Doctoral dissertation of aquaculture effects of nitrite, temperature and hypercapnia on physiological processes and growth in clown knifefish (chitala ornata, gray 1831)

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

LE THI HONG GAM

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

DOCTORAL DISSERTATION OF AQUACULTURE

Can Tho, 2018


MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

LE THI HONG GAM

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

Major: Aquaculture
Major code: 9 62 03 01

DOCTORAL DISSERTATION OF AQUACULTURE

Supervisor
Prof. Dr. NGUYEN THANH PHUONG

Can Tho, 2018


Data sheet

Title:

Effects of nitrite, temperature and hypercapnia on
physiological processes and growth in clown knifefish
(Chitala ornata, Gray 1831)

Subtitle:

PhD Dissertation

Author:

Le Thi Hong Gam, PhD student code: P0613005
Major: Aquaculture, Major code: 9 62 62 03 01

Affiliation:

Department of Nutrition and Aquatic Products Processing,
College of Aquaculture and Fisheries, Can Tho University,
Vietnam

Publication year 2018
Cited as:

Le Thi Hong Gam, 2018. Effects of nitrite, temperature and

hypercapnia on physiological processes and growth in clown
knifefish (Chitala ornata, Gray 1831). Doctoral Dissertation.
College of Aquaculture and Fisheries, Can Tho University,
Vietnam.

Keywords:

Climate change, air-breathing fish, clown knifefish, nitrite,
temperature, hypercapnia, methaemoglobin reductase activity,
acid-base balance, ion exchange

Supervisors:

Prof. Dr. Nguyen Thanh Phuong, College of Aquaculture and
Fisheries, Can Tho University, Viet Nam.
Assoc. Prof. Dr. Mark Bayley, Zoophysiology, Department of
Bioscience, Aarhus University, Denmark.
Assoc. Prof. Dr. Do Thi Thanh Huong, Department of
Nutrition and Aquatic Products Processing, College of
Aquaculture and Fisheries, Can Tho University, Viet Nam.
Assoc. Prof. Dr. Frank Bo Jensen, Department of Biology,
University of Southern Denmark, Odense, Denmark.

i


Result commitment

I commit that this dissertation was investigated based on all the results of my study. All
showed data and results in the dissertation were honest and have never been published

before. The iAQUA project can completely use these data and results.
Can Tho, 18th Nov, 2018

ii


Acknowledgements

Foremost, my sincere thanks go to my principal supervisors Nguyen Thanh
Phuong and Do Thi Thanh Huong, who have given me the opportunity of
studying, enthusiastic guidances and detailed revisions for my thesis as well as
positive encouragements in any situation throughout my academic research
process. I would also like to express my deep gratitude to Mark Bayley, who has
supported, inspired, built my passion in doing researches, also revised the
manuscripts and shared the life experiences for me being more mature. My deep
thanks also give to Frank Bo Jensen, who has taught me the techniques related to
my main research about nitrite toxicity and helped me in manuscript revisions as
well as showed me Odense city in my trip to Denmark. The invaluable supports
from all of them from the first day I stepped into the iAQUA project have brought
to what I have today.
Also, I would like to give my thanks to Tobias Wang, the staff and students in
Zoophysiology Section, Department of Bioscience, Aarhus University for my
stays in Denmark. I sincerely thank for the positive supports from Christian
Damsgaard, who showed me the knowledge related to acid-base regulation. My
thanks also go to Roy, John, Elin and Louise for showing Aarhus city and inviting
me to their home.
My great thanks give to the staff and students in Department of Nutrition and
Aquatic Products Processing, where my project was investigated. I greatly thank
to Nguyen Quoc Thinh, Le Thi Minh Thuy and Tran Minh Phu, who shared me
their experiences about studying PhD, writing and publishing the articles.

I would like to give my thanks to my fellow friends in iAQUA project: Nguyen
Thi Kim Ha, Le My Phuong, Phan Vinh Thinh, and Dang Diem Tuong for their
friendships and what we enjoyed together from the working environment as well
as all the funs outside the campus, especially Phan Vinh Thinh, who has cared me
from meals and movements during my intensively experimental works and the
trips in Denmark.
I also appreciate the positive co-operations from Master students such as Nguyen
Thi Thuy Vu and Tran Trong Nhan; Bachelor students: Ma Thanh Quoc Tri,
Pham Quoc Boong, Dinh Phuc Tai, Ly Thi Ngoc Huynh, Nguyen Ngoc Mai and
Dao Dang Hoang Ngan during our academic activities.
iii


I would like to thank my family and my friends for their love and spiritual
supports during my study. My special thanks give to my sister Le Thi Hong Dao,
who has supported me about both financial and mental sides from Bachelor’s
degree up to date. She has always been my side for sharing happiness and sadness
throughout my whole life.
Last but not least, thanks to all the sacrifired clown knifefish in my project.

iv


Table of contents

Data sheet ............................................................................................................................i
Result commitment ........................................................................................................... ii
Acknowledgements .......................................................................................................... iii
Table of contents ................................................................................................................ v
List of figures ..................................................................................................................... x

List of tables .................................................................................................................... xii
List of abbreviation .........................................................................................................xiv
Summary .........................................................................................................................xvi
Tóm tắt ......................................................................................................................... xviii
Chapter 1 ............................................................................................................................ 1
INTRODUCTION ............................................................................................................. 1
1.1 Introduction .............................................................................................................. 1
1.2 The objectives of dissertation ................................................................................... 3
1.3 The main projects of dissertation ............................................................................. 3
1.4 The hypotheses of dissertation ................................................................................. 3
1.5 New findings of the dissertation ............................................................................... 4
1.6 Significant contributions of the dissertation ............................................................. 5
References .......................................................................................................................... 5
Chapter 2 ............................................................................................................................ 7
LITERATURE REVIEW................................................................................................... 7
2.1 The status and importance of aquaculture and fisheries ........................................... 7
2.2 Climate changes and impacts on aquaculture and fisheries ..................................... 9
2.3 The status of farming clown knifefish (C. ornata) in MD ..................................... 10
2.4 Background about effects of some key environmental parameters on physiological
processes and growth in aquaculture ............................................................................ 11
2.4.1 Temperature........................................................................................................ 11
2.4.2 Nitrite (NO2-) ...................................................................................................... 14
2.4.3 Hypercapnia (elevated level of carbon dioxide) and acid-base balance ............ 18
References ........................................................................................................................ 20
Chapter 3 (Paper 1) .......................................................................................................... 29
v


EXTREME NITRITE TOLERANCE IN THE CLOWN KNIFEFISH CHITALA
ORNATA IS LINKED TO UP-REGULATION OF METHAEMOGLOBIN

REDUCTASE ACTIVITY .............................................................................................. 29
3.1 Introduction ............................................................................................................ 30
3.2. Materials and methods........................................................................................... 32
3.2.1 Experimental animals ......................................................................................... 32
3.2.2 Determination of acute nitrite toxicity (96 h LC50) ............................................ 32
3.2.3 Sub-lethal exposures and blood sampling .......................................................... 33
3.2.4 Analysis of haemoglobin derivatives ................................................................. 34
3.2.5 Plasma ion and protein analysis ......................................................................... 34
3.2.6 Measurements of whole body water content ...................................................... 35
3.2.7 Methaemoglobin reductase activity.................................................................... 35
3.2.8 Statistics.............................................................................................................. 36
3.3. Results ................................................................................................................... 36
3.4. Discussion ............................................................................................................. 45
3.4.1 Nitrite tolerance .................................................................................................. 45
3.4.2 MetHb reductase activity.................................................................................... 46
3.4.3 Plasma ions ......................................................................................................... 47
3.5. Conclusions ........................................................................................................... 49
References ........................................................................................................................ 49
Chapter 4 (PAPER 2) ....................................................................................................... 54
EFFECTS OF NITRITE EXPOSURE ON HAEMATOLOGICAL PARAMETERS
AND GROWTH IN CLOWN KNIFEFISH (Chitala ornata, GRAY 1831) .................. 54
4.1 Introduction ............................................................................................................ 55
4.2 Materials and methods............................................................................................ 56
4.2.1 Effects of nitrite on haematological parameters in C. ornata ............................ 56
4.2.2 Effects of nitrite on growth of C. ornata ............................................................ 57
4.2.3 Data analysis....................................................................................................... 57
4.3 Results and discussion ............................................................................................ 58
4.3.1 Effects of nitrite on haematological paramters in C. ornata .............................. 58
4.3.2 Effects of nitrite on growth parameters in clown knifefish C. ornata ............... 62
4.4 Conclusions ............................................................................................................ 64

References ........................................................................................................................ 64
vi


Chapter 5 (PAPER 3) ....................................................................................................... 69
THE EFFECTS OF ELEVATED ENVIRONMENTAL CO2 ON NITRITE UPTAKE IN
THE AIR-BREATHING CLOWN KNIFEFISH
CHITALA ORNATA .................. 69
5.1 Introduction ............................................................................................................ 71
5.2 Materials and methods............................................................................................ 73
5.2.1 Animal holding ................................................................................................... 73
5.2.2 Experimental protocols....................................................................................... 74
5.2.3 Analytical procedures ......................................................................................... 74
5.2.4 Statistics.............................................................................................................. 76
5.3 Results .................................................................................................................... 76
5.3.1 Acid-base parameters and plasma ions .............................................................. 76
5.3.2 Nitrite uptake and levels of Hb derivatives ........................................................ 81
5.4 Discussion .............................................................................................................. 85
5.5 Conclusions ............................................................................................................ 88
References ........................................................................................................................ 88
Chapter 6 (Manuscript 1) ................................................................................................. 93
THE COMBINED EFFECTS OF NITRITE AND ELEVATED ENVIRONMENTAL
CO2 ON HAEMATOLOGICAL PARAMETERS IN SMALL-SIZED CLOWN
KNIFEFISH (CHITALA ORNATA) ................................................................................. 93
6.1 Introduction ............................................................................................................ 94
6.2 Materials and methods............................................................................................ 95
6.2.1 Animal handling and experimental protocols .................................................... 95
6.2.2 Statistics.............................................................................................................. 96
6.3 Results .................................................................................................................... 97
6.3.1 Combined effects of nitrite and carbon dioxide on haematological parameters in

small-sized C. ornata .................................................................................................. 97
6.3.2 Combined effects of nitrite and carbon dioxide on acid-base parameters and
plasma ions in small-sized C. ornata ........................................................................ 103
6.4 Discussion ............................................................................................................ 107
6.5 Conclusions .......................................................................................................... 111
References ...................................................................................................................... 111
Chapter 7 (Manuscript 2) ............................................................................................... 115
EFFECTS OF DIFFERENT TEMPERATURES ON HAEMATOLOGICAL
PARAMETERS IN CLOWN KNIFEFISH (CHITALA ORNATA) ............................... 115
vii


7.1 Introduction .......................................................................................................... 116
7.2. Materials and methods......................................................................................... 117
7.2.1 Experimental animals ....................................................................................... 117
7.2.2 Determination of temperature limits in the clown knifefish ............................ 118
7.2.3 Effect of different levels of temperature on haematological parameters ......... 118
7.3. Results ................................................................................................................. 120
7.3.1 Temperature tolerance in C. ornata ................................................................. 120
7.3.2 Effects of different temperatures on physiological parameters in small-sized C.
ornata ........................................................................................................................ 121
7.3.3 Effects of different temperatures on physiological parameters in large-sized C.
ornata ........................................................................................................................ 127
7.4 Discussion ............................................................................................................ 134
7.5 Conclusions .......................................................................................................... 136
References ...................................................................................................................... 136
Chapter 8 (Manuscript 3) ............................................................................................... 141
EFFECTS OF NITRITE AT DIFFERENT TEMPERATURES ON
HAEMATOLOGICAL PARAMETERS AND GROWTH IN CLOWN KNIFEFISH
CHITALA ORNATA ....................................................................................................... 141

8.1. Introduction ......................................................................................................... 142
8.2. Materials and methods......................................................................................... 143
8.2.1 Experimental animals and general experimental design .................................. 143
8.2.2 Determination of acute nitrite toxicity (96 h LC50) at 30ºC and 33ºC in C.
ornata ........................................................................................................................ 144
8.2.3 Sub-lethal nitrite exposures at different temperatures and blood sampling in C.
ornata ........................................................................................................................ 144
8.2.4 Analysis of haemoglobin derivatives ............................................................... 146
8.2.5 Effects of nitrite at different temperatures on growth and digestive enzyme
activities in C. ornata ................................................................................................ 146
8.2.6 Calculations ...................................................................................................... 147
8.2.7. Statistics........................................................................................................... 147
8.3 Results .................................................................................................................. 148
8.4. Discussion ........................................................................................................... 159
8.4.1 Values of 96 h LC50 for nitrite at different temperatures in C. ornata ............. 159
8.4.2 Effects of nitrite at different temperatures in C. ornata ................................... 161
viii


8.4.3 Effects of nitrite at different temperatures on growth and digestive enzyme
activity in C. ornata................................................................................................... 163
8.5 Conclusions .......................................................................................................... 165
References ...................................................................................................................... 165
Chapter 9 ........................................................................................................................ 173
A SURVEY ON SOME ENVIRONMENTAL PARAMETERS IN CLOWN
KNIFEFISH (Chitala ornata, Gray 1831) PONDS ....................................................... 173
9.1 Introduction .......................................................................................................... 174
9.2. Materials and methods......................................................................................... 174
9.2.1 Materials ........................................................................................................... 174
9.2.2 Methods ............................................................................................................ 174

9.3 Results and discussion .......................................................................................... 175
9.4 Conclusions .......................................................................................................... 177
References ...................................................................................................................... 177
Chapter 10 ...................................................................................................................... 178
GENERAL DISCUSSIONS .......................................................................................... 178
10.1 Effects of nitrite exposure to physiological functions in C. ornata ................... 178
10.2 Effects of nitrite exposure on growth in C. ornata............................................. 179
10.3 Effects of elevated temperatures to physiogical parameters in C. ornata .......... 180
10.4 Combined effects of hypercapnia and nitrite on nitrite uptake and acid-base
regulation in C. ornata ............................................................................................... 180
References ...................................................................................................................... 181
Chapter 11 ...................................................................................................................... 185
CONCLUSIONS AND RECOMMENDATIONS ........................................................ 185
11.1 Conclusions ........................................................................................................ 185
11.2 Recommendations .............................................................................................. 186
11.2.1 Recommendations for intensive farming systems .......................................... 186
11.2.2 Recommendations for further studies............................................................. 186
List of appendices .......................................................................................................... 187
Appendix 3.2.1. Information in the C. ornata culture ponds ..................................... 187
Appendix 9.3: Determing the values of 96h LC50 for nitrite at 27, 30 and 33ºC in C.
ornata (SPSS analysis) ............................................................................................... 188
List of pictures about experimental setup, blood sampling and devices of analysis used
in the studies................................................................................................................... 189
ix


List of figures

Figure 3.3.1

Mortality of C. ornata (8-10g) by a function of nitrite concentration . 37

Figure 3.3.2

Extinction coefficients for the four haemoglobin species at wavelengths
from 480 to 700 nm and spectrum from a fish exposed to 1 mM nitrite
for 2 day and the fitted curve ............................................................... 38

Figure 3.3.3

Plasma NO2-, plasma NO3-, percentage metHb, percentage HbNO,
functional Hb and total plasma nitrite and nitrate after exposure to nitrite
.............................................................................................................. 41

Figure 3.3.4

Plasma chloride, plasma sodium, plasma HCO3-, plasma osmolality,
blood lactate after exposure to nitrite ................................................... 43

Figure 3.3.5

Plasma protein and whole body water content after exposure to nitrite
.............................................................................................................. 44

Figure 3.3.6

Davenport diagram, blood PCO2, pHe after exposure to nitrite ........... 45

Figure 3.3.7

Rate constant (k, min-1) for erythrocyte metHb decline via metHb
reductase in fish exposed to nitrite ....................................................... 45

Figure 4.3.1

Haematological paramters in C. ornata after 14 days exposed to nitrite.
.............................................................................................................. 61

Figure 4.3.2

Growth paramters in C. ornata after 90 days exposed to nitrite .......... 64

Figure 5.3.1.1 Time-dependent changes in pHe, plasma bicarbonate, plasma Cl-, PCO2,
plasma Na+, and plasma osmolality during exposure to nitrite and
hypercapnia ..........................................................................................77
Figure 5.3.1.2 Davenport diagram showing changes in acid-base status during exposure
to nitrite and hypercapnia ................................................................... .81
Figure 5.3.2

Time-dependent changes in plasma NO2-, metHb percentage, HbNO
percentage, functional Hb, plasma NO3-, and the sum of plasma nitrite
and nitrate during exposure to nitrite and hypercapnia ....................…84

Figure 6.3.1

Plasma NO2-, metHb, HbNO (C), functional Hb, plasma NO3- (E), and
total nitrite and nitrate after exposure to nitrite and carbon dioxide…100

Figure 6.3.2.1 pHe, plasma HCO3-, PCO2, plasma Na+ and osmolality after exposure to
nitrite and carbon dioxide................................................................... 104

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Figure 6.3.2.2 Davenport diagram presenting the changes in acid-base status after
exposure to nitrite and carbon dioxide ............................................... 107
Figure 7.3.2.1 Plasma Na+, plasma osmolality plasma glucose, plasma K+ in smallsized C. ornata after exposed to five different temperatures 24ºC, 27ºC,
30ºC, 33ºC, 36ºC ................................................................................ 125
Figure 7.3.2.2 pHe, blood PCO2, plasma HCO3-, plasma Cl- in small-sized C. ornata
after exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC,
36ºC .................................................................................................... 127
Figure 7.3.3.1 Plasma Na+, plasma osmolality, plasma glucose, plasma K+ in largesized C. ornata after exposed to five different temperatures 24ºC, 27ºC,
30ºC, 33ºC, 36ºC ................................................................................ 132
Figure 7.3.3.2 pHe, PCO2, plasma HCO3-, plasma Cl- in large-sized C. ornata after
exposed to five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC
............................................................................................................ 133
Figure 8.3.1

Mortality (96 h LC50 for nitrite) of C. ornata (8-10 g) at three different
temperatures: 27ºC, 30ºC, and 33ºC .................................................. 149

Figure 8.3.2

Plasma NO2-, metHb, HbNO, functional Hb, plasma NO3-, and total
NO2- and NO3- after exposed to nitrite at five different temperatures
24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 153

Figure 8.3.3

Plasma Na+, plasma osmolality, plasma Cl-, plasma HCO3- after exposed
to nitrite at five different temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC

............................................................................................................ 154

Figure 8.3.4

Davenport diagram presenting the changes in acid-base status, blood
PCO2, and pHe after exposed to nitrite at five different temperatures
24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 156

Figure 8.3.5

Survival rate and 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 .................................................................................................... 157

Figure 9.3

Temperature, pH, PCO2, PO2, NO2- (E), NO3- (F) in the water at the C.
ornata ponds ...................................................................................... 176

xi


List of tables

Table 3.3.1

Hct, Hb and MCHC after exposure to nitrite ..................................... 40

Table 4.3.1

RBCs and WBCs after 14 days exposed to nitrite ............................. 59

Table 4.3.2

Initial weight (W0), weight at day 90 (W90), WG, SGR, and DWG after
90 days exposed to nitrite................................................................... 63

Table 5.3.1.1

Plasma K+, plasma glucose during exposure to nitrite and hypercapnia
............................................................................................................ 79

Table 5.3.1.2

Hct, Hb and MCHC during exposure to nitrite and hypercapnia ....... 83

Table 6.3.1.1

RBCs and WBCs after exposure to nitrite and carbon dioxide.......... 98

Table 6.3.1.2

Hct, Hb and MCHC after exposure to nitrite and carbon dioxide ..... 102

Table 6.3.2

Plasma potassium and plasma glucose after exposure to nitrite and
carbon dioxide .................................................................................... 106

Table 7.3.2.1

RBCs and WBCs in small-sized C. ornata after exposed to five different
temperatures 24ºC; 27ºC; 30ºC; 33ºC 36ºC ....................................... 122

Table 7.3.2.2

Hct, Hb and MCHC in large-sized after exposed at five different
temperatures 24ºC; 27ºC; 30ºC; 33ºC; 36ºC ...................................... 124

Table 7.3.3.1

RBCs and WBCs in large-sized C. ornata after exposed to five different
temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 129

Table 7.3.3.2

Hct, Hb and MCHC in large-sized after exposed at five different
temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 131

Table 8.3.1

RBCs and WBCs after exposed to nitrite at five different temperatures
24ºC, 27ºC, 30ºC, 33ºC, 36ºC ............................................................ 150

Table 8.3.2

Hct, Hb and MCHC after exposed to nitrite at five different
temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 151

Table 8.3.3

Plasma glucose and potassium after exposed to nitrite at five different
temperatures 24ºC, 27ºC, 30ºC, 33ºC, 36ºC....................................... 155

Table 8.3.4

Initial weight (W0), final weight (W90), GW, DWG, and SGR after 30,
60, and 90 days exposed to 27ºC (control), 30ºC, 33ºC, 1 mM nitrite at
27ºC, 1 mM nitrite at 30ºC, 1 mM nitrite at 33ºC .............................. 158

Table 8.3.5

Activities of digestive enzymes: pepsine (in stomach), trypsine (in
intestine), chymotrysine (in intestine), α-Amylase (in stomach and
xii


intestine) 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 ..... 159
Table 8.4.1

The values of 96h LC50 for nitrite in some fish species ..................... 160

xiii


List of abbreviation

[CO2]total

total plasma CO2 concentration

96 h LC50

Lethal concentration in 96 hours

Cl-

Chloride

CO2

Carbon dioxide

DARD

Department of Agriculture and Rulral Development

DeoxyHb

Deoxygenated haemoglobin

DWG

Daily weight gain

FAO

Food and Agriculture Organization

FCR

Feed conversion ratio

Fig.

Figure

GW

Gained weight

Hb

Haemoglobin

HbNO

Haemoglobin nitrosyl

HCO3-

Bircabonate

Hct

Haematocrit

IPCC

Intergovernmental Panel on Climate Change

K+

Potassium

MCHC

Mean corpuscular haemoglobin concentration

MD

Mekong Delta

metHb

Methaemoglobin

Na+

Sodium

NH3+

Ammonia

NO2-

Nitrite

NO3-

Nitrate

OxyHb

Oxygenated haemoglobin

PCO2

Partial pressure of carbon dioxide

pHe

Extracellular pH
xiv


PO2

Partial pressure of oxygen

RBCs

Number of red blood cells (erythrocytes)

SEM

Standard error of mean

SGR

Specific growth rate

SR

Survival rate

WBCs

Number of white blood cells (leukocytes)

αCO2

CO2 solubility in trout plasma

βNB

Non-bicarbonate buffer effect

xv


Summary

This dissertation investigated the isolated and combined effects of environmental
factors such as nitrite, temperature and hypercapnia (high concentration of
carbon dioxide) on physiological parameters, growth and digestive enzyme
activity in clown knifefish (Chitala ornata) in Mekong Delta, Vietnam. This airbreathing species, which is one of the most popular species has been culturing in
the South East Asia with high protein quality and ornamental purposes, typically

high environmental resistance under intensive culturing systems. The current
situation of climate change has been seriously affecting almost all fields of living
organisms including: human, plants, animals, particularly aquatic animals –
pokilothermic species. Therefore, the studies in the dissertation about changes of
aquatic environment related to fish health and growth, including physiological,
biochemical processes in fish have been one of the pressing and necessary issues
in order to provide a better physiological understanding as well as
recommendations and solutions for minimizing nitrite toxicity and its
combination with other environmental elements in aquaculture ponds under
global climate change at the present.
We discovered that C. ornata has become the most tolerant air-breathing species
of nitrite with 96 h LC50 of 7.82 mM at 27ºC. Behind the effective mechanism of
denitrification coverting nitrite to nitrate in sub-lethal nitrite exposure, this is also
the first study to show that up-regulation methaemoglobin reductase activity in
metHb reduction in fish increased almost 5 folds (the rate constant from 0.01 in
controls to 0.046 min-1 after 6 days of nitrite exposure for converting metHb to
functional Hb). Interestingly, C. ornata had an incomplete acid-base regulation
with 50% of extracellular pH compensated during 96 h exposed to 21 mmHg
PCO2 by plasma bicarbonate accumulation while it is considered that the airbreathing species with the reduced surface area of gills may cause limitations on
transepithelial ion exchange, leading to low capacity of pH regulation. Morever,
in combined exposure of acclimated hypercapnia and nitrite, acid-base regulation
mainly resulted in chloride-mediated (reduced Cl- influx via the branchial HCO3/Cl- exchanger) reduced significantly the nitrite uptake across the gill during 96
h.

xvi


In addition, C. ornata had rather high temperature tolerance among various
tropical species with upper and lower limits of temperature (41ºC and 12ºC,
respectively). There were no significant impacts of various temperatures (24ºC,

27ºC, 30ºC and 33ºC) to physiological parameters in both 2 sizes of C. ornata
(small-sized and large-sized), but the appearance of mortality after 2 days
exposed to 36ºC in commercial fish accompanied with the sudden declines in
extracellular pH, haematocrit and haemoglobin concentration may resulted from
insufficient oxygen carrying in the blood.
In three different temperatures of 27ºC, 30ºC, 33ºC, C. ornata had the highest
nitrite tolerance at 30ºC with 96 h LC50 of 8.12 mM, where the values of 96 h
LC50 at 27ºC and 33ºC were 7.82 mM and 6.75 mM, respectively. After 2 weeks
in nitrite exposures at 5 different temperatures (24ºC, 27ºC, 30ºC, 33ºC, 36ºC),
the significant decrease in methaemoglobin via the recovery in functional
haemoglobin to 80-85% of total haemoglobin despite of the peak of
methaemoglobin of 55% after 2 days exposed to 36ºC. Also nitrite exposure at
elevated temperatures caused significant effects to acid-base regulation compared
to this at low temperature, e.g. the significant rises of PCO2 and reduction in
extracellular pH at the first day. However, extracellular pH was recovered more
than 50% for all groups with accumulation of plasma bicarbonate via a HCO3-/Clexchanger after 14 days. In addition, we found that long-term exposure of nitrite
significantly affected growth parameters. The treatment of 30ºC had the highest
survival rate and the lowest FCR compared to other treatments (27ºC, 30ºC,
33ºC, 1 mM nitrite at 27ºC, 1 mM nitrite at 30ºC, and 1 mM nitrite at 33ºC). The
activities of digestive enzyme were influenced by nitrite and temperature, where
chymotrypsine in intestine in the group of isolated temperature reached the
highest values 30ºC compared to this in other groups after 90 days culturing.
Key words: Chitala ornata, growth, hypercapnia, metHb reductase, nitrite,
physiological processes, temperature.

xvii


Tóm tắt

Luận án này được thực hiện để tìm hiểu ảnh hưởng đơn lẻ và kết hợp của một số
yếu tố môi trường như nitrit, nhiệt độ và hypercapnia (nồng độ carbon dioxide
cao trong nước) lên các chỉ tiểu sinh lý máu, tăng trưởng và hoạt động của
enzyme tiêu hóa trên cá thát (Chitala ornata) ở đồng bằng sông Cửu Long, Việt
Nam. Loài cá hô hấp khí trời này là một trong nhưng loài được nuôi phổ biến
nhất ở vùng Đông Nam Á với chất lượng thịt cao và có giá trị làm cảnh, đặc biệt
là khả năng chịu đựng môi trường cao trong hệ thống nuôi thâm canh. Tình trạng
biến đổi khí hậu (sự tăng nhiệt độ đã và đang ảnh hưởng nghiêm trong đến tất cả
các sinh vật sống bao gồm con người, cây trồng, các loài động vật, đặc biệt là
động vật thủy sản, loài chịu ảnh hưởng trực tiếp từ sự thay đổi nhiệt độ môi
trường. Vì vậy, những nghiên cứu trong luận án này về sự thay đổi của môi
trường nước liên quan đến sức khỏe và sinh trưởng của cá bao gồm các quá trình
sinh lý, hóa sinh của cá là một trong các vấn đề cấp thiết để cung cấp những kiến
thức sinh lý tốt hơn cũng như là các đề xuất và giải pháp nhằm hạn chế tối thiểu
tính độc của nitrite và ảnh hưởng kết hợp của nó với các yếu tố môi trường khác
trong ao nuôi thủy sản dưới tác động biến đổi khí hậu ngày nay.
Nghiên cứu đã phát hiện ra cá thát lát còm là loài cá hô hấp khí trời có khả năng
chịu đựng nitrit cao nhất hiện nay với giá trị LC50 96 h là 7.82 mM ở 27ºC. Bên
cạnh cơ chế giải độc nitrit là quá trình nitrat hóa bên trong cơ thể cá chuyển đổi
nitrit thành nitrat khi tiếp xúc với nồng độ nitrit bán cấp tính, đây cũng là nghiên
cứu đầu tiên thể hiện sự tăng hoạt động của enzyme khử nitrit methaemoglobin
reductase gấp 5 lần (hằng số hoạt động của enzyme này tăng từ 0.01 ở nghiệm
thức đối chứng lên 0.046 min-1 sau 6 ngày tiếp xúc 2.5 mM nitrite). Quá trình cân
bằng acid-base ở cá thát lát còm cũng khá hiệu quả với 50 % giá trị pH ngoại bào
được đền bù sau 96 h tiếp xúc 21 mmHg CO2 nhờ vào sự tích lũy đáng kể của ion
HCO3- trong huyết tương trong khi các loài hô hấp khí trời với sự tiêu giảm diện
tích mang có thể làm hạn chế quá trình trao đổi ion qua lớp biểu mô, dẫn tới khả
năng điều hòa pH ngoại bào thấp. Hơn nữa, trong sự tiếp xúc kết của hypercapnia
và nitrit, quá trình cân bằng acid-base chủ yếu từ cơ chế trao đổi ion chloride gián
tiếp (giảm ion Cl- qua sự trao đổi HCO3-/Cl-) đã làm giảm đáng kể lương nitrit

qua mang cá suốt 96 h tiếp xúc.

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Ngoài ra, cá thát lát còm có khả năng chịu đựng nhiệt độ khá cao so với các loài
cá nhiệt đới khác với nhiệt độ ngưỡng trên là 41ºC và ngưỡng dưới là 12ºC. Các
yếu tố sinh lý máu ở cả hai kích cỡ cá giống và thương phẩm đểu không bị ảnh
hưởng đáng kể sau 2 tuần tiếp xúc với các mức nhiệt độ (24ºC, 27ºC, 30ºC and
33ºC), nhưng tỷ lệ chết ở cá thương phẩm đã xuất hiện sau 2 ngày tiếp xúc nhiệt
độ 36ºC cùng với sự giảm xúc đáng kể của pH ngoại bào, haematocrit và nồng độ
có thể xuất phát từ sự thiếu oxygen trong máu.
Khi xác định vì khả năng chịu đựng nitrit của cá thát lát còm ở các nhiệt độ khác
nhau như 27ºC, 30ºC, 33ºC, kết quả đã cho thấy cá thát lát còm chịu đựng nitrit
tốt nhất ở nhiệt độ 30ºC với 96 h LC50 là 8.12 mM trong khi lần lượt ở 27ºC và 33
ºC là 7.82 mM và 6.75 mM. Sau 2 tuần tiếp xúc nitrit ở 5 mức nhiệt độ (24ºC,
27ºC, 30ºC, 33ºC, 36ºC), sự giảm đáng kể của methaemoglobin qua sự phục hồi
của haemoglobin chức năng về 80-85% trong tổng số haemoglobin mặc dù
methaemoglobin đạt giá trị cao nhất là 55% sau 2 ngày ở 36ºC. Nitrit ở nhiệt độ
cao ảnh hưởng nhiều đến quá trình cân bằng acid-base nhiều hơn nitrit ở nhiệt độ
thấp như sự tăng mạnh của PCO2 và giảm sút của pH ngoại bào vào sau 1 ngày.
Tuy nhiên, pH ngoại bào đã được phục hồi hơn 50% cho tất cả các nhóm thí
nghiệm sau 14 ngày. Chúng tôi cũng tìm ra ảnh hưởng mãn tính của nitrit và nhiệt
độ đến các chỉ tiêu tăng trưởng. Nghiệm thức 30ºC có tỷ lệ sống cao nhất và hệ số
chuyển đổi thức ăn FCR thấp nhất so với các nghiệm thức khác (27ºC, 30ºC,
33ºC, 1 mM nitrit ở 27ºC, 1 mM nitrit ở 30ºC, và 1 mM nitrit ở 33ºC). Hoạt động
của enzyme tiêu hóa cũng bị ảnh hưởng đáng kể bởi nhiệt độ và nitrit, cụ thể là
hoạt động của chymotrypsine trong ruột đạt giá trị cao nhất cũng ở nghiệm thức
30ºC sau 90 ngày nuôi.
Từ khóa: Chitala ornata, chỉ tiêu sinh lý, CO2 cao, metHb reductase, nitrit, nhiệt

độ, tăng trưởng.

xix


Chapter 1
INTRODUCTION

1.1 Introduction
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 27th 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. Climate change with the elevation of temperature, drought, sea-level rise,
season and precipitation amount causes serious consequences to all fields,
especially agriculture and aquaculture. Production of aquatic animals from
aquaculture reached 73.8 million tons in 2014, with an estimated first sale value
of US$ 160.2 billion. China accounted for 45.5 million tons in 2014 or more than
60 percent of global fish production from aquaculture. Other major producers
were India, Viet Nam, Bangladesh, and Egypt (FAO, 2014). Growth of fish
supply for human consumption has outpaced the growth of population in the past
five decades, reaching in the period 1961-2013, double that of population
growth, leading to the increase of average per capita availability with 9.9 kg in

the 1960s to 14.4 kg in the 1990s and 19.7 kg in 2013 to 20 kg with preliminary
estimates in 2014 and 2015 (FAO, 2014). This significant growth in fish
consumption has improved people’s diets around the world through diversified
and nutritious food. Fish accounted for 17 percent of the global population’s
intake of animal protein and 6.7 percent of all protein consumed. Viet Nam
which is tropical country with significant contribution of fish production has
been under various problems for aquatic system by global warming. The
increases of temperature induce the rise of metabolism of organism and aquatic
1


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 with 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
about physiology 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 air-breathing species.
Moreover, there is obviously not only one toxin existing in aquatic environment;
the best assumption is that the combination of a variety of toxin may cause more
bad effects by competition to uptake into fish blood. However, the studies about
combinative effects of environmental parameters to bio-chemical and
physiological processes have not been carried out popularly. There have been
two studies about the combined effects of nitrite and carbon dioxide until now,
including (i) the study of Jensen (2000) in crayfish (Astacus astacus) and (ii) the
study of Hvas et al., 2016 in air-breathing striped catfish with different responses
in exposure of these environmental factors.
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
2


an understanding about effects and adaption mechanisms of this air-breathing
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

1. Conducting a
ornata ponds

survey on some selected environmental parameters in C.

2. Determining the 96 h LC50 of nitrite and examining the effect of nitrite on
haematological parameters and growth in C. ornata
3. Determining the activity of metHb reductase in metHb reduction in sub-lethal
nitrite exposures in C. ornata
4. 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
5. Determining the temperature tolerance and the effect of various levels of
temperature on haematological parameters in small-sized and large sized C.
ornata
6. Determining 96 h LC50 of nitrite at elevated temperatures and investigating
the effects of nitrite at different temperature on haematological paramters in
C. ornata
7. 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
1) During nitrite exposure, C. ornata reduce their branchial HCO3-/Clexchanging rate and/or increase the activity of erythrocyte NADH metHb

3


reductase for metHb reduction and experience significant changes in exchanging
rate of other branchial ions for recovery.
2) pH regulation under a respiratory acidosis stimulate a reduction in branchial

HCO3-/Cl- exchanger and thereby protect against nitrite toxicity C. ornata
3) Chronic exposures of nitrite cause negative impacts to growth parameters such
as low weight gain, low survival rate and high FCR C. ornata
4) 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 C. ornata
5) 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 New findings of the dissertation
The dissertation showed that C. ornata is the most nitrite tolerant species up to
date with the values of LC50 at 27ºC of 7.82 mM by effective denitrification
process converting nitrite to nitrate, and typcially the increase in rate constant of
erythrocyte metHb reductase enzyme for metHb reduction which is the first
experimental evidence found in fish.
The dissertation also indicated that exposure of high nitrite concentration
(50%*96 h LC50 at 27ºC) caused negative physiological impacts to the number of
blood cells, metHb, Hct, Hb concentration during 14 days, and significantly low
survival rate and high FCR value during 3 months.
Similar to P. hypophthalmus, the dissertation illustrated that C. ornata is the
second air-breathing fish having high capacity of acid-base regulation in
hypercapnic conditions with 50% of pH compensation after 96 h exposed in 21
mmHg. Interestingly, nitrite uptake in this species was significantly reduced after
reaching pH regulation during acclimated hypercapnia by an apparent reduced
transport rate of the branchial HCO3-/Cl- exchanger.
The dissertation also demonstrated that C. ornata is one of the most high
temperature tolerant species with temperature limits (12ºC and 41ºC for lower
and upper limit, respectively). And, there were no significant impacts in
haematological parameters and acid-base status in the elevation of temperature
although mortality appeared at the temperature of 36ºC during physiological

experiment of 7 days.
4