STUDY ON PESTICIDE RESIDUE IN FRUITS AND

STUDY ON

PESTICIDE RESIDUE

IN

FRUITS AND VEGETABLES

in partial fulfillment for the MSc (Agr. Vet. Pharm)

Therese Visanich

May 2001

Dedicated to baby

Martha

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.


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.


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.


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.


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


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


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.


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.


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


- 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


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.


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.


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.


Chapter 2

Classification of Insecticides

Their Composition and Properties

The popular outcry

againsl llleir use (peslicides)

cannot ovrershadow

their use

Food Agriculture Organization


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.


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


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).


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.


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


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.


Chlorpyrifos

Organophospate (OP)

Chapter3

It is the world's leading insecticide

Agrow's Top Twenty Five 1


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.


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.


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.


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.


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.


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.


Chapter 5

Metalaxyl

From Phenylamides Group

Extensively used to control

Into potato blight

now registered for use on

other crops

US -Environment Protection Agency


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.


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.


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



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


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·


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.


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.


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.


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


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


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


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:


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



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


Materials used for the Synthetic Pyrethrold .kit:


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 ~~~--~~~~~--~~


1 bottle of Cypermethrin enzyme conjugate

1 bottle of Sodium Hydroxide Solution (0.2N)



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


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.


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


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


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


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.


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 ~~~~~~~~~~~


Results obtained for the Chlorpyrifos kit

The %Bo is found out as

%Bo = average OD of calibrator or sample X 100


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


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.


Results obtained for the Metalaxyl kit


%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


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


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


Results obtained for the Synthetic Pyrethroid kit


%Bo: average OD of calibrator or sample X 100


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.


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.


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)


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



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


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


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


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


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)


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.


• 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 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.


8. Add 100 µL of Stop Solution to each well and mix thoroughly. 'l11is will tum the well contents yellow.


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.


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. ·


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


100

90 ---- ·-------t-----t

80 t----- ------- ----

70

60

o;. Bo 50

40 --· ~

30

20

10

0 +------.---~-....-~--........

001 0.1 10

Metalaxy1 Concentration (ppb)


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


• 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 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·


• 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.


8. Add 100 µL of Stop Solution to each well and mix thoroughly. This will tum the well contents yellow.


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.


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


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).


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



%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


The number in each box refer to the PQ~t!cid~~ ~\;!Y at the foot of page 8.

APPENDIX ..

2, 4, 7, 8, 12, 15, 16, 19, 20

.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

Documents

STUDY ON PESTICIDE RESIDUE IN FRUITS AND