CAN THO UNIVERSITY
COLLEGE OF AQUACULTURE AND FISHERIES
NGUYEN QUANG TRUNG
EFFECTS OF QUINALPHOS INSECTICIDE ON
PHYSIOLOGY, BIOCHEMICAL CHANGES
AND GROWTH OF COMMMON CARP
(Cyprinus carpio Linnaeus, 1758)
Major: AQUACULTURE
Code: 62.62.03.01
ABSTRACT OF THESIS OF DOCTOR DEGREE OF
AQUACULTURE

Can Tho, November 2013
1
The study was conducted at Department of Nutrition and Fisheries
Processing, College of Aquaculture and Fisheries, Can Tho University
Supervisor: Asso.Dr. Đo Thi Thanh Huong – Can Tho University
Commentator 1:
Commentator 2:
Commentator 3:
Thesis will be defensed at final thesis commentary council at:
………………………………………………………………………
At: o’clock date
i
TABLE OF CONTENTS
AT: O’CLOCK DATE I
TABLE OF CONTENTS II
LIST OF FIGURE VI
VI
CHAPTER 1: INTRODUCTION 1
1.1 Introduction 1

75% value of LC50-96 hrs., six replicates, 4 fish/2 L. Oxygen
comsumption was determined by method of closed-vessel. There were
two cases: contaminating design before 24 hours and direct design.
When observed mortality of 50% in vessel, sampling oxygen to
determine oxygen threshold 6
3.7.4 Effects of quinalphos on ChE sensitivity and inhibition threshold of
common carp 6
3.7.5 Effects of quinalphos on haematological parameters, ChE activity,
digestive enzymes and growth of common carp 7
3.7.5.1 Effects of quinalphos on haematological parameters, and enzyme
activities 7
3.7.6 Effects of quinalphos on cholinesterase activity and growth
performances of common carp cultured in rice-field 8
3.7.6.1 Implementary method 8
3.8 Methods of analysis 8
3.8.1 Measurable method of haematological parameters 8
3.8.2 Measurable method of oxygen consumption 8
3.8.3 Analytic method of biochemical parameters 8
3.8.4 Measurable methods of growth parameters 9
iii
3.8.5 Measurable method of environmental parameters 9
3.8.6 Measurable method of quinalphos residue 9
3.9 Method of data treatment 9
VALUE OF LC50 WAS DETERMINED BY METHOD OF
PROBIT. ONE-WAY ANOVA AND TWO-WAY ANOVA
WERE USED TO COMPARE THE DIFFERENCES
BETWEEN TREATMENTS AT A 5% SIGNIFICANT
LEVEL, USING SOFTWARE SPSS 11.5 9
CHAPTER 4: RESULTS AND DISCUSSIONS 10
4.1 Survey on pesticide use in Can Tho city 10

activity, digestive enzymes and growth performances of common carp
cultured in tank 16
4.5.2.1 Environmental parameters and quinalphos residue 16
4.5.2.2 Effects of quinalphos on haematological parameters 16
4.5.2.3 Effects of quinalphos on ChE activity and digestive enzymes
activities of common carp 17
4.6 Effects of quinalphos on ChE activity and growth of common carp
cultured in rice-field 19
4.6.1 Environmental paramters during the experiment 19
4.6.2 Effects of quinalphos on ChE activity 19
4.6.3 Effects of quinalphos on growth, survival and yield of common
carp cultured in rice-field 19
4.7 General discussion 20
CHAPTER 4: CONCLUSIONS AND
RECOMMENDATIONS 22
4.1 Conclusions 22
4.2 Recommendations 22
REFERENCES 23
LIST OF RESEARCH WORK 27
LIST OF TABLE
Table 4.9: Survival of common carp exposing to quinalphos 10
Table 4.25: Growth of common carp …………………………. 17
v
LIST OF FIGURE
Figure 2.3: Active mechanism of AChE 3
Figure 4.1: Changes of brain ChE activity after 28 days ………. 12
Figure 4.2: Brain ChE inhibition after 28 days………………… 12
Figure 4.3: Changes of muscle ChE activity after 28 days ……. 12
Figure 4.4: Muscle ChE inhibition after 28 days ………………. 12
Figure 4.5: Changes of gill ChE activity after 28 days………… 13

change physiological parameters (haematology and respiratory
physiology), biochemical changes (enzyme activities) and growth
performances of common carp. As a result, physiological parameters
or biochemical changes as a biomarker for organophosphorus
insecticides like quinalphos in rice-filed.
1
1.3 Contents
a) Survey on pesticide use in Can tho city.
b) Determination of LC
50
-96 hrs. of quinalphos on common carp.
c) Effects of quinalphos on respiratory physiology of common
carp Cyprinus carpio.
d) Effects of quinalphos on ChE sensitivity and inhibition
threshold of common carp.
e) Effects of quinalphos on haematological parameters,
cholinesterase activity, digestive enzymes and growth performances of
common carp cultured in tank.
f) ) Effects of quinalphos on cholinesterase activity, digestive
and growth performances of common carp cultured in rice-field.
1.4 Signification of thesis
The results of thesis are scientific basic to recommend farmers
suitable pecticide use in rice-field.
1.5 New results of thesis
Thesis determined quinalphos changed haematological parameters
and respiratory physiology (oxygen consumption and threshold).
Quinalphos considerably inhibited the brain, muscle, gill and liver
ChE activities of common carp exposed to sublethal concentrations in
96 hours of acute test and 28 days and 60 days of long test in tank and
44 days in rice-filed. ChE activity was completly recovered at least 21

PS
- Molar mass: 298,3 g/mol
- Chemical formula:
2.2 Active of mechanism of AChE
AChE serves as a regulating agent of nervous transmission. AChE
catalyzed hydrolysis of ACh into choline (Ch) and acetic acid (A). When
AChE is inactivated by an organophosphorus, the enzyme is no longer
able to hydrolyze ACh; the concentration of ACh in the junction remains
high, and continuous stimulation of the muscle or nerve fiber occurs,
resulting eventually in muscle twitching, tremors or exhaustion
Figure 2.3: Active mechanism of AChE (A) and mechanism
of inhibition of organophosphorus pesticide (B)
(Richard and David, 2008)
4
CHAPTER 3: METHODOLOGY
3.1 Experimental site
The study was carried out at College of Aquaculture and Fisheries,
Cantho University and Thoi Hung commune, Co Do district, Can Tho
city from May 2009 to July 2012.
3.2 Materials
The study used main materials such as:
- Composite tank (capacity of 500 L, 2 m
3
, 4 m
3
), glass tank 84 L.
- Spectrophotometer, centrifugal machine and so on.
3.3 Experimental fish
Common carp (8-12 grams/individual) were selected from
hatcheries in Can Thome. Fish were acclimated in composite tank at

LC
50
-96 hrs. definitive test
Experiment was designed 7 concentrations (three replicates for
each) from 0-1,8 mg/L. The mortality was recorded in 96 hours.
Temperature, pH and oxygen were measured twice daily.
3.7.3 Effects of quinalphos on respiratory physiology of common carp
3.7.3.1 Oxygen comsumption
Experiment included in five treatments: control 10%, 20%, 50%
and 75% value of LC
50
-96 hrs., six replicates, 2 fish/2 L. Oxygen
comsumption was determined by method of closed-vessel. There were
two cases: contamination design before 24 hours and direct design.
3.7.3.2 Oxygen threshold
Experiment included in five treatments: control 10%, 20%, 50%
and 75% value of LC
50
-96 hrs., six replicates, 4 fish/2 L. Oxygen
comsumption was determined by method of closed-vessel. There were
two cases: contaminating design before 24 hours and direct design.
When observed mortality of 50% in vessel, sampling oxygen to
determine oxygen threshold.
3.7.4 Effects of quinalphos on ChE sensitivity and inhibition
threshold of common carp.
3.7.4.1 ChE sensitivity
a) Experimetal design
Experiment was conducted based on two way anova. Two factors
were sampling time and concentrations including in control, 1%, 10%,
20%, 50% and 75% value of LC

b) Management of experimental fish
Renewal water was periodically carried out. No aeration and
siphon daily were applied. Fish were feeding on satiation.
c) Method of sampling
- Fish were sampled before exposure (day 0), day 1, 7, 14, 21 and 28
after exposure. Each sampling time, six fish per treatment. Blood was
collected to measure haematological parameters such as erythrocyte,
hemoglobin, hematocrit, MCV, MCH and MCHC. Brain, muscle, gill and
liver was collected to measure ChE, GST, CAT and LPO activities.
- Temperature, pH and oxygen was measured daily.
3.7.5.2 Effects of quinalphos on haematological parameters, ChE,
activity, digestive enzymes and growth performances of comon carp
a) Experimetal design
Experiment was conducted based on two way anova (biochemical
experiment). Two factors were sampling time and concentrations
(control, 10%, 20%, 50% and 75% value of LC
50
-96 hrs.), three
replicates, 80 fish/tank 380 L. Experimental time was 90 days.
Quinalphos insecticide was applied twice.
b) Method of sampling
Fish were collected at day 0 (before exposure), 6 hours, day 3, day 30
after one exposing time; day 31 (6 hours), day 33 and day 60 after two
exposing time. Blood was collected to measure haematological parameters
(erythrocyte, hemoglobin, hematocrit, MCV, MCH and MCHC).
Brain, muscle, gill and liver was collected to measure ChE activity.
Gut was collected to determine digestive enzymes (trypsin,
chymotrypsin, alpha-amylase). Each time collected 5 fish/tank.
c) Management of experimental fish
Renewal water was periodically carried out. Tanks were slightly

30 fish/plot. Recorded parameters were weight gain, DWG, SGR,
survival rate and productivity. Temperature, pH and oxygen was
measured once a week. Ammonia was measured twice a month.
3.8 Methods of analysis
3.8.1 Measurable method of haematological parameters
- Erythrocyte was counted by Neubauer method.
- Blood parameters related to erythrocyte (MCV, MCH, MCHC)
were measured by Svobodova et al .(1991).
- Hemoglobin was measured by spectrophotometer 540 nm.
- Hematocrit was measured by percent of plasma as compared to
total of volume
3.8.2 Measurable method of oxygen consumption
Oxygen consumption was measured by Đo Thi Thanh Huong, 1997:
3.8.3 Analytic method of biochemical parameters
- Cholinesterase (ChE) was determined by Ellman et al. (1961).
8
- Catalase (CAT) was determined by Baudhuin et al. (1964).
- Glutathione S-transferase (GST) was determined by Habig et al. (1974).
- Lipid peroxidation (LPO) was determined by Fatima et al. (2000).
- Digestive enzymes: Trypsin activity was determined by Tseng et
al. (1982). Chymotrypsin activity was determined by Worthington
(1982). Alpha-amylase was determined by Bernfeld (1951).
- Protein was determined by Lowry et al. (1951) and Bradford (1976).
3.8.4 Measurable methods of growth parameters
Weight gain – WG
WG(g) = W
final
– W
initial
Specific growth rate –SGR

- Oxygen content were measured by method of Winkler.
3.8.6 Measurable method of quinalphos residue
Quinalphos insecticide residue was measured by method of GC-
ECD (Nguyen Quoc Thinh et al., 2012).
3.9 Method of data treatment
Value of LC
50

was determined by method of probit. One-way Anova
and Two-way Anova were used to compare the differences between
treatments at a 5% significant level, using software SPSS 11.5.
9
CHAPTER 4: RESULTS AND DISCUSSIONS
4.1 Survey on pesticide use in Can Tho city
4.1.1 General information
Area of rice cultivation was 1.93 ha (70% of total area), area of
rice-fish was 2.02 ha (75%).
4.1.2 Information on Autumn-Summer crop
OM 2514 was the most common variety (26,7%). Most of farmers
began Autumn-Summer crop from Lunar March to June (88,9%). Rice
growth duration was 95 days.
4.1.3 Technical parameters on rice-fish system
Common carp was the most common species in rice-fish system
(100% household). Density of fish was 0.42±0.25 con/m
2
. Culture
duration was 149 days. Fish productivity was 577±322 kg/ha.
4.1.4 Economic efficiency of rice and rice-fish
Productivity of mono-rice was 4.5 tons/ha and rice crop in rice-fish
model was 4.4 tấn/ha. Profit of rice crop in rice-fish model was 7.7

o
C; fluctuation of temperature was under 1
o
C. pH ranged 7.8-7.9.
Oxygen dissolve ranged 3.6

mg/L.
4.2.2 Acute toxicity of quinalphos insecticide on common carp
Mortality and value of LC
50
of quinalphos on common carp were
presented in Table 4.9. After 1 hour, died fish (13,3%) appeared at
concentration of 1.8 mg/L; 80% after 3 hours and 100% after 72 hours.
Value of LC
50
of quinalphos on common carp (size of 8.6 g) was determined
1.16, 0.76 and 0.76 mg/L after 24, 72 and 96 hours, respectively.
Table 4.9: Mortality of common carp exposed to quinalphos in 96 hours
Time
(hour)
Concentrations of quinalphos (mg/L)
LC
50
(mg/L)
0
0.2 0.5 0.8 1.1 1.5 1,8
1 0 0 0 0 0 0 13.3
3 0 0 0 0 0 23.3 83.3
24 0 0 13.3 26.7 36.7 56.7 90 1.16 (0.96-1.44)
72 0 0 23.3 53.3 70 93.3 100 0.76 (0.66-0.85)

4.4.1 Environment parameters during the experiment
pH in the morning and afternoon was 6.8±0.1 and 6,9±0,1,
respectively. Temperature in the morning and afternoon was 25.4±0.1
o
C
and 26.9±0.1
o
C, respectively. Oxygen dissolve ranged 5.9-6.3 mg/L.
4.4.2 ChE sensitivity
The study showed that brain ChE activity decreased with increased
concentrations and time. At 0.0076 mg/L, ChE activity was significantly
inhibited (25%) as compared to the control (p<0.05). The most inhibited
ChE activity was recorded at 0.57 mg/L (91,6%). During the experiment,
ChE inhibition was 74.8% and there was no sign of complet recovery as
compared to the control after 96 hours (p<0.05).
4.4.3 ChE inhibition threshold
The present study reported that ChE inhibition affected swimming
behaviours such as imbalance (inhibited 82.8%), dull swimming
(91.8%) and died fish (95.1%).
Fleming and Grue (1981) reported that brain AChE activity
decreasing 20% as compared to the control considered as fish exposing
to organophosphorus insecticide. The relation between ChE inhibition
and dead fish was unknown because some species could live when brain
ChE inhibition was 90-95% (Fulton and Key, 2001; Ferrari et al., 2004).
ChE inhibition causing change of behaviours might affect to fish
survival in experimetal condition (Ferrari et al., 2007).
4.5 Effects of quinalphos on haematological parameters, ChE
activity, digestive enzymes and growth of common carp
4.5.1 Effects of quinalphos on haematological parameters and
enzyme activities of common carp

ChE activity had a sign of incompletly recovery at all concentratons Figure 4.3: Changes of muscle ChE activity Figure 4.4: Muscle ChE inhibition
a
a
a
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bc
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0

abc
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de
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bc
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defg
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0
20
40
60
80
100
120
140
0 1 4 28
Experimental time (day)
nmol/min/mg protein
0 mg/L

77.5 and 61.3%, respectively. As a result, brain ChE inhibition was the
highest or brain was the most sensivtive organ of common carp exposed
to quinalphos.
b) Glutathione-S-transferase activity (GST)
- Brain: GST activity (140-212 nmol/min/mg protein) at 0.57
mg/L was significant difference as compared to other concentrations.
After 1 day, GST activity significantly increased as compared to the
control. GST activity completely recoverd after 14 days.
- Muscle: GST activity was no significant difference at all
concentrations and ranged 32-41 nmol/min/mg protein. After 28 days,
GST activity didn’t significantly changed (32-40 nmol/min/mg protein).
- Gill: GST activity was no change at all concentrations, ranged
101-122 nmol/min/mg protein. After 28 days, GST activity had no
significant difference (p>0.05).
ab
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0,38 mg/L
0,57 mg/L
14
- Liver: GST activity was the highest and ranged 559-630
nmol/min/mg protein. There was no significant difference of GST
activity during the experiment.
c) Catalase activity (CAT)
The present study presented that brain and gill CAT activity had a
tendency to increase significantly; liver CAT was decreased at 0.57
mg/L as compared to the control (p<0.05). Meanwhile, quinalphos
didn’t affect to muscle CAT activity (p>0.05).
d) Lipid peroxidation activity (LPO)
- Brain: LPO activity at concentrations fluctuated 162-179 nmol/g
protein (p>0.05). After 4 days, brain LPO activity was significantly
decreased (p<0.05). From day 7 to ending the experiment, brain LPO
completely recoverd as compared to the control (p>0.05).
- Muscle: Muscle LPO activity at 0.57 mg/L significantly differed as
compared to other concentrations (p<0.05). After 21 days, muscle LPO
activity was sinificant differrence (p<0.05) and after 28 days, muscle LPO
had a sign of complet recovery as compared to the control (p>0.05).
- Gill: Gill LPO activity was significantly increased at 0.57 mg/L
as compared to other concentrations (p<0.05). After 4 days, gill LPO
was significantly increased and completely recoverd as compared to
the control after 28 days (p>0.05).
- Liver: After 7 days, liver LPO was significanly increased as
compared to the control (p<0.05). After 28 days, liver LPO had a sign
of complet recovery as compared to the control (p>0.05).
Tran Thien Anh (2012) reported that quinalphos considerably
decreased brain, muscle and gill of silver barb Barbonymus gonionotus. Đo
Van Buoc (2010) stated brain GST of tilapia Oreochromis niloticus

ranging 1.7-2 million cells/mm
3
. After 3 days, erythrocyte significantly
decreased as compared to the control (p>0,05). After 30 days, erythrocyte
had a tendency of complete recovery (p>0.05). At the second exposing
time, erythrocyte tended to decrease but there was no significant
difference as compared to the control (p>0.05).
- Hemoglobin: Hemoglobin content was no change at concentrations
(p>0.05). After 3 days, hemoglobin was decreased at both of two exposing to
insecticide but no significant change was recorded (p>0.05).
- Hematocrit: Hematocrit was significantly decreased at most of
concentrations as compared to the control (p<0.05). After 3 days,
hematocrit was significantly decreased as compared to the control
(p<0.05). Hematocrit completely recoverd as compared to the control
after 30 days (p>0.05).
- MCV: MCV (ranging 177-198 fl) tended to increase with
increased concentrations but there was no significant change as
compared to the control (p>0.05). During the expriment, MCV didn’t
significantly change (p>0.05), ranging 159-210 fl.
- MCH: MCH (41-56 pg) significantly increased with increased
concentrations as compared to the control (p<0.05). After 3 days,
MCH (62 pg) significantly increased and complete recovery as
compared to the control after 30 days (p>0.05).
16
- MCHC: MCHC (26-30 g/dL) was significantly increased at all
concentrations as compared to the control (p<0.05). After 3 days,
MCHC was no significant difference (p>0.05). After 6 hours at the second
exposing time, MCHC was significantly decreased (p<0.05). MCHC was
no complet recovery as compared to the control after 60 days (p<0.05).
4.5.2.3 Effects of quinalphos on ChE activity and digestive enzymes

the most sensivtive organ of common carp exposing quinalphos.
b) Digestive enzymes activities
17
- Trypsin: Trypsin activity (12-22 mU/min/mg protein) was
significatly inhibited 20-45% at all concentrations as compared to the
control (p<0.05). After 30 days, trypsine activity (21-28 mU/min/mg
protein) completely recovered as compared to the control (p>0.05).
However, trypsin activity (17-28 mU/min/mg protein) had a sign of
incomplete recovery after 60 days.
- Chymotrypsin: Chymotrypsin activity (881-1.058 mU/min/mg
protein) insignificantly decrease at all concentrations as compared to
the control (p>0.05). After 60 days, chymotrypsin activity was no
change, ranging 897-1.190 mU/min/mg protein (p>0.05).
- Alpha-amylase: Alpha-amylase activity (108-148 mU/phút/mg
protein) was significantly inhibited 21-27% at all concentrations as
compared to the control (p<0.05). After 6 hours at both of two exposing
time, alpha-amylase activity was significantly inhibited 48-50%.
Alpha-amylase activity incompletely recovered after 30 days and 60
days (p<0.05).
Nguyen Thi Kim Ha et al. (2012) realized that trypsin, chymotrypsin
and alpha-amylase activity of silver barb Barbonymus gonionotus
exposing to quinalphos was significantly inhibited after 6 hours. Tran
Thien Anh (2012) reported that quinalphos considerably affected to
growth parameters of silver barb Barbonymus gonionotus.
4.5.1.4 Growth, survival and FCR
Fish weight at the end of experiment (18.9 g/con), day weight gain
(DWG) (0.12 g/ngày) and specific growth rate (SGR) (1,26%/ngày) at
0.57 mg/L were the lowest and significant difference as compared to
the control (p<0.05).
Table 4.25: Growth of common carp

a
0.38 10.51±0.56
a
20.98±0.79
bc
0.15±0.02
b
1.55±0.16
a
0.57 10.52±0.36
a
18.89±1.60
c
0.12±0.01
b
1.26±0.06
a
Values represent mean ±standard deviation
Vaues in a column with the same letter (a, b, c) are not different to each other (p>0.05)
During 90 days, the highest survival rate was at the control (98.8%) and
significant difference as compared to other treatments (p<0.05), next to at
0.076 mg/L (89.2%), 0.152 mg/L (73.3%), 0.38 mg/L (67.5%) and the
18