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)

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 CO 2 , N 2 0, CH 4 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. 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 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 an understanding about effects and adaption mechanisms of this air-breathing fish under climate change.

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

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

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

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

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

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

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

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 small-

sized 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 large-

sized 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

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

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

List of abbreviation

[CO2]total total plasma CO2 concentration

96 h LC50 Lethal concentration in 96 hours

DARD Department of Agriculture and Rulral Development DeoxyHb Deoxygenated haemoglobin

FAO Food and Agriculture Organization

PCO2 Partial pressure of carbon dioxide

PO2 Partial pressure of oxygen

RBCs Number of red blood cells (erythrocytes)

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

air-breathing 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 air-breathing 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

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-/Cl-

exchanger 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

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

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

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

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

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 survey on some selected environmental parameters in C ornata ponds

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-/Cl

-exchanging rate and/or increase the activity of erythrocyte NADH metHb

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

The dissertation also discovered that growth parameters of C ornata had no

significantly negative effects in exposure of isolated elevated temperature (33ºC) and combined exposure of elevated temperature and nitrite despite of high FCR values

1.6 Significant contributions 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 and other air-breathing species or

comparing physiological responses of this species to those in other aquatic animals under extreme environmental changes

FAO, 2014 The State of Food and Agriculture Innovation in family farming

Hvas, M., Damsgaard, C., Gam, L.T.H., Huong, D.T.T., Jensen, F.B., Bayley, M., 2016 The effect of environmental hypercapnia and size on nitrite toxicity in the striped

catfish (Pangasianodon hypophthalmus) Aquatic Toxicology 176: 151–160

IPCC, 2013 Climate Change 2013: The Physical Science Basis Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel

on Climate Change Intergovernmental Panel on Climate Change, Working Group

I Contribution to the IPCC Fifth Assessment Report (AR5) (Cambridge Univ Press, New York), 1535 pp

Jensen, F.B., Koldkjaer, P., Bach, A., 2000 Anion uptake and acid-base and ionic effects during isolated and combined exposure to hypercapnia and nitrite in the

freshwater crayfish, Astacus astacus Journal of Comparative Physiology, Part B

170, 489–495

Lefevre, S., Jensen, F.B., Huong, D.T.T., Wang, T., Phuong, N.T., Bayley, M., 2011 Effects of nitrite exposure on functional haemoglobin levels, bimodalrespiration,

and swimming performance in the facultative air-breathing fish Pangasianodon

hypophthalmus Aquatic Toxicology 104: 86–93

Lefevre, S., Jensen, F.B., Huong, D.T.T., Wang, T., Phuong, N.T., Bayley, M., 2012 Haematological and ion regulatory effects of nitrite in the air-breathing snakehead

fish Channa striata Aquatic Toxicology 118-119: 48–53

Kroupova, H., Machova, J., Svobodova, Z., 2005 Nitrite influence on fish A review Vetenary Medicine, 50: 461- 471

UNFCCC, 1992 Introducing the United Nations Framework Convention on Climate

Change

Chapter 2 LITERATURE REVIEW 2.1 The status and importance of aquaculture and fisheries

The world human population is increasing with an average rate of 1.2% annually and reached 6.92 billions in 2010 (Bongaarts and Sinding, 2011) According to the Department of Economic and Social Affairs – United Nations (United Nations, 2013), the world population is predicted to grow up to 10 billions by

2100 The development of aquaculture and fisheries provides a number of benefits for human-being lives; however, these sectors are now dealing with some difficulties from rapid growth of human population The overfishing spread-out with biologically unsustainable levels is currently causing gradual depletion of wild fish population For effective protection of wild fish resources, people should both enhance cultured fish productivity, and guarantee that the sustainable growth of aquaculture and capture fisheries are maintained for food world demand (FAO, 2014) Dramatic growth of population and the increase of urbanization affect to human-being diet, and the requirement of high food quality leads to a serious food crisis To deal with these issues, researches about the enhancement solutions of crop yields, livestock productivity as well as technical

developments should be currently carried out Predicted by Parry et al (2013),

there have been 370 million hungers by 2060 with approximately 5% of the total human population in developing countries although food productivity has gradually improved

With an average annual rate of 6.6% since 1995, global aquaculture production achieved 106 million tons in 2015, including 76.6 million tons of aquatic animals and 29.4 million tons of aquatic plants (FAO, 2017) Thus, aquaculture sector plays a key factor in food contribution, poverty alleviation and economic development for the poor people However, in order to maintain the at least level

of per-capita food consumption of 19.7 kg in 2013 (FAO, 2016), an additional 23 million tons mainly from aquaculture needs to be provided over the world by

2020 (FAO, 2017) From 1995 to 2015, there was a rise in production of dependent aquaculture from 12.2 to 50.7 million tons; therefore, market opportunities for increasing production efficiency were expanded from the use of aquatic groups such as carps, tilapias, shrimps, and salmonids with established aquaculture technologies (FAO, 2017)

feed-Fisheries and aquaculture not only support to human nutrition but also to job opportunities, particularly they provide ten million jobs and produce hundreds of millions livelihoods with fish, especially in most developing countries (FAO, 2014) Following to the new dataset of FAO (2017), there are 591 aquatic species and species groups farmed in inland freshwater, inland saline water, coastal brackish water and marine water The world aquaculture production continuously increased to 106 million tonnes in live weight in 2015, achieving US $ 163 billion for a total estimated first-sale value (FAO, 2017) Capture fisheries and aquaculture become the primary job sector for 58.3 million people (37% full time) in developing countries with 84% of employees in Asia and 10% of employees in Africa (FAO, 2014) During 2001 – 2015, the aquaculture growth averaged at 10.4% in Africa, 6% in Asia, 5.7% in Americas, only 2.5-2.9% in Europe (FAO, 2017) In 2015, the finfish farming representing for the major aquaculture product accounted for 63-68% whereas mollusks farming and crustacean farming accounted for 30% and 23.7 and 10% of the total food fish farming production, respectively (FAO, 2017) In the world fish supply, aquaculture (including captured and farmed combined) has been increasing its contribution from 25.7% in 2000 to 45.3% in 2015 (FAO, 2017) For human consumption, aquaculture supplied 10.42 kg of food fish on world average in

2015, which increased by 0.28 kg compared to 10.14 kg in 2014 (FAO, 2017) Generally, fisheries and aquaculture are considered to be as a source of protein and income for 540 million people, and fish production supplies more than a half

of their animal protein and dietary minerals for 400 million people (FAO, 2017) The Asia-Pacific region (mainly China, South Asia and South-East Asia sub-regions) has dominated global aquaculture production, contributing 65.2 million tonnes of aquatic animals, approximately to 88 percent of the global total in 2014 with the value of US$ 127 billion (FAO, 2017) China which is the main fish producer and largest exporter of fish and fishery products is also the main importer from outsourcing of processing from other countries and growing domestic consumption of species not produced locally However, the fishery trade of this country experienced a decline in its processing sector in 2015 while Norway, the second major exporter, posted record export values in 2015 (FAO, 2016) In 2014, Vietnam which became the third major exporter (overtaking Thailand) has experienced a substantial reduction in export since 2013due to the decreased shrimp production from disease problems (FAO, 2016)

In 2012, Vietnam was ranked the 9th rank among the top ten producers of marine capture fisheries with the production of 2.42 million tons of fish, a significant increase of 46.8% compared to the previous decade In addition, this country also ranked the 3rd position among the top producers of fish food, supplying 3.09 million tons, accounting for 4.6% of the world fish production (FAO, 2014) Therefore, fish products maintain an important role in Vietnamese economics, particularly the average annual consumption of Vietnamese people was 14.6 kg/capita of fish, presenting 8.5% of total protein consumed Generally, fish and fish products have the significant importance in all areas of Vietnam, especially

in the Mekong Delta, where people consume up to 24.4 kg/capita of fish in their diets (FAO, 2014)

2.2 Climate changes and impacts on aquaculture and fisheries

According to FAO (2008), climate change is significant changes of weather in a long-term time period from several decades to a million of year Climate change may originate from the internal changes in nature or external forced changes or continuous changes of human-being on the atmosphere or land usage

Climate change affects to marine and land ecosystem, causing changes in use such as sediment loads, water flow regimes and physic-chemical effects (hypoxia, hypercapnia, and salinity change) These processes cause complicated consequences that can disturb aquatic community productivity, composition and seasonality processes of both fish and plankton population (Barange and Perry, 2009) There have been many studies about effects of different temperatures on

land-biochemical and physiological processes (Imsland et al., 2001; Imsland et al.,

2007; Kemp, 2009; Portner, 2001; Somero and Hochachka, 1968; Wright and Tobin, 2011)

MD, which is a low topographic region in the South of Vietnam, has the area of 4.06 million ha and the population of 17.3 million people (rural area accounting for 79%) (VGSO, 2012) In addition, the MD has a total freshwater area of 641,350 ha, accounting for 67.2% of the total surface area in this region (Phuong and Oanh, 2010) According to IPCC (2007), the MD has been predicted to be one of the most three affected regions by climate change over the world However, this region is the most important area of rice and fish production in Vietnam The temperature is predicted to increase from 1-4ºC in the next century (IPCC, 2013), the maximum level of temperature in the lower Mekong River was

predicted to reach over 32ºC by 2050 (Mainuddin et al., 2010)

There have been a significant number of studies that investigate the predicted effects of climate change on various sections of agriculture, aquaculture, fisheries, economy-society, and diseases in plant, animal and human-being Continued emissions of greenhouse gases will lead to further warming and serious changes in all climate change system It is required substantial and sustained reductions of greenhouse gas emission in order to limit the consequences of climate change (IPCC, 2013) Atmospheric CO2 concentrations are predicted to be higher in 2100, resulting from a further rise of cumulative

CO2 emissions during the 21st century

2.3 The status of farming clown knifefish (C ornata) in MD

With geographical and natural advantage conditions, aquaculture and fisheries in

MD have been strongly developed and contributed a significant income for farmers in this region The coast of 700 km stimulates the development of seafood source as well as mangrove for culturing both brackishwater and marinewater species This area has a large area of water surface with various aquatic systems such as rivers, canals, ponds, lakes for freshwater aquaculture Tropical climate facilitates for aquaculture and fisheries during the year The annual flood in MD provides huge resource of freshwater species, abundant gene resource of aquatic animals, food for agriculture and breeding Following to Southwest Steering Committee (2014), the area for aquaculture in MD was expanded to 800,000 ha water surface (increased by 5,000 ha compared to 2013), reached 2.4 million tons (increased by 400,000 tons compared to 2013 Beside the main species such as black tiger shrimp, white leg shrimp, freshwater prawn, striped catfish, MD also develops some other freshwater species such as Nile tilapia, climming perch, red tilapia, snakehead, Especially, clown knifefish, which is high-economical species with high nutrition quality is significantly consummed by a numerous people This species has fast growth, high tolerance

with environments and survive with high stocking density C ornata is

popularly farmed in Can Tho city, and Dong Thap, Vinh Long, An Giang provinces with different systems including ponds, hapas, tanks, typically in Hau Giang province (mainly in some districts: Phung Hiep, Long My, Vi Thuy and Vi Thanh city) According to (DARD, Hau Giang 2013), the total aquaculture area in Hau Giang was 10,700.05 ha (75,115.83 tonnes), including 10,212.68 ha for extensive and improved extensive systems, and 487,37 ha for

intensive and semi-intensive systems, whereas C ornata accounted for 38.55 ha

with the production of 1,022.40 tonnes However, the total aquaculture area in this province decreased to 7,025.41 ha with the production of 63,599 tonnes (DARD, Hau Giang 2017)

2.4 Background about effects of some key environmental parameters on physiological processes and growth in aquaculture

2.4.1 Temperature

Temperature is one of the most important environmental factors affecting aquatic animals, which are pokilotherms with sensititive reactions in the changes of temperature levels (Fry, 1971) Every aquatic organism has their specific range

of environmental temperatures that can be tolerated more or less indefinitely; therefore, this tolerance range is determined by an interplay of developmental, genetic and environmental impacts such as the developmental stage of living organisms, their age, physiological condtions as well as the stages in the history

of their lives (Cairn et al., 1975) The tolerance zone may be respectively moved

upward or downward within genetic limits of a species by acclimating them to

higher or lower temperature (Brett et al., 1969; Fry and Hochachka, 1970)

Temperatures above or below the tolerance range are considered to be in the ranges of resistance Thermal resistance times (time to death at a given lethal temperature) have been measured for comparing between fish species (Fry, 1967) Tissue anoxia appears at elevated temperatures, thus the impacts of any toxicant that either rises metabolic demand (e.g., copper) or blocks oxygen uptake at the gill level for fish (e.g., zinc) by increased temperatures (Heath and Hughes, 1973) In addtion, it has been suggested that the causes of thermal in all aquatic organisms include failure of osmoregulatory processes and alterations in cellular enzymes and membrane lipids (Coutant and Pruderer, 1973) Protein may be denatured at high temperature, but many organisms die at temperatures, where protein denaturation does not exist The most cellular enzymes indicate sharp increases in the Michaelis-Menten (Km) constant at both low and high temperatures The Km is inversely proportional to the affinity of an enzyme with its substrate, thus a rise in Km is considered to be a decreased substrate affinity

If the Km increases excessively, the reactions catalyzed by that enzyme might decrease to a lethal level This hypothesis is related to chemical toxicity, where the toxicant apparently accelerates or limits specific cellular enzymes (Bostrom and Johansson, 1972)

The effects of temperature on chemical toxicity is a complicated matter because temperature may be a lethal factor and toxicants may replace lethal themal limits (Brett, 1969) In the thermal tolerance zone, temperature might act as a controlling factor via its effects on metabolism, then leading the limits on maximum activity (Brett, 1964) With the exception of marine mammals, birds, tuna fish, especially all aquatic animals which are ectothermic have the body temperature almost the same as that of the environment Being ectothermic also means that the rate of metabolism in aquatic animals undergoes an approximately two-fold increase with every 10ºC increase in temperature (Boyd and Tucker, 1998) Depending on the species, there is commonly an eventual stabilization of

metabolism for non-photosynthetic organisms (e.g., oxygen consumption) at an

intermediate level Therefore, acclimation to a temperature change tends to modulate the direct effects of this environmental variety on the ectothermic organisms (Fry, 1971) The natural environment produces conditions, which may

be historical stresses such as temperature, evolving physiological mechanisms to either moderate the stress, tolerate or probably compensate for it (Todd et al., 1972) When appearing the combination of two or more stresses, the effects may

be synergistic, additive, or antagonistic (the combination has a lesser effect than the individual effects); therefore, this zone temperature acts a controller of response in the combined effects of temperature with chemical toxicants (Cairn

et al., 1975) Morever, a change in temperature can induce a given chemical more

or less toxic to an aquatic animal, resulting to the rise or reduction in the minimum lethal concentration, and thus the survival time in a lethal

concentration may be lasted longer (Cairns et al., 1975)

Aquatic animals such as fish are extremely sensitive to environmental temperature, not only about high or low temperature but also about the changing speed of temperature The fish will be seriously shocked when they are moved from this environment into another environment (Beitinger and Lutterschmidt, 2011) Temperature significantly affects to the habitat of aquatic animals; its fluctuation may lead to the changes in physiological, metabolism processes, oxygen consumption; and activities of digestive enzymes are also changed Among the various physical factors affecting the aquatic environment, temperature plays an important role in the life of aquatic poikilotherms Temperature is considered as an abiotic master parameter (Fry, 1971) Water temperature influences physiological processes such as food consumption,

digestion, and immunity (Zeng et al., 2009) Low temperatures in aquatic system

show profound immunosuppressive effects on ectothermal animals like fish (Bly

et al., 1986) Temperature increases, metabolism and activities of digestive

enzyme increase; aquatic animals can absorb nutrients from environment and food better, the fish growth also increases and the protein need also boosts (Kemp, 2009) For most species in normal range of temperature, a slight increase

in temperature benefits to growth because it results in more energy, leading to higher reaction rates for growth This is commonly due to how the molecular structure of mitochondria if influenced by changes in temperature (Guderley, 2004) The rate of biochemical processes rises by two-fold for every 10ºC increase in temperature (Boyd and Tucker, 1998) The optimal range of temperature for most of tropical fish species is 25-32°C (Boyd and Tucker, 1998)

Temperature is an environmental stressor where the elevation of temperature causes a decrease of oxygen solubility in water (Cech and Brauner, 2011) The oxygen solubility reduces as water temperature soars while the simulation by temperature of metabolism processes cause an increase in the need of oxygen delivery Growth basically increases with temperature, this is indicated in the

study of striped bass (Morone saxatilis) and white sturgeon (Acipenser transmontanus) which also presents the typical Q10 (Cech et al., 1984)

Depending on temperature and salinity, water contains 20-40 times less oxygen

by volume and diffuses about ten thousand times more slowly through water than air (Graham, 1990) The increase of temperature causes a lowering of HbO2

affinity which must be compensated when fish deal with a reduction in water oxygen solubility and a rise in oxygen demand (Salama and Nikinma, 1990) In

the tambaqui (Colossoma macroponum), HbO2 affinity at pH 7.1 is reduced 3 fold as the elevation of temperature from 19 to 29ºC With condition above 30ºC,

the oxygen consumption in C macropomum was decreased, reporting an overall

reduction in metabolic rate (Saint-Paul, 1983) During exposure to elevated temperature, a rise in oxygen-carrying capacity of the blood benefits for oxygen delivery at a period time when metabolic rate becomes increased, it has been proposed that changes in hemoglobin content of the blood may be more significant than changes in P50 in ensuring oxygen transport under a number of environmental conditions (Brauner and Wang, 1997)

In other hand, there have been a numerous of studies about the effect of

temperature on growth rate such as in channel catfish (Ictalurus punctatus)

(Buentello et al., 2000), silversides (Odontesthes bonariensis) (Carriquiriborde et al., 2009), and two rock pool fishes (Caffrogobius caffer and Diplodus sargus)

(Kemp, 2009) Generally, these studies show that temperature has a close relationship with growth rate, survival and metabolism, typically during juvenile stage The regulatory mechanisms underlying the relationship between growth rate and temperature are associated with enzymatic modulation of metabolic

processes (Buentello et al., 2000) In most of warm water species, growth rate

increases with the elevation of temperature to a few degrees below the upper lethal limit, thus food supply is not limited (Talbot, 1993) Increasing temperature in culture systems can also increase some haematological parameters

such haematocrit in sturgeon (Huso huso) (Zarejabad et al., 2010), and numner of red blood cell and haemoglobin in neotropical fish (Prochilodus scrofa)

(Carvalho and Fernandes, 2006)

nitrite exposure (Jensen, 2007)

4Hb(Fe2+ ) O2 + 4NO2- +4H+  4Hb(Fe3+) + 4 NO3- + O2 +2 H2O (1)

4Hb(Fe2+ ) + NO2- + H+  Hb(Fe3+) + NO + OH- (2)

Hb(Fe2+ ) + NO  Hb(Fe2+)NO (3)

According to Jensen (2003), nitrite not only causes oxygen deficiency by metHb formation, but also affects to other organs by different mechanisms Nitrite exposure causes a rapid boost in heart rate in rainbout trout; nitrite generates hyperventilation from an increase of arterial PO2 (and a decrease of arterial PCO2) in carp (Jensen et al., 1987) as well as an elevation in ventilation rate in rainbout trout (Aggergaard and Jensen, 2001) Margiocco et al (1983) reported

that nitrite not only causes consequences in gills and blood but also accumulates

in liver, brain and muscle; at the beginning, the amount of nitrite entering fish body transformed to nitrate; and this denitrification process happens in liver

Since nitrate is relatively non-toxic, its production in the reaction between nitrite and oxygenated Hb can be viewed as a detoxification mechanism for nitrite, because the formed metHb can be reduced to functional Hb by erythrocytic NADH-metHb reductase (Doblander and Lackner, 1997; Jensen, 2003) However, when ambient concentration of nitrite is extremely high, fish may die

by the acute increase of metHb in blood The formation of metHb causes blood phenomenon’’ It is a typical syndrome in nitrite exposure (Kroupova, 2005)

“brown-Nitrite which is an intermediate product of nitrogen cycle in the ecosystem occurs

at low concentration (typically <1 µM) in aquatic environment The imbalance in bacterial nitrification and denitrifcation processes are likely from elevated nitrite

by nitrite uptake via gills, especially with freshwater species this uptake becomes more active because ion Cl- in freshwater is low, nitrite has an affinity for uptake mechanism of Cl-, changing the ratio Cl-/HCO3- and nitrite will shift Cl- to uptake

to fish body with an ambient NO2- in water, causes the reduce of chloride in plasma although small ambient concentrations of nitrite in the water cause the increase of internal nitrite levels (nitrite in the plasma) (Jensen, 2003) Freshwater species are hyper-osmotic to their habitat; they need an uptake channel of active ions such as Na+ and Cl- across the gills to compensate passive ions lacked across the gills and in the urine also Differently, marine fish are hypo-osmotic to their environment and excrete NaCl across the gills actively In addition, high concentration of ion Cl- in seawater (550 mM) will compete with nitrite through shared transportation (Jensen, 2009)

NO is produced from nitrite by low pH, hypoxia and high [NO2-] (Zweler et al.,

1999) NO interferes with processes regulated by local hormones Steroid hormone synthesis may be inhibited, while changes in ammonia and urea levels and excretion rates reflect an influence of nitrite on nitrogen metabolism The growth, sex hormone and maturation are also affected by long-term exposure of

nitrite (Ridnour et al., 2004) In water with nitrite contamination, nitrite

concentrations in plasma can reach in the millimolar range; nitrite becomes toxic

at high concentration with critical disturbances for physiological processes that affect respiration, ion regulation and endocrine system (Jensen, 2003) In zebro

fish (Danio rerio), HbNO levels in the blood change with variable ambient

concentrations of nitrite at different time periods This indicates a large NO production from nitrite and the action of nitrite toxicity at high level leads to

disturbance of NO homeostatic (Jensen, 2007) The NO formation with a massive number both influences in physiological processes and causes nitrosative stress in tissues; as the result, cellular structures may be damaged by high levels of S-nitrosylated proteins (Jensen, 2007) Morever, the high levels of HbNO result in confusion of O2 transport because higher levels of metHb and a lowering of functional Hb exists at the same time in nitrite-contaminated fish (Jensen, 2007) Elevated nitrite in freshwater ecosystem is originated from the imbalances of bacterial nitrification and denitrification processes in the environment (Jensen, 2003) Accordingly, the internal concentrations of nitrite in freshwater fish may modestly be increased compared to mammals and seawater fish; however, they are not commonly high; additionally, the hypoxic milieu activates the endogenous nitrite basin; and it habitually coincides with the regular elevation of ambient nitrite levels (Jensen, 1987) Consequently, freshwater fish normally deals with attainable nitrite levels during elongated hypoxic periods In fact, some freshwater species can naturally tolerate several weeks in hypoxic habitat; and possibly having approach to environmental nitrite as well as an internal NO levels generated from uptake across the gills (Eddy and William, 1987) One of the most important methods to reduce nitrite toxicity is to add a suitable amount of chloride (Jensen, 2003; Kroupova, 2005) This method was first studied in catfish and salmon from 1970s (Lewis and Morris, 1986) and this is the simplest and effective method to limit nitrite absorbtion and toxicity to fish

The species that have low ratio of chloride absorption will have higher

accumulation of nitrite (Jensen et al., 1987; Jensen, 1996, Jensen, 2003)

Crawford and Allen (1977) showed that the effect of nitrite on rainbout trout depends on salinity because nitrite and chloride have the same absorption mechanism to fish body; chloride competes and limits the nitrite effects These authors indicated that the fish mortality by nitrite in freshwater is higher than this

in marine water from 50 to 100 times For example, the effects of nitrite in

freshwater milkfish (Chanos chanos) are 56 fold higher if compared to milkfish

C chanos in marine water 16‰ (Almendras, 1987) When adding ion [Cl-] in water with 2 mg/L [NO2-], fish can survive after 5 days exposed (Jensen, 2003)

Following Kroupova et al (2010), common carp (Cyprinus caprio) exposed to

1.45 mM [NO2-], and 0.31 mmol/L [NO2], metHb was very high (90%) at 48 hr while metHb decreased to 10% when [Cl-] in water was 3.47 mmol/L According

to Costa (2008) in Trachinotus marginatus, salinity also reduced the effect of

nitrite; 96 h LC50 at two different salinities 5 and 10‰ was respectively 39.9 and

117 mg N-NO2-/L Rainbow trout (Oncorhynchus mykiss) can live well with the

ratio [Cl-]:[NO2-]=17:8 In channel catfish (Ictalurus punctatus), metHb was 80%

if cultured in the pond with [Cl-]:[NO2-]=1:1; however, metHb was 25% if cultured in the pond with [Cl-]:[NO2-]= 3:1 and fish were completely isolated from diseases with [Cl-]:[NO2-]=20:1 (Schwedler and Tucker, 1983) Milk fish in the water with [Cl-]:[NO2-] = 15:1 only generates a small amount of metHb; however, metHb reaches many fold higher with [Cl]:[NO2-] = 8:1 in the water (Almendras, 1987) No effect to rainbow trout juveniles exposed to 29.8 mg/L [NO2-], and 261 mg/L [Cl-] in 48 h was found; while fish died 58.3% in 12 h with 3.8 mg/L [NO2-], and 2.5 mg/L [Cl-] (Perone and Meade, 1977) Some studies showed that the ratio [Cl-]:[NO2-]=6:1 is safe for any nitrite concentration in channel catfish ponds (Boyd, 1998) Therefore, fish with high branchial uptake rates of chloride (such as rainbow trout, pike and perch) are more sensitive to nitrite exposure than species with low Cl- uptake rate (such as eels, tench and carp) (Williams and Eddy, 1986) Therefore, a presence of nitrite is a great concern in intensive freshwater aquaculture system and natural watercourses (Eddy and Williams, 1987; Hagreaves, 1998; Jensen, 2003)

Nitrite also causes negative effects on growth performance Following to Huong

and Vi (2013), nitrite led to the decrease of growth in snakehead Channa striata Similarly, the growth in Danio rerio was also reduced in exposure of 107 mg/L

NO2- (2.3 mM NO2-, 96 h LC50) compared to that in control treatment (Voslářová, 2008) The study of Siikavupio and Sæther (2006) showed that the growth of

snow fish Gadus morhua decreased significantly after 31 days in acute nitrite

exposure and decreased in all lower nitrite exposures in chronic period However,

there were unaffected changes in the growth of salmon Salmo gairdneri in nitrite

exposure with the concentration of 10%*96h LC50 (Wedemeyer and Yasutake,

1978) Kamstra et al., 1996) documented that nitrite concentrations under lethal

range including 0; 1; 5; 10; 20 mg/L NO2- (0; 0.07; 0.36; 0.72; 1.45 mM NO2-) had no effect to the growth of Europe eel Anguilla Anguilla because of limited absorbtion of branchial Cl- ion In addition, FCR in the fish exposed to nitrite was

siginificantly higher that in control fish (Kamstra et al., 1996; Frances et al.,

1998)

2.4.3 Hypercapnia (elevated level of carbon dioxide) and acid-base balance

Environmental hypercapnia basically occurs in tropical freshwater systems, particularly in areas covered with high density of vegetation where CO2

concentrations can increase as high as 60 mmHg (Heisler, 1982) High CO2

concentration may directly affect to fishes via acute and lethal impacts on fish life cycle, although there have been a limited number of studies about CO2

effects on aquatic animals High CO2 levels in the water will indirectly influence fishes through disturbances on the aquatic environment such as rising water temperature as well as ecosystem structure and function On the timescale of 100-200 years, shallow water fishes will be under greater impacts by CO2

diffused from the atmosphere, whereas deep sea species may be affected by high

CO2 levels when CO2 is generated into the deep sea to reduce the rapid increase

of atmospheric CO2 level (Ohsumi, 2004) Over longer timescale of 500-1000 years, the ocean CO2 will equilibrate with the atmosphere, and affecting all marine biota However, there was very limited information about the effects of

CO2 on marine fishes as compared to that on freshwater fishes, and unknown conclusion for deep sea species (Ishimatsu and Kita, 1999) Unfortunately, CO2

ocean sequestration has been increasing as a potential mitigation method in recent years, the effect of CO2 on marine organisms was feasibly discussed by this strategy (Seibel and Walsh, 2001; 2003)

Environment hypercapnia causes disturbances in acid-base and ionoregulation for cellular signaling and processes, particularly volume regulation via organ function and animal performance (Putnam and Roos, 1997) The ecosystems are featured by daily high organic leading resulting in combined hypoxia and hypercapnia with PCO2 of 65 mmHg (Furch and Junk, 1997; Ulsch, 1987) Environmental hypercapnia causes to a respiratory acidosis inside fish body, which induces a compensatory branchial transfer of acid-base equivalent ions (H+excretion and HCO3- retention) to deal with respiratory acidosis

In verterbrates, the compensation of an acid-base disturbance can be limited by some mechanisms such as (i) physicochemical buffering with bicarbonate and non-bicarbonate buffers; (ii) changes in ventilation to cope with high PCO2 and low pH via the CO2-HCO3- buffer system; or (iii) equivalence in the net transport

of acid-base between the cell and the blood compartment or the blood

compartment and the environment (Evan et al., 2005) In terrestrial air-breathers,

the second mechanism of bicarbonate buffering through ventilator changes in

blood PCO2 levels in princible plays a short-term role in acid-base regulation Differently, in water breathers, blood PCO2 is relatively low compared to that in air breathers because of high requirement of ventilation for oxygen uptake, which relates to the high CO2 capacitance of water, the ventilator changes can only partly alter CO2 tensions (Dejours, 1988) As a result, Water-breathing fishes are more sensitive to an elevation of CO2 concentration than terrestrial animals due

to the lower CO2 partial pressures (PCO2) of their body fluids (Ultsch and Jackson, 1996) Ventilation of water-breathing fishes is often depended by O2

stimuli because of the low O2 concentration in the water, and in equilibrium with air (O2 concentration in the water is 30 fold lower than that in the air at the same

PO2) (Dejour, 1988)

There have been a limited number of studies about bimodal breathing fishes up to date while acid-base and ionregulation was examined in a few species of water-

breathing fishes (Evans et al., 2005; Perry and Gilmour, 2006); Gilmour and

Perry, 2009) In water-breathing fishes, acid-base regulation mainly happens at the gills, which generally remain 90% of total acid-base ion transport during pH regulation, where kidney only accounts for 10% of that total (Heisler, 1984; Evan

et al., 2005) Acid-base and ionoregulatory disruption may result from a

consequence of air-breathing via the reduced gill, the physical difference and

water interaction between air and water by respiratory media Gonzalez et al

(2010) presented that there are some differences between water and bimodal breathers affecting acid-base and ionoregulation such as the changes in gill

morphology related to the changes in ion exchange rates (e.g pirarucu (Arapaima gigas), or a reduction in gill surface area and gill ventilation

disturbing CO2 excretion and pH compensation (e.g spottoted gar Lepisosteus oculatus) (Smatresk and Cameron, 1982) Therefore, it is presented that air-

breathing fishes may have low capacity of pH regulation with the reduced surface area of gills inducing to limitations on transepithelial ion exchange (Heisler,

1982; Brauner et al., 2004; Brauner and Baker, 2009; Harter et al., 2014; Shartau

and Brauner, 2014) The level of pH compensation changes by species, and also depends on water ionic composition, leading to slower and less complete acid-base compensation in soft and ionic poor water than in a harder and ionic rich

water (Larsen and Jensen, 1997) However, the recent study of Damsgaard et al (2015) showed that the freshwater air-breathing fish Pangasianodon hypophthalmus had extremely high capacity of acid-base regulation with

complete pH compensation during a respiratory acidosis of 34 mmHg CO2 with

plasma HCO3- elevation of 40 mM This is completely opposite to the finding of Heisler (1986), where freshwater fishes are impossible to increase plasma HCO3-

beyond 25-35 mM due to limits in the capacity for bicarbonate accumulation

The new finding is that P hypophthalmus apparently has higher capacity in pH

regulation in comparison with some other air-breathing species including the

marbled swamp eels (Synbranchus marmoratus) (Heisler, 1982), the amoured catfish (Lipocar pardalis) (Brauner et al., 2004), the South American lungfish (Lepidosiren paradoxa) (Sanchez et al., 2005), or the bowfin (Amia calva)

(Brauner and Baker, 2009)

Beside the impact to acid-base status, CO2 also caused influence to the growth in fish An explaination is that the lower growth performance might result from the

process of acid-base regulation which costs significantly energy (Evans et al.,

1999) There was slow growth rate due to the reduction in feed intake and the rise

of protein used as a fuel source (Stiller et al., 2015) Similarly, the decreases in the growth were found in some fish species in CO2 exposures such as: Atlantic

salmon Salmo salar L (Fivelstad et al., 2015), rainbow trout Oncorhynchus mykiss (Hafs et al., 2012) SGR values of these species also significant increased

in CO2 exposures compared to those in controls Mortality of salmon Salmo salar

L in the medium and high CO2 groups rose siginificant in comparion with

control, 1.1 % and 4.3%, respectively (Fivelstad et al., 1998)

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