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Occurrence and Distribution of Nemacur Residues in
Soil, Water, and cucumber Plant in the Middle
Governorate, Gaza Strip, Palestine
الخيار نباتت النيماكور في التربة والماء و تواجد وتوزيع متبقيا في المحافظة الوسطى، قطاع غزة، فمسطين.
Mohammed Ouda AL-Louh
Supervised by
Dr. Yasser El-Nahhal
Associat Prof. of Pesticides
Chemistry and Toxicology
Dr. Said AL-kurdi
Assisstant Prof. of Inorganic
Chemistry
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree
of Master in Environmental Science – Environmental
Monitoring and Management
October/ 2016
سةــغ – تــالميــــــت اإلســـــــــبمعـالج
شئون البحث العلمي والدراسبث العليب
ـــــومـــــت العـلـــــــــــــــــــــــــــــليـك
تـــوم البيئيــــــــمبجستيـــــــر العلــــ
تـاإلدارة والمراقبةةةةةةةةةةةةةةةةت البيئيةةةةةةةةةةةةةةةة
The Islamic University–Gaza
Research and Postgraduate Affairs
Faculty of Science
Master of Environmental Sciences
Environmental Monitoring and Management
III
Abstract
Agricultural activities in the Gaza Strip have been associated with excessive and
uncontrolled use of dozens of pesticides. Application of Nemacur provided good
distribution of residues in soil to the plant tissue, and leaching to ground water. The
objectives of this study were to determine the concentration of Nemacur in cucumber
fruits, cucumber plants, soil matrix and water filtrate, and to compare methods of
Nemacur detection. Nemacur was applied in cucumber seedlings of different rates
(0.0, 0.5F, 1F, 2F). Plants were irrigated with regular water. The filtrate water was
collected in special bottles. Cucumber fruit's, plants, water and soil matrix were
collected during the study and analyzed for Nemacur concentration. Chemo-assay
and bio-assay methods were used. Results showed that considerable concentrations
of Nemacur were determined in cucumber fruits', cucumber plants, filtrate water and
soil matrix. Concentration of Nemacur was higher in water samples collected from
sandy soil(0.0072mg/L) than from clay soil(0.0034mg/L), and the concentrations in
sandy soil(0.00023mg/kg),were lower than those in clay soil(0.0013mg/kg). The
concentration in top layer in clay soil 1.87mg/kg was lower than other layers. And
the concentration in cucumber fruits by sandy soil(0.04mg/kg) were lower than those
in cucumber fruits by clay soil(0.80mg/kg). Concentration of Nemacur was higher in
cucumber plant by clay soil(2.02mg/kg) than sandy soil(1.3mg/kg). Nemacur
residues does not exist and none of traces in the six random samples by using HPLC
technique, were collected randomly from the market. Chemo-assay techniques
determined lower concentration of Nemacur than bioassay techniques. It can be
concludes that considerable concentration of Nemacur was found in all tested
samples. Comparing with maximum residues limits (MRLS), The concentration of
Nemacur in various environmental samples less than the maximum residues limits.
Application of Nemacur is a potential health risk. It is recommended to exclude
Nemacur application from cucumber and related crops.
Keywords: Nemacur, Fenamiphos, OP, HPLC, AchE, Colorimetric, Deir Al-Balah,
middle Governorate, Palestine
IV
ممخص الدراسة
انبذاث ي نهؼششاث انؼشائ انفشط بالسخخذاوب غضة لطبع ف انضساػت األشطت اسحبطج
انببحت، األسدت إن انخشبت ف انخبمبث ي خذ حصغ انبكس حطبك شف. انسششت
كس ف ثبس انخبس، ب حسذذ حشاكض ان ،أذاف ز انذساست. اندفت انب إن انششر
نهكشف ستانكبئت ان ، يمبست ب انطشقشاشستانانخبس، طبمبث انخشبت، انب ببث
ببث انخبس بؼذالث ألم يسبت ضؼف انخشكض كس ػه بكس. حى حطبك انبػ ان
حى ،سج انببحبث ببنب انؼبدت (0.5F, 1F, 2F ,0.0).انط ب ي صاسة انضساػت
طبمبث انخشبت خالل ،ببث انخبس ،ثبس انخبس، خغ انبء انشاشر ف صخبخبث خبطت
أظشث .اسخخذيج طشق انفسض انكبئ انس .كسبانذساست نخسذذ حشكض ان
،انبء انشاشر ،انببحبث ،كس زذدث ف ثبس انخبسبي ان يؼخبشةانخبئح أ حشكضاث
0.0072 كس ف انبء انشاشر ي خالل انخشبت انشيهتبحشاكض ان .طبمبث انخشبت انخخبنت
كبج ،يهدشاو/نخش 0.0034 ر ي خالل انخشبت انطتكب أػه ي انبء انشاش يهدشاو/نخش
ت حهك انخدة ف انخشبت انطألم ي ،يهدشاو/نخش 0.00023 انخشكضاث ف انخشبت انشيهت
1.87، حشاكض انبكس ف انطبمت انؼهب نهخشبت انطت )سطر انخشبت(يهدشاو/نخش0.0013
يهدشاو /نخش باسطت انخسهم انس كبج ألم ي حشاكض انبكس ف انطبمبث األخش .
م ي انخبمبث يهدشاو/نخش أل 0.04حشاكض انبكس ف ثبس انخبس ي خالل انخشبت انشيهت
يهدشاو/نخش، كزنك حشاكض انبكس ف ببث 0.80انخشبت انطت ي خاللف ثبس انخبس
ف ببث انخبس ي انبكسحشاكضش ألم ي يهدشاو/نخ 1.3 انخبس ي خالل انخشبت انشيهت
ي خالل انخسهم انكبئ نؼبث ثبس انخبس انخ حى يهدشاو /نخش. 2.02 خالل انخشبت انطت
ي انسق انشكض ف دش انبهر نى ظش أ يخبمبث نهبكس. (6ػشائب ػذدب ) خؼب
أ خخى ك ،كس ألم ي حمبث انفسض انسبحمبث انفسض انكبئ زذد حشاكض ان
بمبست انخشكضاث انسظم ػهب يغ كس يخد ف خغ انؼبث انخ حى فسظبببأ ان
، كبج حشاكض انبكس ف انؼبث انبئت (MRLSلت انسذ األػه انسذ ب ػبنب )
سبب يخبطش بكس لذفئ اسخخذاو ان انخخهفت ألم ي انسذ األلظ انسذ ب ػبنب،
.انسبطم راث انظهت ،انخبس كس ف صساػتبف انسخسس اسخبؼبد حطبمبث ان ،طست
V
رب يئ ىئ نئ مئ زئ رئ ٹٱٹٱٱ مث زث رث يت ىت نت مت زت رت يب ىب نب مب زب اك يق ىق يف ىف ىثيث نث
[205-204:انبمشة ]
VI
Dedication
To my parents whom I owe everything since I was born.
* * * * *
To my wife who supported me at all stages of my study.
* * * * *
To my beloved sons Yazan, Kenan, and beloved daughter Faten who have been a
great source of motivation and inspiration to me.
* * * * *
To my brothers, sisters, friends, and all those who believe in the richness of learning.
VII
Acknowledgment
First of all, I praise Allah for blessings and guidance in fulfilling this goal. I would
like to express my sincere gratitude to my supervisors Dr. Yasser EL-Nahhal
Associate professor, Environmental Chemistry of Pesticides, Environmental and
Earth Science Department- Faculty of Science- Islamic University, Gaza strip,
and Dr. Said AL- Kurdi Assistant Professor, Chemistry Department, and Faculty of
Science, Co- Supervisor.
I would like to express gratitude to the Palestinians Relief and Development Fund
( Interpal ) – Award of Scholarships for Post-Graduate Students In The Gaza Strip
and supervised by Research & Graduate Affairs at the Islamic University – Gaza, for
financially supporting this work.
Mohammed AL-Louh
VIII
Table of Contents
Declaration ................................................................................................................... II
Abstract ....................................................................................................................... III
Dedication .................................................................................................................. VI
Acknowledgment ...................................................................................................... VII
Table of Contents ..................................................................................................... VIII
List of Tables ............................................................................................................... X
List of Figures ............................................................................................................ XI
List of Abbreviations ................................................................................................ XII
Chapter (1) Introduction ........................................................................................ 2
1.1 Overview .................................................................................................................... 2
1.2 Environmental Fate of Pesticides ........................................................................... 3
1.3 Pesticides in Palestine .............................................................................................. 3
1.4 Nemacur (Fenamiphos) .......................................................................................... 4
1.5 Health Effect ............................................................................................................. 5
1.6 Problem Identification ............................................................................................. 7
1.7 Objectives: ................................................................................................................. 7
1.7.1 General Objectives: ..................................................................................... 7
1.7.2 Specific Objectives: ..................................................................................... 7
Chapter (2) Literature Review .............................................................................. 9
2.1 Introduction ............................................................................................................... 9
2.2 Environmental Fate of Nemacur ........................................................................... 10
2.2.1 Nemacur Fate in Soil ................................................................................. 10
2.2.2 Hydrolysis and Photolysis of Nemacur ..................................................... 12
2.2.3 Nemacur Fate in Water ............................................................................. 13
2.2.4 Nemacur Fate in plant ............................................................................... 13
2.2.5 Nemacur Fate in Air .................................................................................. 14
2.3 Toxicity of Nemacur .............................................................................................. 14
2.4 Eco-toxicity of Nemacur........................................................................................ 15
2.5 Health Effects on Humans ..................................................................................... 16
2.6 Inhibition of AchE by Nemacur ........................................................................... 16
Chapter (3) Material and Methods .................................................................... 19
3.1 Study area ................................................................................................................ 19
3.1.1 Planting ...................................................................................................... 20
3.2 Chemo assay technique .......................................................................................... 20
3.2.1 Chemicals .................................................................................................. 20
IX
3.2.2 Soil collection ............................................................................................ 21
3.2.3 Water collection ........................................................................................ 21
3.2.4 Cucumber fruits and plant collection ........................................................ 22
3.2.5 Cucumber fruits samples from the central market in Deir Al-Balah. ....... 22
3.2.6 HPLC-Measurement .................................................................................. 22
3.2.7 Standard Curve of Nemacur ...................................................................... 23
3.3 Bio assay technique ................................................................................................ 23
3.3.1 Tested Organisms ...................................................................................... 23
3.3.2 Assay procedures (Colorimetric measurement) ........................................ 24
3.4 Statistical Analysis: ................................................................................................ 25
Chapter (4) Results and Discussion.................................................................... 27
4.1 Results: ..................................................................................................................... 27
4.1.1 The spectrum of Nemacur ......................................................................... 27
4.1.2 HPLC-Chromatogram of Nemacur .......................................................... 28
4.1.3 Standard curve of Nemacur ....................................................................... 28
4.1.4 Occurrence and Distribution of Nemacur residues ................................... 30
4.2 Discussion ................................................................................................................ 41
4.2.1 Occurrence and Distribution of Nemacur Residues in water, soil matrix,
cucumber fruits, and cucumber plant. ................................................................ 41
Chapter (5) Conclusion and Recommendations .............................................. 48
5.1 Conclusions: ............................................................................................................ 48
5.2 Recommendation: ..................................................................................................... 49
References ................................................................................................................ 51
X
List of Tables
Table (1.1): Quantities (L) of pesticides used in the past years in Gaza Strip ............ 4
Table (1.2): Quantities (Liter) of Nemacur used in the last seven years in Gaza strip 5
Table (1.3): Selected cha`racteristics of Nemacur ...................................................... 6
Table (3.1): Global and Palestinian coordinates ....................................................... 20
Table (4.1): Nemacur concentration in filtrate water average and standard deviation
mg/l in 30/11/2015 by HPLC. .............................................................. 30
Table (4.2): Nemacur concentration in filtrate water average and standard deviation
mg/l in 25/12/2015 by HPLC ............................................................... 31
Table (4.3): Nemacur concentration in sand soil average and standard deviation
mg/kg soil in 29/12/2015 by HPLC ...................................................... 31
Table (4.4): Nemacur concentration in clay soil average and standard deviation
mg/kg soil in 30/12/2015 by HPLC ...................................................... 32
Table (4.5): Nemacur concentration in clay soil average and standard deviation
mg/kg soil in 30/12/2015 ..................................................................... 35
Table (4.6): Nemacur concentration in sandy soil average and standard deviation
mg/kg soil in 29/12/2015 ...................................................................... 35
Table (4.7): Nemacur concentration in water average and standard deviation mg/L.
.............................................................................................................. 36
Table (4.8): Nemacur concentration in water average and standard deviation mg/L.
.............................................................................................................. 37
Table (4.9):.Nemacur concentration in cucumber fruits average and standard
deviation mg/kg. ................................................................................... 38
Table (4.10): Nemacur concentration in cucumber average and standard deviation
mg/kg. ................................................................................................... 39
Table (4.11): Nemacur concentration in cucumber plant average and standard
deviation mg/kg. ................................................................................... 40
XI
List of Figures
Figure (1.1): Fate of pesticide residues in soil ............................................................ 3
Figure (2.1): Metabolic pathway of Nemacur by the isolated bacteria .................... 11
Figure (3.1): The middle Governorate (Deir Al- Balah) in Gaza strip ..................... 19
Figure (3.2): Column techniques used to collect sandy and clay soil samples from
the pots. ................................................................................................... 21
Figure (3.3): The cucumber fruits from the market in the middle governorate. ....... 22
Figure (4.1): Absorbance spectrum of Nemacur, Me OH: water, 80:20, ................. 27
Figure (4.2): HPLC-Chromatogram of Nemacur ..................................................... 28
Figure (4.3): Relationship between peak area and standard concentration of
Nemacur .................................................................................................. 29
Figure (4.4): Cucumber plant in greenhouse ............................................................ 29
Figure (4.5): Nemacur concentration in filtrate water average and standard deviation
mg/L . ...................................................................................................... 30
Figure (4.6): Nemacur concentration in filtrate water average and standard deviation
mg/L. ....................................................................................................... 31
Figure (4.7): Nemacur concentration in sand soil average and standard deviation
mg/kg soil. ............................................................................................... 32
Figure (4.8): Nemacur concentration in clay soil average and standard deviation
mg/kg soil. ............................................................................................... 33
Figure (4.9): Relationship between AchE activity and Nemacur concentration and
values in log scale ................................................................................... 34
Figure (4.10): Nemacur concentration in clay soil average and standard deviation
mg/kg soil ................................................................................................ 35
Figure (4.11): Nemacur concentration in sand soil average and standard deviation
mg/kg soil. ............................................................................................... 36
Figure (4.12): Nemacur concentration in water average and standard deviation mg/L
................................................................................................................. 37
Figure (4.13): Nemacur concentration in water average and standard deviation
mg/L. ....................................................................................................... 37
Figure (4.14): Nemacur concentration in cucumber fruits average and standard
deviation mg/kg. ..................................................................................... 38
Figure (4.15): Nemacur concentration in cucumber fruits average and standard
deviation mg/kg. ..................................................................................... 39
Figure (4.16): Nemacur concentration in cucumber plant average and standard
deviation mg/kg. ..................................................................................... 40
XII
List of Abbreviations
Acetyl Choline Esterase AchE
Diode Array Detector DAD
5,5'-dithiobis-(2-nitrobenzoic acid) DTNB
Distilled Water D.W
European Food Safety Authority EFSA
Environmental Protection Agency EPA
Food and Agriculture Organization FAO
Fenamiphos FEN
Fenamiphos sulfoxide FSO
Fenamiphos sulfone FSO2
High Performance Liquid Chromatography HPLC
International Union of Pure and Applied Chemistry IUPAC
Lethal Concentration 50 LC50
Lethal Dose 50 LD50
Ministry of Agriculture MOA
Methanol Me OH
Fenamiphos Sulfoxide MO2
Fenamiphos sulfone MO1
Maximum Residue Limits MRLs
Organ phosphorus OP
Acid dissociation constant (- Log) pKa
United Nations Environment Programme UNEP
Unites States of America USA
United States Environmental Protection Agency USEPA
Global Coordinates UTM WGS84
Ultra Violet UV
World Health Organization WHO
Chapter (1)
Introduction
2
1 Chapter (1)
Introduction
1.1 Overview
Pesticides are widely used to control pests that affect agricultural crops and pests in
homes, yards and gardens. These chemicals generally are manmade organic
compounds. The principal processes that influence their potential for loss from soil to
groundwater is leaching decomposition, retention by the soil, and transport by water
(EL-Nahhal, Nir, & Polubesova., 1997). The problem of agricultural pesticides in
the Arab countries is not only an issue of uncontrolled use, but also pertains
to the handling, misuse and disposal of unwanted pesticides. This is exacerbated
by undeveloped national laws and regulations in regard to potential fate and residuals
impacts of pesticides on groundwater, food safety and public health (Bashour, Tolba,
& Saab., 2008).
Nemacur is an organophosphate that is an active in some insecticides to control
nematodes. However, Nemacur is toxic to both people and the environment (EPA,
2013). Nemacur is applied to the soil before planting or at planting time. It can also
be applied to established plants. Nemacur is toxic to mammals (LD50= 8 mg/kg body
weight male rats, and to fish at concentrations above 3mg /liter. It is moderately
persistent in soil, and half-life ranges from 10 to 67 days and in another study half-
life from12 to 87, and an average half-life of 30 days (USEPA, 1984).
Nemacur used widely in the Palestinian agriculture, and statistics that have been
obtained on 2015 from the Ministry of Agriculture indicate that there is an extra
usage of this material in Gaza strip. Nemacur is an a systemic pesticides, that’s
absorbed from roots and transports into plant parts to reach plant tissue. Nemacur
provides crop protection against nematode damage through systemic activity in the
plant and also via contact action in the soil (Caceres, Megharaj, Venkateswarlu,
Sethunathan, & Naidu., 2010).
3
1.2 Environmental Fate of Pesticides
The fate of pesticides in soil is controlled by chemical, biological and physical
dynamics of this matrix. Pesticides may be transformed by degradation processes or
transported from the site of application by several processes (Navarro, Vela, &
Navarro., 2007). Pesticides are distributed in the environment by physical processes
such as, adsorption, volatilization, leaching, and photochemical degradation, in
addition dissipation of particles in the environment (EL-Nahhal, et al., 1998), shown
in Figure 1.1.
Figure (1.1): Fate of pesticide residues in soil
Source: (Field, 2011)
1.3 Pesticides in Palestine
The consumption of pesticides were annually estimated in the West Bank
governorates as 979 tons of pesticides during the year 2006 (MOA, 2008). However,
in recent years, Gaza strip applied more than 400 tons of pesticides annually as
shown in (Table 1.1).
4
Table (1.1): Quantities (L) of pesticides used in the past years in Gaza Strip
Total(L)
Fumigants
and
Hormones
Fungicides Insecticides Soil
sterilizer Herbicides Year
453170 980 74336 56714 300700 20440 2005
248315 855 55650 55270 111600 24940 2006
185950 3500 34270 35580 93800 18800 2007
364478 60828 42200 49650 193600 18200 2008
711802 10771 123694 139337 394392 39432 2009
486819 61327 99630 144682 162400 18780 2010
484164 7429 136477 220169 93035 27054 2011
544427 5209 137911 232488 143210 25609 2012
443887 8577 104705 180664 125690 24251 2013
Source: (MOA, 2014)
It seems from the above table, there are an oscillatory increasing usage of pesticides
quantity in Gaza strip .And this is an evidence of an excessive usage in the
agriculture this would has a negative effect on human and environmental health.
1.4 Nemacur (Fenamiphos)
Nemacur (ethyl 4-methylthio-m-tolyl isopropylphosphoramidate) is an organ
phosphorus OP nematicide used to control a wide variety of nematode (roundworm)
pests. The pesticide is registered for use in more than 60 countries. It provides
effective control of free living root-knot and cyst-forming nematodes. This pesticide
was commercialized by the company Bayer under the name of Nemacur and is
formulated as a granular product or an emulsifiable concentrate at 400g active
ingredient per liter (Caceres, 2010). Nemacur belongs to the class of
phosphoramidate insecticides and organophosphate nematicides. It is a systemic
compound which is absorbed by the roots of the plant and trans located to the
leaves. It is a cholinesterase inhibitor used for the control of nematodes , with a
secondary effect against sucking insects.( EFSA, 2009). Nemacur quickly oxidizes to
fenamiphos sulfoxide, which in turn, slowly oxidizes to fenamiphos sulfone
(Fenoll, et al., 2011).
5
In the last seven years the quantity of Nemacur used in agriculture in Gaza
Governorate increased from 18120 L in 2009 to 39355 L in 2015 (MOA, 2016) .
Historical applications of Nemacur in Gaza as shown in (Table 1.2).
Table (1.2): Quantities (Liter) of Nemacur used in the last seven years in Gaza strip
(MOA, 2016).
Amount (L/Year) Year
2009 18120
2010 13836
2011 30440
2012 19968
2013 23015
2014 27618
2015 39355
Source: ( MOA, 2016).
It seems from the previous table, that there is a clear increasing of Nemacur usage in
Gaza strip.
1.5 Health Effect
Nemacur can cause cholinesterase inhibition in humans; that is, it can over stimulate
the nervous system causing nausea, dizziness, confusion, and at very high exposures
(e.g. accidents or major spills), respiratory paralysis and death (EPA, 2002).
6
Table (1.3): Selected characteristics of Nemacur
Information Identity
Fenamiphos Common name
Nemacur Trade name
Ethyl 3-methyl-4-(methylthio) phenyl-(1-methylethyl)
phosphoramidate
IUPAC name
Organophosphate Chemical family
C13H22NO3PS Empirical formula
Structural formula
303.4 Molecular weight
Physical/chemical property Value Source
Water solubility at 20 °C 0.4 g/L Tomlin (2000)
Solubility in hexane at 20°C 10–20 g/L Patrick et al (2001)
Molecular weight 303.4 Tomlin (2000)
Melting point 49°C Tomlin (2000)
pKa dissociation constant 10.5 National Library of
Medicine (2004)
Specific gravity/density at 23°C 1.191 Tomlin (2000)
Vapor pressure at 25°C 0.12 m Pa at 20°C Extoxnet (1996)
Henry’s Law constant at 20°C 9.1×10-5
Pa m3
/mol Tomlin (2000)
Source ( APVMA, 2008) and ( Caceres, et al ., 2010)
Physicochemical properties of pesticides have a fundamental influence on the residue
levels in the environment (Kennedy, Skerritt, Johnson, & Highley., 1998).
7
1.6 Problem Identification
Large amount of Nemacur are applied in Gaza annually. The quantity increased from
18120L in 2009 to 39355L in 2015. This may result in accumulation of Nemacur
residues in soil, water, fruit, and vegetable samples. And its effects on human life,
Soil zooplankton and microbial community.
In addition, their residues may transport to the next plant in the crop rotation, and
leading to further hazards to the eco-systems.
1.7 Objectives:
1.7.1 General Objectives:
The purpose of this study is focused on the occurrence and distribution of the
Nemacur in the soil, water and plant in the middle governorate in Gaza strip.
1.7.2 Specific Objectives:
To evaluate the Nemacur residues in soil, water and plant tissue.
To examine the behavior of the Nemacur.
Chapter (2)
Literature Review
9
2 Chapter (2)
Literature Review
2.1 Introduction
Pesticides are chemicals, synthetic or natural substances that are widely used in
agriculture to control of insects, fungi, bacteria weeds, nematodes, and other pests
that damage fruits and vegetables. Thus, the use of pesticides during the cultivation
plays an important role in harvest quality and food protection. These compounds and
the products of their degradation or metabolism may spread through the environment
and frequently contaminate different environmental compartments, including
groundwater and surface water( Fenoll, et al., 2011). Application of pesticides
created many health problems in Gaza strip (Safi, EL-Nahhal, Soliman, and El-
Sebae., 1993; Safi, Mourad, and Yassin, 2005; EL- Nahhal, & Radwan, 2013; EL-
Nahhal, 2016).
(Alyaseri, & Bahi, 2012). A study was carried out in the laboratories of the National
Center for Pesticides Control/State Board for Plant Protection Ministry of
Agriculture in 2010-2011. Pesticides residues have been determined in many Arab
countries (EL- Nahhal, 2004). The aim was to determine the residues of some
pesticides in fruits and vegetables imported to Iraq from neighboring countries
(Jordan, Syria, Turkey and Iran). Two types of fruits (apples and oranges) and two
types of vegetables (tomatoes and cucumber) were selected for the purpose of this
study. These fruits and vegetables are the most agricultural products imported for
human consumption in Iraq. Samples were taken from different border points and
from local markets. Extraction, clean up and analysis were then processed. The
results indicated the presence of small amounts of residues of certain. Pesticides such
as Deltamethrin and Abamectin and Thiamethoxamin some samples. However, these
amounts were less than the limit allowed internationally. No indication was observed
for the presence of other pesticides residues such as Bifenthrin Trticonazol and
Imidacloprid.
Several studies in Gaza strip determined pesticides residues in vegetables and fruits
(Safi, Abou‐Foul , El‐Nahhal, and El‐Sebae, 2002; Schecter, et al., 1997; EL-Nahhal,
& Safi, 2008).
10
(Baig, Siddiqui, Chakravarty, & Moatter., 2009) determined residues of three
pesticides in vegetables, belonging to Organophosphate group, which was
extensively used in the Southern part of Punjab province in Pakistan. Triazophos,
profenofos and chlorpyrifos Organ phosphorus insecticides, were commonly used on
the summer vegetables like: eggplant, Pumpkins and okra cultivated in study area.
Vegetables were analyzed for pesticide residues using multi residue analysis by
High-Performance Liquid Chromatography (HPLC) equipped with UV detector. It
was observed that 33.0% of the samples were contaminated with any of the above
three pesticides.
Moreover, application of pesticides has been damage the ecosystem ( EL-Nahhal,
Hamdona, and Alshanti 2016; EL-Nahhal, ,2014; EL-Nahhal, & AL-Dahhdouh,
2015).
Several attempts have been made to reduce pesticides contamination in soil. This
includes organoclay formulations (EL-Nahhal, et al., 2016; EL-Nahhal, & Logaly,
2005; EL-Nahhal, et al., 2001, 1998, 1997; EL-Nahhal, & Safi, 2005).
2.2 Environmental Fate of Nemacur
2.2.1 Nemacur Fate in Soil
Nemacur (FEN) is an organophosphate nematicide used in protected horticultural
crops. In soil, FEN is gradually oxidized to its sulfoxide (FSO) and sulfone (FSO2),
which also possess high nematicidal activity and is equally toxic to non-target
vertebrates.(Porto, Melgar, Kasemodel, & Nitschke., 2011) Degradation studies of
FEN in a range of soils showed half-life values ranging from 12 to 87 days. Nemacur
and its oxidation products FSO and FSO2 showed low to moderate affinity for soil
adsorption and their soil accumulation may result in their eventual downward
movement into groundwater because specific surface area of clay, and binding with
organic matter. (Kelley, and wales., 1997) In soil, Nemacur oxidizes to the
corresponding sulfone and sulfoxide. Its half – life time in Arredondo soil is 38-
67days. Nemacur and its oxidation breakdown products were more persistent in soil
and leached deeply into the soil profile (Weaver, Mckenry, & Marade., 1990). The
degradation of Nemacur in soil is also influenced by microbial activity. A fraction of
11
the soil biota present in the soil can develop an enhanced ability for degrading
pesticides, after successive applications to the soil (Singh, &Walker., 2006).
Accelerated microbial degradation of organophosphate and carbamate nematicides is
a phenomenon whereby biodegradation in the soil is increased, leading to a
dramatically shortened persistence of nematicides. Nemacur is a non-volatile,
systemic organophosphate nematicide. In soil, it is rapidly oxidized to fenamiphos
sulphoxide, which is slowly oxidized into fenamiphos sulphone (Leonard,
Shirmohammadi, Johnson, & Marti., 1998). Both the primary oxidation products,
fenamiphos sulphoxide and fenamiphos sulphone, have nematicidal activity and
toxicity similar to that of fenamiphos with both products being more mobile and
persistent ( Hugo, Mouton, & Malan., 2014). According to sorption and leaching data,
fenamiphos can be classified as a low soil mobility chemical (Tomlin 2000) ( Cáceres, et al
., 2010) .
Figure (2.1): Metabolic pathway of Nemacur by the isolated bacteria (Chanika, et al., 2011)
Two bacteria identified as Pseudomonas putida and Acinetobacter rhizosphaerae,
able to rapidly degrade the organophosphate fenamiphos, were isolated. The bove
figure describe the movement of total Nemacur residues (parent + M01 + M02)
12
through the soil profiles. Nemacur itself was the least mobile of the analyses. Both
strains hydrolyzed FEN to fenamiphos phenol and ethyl hydrogen isopropyl
phosphoramidate (IPEPAA). It was found in the top 0-15 cm soil layer in both test
plots only with no detections below this layer at any sampling time (APVMA, 2008).
Biological mechanisms in soil and living organisms utilize oxidation, reduction,
hydrolysis and conjugation to degrade chemicals.(Linde., 1994).
Nemacur is low persistent in soil under aerobic conditions being rapidly oxidized to
the major metabolites fenamiphos-sulfoxide and fenamiphos-sulfone. Fenamiphos –
sulfoxide is moderate to high persistent in soil and fenamiphos sulfone low to
moderately persistent. (EFSA, 2006).
Soil microorganisms have adapted in such a way that they can rapidly metabolize
specific nematicides. The degradation products (metabolites) of nematicides have
been implicated in an enrichment emergence of soil micro flora capable of
accelerated degradation of parent compounds(Nemacur)(Motta, 2009).
The more rapid degradation of Nemacur in no autoclaved soil indicates that
Nemacur degradation is biologically mediated (Johnson, 1998).
HPLC is an analytical tool adequate for the determination of pesticides that are not
thermally stable or not volatile. Reversed-phase HPLC has been widely used in the
analysis of pesticides as most of these compounds present a low polarity. The HPLC
methods developed for the determination of pesticides in soil. Ultraviolet (UV)
detection has been the most frequently used technique in liquid chromatography
(Tadeo, 2008).
2.2.2 Hydrolysis and Photolysis of Nemacur
Nemacur metabolites are hydrolyzed to the major metabolites fenamiphos-sulfoxide-
phenol and fenamiphos-sulfone-phenol. These metabolites are subsequently
transformed to non-extractable residues and mineralized to CO2. Photolysis may
contribute to the environmental degradation of Nemacur (EFSA, 2006). The reaction
is very slow at low temperature and in neutral water, but complete hydrolysis of the
product is observed in basic water at 50°C. So, the more alkaline soil, the faster the
hydrolysis. However, the soils of a neutral nature favor the accumulation of residues
13
of this pesticide for a longer time which may contribute to groundwater pollution (El-
Yadini, et al., 2013).
The bacterium also, Hydrolyzed Nemacur and its oxidies in soil and ground water
Interestingly, fenamiphos phenol, fenamiphos sulfoxide phenol and fenamiphos
sulfone phenol, formed during bacterial hydrolysis of Nemacur and its oxides,
persisted in the mineral salts medium, but were transitory in soil and groundwater
due to their further metabolism by indigenous micro-organisms ( Megharaj, et al.,
2003).
2.2.3 Nemacur Fate in Water
Until recently, environmental fate data have not been made available by either the
US Environmental Protection Agency (USEPA) or product manufacturers that can be
used to assist pesticide users in selecting appropriate products to minimize potential
adverse impact on surface or groundwater (Hornsby, Buttler, & Brown., 1993).
Quality Twenty-four wells were sampled in 16 sections in Fresno County, 12
wells were sampled in 8 sections in San Joaquin County and 5 wells were
sampled in 3 sections in Kern County. No residues of fenamiphos or its
sulfoxide and sulfone metabolites were detected in any of the samples. These
results may be due either to Nemacur environmental fate or to current use
patterns. With respect to environmental fate, the soil half-life of Nemacur and
its metabolites may be short enough to allow for degradation before recharge
into ground water occurs (Troiano, Turner& Miller., 1987). Residues of this
pesticide were found in groundwater samples from California, Georgia, and Florida
in the USA (Cáceres, et al., 2010). Due to the high solubility of Nemacur in water
(0.4 g/L) and moderate ability to adsorb onto soils it can be readily leached from
A ).3201 et al., Yadini,-El( bodieswater cation to surface and ground appli sites of
and its two metabolites Nemacurmethod for the simultaneous determination of
, in water is described. The sulphoxide and fenamiphos sulfone fenamiphos
application of the method to ground waters showed that a minimum detection level
.)1989Singh, ( 10 mg/Lof the order of
2.2.4 Nemacur Fate in plant
The behavior and metabolism of Nemacur was investigated in a many crops
representing fruity crops, root and tuber crops, leafy crops, oilseeds and pulses. All
14
studies showed a similar metabolic path way in the plants. Nemacur is only found
directly following application. A short period after treatment, fenamiphos-sulfoxide
is the dominant metabolite. In some plants, fenamiphos-sulfone is increasing in time.
Therefore, fenamiphos, fenamiphos-sulfoxide and fenamiphos-sulfone form the
toxicological relevant residue. (EFSA, 2006) .
In general, MRLs for these compounds are in the range of 0.01–10 mg/kg but can be
lower for infant food where there is no approved use (Limit of Determination MRL,
typically between 0.01 and 0.05 mg/kg). Although EU regulation of residues in
drinking water does not contain detailed residue definitions (Tadeo, 2008).
2.2.5 Nemacur Fate in Air
The calculated Henry’s Law Constant of 9.1x10-10
atm.m3/mol (calculated from
VP/Sol) is indicative of very slight volatility from water. Therefore, when released to
either soil or water, the compound is unlikely to partition to any significant extent to
the atmosphere. While no experimental data for degradation in the atmosphere were
provided, this was considered through modeling (APVMA, 2008). Field blank and
spike samples should be collected at the same environmental conditions (e.g.,
temperature, humidity, exposure to sunlight) and experimental conditions (e.g., air
flow rates) as those occurring at the time of ambient sampling (Kelley, &Wales,
1997).
2.3 Toxicity of Nemacur
The magnitude of the toxic effect will be a function of the concentration of altered
molecular targets, which in turn is related to the concentration of the active form of
the toxicant( biologically effective dose) at the site where the molecular targets are
located(Trush, 2008).
The toxicity of Nemacur is integral to assessing the occupational risk. The Agency’s
occupational risk findings show that many Nemacur uses do not pose risks of
concern. Some uses, however, pose occupational risks exceeding the Agency’s level
of concern for certain handlers and workers. All risk calculations are based on the
most current toxicity information available for Nemacur, including a 21-day dermal
toxicity study and a 21-day inhalation toxicity study (EPA, 2002). Nemacur dose not
15
accumulate in body tissues but accumulation of effects was demonstrated during
exposure.
Plasma and erythrocyte chE activity remain inhibited, but clinical symptoms such as
behavioral changes and tremors appear only during the early period of exposure
(WHO/FAO, 1994). Nemacur is toxic to the environment, especially to insect, birds,
fish and other aquatic organism. Inhibition of acetyl cholinesterase activity is the
basis for its majors toxic effects(EPA, 2013).
2.4 Eco-toxicity of Nemacur
The application of pesticides may cause adverse effects among the different forms of
life and among the ecosystems; this will depend on the sensibility grade of the
organisms and the pesticide (Ortize-Hernández, Sanchez, Olvera, & Folch., 2011).
Nemacur is applied to the soil before planting or at planting time. It can also be
applied to established plants. Nemacur is toxic to mammals (LD50= 8 mg/kg body
weight male rats (USEPA, 1984). Nemacur is extremely hazardous after single oral
doses to rats, mice, rabbits, cats, dogs, and chickens (LD50 values 2.4-23 mg/kg) and
highly hazardous after dermal administration to rats and rabbits (LD50 values 75-230
mg/kg). It is moderately hazardous after inhalation in rats and mice (LC50 values
≤100ìg/l, 4 h). WHO has classified Nemacur as “extremely hazardous” (Lyon, 1997).
The toxicity of Nemacur and its metabolites to the aquatic alga Pseudokirchneriella
subcapitata and the terrestrial alga Chlorococcum sp. (Cova, et al., 1989) Pesticides
can enter the body of an animal through the respiratory system, skin and alimentary
canal. Whatever the route of entry, these compounds are initially distributed to the
various tissues by the blood stream(Cáceres, Megharaj, & Naidu., 2008).
The toxicity of Nemacur has been studied in aquatic and terrestrial organisms
Recently, (Cáceres, et al., 2008). Determined the toxicity of Nemacur and its
metabolites to the aquatic alga Pseudokirchneriella subcapitata and the terrestrial
alga Chlorococcum sp. The toxicity followed the order fenamiphos phenol
>fenamiphos sulfone phenol > fenamiphos sulfoxide phenol > fenamiphos.
(caceres.et al., 2010).
16
2.5 Health Effects on Humans
Nemacur can cause cholinesterase inhibition in humans; that is, it can over stimulate
the nervous system causing nausea, dizziness, confusion, and at very high exposures
(e.g., accidents or major spills), respiratory paralysis and death. (EPA, 2002).
Furthermore, Reviewing the acute poisonous cases in health records indicates that
the reported acute toxic cases were among local farmers in Gaza and the number
of acute toxic cases increased annually indicating direct health risks associated
with pesticide use. In addition, the increased number of congenital malformation
among the newborns indicates indirect health risks. Moreover, the number of
cancer cases in Khan Younis governorate indicates a positive association with
pesticide use. As indicated in the study, (El-Nahhal, and Radwan., 2013). During the
risk assessment process, a chronic exposure estimate is always made for parent
compounds. A specific chronic exposure estimate for the metabolites is therefore
also needed in the process of evaluating their toxicological relevance. An acute
exposure estimate for parent compounds is done if the compound has acute toxicity,
and the possibility of acute toxicity of the related metabolites should also be
considered (EFSA, 2012).
2.6 Inhibition of AchE by Nemacur
The OP pesticides are acutely neurotoxic. They are designed to be effective
inhibitors of the enzyme acetyl cholinesterase located at neuromuscular junctions in
the central and peripheral nervous system (Walker, Hopkin, Sibly, & Peakal., 2001).
Cholinesterase's can also be found in plasma or haemolymph, in mammalian red
blood cells and in other organs although the physiological functions of AChE in
tissues other than nerves are not known (Carlock, et al., 1999).
Recent study ( EL-Nahhal, 2016) indicated the inhibition of AchE among farmers
have long term working with pesticides application under field condition.
Acetyl cholinesterase (AchE) is an important enzyme of the nervous system
that splits a neurotransmitter – acetylcholine. Inhibition of the enzyme is carried
out to extend the nerve impulse generation in various diseases, including
neurodegenerative ones, such as Alzheimer’s disease (Andrianov, Akberova,
Ayupov., 2015). Acetyl cholinesterase (AchE) activity was suppressed by > 95% in
17
nematodes treated with carbofuran or Nemacur. Nematodes that had been treated
with Nemacur showed only slight AchE recovery (Pree, Townshend, &Cole., 1990).
Nemacur is an organ phosphorus compound and virtually all its toxicological effects
are due to acetyl cholinesterase inhibition. Clinical signs of cholinergic toxicity were
seen at 0.5 mg/kg and above, while plasma cholinesterase activity was significantly
inhibited at 0.12 mg/kg and above and also transiently at the lowest dose
( FAO/WHO, 2003).
The AChE is responsible for termination of neuronal transmission via degradation of
acetylcholine in the synaptic cleft. This irreversible AChE inhibition causes the
accumulation of acetylcholine in the synaptic cleft and thus permanent activation of
cholinergic (muscarinic or nicotinic) receptors (Bajgar, 2004).
(Kao, Tzing, & Cheng., 2003) determined anticholinergic residue levels in the rice.
The residues most commonly detected in rice are those of anticholinergic agent such
as, methamidophos, ethion and methomy. A rapid and highly sensitive acetyl
cholinesterase the rapid bioassay of pesticides residues (RBPR), that has currently
been used to detect residues organophosphate and carbamate insecticides in fruit and
vegetables. The acetyl cholinesterase inhibition correlated positively with the
chemical analysis of organ phosphorus and carbamate residues.
Nemacur poisoning can be treated non-pharmacologically and pharmacologically.
The non-pharmacologic treatment includes resuscitation, oxygen supply or
decontamination depending on the OP entrance to the human body (e.g. skin, eye,
gastric decontamination), whereas the pharmacologic treatment is based on the
administration of symptomatic and causal drugs. Parasympatolytics (usually
atropine, Fig. that reduce the effects of the accumulated ACh on the cholinergic
receptors and anticonvulsives (usually diazepamdiminishing neuromuscular seizures,
are used as the symptomatic treatment. The causal treatment comprises AChE
reactivators that, unlike the symptomatic drugs, regenerate the enzyme native
function by cleaving OP moiety from AChE serine active site( Colovic, et al., 2013).
Chapter (3)
Material and Methods
19
3 Chapter (3)
Material and Methods
3.1 Study area
Gaza Strip is an important part of State of Palestine. As shown in (Figure3.1) it
consists of five Governorates, the northern area, Gaza, the middle (Deir al-blah),
Khan Yunis and Rafah Governorates. The Gaza Strip, as one of the most densely
populated areas in the world (2638 people/km2), has limited and declining resources
and has already started to experience deterioration of environmental quality. Two
thirds of the Gaza Strip (total 365 km2) is an agricultural area (Shomar, et al., 2005).
Figure (3.1): the middle Governorate (Deir Al- Balah) in Gaza strip
Source: Researcher
20
The population of the middle governorate (281 thousand people) (MOI, 2016). Its
famous of vegetables and palm cultivation.
The global and local coordination (GPS) were specified as shown in table 3.1
Table (3.1): Global and Palestinian coordinates
samples UTM WGS 84 – Coordinates PALESTIN GRID – Coordinates
(East , North) (East , North)
Sandy soil
Clay soil
34 20 41 E 31 24 17 N
34 20 25 E 31 24 22 N
87682.00 E 90620.00 N
87274.00 E 90774.00 N
3.1.1 Planting
the sandy and clay soils were free from Nemacur. The selected Soil samples were
dried for 48 h, and then passed through a 2-mm sieve, as described in study by
(Shomar, et al., 2005). Sandy soil were placed in 16 pots , and clay soil put in 16
pots, each pots size was 8 L. In the middle governorate (Deir al-balah) in Gaza Strip,
cucumber seedlings were planted in the sandy and clay soil in 32 pots, at greenhouse,
to protect the seedlings from the weather conditions. Cucumber seedlings were
planted in a specified 32 pots. The applied Nemacur concentrations were used as
follow, (0.0,0.5F,1F, 2F). According to ministry of agriculture recommendation, the
field concentration (F) 1L to 2L / 1000 m2. The field concentration were calculated
for each pot according to surface area for the pot. The concentrations are as follows,
(0.0, 6.586, 13.173, 26.345)mg/L respectively. Four pots with each Nemacur
concentration were used for each type.
8.5 L of a regular water were irrigated for each pot along 3 months. Cucumber plant
had a normal growth under a normal conditions at specified greenhouse.
3.2 Chemo assay technique
3.2.1 Chemicals
Commercial emulsifiable formulation of Nemacur, from Dow Agro Sciences Co.,
USA, was purchased from Gaza , Commercial Nemacur were extracted and
converted into a standard substance, 10 mL of commercial Nemacur were suspended
in 20 mL of water, and extracted with 3*20 mL of dichloromethane, organic layer
21
was separated and dried with anhydrous sodium sulfate. Dichloromethane was
evaporated under stream of nitrogen gas, and the residue was recrystallized using
methanol\water system. The white solid was collected by filtration. The purity of
Nemacur was tested using HPLC and GC-MS. And Methanol of HPLC grade, purity
99.9% , was purchased from Sigma Aldrich Co., Germany ,was purchased from
Gaza.
3.2.2 Soil collection
The Samples were collected from the sandy and clay matrix by soil column from a
depth of 0 to 15 cm, then the soil were divided into three different depths as show in
the figure 3.2. And then the samples weight , and distilled water were added as the
same weight of the soil sample, then the samples were placed in ultrasonic
instrument for 6 minutes (Sun, Littlejohn, & Gibson., 1998). Then filtrate of the
sample by filter papers, and then the sample were injected in the HPLC .
Figure (3.2): Column techniques used to collect sandy and clay soil samples from the pots.
3.2.3 Water collection
Water filtrate were collected in closed bottles randomly from the pots, at different
periods (1.5-3 months). And the samples were transported immediately to analysis
(Wilde, Radtke, Gibs, & Iwatsubo., 1998).
22
3.2.4 Cucumber fruits and plant collection
The cucumber fruit and cucumber plants samples were collected from planting pots.
The selected Cucumber samples, It was weighing 100 grams of sample, and 100 ml
distilled water were added, were put in a blender, and then transferred to the
centrifuge (high speed centrifuge, model TGL- 16G) for 10 minutes, then filtred.
Finally, the samples were injected into the HPLC (Agilent 1620 model) under
specific condition (Takatori, et al 2011).
3.2.5 Cucumber fruits samples from the central market in Deir Al-
Balah.
Six samples of cucumber fruits were collected randomly from the market . The
samples were transferred to the laboratory and prepared as described above for
analysis on HPLC. The picture below shows the samples collected from the market
in the middle governorate (Deir Al-Balah).
Figure (3.3): the cucumber fruits from the market in the middle governorate.
3.2.6 HPLC-Measurement
HPLC (Agilent 1620) analyses were performed on isocratic system, It was developed
way. Nemacur concentrations in the supernatant were determined by Diode Array
23
Detector (DAD) equipped with manual-injection system. The column was Reverse-
phase. Packing ODS-BP5m (C18), and a 150 mm X 4.6 mm (i.d.) . Injection
volume is 50l and wave length of detection was 250 nm, Mobile phase is water:
methanol 20:80. The flow rate was maintained at 2 ml min1.other conditions were as
used for the silica gel column. External calibration was used for quantification of
Nemacur.
3.2.7 Standard Curve of Nemacur
As described (Brown, 2001) a dose response curve was prepared by a volume of the
stock solution 100ml, containing 1mL Nemacur, was transferred to a 100 ml
volumetric flask and diluted in Me OH (methanol) up to the mark as working
standard. A series of Nemacur standards of 0.0, 0.05, 0.1, 0.2, 0.6, 0.8 and 1 mg/L
were prepared. The absorption was measured by HPLC at wavelength 250 nm and
retention time 2.004 min.
3.3 Bio assay technique
3.3.1 Tested Organisms
The organisms used in this study fish larvae weight of 5 g , which were collected
from environmental labs, Islamic university. Five fishes were transferred to several
beaker containing different concentrations of Commercial Nemacur 0.004, 0.04, 0.4,
0.8, 1.6 mg/L. and remain 24 hour at lab, And monitoring the effects of Nemacur on
the fishes and the number of death ( Hayasaka, Korenaga, Sánchez-Bayo, & Goka,
2012). As shown in Figure 3.3.
24
Figure (3.4): fishes larvae in different concentration of Nemacur
The biological activity of Nemacur was tested in the laboratory to insure this
Nemacur is fresh, effective, and stable, in this test.
3.3.2 Assay procedures (Colorimetric measurement)
According to study carried out by (Ben Oujji, et al., 2012), 200µl of phosphate buffer
PH8 were added in each well containing AchE – modified magnetic beads
suspension in order to equilibrate the enzyme, 200 µl of phosphate buffer containing
2mM ACTh-I and 6% DTNB were added and the micro plate was incubated for 30
min under constant orbital stirring (300 rpm). The absorbance was then measured at
405 nm using 100 µl of the solution taken from each samples. Inhibition experiments
were performed by incubating magnetic beads (1µl) with 100µl of different
concentration of different samples during 10 min. according to the procedure
described by Ell man et al, The thiocholine reacts with 5,5'-dithiobis- (2-nitrobenzoic
acid) (DTNB) yielding a yellow complex absorbing at 412 nm. The inhibition rate
was calculated to the following formula :
I and I0 being respectively the current after the and before inhibition.
25
The intensity of yellow color indicates the free activity of AchE. Subtracting the free
enzyme from the added are the gives bond enzyme. Application with standard curve
gives the concentration of Nemacur in the samples.
3.4 Statistical Analysis:
The occurrence and distribution of Nemacur residues in sandy and clay soil, water,
and cucumber fruit at three applied rates, with average and standard deviation were
determined from each concentration by Microsoft Excel, Means of Nemacur residues
in tested samples were compared by Turkey's test at = 0.05.
Chapter (4)
Results and Discussion
27
4 Chapter (4)
Results and Discussion
4.1 Results:
4.1.1 The spectrum of Nemacur
The absorbance spectrum of Nemacur extracted from the commercial formation is
shown in Figure (4.1). The peak was observed at wave length 250 nm.
λ
Figure (4.1): Absorbance spectrum of Nemacur, Me OH: water, 80:20,
λ Max =250 nm, flow rate=2ml
Ab
s.
28
4.1.2 HPLC-Chromatogram of Nemacur
The HPLC chromatogram of standard Nemacur is shown in (Figure 4.2). It is
obvious that a sharp peak with considerable peak area were obtained at 2.043 min.
This indicates the accuracy of the used method for determination.
Retention Time (min.)
Figure (4.2): HPLC-Chromatogram of Nemacur
4.1.3 Standard curve of Nemacur
The relationship between peak area and gradient concentration of Nemacur are
shown in (Figure 4.3). It is obvious that the peak area increased linearly as the
concentration of Nemacur increased in the solution. These findings indicate a linear
relationship. Regression analysis showed a correlation coefficient (R2) of 0.9951
This linearity indicates the validity and the suitability of the used method and allows
direct measurements of Nemacur in the supernatants by HPLC.
29
Figure (4.3): Relationship between peak area and standard concentration of Nemacur
( figure 4.4) clearly show the normal growth of cucumber plants and field conditions
in greenhouse.
Figure (4.4): Cucumber plant in greenhouse
y = 81.609x R² = 0.9951
0
10
20
30
40
50
60
70
80
90
0 0.2 0.4 0.6 0.8 1 1.2
Pea
k ar
ea
Nemacur mg/l
30
4.1.4 Occurrence and Distribution of Nemacur residues
Occurrence and distribution of Nemacur residues in water filtrate, soil, and plants at
different applied rates by chemo assay (HPLC) and bioassay (colorimetric method),
as shown in Tables and Figures below.
4.1.4.1 Chemo assay Technique:
Table (4.1): Nemacur concentration in filtrate water average and standard deviation mg/L .
2F 1F 0.5F
clay 0.0034±0.00093 0.0023±0.0002 0.0014±0.00014
sand 0.0072±0.00026 0.0054±.00019 0.0022±0.00035
Figure 4.5 shows a high concentration of Nemacur in water filtrate by sandy soil, and
low concentration in water filtrate by clay soil .
Figure (4.5): Nemacur concentration in filtrate water average and standard deviation mg/L .
It can be seem that concentration of Nemacur in both soil water extracts are higher at the
highest applied rate (2F) than lower applied rate 0.5F.
0
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.01
2F 1F 0.5F
co
nc
( m
g/l
)
Applied rates
water in 30/11/2015
clay
sand
31
Table (4.2): Nemacur concentration in filtrate water average and standard deviation mg/L .
0.5F 1F 2F
clay 0.0058±0.00016 0.0042±0.0025 0.0014±0.0021
sand 0.0090±0.00097 0.0061±0.0023 0.0016±0.0035
Figure 4.6 shows, a high concentration of Nemacur in water filtrate by sandy soil.
Figure (4.6): Nemacur concentration in filtrate water average and standard deviation mg/L.
It can be seem that concentration of Nemacur in water filtrate obtained in 25/12/215 are
higher than those obtained 30/11/2015.
Table (4.3): Nemacur concentration in sand soil average and standard deviation mg/kg soil .
soil depth 0.5F 1F 2F
0-5 cm 0.011±0.013 0.0029±0.00022 0.0042±0.00040
5-10 cm 0.0064±0.0086 0.0015±0.00037 0.0027±0.00057
10-15 cm 0.0023±0.00075 0.0017±0.00012 0.0020±0.00020
0
0.002
0.004
0.006
0.008
0.01
0.012
0.5F 1F 2F
con
c m
g/l
Applied rates
water in 25/12/2015
clay
sand
32
(Figure 4.7) shows .a high concentrations in all layers, but in top layer its higher
than other layers.
Figure (4.7): Nemacur concentration in sand soil average and standard deviation mg/kg soil.
It can be seem that Nemacur concentration in sandy soil is higher in the top soil layer 0-5 cm
than deeper layers.
Moreover, the concentration of 0.5F is the highest among all applied rates. The trend is
similar in all layers. Statistical analysis indicates significant difference > 0.05.
Table (4.4): Nemacur concentration in clay soil average and standard deviation mg/kg soil.
soil depth 0.5F 1F 2F
0-5 cm 0.002886±0.00015 0.0011±0.0025 0.0015±0.00024
5-10 cm 0.0019±0.0014 0.0021±0.00027 0.0022±0.00072
10-15 cm 0.0013±0.00030 0.0018±0.0025 0.00072±0.0096
-0.002
0
0.002
0.004
0.006
0.008
0.01
0.012
0.014
0.016
0.5F 1F 2F
con
c m
g/k
g
Applied rates
Nemacur in sand
0-5 cm
5-10 cm
10-15 cm
29/12/2015
33
(Figure 4.8 ) shows, low concentration of Nemacur in top layer(0-5 cm), but high
concentration of Nemacur in other layers.
Figure (4.8): Nemacur concentration in clay soil average and standard deviation mg/kg soil.
Concentration of Nemacur in clay soil has different behavior. It can be seem that the
concentration is higher in layers 5-10 cm in all applied rate. This trend is similar in all layers.
4.1.4.1.1 Nemacur concentration in cucumber fruits average and standard
deviation mg/kg from market in the Middle Governorate (Deir Al- Balah).
Nemacur residues does not exist and didn’t show any traces in the six random
samples by using HPLC technique.
4.1.4.2 bioassay technique:
The relationship between AchE activity and gradient concentration of Nemacur are
shown in (Fig. 4.9.a & 4.9.b). It is obvious that the AchE inhibition increased
linearly as the concentration of Nemacur increased in the solution. Converting the
data to log scale gives better fitting to the linear regression. These findings indicate a
linear relationship, regression analysis showed a correlation coefficient (R2) of
0.9383 This linearity indicates the validity and the suitability of the used method and
allows direct measurements of Nemacur in the supernatants by spectrophotometer.
0
0.0005
0.001
0.0015
0.002
0.0025
0.5F 1F 2F
Co
nc
mg/
kg
Applied rates
Nemacur in clay
0-5 cm
5-10 cm
10-15 cm
30/12/2015
34
(4a): Relationship between AchE activity and Nemacur concentration
(4b): values in log scale
Figure (4.9): Relationship between AchE activity and Nemacur concentration and values in
log scale.
As shown in figure (4.9.b) a good linear correlation between concentration and
inhibition of AchE was investigated. The residual activity of AchE was calculated by
comparing the slope of obtained kinetics before and after inhibition.
0
10
20
30
40
50
60
70
80
90
100
0 0.05 0.1 0.15 0.2
% A
CH
E In
hib
itio
n
Nemacur Conc. mg/l
% inhibition
y = 0.21x + 1.7671 R² = 0.9383
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
-3-2.5-2-1.5-1-0.50
Log scale concentration
Lo
g %
in
hib
itio
n
35
Table (4.5): Nemacur concentration in clay soil average and standard deviation mg/kg soil.
2 F 1 F 0.5 F soil depth (cm)
1.87±0.23 1.87±0.11 2.01±0.18 0-5 cm
2.21±0.29 2.91±0.75 2.16±0.07 5-10 cm
2.22±0.07 2.33±0.45 2.14±0.69 10-15 cm
(Figure 4.10 ) shows, a low concentration of Nemacur in top layer (0-5 cm), but high
concentration in deeper layers.
Figure (4.10): Nemacur concentration in clay soil average and standard deviation mg/kg soil
P-values for clay soil columns ranged between 0.06-0.5 indicating no significant
difference. However, the p-value of 0.06 is very low to the border of significant
difference.
Table (4.6): Nemacur concentration in sandy soil average and standard deviation mg/kg.
soil depth(cm) 0.5F 1F 2F
0-5 cm 4.01±0.0031 3.95±0.070 3.87±0.17
5-10 cm 4.36±0.0026 4.12±0.09 3.50±0.0033
10-15 cm 4.34±0.49 4.02±0.52 5.80±0.0025
0
0.5
1
1.5
2
2.5
3
3.5
0.5F 1F 2F
con
c m
g/L
Applied rates
30/12/2015
0-5cm
5-10cm
10-15cm
36
(Figure 4.11 ) shows, high concentration of Nemacur in deeper layers, and low
concentration rate in top layer(0-5 cm).
Figure (4.11): Nemacur concentration in sand soil average and standard deviation mg/kg
soil.
Table (4.7): Nemacur concentration in water average and standard deviation mg/L.
Water filtrate 0.5 F 1 F 2 F
clay 1.00±0.0053 4.30±0.17 4.01±0.059
sand 4.01±0.50 4.56±0.0058 4.45±0.041
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
0.5F 1F 2F
con
c m
g/kg
Applied rates
0-5 cm
5-10 cm
10-15 cm
29/12/2015
37
(Figure 4.12 ) shows , a high concentration of Nemacur in water filtrate by sandy soil ,
more than by clay soil.
Figure (4.12): Nemacur concentration in water average and standard deviation mg/L
Table (4.8): Nemacur concentration in water average and standard deviation mg/L.
Water filtrate 0.5 F 1 F 2 F
clay 1.21±0.23 3.53±0.56 3.21±0.28
sand 3.84±0.70 3.87±0.56 3.63±0.77
(Figure 4.13) shows, high concentration of Nemacur in water filtrate by sandy soil.
Figure (4.13): Nemacur concentration in water average and standard deviation mg/L.
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
0.5 F 1 F 2 F
con
c m
g/L
Applied rates
caly
sand
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0.5 F 1 F 2 F
Co
nc
mg/
L
Applied rates
calysand
38
p-values of collected water are in the range of 0.14-0.47 indicating no significant
difference.
Table (4.9):.Nemacur concentration in cucumber fruits average and standard deviation
mg/kg.
cucumber 0.5 F 1 F 2 F
clay 0.24±0.168 0.80±0.0526 1.26
sand 0.74±0.132 0.04±0.0391 0.94
(Figure 4.14 ) shows, low concentration of Nemacur in cucumber fruits in sandy soil
and the concentration its low in cucumber in general in sandy soil .
Figure (4.14): Nemacur concentration in cucumber fruits average and standard deviation
mg/kg.
-0.40
-0.20
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
0.5 F 1 F 2 F
con
c m
g/k
g
Applied rates
caly
sand
Cucumber 9/12/2015
39
Table (4.10): Nemacur concentration in cucumber fruits average and standard deviation
mg/kg.
cucumber 0.5 F 1 F 2 F
clay 1.93±0.46 2.09±0.22 2.26±0.46
sand 1.60±0.61 2.97±0.36 1.93±0.21
(Figure 4.15) shows, high concentration of Nemacur in cucumber fruits by clay soil.
Figure (4.15): Nemacur concentration in cucumber fruits average and standard deviation
mg/kg.
p-values in cucumber fruits are significantly different and ranged between 0.007-
0.017 indicating significant difference. P-values between different dates of fruit
collection is 0.0051 indicating significant differences.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0.5 F 1 F 2 F
con
c m
g/k
g
Applied rates
caly
sand26/12/2015
40
Table (4.11): Nemacur concentration in cucumber plant average and standard deviation
mg/kg.
Plant 0.5 F 1 F 2 F
clay 1.16±0.02 1.14±0.12 2.02±0.30
sand 0.95±0.29 1.15±0.04 1.30±0.05
(Figure 4.16 ) shows, a low concentration of Nemacur in the cucumber plant by
sandy soil, but high concentration of Nemacur in cucumber plant by clay soil .
Figure (4.16): Nemacur concentration in cucumber plant average and standard deviation
mg/kg.
p-values for plant samples in grown in different soils or applied rate are in the range
of 0.14-0.28 indicating no significant differences.
0.00
0.50
1.00
1.50
2.00
2.50
0.5 F 1 F 2 F
con
c m
g/k
g
Applied rates
caly
sand
41
4.2 Discussion
4.2.1 Occurrence and Distribution of Nemacur Residues in water, soil
matrix, cucumber fruits, and cucumber plant.
The data in ( figure 4.1) Clearly shows, that Nemacur absorb UV- radiation at 250
nm, accordingly this wave length is used in developing the HPLC method for
detecting Nemacur concentration in environmental samples. Using the wave length
250 nm enabled us to detect HPLC concentration at low concentration. This is
obvious (in figure 4.2) very sharp peak is obtained at 2.043 minute indicating
accuracy of the method. Accordingly HPLC method was used to determine Nemacur
concentration in different samples.
Moreover, response of HPLC to gradient concentration of Nemacur gave linear
relationship at a log scale relationship with high value of R2 ( 0.9951), as clearly in
figure(4.9.b). This indicates strong positive association between Nemacur
concentration and peak area. Accordingly Nemacur was determined at minimum
concentration, and a dilution factor was used whenever necessary.
Moreover,( figure 4.4) clearly show the normal growth of cucumber plants under
field conditions regardless to the fact that higher concentrations of Nemacur was
used than regular concentration. These data indicate Nemacur is not harmful to
cucumber plants (Tomlin, 2000).
4.2.1.1 Chemo assay results:
The data in Tables 4.1,4.2,4.3 and 4.4 clearly shows the concentration of Nemacur in
filtrate water at different collection times, and in different depth in sand and clay
soils. It can be seem that the concentration in sandy soil is lower than the
concentration in clay soil, regardless to some discrepancy. The explanation of these
variations is that clay soil can adsorb Nemacur more than sandy soil, Because saving
of water more than sandy soil, it is remarkable, that there is a gradual increasing of
adsorbed amount of Nemacur on sand soil. The retention of pesticides in soils is
mainly due to the adsorption. After the Nemacur associate with the organic matter,
other binding process make take place either with the organic matter functional
groups or with the intimately associated clays (Felsot, & Dahm., 1979).
Structure is characterized by the bulk density and pore geometry which depend on
agricultural practices on the climate (Alletto, et al., 2010). In static conditions, the
42
rate of pesticides adsorption decreases when the density of soil aggregates increases
(Chaplain, et al., 2011).
Figure(4.8) shows, the results obtained at the surface of the clays(top layers 0-5 cm)
its lower rate in concentration of Nemacur than deeper layers(5-10 and 10-15
cm),according to (Felsot &Dahm, 1979), the specific surface area on clay soil, there
are many process include van der Waals interactions and hydrophobic bonding,
physical adsorption, chemisorption(ligand exchange, cation exchange, coordination),
hydrogen bonding. that’s agree with study was conducted by ( Tajeddine, et al.,
2010) who found that the Nemacur degradation process clearly depends on the
amount of humic substances and iron, the latter component accelerated the
disappearance of Nemacur, while degradation rate increased in the presence of water
and was mainly due to the involvement of the photo hydrolysis process leading to the
scission of the P-O bond. disappearance of Nemacur when the irradiation was
performed at the surface of montmorillonite and kaolin at the surface of soils and
clays. The adsorption interactions of pesticides in soil may involve either the mineral
or organic components, or both. In soil that have higher organic matter levels( <5 ( % ,
pesticide adsorption depend on organic matter contents (Spark, and Swift, 2009).
Also micro-organisms play an important role in breaking down the pesticide in the
soil surface.
In general, the adsorption of pesticides decreases when their water solubility
increases because of their high affinity for the water phase, and conversely, the
adsorption increases with the hydrophobicity of pesticides. However, it also depends
on the hydrophilic/hydrophobic balance of the soil adsorbent (Calvet, 1989).
The results indicate that soils with different physico-chemical properties have
different effects on the adsorption of Nemacur, especially at higher concentration
levels. And the degradation of Nemacur was faster in the alkaline soils, followed by
neutral and acidic soils. The above theoretical principles supported the obtained
results.
Concentration of Nemacur was more percent in water samples collected from sandy
soil than from clay soil. Water filtrate does not happen to him degradation rapidly to
not stay in the sandy soil for a long time. Soil and environmental conditions present
in the areas studied appear to favor persistence of Nemacur residues and permit down
ward leaching of the pesticides over time. Due to the high solubility of Nemacur in
water (0.4 g/L) and moderate ability to adsorb onto soils it can be readily leached
43
from sites of application to surface and ground water bodies (El-Yadini, 2013). The
Nemacur residues in cucumber fruit by HPLC after three months from planting it
was under detection limits. Our results agree with previous reports ( Safi, et al.,
2002), who found low pesticides residues in vegetables and fruits collected from
different farms in Gaza Strip. Moreover similar observation were found in Arab
countries (EL-Nahhal, 2004). Additionally, in soil Nemacur and its metabolites have
been shown to leach to ground water where conditions favorable for leaching exist
(APVMA, 2013).In another study the potential minimize leaching of Nemacur by
reduced application rates and limited irrigation during rainy period was evident from
nematicide concentration profiles measured on three commercial pin apple field on
oahu, little Nemacur was detectable mellow 1m. In a study by center of analytical
chemistry in California department of food and agriculture that’s indicated the limit
of Nemacur in the well is 0.05µg/l. In other study, ground water samples collected
from the bottom of the deep core hole contained no Nemacur residues (Weaver, et
al., 1990).
The Nemacur residues does not exist and disappear in six random samples, that were
taken from the market. The explanation of these results, It seems that farmers are
committed to the recommendations of the Ministry of Agriculture. And a rapid
leaching for water through the soil, and degradation of Nemacur in soil by
microorganisms, physical and chemical decomposition.
4.2.1.2 Bio assay results
The indicators by using fish larvae in figure 3.3, clearly show an increase in the death
of fish larvae at different concentration of Nemacur. This indicates acetyl
cholinesterase inhibition by due to exposure to Nemacur. The use of AchE as a
biomarker of Nemacur in environmental samples is very important. In all cases, it
appears that significant levels of brain AchE inhibition can be measured in fish
exposed to Ops at concentrations well below those causing mortality (Fulton, and
Key., 2001).
Moreover, responses of UV- spectrophotometer to gradient concentration of
Nemacur gave linear relationship, with high value of R2
(0.9383). This indicates
strong positive association between Nemacur and percent AchE inhibition,
accordingly to figures (4.9.a, 4.9.b).
44
Bioassay techniques have previously been used to determine concentration of
herbicides in Gaza soils (EL-Nahhal 2003,a,b; EL-Nahhal, et al., 2005, 2013; Safi, et
al., 2014). This techniques has been more developed recently (EL-Nahhal, et al.,
2016). And ( Ben Oujji, et al 2012) who found three of pesticides by using
colorimetric method for the detection of insecticides widely used in the treatment of
olive trees.
The data in tables 4.5, 4.6, 4.7, and 4.8 clearly show, the concentration of Nemacur
in different depth in sandy and clay soil, and in filtrate water, at different collection
time. It can be seem that the concentration in filtrate water by sandy soil is higher
than the concentration by clay soil, because the sandy soil is high permeability more
than clay soil. Our results agree with (EL-Nahhal, et al., 2014; Safi, et al., 2014) who
found low concentration of pesticides in sandy soil. Leaching potential is dependent
on soil clay fraction, soil organic matter, and soil PH.
It can be seem that the concentration rate of Nemacur in sandy soil matrix is lower
than the concentration in clay soil matrix. And can be seem that, the concentration of
Nemacur in top layer (0-5 cm) is lower than the concentration in deeper layers (5-10,
10-15 cm), this results that’s agree with study was conducted by ( Tajeddine, et al.,
2010).
The results from the present experiments, suggest that alkaline pH in soil supports
higher microbial biomass and enzymatic expression, which in turn helps the
microbial community to adapt and develop gene-enzyme systems for the enhanced
degradation of pesticides (Singh, et al., 2003).
In study were conducted by (Abou-Aly, and Nasr., 2009)The rate of Nemacur
decomposition by the bacterial strains was higher in cultivated soil than in
uncultivated soil, and cultivated soil exhibited the most rapid disappearance of
Nemacur with the mixture of the three strains, Paenibacillus polymyxa,
Pseudomonas fluorescens and Streptomyces aureofaciens. When soil samples were
analyzed for the Nemacur biodegradation after 22 days of the pesticide addition and
15 days after inoculation with the biodegradants, three metabolites of Nemacur were
detected. Results show that Nemacur degradation can be effectively accelerated in
soil by these microorganisms.
45
(Tables 4.5,4.6 ), indicates that the inhibition of AchE are present in all layers of the
sandy and clay soil matrix with different applied rates. The highest ratio of Nemacur
residues in the deeper soil layer(5-10 and 10-15 cm); but a low concentration in top
layers(0-5 cm), this results agree with (Padula, et al., 2008), who found high
concentration of Nemacur in the top 0-15 cm soil layer in both test plots only with no
detections below this layer at any sampling time. Nematodes that had been treated
with Nemacur showed only slight AChE recovery (pree, 1990).The Nemacur
residues are determined by colorimetric measurement, As it is illustrated in (figure
4.11,4.12) ),illustrate the high concentration of Nemacur in water filtrate by sandy
soil more than in water filtrate by clay soils. The concentration of Nemacur in the
water depends on the soil nature and soil contents, other factors such as the bio-
degradation in the soil, the Nemacur metabolite to bi- products, first oxidized quickly
to fenamiphos sulfoxide and slowly to fenamiphos sulfone, they are more toxic.
After the Nemacur associated with the organic matter, other binding process may
take place either with the organic matter functional groups or with intimately
associated clays (Felsot, & Dahm., 1979).
Nemacur moved slowly through the soil and was effective 25 to 30 cm below the soil
surface. Furthermore Nemacur was present for up to 12 Weekes after application,
indicating good persistent activity (Lolas, 1991).
After 45 days of planting, the concentration of Nemacur in sand are a little and
traces, This was achieved through previous studies.
The bioassay technique provides data higher than chemo assay. The explanation of
the results is that in bioassay technique acetyl cholinesterase is not Nemacur specific
inhibitor. Acetyl cholinesterase is inhibited by nearly all organ phosphorus
insecticides and carbamate insecticides . Accordingly any traces of the compounds in
soil or water can inhibit acetyl cholinesterase accordingly higher concentration of
Nemacur than expected was found in soil, water, and fruit compounds at HPLC
which is Nemacur specific determined, our results agree with (Kao, Tzing, and
Cheng., 2003; EL-Nahhal, 2004) who found similar result.
As it is illustrated in (figure 14,15,16) the Nemacur residues was detected in
cucumber fruit and plant, a high residues of Nemacur in cucumber fruits and
cucumber plants by clay soils, more than by sandy soil. These results agree with
46
chemo assay results and previous studies (Kao, et al ., 2003) who found organ
phosphorus and carbamates in fruit and vegetables by a rabid bioassay of pesticides
residues(RBPR). And our results agree with previous study (safi, et al., 2002; EL-
Nahhal, 2004) who found low pesticides residues in fruit and vegetables collected in
Arab countries.
Chapter 5
Conclusion and
Recommendations
48
5 Chapter 5
Conclusion and Recommendations
5.1 Conclusions:
In the present study, The results show that Nemacur effectively for treating the pest
and showed the normal growth of cucumber plant under field conditions. Occurrence
and distribution of Nemacur residues in environmental samples were determined at
different applied rates, it can be concludes considerable concentration of Nemacur
was found in all extracted tested samples. The result showed that lower a
concentration of Nemacur in sandy soil than clay soil, the concentration in sandy soil
is higher in the top soil layer 0-5cm than deeper layer, and the concentration of 0.5F
is the highest among all applied rates .The concentration in clay soil is lower in the
top layer 0-5cm (1.87 mg/kg) than deeper layer (2.22 mg/kg), and the concentration
is higher in layers 5-10 cm in all applied rates. And the concentration in sandy soil
(0.0003mg/kg) lower than clay soil (0.0013mg/kg), p-value for clay soil ranged
between 0.06 -0.5 indicating no significant difference And the concentrations in
filtrate water by sandy soil(0.0072mg/L) more than by clay soil (0.0034mg/L), p-
value ranged between 0.14-0.47 indicating no significant difference. The
concentration in cucumber fruits extracts by clay soil(0.80mg/kg) more than by
sandy soil(0.04mg/kg),at different times. And p-value in cucumber fruits are
significantly different and ranged between 0.007-0.017. The concentration in
cucumber plants extracts by clay soil (2.02 mg/kg) more than by sandy soil (1.3
mg/kg), p-value for cucumber plant in different soils or applied rates in the range of
0.14-0.28 indicating no significant difference. Nemacur residues does not appear in
random samples taken from the market. The biological activity of Nemacur was
tested in the laboratory by fish larvae. The number of dead fishes larvae increased as
the concentration of Nemacur increased in the solution. Chemo assay by HPLC and
bioassay techniques were effective and strong tools for determination of Nemacur
residues in environmental samples. Nemacur concentrations determined by bio-assay
technique were higher than those determined chemo-assay technique . the results by
chemo-assay and bio-assay were positive.
49
5.2 Recommendation:
The following recommendations are resulted from the current study in order to
enhancing the terms and concepts of Nemacur using harms in planting at Gaza strip.
1- Further studies must be encouraged to generate better understanding of
occurrence and distribution of Nemacur residues in environmental samples, at Gaza
strip, Palestine.
2- Application of Nemacur should be used under monitoring by decision makers to
avoid the negative effects on human health and ecosystem.
3- It is recommended to exclude Nemacur application from cucumber and related
crops.
4- It is recommended to avoid using Nemacur application before planting directly, at
least three months before planting as Nemacur residues disappear widely in plants
and ecosystem.
5- It is recommended that farmers must avoid using high concentration of Nemacur
in planting and comply with regulations and recommendations that issued by
ministry of agriculture.
50
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51
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