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1.1 Introduction to Pesticides

“The most intelligent species on earth poisons its food before eating it.” These

startling words by Rishi Miranhshah [1] reveal the stark reality of destruction that we

face today due to the relentless and injudicious use of pesticides. We are continuously

fed on pesticide diet as they reach our food, drinking water, air, etc. Their continued

use on pests is also known to develop pest resistance, thus arises the need to probe

and develop new alternatives. United States federal law under Federal Insecticide,

fungicide and Rodenticide Act (FIFRA) defines pesticide as [2] "any substance or

mixture of substances intended for preventing, destroying, repelling or mitigating any

insects, rodents, nematodes fungi or weed or any other form of life declared to be

pest, and any substance or mixture of substances intended for use as a plant regulator,

defoliant, or desiccant."

As India is a tropical country, it suffers severe losses in agriculture due to pests [3].

This necessitates the use of pesticides to protect our crops against the attack of several

pests. Pesticides are the chemicals that kill or destroy the pests. Even though the

pesticides are poisonous chemicals, they are a major savior of humankind as they

have helped us to keep up with the food demands of the growing population.

Pesticides are our evil necessity which we cannot do without. Pesticides are relied

upon for various purposes namely increasing agriculture production, forestry, public

health, etc. In the absence of pesticides the humans, plants and animals would suffer

from various diseases [4]. The first known pesticide is Arsenic and the most popular

one has been DDT (dichlorodiphenyltrichlorethane) which was introduced in 1934 by

a scientist Paul Mullah during World War II. It was used as an effective tool to

eradicate malaria. The effective control of these pests and diseases require the use of

chemical pesticides whose usage depends upon the price, risk, credit availability and

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cropping patterns [5]. But, it is also true that their excessive usage has made the planet

unlivable. As per WHO guidelines [6] every pesticide should be used according to the

recommended method of application. A set of guidelines should also be followed by

the manufacturers so that different companies manufacturing the same pesticide

should produce pesticide of same efficacy. The specification is even more important

as pesticides are no longer protected by patents but have become ordinary

commodities. The impurities present in the active ingredient also demand serious

attention as this may affect the toxicity and action of pesticide. Analytical methods

may also be needed to detect the impurities in the formulations. But all these steps

entail high expenditure which the developing countries like India may not be able to

afford. Hence, it is recommended that the funds, equipments and resources may be

shared between different centers in a country or with other countries. As pesticides are

meant for controlling harmful or unwanted organisms we must ensure that safeguards

are in place to protect the people, environment and wildlife from their ill-effects.

Though, the pesticides are very useful in protecting crops but these are risky by way

of their very nature. Their nature to kill the living organisms has become a bane from

living beings and environment [7]. Hence, the research is on for funding safe and

efficient methods. It is important to identify the pest to be eliminated and chose a

suitable pesticide for it. Using a general pesticide for an unsuitable pest will be a sheer

wastage of pesticide. Pesticide labels should never be ignored as they clearly state the

risks involved and the necessary precautions to be followed. The issue of pesticide

usage is a very touchy one involving $ 500 billion agro industry, environmentalists,

health concern advocates and common people. But the truth is pesticides are toxic and

need to be eliminated from the environment.

1.1.1 Pesticides Use

Agriculture is the mainstay of the Indian economy. It is a formidable task to ensure

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food security for more than 1 billion Indian people with shrinking cultivable land

resources and this necessitates the use of high yielding variety of seeds, balanced use

of fertilizers and judicious application of quality pesticides along with education to

farmers for the implementation of modern farming techniques. One of the eight

targets marked by United Nations Hunger Task Force is to conserve the nature and

safeguard our environment [8]. As per estimates, India approximately loses 18% of

the crop yield valued at Rs.900 billion due to pest attack each year [9]. The average

pesticide consumption in India is a low 288 g/ha as compared to the global average of

900 g/ha. [10]. The use of pesticides helps to reduce the crop losses [11], provide

economic benefits to farmers, reduce soil erosion and help to ensure food safety and

security for the nation. Pesticide use is high in the cultivated regions with better

irrigation facilities and where commercial crops are planted [12], e.g., cotton is grown

in 5% of cropped area but uses up whopping 45% of total pesticides. Over 56% of

India’s population are engaged in agricultural practices and have exposure to the

pesticides used in agriculture [13]. Pesticides being used in agricultural tracts are

released into the environment and come into human contact directly or indirectly. The

production of food grains has almost quadrupled from 50 million tonnes in 1948-49 to

198 million tonnes in 1996-97 and the credit goes to increasing use of pesticides along

with betterment of technology, seeds and use of fertilizers [14]. Punjab is one of the

highest users of these pesticides since the advent of green revolution. Punjab accounts

for 1.5% landmass of the country, but its pesticide consumption is 17% of India, the

per hectare consumption being 923 g/ha, which is the highest among the other

agriculturally advanced states like Haryana, Andhra Pradesh, Tamil Nadu, Karnataka

and Gujarat. India suffers agricultural export losses worth Rs. 1000 crore due to

rejection of produce on account of excess pesticide residues [15]. India accounted for

85,000 MT of pesticide production worth Rs.74 bn inclusive of exports worth Rs. 29

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billion during FY 07 which is the second highest in Asia (behind China) and twelfth

globally [11]. But the spread of pesticides has been uneven, the world over [16], as

North America and Western Europe account for more than 50% of global

consumption but comprise only 25% global cropland. Developing countries, which

have 55% of the world’s agricultural land use 20% of the pesticides. Proper use of

pesticides has brought many rewards. Not only has it increased the crop yields and the

income of farming community but also the quality of cereals and seeds. Pesticides

have played a crucial role in taming several vector borne diseases like malaria and

typhus, which had gained epidemic proportions. Malaria alone accounted for 300

million cases and 3 million deaths. DDT was instrumental in bringing the menace of

malaria under control in several countries of Mediterranean, India, Sri Lanka, Java,

Bali, Far East, Central and South America. As far as the gains for the mankind with

the use of DDT are concerned in the war against malaria and typhus are concerned,

they far outweigh the detrimental effects of DDT. In this light, problems of pesticide

safety, regulation of pesticide use, use of biotechnology, and biopesticides, and use of

pesticides obtained from natural plant sources such as neem extracts are some of the

future strategies for minimizing human exposure to pesticides. The pesticides have

been classified into several sub-divisions as per their chief target pest. But this doesn’t

imply that its effect will be limited to that particular class of pests. Many herbicides

can be toxic to mammals or insects and some lethal pesticides are broadly called

biocides [17].

Developed Countries follow the intensive commercial model of agriculture. In the

United States, the use of synthetic pesticides since 1945 has grown thirty-three-fold to

about 0.5 billion kilograms (kg) per year or 3 kg per hectare per year

(http://www.pollutionissues.com/A-Bo/Agriculture.html). The toxicity of modern

pesticides has increased by more than ten-fold over those pesticides used in the early

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1950s and hence the risk posed has also increased manifold. Ground and surface

water contamination from pesticides is a serious issue. India is one of the only two

countries worldwide (along with the United States) to have applied more than 100,000

tons of dichlorodiphenyl trichloroethane (DDT) since its initial formulation. Because

of the excessive and indiscriminate use of pesticides in India, the total intake of

organochemicals per person in our country is the highest in the world.

Table 1.1: Classification of Pesiticides17

S. No. Sub-Division Target Pest

1. Acaricides Mites, spiders

2. Algacides Algae

3. Antibiotic Bacteria and virus

4. Avicide Birds

5. Dessicants Dry up animals or plants

6. Fungicides Fungi

7. Herbicides Plants

8. Insecticides Insects

9. Molluscicids Molluscs

10. Nematocides Nematodes

11. Piscicides Fish

12. Plant Regulators Retrard or speed plant growth

13. Repellants Drive away pests

14. Rodenticides Rodents

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15. Sterilants Stop Reproduction

1.1.2 Creating Awareness

Most Indian farmers rely on chemical pesticides even though being aware of their

hazardous effect on health. Many of them do not take adequate precautions while

handling these dangerous chemicals thus creating a precarious situation in rural

environment where these are abundantly used. Knowledge is power, therefore, when

farmers have knowledge of all the available options, they can reduce the risks by

avoiding most toxic substances and use the less toxic formulations. The frequency of

spraying and the volume of pesticides used should also be monitored. The farmers

should be taught how pesticides may enter our bodies and what precautions are

needed to avoid life-threatening situations. Information about the correct storage and

disposal practices must be disseminated so as to avoid contamination of food, water,

etc. The knowledge about signs and symptoms of pesticide poisoning is another

important aspect to be taken care of. In view of the above discussion, it becomes

amply clear that awareness about pesticide use needs to be spread widely and at the

same time better and less toxic alternatives must be explored. A pesticide passes

through many checks and regulations before it enters the market. Then it must be

registered with the appropriate Government Authority as mentioned under the

Insecticides Act 1968 before it is allowed to be used. The Ministry of Agriculture

administering this Act, recommends the type and dosage of a pesticide for use on a

particular crop. The Ministry of Health and Family Welfare prescribes standards for

different pesticides which can be allowed to remain in the case of particular

agricultural commodity [7]. Under the Prevention of Food Adulteration Act, 1954

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MRLs (Maximum Residual Limits) are prescribed for pesticide residues in

agricultural commodities. MRL is expressed in mg/kg.

1.1.2.1 New Alternatives to Conventional Pesticides

The chemical control of disease vectors has been remarkably successful, but it is clear

that if our aim is sustainable development, the dependence on chemical methods has

to be reduced and alternative methods of biological control and environmental

management must be implemented. Hence, Integrated Pest Management (IPM) must

be followed to develop safe and effective methods for crop protection while

preserving the environmental balance and resources [18-21].Various new alternatives

for pesticides have emerged lately:

1. Biopesticides are green pesticides which are derived from plants or other

organisms. Biopesticides generally include: microbial living systems-bacteria, fungi,

virus, entomopathogenic nematodes, insect predators and natural parasites, plant-

derived products, insect pheromones and metabolites. Biopesticides are being seen as

a possible alternative as these are safer, cost-effective, target specific, biodegradable

and ensure sustainable agriculture.

i.) Botanical Pesticides: are derived from the plant sources, examples include

Azadirachtin from neem, pyrethrum daisy extract; eucalyptus globules etc.

ii.) Microbial Insecticides: comprise of microscopic living organisms (virus,

bacteria, fungi, protozoa or nematodes) [22-29]. Greatest progress has been made with

biological control agent, e.g., Bacillus thuringiensis serotype H-14.

iii.) Fly-ash based herbal pesticides [30]: derived from waste material from thermal

power plants has proved to be a useful insecticide against several pests infesting the

rice, vegetables and other field crops. It is being developed as a potential dust

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insecticide as well as an active carrier in many insecticide formulations like dust,

wettable powder and granules.

2. Other substitutes for Pesticides

Some other viable alternatives to pesticides can be the use of physical barriers

(www.oznet.ksu.edu/dp_hfrr/extensn/Hort_Tips/Garden_Tips/Alternative%20Pesticid

es.htm) to block entry of pests and crop rotation and diversification can be used as a

means to restrict pest attacks and movement.

1.1.2.2 Future Outlook

It is being projected that the continued regulatory pressures will drive the need for

safer, cost-effective disease and pest management [31-34]. This in turn shall help

newer products and GMOs to emerge. The adoption of biopesticides is bound to

increase due to better grower understanding of customer value equation and consumer

demand. These factors along with the continued integration of cost effective

alternatives will bring a paradigm shift in the mindset. Biopesticides are here to stay

and will remain an integral part of crop production as "life-extenders" or "stand-

alone" entities in commercial agriculture.

1.1.3 Pesticide Hazards

In India, 63% of the pesticide usage is for the agricultural sector and rest of it is used

in the non-agricultural sector [35]. The impact of pesticides in agricultural output is

remarkable, for example, in United Kingdom, the yields of wheat and barley crops

doubled between 1950 and 1975, with the use of pesticides along with fertilizers and

other agricultural inputs. Although the use of pesticides has helped to enhance

economic gains through crop protection but these gains cannot nullify the hazards of

pesticides. They have had serious implications to human health and non-target plants

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and animals by accumulating in food and water. Widespread use of pesticides has

resulted in their bioaccumulation due to their lipophilic properties. Dikshith has

studied the effect of pesticides on the behavior of animals, the morphological and

biochemical lesions and the metabolic pathways of pesticides have been studied in

aquatic environment and various fauna [36]. The path breaking work of Rachel

Carlson, “Silent Spring” in 1962 warned that OC compounds could pollute the tissues

of virtually every life form on the earth, the air, the lakes and the oceans, the fishes

that live in them and the birds that feed on the fishes [37]. The US National Academy

of Sciences made a shocking expose that the DDT metabolite, DDE causes eggshell

thinning and that the bald eagle population in the United States declined primarily

because of exposure to DDT and its metabolites [38]. Many pesticides act as

endocrine disruptors which act by mimicking or destroying the natural hormones in

the body and their long-term, low-dose exposure is responsible for many dreaded

conditions like immunosuppression, hormone disruption, diminished intelligence,

reproductive abnormalities and cancer [39-41]. The first case of pesticide poisoning in

India was reported from Kerala in 1958 where, over 100 people died after consuming

wheat flour contaminated with parathion [42]. Even though the consumption of

pesticides in India is still very low yet the food commodities are widely contaminated

with pesticide residues, mainly because of the non-judicious use of pesticides. It has

been found that 51% of food commodities in India are contaminated with pesticide

residues and 20% of these residues are above the maximum residue level values.

Many vegetable samples taken from the farm gate in West Bengal were found to

contain various pesticides (0.01-2.23 ppm) [42-44]. On the contrary, the UK Pesticide

Residue Committee annual report (2002) showed that over 70% of the food in the UK

was pesticide free and only 1.09% contained residues above the statutory maximum

residue levels (MRLs) [44].

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In this light, problems of pesticide safety, regulations of pesticides use, use of

biotechnology, and biopesticides are some of the future strategies for minimizing

human exposure to pesticides. U.S. data show that 18 percent of all pesticides and

about 90 percent of all fungicides are carcinogenic. In addition to humans, thousands

of domestic animals are also poisoned by pesticides in the United States. The

destruction of natural predators and parasites is costing the USA more than $ 500

million each year and resulting in the development of pesticide resistance

[http://www.pollutionissues.com/A-Bo/Agriculture.html].

Indiscriminate aerial spraying will make the atmosphere a major reservoir for drift

prone finely divided particulate pesticides and highly volatile compounds. After

spraying, the pesticides can evaporate from soil and foliage and contaminate the

environment [45]. The volatilization rate can be as high as 80-90 percent of the

applied pesticide within a few days of application [46]. Many studies have

consistently confirmed the presence of pesticide residues in air. Almost all the

investigated pesticides have been detected in rain, air, fog, or snow across the USA at

different times of the year [47]. Pesticide drift can account for wastage of 2 to 25% of

the chemical being applied; the some of the pesticides have the capacity to spread

over a distance of several miles.

Pesticides are responsible for severe environmental imbalance as some microbe

populations may perish and others may flourish like the saphrophytic and spore

forming [48]. One spoonful of healthy soil has millions of tiny organisms which

include fungi, bacteria, and a host of others. These microorganisms act as facilitators

for the plants to help them utilize the soil nutrients. Microorganisms also play a vital

role in storage of water and nutrients in the soil and regulating water flow [49]. The

accumulation of residual pesticides and their metabolites can drastically alter the soil

chemical properties, further disrupting delicate balance in the soil microenvironment,

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which may reduce the soil fertility and its ability to support life. The population of

some of the useful soil invertebrates like earthworms, mites, centipedes and beetles

may dwindle due to non-target or residual pesticide toxicity [48]. The study carried

out by the U.S. Geological Survey (USGS) on major river basins across the country in

the early to mid- 90s yielded shocking results as it was stated that more than 90

percent of water and fish samples from all streams were found to be contaminated

with one, or more often, several pesticides [50]. A survey in Bhopal has revealed that

58% of drinking water samples drawn from various hand pumps and wells around

Bhopal are contaminated with organochlorine pesticides above the EPA standards of

[51]. The ground water which is once polluted with toxic chemicals may involve a lot

[51]. The ground water which is once polluted with toxic chemicals may involve a lot

money and time and yet the clean up may not be completely possible [52-54].

1.1.3.1 Pesticide Poisoning

Some 500,000 people are either killed or incapacitated by poisoning every year and

developing countries make most of these casualties due to the extensive and non-

judicious use [55, 56]. As per an estimate, worldwide nearly 10,000 deaths occur

annually, worldwide, owing to the use of chemical pesticides, with about 75% of

these casualties being from developing countries [57]. At present, India is the largest

producer of pesticides in Asia and ranks twelfth in the world for the use of pesticides

with an annual production of 90,000 tons. Humans are exposed to pesticides found in

environmental media (soil, water, air and food) by different routes of exposure such

as inhalation, ingestion and dermal contact. Exposure to pesticides results in acute and

chronic health problems. These range from temporary acute effects like irritation of

eyes, excessive salivation to chronic diseases like cancer, reproductive and

developmental disorders, etc [58]. Pesticides are harmful in another way also, as their

use also leads to increased and unnecessary pest outbreaks causing additional crop

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losses, necessitating more pesticide use and thus destroying enemies of pests and

emergence of pesticide resistant and secondary pests. This pulls the farmers into a

vicious cycle as it creates the need to spray increased doses of stronger pesticides to

kill the mutilating pests [59]. This is supported by the fact that in 1938, 7 species of

insects and mites were reported to be pesticide resistant, but in 1984, this figure

rocketed to 477 with the increase in pesticide use. Hence, it is wrong to assume that

more pesticide use will usher us into pest free society, rather we get resistant pests in

bargain, which are highly difficult to control by the prevailing methods. A PGIMER

(Post Graduate Institute for Medical Education and Research), Chandigarh, research

has linked the use of large amounts of pesticides to the ever increasing cancer cases in

the Bathinda cotton belt. A house-to-house survey of cancer cases in Punjab has put

the figures at an alarming 31 cases per lakh of population [60, 61]. Murali et al

conducted a study on acute poisoning cases in PGIMER, Chandigarh covering 15

years (1990 to 2004). The most common agents were anticholinesterases (35.1%) and

aluminum phosphide (26.1%). Maximum case fatality ratio was due to aluminum

phosphide exposures followed by anticholinesterase agents. This calls for integrated

pesticide management and training of farmers in spraying techniques [61]. The link

between unsafe and indiscriminate pesticide use has further been validated by a study

conducted by Grace et al among farmers of Thanjavur district (South India) [62].

Silva et al conducted a study on groundwater evaluation in oryziculture areas in

Portugal and the groundwater was found to be contaminated with several pesticides

which call for sustainable use of pesticides to preserve aquatic and soil resources

[63].There is strong evidence now that pesticides have entered our food chains

through ground and surface waters. CSE (Centre for Science and Environment), New

Delhi in 2004 detected pesticides in samples of renowned soft drink brands; Coke and

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Pepsi, and these were thought to be contaminated by the water used for the production

of these drinks [7].

The pesticide invasion into US homes has been studied by Landrigan et al have

studied the [64]. As per this research, 90% of the US households use pesticides and

this makes up for $ 2 billion industry. Children are particularly a high risk group as

they are exposed to pesticides through their homes and also through the diet including

drinking water. Endosulfan is an organochlorine pesticide which has become a

leading chemical used against pests in agriculture, to kill fish in water bodies and as

an insecticide but is not recommended for household use as it is known as a potent

poison. Endosulfan got publicity when it was aerially sprayed at Kasargad in Kerala

and caused physical and mental defects in poor farmers and their families. It is proven

to accumulate in human breast milk and is the cause of heinous congenital defects,

which are still being observed at Kasargad, “Kerala’s Bhopal”. After this, 62

countries have either banned or restricted Endosulfan use, but still, India continues to

be the largest producer and exporter of this pesticide. The government has not woken

up to its dangers and succumbed to the pressure of industrialists’ lobby, and continue

to call it safe, so far, as a result of these, only the state of Kerala has banned it [65].

In rural Asia, the easy access to pesticides is turning out to be bane as the cases of

self-poisoning with agricultural pesticides has become the most common means of

suicide. Rao et al conducted a study in district government hospital of Warangal in

Andhra Pradesh [66] for the years 1997 to 2002 on 8040 patients admitted with

pesticide poisoning with the fatality rate being 22.6% and 96% had intentionally

poisoned themselves. Monocrotophos and endosulfan, accounted for the majority of

deaths in the study. Inappropriate and low doses of antidotes used were some of the

reasons for high mortality rate. As per estimates every year 250,000 to 370,000

suicides occur due to pesticide poisoning worldwide [67]. In Asian countries like

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China, Malaysia and Sri Lanka, pesticide self-poisoning makes up 60% to 90% of all

suicides cases. The study conducted by Mohamed et al in Sri Lanka concluded that

pesticides were readily available to the people inflicting self-poisoning within their

homes or nearby. In this context it becomes imperative to reduce the accessibility of

pesticides in the domestic environment as it can have a direct bearing on reducing the

number of pesticide related suicides [68]. It is important that the pesticides be stored

safely and efforts must be made to make the sale of pesticides safer from the shops. In

a study conducted on workers (N=356) in four units manufacturing HCH by Nigam et

al revealed that 21% of the studied workerses had neurological symptoms related to

the intensity of exposure [69]. The National Institute of Occupational Health (NIOH)

studied the magnitude of the toxicity risk involved in the spraying of methomyl, a

carbamate insecticide, in field conditions [70]. The study highlighted immense

changes in the ECG and the levels of serum LDH and ChE activities in the spray men

thus underlining the cardiotoxic effects of methomyl. The detrimental impact of

sprayed pesticides like OC, OP and carbamates was studied for reproductive toxicity

in 1,106 couples when the males were associated with the spraying of pesticides (OC,

OP and carbamates) in cotton fields [71]. Another study brings to the fore the harmful

effects of HCH in malaria spray men (N=216) over a period of 16 weeks in field

conditions [72].

1.1.4 Pesticide Regulations in India

1.1.4.1 Use and regulation of insecticides and pesticides in India

The Ministry of Agriculture has the powers to regulate the manufacture, sale, import,

export and use of pesticides through the ‘Insecticides Act, 1968’ [7] and the rules

framed in it. A Central Insecticides Board (CIB) was constituted under Section 4 of

this 1968 Act, which advises Central and State Governments on pesticide related

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matters. The Act also comprises a Registration Committee (RC) that is responsible for

granting approval to the use of pesticides and new formulations. While, the Ministry

of Health and Family Welfare, monitors the pesticides residue levels in food, a

Central Insecticide Board (CIB) was constituted under Section 4 of the Insecticides

Act, 1968. Its task was to advise Central Government and State Governments on

technical matters regarding pesticides viz:

(i) Requisite safety measures to prevent risk to human beings or animals in

manufacture, sale, storage, distribution and application;

(ii) Decide suitability for aerial use;

(iii) Ascertain and specify shelf-life;

(iv) Specify residue tolerance limit and waiting period;

(v) Suggest colorization;

(vi) Recommend which chemicals/substances should be included in the Schedule or

insecticide list;

(vii) Other functions pertaining to these matters, as per the need.

A Registration Committee (RC) was constituted under Section 5 of the Insecticides

Act, 1968 to register insecticides for their usage after scrutinizing different aspects of

pesticides. The Committee was informed that so far 181 pesticides have been

registered for regular use in the country. The Ministry of Health and Family Welfare

under PFA (Act), 1954 prescribes the MRL values of pesticides in food and

commodities. The Registration Certificate mandates that a label be put on the

packaging, indicating the nature of the insecticide (Agricultural or Household use),

composition, active ingredient, target pest(s), recommended dosage, caution sign and

safety precautions. Hence, a pesticide labeled for agriculture should not be used in a

household. The CIB & RC periodically reviews all pesticides and their application

and may ban some pesticides before registration. Internationally, several aid

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organizations help to increase the skills and resources available to pesticide regulatory

authorities in developing countries. The Food and Agriculture Organization (FAO),

United Nations Environment Programme (UNEP), The US Agency for International

Aid (USAID), World Health Organisation (WHO) and others provide training,

information systems, support, analytical equipment to study risks, benefits and help

for the legislative reforms [73]. The pesticides and their residues in food products are

monitored and regulated through concepts like MRL, ADI and GAP [74].

MRL: It is the maximum concentration of pesticide residue present in a food item

or crop, when the pesticide is applied according to GAP (Good Agricultural

Practices). MRL is expressed in milligrams of pesticide residue per kg of commodity

or animal food. No Observed Adverse Effect Level (NOAEL) is calculated from

evaluation of MRL and the chronic toxicity studies. NOAEL is expressed in

milligrams of the substance being considered per kg of body weight.

ADI: ADI is calculated by dividing NOAEL normally with a safety factor of 100

(10x10), the first 10 provides interspecies variation while the 2nd

10 is for intraspecies

variation.

Good agricultural Practices: Some of the Good agricultural Practices (GAP) include

adhering to the provisions of the Insecticides Act, 1968, education of farmers

regarding the dangers of pesticides, need-based use of pesticides as per the prescribed

dosage, application technique and the recommended waiting period. Observing the

IPM (Integrated Pest Management) practices and benefiting from organic farming.

1.2 Introduction to Sample Preparation Techniques Applied

1.2.1 Microextraction by Packed Sorbent (MEPS)

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Microextraction by packed sorbent or MEPS is a novel sample preparation and clean

up technique which can be connected online to LC and GC [75]. This technique is a

miniaturized version of solid phase extraction (SPE).The MEPS syringe has

approximately 1 mg of the packing material inserted on the bed. The sample is passed

through the solid support; the analytes get adsorbed on the sorbent. The process of

drawing in the sample is repeated several times (10 to 40) for optimum adsorption

onto the syringe bed. The solid phase is given washing with water to remove the

interferences. The analytes are then eluted with a solvent like methanol or the mobile

phase. The process can be made fully automated [76]. The elution volume of MEPS is

compatible with the injection volumes for LC and GC systems. Additionally, the

MEPS technique can be used with as low sample volumes as 10µL which is

particularly useful for analyzing biological samples.

Figure 1.1: MEPS needle with 30µm C18 Packed bed SPE device [www.sge.com]

MEPS also helps to suppress the interferences due to complex biological and

environmental matrices [75]. Another advantage of this novel technique is that the

MEPS syringe can be used over 100 times for urine and plasma samples; on the

contrary, the SPE syringe can only be used once.

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This new approach to sample preparation and clean-up is rapid, convenient and more

cost effective than the SPE procedure. Several factors like solvent volume, number of

times the sample is drawn through syringe, washing volume etc. affect the

performance of MEPS [77].

1.2.1 Solid Phase Extraction

Solid phase extraction is a technique developed in 70s to replace liquid-liquid

extraction technique for clean-up, separation and pre-concentration of solutes in a

given sample [78-80]. SPE and LLE are similar in principle as both involve

partitioning of solute between two phases [81], but in the SPE, one of the phases is

liquid (matrix) and the other is solid sorbent of the cartridge.

The SPE operation includes three or four consecutive steps. These steps are illustrated

in the figure 1.2. The first step is conditioning whereby, the solid sorbent is

conditioned with an appropriate solvent depending on the type of solid sorbent. The

solvent is used to wet the packing material and also helps to remove the initial

impurities present on the packing bed. By this step, air is removed from the column

and void volume is filled with solvent. It is imperative that the solid sorbent is not

allowed to dry in between various steps, as drying up will cause poor retention of

analyte onto the sorbent. The second step is the loading of the sample on the sorbent

bed through percolation. The volume for sample loading can vary from 1 mL to 1 L,

depending on the choice of analysis selected and other factors.

Washing/

Loading

Washing Elution

Conditioning

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Figure 1.2: Stepwise working of SPE procedure [82]

The sample flow rate must be adjusted so as to strike a balance to have optimum

retention and to avoid over retention. During this step, many matrix components pass

through the cartridge, while some are retained on the sorbent. This step entails the

concentration of analyte on the sorbent. The third step is washing of the loaded

sorbent with a suitable solvent to eliminate the matrix components without the elution

of analytes. This is an optional step; it may be followed by drying step for aqueous

matrices to remove traces of water. The last step is to elute the adsorbed analytes with

an appropriate solvent which has higher affinity for analyte particles rather than the

matrix. Solvent volume and solvent flow should be adjusted to ensure high pre-

concentration of the analyzed substance.

A variety of sorbent materials are available for the SPE discs depending upon the type

of analyte to be pre-concentrated. Reversed phase sorbents like C18, C2, C8,

Normal/Reverse phase sorbents like cyanopropyl, aminopropyl and normal phase

sorbents like silica, florisil, alumina, etc. are available for various requirements [83].

1.3 Introduction to Derivative Spectrophotometry

20

Spectrophotometry is the measurable or the quantifiable study of electromagnetic

spectra. Derivative spectroscopic technique was used to further enhance the

information obtained from simple absorbance spectra [84]. The concept of derivative

spectroscopy was first introduced in 1950s and it was found to be very advantageous.

With the development of technology and the advent of microcomputers, it became

highly practicable and easily reproducible thus, becoming a very popular tool for

analytical studies. Derivative spectrophotometry (DS) involves the differentiation of a

normal spectrum and helps in enhancing the resolution of mixtures by increasing the

detectability of minor spectral features [85-90]. Derivative spectrophotometry (DS)

makes use of first and higher derivatives of absorption spectra with respect to

wavelength for qualitative analysis and for quantification. The derivatization makes

the absorbance spectra more meaningful as it allows to remove broad band

interferences caused by turbidity or matrix absorption. Derivative spectra can be

obtained by optical, electronic and mathematical methods. The mathematical

technique has superseded the other techniques. In this approach, the spectrum is

digitalized having a sampling interval of Δλ [91]. The size of Δλ depends upon the

natural bandwidth of the bands being processed and on the bandwidth of the

instrument being used. The derivative spectra are complex as compared to the zero

order spectra. The 1st derivative is the rate of change of absorbance against

wavelength. This spectrum starts at zero and then passes through zero at the

wavelength same as the λmax of the original spectrum. The 1st derivative has a positive

and a negative band. The second order derivative is characterized by a negative band

with the minimum at the wavelength corresponding to λmax of the zero order bands.

Two positive satellite bands are also present on the either sides of the main band. The

1st and the 2

nd derivatives are very sensitive to the variation in slope of the absorption

spectrum, thus DS is suitable to analyze shoulders or the overlapping bands. Hence,

21

DS is quite handy for trace detections and quantitative determinations in analytical

chemistry.

Lambert –Beer Law 92

A = ε.c.d

1st derivative dA/dλ = c x d dε/dλ

2nd

derivative d2A/ d

2 λ = c x d d

2ε / d

2 λ

4th

derivative has a positive band. The even order spectra have a strong positive or

negative bands with the minimum or maximum at the same wavelength as λmax. The

number of bands obtained in the derivative spectrum is one more than the order of the

spectrum [93, 94].

1.3.1 Peak Measurement

Derivative Spectrophotometry (DS) technique has been used for the determination of

pesticides [95,96] and for simultaneous determination of metal ions [97-99]. The

various aspects of the DS technique have been discussed by several authors [100-

106]. Zero-crossing derivative spectra are used most frequently to simultaneously

analyze the binary mixtures showing overlapping spectra [107]. But the zero-crossing

method is not appropriate for the ternary mixtures of compounds showing overlapping

in spectra as the zero-crossing process mandates the selection of a critical wavelength.

This selection leads to lesser sensitivity and precision. For ternary mixtures, “ratio

spectra derivative” helps to avoid the above problems [108]. Salinas et al have also

provided solutions for similar problems [109]. DS can also be combined with

Fourier-Transform smoothing with ratio spectra derivative spectrophotometry for the

analysis of binary and ternary mixtures [110]. Several other advancements have been

made in the field of DS like applying flow-injection through sensor with diode-array

detector and first derivative spectra [111].

22

1.3.2 Features and Applications

Graphics

As the order of derivative increases, the number of bands also increases. The

increasing complexity can be useful for qualitative analysis involving characterization

and identification. The overlapped peaks of absorption spectra can separate out by

derivatization.

Resolution

It is observed that the derivative centroid bandwidth of even order derivatives

decreases with increasing order. In comparison with the zero-order spectrum, the

derivative centroid band-width for a Gaussian band decreases to 53%, 41% and 34%

of the original bandwidth in the 2nd

, 4th

and 6th

orders respectively. This feature can be

employed for resolution enhancement of two analytes with similar λmax values and

which are not resolved in the absorbance spectrum.

Background elimination

Baseline shift is a common unwanted effect in spectroscopy. It can be eliminated

through the 1st order derivative, thereby improving the efficiency of the

quantification. Some other background effects are directly proportional to higher

orders of the wavelength as:

A = ao + a1 λ1

….+ an λn

can be eliminated by using higher orders of derivative.

Discrimination

A very useful aspect of the DS is that it suppresses the broad bands more as compared

to the sharp ones and this feature increases with increasing derivative order. This

effect arises due to the fact that the amplitude, Dn

of a Gaussian band in the nth

derivative is inversely proportional to the original bandwidth, W, raised to the nth

degree:

23

Dn =1/W

n

Hence, for two coincident bands of equal intensity but different bandwidth in the zero

order, the nth

derivative amplitude of the sharper band, X, is greater than that of the

broader band, Y, by a factor dependent on the relative bandwidth and the derivative

order:

Dn

X/Dn

Y = Wn

Y/Wn

X

This property is used to improve the accuracy of quantification of a narrow band

component in the presence of a broad component and to reduce the error caused by

scattering. In general, the instrumental requirements for the derivative as well as

absorbance spectroscopy are the same but for DS the wavelength reproducibility and

signal to noise ratio are quite important as even small wavelength errors can cause

great large signal errors in derivative mode. The instrument should ideally have low

noise levels because of the negative impact of derivatization on the S/N ratio. The

spectrophotometer should preferably be able to scan and average multiple spectra

before derivatization. Controlling the degree of smoothing is also an important aspect

of derivatization which can be done with Savitzky-Golay method whereby the

smoothing is changed by varying the order of the polynomial and the number of data

points used. A new fractional DS technique is proposed by Mo et al [112].

1.4 Introduction to High Performance Liquid Chromatography

(HPLC)

Russian Botanist Tswett

is considered to be the inventor of chromatography. In 1903

he succeeded in separating leaf pigments using a solid polar stationary phase.

Chromatography is a separation technique where a mobile phase carrying the analytes

is moved in contact with adsorbent stationary phase. The compounds can be identified

by the different rate of flow and interaction with the mobile phase. High Performance

24

Liquid Chromatography (or high pressure liquid chromatography) has become the

analytical technique of choice for analysts for the past more than 25 years bring used

in about 75% of the laboratories [113]. HPLC is a type of column chromatography

used to separate, quantify and identify compounds based on their unique polarities

and interactions with the column’s stationary phase [114-119].

1.4.1 Principle

Chromatography is an analytical method based on the separation of components in a

mixture (the solute) due to the difference in migration rates of the components

through a stationary phase by a gaseous or liquid mobile phase. High performance

liquid chromatography, which is a highly improved version of column

chromatography, uses high pressures of up to 400 atmospheres to force the solvent

through the column, instead of a solvent being allowed to drip through under gravity,

thus greatly increasing the speed. This allows the use of very minute particles for the

column packing material and hence increasing the surface area for interactions

between the stationary phase and the molecules flowing past it. All these factors

contribute to better separation of the components of the analyzed mixture. The small

volume of the sample to be analyzed is introduced into the stream of mobile phase.

The analyte undergoes specific physical and chemical interactions with the stationary

phase and elutes at a particular time called its retention time which is considered to be

its unique identification feature. Most common solvents used as mobile phase are

acetonitrile and methanol along with water, to which buffers may be added to increase

the separation of analytes. Gradient elution can also be used to further resolve the

multi component samples. In gradient elution, the composition of the mobile phase is

changed over the course of analysis. The other major improvement over column

chromatography concerns the detection methods which can be used.

25

These methods are highly automated and extremely sensitive. A host of developments

in the field of HPLC instrumentation are responsible for the popularization of this

technique. Apart from the commonly used UV detectors, several new and improved

options like photodiode array, electron light scattering, scanning fluorescence,

electrochemical, conductivity and mass spectrometer detectors. Better and more user-

friendly system control programs are also available, making HPLC the obvious choice

of analytical chemists around the world.

1.4.2 Pumps

Apart from this, the pump systems with better reproducibility and reliability, and the

possibility of using wide range of solvents, use of gradient systems with multistep and

non-linear profile abilities have made the analysis with HPLC a better option. Most of

the analytical pumps are designed in a way to operate efficiently between 0.1 to

10ml/min [120].The latest auto sampler systems can deliver precise amounts of

samples of the required concentrations at hot or cold temperatures and can even rinse

or clean the needles. This combined with the availability of wide range of solvents

and columns have been instrumental in increasing the popularity of this technique.

The U.S. Food and Drug Administration (FDA) have adopted and recommended

HPLC for the analysis of thermally labile, nonvolatile, highly polar compounds as

HPLC carries out the separations and detection at ambient temperatures [121].

1.4.3 Columns

The columns used in HPLC can be open, tubular or packed ones. Packed bed columns

are being used frequently. The size of packing particles in such columns could be 3 to

10 μm (diameter) or 20-50 μm (diameter). These particles are generally made of

incompressible substances like silica, alumina, C or polymers [122,123]. Apart from

26

this latest packings used are cellulose and cyclodextrins [124]. Silica is the most

commonly used material for chromatographic support of bonded phases. Bonded

phases are generally made up of alkyl silanes. The packing particles can be permeable

or impermeable to the flow of the solvent. Several commercially available columns

have polystyrene-divinyl benzene as chromatographic phase in homogeneous form

and it’s useful for RP and gel permeation chromatography. Alkyl silane, phenyl and

cyano columns have bonded phases are useful for RP chromatography, whereas,

amino column is a normal phase or ion-exchange column. Poly (methylmethacrylate)

is a homogeneous phase column employed in gel permeation chromatography.

1.4.4 Types of HPLC [125,126]

1.4.4.1 Partition Chromatography

Partition chromatography uses a retained solvent on the surface of or within the grains

of an inert solid matrix. The equilibration or partition of the molecules between a

liquid stationary phase and the eluent is called Hydrophilic Interaction Liquid

Chromatography (HILIC) in HPLC and it separates analytes based on polarity

differences. HILIC is advantageous as it can separate acidic, basic and neutral solutes

in a single chromatogram.

27

Figure 1.3: Types of chromatographic techniques [http://people.whitman.edu]

1.4.4.2 Normal Phase Chromatography

Normal Phase HPLC or NP-HPLC or adsorption Chromatography is based on the

adsorption of the variable adsorption of the analytes on a polar stationary surface as

per the polarity of the analyte. The prominent feature of normal phase

chromatography is the H-bonding or polar interactions between the analyte and the

stationary phase [127]. This phase uses non-aqueous or non-polar solvents and is used

for the separation of organic or non-polar compounds

The mobile phase used is non-aqueous and non-polar whereas the stationary phase is

polar in nature. Higher the polarity of the analyte more will be its adsorption on the

stationary phase. Hydrophilic solvents will increase the retention time whereas the use

of hydrophobic solvents will cause a decrease in the retention time.

1.4.4.3 Reverse Phase Chromatography (RPC)

28

RPLC or reversed phase liquid chromatography is the most widely used form of

HPLC. A solute adsorbed on the reversed phase column undergoes hydrophobic

interactions. Its working is just opposite to that of the normal phase chromatography.

In reverse phase HPLC a non polar stationary phase and an aqueous moderately polar

mobile phase is used. The stationary phase is Silica treated with RMe2SiCl, where R

has 18 or 8 Carbon chain. In these stationary phases polar molecules elute faster RPC

operates on the principle of hydrophobic forces due to the high symmetry in the di-

polar water structure.

Figure 1.4: The schematic diagram of HPLC system [www.waters.com]

The polar solvent and polar molecules in the mixture being passed through the

column show high attraction towards each other. There won't be as much attraction

between the hydrocarbon chains attached to the silica (the stationary phase) and the

polar molecules in the solution hence these will mostly move with the solvent.

Whereas, the non-polar compounds in the mixture will tend to form attractions

with the hydrocarbon groups because of van der Waals dispersion forces. They

therefore, spend less time in solution in the solvent and this will slow them down on

29

their way through the column. Analyte adsorbs and desorbs on the stationary phase

hence its rate of movement through the column is lesser than that of the solvent. The

solvent system used has water (polarity, p’=10.2) and miscible organic solvents like

methanol (p’=5.1) or acetonitrile (p’=5.8) or tetrahydrofuran (p’=4.0). The

proportions of water to non-polar solvent are optimized such that the capacity factor

of the last eluted analyte gets a value of nearly 2 [128]. Capacity factor, k’= (tr – to)/t’0.

1.4.4.4 Size-Exclusion Chromatography or Gel Permeation Chromatography

The property of gel permeation chromatography is the separation of particles on the

basis of size. Since it is a low resolution chromatography hence is mainly used for the

final step of resolution. It can also be employed to determine the tertiary structure of

purified proteins and large molecules like polymers. In this type of chromatography,

the stationary phase consists of pores of particular size. Analyte particles which are

bigger in size than the pores are removed from these cavities and hence are able to

pass through the column at a faster rate than the smaller analyte particles [129].

1.4.4.5 Ion-exchange Chromatography

In this kind of chromatography, the basis of retention of analyte is the attraction

between the solute ions and the charged sites attached to the stationary phase. The

different types of ion exchangers used are:

Polystyrene resins, Gels (Cellulose and dextran ion exchangers), Porous silica or

Controlled-pore glass.

30

UV Visible

detector

Pump

HPLC-UV System at Punjabi University, Patiala

Solvent containers

Figure1.5: The HPLC-UV System (Dionex, Germany) at Chemistry Department, Punjabi University, Patiala

31

1.5 Pesticides Selected for Present Study

Five phenylurea herbicides (linuron, diuron, monuron, metoxuron and metazachlor),

four dithiocarbamtae pesticides (zineb, maneb, ziram and propineb), one

organophosphorous compound (dimethoate), two triazines (propazine and terbutryn),

one carbamate (aldicarb) and a nature derived pesticide i.e., biopesticide

(azadirachtin) were selected for this study.

32

Table 1.2: Characteristics of various Pesticides studied during this work

Common name Structure Class Use Commercial name

Zineb

Dithiocarbamate fungicide Used against potato and tomato blight,

downy mildew, rust etc.

Dithane Z 78,

Amitan

Maneb

Dithiocarbamate fungicide Used to control the early and late

blights of potatoes and tomatoes and

various diseases of fruits, vegetables,

field crops, and ornamentals

Dithane M-45,

Dithane M-22

Propineb

Dithiocarbamate fungicide Protectant fungicide, used against

potato and tomato blight, downy

mildew, apple scab, blue mould of

tobacco etc.

Antracol, Propineb

70% W. P.

Ziram

Dithiocarbamate fungicide Fungicide on stone fruits, pome fruits,

nut crops, commercially grown

ornamentals for several diseases like

scab, leaf curl, early blight, leaf hole,

brown spot etc.

Ziram 85G

33

Dimethoate

Organophosphorous

pesticide

Contact insecticide for mites and

insects on ornamental crops, alfalfa,

apples, corn, cotton, grapes etc.

Rogor, Dimethoate

tech 95%,

Aldicarb

Carbamate pesticide Insecticide, acaricide and nematicide

against several organisms like aphids,

mites, whiteflies, nematodes etc. on

crops like beans, potatoes, onions,

coffee, banana, citrus fruits, soy etc.

Temik

Propazine

Triazine herbicide Used as a systemic herbicide in the

soil absorbed by target plants

Gesamil, Milogard

Terbutryn

Triazine herbicide Preemergent and postemergent control

agent for grass and weeds

Terbutryn 80%

W.P., Terbutryn

50% W. P.

Prebane

Diuron

Phenylurea herbicide Broad spectrum residual herbicide

used for grass and broad leaved weeds

Diater, Carmex

34

Linuron

Phenylurea herbicide Used to check growth of annual and

perennial weeds at crop and non-crop

sites.

Afalon, DuPont 326

Metazachlor

Phenylurea herbicide Used to control pre-emergence and

early post-emergence grasses and

broad-leaved weeds in broccoli,

asparagus, cabbage, cauliflower, garlic

etc.

Clayton Metazachlor

50 SC

Monuron

Phenylurea herbicide Used for the control of weeds and

grasses in non-croppable areas

Monuron (Telvar)

Metoxuron

Phenylurea herbicide It is a contact herbicide used to kill

broadleaf weeds in carrot, wheat,

barley

Dosaflo

Azadirachtin

Neem derived natural

pesticide

Tetranortriterpenoid

Effective for control of over 200

species of insects

Neem Extract

35

1.6 Objectives and Scope of Present Study

The main objectives of this study are:

a) To development of accurate, efficient and selective methods for the

simultaneous determination of pesticides in various food stuffs and

environmental matrices via liquid chromatographic and spectrophotometric

techniques.

b) To apply and validate the developed methods on the analysis of pesticide

residues in food stuffs and different environmental matrices.

c) To determine various active ingredients in commercial pesticide formulations.

36

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