CHAPTER 4: RESULTS AND DISCUSSIONS ...94.1 Survey on pesticide use in Can Tho city...9 4.1.1 General information...9 4.1.2 Information on Autumn-Summer crop...9 4.1.3 Technical parameter
Trang 1CAN 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
Trang 2The study was conducted at Department of Nutrition and Fisheries Processing, College of Aquaculture and Fisheries, Can Tho UniversitySupervisor: Asso.Dr Đo Thi Thanh Huong – Can Tho University
Trang 3TABLE OF CONTENTS
TABLE OF CONTENTS ii
LIST OF TABLE iv
LIST OF FIGURE iv
CHAPTER 1: INTRODUCTION 1
1.1 Introduction 1
1.2 Objective 1
1.3 Contents 2
1.4 Signification of thesis 2
1.5 New results of thesis 2
CHAPTER 2: LITERATURE 3
2.1 Chemical 3
2.2 Active of mechanism of AChE 3
CHAPTER 3: METHODOLOGY 4
3.1 Experimental site 4
3.2 Materials 4
3.3 Experimental fish 4
3.4 Experimental chemical 4
3.6 Feed 4
3.7 Methods 4
3.7.1 Survey on pesticide use in Can Tho city 4
3.7.2 Determination of LC50- 96 hrs of quinalphos on C.carp 4
3.7.3 Effects of quinalphos on respiratory physiology of C.carp 5
3.7.4 Effects of quinalphos on ChE sensitivity and inhibition threshold of common carp 5
3.7.5 Effects of quinalphos on haematological parameters, ChE activity, digestive enzymes and growth of common carp 6
3.7.6 Effects of quinalphos on cholinesterase activity and growth performances of common carp cultured in rice-field 7
3.8 Methods of analysis 7
3.8.1 Measurable method of haematological parameters 7
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 8
3.8.5 Measurable method of environmental parameters 8
3.8.6 Measurable method of quinalphos residue 8
3.9 Method of data treatment 8
Trang 4CHAPTER 4: RESULTS AND DISCUSSIONS 9
4.1 Survey on pesticide use in Can Tho city 9
4.1.1 General information 9
4.1.2 Information on Autumn-Summer crop 9
4.1.3 Technical parameters on rice-fish system 9
4.1.4 Economic efficiency of rice and rice-fish 9
4.1.5 Present status of pesticide use 9
4.1.6 Information on agents of pesticide trade 9
4.2 Acute toxicity of quinalphos insecticide on common carp 10
4.2.1 Environment parameters during the experiment 10
4.2.2 Acute toxicity of quinalphos insecticide on common carp 10
4.3 Effects of quinalphos on respiratory physiology 10
4.3.1 Oxygen consumption 10
4.3.2 Oxygen threshold 10
4.4 Effects of quinalphos on ChE sensitivity and inhibition threshold 11
4.4.1 Environment parameters during the experiment 11
4.4.2 ChE sensitivity 11
4.4.3 ChE inhibition threshold 11
4.5 Effects of quinalphos on haematological parameters, ChE activity, digestive enzymes and growth of common carp 11
4.5.1 Effects of quinalphos on haematological parameters and enzyme activities of common carp 11
4.5.2 Effects of quinalphos on haematological parameters, cholinesterase activity, digestive enzymes and growth performances of common carp cultured in tank 15
4.6 Effects of quinalphos on ChE activity and growth of common carp cultured in rice-field 18
4.6.1 Environmental paramters during the experiment 18
4.6.2 Effects of quinalphos on ChE activity 18
4.6.3 Effects of quinalphos on growth, survival and yield of common carp cultured in rice-field 18
4.7 General discussion 19
CHAPTER 4: CONCLUSIONS AND RECOMMENDATIONS .21
4.1 Conclusions 21
4.2 Recommendations 21
REFERENCES 22
iii
Trang 5LIST OF RESEARCH WORK 25
Trang 6
CHAPTER 1: INTRODUCTION 1.1 Introduction
Quinalphos is among the most widely used organophosphorusinsecticides (OPs) in agriculture in India and some other countries
among them Viet Nam (Chebbi et al., 2009)
Haematological parameters such as hemoglobin, hematocrit,erythrocyte,…can be used to find physiological reaction in fish when
environment was pollution (Dethloff et al, 2001) AChE inhibition
affected to respiration, swimming activity, feeding and aquatic animalbehaviours leading to imbalance, convulsion and even death (Peakall,1992) Digestive enzymepatterns can reflect the feeding rate anddigestive capacity of fish; hence, digestive enzyme activity can be used
as bio-indicators of growth and health status of fish (Debnath et al., 2007; Suarez et al., 1995; cited by Li et al., 2010).
Common carp Cyprinus carpio was the most common species in rice-field in Mekong Delta and Can Tho (Nguyen Van Hảo et al., 2001; Vromant et al., 2002) Quinalphos was widely used in agriculture and residue of quinalphos can affect to fish (Das et al.,
2000) In order to study effects of insecticide on physiologicalparameters, biochemical changes in tank and rice-field, this study wasconducted at College of Aquaculture and Fisheries – Can ThoUniversity
1.2 Objective
- General objectives: Search scientific data of toxicity of insecticide
on aquatic animals in order to recommend farmers suitable pecticide use
in rice-field basically
- Specific objectives: Search effects of quinalphos insecticide to
change physiological parameters (haematology and respiratoryphysiology), biochemical changes (enzyme activities) and growthperformances of common carp As a result, physiological parameters
or biochemical changes as a biomarker for organophosphorusinsecticides like quinalphos in rice-filed
1
Trang 71.3 Contents
a) Survey on pesticide use in Can tho city
b) Determination of LC50-96 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 inhibitionthreshold of common carp
e) Effects of quinalphos on haematological parameters,cholinesterase activity, digestive enzymes and growth performances ofcommon carp cultured in tank
f) ) Effects of quinalphos on cholinesterase activity, digestiveand growth performances of common carp cultured in rice-field
1.4 Signification of thesis
The results of thesis are scientific basic to recommend farmerssuitable pecticide use in rice-field
1.5 New results of thesis
Thesis determined quinalphos changed haematological parametersand respiratory physiology (oxygen consumption and threshold) Quinalphos considerably inhibited the brain, muscle, gill and liverChE 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 21days in tank and 14 days in rice-field
Thesis also determined GST activity of common carp didn’t play
an important role for decontamination Quinalphos considerablyinhibited digestive enzymes Thesis determined brain was the mostsensitive organ of common carp ChE inhibition affected to swimmingbehaviour of fish ChE inhibition threshold causing died fish was 95%.Thesis determined quinalphos affected to growth performmances,feed conversion rate and survival rate of common carp cultured in tank.Recommended dose affected to growth performmances, survival rate andproductivity of common carp as compared to the control in rice-field.Concentrations of quinalphos which gradually decreased during theexperiment were under the limit of determination (<LOD) at least 14days
Trang 9CHAPTER 2: LITERATURE
2.1 Chemical
- Trade name: Kinalux 25EC
- Chemical name: 0,0-diethyl 0–2 quinoxalin
phosphorothioate
- Active ingredient: Quinalphos
- Source of chemical: organophosphosphat
- Molecular formula: C12H15N2O3PS
- Molar mass: 298,3 g/mol
- Chemical formula:
2.2 Active of mechanism of AChE
AChE serves as a regulating agent of nervous transmission AChEcatalyzed hydrolysis of ACh into choline (Ch) and acetic acid (A) WhenAChE is inactivated by an organophosphorus, the enzyme is no longerable to hydrolyze ACh; the concentration of ACh in the junction remainshigh, 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)
Trang 10CHAPTER 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 m3, 4 m3), glass tank 84 L
- Spectrophotometer, centrifugal machine and so on
3.3 Experimental fish
Common carp (8-12 grams/individual) were selected fromhatcheries in Can Thome Fish were acclimated in composite tank atleast 7 days before experiment
3.7.1 Survey on pesticide use in Can Tho city
- Secondary data collection: The secondary data was collected
from local management office
- Primary data collection: Total of samples was 105 including
three groups: 45 rice cultivation farmers, 45 rice-fish farmers and 15agencies of pesticides in Co Do, Thoi Lai and Vinh Thanh district
3.7.2 Determination of LC 50 - 96 hrs of quinalphos on common carp
Experiment was conducted in glass tank of 50 L, 10 fish/tank,weight from 8-10 g During the experiment, no exchange of water, noaeration and no feeding was applied
Range-finding toxicity test
Experiment was designed 10 concentrations (one replicate for
each) from 0-4 mg/L The mortality was recorded in 96 hours finding toxicity was calculated to find value of LC50-96 hrs
Range-5
Trang 11LC 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 LC50-96 hrs., six replicates, 2 fish/2 L Oxygencomsumption was determined by method of closed-vessel There weretwo 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 LC50-96 hrs., six replicates, 4 fish/2 L Oxygencomsumption was determined by method of closed-vessel There weretwo cases: contaminating design before 24 hours and direct design.When observed mortality of 50% in vessel, sampling oxygen todetermine 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 factorswere sampling time and concentrations including in control, 1%, 10%,20%, 50% and 75% value of LC50-96 hrs., three replicates, 30 fish/tank
100 L Experimental time was 96 hours
b) Method of sampling
Three fish per tank were collected in 96 hours Brain was collected
to determine sensitivity of ChE Temperature, pH and oxygen wasmeasured twice daily
3.7.4.2 ChE inhibition threshold
a) Experimetal design
Treaments consisted of control, 1%, 10%, 20%, 50%, 75% and100% value of LC50-96 hrs., three replicates, 10 fish/tank 50 L.Experimental time was 96 hours
b) Method of sampling
Brain was collected when obversed abmormal swimming activitiessuch as imbalance, dull swimming and died fish to determine ChEinhibition threshhold
Trang 123.7.5 Effects of quinalphos on haematological parameters, ChE activity, digestive enzymes and growth of common carp
3.7.5.1 Effects of quinalphos on haematological parameters, and enzyme activities
a) Experimetal design
Experiment was conducted based on two way anova Two factorswere sampling time and concentrations including in control, 10%,20%, 50% and 75% value of LC50-96 hrs., six replicates, 15 fish/tank
60 L Experimental time was 28 days
b) Management of experimental fish
Renewal water was periodically carried out No aeration andsiphon 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 28after exposure Each sampling time, six fish per treatment Blood wascollected to measure haematological parameters such as erythrocyte,hemoglobin, hematocrit, MCV, MCH and MCHC Brain, muscle, gill andliver 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 (biochemicalexperiment) Two factors were sampling time and concentrations(control, 10%, 20%, 50% and 75% value of LC50-96 hrs.), threereplicates, 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 30after one exposing time; day 31 (6 hours), day 33 and day 60 after twoexposing 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 slightlyaerated Fish were fed on commercial feed (30% crude protein) based onsatiation Feed were weighted before and after feeding to determine FCR
7
Trang 13d) Recorded parameters
Fish growth were measured at day 30, 60 and 90, sampling 30 fish/tank Data was collected to determine growth parameters such asweight gain, DWG, SGR, FCR and survival rate Temperature, pH andoxygen was measured daily
3.7.6 Effects of quinalphos on cholinesterase activity and growth performances of common carp cultured in rice-field
3.7.6.1 Implementary method
Experiment was conducted based on two way anova (enzymeactivity) Two factors were sampling time and concentrations (control andrecommended dose), three replicates, 2 fish/m2 (each plot of 1.000 m2approximately Experimental time was 132 days Quinalphos insecticidewas applied twice: OM 4218 variety was used in this experiment
3.7.6.4 Recorded parameters
Fish growth were measured at day 50, 100 and 132 Sampling
20-30 fish/plot Recorded parameters were weight gain, DWG, SGR,survival rate and productivity Temperature, pH and oxygen wasmeasured 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 tototal 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).
Trang 14- 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) = Wfinal– Winitial
Specific growth rate –SGR
Daily weight gain – DWG
Wfinal : Fish weight of ending experiment
T : Experimental time (day)
Feed conversion ratio - FCR
FCR= Dry food fed/ Weight gain of fish
3.8.5 Measurable method of environmental parameters
- Temperature, pH and oxygen were measured by oxygen meter
- Ammonia was measured by method of Indophenolblue
- Oxygen content were measured by method of Winkler
3.8.6 Measurable method of quinalphos residue
Quinalphos insecticide residue was measured by method of ECD (Nguyen Quoc Thinhet al., 2012).
GC-3.9 Method of data treatment
Value of LC50was determined by method of probit One-way Anovaand Two-way Anova were used to compare the differences betweentreatments at a 5% significant level, using software SPSS 11.5
9
Trang 15CHAPTER 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 ofrice-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/m2 Cultureduration 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-fishmodel was 4.4 tấn/ha Profit of rice crop in rice-fish model was 7.7millions VND/ha and significant difference as compared to mono-rice(4,3 millions VND/ha)
4.1.5 Present status of pesticide use
The results showed that number of spray ranged from 2.7 from 2.9times/crop The most extensive spray time was from day 31 to day 60
of mono-rice (89.7%) and rice-fish (73.7%) Chess was the mostwidely used organophosphorus insecticides in mono-rice (48.9%household), next to Basa (26,7%) and Kinalux (active ingredient ofquinalphos) (20%) For rice-fish system, Basa was the most widelyused insecticides (37.8% household), Kinalux 6.7% In agents ofpesticide trade, Dragon was the most widely consumed insecticide(80%), next to Kinalux (66.7% household)
4.1.6 Information on agents of pesticide trade
There were about 133 products of pesticides which bought at 15agents in Can Tho city. The most consumingvolume of pesticide wasBasa 3,041 liters/year, next to Kinalux 1,497 liters/year
Ngo Van Ngoc et al (2001), Nguyen Van Hao et al (2001) indicated
that common carp were among the most common species in rice-field inMekong Delta According to survey on pesticide use in Can Tho city in
2007 (Tam, 2008) showed that fenobucarb (Basa),Chlorpyriphos+Cypermethrin (Dragon) and quinalphos (Methink) were
Trang 16among the most widely used insecticides The present study recorded thatquinalphos was one of the most extensively used insecticides.
4.2 Acute toxicity of quinalphos insecticide on common carp
4.2.1 Environment parameters during the experiment
Temperature in the morning was 26.9oC and in the afternoon was27.6oC; fluctuation of temperature was under 1oC pH ranged 7.8-7.9.Oxygen dissolve ranged 3.6mg/L
4.2.2 Acute toxicity of quinalphos insecticide on common carp
Mortality and value of LC50 of quinalphos on common carp werepresented in Table 4.9 After 1 hour, died fish (13,3%) appeared atconcentration of 1.8 mg/L; 80% after 3 hours and 100% after 72 hours.Value of LC50 of quinalphos on common carp (size of 8.6 g) was determined1.16, 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 96hours
Value of LC50-96 hrs of quinalphos on tilapia (Oreochromis
niloticus) was 0.84 mg/L (Đo Van Buoc, 2010) and silver barb
(Barbonymus gonionotus) was 0.856 mg/L (Tran Thien Anh 2011)
4.3 Effects of quinalphos on respiratory physiology
4.3.1 Oxygen consumption
In case of direct exposing, oxygen consumption significantlyincreased with increased concentrations, ranged 382-518 mgO2/kg fish/hour (p<0.05) In case of contamination 24 hours, oxygen consumptiontended to decrease but there was no significant difference, ranged 356-
371 mgO2/kg fish/hours (p>0.05)
4.3.2 Oxygen threshold
Oxygen threshold significantly increased with increasedconcentrations in case of direct exposing, ranged 0.13-0.45 mg/L(p<0.05) In case of contamination 24 hours, oxygen threshold
11