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STUDY ON
PESTICIDE RESIDUE
IN
FRUITS AND VEGETABLES
in partial fulfillment for the MSc (Agr. Vet. Pharm)
Therese Visanich
May 2001
ACKNOWLEDGEMENTS
I would like to thank all those who in some way or other helped in this dissertation.
Special thanks go to my tutors, Dr. Everaldo Attard and Dr Antoine Vella for their professional
help, guidance and assistance throughout the research.
I am also very much indebted to Profs A Scicluna Spiteri Head of Institute of Agriculture for his
invaluable support throughout this dissertation.
Special thanks goes to Janet Groom and staff from Envirologix UK for all her help throughout the
research of this project.
I would also like to express all my gratitude to the officials and staff at the Ghammieri Government
Experimental Farm.
Special gratitude goes to all my family for all their continuous assistance and support throughout
the years.
THERESE V ISANICH
May 2001
Abstract
The term pesticides includes insecticides, fungicides and herbicides together with rodenticides,
nomaticides, molluscidos, and acmicidos. Those me ngroohomionb designed to control the vnri
ous attacks of pests in agriculture and horticulture.
Modern pesticides exert a systemic mode of action. They enter the plant's vascular system
via its cuticle. Man's fight against pests dates back to ancient times, and we note various land
marks in history when pesticides were introduced. The introduction of Paris green and Bordeaux
Mixture in 1800s to control the Colorado Beetle and other pests I diseases led to the enactment of
first law, in the USA in 1900, about pesticides. However it was not until the second world war that
the revolutionary DDT was introduced. It was one of the first synthetic organochlorine. Later on a
new generation organophosphates based on nerve gasses came into being. In 1959 both the
FAO and the WHO then started to issue regular studies and regulations regarding pesticide use
and their effects on man, animal and environment. Later on both organizations complied the
concepts of ADI, MRL and GAP .
Pesticides have served to increase the world's food supply and decrease pests. However
their environmental impact has generated controversy regarding their use. Today we note a de
mand for biologically grown foods. However still pesticides are being used to protect our crops.
Thus the amount of pesticide used must be kept to a minimum so as to have the minimal residual
levels possible in the crops we consume. We must not forget another factor that of pest and vector
resistance to pesticides, which have given rise to new pesticides to be formulated.
Aim The main aim of the study was to determine the presence or absence of any of the pesti
cides being investigated in a number of crops and fruits, both produced locally or imported but
consumed locally. Detection of pesticide residue was done using ELISA kits supplied by the
Institute of Agriculture at the University of Malta.
Methodology Twenty-four different samples were collected from different vegetable hawkers found in vari
ous places in Luqa, Zabbar, M'Xlokk, St. Paul's Bay, Zebbug, Mgarr (Malta), and B'Buga.
Any pesticide residue present was extracted in Methanol. During extraction 20 grams of
finely chopped crop material was added to 1 OOml of Methanol ANALAR grade. A 1:100 dilution in
distilled water was then performed. The outcome was run as the sample on the plate.
The samples collected, were tested using three different Envirologix ELISA kits - Chlorpy
rifos Kit, Metalaxyl Kit and the Synthetic Pyrethroid Kit. In these kits any pesticide residue present
competes with enzyme (Horseadish-peroxidase)- labelled pesticide for a limited number of bind-
ing sites on the inner surface of the test wells. The outcome was visualised as a colour develop
ment step where sample concentration is inversely proportional to colour development. He
samples were run as two differnt trials. In Trial1 samples 1-8 were tested whereas 16 other
different samples were run during Trial 2.
For both trial crops and fruits were bought from different outlets - farmers and vegetable
hawkers from different parts of the island. From a number of kilos, the various parts were taken to
form the final kilo of sample material and then it was liquidized using a normal kitchen liquidizer.
Then part of it was added to Methanol. It was then added to 20-30 grams of Anhydrous Sodium
Sulphate. This was done to eliminate the extra water. Then a subsample was taken and added to
1 OOml of distilled water. Again a subsample was taken and mixed with the materials provided with
each kit before runnino the test pl8te on a reader to obtain the finals results.
The apparatus and materials used as well as the procedure followed varied according to the
kit being used.
Results and Calculations
The results obtained were entered in a database and the average absorbance determined. For all
kits the %Bo was determined as:
% Bo = average OD of Calibrator or sample x 100
average OD of Negative Control
A graph of% Bo (on the Y axis) against concentration (on the X axis) was plotted. After
determining the values of the slope( m) and intercept (c), the value for the corresponding %Bo of
each sample was calculated. The results were converted to parts per million (ppm) and compared
to standards for each crop. Results obtained are summarised below.
Type of Sample Chlorpyrifos Metalaxyl S. Pyrethroid
Actual EPA Actual EPA Actual EPA
1 Apples (Sicily) 3.46 1.5 0.09 0.2 Nil 0.05
2 Tomatoes NIL 0.05 0.18 3 7.71 2
3 Tomatoes NIL 0.05 0.14 3 15.27 2
4 Oranges (Sicily) NIL 1 0.17 1 8.11 NA
5 Courgettes Nil NA 0.08 NA 19.37 NA
6 Cauliflower Nil 1 0.05 1 12.40 1
7 Lettuce Nil 2 0.38 5 10.42 20
8 Cauliflower Nil 1 NIL 1 11.05 1
II
Type of Sample Chlorpyrifos Metalaxyl S. Pyrethroid
Actual EPA Actual EPA Actual EPA
1 Oranges (Egypt) Nil 1 0.006 1 Nil NA
2 Banana ( Ecuador) 0.53 0.1 0.41 NA Nil NA
3 Cabbages 0.34 1 0.61 1 Nil 6
4 Potatoes (Cara type) 0.261 0.05 0.02 0.5 Nil 0.05
5 Lettuce (Cabbage form) Nil 2 Nil 5 Nil 20
6 Cauliflower 2.751 1 Nil 1 Nil 1
7 Apples (Sicily) 4.721 1.5 Nil 0.2 12.071 0.05
8 Oranges (Sicily) Nil 1 0.70 1 Nil NA
9 Strawberry 7.061 0.2 Nil 10 Nil NA
10 Banana (Ecuador) 1.51 0.1 0.49 NA Nil NA
11 Cabbages Nil 1 0.76 1 Nil 6
12 Potato {Alpha) Nil 0.05 0.13 0.5 Nil 0.05
13 Lettuce Nil 2 Nil 5 Nil 20
14 Cauliflower 1.02 1 Nil 1 Nil 1
15 Apples (USA) Nil 1.5 Nil 0.2 0.861 0.05
16 Strawberry Nil 0.2 Nil 10 Nil NA
1 These values atre somewhat high when compared to the US Environment Protection Agency.
The difference, however may be due to various factors. These may include a different pesti
cide testing positive with the ELISA Kits. The values supplied by the EPA are those for the
pesticide tested as indicated at the top of the list.
To verify the actual results which the ones obtained locally, one should have made
an HPLC test to confirm that the pesticide is the same one indicated by the test kit or those
which could be ddtected by the kit.
Conclusion and Recommendations The test carried out show a diffinite pattern of pesticide residuue in all three kits. This is
alarming even though the levels mostly bdo not exceed those established by the EPA. A mistake
of evaluation by the farmer may lead him to use a double dose, thus exceeding the residue level.
This miscalculation may arise out of lack of knowledge of the fact that a pest or vector may
adapt itself to a given pesticide, or that the pesticide was out dated even if bought recently. this is
due to the fact that excess himidity or heat or excessive cold can disable the effectiveness of the
pesticide.
Therefore one concluds be recommending that in future pesticides should not only have
claer (possinbly in Maltese) indications on the way they should be used but also the time needed
between each spraying session and other precautions which might be useful not to endanger the
farmers health and the enviroment.
Ill
The rational farmer must ...
* examine whether his methods are in harmony with certian truths and natural
laws, or whether they hurt them,
* he must constantly keep in mind that the goal of true practice is not only to work
towards the highest yields, but also to make possible that these high yields
always remain just as high year after yea1:
Justus von Liebig
1803 -1873
Contents
Introduction 5-14
Chapter 1: The Emergence of Pesticides Residue Test Kits 15- 22
Chapter 2: Classification of Pesticides 23- 29
Chapter 3: Chlorpyrifos 30- 36
Chapter 4: Metalaxyl 37- 41
Chapter 5: Synthetic Pyrethroids 42-48
Chapter 6: Aims and Methodology of the Study 49-56
Results and Calculations 57- 66
Interpretation of Results 67- 69
Discussion and Conclusion 70-73
Appendices 1 - 3 (Inserts about the Kits) 74- 86
4. A=Apple P=Pesticides the chemicals & pesticides usually used on crops and fruit. 87- 95
Study - Pesticide Residue in Fruits and Vegetables 3
Introduction
Under Maltese Law (Act XI of 2001) the term pesticide includes all plant protection
and biocidal products1• Biocidal products, in this same law means the active
substances and preparations containing one or more active substances, in the form
in which they are supplied to the user, intended to destroy, deter, render harmless,
prevent the action of, or otherwise exert a r;nntmlling effect on any harmful organism
by chemical or biological means2·
Kenneth A. Hassall includes insecticides, fungicides and herbicides together
with rodenticides, nematicides, molluscides and acaricides under the general name
of pesticides. These are agrochemicals designed to control and combat the various
attacks of pests in agriculture and horticulture3 •
Pesticides may be divided into two types - non-systemic and systemic. The
non-systemic were developed to exert their action on the outside of the plant and
thernfore do not penetrate the plant tissue itself. These types of pesticides, tough
they offered protection and relief from diseases, had a major drawback as they were
susceptible to the effects of the elements especially wind and rain. Thus large
amounts of non-systemic pesticides had to be used so as to obtain the desired goals.
Moreover newly grown plants were unprotected against the attacks of pests unless
sprayed repeatedly. These non-systemic pesticides were introduced followlng a rush
to produce more food to cater for the lack of food especially in Third 'Norld Countries.
Today's pesticides are systemic which are designed and produced in such
a way that they can enter the plant's vascular system through the plant's cuticles e.g.
benomly (1967).
But man's fight against insects and crop dise:::tses dates back to the first days
of civilisation. We find for example, various referencP.s in the Old Testament, to crop
diseases and pest which still today ran havoc with our crops, such as the locusts
mentioned as a plague used by Moses against the Pharaoh in Egypt, so that he will
be forced to liberate the Israelites from slavery. These locusts still today destroy huge
areas for crops in Egypt and other parts of North Africa. We also have reference to
1. Act No.XI of 2001 of the Laws of Malta to provide for the control of pesticides, and for other purposes connected therewith or incidental thereto, paragraph - "pesticide"
2. Ditto paragraph - "biocidal products" 3. Kenneth A. Hassall, The Chemistry of Pesticides, their metabolism, mode of action and uses in crop protection, 1982.
-------- ---~-·~
Study - Pesticide Residue iri Fruits and Vegetables 5
the rust diseases - blasting - mentioned by Prophet Amos (760BC)4 and which is
still today responsible for many crop losses. Later on the Greek Theophrastus
(300BC) considered as the father of Botany described may plant diseases such as
scorch, rot, scab and rust5•
The first farmers simply picked off the bugs and pests such as locusts by hand.
In Ancient Rome and Greece, besides magic rituals to protect the vine from moth
attacks, we find mention of the use of chemicals to control insects and pests
destroying their crops. Both Pliny the Elder and Homer make reference to the use of
sulphur, arsenic, soda and olive oil for the seed treatment of legumes. In Medieval
France caterpillars were excommunicated and grasshoppers tried in courts for
attacking crops6•
In the 16th Century the Chinese used arsenicals and nicotine as insecticides.
They also used nicotine in the form of tobacco to control plum curcilic. In the
19th century we find reference to the use of pyrethrum and soap to combat insect
infections as a mixture of tobacco, sulphur and lime against both fungi and insects.
LriffH' on, in tile 17tl1 Century, Justus von Leibig7 and Freid rich Wohler8
established the empirical formulas of many organic compounds, the discovery of new
4. Hough Peter, The Global Politics of Pesticides, Forging Consensus from Conflicting Interests, EarthScan 1998. 5. Debona Helen, Residue of Cypermethrin, Umethoate and Metalaxyl in Locally Harvested Carrots, Univ. Malta, May
1998. 6. Fishbein L., Pesticides Biochemistry and Physiology, 1976, p.555 7. Liebig Justus, Baron von b. May 12, 1803, d. Apr. 18, 1873, was a German chemist whose chief contributions were
in the relatively new field of organic chemistry. They included the analysis and the establishment of the empirical formulas of many organic compounds, the discovery of new compounds, the theory of chemical RADICALS, the hydrogen theory of acids, and agricultural and physiological chemistry. Liebig became a full professor at the University of Giessen at the age of 23, and for the next 28 years the chemistry department there was famous throughout the world. Many of his students, such as August von HOFMANN, Friedrich KEKULE and Charles Adolphe WURTZ, also became famous chemists.
Among Liebig's earliest research was the study of the compounds known as fulminates, which he showed to be isomers of cyanates. He and Friedrich WOHLER collaborated on their famous paper on the benzoyl radical (1832), the first to describe the persistence of a group of atoms in a series of reactions. The also worked on uric acid. Another of Liebig's earlier chemical investigations had to do with perfecting the methods of organic analysis. He devised a procedure of quantitative organic combustion that was used well into the 20th century. In addition, the soil fertility researches of Liebig and others created an interest in artificial mineral manures and led to the rise of the fertilizer industry, even though the fertilizer devised by Liebig was not successful because it was too insoluble. The requirement of microorganisms for growth-accessory factors was postulated by Liebig in 1871.
Before Liebig began to publish his views on physiological chemistry, physiologists paid scant attention to the chemical aspects of their subject. His book Animal Chemistry (1842) drew attention to the need for research in this area even though it contained many erroneous ideas. The most important of Liebig's publications was Annalen der Pharmacia (Annals of Pharmacy) founded in 1832, which became the preeminent chemistry journal. It carried reports on current major developments, particularly those in organic chemistry. Wohler became coeditor in 1840. In 1873, following Liel:>ig's death, the name nf hi<; p1ihlir.atinn wa-; r.hanonrl tn .Justus I inhio's Annnlnn rlnr r.homifl
8. Wohler Friedrich, b. July 31, 1800, d. Sept. 23, 1882, the Germany scientist, discovered the salts known as cyanates in 1822 and within 6 years carried out the first synthesis of an organic compound, urea, by the rearrangement of ammonium cyanate. This achievement began the decline of the vitalistic theory that had dominated organic chemistry and that had requirerl thfl existenc:e of a "life force" for organic synthesis. Wohler's work on cyanates brought him in contact with Justus von LIEBIG. In 1832 they published a memorable investigation or benzaldehyde, important because it showed the existence of a radical in a series of compounds. Together they edited the Annalen der Chemie. Wohler later published many analyses of minerals; prepared phosphorus, nickel arsenide, crystalline boron, and silicon; and discovered silicon hydride.
9. The mixture known as Paris Green was discovered in Paris (France) and is a very poisonous copper based solution of a bright green colour. It was used as an insecticide and for pigmentation as early as 1868. It was found that it could control leaf eating insects. Together with Bordeaux Mixture was among a number of pestiddes discovered in the 1880's and consist of copper, arsenic salts and copper oxide.
Study - Pesticide Residue in Frutts and Vegetables 6
compounds, the theory of chemical radicals, the hydrogen theory of acids, and
agricultural and physiological chemistry.
The use of an impure copper arsenate known as Paris green (1867)9 to control
the Colorado beetle was so widespread in the second half of the 19th Century that
in 1900 in the USA, we find probably the first law in the world, enacted to control the
use of pesticides.
The Bordeaux Mixture10 was introduced in 1885 to control downy mildew
in vines, while a few years later the same mixture was used to control weeds. As time
passed, more and more inorganic substances were introduced to control pests and
diseases encountered in agriculture.
Other important dates worth noting was the use of organic seed dressings in
1913 Dlnitro orthocrosol was patented in France in 1932 , while thiriam was
patented in the USA in 1934. In 1945 the first soil acting carbonate insecticide was
discovered in Britain. However it was not until the Second World War that another
revolution occurred in bug and insect killing. DDT11, one of the first synthetic
organochlorine pesticide was developed. Its inventor Dr. Paul Muller (Swiss) won the
Nobel Prize, such was the euphoria with which this new pesticide was hailed.
However five years later, it was noted that, insects started to develop resistance to
these pesticides. Thus a new generation or organophosphates based on nerve
gases came into being. Unfortunately these chemicals were first used in the Nazi
Concentration Camps of Aushwiz (Poland). "What worked against people could also
kilf bugs" 12 •
These chemicals deferred from their predecessors in two ways:-
a). they were entirely synthetic (developed in the laboratory),
b). they had the ability to attack the nervous systems, killing or disabling
the pest with deadly precision.
Between 1950-55 derivations of the herbicide urea were developed in USA.
During these years captan glyodioros and malathion were introduced. In the 60s we
come across triazine, trifliudin and bromxyltogether with benomyl and the soil acting
glycophosphAtA.
In the post war era such was the extent of development of synthetic pesticides
that it became clear that out of 800 or more substances developed only one was a
10. Bordeaux Mixture is made up of slaked lime and copper sulphate. It was discovered in 1882 and was later used on a broad range of plants and fruit trees. It was discovered by the French botanist Millardet Alexis. It was used as a preventive fungicide and insect repellent and is still in use.
11. DDT = dichlor-+diphenyl-+tricholr- (fr.tri-+chlor) [C 14H9CI 15] is a colourless, odourless water-insoluble crystalline insecticide that tends to accumulate in ecosystems and has toxic effects on many invertebrates was recognised a potent nerve poison on insects. It was used to combat yellow fever, typhus an elephantiasis and other insect-vectored diseases. In India malaria was reduced from 75 million cases to a 5 million cases in a decade. However after the publication of Carsons Silent Spring in 1962 suspicion grew that DDT by entering the food chain caused reproductive dysfunctions such as thin egg shells in certain birds. Some insects gradually became resistant to it with their population growing unchecked while their natural predators were being destroyed by spraying.
12. Nikki van der Gaag, Pick your Poison, New Internationalist, May 2000.
Study - Pesticide Residue in Fruits and Vegetables 7
winner. Moreover the cost of its development was such that the manufacturer was
lucky to break even and earn back his expenses before ten years.
After World War II, when the use of synthetic pesticides and chemicals in
agriculture grew to such an extent that it became obvious that they were causing harm
to man and the environment, The Food and Agriculture Organization (FAO)
together with most governments introduced rules and regulations to prevent the
indiscriminate use of pesticides and at the same time to protect the farmer, his family
and those around them, against health hazards. Later on these rules and regulations
were extended to protect domestic animals and the environment.
Back in 1959 both FAO and The World Health Organization (WHO), insisted
that governments should include in their respective countries, bodies or organizations,
to be involved in agricultural rmstir.ido, plant and animal protection, advising them
about the levels of synthetic pesticide residue levels in food and animals for human
and animal consumption. These bodies were created alongside the Public Health
Authorities that each and every country already had.
New Concepts Moreover the Rome meeting of October 1961 recommended to governments
the setting up of principles to establish pesticide residue levels in food. This meeting
also established the concept of Acceptable Daily Intake (ADl's), together with the
principle of tolerance compatible with the Maximum Residue Levels (MRL's).
These have to be estimated taking into account the range of residue actually
remaining when the food is first offered for consumption following Good Agricultural
Practice (GAP).
During November 1962, the Conference on Pesticides in Agriculture convened
by FAQ in Rome, expressed concern about the differences in residue tolerance not
only between different countries but also between the regions of the same country.
This led the meeting to setup a working party to pay particular attention to ...
a. the toxicity of pesticides and test methods,
b. t11e possible unification of tolerances,
c. coordination of methods of analysis,
d. surveys collecting residue data,
e. the establishment of a list of pesticides to which interested
governments should give research priority13•
But it was in Geneva 1963 that for the first time the FAQ Committee studied a
number of pesticides and established a few ADl's. In December 1963, the working
13. Food and Agriculture Organization, FAO Manual on the Submission and Evaluation of pesticides Residue Data. Rome 1999.
Study - Pesticide Residue in Fruits and Vegetables 8
party on Pesticide Residue studied ways and means to arrive at recommendations for
levels of residue tolerance. The following were considered essential.
i. residue levels resulting from Good Agricultural Practice (GAP) should
be obtained by FAO from governments and pesticide manufacturers.
These data should be considered by the FAO Working Party on
Pesticide Residue. After consideration of the ADI and of the national
nutritional patterns as stated in the FAO Food Balance Sheets, the
Working Party would propose tolerances for residue on individual crops
for consideration by governments and by the Expert Committee on
Pesticide Residue of the Codex Alimentarius Commission;
ii. residue found in surveys of marketed commodities;
Ill. ADls to be estimated by joint meetings of the WHO Committee on
Pesticide Residue and FAO Committee on Pesticides in Agriculture;
1v. national nutrltlonal patterns and
v. acceptable analytical methods for residue. These methods should be
adopted by the Pesticide Committee of the Codex Alimontarius.
Following this, the FAO Panel was entrusted with the definitive responsibility of
reviewing, from time to time, pesticide use patterns in GAP; the data and the
composition of pesticides; environmental fate; metabolism in farm animals and crops;
methods of analysis for pesticide residue and for estimating MRLs and Supervised
Trial Median Residue values (STMRs). Where no ADI was not yet estimated the FAO
Working Party has to propose provisional tolerances.
It must be emphasised that residue levels deriving from supervised field trials
can only be used for estimating MRLs, if the trial conditions can be matched with
relevant national GAP.
The current committee studying the effects and results of studies on pesticide
residue formed by the WHO Toxicological and Environmental Core Assessment
Groups and the FAO Panel of Experts on Pesticide Residue in Food and the
Environment.
The WHO group reviews pesticides toxicological and related data and
estimates No Observed Adverse Effect Levels (NOAELs) of pesticides and Acceptable
Daily Intakes (ADI) of their residue in humans. The group also estimates Acute
reference dose (acute Rfd) and characterises other toxicological criteria such as non
dietary exposures. It also identifies the risks to organisms in the environment.
A New Phase: Organic Agriculture The name organic chemistry originated at the beginning of the 19th century, when
----·------------·------Study - Pesticide Residue in Fruits and Vegetables g
scientists wished to differentiate between those substances derived from plant and
animal (organic) sources and those derived from inanimate (inorganic) materials.
Organic substances generally had more complicated compositions than did inor
ganic materials, and the scientists of the day were unable to synthesize any of these
organic substances in the laboratory. It was believed that organic substances had
special qualities and could be created only in the presence of the vital force found
in living organisms. Even though the vital force theory was eventually disproved, the
classification of chemical substances as organic or inorganic has continued to the
present.
The modern usage of organic chemistry refers to the chemistry of compounds
containing carbon, but this definition should be further clarified, because compounds
such as carbon monoxide, carbon dioxide, and calcium carbonate, are considered to
be inorganic. A better definition of organic substances is that they are generally
characterized by chains of connected carbon atoms. More than two million of such
organic compounds are known.
Many ofthoso arc natural products or compounds found in nature. The study
of the large organic molecules found in living systems and their reactions, which make
up the life processes, has come to be called biochemistry. A large number of the
known organic chemicals have been synthesized in the laboratory, and our society
is dependent on such synthetic materials as plastics, synthetic fibres, dyes, deter
gents, and insecticides.
The chemical and allied product industries contribute a large portion of the
gross national product of the United States, and more than 150,000 people are
employed as chemists. The vast majority of synthetic products are derived from
petroleum, and as the world's supply of petroleum decreases, new sources of carbon
containing raw materials will have to be found. Also, it has become apparent that
many synthesized compounds have deleterious effects both on the environment and
on living organisms. Future developments in organic chemistry must take these
effects into account.
At first il µroved lo be impossible to prepare any of these compounds in the
laboratory, and the belief was held that their synthesis involved a vital force present
only in living organisms. But in 1828, the German chemist Friedrich Wohler uninten
tionally converted ammonium cyanate, a purely inorganic substance, into urea,
which is an end product of animal metabolism. This discovery set the stage for the
eventual ove1ihrow of the vital force theory. Over the next 20 years other organic
compounds were synthesized, and as evidence accumulated, the vital force theory
was slowly abandoned.
Wohler's preparation of urea had another important consequence in that it
Study - Pesticide Residue in Fruits and Vegetables 1 O
furnished an example of two substances, urea and ammonium cyanate, which had
the same chemical composition but very different properties. As experimental
techniques improved, it became clear that there were other examples of more than
one substance having the same composition. In the 1830s Berzelius14 used the term
isomerism (composed of equal parts) to describe this phenomenon, but an
understanding of isomerism was not possible until the structure of these compounds
became understood later in the century.
The mid-19th century saw the steady development of systematic organic
research. A key step in this development was the establishment of the idea of radicals
as the organic equivalent of atoms. Radicals were groups of atoms that retained their
chemical identity during a chemical reaction. In simpler compounds these radicals
contained only hydrogen and carbon (hydrocarbon radicals). In 1832, Wohler and
Justus von Lei big produced a series of compounds all containing the benzoyl radical,
and their worl~ cncoumgcd othom to discover new radicals by systematic reactions
of organic compounds.
Organic Chemistry and the Environment
Peter Rosset15 says that our dependence on pesticides is focused on the obvious
problem, that is getting rid of pests without looking deeper into a farming system out of
balance. The search for better pesticides has led us to forget that nature itself by using the
crop rotation system and predator insects can actually control pests and diseases.
Organic research has produced many compounds that have benefited society as a
whole. At the same time many of these synthetic substances have been found to have
deleterious effects not only on the Earth's environment but on animals and human beings as
well.
An ever-increasing number of organic substances have proved to be carcinogenic or
cancer-causing 16• Since the induction period between exposure to a carcinogen and the onset
of cancer can often be years, determining whether or not a substance is a carcinogen is not
always a simple matter. Certain classes of compounds are known carcinogens. A particularly
dangerous group of compounds includes the aromatic amines, which have been widely used
14. Berzelius, Jons Jakob, 1779-1848 was a Swedish chemist. Taught medicine and pharmacy at Stockholm (from 1807) and chemistry (1815-32). Created baron ( 1835). Determined atomic and molecularweights of thousands of substances, using oxygen as a standard; experimented in electrolysis and developed the dualistic theory originated by Lavosier; discovered the elements cerium (1803), selenium (1817) and thorium (1826, and first isolated silicon (1823), zirconium)1824), titanium (1825); introduced present system of writing chemical symbols and formulas; improved analytical methods, esp., the blowpipe method; studied and named phenomena of isomerism and catalysis.
15. Peter Ros set, Killing the chemical Habit, New Internationalist, May 2000. He is also the Executive Director of Food First - Institute for Food and Development Policy in Oakland, California.
16. Dr. Theo Colborn, Crossed Bills and Broken eggs .. New Internationalist, May 2000. By accident while working on a project about the environment of the Great Lakes around Michigan, she noted that many animals had sexual problems and that their babies died unexpectedly. This rang the alarm bells leading her to believe that the chemicals used in the area had the ability to cross the placenta. Later on studies carried out on people who ate from the fish of the Great Lakes showed that they had children with lower IQ.
Study - Pesticide Residue in Fruits and Vegetables 11
in the production of dyes. An example is 2-naphthylamine, which has been banned in many
parts of the United States because it is known to produce cancer in at least 50% of the people
exposed to it.
Another group of carcinogens, also important in the dye industry, includes some of the
azo dyes, which contain a doubly bonded pair of nitrogen atoms attached to aromatic rings.
An example of acarcinogenicazo dye is methyl(butter) yellow (4-Dimethylaminoazobenzene),
used in the past to colour butter.
A widely publicised example of a carcinogenic organic molecule is diethylstilboestrol
(DES), a synthetic female sex hormone whose use has now been severely restricted. DES
was commonly used in the United States as an addition to cattle feed because it caused cattle
to fatten faster, even though it had been known since 1940 to cause cancer in animals. DES
was also taken by a number of pregnant women to prevent miscarriage, and it has now been
found that DES can produce a rare form of cancer in these women's daughters.
A group of chemicals that are not carcinogens but are still dangerous includes various
pesticides. As already argued the use of pesticides has served to increase the world's food
supply and decrease disease-cArrying pests, but many of these pesticides, particularly the
polychlorinated hydrocarbons, have generated controversy because of their persistence
in the environment. An example of such a hard insecticide is 2,2-di(p-chlorophenyl)-1, 1, 1-
trichloroethane (DDT). DDT is extremely stable and persists for years in the environment.
Consequently, it moves through the food chain and gradually builds up in plant and animal
tissues. Although there is no evidence that normal use of DDT has ever harmed humans, it
has proved harmful to fish and birds of prey, and the use of DDT has now been severely limited.
Two other classes of pesticides are now widely used and are known as soft
insecticides because they decompose more rapidly in the environment than the polychlorinated
hydrocarbons.
These groups are the organophosphates, an example of which is Malathion and
Chlorpyrifos, and the carbamates, an example being Sevin. Even these must be used with
care, however, since they are highly toxic.
Andrew Watterson call pesticides the The 13 Klllers17• He includes the following
under the term pesticide.
weed killers
Insecticides
fungicides
aracldes
nematocide
rodenticides
also known as herbicides
which kill insects
which kill fungi, including mould.
which kill spiders
which kill, round, thread or eel worms
which kill mice and rats
17. Andrew Watterson, Pesticides and your food, Green Print 1991.
Study - Pesticide Residue in Fruits and Vegetables 12
alglcldes
mlticldes
which kill algae
which kill mites
molluscldes
growth regulators
which kill snails and slugs
which stimulate or retard plant growth
defoliants
desiccants
attractants
Conclusion
which remove plant leaves
which speed plant drying
which attract insects e.g.. phoromes.
In concluding we must say that man seems to have learned to look around him and
having observed profoundly, is succeeding to ask questions which take him I her beyond what
we see around. From past experience mankind may perhaps have learnt a lesson.
Today we are confronted with the Mad Cow disease -BSE- or Bovine Spongiform
Encephalopathy18• This disease was first evidenced in the United Kingdom about 10 to 15
years ago. It was brought about, according to many, especially in Europe by the fact that man
have started to feed cattle to cattle19, th;:it is WA fA<i thA vAgAt;:iri;:in cow with dead anim;:il
carcases as if it was a carnivorous animal. This we did by feeding the cows with animal
remains under the form of flour. As Colin Tudge outlined recently in an interview in a local
paper, we have created an new food chain when we changed the cow's natural pastures to
synthetic feeding even though they contained animal remains. At least this is what most are
thinking. And there is ample proof that there is the human version of BSE- Creutzfeldt-Jackob
disease. Most think that man was infected with the animal version.
We are therefore not surprised to see people rushing and demanding healthier,
cleaner and safer foods and animals produced or grown biologically or organically. This is
nothing new but using the past methods with the past experience in mind, mostly leaving
nature to take its course helping it by using what nature itself produced without the need to
synthesize it in the laboratories.
What is biologically grown is on the increased. So much so that last May (2001) many
European experts meet in Denmark to launch the European Organic (biological) Plan for the
Old Continent.
And this is not surprising when only a few weeks ago we noted England and Germany,
among the first countries to denounce the factory farms - started at the turn of last century in
England - and taking steps to abolish them in favour of going back to the old natural ways by
which our fathers used to provide us with fruits and vegetables. Yet keeping in mind the
lessons we learnt from the past. 18 Creutzfeldt-Jakob disease also Creutzfeldt-Jakob disease. Hans G. Creutzfeldt was a German psychiatrist. It is a rare
progressive fatal encephalopathy caused by a slow virus and marked by development of porous brain tissue dementia in middle age, and gradual loss of muscular coordination.
19. Colin Tudge, Back to Basics, Malta Independent, 28th April 2001. And Press Conference at Bay Street Complex, St. Julians 26th April 2001. Tudge is an expert on Organic Agriculture, studied zoology and wrote 12 books on the subject.
20. Press Release by Agenr.y France Presse, Intensive 18.rming Blamed for Deteriorating Water Quality in France, May 18,2000
Study - Pesticide Residue in Fruits and Vegetables 1 3
In France, Jean-Claude Lefeuvre who conducted a study about the pig population in
Brittany for the World Wildlife Fund (WWF) was cited as saying that the water quality is
detoriorating because of intensive farming methods when a population of 8 million pigs
produce as much excrement as a city of 24 million people.
Moreover in his report conducted in 1981, in the Northwest of France, he concluded that
the heavy use of nitrate fertilizers and manure was responsible for the sharp fall in the quality
of water in streams, rivers and lakes20•
In this scenario therefore it is not surprising to find companies, associations, universi
ties and Non-Governmental Organization (NGOs) working to produce better and healtier
food. As time goes by the studies regarding pesticide residue and pest I vector resistance
are on the increase. Not only so but reviews are carried out from time to time to check and
re-check the results and findings.
These organizations are from time to time developing better equipment which
would be faster, efficient, reliable nnd ohenp. Among these are the lmmunosorbent
Assays Test Kits (ELISA), These are being developed to give quick results on the
field and stores where food is produced or stored. In sn rlning w;:,stP. of timA is
reduced. These kits give results minutes as compared to laboratory tests which often
take days.
Throughout this study I have used ELISA kits for three pesticides which I think
are widely used locally. These are Chlorpyrifos, Metalaxyl and Synthetic Pyretroids.
The subject and studies about these pesticides are so vast that it is almost impossible
to summarizes them in this study. However I did my utmost to give a short clear
picture about them, the way they work and their impact on plants, vegetables, fruit
and the environment in general.
20. Press Release by Agency France Presse, Intensive farming Blamed for Deteriorating Water Quality in France, May 18,2000
Study - Pesticide Residue in Fruits and Vegetables 14
The Emergence
of
Chapter1
Pesticide Residue Test Kits
Every person on earth
has absorbed at least
250 synthetic chemicals
into their body during his I her
lifespan
Niki van der Gaag
Study - Pesticide Residue in Fruits and Vegetables 15
Introduction
In recent years, new relatively inexpensive analytical test kits have been developed for the
rapid testing of pesticides in foods and water. Many manufacturers of these kits boast that
users can get results faster and cheaper than ever before, and thatthe kits can detect pesticide
residue at levels as low as or lower than the conventional solvent extraction/gas chromatography
based multi-residue methods.
But are these new methods as good as kit manufacturers claim? What are their best
application? What are their limitations, if any? Have new kits made expensive instruments
tests obsolete? For that matter, do food companies really need to worry much about
performing routine pesticide residue testing?
While there are no simple answers to these questions, considP.n:ition of GP.nP.ral Mills
Inc., recent pesticide fiasco can give some useful insight into the need for pesticide testing
and how to do it.
In 1995 General Mills1 learned that some 21 million bushels of oats were tainted with
an unauthorized pesticide. As a result, GM destroyed approximately 50 million boxes of
Cheerios and Lucky Charms and 15 million bushels of raw oats. The pesticide that was
applied by one of the farm workers who supplied the oats to GM, Chlorpyrifos-ethyl, is
approved for use with some grains but not oats. This episode is said the have costed General
Mills Inc. about $140 million or Lm 52 millions.
This suggests that food companies should pay morn alle11Lio11 Lo peslicide con lamination
problems and how expensive such a problem can be. For this reason a number of companies
have developed a series of multi-residue test methods and Enzyme-Linked Im mu no Sorbent
Assays (ELISA). Such kits provide a more or less exact indication of the presence or absence
of most of the known pesticides commonly used worldwide in agriculture.
These ELISA kits have been produced in order to secure health safety to the farmer,
his family and the environment before much hazards have been produced which might
become unrepairably. As Nikki van der Gaag2 says man is believed to absorb about 120
synthetic pesticides during his lifetime.
Health safety was the spin-off invention leading to the development of these kits. As
a matter of fact the first !{its wore invented and teGted by the /\merioan /\rmod Forooa,
especially the Infantry, in order to supply their members with a quick indicator showing the
1. Villani Joe, Pesticide Testing - What's the Best way? The Scientist 7 Feb. 1994 2. Nikki Van der Gaag, Pick your Poison, the price we pay for using pesticides, New Internationalist, May 2000. 3. Villani opus cit.
Study - Pesticide Residue in Fruits and Vegetables 16
presence of poison in drinking water when they are cough behind enemy lines. The stranded
pilot, sailor or soldier wanted to know immediately whether his supply of drink found on
enemy territory was fit for drinking, surviving to tell his story on his return home.
Before the advent of these test kits one had to take his produce to a laboratory, where
one had to wait for a couple of hours if not days before getting a clear indication of the residue
and their levels in a given product. These kits are qualitative because they just indicate the
presence or not of pesticide residue even though these can be planned to give as accurate
a reading as possible. The need for these kits rose from the great number of pesticides being
produced to counter each and every pest, insect or vector on earth so as to produce more food.
It is estimated that there are about 11,000 registered pesticides in use world wide.
These assays are generally designed for measuring specific analytes in water. The
human exposure or Biological Exposure Monitoring (BEM) assays are optimised for urine
samples, while the agricultural/crop/food assays may be designed for soil, seed or raw plant
samples. The product insert that accompanies each assay specifies the design matrl')C in
which the analyte is expected to be found. This is important to understand in immunoassays
as the matrix, or the solwmts used to extract the analyte from the matrix, may have an effect
on running or properly interpreting the assay. These influences on the assay are called matrix
effects. They may result in false positive or false negative results.
When properly applied, rapid test kits for pesticide residue analysis are an invaluable
tool for food companies. For Example Newton, PA-based Oh micron has developed a panel,
of immunoassay kits for residue that are very difficult and costly to detect by traditional
techniques. These include 1,4-D, benomy/, paraquat and captan. The company has several
magnetic separation immunoassays kits available for analysing herbicides, insecticides,
fungicide, and environmental contaminants in food. For most kits, detection limits are in the
10 to 50 ppb range and analysis time is less than 60 minutes.
Enzyme-linked immunosorbent assays combine selective antibodies with sensitive
enzyme reactions to produce analytical systems capable of detecting very low concentrations
of chemicals.
If a company has a potential pesticide crises and it has identified the pesticide
contaminant, then the screening tests are extremely useful. For example, once General Mills
Inc. had learned that its oats supply was potentially contaminated with Chlorpyrifos-ethyl, it
could have used one of the kits to screen its oats and cereal supplies to decide which ones
had to be destroyed.
In crises-management situations, companies are often forced to test hundreds of
samples and make decisions rapidly and it could be impossible to keep up with analytical
testing demands with traditional solvent extraction/gas chromatography methods. These test
kits provide the benefits of reduced testing time, reduced solvent consumption and disposal,
and reduced cost per test.
Study - Pesticide Residue in Fruits and Vegetables 17
One limitation of the ELISA based kits is specificity. The specificity of the ELISA tests
is described in terms of its antibody cross-reactivity to other related compounds. For example
Envirologix's4 Chlorpyrifostest kits measurers not only this pesticide but other analogues as
well, including Chlorpyrifos-methyl, Diazinon, Quinalphos, Primiphos-methyl and others.
Each of these chemicals responds differently to kit reagents.
What all this means is that it is difficult to get accurate quantitative results for specific
pesticides. Never the less, the kits are an excellent screening tool. Frequently, companies
use ELISA screening test to find potentially contaminated samples and then test for
confirmation. Compared to ELISA tests, gas chromatography-based tests offer greater
specificity but are more costly, time consuming and require larger volumes of samples.
Another limitation of ELISA tests is that there may not be a test kit developed for your
particular contaminant. There is for example, no kit to test for alar, the pesticide used on
apples, that caused such an uproar a few years ago in the United States.
Another company markets a pesticide test l{it based on Ml Neogen. Its AgriScreen™
Ticket is an enzyme-based test method that rapidly detects the presence of approximately 50
different carbamate, thiophospahate and organophosphate pestir,i<iP.s.
The test is less specific than ELISA tests and is not quantitative. It has been used for
measuring pesticide levels in air, water, soils, crops, spills, solvents, surfaces and produce.
The test is based on the reaction between the pesticide and an enzyme impregnated on a filter
disc. It is very sensitive to choline esterase inhibitors like carbamates.
Since other chemicals besides the 50 pesticides will inhibit choline esterase, testing
may provide a false positive. If you're unfamiliar with the samples you are testing, there is a
chance of getting erroneous results. However if the test kits provide a positive result the
traditional methods of confirmation should be used.
The first evaluations of ELISA kits were carried out under the EPA Superfund Innovative
Technology Evaluation (SITE) programme in 1989. Commercial tube and 96-well plate kit
format immunoassay kits for the determination of pentachlorophenol (PCP) were evaluated
by comparison of kit results with conventional gas chromatography (GC) and GC-mass
spectrometry results. The results showed both the tube and plate immunoassays to be
sensitive screening methods. Though slightly less accurate, they were faster, less expensive
and had a higher sample throughput than GC analysis5•
Extending the Assay Range Each product insert found with tho tost kit, spocifios tho range of nnnlyto concentrations
over which the assay is most effective. One may wish to decrease the sensitivity of an assay
4. Envirologix's leaflets supplied with ELISA Kits .... See appendix I. 5 Environmental Sciences Division, Las Vegas [online} http://www.epa.gov/esd/chemistry [as dated] 23rd Feb. 2000
Study - Pesticide Residue in Fruits and Vegetables 18
- testing samples with 1 Oto 100 parts per million (ppm) rather than the 1 to 10 parts per billion
- around which the assay may have been designed. In cases where the one prefers the assay
to detect an analyte at even lower concentrations than the limit of detection, it may be possible
through concentration of the analyte.
As already argued different types of assays have been developed in pesticide residue
analysis. Such assay can also detect human exposure to a pesticide.
Human Exposure Companies developing these kits have long believed that detecting and measuring
human exposure to potentially toxic materials is a significant and largely unmet need. Current
methods generally rely on broad spectrum analysis through gas chromatography or high
performance liquid chromatography analysis. These techniques are certainly we II documented,
but they are also time consuming and expensive, and usually require rather large samples for
cleanup, concentration and extraction.
To meet this need, they have developed several low-cost, rapid and sensitive
imm1 mo;:iss;:iys for rnutinA usA with urine samples. The initial suite of human exposure assays
monitors the urinary metabolites of three of the most widely used herbicides in the United
States, as well as DEET, the very popular insecticide which, among other applications, was
widely used for troop protection during the Gulf War.
Human exposure to pesticides and industrial chemicals can arise in a number of ways,
either through direct exposure in the workplace or through unaware downwind or downstream
intake. Chemicals may be internalized in the body by ingestion, inhalation, absorption through
the skin or by introduction through cuts and abrasions in the skin.
Work place Exposure Work place exposure may arise through applying pesticides or industrial chemicals,
through merely handling them or mixing them for later application, through premature re-entry
into recently sprayed areas, through contact with plants or soil, or in the process of cleaning
up contaminated sites and transporting or disposing of contaminated soil and water.
Indirect Exposure Indirect airborne exposure can materialize from over spraying, wind drift, and home
or work place application of control chemicals. Indirect water-borne exposure can result from
over spraying settling onto a stream or lake, from chemicals leaching into surface water from
nearby sprayed or contaminated areas, or from leaching into underground water supplies
which become part of public or private water supplies. Tertiary or more remote, but still
potentially significant, exposure can arise through evaporation of moisture from sprayed fields
Study - Pesticide Residue in Fruits and Vegetables 19
which yields precipitation many miles downwind from the spray site in areas of human use or
in otherwise protected water supplies.
Studies have shown that many people were contaminated through this kind of
exposure even many miles away6. High levels of pesticide residue were found in the Arctic
in the bodies of Inuit people, polar bears and other wildlife who have never gone near such
chemicals. This could have happened only through water movements in the rivers and seas
and air which flow from places long distances away. Take the flood in Mississippi in 1966.
From aerial photos one can see the floodwater flowing down the rivers into the Gulf of Mexico.
The currents took them across the North Atlantic via the Gulf stream. Six weeks later they were
off the coast of Newfoundland. The rivers, seas and oceans are not separate. They mix
together. The same happens with air and air currents. Many of the chemicals take a long
timo to break down in the body. DDT for example has a half life of 57 years in temperate zones.
Why test metabolites? Many chemicals break down into harmless metabolites after exposure to sunlight.
Many others, however, remain intact until they are processed within the human system where
they form metabolites or combine with other elements to form new compounds. Frequently
the original pesticide or industrial chemical is not detectable in human samples such as urine,
saliva or serum, but one or more metabolites can be detected as markers of the human
exposure.
Specific benefits from immunoassay testing Until recently, regular prevention and testing programmes were limited to the very
highest risk exposures and bare minimum testing intervals. This was due to the high cost of
tests per individual, slow turnaround of results (typically expressed in weeks) and the resulting
inability to relate increased exposure to specific work conditions or practices.
With immunoassay test programs, the results can be documented and fed back to
supervisors or employees on the spot or overnight, allowing corrective action immediately,
and encouraging timely follow-up to reinforce the positive risk control benefits of appropriate
procedures. Moreover costs for immunoassay tests start from Lm 1.75c to Lm3.50 pertest,
in comparison to Lm30 and Lm80 per sample for historical analytical methods, the economic
barrier to effective risk control is essentially removed.
6. Dr. Theo Colborn, Crossed bills and Broken eggs, New Internationalist May, 2000.
Study - Pesticide Residue in Fruits and Vegetables 20
Food and Beverages Selected assays have been designed specifically for use in detecting pesticide residue
in foods. The kits targeted for foods include several of the more commonly used fungicide and
insecticides and the Broad Screen Cholinesterase and Organochlorines assays.
Fruits and vegetables are frequently treated with pesticides during the growing season
and specificallywith fungicide to protect them during storage and shipment. Some Envirologix
kits were developed in conjunction with the Commonwealth Science and Industry Research
Organization (Australia), the Grape and Wine Research and Development (Australia) and the
Dried Fruits Research and Development Council of Australia. Thousands of successful
assays have been run with samples of dried tree fruits (such as pears, peaches and apricots)
and dried vine fruits (such as raisins and sultanas), as well as wine and juices.
Crops & Plants From pre-planting diagnostics to hybridization and plant genomics; to seed protection;
to in-store labelling support, these immunoassay kits play a significant role in protecting crop
quality and reducing production costs. Immunoassay kits are effective in fungal and toxin
detection, pesticide monitoring, genetic trait detection, and seed coating monitoring. As in
other applications, the characteristics of low cost, simplicity, high specificity, high sensitivity
and speed permit extensive screening with real-time results.
Typical applications include:
*
*
*
*
*
Detecting Transgenic Markers
Pythium Detection
Glycoalkaloid Monitoring
Seed Coating Quality Assurance
Pesticide Monitoring as they are used in this project
Pesticide Monitoring Envirologix pesticide kits can also be used to determine active pesticide levels in soil
before planting or before further application of control chemicals. Recent studies on aldicarb
and metalaxyl have documented decreasing effectiveness of the pesticides in certain
conditions of heavy and repeated use. Although still preliminary, these data suggest the cause
may be pesticide resistance in tile target species (long a concern of producers and users
alike). Another phenomenon may also be developing; this is enhanced degradation in which
nontarget bacteria in the soil actually develop a preference for the fungicide as a nutrition
source, metabolize it, and compromising the fungicide's ability to attack fungi.
Study - Pesticide Residue in Fruits and Vegetables 21
EnviroLogix pesticide assays, with their low cost and rapid results, can be used to screen
both active fields and adjacent buffer zones, for example, to compare the effectiveness of
fungicide in target and control soils. Results of the assays can help predict effectiveness of
additional applications and point to either alternative treatments or crop rotation to fields where
the degradation effects and mutant bacteria are not found.
Study - Pesticide Residue in Fruits and Vegetables 22
Chapter 2
Classification of Insecticides
Their Composition and Properties
The popular outcry
againsl llleir use (peslicides)
cannot ovrershadow
their use
Food Agriculture Organization
Study - Pesticide Residue in Fruits and Vegetables 23
Introduction
Despite the increase in food production due to technological advances in agriculture,
enormous amounts of harvested foods are wasted due to inadequate protection of stored
products. According to an FAO estimate, the losses due to insect infestations in Third World
countries amount to about 10% or more. These can potentially be greater now that many
countries are establishing national or international buffer stocks of foodstuffs to guard against
irregularities in production due to the unpredictable climatic conditions.
The popular outcry against the use of synthetic pesticides in agriculture cannot
overshadow the necessity of their use so L11al µr oleellon of Lile eonsumers of lrealecl µrocluee
and education of the users of the chemicals is imperative1•
Chemical pest control methods, if carried out intelligently and knowledgeably, can be
both effective and safe. But it is extremely important for users to have a knowledge of the
classification, mode of action, properties, metabolism and residue of the pesticides, to enable
them to make proper appraisal of the benefits and potential hazards of the pesticides. Thus,
they should be able to choose insecticides judiciously and formulate efficient control
measures in any particular set of circumstances. Moreover field operators must adhere to
the safety measures required before, during and after the application of pesticides. These
include the wearing of masks, protective clothing and observation of entry times especially
in closed quarters such as green houses.
Besides studies must go on to have a clear picture of each and every pesticide so as
to evaluate the physical and chemical properties of the pure active ingredient in order to
recognise the influence these properties have on the behaviour of the pesticide during and
after its application on crops and animals.
CLASSIFICATION OF INSECTICIDES Insecticides are classified according to their mammalian toxicity, chemical origin or
composition, mode of entry, and formulation.
Mammalian Toxicity
Toxicological studies are conducted to determine the threshold limit of a chemical
which an animal or human is capable of handling without significant biological effects. The
1. Food Agriculture Organization, Manual on the Submission and Evaluatiom of pesicide Residue Data., Rome 1999.
Study - Pesticide Residue in Fruits and Vegetables 24
usual beginning in any toxicological evaluation is the assessment of the acute toxicity, i.e. the
effects of a single dosage of the chemical. The general technique is the determination of the
LD50 (the dosage necessary to produce death or reproducible effect in 50% of the animal
population tested). The compound is administered on a weight/weight basis (milligram or
gram of compound per kgm of body weight of test animals) in a suitable solvent or suspension
system.
This is evaluated by
acute tests, orally (AO) or
dermal (AD);
chronic oral tests (CO),
vapour toxicity tests (VA) and
chronic vapours tests (VC) or
inhalation tests (IT).
Insecticides can be classified according to their toxicity based on the LD50 values:
1. Highly toxic
AO LD50 = 0-50 mg/kg
AD LD50 = 0-200 mg/kg
IT LD50 = 0-2000 ug/I
Danger, skull and crossbones and poison on label.
2. Moderately toxic
AO LD50 = 51-500 mg/kg
AD LD50 = 201-2000 mg/kg
IT LC50 = 2,001-20,000 ug/I
Warning on label.
3. Slightly toxic
AO LD50 = 501 -5000 m!'.]/k!'.]
AD LD50 = 2000-20,000 mg/kg
IT LC50 = more than 20,000 ug/I
4. Relatively nontoxic
AO LD50 = 5000 + mg/kg
AD LD50 = 20,000 + mg/kg
Generally, the insecticide used in stored product treatment is of low mammalian
toxicity (Table 1 below) in a formulation that is likely to be effective against the species
Study - Pesticide Residue in Fruits and Vegetables 25
involved, persistent for the required period of time under given storage conditions and will not
alter the flavour, colour and odour of the stored commodity.
Table 1. Acute oral LOSO (mg/kg body wt. rat) of Insecticides used for storage pest control.
INSECTICIDE ORAL (RAn DERMAL
1. Malathion*** 1375-2800 4000-4800
2. Pirimiphos methyl*** 2050 2000
3. Chlorpyrifos methyl* 1650-2100 3000
4. Tetrachlorvinphos* 4000-5000 5000
5.Bromophos* 4000-8000 2188
6. Dichlorvos* • 80 107
7. Fenitrothion* • 250-500 3000
8. Diazinon* 300-850 2150
9. lodofenphos* 2100 -10. Phoxim* 1845 7100
11. Etrimfos* 1800-2040 -
12. Lindane* 88 1000
13. Methoxychlor* 5000-7000 2820-6000
14. Carbary! • 400-850 4000
15. Pyrethrum • • 1500 1800
16. Bioresmethrin*** 9000 10,000
17. Deltamethrin 1290 2940
10. Fenvalerate* 450 3700-5000
19. d-Phenothrin* 5000 5000
20. Resmethrin* 1500 3040
21. Permethrin * 4000 4000
22. Methoprene* 5000 relatively nontoxic
23. Piperonyl butoxide• relatively nontoxic relatively nontoxic
*Occasional Use **Moderate Use ***intensive Use •Used as synergist
Chemical Origin of Composition
Inorganic compounds - The toxicity of these compounds (arsenicals, fluo
ridos) are usually associated with the concentrations of elements. They arc highly toxic to
man, domesticated animals, and plants. They accumulate in the soil.
Organic Insecticides - These groups are characterized by organic carbon
bonding (i.e. c-c; c = c).
Study - Pesticide Residue in Fruits and Vegetables 26
a. Botanicals - The toxic prin,ciples are extracted from plants such as pyrethrum
from the flowers of Chrysanthemum cinerariafolium and C. cocciniu. It
has remarkable low toxicity to mammals but toxic to insects.
b. Synthetic insecticides - They are syntihesized in the laboratory and are
classified into three main groups:the organochlorine, organophosphates,
and carbamates.
b-1. Organochlorines -
These are chlorine (C1) - containing compounds further subdivided into
DDT type, Hexachloro-cyclohexane type and Cyclodicnes. Most of them are quite toxic to man
and are not used on stored food commodities.
c. Organophosphates - This is a generic term for all pesticides containing
phosphorus which can be an ester of phosphoric acid (P = 0) or phosphorothioate acid (P =
S) and can be represented by this formula:
Formula
The formula implies that sulphur (S) or oxygen (0) is directly linked to phosphorus. R1 and R2
may be alkyl or aryl groups or amine radical whereas X is the acyl or leaving group which may
be radical of an inorganic acid. This is a very large class of compounds which features a greatly
varying activity despite the very uniform mechanism of action. The organophosphorus
compounds, however, decompose rapidly and recent advances in understanding the
mechanisms of selective toxicity of insecticides such as malathion have led to safer
insecticides such as malathion, bromphos, pirimiphos methyl, chlorpyrifos methyl, etc.
Formula
d. Carbamates - These are esters of carbamic acid, HOC (0) NH2 and can be
represented by this tormula:
R1 can either be an aliphatic or aryl radical. This contains compounds of high to
low mammalian toxicity such as carbaryl and have so far shown limited application in stored
product pest control.
e. Insect Growth Regulators -These groups of compounds are synthetic analogues
of naturally occurring hormones in insects, ecdysone and juvenile hormones. Ecdysone
regulates metamorphosis by initiating the moulting process while juvenile hormones regulate
growth and development under normal concentrations. These growth regulators (IGRs) also
control other developmental processes in insects such as sexual maturation, colour
differentiation and reproduction. Examples arc difludenzuron and methoprcne.
Mode of Entry Insecticides can be divided into three main groups depending upon the way they
penetrate into the body of the insect.
Study - Pesticide Residue in Fruits and Vegetables 27
Contact Insecticides-These insecticides are applied in such a mannerthatthey come
in contact with some part of the body of the insect; the compound is able to penetrate the
exoskeleton and is transported to the tissues via the circulatory system. Most of the
insecticides used in storage belong to this group. The inert insecticidal dusts which disrupt the
thin epicuticle leading to the desiccation and death of the insect are included in this group.
Stomach Insecticides - These materials exert their toxic action only when they are
consumed and absorbed through feeding on treated surfaces through the guts.
Systemic Insecticides - These are translocated to the untreated parts of plants or
animals in concentration that makes the final translocation site toxic to insects. These are not
used in stored product pest protection.
Fumigants - These are insecticidal gases at normal temperatures penetrating
through the tracheal system into bony tissues and are used in enclosed spaces. Examples
are phosphine and methyl bromide. Some contact insecticides like dichlorvos, vaporize
partially in warm ambient conditions, thus having fumigant and contact actions.
Mode of Action Physical Insecticides - These materials such as the heavy mineral oils and inert
dusts, characteristically exert a physical rather than a biochemical action. Mineral oils exert
a purely asphyxiant effect and dusts affect loss of body moisture by abrasion (aluminium
oxide) or by absorbing moisture (charcoa~.
Protoplasmic insecticides - The action of these compounds is associated with the
cellular destruction of the midgut epithelium such as inorganic insecticides.
Respiratory poisons-These include the fumigants and those that block cellular
respiration such as rotenone. Rotenone inhibits the catalytic action of cytochrome oxidase
and other Fecundating oxidase.
Cholinesterase (CHE) Inhibitors - The OPs and carbamates, inactivate the
cholinesterase, consequently causing sickness or if applied at correspondingly high dosages,
death of the affected organisms.
The CHE are a woup of esterases which are capable of hydrolysing acetylcholine, a
chemical transmitter of nerve impulse. Normally, acetylcholine is rapidly hydrolysed to
acetate and choline in insect or in vertebrates in the presence of cholinesterase. But, with an
inhibitor (either OP orcarbamates) there is an accumulation of acetylcholine, which will cause
an excessive stimulation (twitching) and finally complete block of the system (paralysis).
Depression of the cholinesterase of the central nervous system results in restlessness,
discomfort, giddiness and anxiety, followed by a headache, sleeplessness, ataxia, tremors,
ultimately resulting in coma, generalized spasms and disappearance of reflexes.
Neurotoxlcant - These materials act on the central and peripheral nervous system
by changing the required balance between input and events. There is an excessive
Study - Pesticide Residue in Fruits and Vegetables 28
stimulation which later will cause excitation, convulsion, paralysis and death - these are
characteristic symptoms of nerve poisoning. To this group belong the organochlorine
insecticides, nicotinoids and pyrethroids.
Enzyme Inhibitors - These compounds (fluorides, arsenates) inhibit enzymes neces
sary for normal metabolism. Flouroacetate blocks the tricarboxylic cycle by combining with
acetyl CoA forming fluorocitrate which inhibits aconitase which convert citrate to succinate.
The blockage leads to reduced energy production and 02 utilization resulting in respiratory
disorders causing death. Arsenates and nitrophenols kill primarily by inhibiting respiratory
enzymes which block the production of AIP (energy).
Insect growth regulators- There are two types of IGRs: the chitin synthesis inhibitors
such as diflubenzuron and the anti juvenile hormone such as methoprene that disrupts the
mo1Jlting process. Chitin which is essentially a polymer of N-acetyl glucosamine is a structural
component of the insect cuticle essential for proper insect development. Diflubenzuron
interferes with the larval cuticle deposition and disrupts the moulting process by inhibiting the
synthesis of chitin. The function of hormone is to maintain the larval tissues at a moult. The
presence of JH mimics such as mithoprene in the insect when the natural hormone level is
low would be expected to disturb normal morphogenesis.
Insects exposed experimentally to large concentrations of IGRs during the life cycle
when they are not normally active, inhibit various developmental and morphological
abnormalities including a juvenilizing effect.
General Properties Of Insecticides. Metafaxvf. Chforpvrifos. Svnthetic oesticides
The selection of insecticides for treatment of edible commodities is based mainly on the
toxicological data (low mammal fan toxicity), effectiveness and persistence under certain
storage conditions and absence of side effects such as discoloration, flavour alternation and
odour.
The common insecticides used in stored product pest control belong to four groups:
the pyrethroids, the organophosphorus; the organochlorines, and the carbamates.
Study - Pesticide Residue in Fruits and Vegetables 29
Chlorpyrifos
Organophospate (OP)
Chapter3
It is the world's leading insecticide
Agrow's Top Twenty Five 1
Study - Pesticide Residue in Fruits and Vegetables 30
What is Chlorpyrifos (OP)
Chloropyrifos1 is a broad spectrum organophosphate insecticide. It was originally mar
keted in the US market in the 1960's. Now 36 years later the US EPA - Environment
Protection Agency , has launched a Risk Reduction Plan for Chlorpyrifos which includes a
number of restrictions of its use because of alleged reports of ill health connect to it. It is
registered for use in 88 countries, and is one of the most researched and evaluated pesti
cides with more than 3,600 studies.
Chlorpyrifos is toxicity class II - moderately toxic. Products containing chlorpyrifos bear
the Signal Word WARNING or CAUTION, depending on the toxicity of the formulation. It is
classified as a General Use Pesticide (GUP).
Other trade names include Brodan, Dctmol U/\, Dowco 179, Dursban, Empire, !::radex,
Lorsban, PriqP.r:mt, PiricfanP., Scout, and Stipend. It is available as granules, wettable pow
der, dustable powder and emulsifiable concentrate2•
The EPA has also established a 24-hour re-entry interval for crop areas treated with
emulsifiable concentrate or wettable powder formulations of chlorpyrifos unless workers wear
protective clothing. This Agency considers this pesticde as the leading cause of acute in
secticide poisoning in the US. Therefore concerned with reports of ill health following its
. use, it is being banned from being used on grapes, tomatoes and apples. This is being done
to reduce acute dietary risks. The reports which brought about this Risk Reduction Plan
were made in 1997 when 22 cases of alleged nervous system disorders and 35 cases of
sensitivity to the chemical were made.
Its symptoms are very much like those of the common flu with headache, nausea,
dizziness, muscle twitching, weakness, increased sweating and salivation. These occur
when cholinesterase activity has been reduced to 50%. Unconsciousness, convulsions and
death can result with sufficient exposure.
While originally used to kill mosquitoes, it is no longer registered for this use. It is
effective in controlling cutworms, corn rootworms, scale insects, aphids, massots, leafrollers,
cockroaches, grubs, flea beetles, flies, termites, fire ants, and lice. It is used as an insecti
cide on grain, cotton, fruit, nut and vegetable crops, as well as on lawns and ornamental
plants. It is also registered for direct use on sheep and turkeys, for horse site treatment, dog
1. Agrow's Top Twenty Five, PJB. PubL 1992 2. US Environment Protection Agency, [on-line] http://www.epa.gov/ [as dated} 9th January 2001.
Study - Pesticide Residue in Fruits and Vegetables 31
kennels, domestic dwellings. farm buildings. storage bins. and commercial establishments.
Mode of Action This pesticide acts on pests primarily as a contact poison, with some action as a
stomach poison. It acts as an inhibitor of anticholinesterase (ACh-ase) which is an enzyme
vital to the nervous system in man and animals.
The transmission of impulses across certain nerve functions in humans involve the
release of a transmitter chemical, acetylcholine (ACh). The stimulant effect of the ACh is
rapidly cancelled by the ACh-ase activity. This results in sustained high levels of ACh with
consequent serious and widespread disruption of the nervous activity.
Toxicological Effects: Acute toxicity: Chlorpyrifos is moderately toxic to humans 3 • Poisoning from chlorpyrifos
may affect the central nervous system, t11e cardiovascular syslern, am:! Lile resµiralory sys
tem. It also posses a risk of serious danger to the eyes and irritates the skin. While some
organophosphates are readily absorbed through the skin, studies in humans suggest that
skin absorption of chlorpyrifos is limited. Persons with respiratory ailments, recent exposure
to cholinesterase inhibitors, cholinesterase impairment, or liver malfunction are at increased
risk from exposure to chlorpyrifos.
In 1966, the Multiple Chemical Sensitivity Referral and Resources 4 in the US cited
more than 4SO cases of adults and children who were poisoned by this pesticide. The most
common symptoms in these cases were chronic headaches, nausea, vomiting, breathing
difficulties, vision problems, and neuromuscular pains. A year later other reports of ill health
followed and these lead to the adoption of the Risk Reduction Plan.
Some symptoms are delayed beginning 1 to 4 weeks after an acute exposure which
may or may not have produced immediate symptoms. In such cases, numbness, tingling,
weakness, and cramping may appear in the lower limbs and progress to incoordination and
paralysis. Improvement may occur over months or years, and in some cases residual im
pairment will remain. Plasma cholinesterase levels activity have been shown to be inhibited
when chlorpyrlfos particles are Inhaled.
Studies have also concluded that he oral LOSO for chlorpyrifos in rats is 9S to 270 mg/
kg 3• The LOSO for chlorpyrifos is 60 mg/kg in mice, 1 OOO mg/kg in rabbits, 32 mg/kg in
chickens, SOO to S04 mg/kg in guinea pigs, and 800 mg/kg in sheep . The dermal LOSO is
greater than 2000 mg/kg in rats, and 1 OOO to 2000 mg/kg in rabbits. The 4-hour inhalation
LCSO for chlorpyrifos in rats is greater than 0.2 rng/L 5 •
3. US EPA, Registration Standard (Second Round Review (for the Registration of Pesticide Products Containing Chlorpyrifos, Washington DC, 1989, pp 4-44.
4. US Environment Protection Agency, [on-line] http://www.epa.gov/ [as dated} 9th January 2001. 5. Gallo, M.A., and Lawryk, N.J.Organicphosphoruspesticides. In Handbook of Pesticide Toxicology. Hayes, W.J. Jr. and
Laws E>R.Jr., Eds. Academic Press, New York, NY, 1991, ppS-3.
Study - Pesticide Residue in Fruits and Vegetables 32
Chronic toxicity. Repeated or prolonged exposure to organophosphates may result in
the same effects as acute exposure including the delayed symptoms. Other effects reported
in workers repeatedly exposed include impaired memory and concentration, disorientation,
severe depressions, irritability, confusion, headache, speech difficulties, delayed reaction
times, nightmares, sleepwalking, and drowsiness or insomnia. An influenza-like condition
with headache, nausea, weakness, loss of appetite, and malaise has also been reported6•
Other studies have also shown that when technical chlorpyrlfos was fed to dogs for
2 years, increased liver weight occurred at 3.0 mg/kg/day. Signs of cholinesterase inhibition
occurred at 1 mg/kg/day. Rats and mice given technical chlorpyrifos in the diet for 104 weeks
showed no adverse effects other than cholinesterase inhibition. Two-year feeding studies
using doses of 1 and 3 mg/kg/day of chlorpyrifos in rats showed moderate depression of
cholinesterase. Cholinesterase levels recovered when the experimental feeding was dis
continued. Identical results occurred in a 2-year feeding study with dogs. No long term health
effects were seen in either the dog or rat study. A measurable change in plasma and red
blood cell cholinesterase levels was seen in workers exposed to chlorpyrifos spray. Human
volunteers who ingested 0.1 mg/kg/day of chlorpyrifos for 4 weeks showed significant plasma
cholinesterase inhibition.
Reproductive effects: Current evidence indicates that chlorpyrifos does not adversely
affect reproduction. In two studies, no effects were seen in animals tested at dose levels up
to 1.2 mg/kg/day7 • No effects on reproduction occurred in a three-generation study with rats
fed dietary doses as high as 1 mg/kg/day. In another study in which rats were fed 1.0 mg/kg/
day for two generations, the only effect observed was a slight increase in the number of
deaths of newborn offspring.
Teratogenic effects: Available evidence suggests that chorpyrifos is not teratogenic.
No teratogenic effects in offspring were found when pregnant rats were fed doses as high as
15 mg/kg/day for 1 O days. When pregnant mice were given doses of 25 mg/kg/day for 1 O
days, minor skeletal variations and a decrease in feta! length occurred. No birth defects
were seen in the offspring of male and female rats fed 1.0 mg/kg/day during a three-genera
tion reproduction and fertility study.
Mutagenic effea,ts~There is no evidence that chlorpyrifos is mutagenic. No evidence of
mutagenicity was found in any of four tests performed.
Carcinogenic effects: There is no evidence that chlorpyrifos is carcinogenic. There
was no increase in the incidence of tamers when rats were fed 10 mg/kg/day for 104 weeks,
nor when mice were fed 2.25 mg/kg/day for 105 weeks. However the Journal of Pesticide
Reform suggests that xylene used in some chlorpyrifos containing product have caused
increased rates of leukaemia among workers exposed to it. These inerts or anything added
6 Official Report on OPs. Report to the Minister of Agriculture, Forests and Fisheries. UK MAFF Pub!. 1998 p.23. 7. The WHO Recommended Classification of Pesticides by Hazards 1996-99. International Programme on Chemical
Safety WHO llPC, 5/96.3.
Study - Pesticide Residue in Fruits and Vegetables 33
to the product which is not the active ingredient like - xylene, clays, wetting agents,
amorphorous silica and1, 2,4-trimethyl-benzene - are now placed on four different lists by
the EPA according to their toxicity. lnerts on list one must either removed from the product or
identified on the label. Those on List 2 are still under monitoring while there no rules yet for
those on List 3 and 48•
Organ toxicity: Chlorpyrifos primarily affects the nervous system through inhibition of
cholinesterase, an enzyme required for proper nerve functioning.
Fate in humans and animals: Chlorpyrifos is readily absorbed into the bloodstream
through the gastro-intestinal tract if it is ingested, through the lungs if it is inhaled, or through
the skin if there is dermal exposure. A recent US research showed that if a child played in his
I her home a week after the application of chlorpyrifos had taken place he I she would be
overexposed to the pesticide. The rcscnrchos found residue on toys two wooks after its
application. So based on this research it was estimated that the exposure levels from indoor
spraying for children was about 21-119 times above the US recommended reference dose of
3mg/kg/day from all sources. To address this residential risk, manufacturers are eliminating
of pl1<1sing out this pesticide from their products, which are designed for use on Loys, curtains
and furniture9•
In humans, chlorpyrifos and its principal metabolites are eliminated rapidly. After a
single oral dose, the half-life of chlorpyrifos in the blood appears to be about 1 day. Chlorpy
rifos is eliminated primarily through the kidneys.
In animals following oral intake of chlorpyrifos by rats, 90% is removed in the urine
and 10% is excreted in the faeces. It is detoxified quickly in rats, dogs, and other animals.
The major metabolite found in rat urine after a single oral dose is trichloropyridinol (TCP).
TCP does not inhibit cholinesterase and it is not mutagenic. Chlorpyrifos does not have a
significant bio-accumulation potential. Following intake, a portion is stored in fat tissues but
it is eliminated in humans, with a half-life of about 62 hours. When chlorpyrifos (Dursban)
was fed to cows, unchanged pesticide was found in the faeces, but not in the urine or milk.
However, it was detected in the milk of cows for 4 days following spray dipping with a 0.15%
emulsion. The maximum concentration in the milk was 0.304 pp. In a rat study, chlorpyrifos
did not accumulate in any tissue except fat10•
Ecological Effects: Effects on birds:Chlorpyrifos is moderately to very highly toxic to birds. Its oral LD50 is
8.41 mg/kg in pheasants, 112 mg/kg in mallard ducks, 21.0 mg/kg in house sparrows, and 32
mg/kg in chickens. The LD50 tor a granular product (15G) in bobwhite quail is 108 mg/kg. At
8. Chlorpyrifos Fact Sheet, prepared by the US Dept. of Agriculture, Forest Service by Information Ventures. Inc. 9. Mace Brown and Woodburn Kent, £cotoxicity of Chlorpyrifos, Reviews of Environmental Contamination and Toxicology,
1995, Vol. 144. 10. American Conference of Govt. Industrial Hygienists Inc. Documentation of the Threshold Limit values and Biological
Exposure Indices, Fifth Edition, Cincinnati Ott. 1986, pp 5-48.
Study - Pesticide Residue in Fruits and Vegetables 34
125 ppm, mallards laid significantly fewer eggs. There was no evidence of changes in weight
gain, or in the number, weight, and quality of eggs produced by hens fed dietary levels of 50
ppm of chlorpyrifos.
Effects on aquatic organisms: Chlorpyrifos is very highly toxic to freshwater fish, aquatic
invertebrates and estuarine and marine organisms. Cholinesterase inhibition was observed
in acute toxicity tests of fish exposed to very low concentrations of this insecticide. Applica
tion of concentrations as low as 0.01 pounds of active ingredient per acre may cause fish
and aquatic invertebrate deaths. Chlorpyrifos toxicity to fish may be related to water tem
perature. The 96-hour LC50 for chlorpyrifos is 0.009 mg/Lin mature rainbow trout, 0.098 mg/
Lin lake trout, 0.806 mg/Lin goldfish, 0.01 mg/Lin bluegill, and 0.331 mg/Lin fathead min
now.
When fathead minnows were exposed to Dursban for a 200-day period during which
they reproduced, the first generation of offspring had decreased survival and growth, as well
as a significant number of deformities. This occurred at approximately 0.002 mg/L exposure
for a 30-day period. Chlorpyrifos accumulates in the tissues of aquatic organisms. Due to its
high acute toxicity and Its persistence In sediments, chlorpyrlfos may represent a hazard to
sea bottom dwellers. Smaller organisms appear to be more sensitive than larger ones.
Effects on other organisms: Aquatic and general agricultural uses of chlorpyrifos pose
a serious hazard to wildlife and honeybees.
Environmental Fate: Breakdown in soil and ground-water: Chlorpyrifos is moderately persistent in soils.
The half-life of chlorpyrifos in soil is usually between 60 and 120 days, but can range from 2
weeks to over 1 year, depending on the soil type, climate, and other conditions. The soil half
life of chlorpyrifos was from 11 to 141 days in seven soils ranging in texture from loamy sand
to clay and with soil pHs from 5.4 to 7.4. Chlorpyrifos was less persistent in the soils with a
higher pH. Soil half-life was not affected by soil texture or organic matter content. In anaero
bic soils, the half-life was 15 days in loam and 58 days in clay soil. Absorbed chlorpyrifos is
subject to degradation by UV light, chemical hydrolysis and by soil microbes. When applied
to moist soils, the volatility half-life of chlorpyrifos was 45 to 163 hours, with 62 to 89% of the
applied chlorpyrifos remaining on the soil after 36 hours. In another study, 2.6 and 9.3% of
the chlorpyrifos applied to sand or silt loam soil remained after 30 days. Chlorpyrifos absorbs
strongly to soil particles and it is not readily soluble in water.
The concentration and persistence of chlorpyrifos in water vary according to type of
soil. For example a large increase in chlorpyrifos concentration occurs when emulsifiable
concentrations and wettable powders are released into water. As the pesticide adheres to
11. Kidd H. and James D.R. Eds., The Agrochemicals Handbook. Third Edition. Royal Society of Chemists Information Services, Cambridge UK. 1991, pp. 5-14.
Study - Pesticide Residue in Fruits and Vegetables 35
sediments and suspended organic matter, concentrations rapidly decline. The increase in
the concentration of insecticide is not as rapid for granules and controlled release formula
tions in the water, but the resulting concentration persists longer. Volatilization is probably
the primary route of loss of chlorpyrifos from water. Volatility half-lives of 3.5 and 20 days
have been estimated for pond water. The photolysis half-life of chlorpyrifos is 3 to 4 weeks
during midsummer in the U.S. Its change into other natural forms is slow. Research suggests
that this insecticide is unstable in water, and the rate at which it is hydrolyzed increases with
temperature, decreasing by 2.5- to 3-fold with each 10 C drop in temperature. The rate of
hydrolysis is constant in acidic to neutral waters, but increases in alkaline waters. In water at
pH 7.0 and 25 C, it had a half-life of 35 to 78 days12•
Breakdown in vegetation: Chlorpyrifos may be toxic to some plants, such as lettuce.
Residue remain on plant surfaces for approximately 10 to 14 days. Data indicate that this
insecticide and its soil metabolites can accumulate in certain crops13•
Physical Properties14 :
A.J.JP-~~r~ac~; .. Technical chlorpyrifos is an amber to white crystalline solid with
a mild sulphur odour.
Chemical Name: 0,0-diethyl 0-3,5,6-trichloro-2-pyridyl phosphorothioate
GAS Number: 2921-88-2
Molecular Weight: 350.62
Water Solubilitv: 2 mg/L@25 C
Solubility in Other Solvents: benzenes.; acetones.; chloroforms.; carbon
disulfide s.; diethyl ethers.; xylene s.; methylene chlorides.; methanol s.
Melting Point: 41 .5-44 C
Vapor Pressure:
Adsorption Coefficient:
Exposure Guidelines15:
ADI:
MCL: RfD:
PEL:
HA:
TLV:
Partition Coefficient:
Adsorption Coefficient:
2.5 mPa@25 C
6070
0.01 mg/kg/day
Not Available
0.003 mg/kg/day
0.2 mg/m3 (8-hour) (skin)
0.02 mg/L (lifetime)
Not Available
4.6990
6070
14. Kidd H and James D.R.., Eds. The Agrochemicals Handbook. Third Edition Royal Society Information Services, Cambridge, UK, 1991 (as updated( 5-14).
15. McEwen, F.L. and Stephenson, G.R., The Use and Significance of Pesticides in the Environment. John Wileyu and Sons. NY, 1979, pp. 5-37.
Study - Pesticide Residue in Fruits and Vegetables 36
Chapter 5
Metalaxyl
From Phenylamides Group
Extensively used to control
Into potato blight
now registered for use on
other crops
US -Environment Protection Agency
Study - Pesticide Residue in Fruits and Vegetables 37
Introduction
Metalaxyl is a systemic, benzenoid fungicide that belongs to the group of phenylamides. It
was introduced in the 1970's and was extensively used to control late potato blight. It was
first evaluated by the WHO and FAO Committee in 1982 with further studies being carried
out in 1989, 1990 and 1992. It is now also registered worldwide for use on strawberries, in
mixtures as a foliar spray for tropical and subtropical crops, as a soil treatment for control of
soil borne pathogens, and as a seed treatment to control downy mildew1• Its predominant
use 1s m the management of potato late bllght2•
Experimental use permits are in effect authorizing the application of metalaxyl on some
food crops, altho11otl tot>Hcco, ornamonlals, conifer, and turf applications are the major uses.
Smokers could be exposed through inhalation3 • Products containing metalaxyl must bear
the signal word "Caution." Other trade names containing metalaxyl include Ridomil, Apron,
Delta-coat AD, Subdue 2E.
Being a systemic fungicide, it enters the plant and acts internally to halt the progres
sion of a disease. Metalaxyl has no effect upon surface inoculum. Even at low concentra
tion metalaxyl immediate halts the development of mycelium and prevents sporulation of the
pathogen upon entering the plant.
Several crops previously treated with metalaxyl have been contaminated with downy
mildew. Metalaxyl had provided the best disease control for downy mildew in lettuce in Italy
until the occurrence of resistant strains have emerged4• This is attributed to the fact that
metalaxyl has a very specific mode of action. Other studies showed that three consecutive
post infection sprays with Ridomil MZ (metalaxyl+mancozeb) at 0.25 percent at an interval
of 1 O days highly effective against downy mildew in onions caused by Peronospora destruc
tor.
Its mode of action is so specific that a small genetic mutation resulted in mutant patho
gens that are unaffected by the compound2• Thus after its initial release, and the subse
quent problems noted in Europe the pure metalaxyl was removed from the market and re
introduced with admixtures with protecting fungicide such as mancozeb. This reduced se-
1. Kimmel, E.C., J.E. Casida, and L.O. Ruzo (1986). Formamidine Insecticides and Chloroacetanilide Herbicides: Disubstituted Anilines and Nitrobenzenes as Mammalian Metabolites and Bacterial Mutagents, J. Agri Food Chem 34:157-161.
2. Systemic Fungicides (On Line) 3 May 2001 (date cited); http://www.plantpath.wisc.edu/pp300/doc/control5.html 3. Food and Drug Administration (1986). The FDA Surveillance Index. Bureau of Foods, Dept of Commerce, National
Technical Information Service, Springfield, VA. 4. Corbelli I., Collina M, Brunelli A. Occurrence in Italy and characteristics of downy mildew resistant to phenylamise
fungicides. Envir J PI Path. July 1998: 105 (5) 449-455.
Study - Pesticide Residue in Fruits and Vegetables 38
lective pressure and provided protection which was due to the development of metalaxyl
resistance. This is achieved as the protecting mode of action is broader and thus almost
inescapable, resulting in a lower incidence of non sensitive strains.
However this strategy resulted in other problems. In a study it was found out that
fungicide applied to potato can enhance green peach aphid outbreaks by interference with
entnopathogenic fungi. In fact late season aphid numbers were highest in potatoes sprayed
with metalaxyl+mancozeb. This is an important factor because of the intensity of spraying
required to protect the crop from metalaxyl-resistant strains of the late blight pathogen5•
Thus to maintain the efficacy of metalaxyl, a set of guidelines were established to
govern the use of metalaxyl in disease management programmes. The most important
criteria being: reducing the number of metalaxyl applications per season; eliminating its use
in a post-infection curative sense, applying it only in admixtures with protecting fungicides,
and restricting its use to foliage only.
TOXICOLOGICAL EFFECTS Acute Toxicity The oral LD50 in rats is 669 mg/kg and the dermal LD50 is >3, 100
mg/kg. Rabbits exhibited slight eye and skin irritation, but guinea pigs displayed no sensitiz
ing.
Chronic Toxicity: A 90-day rat study at levels in the diet of 2.5 to 62.5 mg/kg, showed
some cellular enlargement in the liver at the highest dose. The NOEL was determined to be
12.5 mg/kg/day3.
In a similar study with dogs fed diets of approximately 1 .25 to 25 mg/kg for six months,
the dogs were adversely effected by the highest dose. Manifestations included increased
alkaline phosphatase and increased liver-to-brain weight ratio.
Reproductive Effects: A three-generation rat study where animals were fed up to
62.5 mg/kg, showed no chemical related maternal toxicity or reproductive effects3.
Teratogenlc Effects: Rats and rabbits have been treated by intubation during ges
tation. Rats given a dosage of 120 mg/kg on days 6 to 15 exhibited no embryotoxicity or
teratogenicity, nor rlirl rabhits given a rlosage of 20 mg/k.g on rlays 6 to 18 3
Mutagenic Effect: In vitro rat liver preparations did not produce any detectable ani
line or nitroso compounds from metalaxyl which could be mutagenic1. Dominant lethal stud
ies in male mice also indicated no mutagenic potential.
Carcinogenic Effects: The EPA Cancer Assessment Group decided that metalaxyl
should not be classified as an oncogen because even though a study showed signiticant
parafollicular adenomas of the thyroid in female rats at low and middle doses, this did not
5. Forest Service (19 87). Pesticide Background Statements Vol 111, Nursery Pesticides US, Dept of Agriculture, Agriculture Handbook No. 670.
6. Worthing, Charles R., Editor (1983), The Pesticide Manual, A World Compendium The British Crop Protection Council, The Raverham Press Limited, Raverham, Suffolk, England.
Study - Pesticide Residue in Fruits and Vegetables 39
occur at the higher dose6•
Organ Toxicity: In a long term feeding study with mice at low levels of exposure, the
animals' livers were the primary target for metalaxyl-related effects. No specifics about the
changes in the liver or the doses administered were given in the reference.
Fate In Humans and Animals: Studies with rats and goats showed rapid metabolism
and excretion via urine and faeces. Glucuronic acid conjugates of the metabolites were the
main rat excretion products. There is hydrolysis and oxidative cleavage in warm-blooded
animals7 •
Forty-day feed studies with dairy cattle at 15 ppm, resulted in <0.01 ppm in the muscle
and fat. The liver contained 0.13 to 0.20 ppm and the kidney 0.26 to 0.83 ppm3.
Chickens fed for 28 days at 5 ppm in the diet had <0.05 ppm in the eggs, skin, fat,
breast and thigh and <0.1 ppm in the liver.
ECOLOGICAL EFFECTS Metalaxyl is practically nontoxic to birds8 and freshwater fish.
The 96-hour LC50 for rainbow trout, carp, and bluegill is > 100 mg/19 . rreshwater
aquatic invertebrates are slightly more susceptible to metalaxyl. Daphnia magna, a small
freshwater crustacean, has an LC50 of 12.5 to 28 mg/I, depending on the product formula
tion. This indicates that metalaxyl is slightly toxic to this organisms. Currently the toxicity of
the product is undocumented for marine organisms. The EPA has requested that these stud
ies be done.
There is little tendency for metalaxyl to accumulate in the edible portion of fish. Meta
laxyl did not accumulate beyond seven times the background concentration. It was quickly
eliminated after exposed fish were placed in fresh (metalaxyl free) water.
Metalaxyl is non-toxic to honeybees. The EPA has indicated that metalaxyl poses little
threat to aquatic or terrestrial endangered species, whether plant or animal8 •
ENVIRONMENTAL FATE
Under field conditions, metalaxyl has a half-life of one to eight weeks in soil. Its aver
age half-life in soil is about 70 days. The major breakdown product is from the hydrolysis of
the methyl ester. Although it readily leaches in sandy soil, increased organic matter de
creases leaching 1. In a large scale, national survey, metalaxyl was detected in the groundwater
of several states at concentrations of 0.27 ppb to 2.3 ppb.
At pH's of 5 to 9 and temperatures of 20 to 30 degrees C, the half- life in water was
greater than four weeks. Water exposed to sunlight, however, had a rnsidue halt-lite ot one
week. In water, metalaxyl is not readily degraded by sunlight. In soil, it has a half-life of about
7. Hartley, D., and H. Kidd, Editors (1986). The Agrochemicals Handbook. The Royal Society of Chemistry, The University, Nottingham, England.
8. Walker, Mary M. and Lawrence H. Keith. (1992). U.S. Environmental Protection Agency's Pesticide Fact Sheet Database. Lewis Publishers, Chelsea, Ml.
Study - Pesticide Residua in Fruits and Vegetables 40
2 weeks when exposed to sunlight.
Plants absorb foliar applications through the leaves and stems, and can translocate
the compound throughout the plant. Metalaxyl is not absorbed directly from the soil by plants.
The parent compound is the major residue in potato tubers and grapes, but in potato leaves
and on lettuce, metabolites are the major product. Metalaxyl acts by inhibiting protein syn
thesis in fungi. It has a calculated three-week duration of activity.
Exposure Guidelines:
NOEL: rat: 12.5 mg/kg/day, based on chronic effects; dog: 6.25 mg/kg/day,
based on chronic effects
ADI: 0.03 mg/kg/day
RfD: 0.06 mg/kg/day (EPA)
LEL: 25 mg/kg/day (dog)
Physical Properties:
CAS#: 57837-19-1
Chemical name: methyl-N-(2,6-dimethylphenyl)-N-(2-methoxyacetyl)-
DL-alaninate
Chemical class/use: benzenoid fungicide
Solubility In water: 8,400 mg/I
Solubility In other solvents: methanol 65 g/100 g; benzene 55 g/100 g;
hexane 0.91 g/100 g
Melting Point: 71-72 degrees C
Vapor Pressure: 5.6 x 1 O to the minus 6 power mm Hg10
9. Worthing, Charles R., Editor (1983). The Pesticide Manual, A World Compendium. The E:!ritish Crop Protection Council, The Ravenham Press Limited, Ravenham, Suffolk, Engle.nd.
10. Extoxnet: A Pesticide Information Project of Cooperative Extension Officises of the Universities of Cornell, Michi__ g_an, Oregon and California. Supported and funded by the US Departme~t of Agriculture.
Si.udy - Pesticide Residue in Fruits and Vegetables 41
Chapter 6
Synthetic Pyrethroids
Broad-Spectrum Insecticides
Extracted from Chrysanthemum plants
but
extensively modified in laboratories
to make them
more toxic.
Journal of Pesticide Reform
Study - Pesticide Residue in Fruits and Vegetables 42
Synthetic Pyrethroids
Synthetic Pyrethroids are a diverse class of more than a 1 OOO powerful broad-spectrum
insecticides used both in agriculture, households and stored products. These insecticides are
based on the biological activity of pyrethrumwhich is an extract from the plants in the genus
Chrysanthemum. But their development involve extensive chemical modifications to make
the products more toxic and less rapidly degraded by light. From studies carried out in Europe
and the US thoro is no evidence of pyrethroids residue in ground water due to its uses in
agriculture1•
However inspite of the fact that these Insecticides are widely used in homes and
gardens, there are significant data gaps for older and less persistent pyrethroids such as
alletl11i11, resmet111i11 and pf1e11ot111in and pyrethrins. Tllese gaps include data on many
aspects of acute and chronic toxicity. As for the newer light-stable pyrethroids the information
is more substantial even though we do not yet know how such insecticides as permethrin,
fenalerate, flucythrinate, cypermethrin and tralomethrin behave in the environment. The
information we have indicate that pyrethroids may pose a serious hazard on nontarget
organisms and terrestial invertebrates and possible fish2 ••
The Pyrethrolds
The Pyrethroids are either isolated from plants or synthesized.
Natural Pyrethrins
Pyrethrins remain one of the more widely used materials in stored product pest control.
The rapid knockdown effect, a wide spectrum of activity against insect pests, a general
acceptance of their use associated with foodstuff and an established codex of tolerance have
been the principal reasons for their use. Their use has not been intensive as their cost has
been prohibitive.
The disadvantage of high cost, poor stability, inadequate toxicity to some species and
lack of ovicidal and acaricidal action have been offset somewhat by the use of synergist, i.e.
piperonyl butoxide, sesamin, piperonyl cyclonene, propyl isome, sesamex and sulfoxide.
Synergists are usually present at ratios 1 :3 or 1:10 ( insecticide: synergist).
1. Muller-Beilschmidt Doria, Toxicology and Environmental Fate of Synthetic Pyrethroids/ Journal of pesticide Reform Vol. 10 No.3 fall 1990 [http:// ww.igc/panna/resources] online dated cited 30th April 2001.
2. Subcommittee pn Pesticides and Industrial Organic Pesticides, Pyrethroids. Their Effect on Acquatic and Terrestial Ecosystems. NRCC No. 24376, Ottowa Canada, 1981
Study - Pesticide Residue in Fruits and Vegetables 43
Synthetic Pyrethrolds
The syntheticpyrethroids such as biores-methrin, deltamethrin, permethrin, fenvalerate
and phenothrin are becoming acceptable over pyrethrins because of their high levels of
activity against a wide range of pests and a cost advantage. Bioresmethirn appears to be the
most commonly used. Several other synthetic pyrethroids showing promise against storage
pests are under development.
There have been deficiencies as with pyrethrins. For example, resistance is known
although not extensive, and breakdown to malodorous decomposition products has pre
sented a problem where repeated applications have been made on the same surface. In
addition, like pyrethrins, control of T. castaneum has been less than desirable. Pyrethroids
are highly summarized with piperonyl butoxide.
0 ~
Table 1. Acute oral LOSO (mg/kg body wt. rat) of Insecticides used for storage pest control.
INSl=C JICl[)I= ORAL (RAT) DERMAL
1. Malathion*** 1375-2800 4000-4800
2. Pirimiphos methyl*** 2050 2000
3. Chlorpyrifos methyl* 1650-2100 3000
4. Tetrachlorvinphos* 4000-5000 5000
5.Bromophos* 4000-8000 2188
6. Dichlorvos* • 80 107
7. Fenitrothion* • 250-500 3000
8. Diazinon* 300-850 2150
9. lodofenphos* 2100 -10. Phoxim* 1845 7100
11. Etrimfos* 1800-2040 -12. Lindane* 88 1000
13. Methoxychlor* 5000-7000 2820-6000
14. Carbary! • 400-850 4000
15. Pyrethrum • • 1500 1800
16. Bioresmethrin*** 9000 10,000
17. Deltamethrin 1290 2940
18. FenvaleratP.* 450 8700-5000
19. d-Phenothrin* 5000 5000
20. Resmethrin* 1500 3040
21. Permethrin • 4000 4000
22. Methoprene* 5000 relatively nontoxic
?8 Pireronyl hutoxide• relatively nontoxic relatively nontoxic
*Occasional Use **Moderate Use ***intensive Use (a ) Used as syner·
Study - Pesticide Residue in Fruits and Vegetables 44
The toxicity of a synthetic pyrethroids depends on its stereochemistry or the three
dimensional configuration of the molecules. Each isomer or molecules consisting of the same
atoms, but with different stereochemistry, has its own toxicity. Some pyrethroids may have
as many as eight different isomer. Many pyrethroids have pairs of isomers with different
geometrics known as cis and trans isomers. But commercial formulations have a fixed ratio
of isomers. Those like deltamethrin, which have a single isomer, tend to be more toxic than
those having eight 3•
Acute toxicity A product having a mixture of two isomers depends on the ration of the
amounts of the two isomers in the fomulation. For example , the female rat acute oral LD50
of permethrin increases from 224 milligrams of the pyrethroid per kilogram of bodyweight (mg/
kg) to 6000mg/kg as the proportion of the trans isomer increases from 20% to 80%.
In assessing the toxicity of pyretroids one has to look at the route of exposure, or how
it entered into the body. From laboratory test it was deducted that the introduction of the
compound in the brain is the most toxic followed by introduction through the blood vessels,
the gut, oral exposure, inhalation and dermal. This is due to the metabolic process by
mammals which rapidly detoxity the poisons .
Besides there are other factors that influence the toxicity of pyrethroids. Preexisting
health conditions such as respiratory or skin problems, can increase the toxic effect of some
pyrethroids in humans. Other factors are the halogenated composition of some pyrethroids
such as flucythrinate and tefluthrin and those containing more chlorine, bromine or fluorine.
Diet can also have ab effect on toxicity as well as the temperature in which it is administered
or found. DDT and pyrethroids are some of the few insecticides with toxicity that increases
at lower temperatures4•
Another important factor that should be discussed when analysing the acute toxicity of
these compounds is the effect of inert ingredients and synergists in the formulation. This is
especially important when considering that a formulated product often contains more Inert
(secret) ingredients than the active ingredient perse. Usually the technical grade (chemically
pure) pyrethroid is formulated for use in commercial pest control. The toxicity of these
exponents must be taken into consideration. Studies in mice have shown that there is a tenfold
increase in toxicity between different formulations of the same active ingredient. Pyrethroid
products formulated as emulsified concentrates (oil based formulations) usually have a higher
acute oral LD 50 in rats than wettable powders of the same formulation.
Besides simultaneous contact with substances that inhibit detoxification process called
synergist can increase the acute toxicity of a pyrethroid. High levels of some synergists like
organophosphates and carbamate compounds can block esterases, enzymes that degrade
3. Bradbury, Steven P. and Joel R. Coats, Comparative Toxicology of pyrethroids. Rev. Envirn. Contam. Tocicol. 1989. 108; 133-177.
4. Litchfield M.H., Toxcity to mammals. In J.P. Leahey (ed). The Pyrethroid insecticides. London U.K. Taylor & Francis.
Study ·· Pesticide Residue in Fruits and Vegetables 45
pyrethroids by cleaving the molecule at the double bond between a carbon and an oxygen
atom. Other synergists such as piperonyly, butoxide and sulfoixide block the mixed function
oxidises, enzymes which oxidize and detoxify a wide variety of compounds5•
Acute toxicity to mammals In general pyrethroids are less toxic than organophos
phates, carbamates and organochlorine pesticides. But the third generation of this group
which include deltamethrin, bifenthrin, fenpropathrinand cyhalothrin have higher oral toxicity.
These contain the cyano group or a carbon atom bonded with a nitrogen atom.
Studies have shown that the main effects of the pyrethroids are neurotoxicity at high
doses and liver enlargement- hypertrophy. If death does not occur, the capacity for recovery
from the toxic effects of pyrethroids seems to be a unique characteristic of pyrethroid
poisoning in mammals.
Other effects ot the pyrethroids can be mild to severe irritation of the eyes and skin.
Some pyrethroids can also cause a sensitization of facial skin which has been shown to be
reversible. The dermal toxic of some pyrethroids is greater than that ot the technical grade11 •
Mutagenicity and carcinogenicity: Studies about the ability of pyrethroids to cause
genetic damage have shown that cypermethrfn, allethrfn, cismetflrin, pormotl1rin mHi
fenpropathrin can cause some mutagenic effect. But inspite of these positive results, the
mutagenic potential of pyrethroids is considered to be very low if not nonexistent. Carcinogenicity
studies of permethrin, resmethrin, fenavalerate and deltamethrin have shown increases in
various kinds of cancer.
Human exposure: Studies carried out in China among workers engaged in packaging
fenvalerate and deltamethrin have shown burning sensation, tightness or numbness on the
face, sniffles and sneezes. In five years between 1983 and 1988 when pyrethroids were
started to be used there were 573 cases of acute pyrethroid poising, of which five resulted in
death. The occupation poisoning which were 299 of the reported cases, were attributed to
inappropriate handling7•
Effects on non-target organisms. Birds: Acute toxicity to birds is moderate. They can
be indirectly effected by pyrethroids if the pesticide decimate or substantially change theirfood
supply. Both pyrethrins and deltamethrin have shown to be teratogenic or causing birth
defects in certain birds Resides riyrnthroicfa hnvo 11 c.!Av11ntating AffAct on aquatic inverte
brates with most LC50 vales being less than 1.0ppb. These LC50s are similar to those for
mosquito, blackfly and tsetse fly larvae for which pyrethroids are often used in vector control.
The most effected are fish. About 40% of the LC50 values for fish are less than 1,0ppb.
Deltamethrin is the most toxic, while allethrin is the least toxic of the group. Studies carried
out have shown that pyrethroids are more toxic in laboratory studies than in natural waters
5. Abiola F.A., et al. Cholinesterase depression among Senegalese crop protection workers exposed to organophosphorous pesticides. Bull. Envirn. Contam. Toxicol. 41: ppS 1-85.
6. Roussel Uclaf, Deltamethrin monograph. Avignon France: L'lmprimerie, Aubanel Press 1982. 7. He F. et al. Clinical manifestation and diagnosis of acute pyrethroid poisoning. Arch Toxicol. 63: 54-58. 8. Smith Tara M., and Glenn W. Stratton, Effects of synthetic pyrethroids on non-target organisms. Residue Reviews
97; 93-120.
Study - Pesticide Residue in Fruits and Vegetables 46
because pyrethroids adhere to suspended organic matter in the water and bottom sediment.
Nevertheless pyrethroids have sublethal effects on fish since they damage the gills and cause
behavioural changes. This is due to the fact that they are highly lipophilic, that is are attracted
to the non-water soluble components of the cells, thus low levels of pyrthroids are absorbed
by the gills.
Though less tolerant than most mammals, amphibians and molliusces are much more
tolerant to pyrethroids than fish and Crustacea.
T()rr(}stri?J inv(}rt~lxat~: Pyrethroids are toxic to insects whether they are beneficial
or pests. They cause knockdown followed by recovery or death. Studies have shown that
pyrethroids effect flying and vegetation-inhabiting anthropoids like predatory beetles, more
than soil dwelling anthropoid. Soil applications of pyrethroids seem to decrease the number
of predatory mites and at high rates cause significant reductions in earthworms.
Predator-prey relationship can also be upset by the use of pyrethroids. For example the
caddisfly, a black fly predator, is more susceptible to permethrin than the blacktly. l he
phytoseiid mites, that live on spider mites, need an exposure 15 times lower than the mites
they eat.
Pyrethroids are highly toxic to bees, with the exception of flenvalinate which is used to
control mites in hives. The LD50 for a honey bee is as low as 0.003mg per bee. Studdies
carried out on fields indicate that hazards to the worker bees are reduced because they are
repelled by the smell of pyrethroids. Thus they do not come in contact with recently prayed
plants, and therefore the chances of receiveing lethal dose are diminished. But this also
reduces the foraging activity of the bees8•
Reside in food and water Studies carried out in California between 1982and1995 found
residue of permethrin in cabbage, lettuce, tomatoes ,other vegetable, fruit and meat. Up to
6ppb residue were found on spinach, kale, coloards and turnip greens. The US Food and Drug
Administration established the MRL for leafy vegetables at 200ppm.
In the environment: pyrethroids are usually degraded by one or more biotic and abiotic
processes: metabolic degradation by plant, animal and microorganism and degradation by
light (photolysis). There are three main routes of degradation by light in pyrethroids - ester
cleavage (splitting the molecule where a carbon atom and an oxygen atom are connected
with the double bond), reductive ordehalogenation, (removing chlorine, flourine and bromine
atoms), and isomerization( conversion from one isomer to another). The main product of
pyrethroid photolysis is 3-phenoxybenzoic acid.
In the soil degradation takes place by chemical and microbial action and depends on the
type, climate and species of microbes present and the size of their population. Among the
most commercially use pyrethroids, fenvalerate and deltamethrin are the most persistent in
9. Office of Pesticide and Toxic Substances, US Environment Protection Agency, Pesticide fact Sheet Fenvalerate No. 145 September 1987.
Study - Pesticide Residue in Fruits and Vegetables 47
the soil9•
Pyrthroids are also removed from the site of application by erosion, drift and volatilization.
Heavy spray drift from agricultural uses can cause contamination to neighbouring surface
water. Studies have shown that reside was found months after an application. The
persistence of residue in soil, water and plant tissues varies considerably. The half-life of
pyrethroids in soils varies from 1 day to 16 weeks. Pyrethroids that are not light-stable usually
degrade faster than pesticides that are light-stable and degradation is usually faster in aerobic
(oxygen-containing) soil than anaerobic10•
10. Leahey, J.P., /Metabolism and environmental degradation, in J.P. Leahey (ed) The Pyrethroid insecticides. London U.K.: Taylo & Francis. 1985.
Study - Pesticide Residue in Fruits and Vegetables 48
Chapter 6
Aims
and
Methodology of the Study
Although there is a link
between pesticide resistance
and pesticide residue
intern8tion8lly wo MO still
concerned with the residue problem
putting pest I vector resistance apart
Food and Agriculture Organization
Study - Pesticide Residue in Fruits and Vegetables 49
Aim of the Study
The primary aim of the study was to determine the presence or absence of any of the
pesticides being investigated. This was performed on various fruit and vegetable samples
consumed locally. The samples were either grown locally or imported products. Any pesticide
residue was determined using the ELISA kits supplied by the Institute of Agriculture at the
University Of Malta.
The samples were collected from vegetable hawkers or farmers found in various places
in Luqa, Zabbar, M'Xlokk, St. Paul's Bay, Zebbug, Mgarr (Malta), and B'Buga.
This procedure was chosen Instead of Interviewing farmers and collecling tile product
directly from the field as the information supplied by the farmers was not reliable. For example
some farmers admilled that they did not measure the amount of pesticide being added
accurately. The main cause for this was the lack of any measuring utensil with the bought
product. Generally the farmer or field operator is orally guided to use a spoonful or teaspoon
as a unit of measure. We know that both tablespoons and teaspoons come in various sizes
with varying volumetric capabilities.
This will therefore give rise to either an overdose of pesticide diluted with the water in
the spraying tank or an under-dose if the farm operator is prudent. The latter may however
produce a worst result as the sprayed crop might not be able to resist the pesticide attack and
the farmer will respray his crops. This will then lead to a double dose in which case would be
even worst than the overdose of a bigger spoonful.
As the FAQ says in its Manual on Pesticide Residue Analysis, there is a an over
lapping situation between pesticide residue and pest resistance. "Strange as it may seem,
intensive efforts to control pests on crops by pesticides have in a number of cases diminished
humans' ability to control adequately insect vectors of human release. The ready availability
of pesticides, often accompanied by inadequate controls, has led to exr:esses in their use "
The Manual continues that pests resistance to pesticides triggers a chain reaction, which
through detoriated efficacy leads to high amount of pesticide residue. When resistance starts
to build up, the user often tries to overcome the unsatisfactorily performance of his crops by
increasing the dose or increases the number of spraying sessions. This practice besides the
possibility of contamination of the environment, contributes to the increase of pesticide
residue in food, agriculture commodities and animal feed.
This is confirmed locally when most farmers were not sure on how many times they
sprayed the crops nor could they remember what type of pesticide they used. They keep
~~9rds. Als~ the choice of pesticide used depended on the cost Farmers tend to look
Study - Pesticide Residue in Fruits and Vegetables 50
at the price before they decide which pesticide to use, often going to the bulky and less
expensive product. Bulky products such as powder pesticides bought in bags may deteriorate
from the time they are first bought till the second or third time when the next spraying session
would be necessary. This naturally depends on a number of causes, depending on the place
they are stored, the climatic features of the store and they way it is kept in the store.
Again this will give rise to an inadequate result in pest and vector control leading the
farmer to think that an extra spraying session is necessary. Sometimes little to no thought is
given to the fact that the product may be outdated.
We also know that pesticides may deteriorate by light or excessive heat rendering them
useless, which again may induce the farmer to respray the crops or doubling the dose.
On one occasion a farmer said that he thinks that the products are not as genuine as
they used to be. He said that quite often he has either to increase the dose, or doubles the
spraying session or use another type of pesticide alongside the one he used on the first or
second occasion.
How the kits work The Envirologix's plate kits being used in this project are are a competitive ELISA test.
In these kits the pesticide residue being investigated competed with enzyme (Horseadish
peroxidase)- labelled pesticides for a limited number of binding sites on the inner surface of
the test wells. After a simple wash step the outcome was visualised by a colour development.
Sample concentration was inversely proportional to colour development. The darker the
colour the less pesticide residue is present in the sample tested.
Materials and Apparatus Used
The apparatus used varied according to the kit being operated.
Apparatus used for sample extraction in all three samples, that is Chloryphritos,
Metalaxyl and Synthetic Pyrethroids.
glass vials
Methanol ANALAR grade
Automatic s shaker
common kitchen liquidiser
Anhydrous Sodium sulphate
distilled water
Materials used for the Chlorpyrifos kit:
The kit contained the following items:
8 strips of 12 antibody-coated wells, each in plate frame
1 vial of 1 microgram per millilitre of Chlorpyrifos Calibrator stock
1 bottle of Chlorpyrifos enzyme conjugate
Study - Pesticide Residue in Fruits and Vegetables 51
1 bottle of substrate
1 bottle of stop solution
The following Items were used;
glass vials
pipette with disposable tips capable of delivering 60 microlitres
pipettes capable of delivering 10, 9.5, 7.5, 2.5, and 0.5
disposable tip pipette measuring 50, 100 and 150 microlitres
marking pen
tape to cover the plates while shaking
timer
cool tap water for rinsing wells
microtiter plate reader.
Materials used for the Metalaxyl kit:
The kit contained the following items:
8 strips of 12 antibody-coated wells, each in plate frame
1 vial of Negative control
1 vial of 0.1 ppb Metalaxyl calibrator
1 vial of 0.4 ppb Metalaxyl calibrator
1 vial of 1. 75 ppb Metalaxyl calibrator
1 bottle of Metalaxyl enzyme conjugate
1 bottle of substrate
1 bottle of stop solution
The following items were used;
disposable tip pipette measuring 100 microlitres
marking pen
tape to cover the plate while shaking
timer
cool tap water for rinsino wP.lls
microtiter plate reader.
Materials used for the Synthetic Pyrethrold .kit:
The kit contained the following items:
8 strips of 12 antibody-coated wells, each In plate frame
1 vial of Negative control (neat methanol)
1 vial of 4 ppb Cyfluthrin calibrator
1 vial of 20 ppb Cyfluthrin calibrator
1 vial of 80 ppb Cyfluthrin calibrator ~~~--~~~~~--~~
Study - Pesticide Residue in Fruits and Vegetables 52
1 bottle of Cypermethrin enzyme conjugate
1 bottle of Sodium Hydroxide Solution (0.2N)
1 bottle of substrate
1 bottle of stop solution
The following items were used:
Procedure
Methanol ANALAR grade
distilled water
Glass vials
disposable tip pipette measuring 25 and 100 microlitres
marking pen
tape to cover the plate while shaking
timer
cool tap water for rinsing wells
microtiter plate reader.
Various methods have been analysed to perform Pesticide Residue Analysis. However
several drawbacks and limitations have been encountered. ELISA kits have been chosen in
favour of other analytical methods in this case. This is attributed to the fact that they are rapid,
convenient to use and to carry around, inexpensive and reliable. HPLC could not be used in
this case as it was unavailable due to limited resources.
Literature Review The ELISA kits used in this study were not available locally. After conducting an
extensive literature review, surfing also the internet web pages, Envirologixs Inc. have been
personally contacted and eventually through their catalogues the kits were ordered.
Here a major setback was encountered as the kits supplied were designed to detect
pesticide residue in ground or surface water. Several methods of extraction have been
studied. An example included soil extraction.
Then the samples were chopped and incinerated in a hot air oven and then 5ml of
methanol were added. However as the sample was devoid of its moisture content the
pesticide residue could not be extracted In Methanol and passed through a sieve as suggested
by the enclosed literature.
At one time I also considered using the digester. Having gone through more literature
and even contacting the kits' makers, I opted to the last resort that of using the vegetables and
fruit 9wn juice to test for residue.
Study - Pesticide Residue in Fruits and Vegetables 53
This method of pesticide extraction, common to all three kits, was designed in order
to avoid possible sources of error.
Sample Extraction This was performed as follows:
A kilogram of fruit or vegetable was collected from different sources as specified above.
I opted for this procedure because of the problems encountered and mentioned above while
interviewing the farmers.
The samples were collected from different sources. From each source I was careful
to pick at least one kilogram of each sample taking it from various boxes available at the
vendor's place. This took place in the case of the foreign bo.no.no.G, oranges and apples. I did
this instead of the normal sample collection when fruits have to be collected from various trees
and from different branches of the same in order to ensure a uniform sample of fruits with more
or less the same amount of spray. The fruit at the top is most likely the least to be reached
when spraying takes place, with the h uil HI Ille very bottom, near the grown the most likely to
be heavily sprayed.
In the case of the locally grown crops the same procedure took place. I choose the crops
from different pitkali green boxes available at the particular vendor. Then from the five to six
kilos. I then divided each sample in four parts and then took a final kilo which I chopped and
them passed through the liquidiser to take the final sub-sample to be added to methanol. Care
was taken to ensure that each sample was from edible plant material so as to ensure that
the sample is homogeneous.
The crops were chosen from different boxes in order to simulate the normal procedure
of picking crops from different areas the field. This is usually done following a W pattern so
as to ensure that the sample was uniformly sprayed with the crops in the middle of the field
probably getting an overlapping spray as opposed to those at the end of the field where these
could have hardly been reached with the spraying gun.
From liquidised sample I took 20 grams of the sample and added it to 100 mls of
methanol. This is a 1 :5 w/v dilution.
The mixture was then shaken using a mechanical shaker at 200 shakes per minute for
2 hours.
When the shaking was over, about 20-30 grams of Anhydrous Sodium Sulphate, were
added so as to remove any extra water. The whole mixture was shaken again for a few
minutes. Then the mixture was allowed to settle down and a subsample was diluted to 1:100
v/v in distilled water.
The final sample obtained was run as sample on the plate. The concentration of the
target analyte was assumed to be in the range of 0.1-100 ppb for sprayed on pesticides.
__ In t!'e extraction process it was important to ensure that the samples obtained wore
Study - Pesticide Residue in Fruits and Vegetables 54
homogeneous as the different pesticides tend to distribute differently according to their
chemical nature.
Sample Analysis using ELISA kits The whole procedure adopted for each kit varied. The same sample was used for all
the three kits. A detailed outline of the procedure is available on the package information
leaflets supplied by Envirologix Inc. See Appendices 1-3.
Interpretation of Results and Calculations The results obtained were entered in a database and the average absorbance of each
sample and control was determined.
For all kits the %Bo can be calculated as;
%Bo = average OD of Callbrator or Sample X 100
average OD of Negative Control
A graph of %Bo of calibrator against the concentration of the calibrator was
plotted. %Bo was plotted on the Y Axis and Concentration in ppb (parts per billion) was
plotted on the X Axis. After determining the values of the slope (m) and the Intercept (c) the
concentration of pesticide residue was determined from extrapolation of the graph.
Precautions All samples used were homogeneous as different pesticides distribute themselves in
different plant tissues according to their chemical nature.
All Plate kit components were refrigerated, when not in use, at a temperature between
4-8C. They were not exposed to temperatures above 3TC or less then 2·c . Kit components
were not used after their expiry date. Substrate was not exposed to direct sunlight.
Only borosilicate glassware was used as Chlorpyrifos tends to stick to plastic contain
ers. Care was taken when disposing of samples and kit components.
Limitations and Sources of error Even though maximum care was taken while performing the study, still some limitations
and sources of error have been encountered. Lack of resources and apparatus was the major
drawback encountered during the execution of this study.
Transportation of the test !<its and reagents may have resulted in erroneous results
Study - Pesticide Residue in Fruits and Vegetables 55
Their was a time lag between sample collection and analysis of about 24-30 hours
during which deterioration of the pesticide residue could have occurred.
The Envirologix test kits do not distinguish between the pesticide being tested and
certain other compounds. This is explained on leaflets - see Appendices 1-3. Thus if another
compound was present of the pesticide in question, this was recorded by the kit even though
at a lower concentration.
It would be better to do HPLC multi residue analysis to confirm positive results. Thus
ay residue other than the pesticide being tested would be detected.
Study - Pesticide Residue in Fruits and Vegetables 56
Results and Calculations
All the Samples collected were analysed using the three different ELISA kits, for the three
different pesticide residue. Table 1 and Table 2 below summarise the characteristics of the
samples. Table 1 outlines the first 8 samples referred to as trial 2, whereas the other 16
samples were run in Trial 2. This is important when calculating the results obtained as data
for the two samples will be processed separately.
Sample Type of Locality Local/ Number Sample of collection Imported from:
1 Apples (Sicily) Zabbar Sicily 2 Tomatoes B'Bugia Local 3 Tomatoes Zabbar Local 4 Oranges (Sicily) Luqa Sicily 5 Courgettes Zabbar Local 6 Cauliflower Luqa Local 7 Lettuce Luqa Local 8 Cauliflower Zebbug Local
Table 1. Outlines the characteristics of the samples used in Trial 1
Sample Type of Locality Local/ Number Sample of collection Imported from:
1 Oranges (Egypt) Zabbar Egypt 2 Banana ( Ecuador) B'Bugia Ecuador 3 Cabbages M'Xlokk Local 4 Potatoes (Cara type) Luqa Local 5 Lettuce (Cabbage form) Luqa Local
s Cauliflower M'Xlokk Local V' Apples (Sicily) Luqa Sicily 8 Oranges (Sicily) M'Xlokk Sicily
9 Strawberry Paola Local ~o Banana ( Ecuador) Luqa Ecuador 11 Cabbages M'Xlokk.·· Local 12 Potato (Alpha) Zabbar Local 13 Lettuce M'Xlokk Local 14 Cauliflower Luqa L.ocal
15 Apples (USA) Luqa USA
16 Strawberry Luqa Local -
Table 2. Outlines the characteristics of the samples used in Trial 2 ~~~~~~~~~~~
Study - Pesticide Residue in Fruits and Vegetables 57
Results obtained for the Chlorpyrifos kit
The %Bo is found out as
%Bo = average OD of calibrator or sample X 100
average OD of Negative Control
Thus the % Bo for calibrators using the Chlorpyrifos kit for the first and second trials are
summarised below: A standard curve of Chlorpyrifos concentration on the x axis against%
Bo on the y axis was plotted as illustrated in graph 1 and graph 2, and the values of the
intercept (c) and slope (m) were calculated from the graph.
-
NC C1 C2 C3
(66%. 88%) (40%-57%) (18% - 29%)
~oncentration (ppb) 0 0.3 1.5 6
Abs. Trial1 0.686 0.469 0.300 0.106
0.709 0.476 0.361 0.113
Average Abs 0.6975 0.4725 0.3305 0.1095 -
Yo BO 68% 47% 16%
TABLE 3. % Bo obtained for Calibrators 1-3 for Chlorpyrifos kits for Trial 1
NC C1 C2 C3
(66% - 88%) (40%- 57%} (18% - 29%}
Concentration (ppb) 0 0.3 1.5 6
Abs Trial 2 0.498 0.323 0.213 0.143
0.446 0.307 0.214 0.072
Average Abs 0.472 0.315 0.2135 0.1075 - -%80 100 67 45 50
TABLE 4. % 80 obtained for Calibrators 1-3 for Chlorpyrifos kits for Trial 2
Study - Pesticide Residue in Fruits and Vegetables 58
0
Graph 1: Illustrative Standard Curve for Chlorpyrifos Trial 1
120°/o
100°/o
80°/o
CC 60°/o M
Q c::i
M
40°/o
20°/o
00/o
0 2
Chlorpyrifos
4 6
Concentration
Graph 2: Illustrative Standard Curve for Ch lorpyrifos Trial 2
120%
100%
80%
60%
40%
20%
0% 0 2 4 6
Chlorpyrifos Concentration (ppb)
Study - Pesticide Residue in Fruits and Vegetables
8
8
59
Thus from y = mx + c; The value of the concentration X can be calculated for any % Bo.
Where m = slope of the standard curve
C= intercept of the standard curve.
The calculated concentrations (ppb) of Chlorpyrifos residues in trial 1 are summarised be
low
Samples Absorbanee Average %80 Chlorpyrlfos Cone (ppb) ppm
1 0.007 0.009 0.008 1 6.92 3.46 2 0.746 0.761 0.754 108 NIL 3 0.784 0.718 0.751 108 NIL 4 0.731 0.743 0.737 106 NIL 5 0.792 0.754 0.773 111 NIL 6 0.732 0.748 0.740 106 NIL 7 0.734 0.671 0.703 101 NIL 8 0.718 0.746 0.732 105 NIL
Table 5: The determination of Chlorpyrifos residues in Trial 1.
Similarly the concentration of chlorpyrlfos residues In Trial 2 are shown below.
Samples Absorbances Average %80 Chlorpyrlfos Cone (ppb) ppm
1 0.374 0.428 0.401 85 NIL 2 0.340 0.323 0.332 70 1.06 0.53 3 0.285 0.352 0.319 67 1.58 0.34. 4 0.364 0.327 0.346 73 0.51 0.26 5 0.298 0.505 0.402 85 NIL 6 0.244 0.196 0.220 47 5.49 2.75 7 0.068 0.174 0.121 26 9.43 4.72 8 0.391 0.333 0.362 77 NIL 9 0.286 -0.280 0.003 0.64 14.12 7.06
10 0.288 0.278 0.283 60 2.99 1.5 11 0.398 0.382 0.390 83 NIL 12 0.260 0.546 0.403 85 NIL 13 0.682 0.337 0.510 108 NIL 14 0.328 0.286 0.307 65 2.04 1.02 15 -0.170 -0.152 -0.161 (34.11) NIL 16 -0-178 -0.178 -0, 178 (37.71) NIL
Table 6: The determination of Chlorpyrifos residues in Trial 2.
Study - Pesticide Residue in Fruits and Vegetables 60
Results obtained for the Metalaxyl kit
The %Bo is found out as
%Bo = average OD of calibrator or sample X 100 average OD of Negative Control
Thus the % Bo for calibrators using the Metalaxyl kit for the first and second trials are sum
marised below: A standard curve of Metalaxyl concentration on the x axis against% Bo on
they axis was plotted as illustrated in graph 3 and graph 4, and the values of the intercept ( c)
and slope (m) were calculated from the graph.
NC C1 C2 C3
%Bo 100% (57%-82%) (42%-54%) (21%- 28%)
Concentration In ppb 0 0.1 0.4 1.75
Abs. Trial1 0.554 0.299 0.224 0.101
0.583 0.329 0.204 0.105
AV Abs 0.5685 0.314 0.214 0.103
%BO 55% 38% 33%
TABLE 7 % Bo obtained for Calibrators 1-3 for Metalaxyl kits for Trial 1
NC C1 C2 C3
%Bo 100% (57%- 82%) (42%-54%) (21%- 28%)
Concentration In ppb 0 0.1 0.4 1.75
Abs. Trial 2 0.684 0.515 0.395 0.166
0.438 0.147 ' 0.091 0.137
AV Abs 0.561 0.331 0.243 0.1515
%BO 59% 43% 27%
TABLE 8. % Bo obtained for Calibrators 1-3 for Metalaxyl kits for Trial 2
Study - Pesticide Residue in Fruits and Vegetables 61
0 re &E:
0 Ill ~ 0
Graph 3: Illustrative Standard Curve for Metalaxyl Trial 1
120%
100%
80%
60%
40%
20%
0%
0 0.5 1 1.5
Metalaxyl Cone {ppb)
Graph 4: Illustrative Standard Curve for Metalaxyl Trial 2
120%
100%
80%
60%
40%
20%
0%
0 0.5 1 1.5
Metalaxyl Cone (ppb)
2
2
Study - Pesticide Residue in Fruits and Vegetables 62
Calculations for Metalaxyl: Similarly from y = mx + e;
The value of the concentration x can be calculated for any% Bo.
Where y = 0.338 x + 0.7176 for trial 1.
Mutilply by 500 dilution
Samples Absorbanee Average %80
1 0.394 0.354 0.374 66 2 0.344 0.882 0.338 60 3 0.364 0.346 0.355 62 4 0.335 0.349 0.342 60 5 0.320 0.431 0.3/bb 66 6 0.440 0.337 0.3885 68 7 0.269 0.295 0.282 50
8 0.485 0.507 0.496 87
Tab le 9: Tile determination of Metalaxyl residues in Trial 1.
and y = 0.2972 x + 0.7397 for trial 2
Samples Absorbanee Average %80
1 0.602 0.224 0.413 74 2 0.324 0.234 0.279 50 3 0.221 0.203 0.212 38 4 0.685 0.134 0.410 73 5 0.825 0.512 0.669 119 6 0.430 0.528 0.479 85 7 1.054 O.ObO 0.!:>!:>2 98 8 0.236 0.129 0.183 33 9 0.683 0.299 0.491 88 10 0.162 0.341 0.252 45 11 0.205 0.117 0.161 29 12 0.317 0.427 0.372 66 13 0.306 0.918 0.612 109 14 0.293 1.268 0.781 139 15 -0.087 2.736 1.325 236 16 -0.038 0.149 0.056 10
Table 10: The determination of Metalaxyl residues in Trial 2.
Metalaxyl Cone {ppb) ppm
0.177 0.09 0.364 0.18 0.276 0.14 0.343 0.17 0.169 0.08 0.101 0.05 0.655 0.38
NIL
Metalaxyl Cone {ppb) ppm
0.012 0.006 0.816 0.41 1.217 0.61 0.033 0.02 NIL NIL NIL 1.394 0.70 NIL 0.980 0.49 1.523 0.76 0.258 0.13 NIL NIL NIL
2.1560.18
Study - Pesticide Residue in Fruits and Vegetables 63
Results obtained for the Synthetic Pyrethroid kit
The %Bo is found out as
%Bo: average OD of calibrator or sample X 100
average OD of Negative Control
Similarly to the other kits, the % Bo for calibrators using for the first and second trials were
calculated as above. The standard curves of Cyflurithin concentration on the x axis against
% Bo on the y axis was plotted as illustrated in graph 5 and graph 6, and the values of the
intercept (c) and slope (m) were calculated from the graph
NC C1 C2 C3
Cyflurilhrin Cone 0 4 20 80
Abs. Trial1 0.73 0.493 0.429 0.426
0.612 0.432 0.258 0.212
AV Abs 0.671 0.4625 0.3435 0.319
%80 100% bl:1% 51% 48%
Tab le 11 : The % Bo obtained for Calibrators 1-3 for The Synthetic Pyrethroid
kit for Trial 1.
NC C1 C2 C3
0 4 20 80
Abs. Trial 2. 0.543 0.4 0.258 0.212
0.494 0.218 0.212 0.189
AV Abs 0.519 0.309 0.235 0.2005
%80 100% 60% 45% 39%
Tab le 12: The % Bo obtained for Calibrators 1-3 for The Synthetic Pyrethroid
kit for Trial 1.
Study - Pesticide Residue in Fruits and Vegetables 64
e im ~
0
5
100% 90% 80% 70% 60% 50% 40% 30% 20% 10%
0%
Graph 6: Illustrative Standard Curve for Synthetic Pyrethroids Trial 1
0 20 40 60 80
Cyfluthrln Cone (ppb)
Graph 6: Illustrative Standard Curve for Pyrethroids Trial 2
120%
100%
80%
60%
40%
20%
0%
0
Synthetic
20 40 60
Cyfluthrin Cone (ppb)
80
Study - Pesticide Residue in Fruits and Vegetables
100
100
65
Also from Y = mx + c, the amount of synthetic pyrethroids in the samples was deter
mined. Where y = 0.455 x + 74.18 for the samples in Trial 1.
The dilution factor was 500 (1 :500 dilution) The results were multiplied by 500
and converted to ppm (parts per million).
Samples Absorbance Average %80 S.Pyrethrold ppm Cone (ppb}
1 0.541 0.448 0.495 72 15.42 7.71 2 0.466 0.428 0.447 65 30.53 15.27 3 0.436 0.548 0.492 71 16.22 8.11 4 0.443 0.412 0.428 62 36.73 19.37 5 0.417 0.513 0.465 67 24.80 12.40 6 0.493 0.462 0.478 69 20.83 10.42 7 0.47 0.477 0.474 69 22.10 11.05 8 0.515 0.858 0.687 99 NIL
Tab le 13: The determination of Synthetic Pyrethroid residues in Trial 1.
Also y = 0.507 x + 74.18 for trial 2.
Samples Absorbance Average %80 S. Pyrethroid Cone {ppb} ppm
1 0.538 0.557 0.548 105 NIL 2 0.528 0.521 0.525 101 NIL 3 0.493 0.437 0.465 90 NIL 4 0.791 0.295 0.543 105 NIL 5 0.495 0.583 0.539 104 NIL 6 0.426 0.217 0.322 62 24.13 12.07 7 0.327 0.576 0.452 87 NIL 8 0.628 0.723 0.676 130 NIL 9 0.453 0.402 0.428 82 NIL 10 0.371 0.434 0.403 78 NIL 11 0.400 0.431 0.416 80 NIL 12 0.382 0.345 0.364 70 8.17 4.09 13 0.577 0.502 0.540 104 NIL 14 0.374 0.473 0.424 82 NIL 15 0.504 0.257 0.381 73 1.71 0.86 16 0.243 0.612 0.428 82 NIL
Table 14: The determination of Synthetic Pyrethroid residues in Trial 2.
Study - Pesticide Residue in Fruits and Vegetables 66
Chlorpyrifos
Results interpretation At a first glance at the results from the Chlorpyrifos test kit it transpires that 9 out of 24
samples resulted positive. Remarkable high residue were noted on imported apples from
Sicily and bananas from Ecuador, as well as in cauliflower grown locally. This difference
may be caused by the type of pesticide used (see table below).
It must be noted that in the case of apples the sample was prepared on unpeeled
apples. That is to say that the residue may have resulted from pesticide applications con
ducted on the product while in storage or prior to shipment in order to reduce losses through
rotting.
The same may be said of the bananas. These may have been treated prior to ship
ment from South America, a voyage which takes about three weeks. Product treatment
most probably have taken place during the packing stage.
From the Chlorpyrifos literature review, this pesticide is also indicated for use on veg
etables. This is retlected in sample No. 6 Trial 2 (cauliflower) In Trlal 2 sample 3 (cabbages).
The list below gives the Minimum Residue Level (MRL) indicated by the US Environ
ment Protection Agency (EPA) for Chrloryprifos in crops I fruit tested. The second column
indicates those allowed in the U.K. This is being done to obtain some form of comparison.
One has to keep in mind, however that different pesticides tend to give different results. Not
only so. Pesticides leave different residues according to each crop or plant which react
differently.
I am giving also the values for Malathion which is a product widely used locally almost
on any type of crop and stored products.
Product EPA1 UK2 Malathion EPA1
Apples 1.5ppm NA mg/kg 8 ppm
Banana 0.1 NA NA
Cabbages 1 NA NA
Caullflower 1 NA
Citrus Fruit 1 NA 8 (oranges)
Leafy Vegetables 2 NA 8
Potato 0.05 0.08 8
Strawberry 0.2 1 8
Tomatoes 0.05 2 8
1. the MRL given by EPA is in parts per million
2. the MRL for UK is given in mg/kg of body weight (MAFF- Ministry of Agriculture)
Study - Pesticide Residue in Fruits and Vegetables 67
Metalaxyl
Results interpretation
The most striking feature in results obtained with this kit is that almost all the samples
of locally grown crops had some form of residue. However this is below the level recom
mended by the EPA.
MP.talaxyl is a systemic pesticide used world wide for use on strawberries and lately is
being registered also for use with other crops.
Residue was also noted in banana samples from Ecuador. The residue here may be
the result of applications made prior to packing and shipping .
. . Again the MRL depends also on the pesticide used. Not all pesticides have the same
MRL in crops and fruit. Below is a list of the crops/fruit tested and the Metalaxyl residue
limits.
Product EPA1
Apples 0.2
Banana NA
Cabbages 1
Cauliflower 1
Citrus Fruit 1
Leafy Vegetables 0.1
Lettuce 5
Potato 0.5
Strawberry 10
Tomatoes 3
1. the MRL given by EPA is in parts per million
Study - Pesticide Residue in Fruits and Vegetables 68
Synthetic Pyrethroids
Results interpretation From the results for the Synthetic Pyrethroid test kit is interesting to note that the
majority of the samples tested contained some residue, although none exceeds the 80ppb
of cyflurithrin supplied with the kit. In six out of 24 samples tested residue was recorded.
This is particularly true for samples from Trial 1 ....
No. 2 ( tomatoes - grown locally)
No. 4 (oranges from Sicily)
No. 5 (courgettes - grown locally)
No.6· (cauliflower - grown locally)
and sample No. 6 (cauliflower - grown locally) from Trial 2.
This test kit was designed to test for synthetic pyrethroids which is a broad-spectrum
class of pesticides. The trnces found in thA ahovA samriles confirm the presence of a
synthetic pyrethroid -such as Permethrin - widely used locally to control snails in leafy
crops, left traces in the vegetables tested. When preparing the samples the dry, discoloured
external leaves were removed. The samples (as more than one vegetable of the same type
was collected, was then divided into four parts and mixed together so as to obtain a uniform
sample. The fact that each sample contained more than one crop and the fact that these
were bought from the same hawker but from different boxes, make the sample tested more
reliable.
The locally available pesticide product may have contained other pesticides other than
Permethrin, which might have been the one that tested positive in the microwell plates.
From the test we have a positive result but we cannot be sure of the p0sticido used, unless
the farmer produces the product.
Product
Apples
EPA1
0.05
Banana NA
Cabbages 6
Cauliflower 1
Citrus Fruit NA
Leafy Vegetables 14
Lettuce 20
Potato 0.05
Strawberry NA
Tomatoes 2
1. the MRL given by EPA is in parts per million for Permethrin
Study - Pesticide Residue in Fruits and Vegetables 69
Appendix 1- 3
Inserts about the
ELISA
test Kits
1. Chlorpyrifos
2. Metalaxly
3. Synthetic Pyrethroids
Study - Pesticide Residue in Fruits and Vegetables 7 4
ENVIROLOGIX INC. Catalog No. EP 004
Use of the Kit
"fbe EnviroLogix Chlorpyrifos Plate Kit is designed for the quantitative laboratory detection of Chlorpyrifos pesticide residues in ground and surface water samples, with an assay range from 0.3 to 6 parts per billion (ppb).
How the Kit Works
·n1e EnviroLogix Chlorpyrifos Plate Kit is a competitive Enzyme-Linked fmmunoSorbent Assay (ELISA).
fn the test, Chlorpyrifos pesticide residues in the sample compete with enzyme (horseradish peroxidase)labeled Chlorpyrifos for a limited number of antibody binding sites on the inside surface of the test wells.
After a simple wash step, the outcome , of the competition is visualized with a color development step.
As with all competitive immunoassays, sample concentration is inversely proportional to color development.
Darker co/or= LonJtr concentrati.on Lighter co/Qr = Higher concentration
How the' Kit Performs
Limit of Detection
·nie Limit of Detection (LOD) of the EnviroLogix l,hlorpyrifos Plate Kit is 0.3 ppb. "flie LOD was determined by interpolation at 82.1 % Bo* trom a standard curve. 82.1 % Bo was determined to be 3 standard deviations from the mean of a population of negative water san1ples.
•too% Bo equds the maximum 2.01ount of Cblorpyrifos-<nzymo conjugat.- that is bouud by the antilioJy m the abscn~c ol any C1lo1pynlos m the sample (i.e. negative controQ. %Bo = (OD of S.mpk or Calibrator/OD of Negative ControQ x 100.
Chlorpyrifos Plate Kit
Limit of Quantification
l11e Limit of Quantification (LOQ) of the EnviroLogix Chlorpyrifos Plate Kit was validated at 0.4 ppb (quantification between the 0.3 ppb lowest calibrator and 0.4 ppb may be reliable, but has not been validated). 1be LOQ was determined by fortifying a population of negative water samples at 0.4 ppb. The mean recovery was 95%. with a coefficient of variation [CV, (standard deviation/mean) x 100] of 15.3%.
Precision
Otlo1py1ifos-fortified control solutions were repetitively analyzed both within a single assay, and in different assays on different days. The data is expressed as % CV for both the recmrered concentration and for absorbance (OD).
Recovery OD Yo
Intra-Assay n=7 1 ppb 12.6% 4.3% 5 b 4.6% 2.8%
Inter-Assay n=9 1 ppb 9.4% n/a 5 b 7.4% n/a
Fortification and Recovery
Six ground and surface water samples were fortified ~ith Otlorpyrifos to a concentration of 2 ppb. The average recovery was 111 %, with a CV of 6.4%.
Cross-He activity
"f11e EnviroLogix Chlorpyrifos Plate Kit does not distinguish between Chlorpyrifos and certain other compounds, but det~cts their presence to differing degrees. The following table shows the value for 50% Bo and the vulue for the 82.1% Bo Liniit of Detection for a list of compounds. Concentration is in ppb.
Compound 50%Bo 82.1% Bo LOD
Chlorovrifos 1.5 0.3 Chlorpyrifos- 23 4.1 methvl Diazinon 36 6.5 Quin al ohos 105 17 Azinphos- 106 21 ethyl Azinphos- 426 43 methvl Pirimiphos- 127 33 ethvl Pirimiphos- 223 87 !llethyl , ___ Paratliion 143 32 Parathion- > 1000 165 methvl Fenitrothion 441 95 Malatliion > 1000 233
The following compounds are not detected at 1000 ppb: . Picloram Triclopyr 3,5,6-T richloro-2-pyridinol
Precautions and Notes
• Store all Plate Kit components at 4°C to 8°C (39°F to 46°F) when not in use.
• Do not expose Plate Kit components to temperatures greater than 37°C (99°F) or less
than 2 °C (36°F).
• Allow all reagents to reach ambient temperature (18°C to 27°C or 64°F to 81°F) before use.
• Do not use kit components after the expiration date.
o Do not use. reagents or test well strips from one Plate Kit with reagents or test well strips from a different Plate Kit.
• Do not expose Substrate to sunlight during pipetting or while incubating in the test wells.
Do not dilute or adulterate test reagents or use samples not called for in the test procedure.
• It is reconunended that positive results be confinned by an alternate method (such as liquid or gas chromatography).
• Do not collect or store CWorpyrifos-containing samples or standards in plastic containers. Use only borosilicate glassware to avoid sticking of Chlorpyrifos.
• Observe any applicable regulat:tons when disposing of samples and kit reagents.
Materials
The EnviroLogix Chlorpyrifos Plate Kit contains the following items:
8 strips of 12 antibody-coated wells each, in plate &-..me
1 vial of 1 microgram (J.tg) per milliliter (mL) Oliorpyrifos Calibrator Stock Solution (m methanol)
1 bottle of Oliorpyrifos-e02yme Conjugate 1 bottle of Substrate 1 bottle of Stop Solution
You will need to provide these items:
•
•
•
•
•
distille\i water (or equivalent) for preparation of calibrators
glass vials or tubes for preparation of calibrators (do not prepare or store calibrators in any plastic containers; Oliorpyrifos will stick to plastics).
positive-displacement pipetter with disposable tips and plungers for preparation of calibrators (capable of delivering 60 microliters (µL))
pipette(s) capable of delivering 10, 9.5, 7.5, 2.5 and 0.5 mL
disposable tip, adjustable atrdisplacement pipette which will
•
• •
•
•
•
•
•
measure 50 µL, 100 µL and 150 µL
marking pen (tn~lclible)
tape or Parafilm®
timer ( 1 hour and 30 minutes)
cool tap or distilled water for rinsing wells
microtiter plate reader or strip reader
microtiter plate washer (optional)
twelve-channel pipette that will measure 50 µL, 100 µL and 150 µL (optional)
racked glm dilution tubes for loailiug samples into the plate with a 12 ciianmil pipette (optional)
• orbital pla~ shaker (optional)
Prepa1 alion or Calib1 alors
Working calibrators must .be prepared in distilled water, from the 1 µg/mL Oliorpyrifos Stock Solution supplied in the Kit. Do not use this Stock Solution directly in the assay. To prepare 0.3, 1.5 and 6 ppb calibrators:
1. Using a positive-displacement pipette, add 60 µL ' of the 1 µg/ mL Otlo1pyrifos Stock Solution to 10 mL of water, in a g!iru. tube or vial. This is the 6 ppb Calibrator. Mix thorougltly.
2. Prepare the 1.5 ppb Calibrator by adding 2.5 mL of the 6 ppb Calibrator to 7.5 mL of water. Mix thorougltly.
3. Prepare the 0.3 ppb Calibrator by adding 0.5 mL of the 6 ppb Calibrator to 9.5 mL o~ water. Mix thorougltly. ·
4. Use the distilled water as the Negative Control.
NOTE: These Calibrator solutions are unstable and must be prepared fresh prior to each assay.
2
Chlorpyrifos Plate Kit
How to Run the Kit
•
•
•
•
•
•
•
Read all of these instructions before running the kit.
Allow all reagents to reach room temperature before beginning (at least 10 minutes with un boKed 3trip3 am! reagents at room temperature - do not remove strips from bag with dessicant until they have warmed up).
Organize all samples, reagents and pipettes so that steps 1 and 2 can be performed in 15 minutes or less.
If more than three strips are to be run at one time, the 15 minutes is likely to be exceeded, and the use of a multichannel pipette is recommended (see "Note" below).
It three or tewer strips are to be run, use a disposable-tip air-displacement pipette and a dean pipette tip to add each Calibrator and sample to the well3. Omjugate, Sub3trate, and Stop· Solution may be added in the same manner; alternatively, use a repeating pipette with a disposable tip on the end of the Combitip for these three reagents.
If fewer than all eight strips are used, reseal the unneeded strips and the dessicant in the plastic bag provided.
Use the well identification markings on the plate frame to guide you when adding the samples and reagents. Two strips may be used to run the Negative Control (Nq, three Calibrators (C1-C3) and eight samples, in duplicate. More samples require more strips. For an example plate layout see Figure 1.
Add 150 JlL of Negative Control (Nq, 150 µL of each Calibrator (C1-C3) and 150 µL of each sample (S1-S8) to their respective wells, as shown in Figure 1. Follow this same order of addition for all reagents.
NOTE: In order to minimize setup time it i~ recommended that a multi -d1a1111d pipette be used in steps 1, 2, 6 and 8 when more than 3 strips are used.
2. Immediately add 50 µL of Chlorpyrifos-enzyme Conjugate to each well.
3. 'f110roughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the bcnchtop for a full 20-30 seconds. Be careful not to spill the contents!
4. Cover the wells with tap-:: or Parafilm to prevent evaporation and incubate at ambient temperature for 1 hour. If an orbitnl plate shoker is available shake plate at 200 rpm.
.'i. After incubation, carefully remove the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wrlh romplr.trly with cool tap water, then shake to empty. Repeat this wash step four times. Slap the plate on a paper towel to remove as much water as possible. Alternatively, use a micmtiter plate washer for the wash step.
6. Add 100 µL of Substrate to each well.
7. Thoroughly mix the contents of the wells, as in step 3. Cover the wells with nra tape or Parafilm and incubate for 30 minutes at ambient temperature. Use orbital shaker if available.
Caution: Stop Solution is 1.0 N Hydrochloric acid. Handle carefully.
8. Add HlO µL of Stop Solution to each well and mix thoroughly. Tb.is will turn the well contents yellow.
NOTE: Read the plate within 30 minutes of the addition of Stop Solution.
How to Interpret the Results
Spectrophotometric Measurement and Interpretation
1. Set the wavelength of your microtiter plate reader to 450 nanometers (nm). (If it has dual wavelength capability, use 600, 630 or 650 nm as the reference wavelength.)
2. If the plate re~dcr docs not auto-zero on air, zero the imtrument agaimt 200 µL water in a blank well. Measure and record the optical density (OD) of each wPJl'5 contl'nK. Altl'mativl'ly, ml"asurl" and record the OD in every well, then subtract the OD of the water blank from each of the readings.
3. A semi-log curve fit for the standard curve should be used if the microtiter plate reader you are using has data reduction capabilities. If not, calculate the results manually as described in the next section.
How to Calculate the Results
1. After reading the wells, average the OD of each set of calibrators and samples, and calculate the %Bo as follows:
%Bo= average OD of Calibrator or sample x 100 average OD of Negative Control
The %Bo calculation is used to equalize different runs of an assay. While the raw OD values of Negative Controls, Calibrators, and samples may differ from run to run, the %Bo relationship of calibrators and samples to the Negative Control should remain fairly constant.
3
Chlorpyrifos Plate Kit
The %Bo of each Calibrator should fall within these ranges:
Calibrator 0.3 ppb 1.5 ppb 6 ppb
%Bo 66 - 88% 40 - 57% 18 - 29%
lbe CV for each palr of Calibrator and sample OD values should not exceed 15%.
2. Graph the %Bo of each Calibrator against its Chlorpyrifos concentration on a semi log scale (see Figure 3).
3. Determine the Chlorpyrifos concentration of each sample by finding its %Bo value and the corresponding concentration level on the graph.
4. Interpolation of sample concentration is only possible if the %Bo of the sample falls within the 1ange of %13o's of the Calibrators.
If the %Bo of a sample is higill:!: than that of the ~ Calibrator, the sample must be reported as less than 0.3 ppb.
If the %Bo of a sample is ~ than that of the higlli:.il Calibrator, the sample must be reported as greater than 6 ppb. If a concentration must be determined for these high level samples, dilute the sample 1:10 in distilled water. Run this dilution in a repeat of the immunoassay. If the result now falls within the range of the %Bo's of the Calibrators, you must then multiply the concentration measured in the diluted sample by a factor of 10.
Figure 1. Example of a typical plate setup.
1 2 3 4 5 6 7 8 9 10 11 12 A NC Cl C2 C3 Sl S2 S3 S4 SS S6 S7 S8
B NC Cl C2 C3 Sl S2 S3 S4 SS S6 S7 S8
c 1J E F G II
•1gurc 2 Ill I I . ustrabve ea cu atlons Well OD Average OD %CV %Bo Chlorpyrifos contents ± s<l \...oncentratior '
(ppb)
Negative 1.S19 1.538 ± 0.027 1.7 100 NA Control 1.557 0.3 ppb 1.287 1.284 ± 0.004 0.3 83 NA Calibrator 1.281 1.S ppb 0.8'10 0.310 j_ 0.0.31 3.8 53 NA Calibrator 0.796
I 6 ppb 0.402 0.410 ± 0.011 2.6 27 NA Calibrator 0.417 Sample 0.967 0.944 ± 0.033 3.5 61 0.97 ppb
0.920 _J Actual values may vary; this data 1s for demonstration purposes only.
4
Chlorpyrifos Plate Kit
Figure 3. Illustrative standard curve
100 ·---- -··
9J
80
70
00
o/aBo 50
40
30
~--'"-.._
""' 1-........ "'-
""' .... 20 ~---
10
0 0.1 10
Chlorpyrlfos Concentration (ppb)
ENVIROLOGIX INC. Catalog No. EP 005
Use of the Kit
'!be EnvuoLogix Metalaxyl Plate Kit 1s designed for the quantitative laboratory detection of Metalaxyl pesticide residues in ground and surface water samples, with an assay range from 0.15 to 1.75 parts per billion (ppb).
How the Kit Works
Tur P.nviroLogix Metalaxyl Plate Kit is a competltlve Enzyme-Linked ImmunoSorbent Assay (ELISA).
In the. t-.:.st, Mc.talaxyl pesticide. residuo in the sample compete with enzyme (horseradish peroxidase)-labeled Metalaxyl for a limited number of antibody binding siff'.s on the inside surface of the test wells.
After a simple wash step, the outcome of the competition is visualized with a color development step.
As with all competitive immunoassays, sample concentration is inversely proportional to color development.
Dt11""ker co/Qr = Lower conCt11tmtion Lighter co/Qr = Higher conCt11tration
How the Kit Performs
Limit of Detection
The Limit of Dete<:tion (I DD) of the EnviroLogix Metalaxyl Plate Kit is 0.08 ppb. The LOD was determined by interpolation at 79% Bo"' from a standard curve. 79% Bo was determined to be 3 standard deviations from , the mean of a population of negative water samples.
•too% Bo cqu.Js the maximum amount of Mct.laxyl-.:nzymc conjugate thl.t is bound by tbc 1nfY><>'ly m th• ahs•n<• of any M•u.luyl in tb• sample (Le. ncgt.tivc controQ. %Bo = (OD of 5.mpk or Calibator/OD of Ncgxtivc ControQ x 100.
Limii of Quantification
'l11c l~mit of Quantification (I,OQ) of the EnviroLogi,x Metalaxyl Plate Kit was validated at 0.15 ppb (quantification between the 0.1 ppb lowest calibrator and 0.15 ppb may be reliable, but has not been validated). The LOQ was determined by fortifying a population of negative water samples at 0.15 ppb. The mean recovery was 119% witl1 a coefficient of variation [CV, (standard deviation/mean) x 100] of 4.8%.
Precision
Metalaxyl-fortified control solutions were repetitively analp~d both within a single assay, and in different assays on different days. The data is expressed 11!1
% CV for both the recovered concentration and for absorbance (OD).
Intra-Assay n=7 0.25 ppb 5.1% 1.6% 1.0 b 23% 1.6%
Inter-Assay n=7 0.25 ppb 10.3% n/a 1.0 b 8.9% n/a
Fortification and Recovery
Six ground and surface water samples were fortified with Metalaxyl to a concentration of 0.9 ppb. TI1e average recovery was 118%, with a CV of 8.8%.
Cross-Re:activity
The EnviroLogix Metalaxyl Plate Kit does not distinguish between Metalaxyl and certain other compounds, but detects their presence to diffi:ring degrees. 'Ibe following table shows the value fo1 :>0% l3o and the value for tlie 79% Bo Limit of Detection for a list of compounds. Concentration is in ppb.
Metalaxyl Plate Kit
Compound 79% Bo 50%Bo Metalaxyl 0.08 0.45 Dunethenamid 2.4 11 Mctolachlor 3.2 12 Acetochlor 7.9 36 Propachlor 46 197 Benalaxyl 141 >1000 Alachlor 36 220
Soil Application
'Ilic EnviroLogix Metalaxyl Plate Kit can be used to detect metalaxyl in soil. Metalaxyl must first be extracted from the soil using tlie following procedure:
1. Weigh 5 grams of soil, free from twigs, stone, and gravel, into a screw capped container.
2 Add 5 mL of methanol, replace tlie cap and vigorously shake the container for 3 minutes.
3. Allow to settle, remove an aliquot and filter through a 0.45 µm filter*.
4. Dilute the filtered extract 1:100 into distilled or equivalent water and mix tltoroughly. Run tliis diluted extract in the assay as · a water sample. Remember to multiply results by 100 to compensate for the dilution.
*A suitable filter is the Millex-HV syringe filter, catalog number SLHV025~, available from Millipore Corporaoon (Bedford, MA) US toll-free telephone number 800-645-5476. In Canada, toll free 800 268 ·1881.
Precautions and Notes
• Store all Plate Kit <:omponents at 4°C to 8°C.(39°F to 46°F) when not in use.
• Do not expose Plate Kit wmponents to temperatures
greater than 37°C (99°1') or less than 2 °C (36°F).
Allow all reagents to reach ambient
temperature (18°C to 27°C or 64°F to 81°F) before use.
Do not 11~<' kit components after the expiration date.
• Do not use reagents or test well strips from one Plate Kit with reagents or test well strips from a different Plate Kit.
• Do not expose Substrate to sunlight during pipetting or while incubating in the test wells.
• Do not dilute or adulterate test reagents or use samples not called for in the test procedure,
• l t is recommended that positive results be confirmed by an alternate method (such as liquid or gas chromatography).
• Observe any applicable regulations when disposing of samples and kit reagents.
Materials
The EnviroLogix Metalaxyl Plate Kit contains the following items:
8 strips of 12 antibody-coated wells each, in plate frame 1 vial of Negative Control 1 vial of0.1 ppb Metalaxyl Calibrator 1 vial of 0:4 ppb Metalaxyl Calibrator 1 vial of 1.75 ppb Metalaxyl Calibrator 1 bottle of Metalaxyl-Enzyme Conjugate 1 bottle of Substrate 1 bottle of Stop Solution
You will rwf"d to provide thes<' items:
• disposable tip, adjustable airdisplacement pipette which will
measure 100 microliters (µL).
•
•
•
marking pen (mdelible)
tape or Parafilm®
timer ( 1 hour and 30 minutes)
• cool tap or distilled water for rinsing wells
• microtitcr plate reader or strip reader
• microtiter plate washer (optional)
• twelve-channel pipette that will
measure 100 µL (optional).
• racked dil~tion tubes for loading samples into the plate with a 12-charuiel pipette (optional)
• orbital plate shaker (optional)
How to Run the Kit
• Read all of these instructions before running the kit,
• Allow all reagents to reach room temperature before beginni,ng (at least 30 minutes with un-boxed strips and reagents at room temperature - do not remove strips from bag with dessicant until they have warmed up). ,
• Organize all samples, reagents and pipettes so that steps 1 and 2 can be performed in 15 minutes orless.
• If more than three strips arc to be run at one time, the 15 minutes is likely to be exceeded, and the use of a multichannel pipette is recommended (see "Note" below).
• If three or fewer strips arc to be run, use a disposable-tip air-displacement pipette and a dean pipette tip to add each Calibrator and sample to the wells. Conjugate, Substrate, and Stop Solution may be added in the same manner; alternatively, use a repeating pipette with a disposable tip on the em! of the Combitip for these three reagents.
• If £,ewer than all eight strips arc used, reseal the unneeded strips and the dessicant in the plastic bag provided.
• Use the well identification markings on the plate frame to guide you when adding the samples and reagents. Two strips may be used to run the Negative
2
Metalaxyl Plate Kit
Control (Nq, three Calibrators (C1-C3) and eight samples, in duplicate. More samples require more strips. For an example plate layout see Figure 1.
1. Add 100 µL of Negative Control
(NC), 100 µL of eadi Calibrator (C1~ C3) and 100 µL of eac!I·sample (S1-S8) to their respective wells, as shown in Figure 1. Follow this s~e order of addition for all reagents.
NOTE: Jn order to minimizr. sr.tnp tinw it is recommended that a multi-channel pipette be used in steps 1, 2, 6 and 8 when more than 3 strips are used.
2. Immediately add Metalaxyl-enzyme each well.
100 µL of Conjugate to
3. Thoroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill the contents!
4. Cover the wells with tape or Parafilm ) to prevent evaporation and incubate at ambient temperature for 1 hour. If an orbital plate shaker is available shake plate at 200 rpm..
5. After incubation, carefully remove the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wells completely with cool tap water, then shake to empty. Repeat this wash step fuur times. Slap the plate on a paper towel to remove as mudi water as possible. Alternatively, use a microtiter plate washer for the wash step.
6. Add 100 µL of Substrate to eadi well.
7. Thoroughly mix the contents of the wells, as in step 3. Cover the wells with~ tape or Parafilm and incubatr for 30 minutes at ambient temperature. Use orbital shaker if available.
Caution: Stop Solution is 1.0 N Hydrochloric acid. Handle carefully.
8. Add 100 µL of Stop Solution to each well and mix thoroughly. 'l11is will tum the well contents yellow.
NOTE: Read the plate within 30 minutes of the addition of Stop Solution.
How to Interpret the. Results
Spectrophotomeuic Measurement and Interpretation
1. Set the- wavelength of your micmtiter plate· reader to 450 nllflometers (nm). (If it has dual wavdengtlt- capability; use- 600, 630· or 6-50 nm 8:S thereference wavelength}
2; If ·the- platr· rradrrOOC$- not auto-zero on·a1r, i:ero-the instrumer1t against 200· ~tL water in 8: blank-well. Measure and record the optical density (OD) of eachwell's- contents: Altemntively, measure and. record- the OD- m. e~ry well, then subtract- the OD- gf. the- water blank. from.each.of the..Ielldings..
3: A semi-log curve fit for the- standardcurve should be- used- if·the-microtiter · plate reader yo•r arc· using has chtarcduction capabilities. If not, calculate the results manually as described in the next section.
How to Calculate the Results
1. After reading · the wells, average the OD of each set of calibrators and samples, and calculate the %Bo as follows:
0/oHo=;ru:J;;!gC OD of Calibrator or sample x 100 ••vcrage OD of Neg:itive Control
The %Bo calculation is used to equalize different runs of an assay. While the raw OD values of Negative Controls, Calibrators, and samples may differ from run to run, the %Bo relationship of calibrators and samples to tl11i NrgJlivr lnntrol should remain fairly constant.
1be %Bo of each Calibrator should fall withit1 these rnngesi
Calib.Dlt.o.c 0.1 ppb 0.4 ppb 1.75 ppb
%Bo 67-82% 42-54% 21-28%
TI1e CV for each pair of Calibrator and sample OD values- should- not- exceed· 15%.
2 Graph the 'YoBo of· each Calibrator against its Metalaxyl concentration on a semi~Jog1;cale .(i;ec- Figure 3}.
3
Metalaxyl Plate Kit
3. Determine tl1e Metalaxyl concentration of each sample by finding its %130
value and the corresponding concentration level on the gi;aph.
4. Interpolation of sample concentration is only possible if tl1e %Bo of the sample falls witlun the range of:- %13o's of the Calibrators.
If the %Bo of a sample is hi~ than that of the hLwl:.U Calibrator, the sample must be reported as less than 0.1 ppb.
If the %Bo of a sample is Lill'&!: than that of the bi.ghe.tl Calibrator, thesample must be rcpo1ted as greater· than 1.75 ppb. If a concentration must be determined- fur these high· lf'vrl samples, dilute the sample 1:10-in distilled water. Run this dilution in a repeat of the immunoassay. If thercsult now falls within tl1e range of tbc%Bo's of the Calibrators, yuu ·mustthen · muloply the com.eotrationmeasurcd in the diluted sample by a factor of 10. ·
Figure 1. Example of a typical plate setup.
I 2 3 4 5 6 7 8 9 10 1 J 12 - S6 s7 /\ NC Cl C2 C3 SI S2 S3 S4 SS S8 -B NC Cl C2 0 SI S2 S3 S4 SS S6 S7 S8 ,_ ----c
·--- - --·- - -D ·-- ·-E F -G )' --H
Figure 2. Illustrative calculations
"""~-
Well OD Average OD %CV %B<J Metalaxyl ; contents ± sd Concentration
(ppb)
Ncg:itivc 11?7 1.117±0.015 u 100 NA Control !-!00
-~ .. 0.1 ppb 0.813 0.794 ± 0.028 3.5 71 NA Calibrator O.T74 0.4 ppb 0.504 0.504 ± 0.000 o.o 45 NA Calibrator 0.504 1.75 ppb 0.269 0.261±0.011 4.3 23 NA Calibrator 0.253 Sample 0.344 0.345 ± 0.001 0.4 31 1.1
0.346 Actual values may vary; this data is for d·"!.IDOnstratton purposes only.
4
Metalaxyl Pinto Kit
Figure 3. Illustrative standard curve
100
90 ---- ·-------t-----t
80 t----- ------- ----
70
60
o;. Bo 50
40 --· ~
30
20
10
0 +------.---~-....-~--........
001 0.1 10
Metalaxy1 Concentration (ppb)
ENVIROLOGIX INC. Catalog No. EP 012
Use of the Kit
This kit is designed to quantJtate synthetic pyrethroid residues m methanol extracts ot various samples. 'I11e range of the assay is from 20 to 80 parts per billion (ppb) of cyfluthrin in methanol extracts. These cyfluthrin calibrators may be used to quantitate cyfluthrin, and to approximate deltametJ11in, cypermethrin, and lamhrla -cyhalothrin residues, as the assay uu;;-reactivity fo1 thc3c compounds is nearly equal. To assay quantitatively for other synthetic pyrethroids, contact Envirol ngii< for alternative calibrator materials.
How the Kit Works
'I11c EnviroLogix Synthetic Pyrethroid Plate Kit is a compeutive EnzymeLinked lmmunoSorbent Assay (ELIS1\).
In the test, synthetic pyrethroid pesticide residues in the sample compete with enzyme 010rseradish peroxidase)-labekd cypem1ethrin for a limited number of antibody binding sites on the inside surface of the test wells.
After a simple wash step, the outcome of the cor,npetition is visualized with a color development step.
As with all competitive immunoassays, sample concentration 1s inversely proportional to color development.
Va1ke1 co/01 = L»wt1· conientmlto11..J"L{~hter co/or= Higher concentration/
How the Kit Performs
f:ross-Ri-activity
111e EnviroLogix Synthetic Pyrethroid Plate Kit does not distinguish between the various synthetic pyrethroids, but detects their presence to differing
Synthetic Pyrethroid Plate Kit
degrees. 'I11e following table shows the value for 50% Bo* and the value for the 8)% 130 Limit of Detection for a list of compounds. Concentration is in ppb in methanol extract. Multiplying the numbers in the table by the dilution factor incurred during extraction of a particular sample matrix will give the detection levels in the sample matrix.
Compound 50%Bo LOD 85%Bo
'' --~
Cyfluthrin 17 2.5 Deltamethrin 6 1.0 -·-·-Cyperrnethrin 17 1.7 lambda 6 0.8 Cvhalothrin Bifenthrin 545 125 Allethrin 1680 126 Tralomethrin '118 17 Resmethrin 1890 170 Fenvalerate 3860 560 Permethrin !70 25 Permethric 1110 19 acid Fluvalinate >10,500 >10,500 Pyrcthrins >10,500 >10,500
Precautions and Notes
• Store all Plate Kit components at 4°C to 8°C (39°F to 46°F) when not in use.
• Do not expose components to greater than 37°C than 2 °C (36°f<).
Plate Kit temperatures
(99°F) or less
• 1\llow all reagent~ tu reach ambtent temperature (18°C to 27°C or 64°F to 81 °F) before use.
·~l 00°/o Bo equals the maximum amount of
Synthetic pyrcthroid-enzyme conjugate that is bound by the antibody in the absence of nny Synthetic pyrethroid in the sample (i.e. negative control). %Bo = (OD of Sample or Calibrator/OD of Negative ControQ x 100.
• Do not use kit components after the expiration date.
• Do not use reagents or test well strips from one Plate Kit with reagents or test well strips from a different Plate Kit.
• Do not expose Substrate to sunlight during pipetting or while incubating in the test wells.
• Do not dilute or adulterate test rc>·dgc>nts or m<" sampks not called for in the test procedure.
• It is recommended that positive results be confirmed by an alternate method (such as liquid or gas chromatography).
• Observe any applicable regulations when disposing of samples and ktt reagents.
Materials
The EnviroLogix Pyrethroid Plate Kit following items:
Synthetic contains the
8 strips of 12 antibody-coated wells f'ar·h, in platf' framo'.e
1 vial of Synthetic Pyrethroid Negative Control (neat methanol)
1 vial of~ ppb Cyfluthrin Calibrator, in methanol
1 vial of 20 ppb Cyfluthrin Calibrator, in methanol
vial of 80 ppb Cyfluthrin Calibrator, in methanol
vial of Sodium Hydroxide Solution (0.2 N) bottle of Cypermethrin-enzyme Conjugate
1 bottle of Substrate 1 bottle of Stop Solution
You will need to provide theae items:
• Methanol, ACS Reagent Grade for sample extraction
• Distilled or deionized water for calibrator and sample extract dilution
• Glass vials or test 1ubcs for preparing dilutions
• Positive-displacement pipetter with disposable tips and plungers capable of delivering 100 microliters (µ.L) and 1 milliliter (mL)
• Repeating pipetter, with syringe tip capablf'. of delivering 25 µL and 2 mL (also 3 syringe tips for delivering 100 ~LL if you use the repeater to <idiver conjugate, substrate and stop in the assay)
• Vortex mixer
• Disposable tip, adjustable airdisplacement pipette which will measure 100 µL
• i\!arking pen
• Tape or Parafilm®
• Timer
• Cool tap or distilled water for rinsing wells
• Microtiter plate reader or strip reader
• Miuu1ite1 plate washer (optional)
• Twelve-channel pipette that will deliver 100 p.L (if more than 3 strips• are run per assay)
• Racked dilution tubes for loading samples into the plate with a 12-channel pipette (see previous statement)
• Orbital plate shaker (optional)
Preparation of Sample Extracts and Calibrators
Note: Consult individual apphcation sheets for sample extraction instrnctions specific to the sample material to be analyzed; contact EnviroLogix for further information on applications that are available.
I. Plan out a plate map showing the location of each calibrator and sample to be run (in duplicate) in an assay. A standard curve plus 8 samples may be run m duplicate usmg 2 strips. Additional samples reguire additional strips. (See Figure 1)
2. Label two glass tubes or vials for each sample to be assayed, as well as one glass tube or vial for the Negative Control and each Calibrator. Arrange them in a rack in the order planned on your plate map.
) T Joing a po<>itive-rlisplacement pi petter, dispense 1 mL of each sample extract into the first tube or vial labeled for each sample.
4. I Jsmg a repeatrng p1p~ttf'.r a11d synnge tip, quickly add 25 ~LL of Sodium Hydroxide Solution to each sample extract. Vortex to mix.
5. Cap vials or cover test tubes with Parafilm, and incubate 30 minutes.
6. During the incubation period, use a repeating pipetter to dispense 2 mL of distilled or deionized water into the second tube or vial labeled for each sample extract, and into the Negative Control and Caiibrator tubes or vials.
7. At the end of the 30 mmute mcubat:ion, use a positive-displacement pipetter to transfer 100 pl, of each treated sample extract to its corresponding tube of water. Also dispense 100 µL of the Negative Control and each Calibrator to their tubes of water in the same manner. Vortex to mix.
How lo Run Lhe Kit
• Read all of these instructions before running the kit.
• Allow all reagents to reach room temperature before beginning (at least 30 minutes with un-boxed strips and reagents at room temperature - do not remove strips from bag with dessicant until they have warmed up).
2
J·
Synthetic Pyrethroid Plate Kit
• Organize all prepared Calibrators and samples from step 7 above, reagents and pipettes so that steps 1 a11d 2 can be performed in 10 minutes or less.
• If more than three strips arc to be run at one time, the l 0 minutes is likely to be exceeued, am! the use of a multi channel pipette is strongly recommended (see "Note" below).
• lf three or fewer strips are to be run, use a disposable-tip air-displacement pipette and a clean pipette tip to add each Calibrator and sample to the wells. Conjugate, Substrate, and Stop Solution may be added in the same manner; ;utematively, use a rnp!iating pipette with a disposable tip on the end of the Combitip for these tl reagents.
• H !ewer than all eight suips are u~ed, reseal the unneeded strips and the desiccant in the plasl:i<.. bag prnvidcd.
• Use tlll. wdl iJentification markingn on the plate frame to guide you when adding the samples and reagents. Two strips may be used to run the Negative Control (NC), three Calibrators (ClC3) and eight samples, in duplicate. More samples require more strips. For an example plate layout see Figure 1.
1. Add 100 µL of prepared Negative Control (NC), 100 µL of each prepared Calibrator (C1-C3) and 100 µL of each prepared sample extr (S 1-SS) to their respective wells, as shown in Figure 1. Follow this same order of addition for all reagents.
NOTE: In order to minimize setup time it is strongly recommended that a multichannel pipette be used in steps 1, 2, 6 and 8 when more than 3 suips are used.
2. Immediately add 100 µL of Cypennethrin-enzyme Conjugate to each well.
3. Thoroughly mix the contents of the wells by moving the strip holder in a rapid circular motion on the benchtop for a full 20-30 seconds. Be careful not to spill the contents!
4. Cover the wells with tape or Parafilm to prevent evaporat1on and incubate at ambient temperature for 1 hour. If an orbital plate shaker is available shake plate at 200 rpm.
5 Aftc·r incubation, carefully remove the covering and vigorously shake the contents of the wells into a sink or other suitable container. Flood the wells completely with cool tap water, then shake to empty. Repeat this wash step four times. Slap the plate on a paper towel to remove as much water as possible. Alternatively, use a microtiter plate washer for the wash step.
6. Add 100 µL of Substrate to each well.
'1 J horonghly mix the contents of the wells, as in step 3. Cover the wells with D\'!";Y tape or Parnfilm and incubate for 30 minutes at ambient temperature. Use orbital shakrr if available.
Caution: Stop Solution is 1.0 N Hydrochloric acid. Handle carefully.
8. Add 100 µL of Stop Solution to each well and mix thoroughly. This will tum the well contents yellow.
NOTE: Read the plate within 30 minutes of the addition of Stop Solution.
How to Interpret the Results
Spectrophotometric Measurement and Interpretation
1. Set the wavelength of your microtiter plate reader to 450 nanometers (nm). (If it has dual wavelength capability, use
600, 630 or 650 nm as the reference wavekngth.)
2. If the plate reader does not auto-zero on air, zero the instrument against 200 µL water in a blank well. Measure and record the optical density (OD) of each welt's contents. J\ltematively, measure and record the OD in every well, then subtract the OD of the water blank from each of the readings.
3. A semi-log curve fit for the standard curve should be used if the microtite1 plate reader you are using has data reduction capabilities. If not, calculate the rps11lts manually as described in the next section.
How to Calculate the Results
1. After reading the wells, average the OD of each set of calibrators and sample extracts, and calculate the %Bo as follows:
%Bo=averagi; OD o1Calibrator or sampl<: x 100 average OD of Negative Control
1he %Bo calculation is used to equalize different runs uf an assay. While the raw OD values of Negative Controls, Calibrators, and samples may differ from run to run, the %Bo relationship of calibrators and samples to the Negative Control should remain fairly constant.
1he CV for each pair of Calibrator and sample OD values should not exceed 15%.
3
Synthetic Pyrethroid Plate Kit
2. Graph the %Bo of each Calibrator against its Cyfluthrin concentration on a semi-log scale (see Figure 3).
3. Determine the Synthetic pyrethroid concentration of each sample by finding its %Bo value and the correspondmg concentrauon level on the graph.
4. Interpolation of sample extract concentration is only possible if the %Bo of the sample extract falls within the range of %Bo's of the Calibrators.
If the %Bo of a sample extract is higher than that of the lowe~t
Calibrator, the sample extract must be reported as less than 4 ppb.
If thP. %Bo of a sample extract is !gwi;r than that of the highest Calibrator, the sample extract must be reported as greater than 80 ppb. If a concentration must be detennined for these !ugh level samples, dilute the sample extract 1:200 in 5% methanol in water (instead of diluting 1 :20 in water). Prepare the Negative Control and Calibrator as directed and run this dilution in a repeat of the immunoassay. If the result now falls within the range of the %Bo's of the Calibrators, you musl then multiply the concentration measured in the diluted sample by a factor of 10.
S. To calculate the concentration of synthetic pyrethroid in the original sample material, multiply all results by the dilution factor incurred during extraction of the sample. (See the particular application sheet for further informalJ.on).
Figure 1. Example of a typical plate setup.
1 2 3 4 5 6 7 8 9 10 11 12 A NC Cl C2 C3 Sl S2 S3 S4 SS S6 S7 SS
13 NC Cl C2 C3 St S2 S3 S4 SS S6 S7 SS
c D ~ " "'
c;,':>'
E _() -Cl.. Q_ ( .... ~ \'-"\.'\
F ~ Cl.... '-'- (.__./
,-,
G '+::. ~ 00 H
Figure 2. Illustrative calculations
Well OD Average OI> %CV %Ho Synthetic contents ±sd pyrethroid
Concentration foob)
Negative 1.968 1.996 ± 0.040 2.0 100 NA Control 2.024 4 ppb 1.692 l.6S7 ± 0.049 2.9 83 NA Calibrator 1.623
"~·
20 ppb 1.032 l.OS8 ± 0.037 3.S S3 NA Calibrator 1.084 80 ppb 0.464 0.4SS± 0.013 3.0 23 NA Calibrator 0.44S
-~-•e ~·••
Sample 0.83S O.S29 ± 0.008 0.1 42 32.0 ppb* 0.824
Actual vahws may vary; this cfata is for demonstration purposes only. *If this were a cyfluthrin-contaminated grain sample extract, you would multiply this result by SO (for a l:S dilution during extractjon, and 1: 10 extra dilution of the sample extract) for a fmal result of 1.6 ppm in gram.
4
Synthetic Pyrethroid Plate Kit
Figure 3. Illustrative standard curve
%Bo
100
90
80.
70 60.
50
40
30
20+-~~~~~~~-~~---~~~-<
10+--~~~--~~--t--~~~~~~-<
0- _, ..... , .... , ... ,., ',,, i 'I\ d
1 10 100 Cyfluthrin Concentration (ppb)
Appendix 4
A= Apple P:Pesticide
The most common pesticides used
on various fruits and crops
Study - Pesticide Residue in Fruits and Vegetables 87
.LUSTRATIONS BY ERIC JONES
;st of footnoted pesticides: aldicarb, 2 azinphos methyl, 3 atrazine, captan 6 carbaryl, 7 carbendazim, cypermethrin, 10 DDT, 11 deltametrin, ~ dinocap, 14 the 'drins' (aldrin, dieldrin and endrin), ; endosulfan, 17 heptachlor, 18 gamma-HCH, > paraquat, 21 zineb.
mrces:
4 benomyl, 8 chloropyrifos, 12 dimethoate, 15 diazinon, 19 methyl parathion,
~sticides and your food Andrew Watterson (Green Print), Pesticides News (Pesticides Action Network