedure described in Jensen (2007), Lefevre et al. (2012) and Hvas et
al. (2016) using reference spectra prepared from C. ornata blood.
+ Plasma ions: Plasma was obtained by centrifuging blood at 6000g for
6 min to determine the osmolality and the concentrations of Na +, Cl-,
NO2-, NO3- and protein. Total osmolality was measured on a Fiske oneten osmometer (Fiske® Associates, Two Technology Way, Norwood,

4


Massachusetts, USA). Plasma concentration of Na+ was measured using
a flame photometer (Sherwood Model 420, Sherwood Scientific Ltd.,
Cambridge, UK). Plasma Cl- concentration was measured using a
chloride titrator (Sherwood model 926S MK II Chloride analyzer).
Plasma NO2- and NO3- was measured spectophotometrically using the
Griess reaction (Miranda, 2001; Jensen, 2007; Lefevre et al., 2011).
+ Plasma protein and measurement of whole body water content:
Plasma protein concentration was measured spectrophotometrically with
Bio-rad protein assay (Bio-Rad Laboratory, Richmond, CA), using
bovine serum albumin as standard (Bradford, 1976). Total body water
was calculated from wet and dry weights of the fish. The dry weight
was determined by drying the fish at 60ºC until constant weight (for 4
days).
Methaemoglobin reductase activity: A series of fasted (2 days) fish

(n=216, body mass of 28-30 g) were exposed to 0, 1 and 2.5 mM nitrite,
with each concentration replicated 6 times (6 tanks per treatment). The
fish were sampled for blood at times 0, 2 and 6 days. In control tanks, 6
fish were sampled at each sampling time, whereas three fish were
sampled at each sampling in the nitrite tanks. During the experiment, the
water nitrite concentration was checked twice daily. Blood (1.5 mL)
was withdrawn from each fish of the exposure groups and washed four
times (as above) with Ringer to obtain 3 mL nitrite-cleaned RBC
suspension. This RBC suspension was equilibrated to 1% CO2 with 99%
air, and the MetHb decay was followed (as described above), where
after k in exposed fish was calculated.
Statistics: All figures were made in Sigma plot 12.5. All data were
analyzed with PASW statistics (SPSS 18). Predicted mean, upper and
lower 95% confidence intervals for the 96h LC50 were analyzed in JMP
9.0, using a logistic model. A two- way ANOVA (the Holm-Sidak
multiple comparison method, pair-wise comparison) was used to
identify differences between treatments and sampling times for all
parameters related to nitrite exposure (Hb derivatives, Hct, Hb, MCHC,
plasma nitrite, plasma nitrate, plasma ions, plasma protein, osmolality
and body water content). Normal distribution was tested using the
Shapiro-Wilk test and where necessary data were log transformed to
achieve normality. A p value of less than 5% (p<0.05) was judged
significant. In the methemoglobin reductase activity experiments, the
log of methemoglobin data was analyzed with linear regression to
determine the slope for calculation of the rate constant. All data are
shown as standard error of the mean (S.E.M).
2.2 Project 2: Effects of nitrite exposure on haematological
parameters and growth in clown knifefish (Chitala ornata)

5

Fish: (11.93±0.81 g; 11.53±0.15 g); Chemical: NaNO2 (Merck)
Effects of nitrite on haematological parameters: Fish (initial weight of
11.93±0.81 g, n=800) were randomly collected from 1 m 3 holding tanks
with optimal water quality, and subsequently distributed to 16 500-L
tanks (200 L water contained and 50 fish per tank). The water in these
tanks was continuously aerated in two days prior experimentation. From
the nitrite tolerance of clown knifefish (96 h LC50 of 7.82 mM nitrite,
Gam et al., 2017), the physiological experiment included 4 treatments
such as control, 0.2 mM (9.2 mg/L, recommended concentration), 0.4
mM (18.4 mg/L, 5% of 96 h LC50), and 4 mM nitrite (184 mg/L, 50% of
96 h LC50), with 4 replicates (4 tanks) for each treatment. Nitrite in the
water was recorded twice a day, and extra nitrite was added to maintain
desirable concentrations during experimentation by spectrophotometer
using the Griess reaction (Lefervre et al., 2011; 2012). Three fish per
tank were sampled from each tank at days 0, 1, 3, 7, and 14. The ice was
used for a comatose situation in fish before sampling blood. A total
volume of 300 µL of blood was collected from the caudal vein of each
fish by a heparinised syringe for measuring haematological parameters
including: Hb and metHb (Jensen, 2007; Lefevre et al., 2011, 2012;
Hvas et al., 2016; Gam et al., 2018)
Effects of nitrite on growth of C. ornata:
- Method: Fish (initial weight 11.53±0.15 g, n=600) were randomly
taken from 1 m3 holding tanks with optimal water quality and
subsequently distributed to 12 500-L tanks (300 L water and 50 fish per
tank) with aerated water two days before experimentation. The
experiment included 4 treatments such as 0 mM (control), 0.2 mM, 0.4
mM, and 4 mM nitrite, with 3 replicates (3 tanks) for each treatment in
90 culturing days. Nitrite in the water was recorded every three days

before exchanging water (30%), and subsequently extra nitrite was
added for maintaining the chosen concentrations. The fish were fed with
commercial pellets with feeding rate of 5% of body weight. Humidity of
commercial pellets (Shrimp feed with 38% protein, Tomboy Aquafeed
Company, Vietnam) was less than 10%. The pellets had uniform size (1
g = 203 pellets). Uneaten feed after 30 minutes of feeding was
calculated for determination of feed used. Thirty fish per tank were
sampled on the days 0, 30, 60 and 90 for measuring growth parameters
including WG, DWG, SGR, FCR, and SR.
Statistics: Similar to Project 1. However, a one-way ANOVA was used
to identify differences between control treatment compared to other
treatments for all growth parameters.

6


2.3 Project 3: The effects of elevated environmental CO 2 on nitrite
uptake in the air-breathing clown knifefish Chitala ornata
Fish: C. ornata (571±56.3 g); Chemicals: NaNO2, CO2 gas
Animal holdding and setup: C. ornata from a local intensive farm were
transported to Can Tho University. They were held at ambient
laboratory temperature 27-28ºC in 4 cubic meter tanks with constant
aeration (dissolved oxygen >90%) for 2 weeks before experimentation.
Fish were fed by commercial feed (shrimp feed with 38% protein,
Tomboy Aquafeed company, Vietnam). Thirty percent of tank water
was changed every second day to maintain optimal environmental
condition (NO2- < 1 µM, NO3- < 40 µM and NH3 < 40 µM). Feeding
was stopped 2 days before starting the experiment. The experiment was
performed in accordance with national guidelines on the protection and
care of experimental in Vietnam. A total of 24 fish were used.

- Fish cannulation: They were anaesthetized in 0.05 g L-1 benzocaine
and a polyethylene PE40 catheter (Smiths Medical International Ldt.,
Kent, UK) was inserted into the dorsal aorta through the dorsal side of
the mouth (Soivio et al., 1975), while the gills were irrigated with
well-oxygenated water containing 0.025 g L-1 benzocaine. Fish
recovered in well-aerated water for 24 h before starting
experimentation to allow post-operative normalization of blood gasses
(Phuong et al., 2017a).
- Blood sampling: The experimental set-up included a large 500-L
tank from which water was re-circulated to 6 smaller 120-L tanks
with 1 cannulated fish in each. The water PCO2 was controlled with an
Oxyguard Pacific system coupled with a G10ps CO2 probe and a
K01svpld pH probe (Oxyguard International A/S, Farum, Denmark),
which supplied CO2 to the water when pH changed above a value
corresponding to the desired PCO2 in the water. There were 4 exposure
groups: (i) normocapnia (PCO2 < 0.7 mmHg); (ii) hypercapnia (PCO2 =
21 mmHg); (iii) 1 mM nitrite in normocapnic water and (iv) combined
hypercapnia (acclimated hypercapnia) and 1mM nitrite. In this
combined group, the fish were cannulated then acclimated to
hypercapnia (21 mmHg CO2) for 96 h before adding 1 mM nitrite.
Water temperature was controlled at 27-28ºC throughout experiments
and water PO2 was above 120 mmHg. Nitrite was added as NaNO 2 and
tested after each sampling time. During the exposures, a volume of 0.8
mL blood was withdrawn from the catherter at 0, 3, 6, 24, 48, 72 and 96
h. The blood was divided into two parts. Half was used immediately for
measurements of haematological parameters including: Hct, pH e, PCO2,
and Hb derivatives. The remainder of the blood was centrifuged (6 min
at 6,000g), and the plasma was stored at -80°C for subsequent analysis

7

of plasma ions (NO2-, NO3-, HCO3-, Na+, K+, Cl-), glucose and
osmolality.
- Analytical procedures: all above haematological parameters were
measured with similar protocols described in Project 1.
- Statistics: Similar methods to Project 1.
2.4 Project 4: The combined effects of nitrite and elevated
environmental CO2 on haematological parameters in small-sized
clown knifefish (Chitala ornata)
Fish: C. ornata juveniles (30-40 g); Chemicals: NaNO2, CO2 gas
- Animal holding and setup: Similar to Project 4, with small-size fish,
the density and blood sampling method were different. Each treatment
included 3 replicates (3 tanks, 45 fish/tank).
- Blood sampling: Blood samples (three fish per tank) were taken at 0,
3, 6, 24, 48, 72 and 96 h. Fish were put in the ice for maintaining a
comatose situation before sampling blood by a heparined (400 µL of
blood per fish) for measuring similar haematological parameters as
described in Project 3.
- Analytical procedures: all above haematological parameters were
measured with similar protocols described in Project 1.
- Statistics: Similar to Project 1.
2.5 Project 5: Effects of different temperatures on haematological
parameters in clown knifefish (Chitala ornata)
Fish: (8-10 g, 29.2±3.4 g, 521 ± 32g); Materials: heater, cooler devices
Determination of temperature limits in C. ornata: Twelve fish (8-10 g)
are prepared in plastic bucket (30 L water contained). The experiment
consists of 2 groups: upper limit - increasing temperature and lower
limit - decreasing temperatures with 6 replicates each. The initial
temperature was 27ºC; then it was increased or decreased (1ºC per hour)

by warm water, then using the heaters to maintain the desirable high
temperature; or using iced water to decrease temperature, then using the
coolers to maintain the desirable low temperature. The fish was
recorded swimming performance until 50% of the fish refusing
respiration (no gill ventilation, no moving around). The upper and lower
ranges of temperature were determined at that time.
Effects of different temperatures on physiological parameters in smallsized C. ornata: Fish (n=575, body mass of 29.2±3.4 g) were randomly
taken from the 1 m3 holding tank in a recirculation system with optimal
water parameters and placed in 200-L experimental tanks with
continued aeration. Fish were fasted from this time until experiment
termination. The experiment included five levels of temperature: 24, 27,

8


30, 33, and 36ºC with three tanks per treatment (45 fish per tank). After
collecting the blood samples at day 0, desirable temperatures were
adjusted by heaters or coolers (decreasing or increasing temperature 2ºC
per 12 h) for blood sampling at day 1, 2, 3, 4, 5, 6, 7, and 14 (3
fish/tank). The fish were placed on ice, and then withdrawn by from the
caudal vein (1 mL of blood for each fish), using a heparinized syringe.
The fish were subsequently euthanized by serving the spine for
recognizing with other fish and maintaining the same stocking density.
Effects of different temperatures on physiological parameters in largesized C. ornata: Fish (n=36, body mass of 521 ± 32g)
- Fish canulation: Similar to Project 3.
- Experimental setup and blood sampling: The experiment was similarly
set up in 200L tanks with similar treatments to above experiment in
small-sized C. ornata. However, 1 cannulated fish in each, 6 tanks (6
replicates) per treatment, and a volume of 0.7 mL blood was withdrawn
from the catheters at day 0, 1, 2, 3, 4, and 7.

- Analytical procedures: In the blood: Hct, pH, PCO2; in the plasma:
ions (NO2-, NO3-, HCO3-, Na+, K+, Cl-) and osmolailty. These
parameters were measured with similar methods described in Project 1.
- Statistics: Similar to Project 1
2.6 Project 6: Effects of nitrite at different temperatures on
haematological parameters and growth in clown knifefish Chitala
ornata
Fish: (8-10g, 30-40g); Materials & chemicals: heaters, coolers, NaNO2
- Animal holding: Similar to Project 2.
- Determination of acute nitrite toxicity (96 h LC50) at 30ºC and 33ºC in
C. ornata: Fish (8-10 g, n=1152) The two systems at 30ºC and 33ºC
were conducted by the same concentrations to determination of 96 h
LC50 for nitrite at 27ºC (Project 1, Gam et al., 2017). Heaters were used
for controlling temperatures in systems.
- Sub-lethal nitrite exposures at different temperatures and blood
sampling in C. ornata:
- Experimental method: Fish (30-40 g, n=675) placed in 200- L
experimental tanks with aerated water two days before experimentation.
Fish were fasted from this time until experiment termination. The
experiment was conducted including 5 treatments: 1 mM nitrite at 24,
27, 30, 33, and 36ºC with three tanks per treatment (45 fish per tank).
After collecting the blood samples at day 0, chosen temperatures were
adjusted by heaters (decreasing or increasing temperature 2ºC per 12 h).
Nitrite was subsequently added, and blood samples collected at day 1; 2;

9


3; 4; 5; 6; 7; and 14 (3 fish/tank). After placing the fish on ice (which
causes gentle initiation of a comatose state in this species), 1 mL of

blood was withdrawn by from the caudal vein, using a heparinized
syringe. The fish were subsequently euthanized by serving the spine for
recognizing with other fish and maintaining the same stocking density.
- Analytical procedures: All haematological parameters chosen and their
analyzing methods were similar as described in Project 1.
- Effects of nitrite at different temperatures on growth and digestive
enzyme activities in C. ornata:
- Experimental method: Fish (8-10 g, n=900) were placed in 500-L
experimental tanks with aerated water two days before experimentation.
The experiment was randomly conducted including 6 treatments: 27ºC;
30ºC; 33ºC; 1 mM nitrite at 27ºC; 1 mM nitrite at 30ºC; and 1 mM
nitrite at 33ºC with 3 tanks/treatment (50 fish per tank). After collecting
growth samples at day 0, the temperatures were adjusted by heaters until
reaching the chosen temperatures (2ºC per 12 h). Nitrite was
subsequently added, and growth samples collected at day 30, 60, and 90
for measuring growth parameters such as DWG, SGR, SR, and FCR
(thirty fish randomly sampled in each tank). Three fish per tank were
collected intestine and stomach at day 90 for examining activities of
digestive enzymes. During experimentation, the fish were fed twice a
day (the mixture of trashfish and commercial feed (Shrimp feed with
38% protein, Tomboy Aquafeed company) used in the first month, and
then only commercial feed used until the end of growth experiment.
After 30 minutes feeding, uneaten feed was removed for counting the
total number of pellets and weighed trashed fish used, then subtracting
their humidity to obtain the actual weight of feed used. Dead fish was
removed every day, and thirty percent of the tank water was changed
every third day to maintain optimal environmental conditions. Nitrite
concentration was checked before exchanging water for supplementing
the lacked amount of nitrite.
- Analytical procedures: growth parameters were measured by similar

methods as described in Project 1; digestive enzymes such as pepsine
(Worthington, 1982), trypsine (Tseng et al., 1982), chymotrypsine
(Worthington, 1982), α-Amylase (Bernfeld, 1951).
- Statistics: Similar to Project 1. However, a one-way ANOVA was
used to identify differences between control treatment compared to
other treatments for all growth parameters and digestive enzyme.
2.7 Project 7: A survey on some environmental parameters in clown
knifefish (Chitala ornata, Gray 1831) ponds

10


Sampling method: Examined environmental parameters were sampled
at the C. ornata ponds in Hau Giang province, Viet Nam, including six
factors: temperature, pH, PCO2, PO2, [NO2-] and [NO3-] in the water at
total 9 ponds with 3 different sizes of fish 5-10 g, 200-250 g, 500-700 g
(3 ponds for each size of fish). The environmental parameters were
measured and sampled at 3 positions for each pond (2 corner positions
and central position), 2 layers for each position (surface 30 cm and
bottom 1.2 m) in 24 h at 8 specific time points: 9 am, 12 pm, 3 pm, 6
pm, 9 pm, 12 am, 3 am, 6 am.
Analytical method: Direct measurements on site such as temperature,
pH, PCO2 and PO2 were showed immediately results by the devices.
The water samples for [NO2-] and [NO3-] measurements were
transported to Can Tho University for measuring at the day after
(Lefevre et al., 2011; Miranda et al., 2001).
CHAPTER 3
RESULTS AND DISCUSSION
3.1 Project 1: Extreme nitrite tolerance in the clown knifefish
Chitala ornata is linked to up-regulation of methaemoglobin

reductase activity
96 h LC50 for nitrite in C. ornata: The 96 h LC50 for nitrite in this
species was 7.82 mM (95% CI 6.79-8.85 mM), which make C. ornata
one of the most nitrite tolerant fish species studied (Fig. 3.1.1). The
reference spectra of Hb derivatives were obtained for C. ornata (Fig.
3.1.2A). C. ornata shows very high tolerance compared to 1.65 mM
NO2- in the facultative air-breathing P. hypophthalmus (Lefevre et al.,
2011) and 4.7mM NO2- in the obligate air-breathing C. striata. (Lefevre
et al., 2012). This tolerance is significantly higher than the most nitriteresistant water breathers and more than twice that of the common carp
(96h LC50 = 2.9 mM; Lewis and Morris, 1986).

Fig. 3.1.1. Mortality of C. ornata (8-10 g) by a function of nitrite concentration. Predicted
mean, upper and lower values of LC50 96 h.

11


Fig. 3.1.2 (A) The reference spectra of Hb derivatives in C. ornata at wavelengths from
480 to 700 nm. (B) Spectrum of oxyHb from a fish exposed to 1 mM nitrite for 2 days and
the fitted curve of oxyHb in reference spectra.

The rise in metHb to a maximum on day 2 of nitrite exposure, then
succeeded by a decrease in metHb during continued nitrite exposure
(Fig. 3.1.3), resembles the patterns seen in P. hypophthalmus and C.
striata (Lefevre et al., 2011; 2012). This led these authors to suggest
that the decline of meHb might result from up-regulation of metHb
reductase, which is the enzyme responsible for reducing metHb to
functional Hb. Such up-regulation had also been suggested in carp
recovering from nitrite exposure (Knudsen and Jensen, 1997). The
present study confirms this hypothesis by showing that the rate constant

for metHb reduction via erythrocyte metHb reductase is significantly
elevated during nitrite exposure (Fig. 3.1.4).

Figure 3.1.3. Plasma NO2- (A), plasma NO3- (B), percentage metHb (C), percentage
HbNO (D), functional Hb (E) and total plasma nitrite and nitrate after exposure to 0 mM
nitrite (controls, closed circles); 1 mM nitrite (open circles) and 2.5 mM nitrite (closed
triangles) in 0, 1, 2, 3, 4, 5, 6 and 7 days.

12


Fig. 3.1.4. (A) Rate constant (k, min-1) of metHb reductase in fish exposed to 0 mM
(controls, closed circles); 1 mM (open circles) and 2.5 mM nitrite (closed triangles) in 0, 2
and 6 days. (B) Examples of the decay of metHb in log(metHb) Examples of the decay in
log(metHb) of a control fish and a fish exposed to 2.5 mM nitrite in 6 days.

Acid-base status was significantly affected in the highest nitrite
exposure group (Davenport diagram, Fig. 3.1.5A). In this group, pHe
fell from 7.73 to 7.56 at day 1 (Fig. 3.1.5C), where after it recovered by
0.1 unit. Blood pCO2 rose from 8 mmHg on day 0 to 14 mmHg from
day 1 and onwards (Fig. 3.1.5B). However, the significant respiratory
acidosis during exposure was partially compensated by the associated
increase in ion HCO3-. This is quite contrary to the normal expectation
in nitrite-exposed water-breathing animals, where a respiratory alkalosis
is induced by the hyperventilation brought about by the reduced oxygen
carrying capacity of the blood as methHb increases (Jensen et al., 1987;
Aggergaard and Jensen, 2001; Hvas et al., 2016). Differently, the
facultative C. ornata has the opportunity to change the partitioning of
oxygen uptake when confronted with a waterborne toxicant by
increasing its reliance on air-breathing and reducing gill ventilation

(Lefevre et al., 2014).

Fig. 3.1.5 Davenport diagram (A), blood PCO2(B), pHe (C) after exposure to 0 mM
(controls, closed circles); 1 mM (open circles) and 2.5 mM nitrite (closed triangles) in 0,
1, 3 and 7 days.

3.2 Project 2: Effects of nitrite exposure on haematological
parameters and growth in clown knifefish (Chitala ornata, Gray
1831)
In nitrite exposure, metHb significantly increased and reached the
highest percentages on day 3 (2.55±0.12, 4.30±0.32, and 29.54±0.72%
at the treatments of 0.2, 0.4 and 4 mM nitrite, respectively) (Fig.
3.2.1A). MetHb formation is related to the formation of free peroxide
and changes the properties of essential protein, including Hb and
composition of erythrocyte membrane causing the reduction in Hb
solubility, which damage erythrocyte structures and decompose them
rapidly (Everse and Hsia, 1997; Bloom and Brandt, 2001). Hb and Hct

13


had decreasing trends during nitrite exposures; particularly at the
highest level of nitrite exposure (Fig. 3.2.1B,C). Hb concentration is
converted to metHb and loses capacity with oxygen. Higher
concentrations of nitrite exposure cause higher concentrations of metHb
generated and lower concentration of Hb (Kosaka and Tyuma, 1987;
Jensen, 2009; Huong and Tu, 2010).
The higher concentrations of nitrite were accompanied with the lower
SR in all nitrite treatments. Typically, the treatment of 4 mM nitrite had
the lowest survival rate (59%), which was significantly different from

the treatments of control, 0.2 and 0.4 mM nitrite (95, 92 and 86%) (Fig.
3.2.2A). FCR gradually increased from low to high nitrite levels being
exposed (4.19±0.08 and 4.56±0.11 at the two highest nitrite
concentrations (Fig. 3.2.2B). This may be resulted from the metHb and
HbNO formation, causing the low of oxygen capacity in the blood,
subsequently affecting the fish growth during chronic nitrite exposures
(Jensen, 2007).

Fig. 3.2.1. Haematological paramters in C. ornata after 14 days exposed to nitrite
(control, 0.2 mM, 0.4 mM, and 4 mM). (A) MetHb), (B) Hb concentration, (C) Hct,
and (D) MCHC.

Fig. 3.2.2. Growth paramters in C. ornata after 90 days exposed to nitrite: control, 0.2
mM, 0.4 mM and 4 mM. (A) SR and (B) FCR.

14


3.3 Project 3: The effects of elevated environmental CO 2 on nitrite
uptake in the air-breathing clown knifefish Chitala ornata
Nitrite exposure was associated with nitrite uptake in the plasma, but
plasma NO2- increased significantly less during nitrite exposure in
hypercapnia than in normocapnia (Fig. 3.3.1A). The nitrite uptake
induced a rise of blood metHb to 26% (nitrite group) and 14%
(hypercapnia + nitrite) of total Hb after 48 h, whereupon metHb levels
slowly decreased (Fig. 3.3.1B). Despite lower maximal metHb levels
during exposure to combined hypercapnia and nitrite than nitrite alone,
the rate of metHb formation was highest during the initial hours of
nitrite exposure in hypercapnia (Fig. 3.3.1B). Plasma NO3- significantly
increased, reaching 3.8 mM and 2.5 mM in 96 h in the nitrite and

combined hypercapnia and nitrite groups, respectively (Fig. 3.3.1E).
The sum of plasma nitrite and nitrate (Fig. 3.3.1F) is a good indicator of
the total uptake of nitrite, as nitrate is formed by oxidation of nitrite
(e.g. in the reaction between nitrite and oxyHb). MetHb and HbNO
levels were lower during exposure to combined hypercapnia and nitrite
than during exposure to nitrite alone (Fig. 3.3.1B,C), which is in line
with the reduced nitrite uptake in the combined group. It is notable that
the initial increase in [metHb] is faster in the combined group than in
the nitrite alone group (Fig. 3.3.1). This is to be expected because of the
lower initial pH in the combined group than in the nitrite alone group.
Hydrogen ions are required in the reactions between nitrite and Hb (both
oxyHb and deoxyHb), and a reduced pH will therefore speed up the
reaction rates (Jensen and Rohde, 2010).

15


Fig. 3.3.1 Time-dependent changes in plasma NO2- (A), metHb percentage (B), HbNO
percentage (C), functional Hb (D), plasma NO3- (E), and the sum of plasma nitrite and
nitrate (F) during exposure to normocapnia (open circles), hypercapnia (21 mmHg CO 2,
closed circles), 1 mM nitrite (open triangles), and acclimated hypercapnia and nitrite
(closed triangles).

The changes in acid-base status in the different exposure groups are
illustrated in a Davenport diagram (Fig. 3.3.2). Hypercapnia led to an
acute pH decrease along the buffer line followed by metabolic pH e
compensation via HCO3- accumulation along the PCO2 ~ 21 mmHg
isocline, reaching half-compensation by 96 h. In the combined
hypercapnia and nitrite group a further increase in bicarbonate occurred.
During exposure to nitrite alone there was a minor respiratory acidosis

for some 24 h that subsequently became rectified by a small elevation of
HCO3- (Fig. 3.3.2). This study supports our hypothesis that
environmental hypercapnia reduced branchial nitrite uptake via the
branchial Cl-/HCO3 exchanger, since regulation of a respiratory acidosis
causes a slowing of Cl- uptake via the exchanger and hence also reduces
nitrite uptake. Thus the response of C. ornata to this combined exposure
resembles that of the crayfish Astacus astacus (Jensen et al., 2000), but
is different to that seen in the air-breathing teleost P. hypothalamus,
where nitrite uptake is only transiently decreased and subsequently
increases (Hvas et al., 2016).

Figure 3.3.2 Davenport diagram showing changes in acid-base status during exposure to
normocapnia (open circles), hypercapnia (21 mmHg CO2, closed circles), 1 mM nitrite
(open triangles), and acclimated hypercapnia and nitrite (closed triangles).

3.4 Project 4: The combined effects of nitrite and elevated
environmental CO2 on haematological parameters in small-sized
clown knifefish (Chitala ornata)

16


The data showed that small-sized C. ornata in general had the similar
physiological responses to large-sized C. ornata during combined
exposures of hypercapnia and nitrite in isolation and combination via
branchial chloride/bicarbonate exchanger. Therefore, the results and
discussion had similar trends to those in project 3.
3.5 Project 5: Effects of different temperature on haematological
parameters in clown knifefish (Chitala ornata)
The upper and lower limits for temperature in C. ornata were 41ºC and

12ºC, respectively. This indicated that C. ornata is one of the most
temperature tolerant species in tropical area. The physological responses
described in the values of haematological parameters were similar
between small-sized and large-sized C. ornata in exposures of 24ºC,
27ºC, 30ºC and 33ºC. It is considered that temperature is a critical
environmental factor affecting on physiological processes such as food
digestion, growth, metabolism, immunity, and locomotion (Zeng et al.,
2009). In large-sized fish, plasma Na+ and plasma osmolality
maintained unchanged in the groups of 24ºC, 27ºC, 30ºC while there
were significant declines at the groups of 33ºC and 36ºC (Fig.
3.5.1A,B). Stress indicators such as plasma glucose and plasma K + had
significant increasing values by the elevated temperatures (Fig. 3.5.1C,
D). It is suggested that the levels of glucose in the blood is a stress
indicator and a result of depletion in glycogen storage in the liver
(Ojolick et al., 1995; Click and Engin, 2005).

Fig. 3.5.1 Plasma Na+ (A), plasma osmolality (B) plasma glucose (C), plasma K+ (D) in
large-sized C. ornata after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC,
36ºC in 0, 1, 2, 3, 4, and 7 days.

17


Acid-base parameters were significantly affected by the elevation of
temperature by the decrease of extracellular pH and the rise of PCO 2.
Plasma bicarbonate maintained constant in all temperatures while there
was a rapid reduction in plasma Cl- in elevated temperatures (Fig.
3.5.2). It is obvious that elevated temperatures cause the significant
decreases in pHe (Heisler et al., 1976; Fobian et al., 2014; Damsgaard et
al., 2018; Thinh et al., 2018). The increases in PCO2 in elevated

temperatures may be explained by the increases of air-breathing
frequency (Lefevre et al., 2016), subsequently the metabolic production
of CO2 (Rahn, 1966).

Fig. 3.5.2 pHe (A), PCO2 (B) plasma HCO3- (C), plasma Cl- (D) in large-sized C. ornata
after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4,
and 7 days.

3.6 Project 6: Effects of nitrite at different temperatures on
haematological parameters and growth in clown knifefish Chitala
ornata
The values of 96 h LC50 for nitrite in C. ornata were 8.12 mM at 30ºC,
and 6.75 mM at 30ºC (Fig. 3.6.1) while the 96 h LC50 for nitrite at 27ºC
was 7.82 mM (Gam et al., 2017).

18


Fig. 3.6.1. Mortality (96h LC50 for nitrite) of C. ornata (8-10 g) at three different
temperatures: 27ºC (A), 30ºC (B), and 33ºC (C) as a function of nitrite concentration.
Predicted mean, upper and lower 95% confidence intervals are presented as lines fitted to
the data indicted as dots. 96 h LC50 for nitrite at 27ºC is 7.82 mM (Gam et al., 2017).

MetHb and plasma nitrite significantly peaked to the highest value at the
highest temperature (36ºC) at day 2, but they decreased significantly at
experimental termination (Fig. 3.6.2A, B). This can be explained by the
effective denitrification converting nitrite to nitrate and the effective
activities of metHb reductase enzyme in metHb reduction (Doblander
and Lackner, 1997; Jensen, 2003; Gam et al., 2017; 2018a,b). In
addition, HbNO concentration increased during nitrite exposures at

various temperatures in the present study (Fig. 3.6.2C). The formation
of HbNO is a result of the nitrite reduction to NO through the reaction
between deoxygenated Hb and nitrite (Jensen, 2007).
At the present study, there were significant declined in plasma ions such
as Na+, Cl- and osmolality in elevated temperature(Fig. 3.6.3), possibly
resulting from the rise of nitrite absorption across the gills via Cl -/HCO3exchange (Evan et al., 2005; Jensen et al., 2000). A possibility is that
dilution of body fluids are frequently seen during nitrite exposure
(Jensen et al., 1987; Jensen, 1990a, 1996; Harris and Coley, 1991;
Grosell and Jensen, 2000; Gam et al., 2017).

19


Fig. 3.6.2 Plasma NO2- (A), metHb (B), HbNO (C), functional Hb (D), plasma NO3- (E),
and total NO2- and NO3- (F) after exposed to nitrite at five different temperatures 24ºC,
27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3, 4, 7 and 14 days.

Fig. 3.6.3 Plasma Na+ (A), plasma osmolality (B), plasma Cl- (C), plasma HCO3- (D) after
exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC in 0, 1, 2, 3,
4, 7 and 14 days.

Davenport diagram (Fig. 3.6.4) showed a significant respiratory acidosis
during nitrite exposure at various levels of temperature, and pH
compensated by the elevation of plasma HCO3-. pHe significantly
dropped with lower values in combination of nitrite and elevated
temperatures compared to nitrite exposures alone (Gam et al., 2017;
2018a). Moreover, the air-breathing clown knifefish in this study can
change the the portioning of oxygen uptake via air-breathing and a
reduction in gill ventilation for avoiding environmental toxicants.

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Fig. 3.6.4 Davenport diagram presenting the changes in acid-base status (A), blood PCO2
(B), and pHe (C) after exposed to nitrite at five different temperatures 24ºC, 27ºC, 30ºC,
33ºC, 36ºC in 0, 1, 2, 3, 4, 7 and 14 days.

There were significant changes on growth parameters when C. ornata
were exposed to nitrite at various temperatures. The results showed that
SR decreased in nitrite exposure at elevated temperature (Fig. 3.6.5),
particularly the treatment of 1 mM nitrite at 30 and 33ºC with
significant difference compared to that at 27ºC. In this study, mortality
appeared in the initial culturing stage, subsequently decreasing during
experiment because fish need a period of time to adapt, adjust for
maintaining intercellular activities (Gerlach et al., 1990).

Fig. 3.6.5. Survival rate (SR) and feed conversion ratio (FCR) after 90 days exposed to
nitrite at 27ºC (control); 30ºC; 33ºC; 1 mM nitrite at 27ºC; 1 mM nitrite at 30ºC; 1 mM
nitrite at 33ºC. Different letters (a, b, c) in the same column show significant difference
from control treatment. Showed data are mean±SEM (n=30).

3.7 Project 7: A survey on some environmental parameters in clown
knifefish (Chitala ornata, Gray 1831) ponds
There were significant fluctuations in environmental parameters
between daytime and nighttime (Fig. 3.7 below). IPCC, 2013 predicts a
temperature rise of 1- 4ºC over the next century. Particularly,
temperatures in C. ornata ponds had the highest value at 12-3 pm
(32.5ºC), but it slightly decreased at night time (30-31ºC at 12 – 6 am).
According to Boyd (1990), temperature in the ponds of tropical fish
fluctuates in the range of 20-35ºC and optimal range of 28-32ºC.

Therefore, temperatures in C. ornata ponds were considered to be safe
for their physiological and growth processes. Hypercapnic conditions
appeared in the early morning with PCO2 of 14 mmHg and pH of 6.9
(450-500g) while there were fluctuations of PCO2 and pH around 2
mmHg and 7.5 at 6 am (5-10 g). The CO2 levels were higher in the
ponds of large-sized fish compared to the ponds of small-sized fish.
Contrast to CO2 concentration, oxygen level reached saturated value
with PO2 of 140 mmHg at 3 pm while there was almost no oxygen at 36 am with pond depth of 1.5 m. C. ornata can normally grow in anoxic
condition in the early morning thanks to their air-breathing assessory

21


organs used for taking oxygen directly from the air at hypoxic situations
(Long, 2003). Reviewed by Diaz and Breitburg (2009), oxygen is
commonly limited to the top 1 m of the water column during daytime,
whereas the water is completely anoxic at night in the conditions such
as high pond temperatures (28-32ºC), high organic loading, low water
exchange, and especially the absence of aeration. According to
Damsgaard et al., 2015, P. hypophthalmus pond was completely anoxic
at the water column of 3 m with PCO2 of 28 mmHg in this depth. The
accumulations of nitrite and nitrate levels in the ponds of large sized
fish were higher than those in the ponds of small-sized fish. Particularly,
the concentrations of nitrite and nitrate in the C. ornata ponds were
0.0055-0.0075 mM and 0.058-0.068 mM (450-500 g/fish), whereas they
were only 0.002 - 0.0035 mM (200-250 g/fish) and 0.025-0.035 mM (510 g/fish), respectively. Nitrite and nitrate levels reached the highest
values at 6 pm in ponds of different fish sizes. Boyd (1990) documented
that safe concentrations of nitrogen products: nitrite below 0.3 mg/L
(0.0065 mM in this study) and nitrate 0.2 – 10 mg/L (0.003-0.156 mM
in this study). Therefore, levels of nitrite and nitrate in C. ornata ponds

changed in the optimal ranges.

22