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
i 
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. 
ii 
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 
iii 
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. 
iv 
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. 
v 
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 
vi 
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 
vii 
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 ........................ ... , N., 1987. Blood gases and 
extracellular/intracellular acid-base status as a function of temperature in the 
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Boyd, C. E., 1990. Water Quality in Ponds for Aquaculture. Birmingham Publishing Co. 
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Damsgaard, C., Gam, L.T.H., Dang, D.T., Van Thinh, P., Huong, D.T.T., Wang, T., 
Bayley, M., 2015. High capacity for extracellular acid-base regulation in the air-
breathing fish Pangasianodon hypophthalmus. Journal of Experimental Biology. 
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the pattern for temperature-induced pH regulation in a bimodal breathing teleost. 
Journal of Comparative Physiology. Part B. (in press) 
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selected haematological parameters in fingerling, Catla catla (Hamilton). 
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Ecology, 3: 1–38. 
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certain marine teleosts. Copeia. 3: 586–587. 
Gam, L.T.H., Jensen, F.B., Damsgaard, C., Huong, D.T.T., Phuong, N.T., Bayley, M., 
2017. Extreme nitrite tolerance in the clown knifefish Chitala ornata is linked to 
up-regulation of methaemoglobin reductase activity. Aquatic Toxicology, 187: 9–
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Platichthys flesus. Aquatic Toxicology, 50: 97–107. 
Hargreaves, J.A., 1998. Nitrogen biogeochemistry of aquaculture ponds. Aquaculture, 
166: 181–212. 
Harris, R.R., Coley, S., 1991. The effects of nitrite on chloride regulation in the crayfish 
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Heisler, J. N., Weitz, H. and Weitz, A. M., 1976. Extra and intracellular pH with 
changes of temperature in the dogfish Scryliorhinus stellaris. Respiration 
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Hultquist, D.E., Passon, P.G., 1971. Catalysis of methaemoglobin reduction by 
erythrocyte cytochrome B5 and cytochrome B5 reductase. Nature: New Biology 
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Hvas, M., Damsgaard, C., Gam, L.T.H., Huong, D.T.T., Jensen, F.B., Bayley, M., 2016. 
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The effect of environmental hypercapnia and size on nitrite toxicity in the striped 
catfish (Pangasianodon hypophthalmus). Aquatic Toxicology, 176: 151–160. 
Jaffé, E.R., 1981. Methaemoglobinaemia. Clinical and Laboratory Haematology, 10: 
99–122. 
Jensen, F.B., Andersen, N.A., Heisler, N., 1987. Effects of nitrite exposure on blood 
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Jensen, F.B., Koldkjaer, P., Bach, A., 2000. Anion uptake and acid-base and ionic 
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formation. Journal of Experimental Biology, 152: 149–166. 
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hypophthalmus. Aquatic Toxicology, 104: 86–93. 
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Haematological and ion regulatory effects of nitrite in the air-breathing snakehead 
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Percy, M.J., Lappin, T.R., 2008. Recessive congenital methaemoglobinaemia: 
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Chapter 11 
CONCLUSIONS AND RECOMMENDATIONS 
11.1 Conclusions 
This thesis presents an incredible tolerance to nitrite in C. ornata with 
maintainance of metHb levels at subcritical levels with denitrification process 
converting nitrite to nitrate and nitrite concentration in the plasma maintain lower 
than the ambient nitrite level during sub-lethal time. Although there were the 
related changes in plasma osmolality, plasma ions, protein and whole body 
content in nitrite exposure, this is the first time for showing up-regulation of 
erythrocyte metHb reductase with rate constant for metHb reduction increased 
from 0.01 in controls to 0.046 min-1 after 6 days of 2.5 mM nitrite exposure. 
Growth rate of C. ornata in chronic nitrite exposure of 4 mM (50%*96 h LC50) 
was significantly lower than that in control, 0.2 mM and 0.4 mM nitrite with low 
survival rate (59%) and high FCR (4.5) after 90 days of culture. There were 
negative effects to the number of blood cells; metHb, Hct and Hb concentration 
while MCHC had no effect by these nitrite exposures after 14 days. 
This is the second time about acid-base regulation found in the air-breathing 
species C. ornata in the MD. Although pH regulation capacity in C. ornata (50% 
of pH regulation after 96 h at 21 mmHg CO2) was lower than that in the 
facultative air-breathing P. hypophthalmus (100% of pH compsentation in 72 h in 
34 mmHg CO2), this capacity of pH regulation was significantly better than that 
in other water-breathing species. In addition, acclimated hypercapnia caused a 
decreased nitrite uptake by an apparent reduced transport rate of the branchial 
HCO3-/Cl- exchanger. 
C. ornata had high tolerance of temperature with limitation of 50 % mortality 
(12ºC and 41ºC for lower and upper limit, respectively) due to their living habitat 
in the tropical area MD. Therefore, there were no significant impacts in the 
changes of haematological parameters, but pHe and PCO2 significantly fluctuated 
in the temperature ranges from 24ºC - 33ºC in both juvenile and commercial fish 
size while the mortality after 2 days exposed to 36ºC in commercial size induced 
by sharp decrease in pHe and Hb concentration. 
C. ornata had higher tolerance of nitrite at 30ºC compared to this at 27ºC and 
33ºC. In addition, the number of red blood cells was significantly influenced by 
 186 
combination of nitrite and elevated temperature while metHb and plasma NO2- 
only affected in the initial stage of exposure. In general, growth parameters such 
as GW, SR had no negative effects by elevated temperature and the combined 
exposure of nitrite and elevated temperatures, but FCR reached the highest value 
of 1.95 in 1 mM nitrite at 33ºC compared to that in 27ºC. 
11.2 Recommendations 
11.2.1 Recommendations for intensive farming systems 
From the results of dissertation, some recommendations for optimizing water 
quality and minimizing impacts to physiological processes and growth of fish in 
aquaculture ponds under climate change are showed as below: 
1) Reducing stocking density and changing water regularly for minimizing the 
accumulation of all waste products and generation of toxic gases and supplying 
sufficient levels of dissolved oxygen for the system 
2) Designing ponds with optimal sizes, outlet and inlet cannas of water for 
conveniently for regularly removing the waste products and accumulated toxic 
sediments for good water quality 
3) Supporting aeration systems in situations of elevated temperatures and high 
carbon dioxide levels for supplying sufficient dissolved oxygen in farming 
systems 
11.2.2 Recommendations for further studies 
From the new findings that we found about the impacts of nitrite, temperature and 
hypercapnia on physiological responses and growth in C. ornata in this 
dissertation, some studies are expected to conduct in the future as below: 
1) Chronic effects of nitrite on growth parameters in C. ornata after exposed to 
extremely high nitrite concentration in a short duration in C. ornata 
2) Combined effects of nitrite and hypoxia on haematological parameters, 
metabolic rate and growth in C. ornata 
3) Effects of hypercapnia at different temperatures on acid-base regulation and 
growth in C. ornata 
 187 
List of appendices 
Appendix 3.2.1. Information in the C. ornata culture ponds 
Fish size 
5-10 g/fish 200-250 g/fish 450-500 g/fish 
Area 800 m
2 600 m2 900 m2 
Depth 1.5 m 1.5 m 1.5 m 
Total stocked 
fish 
20,000 fish 4,700 fish 16,500 fish 
Stocking 
density 
25 fish/m2 7.8 fish/m2 18.75 fish/m2 
Feeding 
twice a day 
(5 kg worm+5 kg trash 
fish+2 kg commercial 
pellet)/time 
twice a day 
10 kg/time (pellet) 
Skretting, 40% protein 
once a day 
62.5 kg/time 
Skretting, 40% protein 
Water 
exchange 
Water exchanged daily by 
tide 
twice a month 
25% of water once 
4 times/month 
50% of water once 
Lime adding 
Weekly, 
2-3 kg CaCO3/100 m3 
Weekly, 
 2-3 kg CaCO3/100 m3 
Weekly, 
 2-3 kg CaCO3/100 m3 
Vitamin C 
adding 
Twice a week, 
1 – 2 g/kg feed 
Twice a week, 
1 – 2 g/kg feed 
Twice a week, 
1 – 2 g/kg feed 
 188 
Appendix 9.3: Determing the values of 96h LC50 for nitrite at 27, 30 and 
33ºC in C. ornata (SPSS analysis) 
Confidence Limits 
 At 27⁰C 30⁰C 33⁰C 
Probit 
95% Confidence Limits for nitrite 95% Confidence Limits for nitrite 95% Confidence Limits for nitrite 
Estimate 
Lower 
Bound 
Upper 
Bound Estimate 
Lower 
Bound 
Upper 
Bound Estimate 
Lower 
Bound 
Upper 
Bound 
0.01 -1.8436 -6.07391 0.530851 1.185309 0.003457 2.098659 -0.33004 -1.59976 0.64797 
0.02 -0.71076 -4.48589 1.425663 1.998829 0.938144 2.822752 0.500241 -0.64412 1.385601 
0.03 0.007997 -3.48111 1.996168 2.514981 1.529944 3.283394 1.027026 -0.03888 1.85468 
0.04 0.548688 -2.72708 2.427156 2.903262 1.974338 3.630712 1.423306 0.415732 2.20824 
0.05 0.988497 -2.11512 2.779122 3.219099 2.335218 3.913829 1.745649 0.785005 2.496352 
0.06 1.362845 -1.5954 3.079852 3.487925 2.641891 4.155298 2.020014 1.098891 2.742003 
0.07 1.691074 -1.14071 3.344535 3.723634 2.91036 4.367442 2.260578 1.373746 2.957753 
0.08 1.984964 -0.73449 3.582427 3.934683 3.150367 4.557767 2.475975 1.619527 3.151251 
0.09 2.252245 -0.36587 3.799609 4.126623 3.3683 4.731204 2.671869 1.842763 3.32752 
0.1 2.498278 -0.02734 4.000303 4.303305 3.56859 4.89117 2.85219 2.047984 3.490045 
0.15 3.51692 1.364403 4.841097 5.034813 4.393912 5.557406 3.598768 2.894342 4.166253 
0.2 4.326505 2.455063 5.524789 5.616193 5.043949 6.092812 4.192125 3.562057 4.708626 
0.25 5.021056 3.374774 6.127313 6.114965 5.595893 6.557873 4.701172 4.130096 5.178733 
0.3 5.644785 4.183205 6.685897 6.562878 6.085693 6.981374 5.158312 4.635259 5.605859 
0.35 6.222762 4.912657 7.223188 6.977936 6.533424 7.379953 5.58192 5.098083 6.006939 
0.4 6.771206 5.582601 7.755259 7.371785 6.951804 7.764639 5.983883 5.53153 6.393252 
0.45 7.301832 6.206027 8.294797 7.752839 7.349815 8.143602 6.372787 5.944668 6.773241 
0.5 7.824045 6.792854 8.852494 8.127851 7.734532 8.523542 6.755525 6.344535 7.153927 
0.55 8.346258 7.352055 9.437818 8.502864 8.112194 8.910536 7.138263 6.737253 7.541762 
0.6 8.876884 7.893005 10.05983 8.883917 8.488962 9.310743 7.527166 7.128844 7.943299 
0.65 9.425328 8.426332 10.72852 9.277767 8.871602 9.731168 7.929129 7.525976 8.365927 
0.7 10.00331 8.964695 11.4569 9.692825 9.268329 10.18075 8.352737 7.936863 8.818944 
0.75 10.62703 9.524168 12.26444 10.14074 9.690188 10.67219 8.809877 8.372692 9.315403 
0.8 11.32159 10.12742 13.18342 10.63951 10.15381 11.22558 9.318925 8.850428 9.875815 
0.85 12.13117 10.81171 14.27349 11.22089 10.68795 11.87688 9.912281 9.399476 10.53685 
0.9 13.14981 11.65301 15.66473 11.9524 11.35307 12.70332 10.65886 10.08161 11.37728 
0.91 13.39584 11.85379 16.00317 12.12908 11.51282 12.90382 10.83918 10.24526 11.58138 
0.92 13.66313 12.07106 16.37169 12.32102 11.68604 13.12197 11.03507 10.42265 11.8035 
0.93 13.95702 12.30904 16.77783 12.53207 11.87616 13.36219 11.25047 10.61725 12.04818 
0.94 14.28525 12.57382 17.23243 12.76778 12.08809 13.63087 11.49104 10.83411 12.32192 
0.95 14.65959 12.87463 17.75206 13.0366 12.32935 13.93775 11.7654 11.08088 12.63469 
0.96 15.0994 13.22669 18.36393 13.35244 12.61225 14.29885 12.08774 11.37015 13.00281 
0.97 15.64009 13.65777 19.11787 13.74072 12.95934 14.74347 12.48402 11.72492 13.45621 
0.98 16.35885 14.22837 20.12255 14.25687 13.41973 15.33553 13.01081 12.19532 14.06012 
0.99 17.49169 15.1233 21.71045 15.07039 14.14351 16.27052 13.84109 12.93455 15.01416 
 189 
List of pictures about experimental setup, blood sampling and devices of 
analysis used in the studies 
 190 
 191 
 192