TOXICOLOGICAL STUDIES OF NEEMAZAL ON
Labeo rohita

Z-1022: Dissertation

Submitted to Loyola College (autonomous)
Affiliated to the University of Madras
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE AND ZOOLOGY

By

LEO ANAND .M.A.
WPZ:11

Project guide

Dr. Thresia Matthew, M.Sc., M.Phil., B.Ed., Ph.D.,








POST GRADUATE AND RESEARCH DEPARTMENT OF ZOOLOGY
LOYOLA COLLEGE (AUTONOMOUS)
CHENNAI -600 034

APRIL-2002








DEDICATED TO
MY DEAREST FRIEND
THE EUCHARISTIC JESUS,
AND MY HEAVENLY
MOTHER

















CERTIFICATE

This is to certify that this dissertation entitled
"Toxicological studies of Neemazal on Labeo rohita" submitted by Mr. M.A. Leo Anand (WPZ: 11) in partial fulfillment of the requirement for the Degree of Master of Science in Zoology, During the period of study (2001-2002) under my supervision and it represents an independent work of the candidate.




Dr. D. Sudarsanam,
M.Sc,Ph.D.,M.Ed,DCA
Professor &Head
Department of Zoology
Loyola college,
Chennai -600 034
Dr.K.Thresia Mathews
M.Sc., M.Ed., M.Phil., Ph.D
Faculty of Zoology,
Department of Zoology,
Loyola College,
Chennai- 600 034














INTRODUCTION

Water is an essential component of an Eco-system. It sustains life on earth. A community depends on water for its domestic, agricultural and industrial needs. The total water spread in India is about 4.5 million hectares. Fisheries and aquatic resources are exceptionally valuable assets enjoyed by millions of people. They provide citizens with generous long-term benefits in return for minimal care and protection. These benefits can be direct financial ones that provide employment and profit. For example, the sea food industry provides jobs for the commercial fishers, wholesalers and retailers.

The vigorous development of various chemicals industries and wide spread use of different chemicals in agricultural fields, ultimately find their way in agricultural fields, and ultimately find their way into the aquatic resources resulting in the pollution of water. As a result, it poses a threat to the life of the aquatic organism, by destroying their spawning ground and feeding areas. The growing of human population and activity on aquatic life and water has a direct effect. The aquatic Eco-system is being exploited indiscriminately as the most convenient and cheap water disposed system (Odum, 1971).

Recent studies in the lake of Bangalore city in Karnataka state, showed that there was a large-scale episodal mortality among the fresh water fishes in the year 1995(June)-reported by-Chakrapani et al .,1990.
Most of the pesticides applied for pest control in agricultural and forestry enter the environment through various routes polluting the aquatic Eco-system (Johnson, 1968). Agricultural drainage water entering water body have a fatal effect on fish (Metelev, 1983). In rice eating countries where people suffer from malnutrition, according to FAO statistics the per-capita consumption is high (Shanmugam, 1992).

PESTICIDES
Pesticides are substances used to control pests, including insects, waterweeds and plant diseases. Naturally, occurring pesticides have been used for countries, but wide spread production and use of modern synthetic pesticides have not begun until 1940's. They are one group of toxic compounds linked to human use.

Pesticides are beneficial chemicals. They can protect against forest and farm crop losses and can aid in more efficient food production. They are used to slow the spread of destructive forest insects like the gypsy moth. They are used to establish and maintain lawns and recreational areas. They are used to help reduce malnutrition and starvation of human and animals. Pesticides have also been instrumental in controlling many insect-borne human diseases such as malaria, encephalitis, and bubonic plague. They promote public safety on roads, railroads, power-lines, and rights-of-ways.

Pesticides are (1) relatively easy to apply; (2) generally cost effective and (3) the only practical method of control in some situations. However, the benefits of pesticides are not derived without consequences. Pesticides must be used with great care so that the health of humans, animals, and the environment are protected. Disadvantages of pesticides include their toxicity to some humans, animals, and useful plants, and the persistence (long time) of some of these chemicals in the environment.

TOXICITY IMPACT ON TEST ANIMALS AND HUMANS
All pesticides must be toxic, or poisonous, to be effective against the pests they are intended to control. Because pesticides are toxic, they are also potentially hazardous to humans and animals. It is important for those who use pesticides or regularly come in contact with them to know to the relative toxicity and the potential health effect of the products they use.
Toxicity is a measure of the capacity of a pesticide to cause injury; it is a property of the chemical itself. The toxicity of a particular pesticide is determined by subjecting test animals (usually rats, mice, rabbits, and dogs) to different dosages of the active ingredient and to each of its formulated products. Hazard, or risk on the other hand, is the potential for injury, or the degree of danger involved in using a pesticide and the amount of exposure .An applicator has little or no control over the toxicity of a specific formulated product, but can minimize or eliminate exposure and thus reducing risk by handling the pesticide carefully and by using personal protective clothing and equipment.

Any chemical can be poisonous or toxic if absorbed in excessive amounts. Pesticides injuries involving humans and animals occur when the chemical is absorbed by the body at this level. Pesticides can also cause skin or eye damages (tropical effect) and can reduce allergic responses.
The pesticides have been used to protect foodstuffs, livestock and health of mankind. The development of resistance in insect's population against pesticides poses a severe threat to agricultural productivity of the country. The tremendous advancements in scientific chemistry and other biochemical fields have led to manufacture of various pesticides with quite different mode of action and properties. Men and animals are exposed to the uptake of air and water and through other sources. Pesticides include insecticides, which deals with the examination of insects, herbicides, which is used for extermination of weeds, and fungicides for the control of fungi.
Water containing pesticides, when used for drinking purposes, can lead to harmful effects, ranging from mild headache and skin allergy to cancer of internal organs. The adverse health effects depend upon the degree of toxicity of the pesticides, amount of water intake each day and the individual health can also be caused by impurities in the pesticides. (Internet)

ACUTE TOXICITY AND ACUTE EFFECTS

The acute toxicity of a pesticide refers to the ability of the chemical to cause injury to a person or animal from a single exposure, generally of short duration. Acute toxicity is measured by at least three methods:
(1) dermal toxicity is determined by exposing the skin to the chemicals ;
(2) inhalation toxicity is determined by permitting the test animals to breathe vapors of the chemicals; and
(3) Oral toxicity is determined by feeding the chemicals to test animals.
The harmful effects that occur from a single exposure by any route of entry (dermal, inhalation,oral) are termed acute effects. In addition, the effect of the chemical as an irritant to the eyes and skin is examined under laboratory condition.
Acute toxicity is expressed in LC50 (lethal concentration 50), which is the amount of concentration of a toxicant required to kill 50% of the test population of the animals under a standard set of conditions.
Acute tonicities are the basis for selecting the appropriate signal word (toxic categories) to be used on a product label. These are DANGEROUS-POISON and a skull and cross bones on the package label of pesticides classified as highly toxic, WARNING for those that are classified as moderately toxic, and CAUTION for those that are slightly toxic or "relatively nontoxic". The signal word DANGER without a skull and crossbones symbol implies that the pesticide is a potent skill or eye irritant. (Internet)


CHRONIC TOXICITY AND CHRONIC EFFECTS

Chronic toxicity is determined by subjecting the test animals to long term exposure to the pesticide. The harmful effects that occur from small doses repeated over a period of time ,usually years, are termed chronic effect. Some of the chronic effects found in the test animals exposed to certain pesticides include birth defects (teratogenesis); toxicity to a fetus (fetotoxic effects); production of tumors (carcinogenic), either benign (non cancerous) or malignant (cancerous/carcinogenic); genetic changes (mutagenesis); blood disorders (hemotoxin effects); nerve disorders (neurotoxic effects); endocrine disruption; and reproductive effects. Some pesticides are required to include chronic toxicity warning statements on the label. The chronic toxicity of a pesticide is more difficult to determine through laboratory analysis than the acute toxicity. (Internet)

MODE OF ENTRY

When pesticides enter the aquatic systems, the environmental costs can be high. The cause a large amount of death to aquatic organisms including thousands of fishes, as well of frogs, turtles, mussels, water birds and other wildlife. Frequent usage of pesticides extends their toxic effects. The pesticides from the agricultural and industrial drainage enter the aquatic ecosystem, get enriched in the aquatic food chain through bio-accumulation, bio-concentration and bio-magnification process (Murthy, 1986). Bio availability refers to the amount of pesticide in the environment available to fish and wildlife. Bio-concentration is an accumulation of pesticides in animal tissues at levels greater than those in water or soil to which they were applied. Bio-magnification is the accumulation of pesticides at each successive level of food chain. Many studies were made on the pesticides spills and written reports and journals were published by the Virginia Department of Agriculture. Many research scholars like Stinson, Elizabeth and Peter (1991) has studied about the impacts on animals and their habitats.

Publications released due to the combined efforts of Lutz, Grey, Mayeaux and Grodner, 1992 give an idea about the toxicity of certain an agricultural pesticides to some of the common aquatic organisms in Lourisiance. Remote sensing of the environment and water resources, show the extent to which the pesticides have exploited the ecosystems.
Narayan et al., (1996), has published a report in The Hindu to show the remote sensing can be applied to manage water resources.

IMPORTANT ASPECTS OF PESTICIDES

Chemical pollution has a great impact on aquatic organisms. Due to chemical pollution, the normal functioning of al is disturbed and this in turn, may result in alteration in the fundamental biochemical and physiological mechanisms of animals – Fowler 1972; Larson et al., (1976), Mahaeswari Devi et al., 1991; Sin et al., (1990), Uremia Devi and Balakrishnan (1990).


AQUATIC TOXICOLOGY

Aquatic toxicology is the study of the effects of environmental containments on aquatic organisms, such as the effect of pesticides on the health of fish or other aquatic organisms. A pesticide’s capacity to harm fish and aquatic animals is largely a function of its (1) toxicity, (2) exposure time, (3) dose rate, and (4) persistence in the environment


Toxicity of the pesticide refers to how poisonous it is. Some pesticides are extremely toxic, whereas others are relatively nontoxic. Exposure refers to the length of time the animal is in contact with the pesticide. A brief exposure to some chemicals may have little effect on fish, whereas longer exposure may cause harm.


The dose rate refers to the quantity of pesticide to which an animal is subjected (orally, dermally, or through inhalation). A small dose of a more toxic chemical may be more damaging than a large dose of a less toxic chemical. Dosage can be measured as the weight of toxicant per unit (kilogram) of body weight (expressed as mg pesticide/kg of a body weight) or as the concentration of toxicant in the water or food supply (usually expressed as parts per million, ppm or parts per billion, ppb).


PERSISTENCE OF PESTICIDES


Persistence refers to the length of time a pesticide remains in the environment. This depends on how quickly it breaks down (degrades), which is largely a function of its chemical composition and the environmental conditions. Persistence is usually expressed as the “half life” (T1/2) of a pesticide. Half-life is the amount of time required for half of the pesticide to disappear 9the other half remains). Half-time of pesticides can range from hours or days, to years for more persistent ones.


Pesticides can be graded by sunlight (photodecomposition), high air or water temperatures (thermal degradation), moisture conditions, biological action (microbial decay), and soil conditions (pH). Persistence (long-lasting) pesticides break down slowly and may be more available to aquatic animals.


PESTICIDE FORMULATIONS


The active ingredient (pesticide) is combined with other inert ingredients (carriers, solvents, propellants) to comprise the formulated pesticide product. In some cases the inert ingredients may cause concern for aquatic life. Pesticides may be purchased in solid (granules, powders, dusts) or liquid (water, oil sprays) form. A major concern in using either solid or liquid forms of pesticides is their misapplications.


SUBLETHAL EFFECTS


Not all pesticide poisonings result in the immediate death of an animal. Small “sublethal”doses of some pesticides can lead to changes in behaviour, weights loss, impaired reproduction, inability to avoid predators, and lowered tolerance to extreme temperatures.

Fish in streams flowing through croplands and orchards are likely to receive repeated low doses of pesticides if continuous pesticide applications run-off fields. Repeated exposure to certain pesticides can result in reduced fish egg production and hatching, nest and brood abandonment, lower resistance to disease, decreased body weight, hormonal changes, and reduced avoidance of predators. The overall consequences of sublethal doses of pesticides can be reduced adult survival and lowered population abundance.

A lethal dose is the amount of pesticide necessary to cause death. Because not all animals of a species die at the same dose (some are more tolerant than others), a standard toxicity dose measurement, called a Lethal Concentration 50 (LC ), is used. This is the concentration of a pesticide that kills 50% of a test population of animals within a set period of time, usually 24 to 96 hours.


AIM OF THE STUDY

The objective of the list is to determine the harmful effects of a test material, Neemazal that produces a deleterious effect on a group of Test organism (Fresh water fish Labeo rohita) during a short term exposure under controlled conditions. Although various toxicity tests are conducted by administering the material directly by injection or incorporation of it into food, some are conducted by exposing groups of organisms to several treatments it which different concentration of the material are mixed in water. Histopathological changes are observed and the results are discussed. The acute toxicity of Neemazal to Labeo rohita and the chronic effect of this pesticide on chromosomes and histological studies were carried out.




















REVIEW OF LITERATURE


Water quality, habitat structure, flow regime, energy source and biotic interactions are the major environmental factors that determine water resource integrity (Besch and Scharf, 1985). The physical and chemical attributes of water are the critical components of a water resource. They include temperature, dissolved O , pH, hardness, turbidity, concentration of soluble and insoluble organic and inorganic alkalinity, nutrients, heavy metals, and an array of toxic substances which may have simple chemical properties or their dynamics may be complex and changing, depending upon constituents in the geological strata, soils, and land use in the region (Allan, 1992). Toxicity of pepcidies the toxic effects of the pesticides may be complex damaging different organs, tissues or cells of fishes. Various workers of the field have reported the effect if pesticides at different concentrations.


TOXICITY OF PESTICIDES


Toxicity tests have been conducted with an objective to determine the concentration 9f a test material on the level of an agent that produces deleterious effects over a group of test organisms during a short term exposure under controlled conditions. Aquatic toxicologists have used fresh water invertebrates including water fleas – Daphenia magna, and fish such as blue gills, - Xepomis macrochirus, fat head mimows, - Puriephales promelas and trout – Sabvelinus fontinalis (Eaton, 1963) for toxicity tests. Most of the pesticides applied in agriculture enter the environment through various polluting aquatic ecosystem. Persistence of these toxic chemicals in the aquatic environment is dangerous for the survival of fishes (Johnson 1968, Saunder; 1969, Mawdesley Thomas, 1971). The use of pesticides not only kills fishes but also poses a threat to the wildlife. Many publications and findings have been released in support of this vie ( Palmer, William and Peter Bromely, 1944). The United States Environmental Protection Agency 1944 – have also published issued regarding the usage of pesticides.




TOXICITY OF ORGANIC PESTICIDES / BIOPESTICIDES


Bioassay tests have shown that organo-phosphorus compounds are very toxic to aquatic organisms (Woodward and Mauch 1480, Shukla et al., 1981). The effects of recommended concentration of the organophosphorous compounds edifenphos and glyphosate on the immune response and protein content of T. nilotica were investigated after different time intervals (Yendy, Aly, Sebaae – 1998). An HPLC method for the simultaneous determinations of three organophosphates (malathion, diazionon, quinalphol) accumulated in brain tissue of fish Channa punctatus was determined (Sandhya, Mishra 1997). Determination of organochlourine in fatty fish was detected using multi residue solid-phase extraction (SPE) technique (Scheneck, Calderson, Podhorniak – 1996). The results of studies to determine concentrations of organchlorine pesticides (lindane, heptachlor epoxide, aldrin and isomers of DDT) in rainbow trout from 4 fish farms in Leon, Spain indicated a decline since 1987. Residues were found to accumulate mainly in the brain, and at a lower rate in the kidney. (Sahagun, Teran et al).





























MATERIALS AND METHODS


EXPERIMENTAL FISH

The fresh water teleost Labeo rohita was selected as test fish. Like the Barbels, the Labeos are members of the Carp Family. They can be recognized from the fishes of the genes Barbs by their more elongated bodies. The structure and position of the mouth is also distinctive. It open downwards and not forwards and is situated under the projecting snout. The dorsal side of the body is brownish grey in colour. It is predominantly, a coloumn feeder. L. rohita commonly called Rohu or Bhodhari, is one of the major Carps of India and is one of the most delicious table fish. It occasionly, browses the shallow bottoms having preference to planktonic algae, bottom sand, vegetable debris, decaying leaves of aquatic plants and is most suitable culture. It is one of the major Carp component of the production from island waters under culture. The fish is adaptable to healthy life in the laboratory readily available in number and from the common source. Hence, it has been selected as the experimental fish. The fish have well developed respiratory (gills) system, digestive system and reproductive system.

TAXONOMIC STATUS

Phylum : Vertebrata
Sub-phylum: Craniata
Class : Teleostomi
Sub-class : Actinopterygii
Order : Cypriniformes
Sub-order : Cypriniodei
Family : Cyprinidae
Sub-family : Cyprinini
Genus : Labeo
Species : rohita








MAINTENANCE

Fish (Labeo rohita) weighing 2 to 3 grams were procured alive in a healthy condition from Tamil Nadu Fish Farm, Pattabiram, Chennai and acclimatized in a large glass aquaria in the laboratory for about 25 days. Before stocking, the tank was thoroughly cleansed with potassium permanganate to free the aquaria from fungal infections. The experimental fish was fed with, commercial fish feed daily. The water in the tank was renewed on alternate days. Screening of fish was done often to detect the symptoms of disease, physical damage and mortality (Sprague 1973). The injured or severely infected or dead fishes were discarded immediately to avoid the spread of the disease.


COMPOSITION OF NEEMAZAL

Neemazal is a botanical insecticide made from the kernels of neem. The formulation contains 50,000 pip azadirachtin, the most bioactive compound of neem besides several other compounds. Neemazal is a water soluble concentrate, ready to use with appropriate dilution for pest management. It is manufactured by EID PARRY (INDIA) LTD., Chennai. It is used to control pests attacking vegetables, ornamentals, orchards, greenhouses, home gardens, turf etc.


FEATURES

Common name - Neemazal
Generic name - Azadirachtin A
Chemical family - Tetranortriterpenoids
Empirical formula - C35 H44 O16
Molecular weight - 720
Colour - Brown
Specific gravity - 1.1
pH - 6.5 + 0.02




BIOPESTICIDE – NEEMAZAL


Neem contains several active ingredients, and they act in different ways under different circumstances. These compounds bear no resemblance to the chemicals in today’s synthetic insecticides.

Azadirachtin disrupts the metamorphosis of insect larval. By inhibiting molting, it keeps the larvae from developing into pupae, and they die without producing a new generation. Azadirachtin is structurally similar to insect hormones called “ecdysones” which control the process of metamorphosis as the insects pass from larva to pupa to adult. It affects the corpus cardiacum, and organ similar to thehuman pituitary which controls the secretion of hormones.

Metamorphosis requires the careful synchrony of many hormones and other physiological changes to be successful, and azadirchtin seems to be an “ecdysone blocker” It blocks the insect’s production and release of the vital hormones. Insects then will not molt. This of course breaks their life cycle.

Neem’s ability to repel insects was first reported in the Scientific literature in 1928 and 1929. Two Indian Scientists, R.N. Chopra and M.A. Husain, used a 0.001 percent aqueous suspension of ground neem Kernels to repel desert locust. In 1962, S. Pradhan noted that neem Kernels were even more potent that the conventional insecticides, then available and that neem’s repellency was an important as its toxicity. The azadirachtin was provided by D. Morgan, a Keele University chemist who had been the first to isolate azadirachtin.

Although neem has shown every indication of being safe to mammals in normal use as a pesticide, the possibility of future hazards should not be dismissed. When children under the age of 4 were given doses (5-30 ml) of neem oil, they came down with a disease similar to Reye’s syndrome. This sever disorder involves swelling of the brain, liver, and other organs. Reye’s syndrome is poorly understood. In west Africa of neem-leaf teas possible causing kidney damage when taken over a long period.

The deaths of a few young children in Malaysia in the 1980s have been linked to the doses of neem-seed oil forced on them by their parents. Certainly, no hazard has been observed when neem has been used in tropical treatments (on skin complaints) or in dental uses – which together make up by far the major medical applications. (Noel vietmeyer, 1992).


ACUTE TOXICITY TEST

Stock solutions of Neemazal were prepared using glass distilled water. From the stock solution, desired degrees of concentration were prepared. Based on the progressive bisection of intervals on a logarithmic scale log concentration were selected as an experimental concentration. These concentrations were fixed after conducting the range finding test (APHA 1989). The fish was starved 48 hours prior to the commencement of the experiment with a view to avoid any possible change in situ in the toxicity of the pesticides. After adding the toxicant into the test tank with 20 litres of water having 10 fishes, mortality occurred. Fish showing no respiratory movement and no response to tactile stimuli were considered dead and removed immediately. Percentage of mortality was calculated and values were transformed into probit scale. Probit analysis was carried out as per Finney, (1971). Regression lines of probit against logarithmic transformations of concentrations were made. Slope function (S) and confidental limits (upper and lower) of the regression line with Chi-Square test (UNEP/FAD/IEAE 1987) were calculated as follows.

LC84 LC50
------ X-------
LC50 LC16
S = ______________
2
F = antilog(2.77logS) / ÖN

Where N is the animals tested whose expected effects are between 16 and 84% mortality. Upper confidence limit = LC50 x f , lower confidence = LC50/f .


CHRONIC TOXICITY TEST

Labeo rohita were accordingly exposed to each concentration for a period of 5,10 and 15 days. Sub lethal or safe level concentrations were derived from 96 hours LC50 following APHA (1990) to observe various response of the test fish Labeo rohita on prolonged exposure to neemazal



PREPARATION OF MATERIALS FOR HISTOLOGICAL STUDIES

At the end of each experiment gills, liver and intestine and kidney of control and experimental fish were taken out and histological study was carried out by following Culling (1974) method.


FIXING

The tissues were fixed in Bruin’s fixative (Saturated aqueous solution of Picric Acid (75%), formalin (25%), acetic acid (5%) for 24 hours.


DEHYDRATION

After fixation the tissues were preserved in 50% ethyl alcohol and dehydrated in 70%, 90% and 100% ethyl alcohol and finally in iso propyl alcohol(60% at the interval of 4 hours).





CLEANING

The tissues were cleaned in xylene and embedded in paraffin, heated at 60°C for 2 or 3 changes are given at the interval of 30 minutes.


SECTIONING

Sections of 7 to 8µm thickness were prepared by using rotary microtome. Sections were fixed on albuminoid slides for 24 hours and then deparaffinised in xylene and washed and iso propyl alcohol, absolute alcohol, 90%, 70%, and 50% alcohol series and finally in water.


STAINING

The sections were stained with Erlich’s haematoxylin for 15 minutes and de-stained in dilute hydrocholoric acid. After dehydrating in 50%, 70% and 90% ethyl alcohol and distilled water, counter stained in eosin

DEHYDRATION

The sections were subsequently in upgrading alcohol series (50%, 70% and 90%) and counterstained in eosin. The slides were cleared in xylene and mounted with DPX mountant. Selected slides were photographed by using Carl Zeiss Photomicroscope III.


CHROMOSOMAL PREPARATION IN FISHES

To test the fish Labeo rohita, 0.02% colchicineper gram body weight was injected intraperitoneal, 3 cm away from the opercula region. The fish was then maintained in well aerated glass aquaria with the least disturbance for 3-4 hours. Then the innermost gill arch was taken from the fish and was placed in 0.4% KCl, hypotonic solution for 30 minutes. Conroy’s fixative was used to fix the tissues at different intervals of 30, 20 and 10 minutes. Excessive fixative was removed using blotting paper. Cell suspension was made by tapping the gill tissues gently, in 50% acetic acid. The cell suspension thus made was drawn up in a pasture pipette to be dropped on clean slides, warmed up to a temperature range of 45 to 50ْ C. Thus made, cell smears were dried for a few hours and stained with 6% giemsa for 15 to 18 min. This was done according to the procedure Klingerman and Bloom (1977).













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