INTRODUCTION -...

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INTRODUCTION

Food production is essential to everyone, but sustainable

agriculture developments are especially important for world’s people. In

India more than seventy percent of people live and work in rural areas

and most rely on agriculture directly. These people cannot offer to have

their crops destroyed during production or storage. Worldwide

approximately 9,000 species of insects and mites, 50,000 species of plant

pathogens, and 8,000 species of weeds damage crops. Insect pests cause

an estimated 14% of loss; plant pathogens cause a 13% loss, and weed a

13% loss (Pimentel, 2009). Without pesticide application the loss of

fruits, vegetables and cereals from pest injury would reach 78%, 54% and

32% respectively (Cai, 2008). To minimize these losses different

pesticides are used. The use of pesticides can prevent or reduce

agricultural losses caused due to pests as these pesticides are a powerful

tool against pest they improve yield, as well as help in improving the

quality of the produce in terms of cosmetic appeal often important to

buyers. Crop loss from pests declines to 35% to 42% when pesticides are

used (Pimentel, 1997; Liu and Liu, 1999). About one-third of the

agricultural products are produced by using pesticides (Liu et al., 2002).

Researchers pointed out that if the consumption of pesticides is

prohibited, the food production would drop sharply and the food prices

would soar. In this circumstance, the export of cotton, wheat and soybean

in the United States would decline by 27%, and 132,000 jobs would be

lost. Fungicides are used to 80% fruit and vegetable crops in the United

States. The economic value of the apple has increased 1,223 million

dollars by using fungicides (Guo et al., 2007).

Use of pesticides to protect crops has been reported since before

2500 BC. The first known pesticides were elemental sulfur dusting used

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in Sumeria about 4,500 years ago. By the 15th century, toxic chemicals

such as arsenic, mercury and lead were being applied to crops to kill

pests. In the 16thcentury, ants were controlled with mixtures of honey and

arsenic. In the 17th century, nicotine sulfate was extracted from tobacco

leaves for use as insecticides. The 19th century saw the introduction of

two more natural pesticides, pyrethrum which is derived from

Chrysanthemum and rotenone which is derived from the roots of tropical

vegetables. By the late nineteenth century, U.S. farmers were using

copper acetoarsenite (Paris green), calcium arsenate, nicotine sulfate, and

sulfur to control insect pests in field crops, but often results were

unsatisfactory because of the primitive chemistry and application

methods.

In 1939, Paul Muller discovered that DDT was a very effective

insecticide. It quickly became the most widely used pesticides in the

world. In the 1940 manufacturers began to produce large amounts of

synthetic pesticides and their use became widespread. Some sources

consider the 1940s and 1950s to have been the start of the “Pesticide era”.

Pesticides use has increased 50- fold since 1950. Successful pesticides are

produced in massive quantities. It has been estimated that between 1943

and 1974 the world production of DDT alone reached 2.8-109 kg

(Woodwell et al., 1971). DDT was the first efficient synthetic pesticide

and had all the good properties for an insecticide. It is extremely stable,

and only one treatment may suffice for good control of insect pests. It

was cheap to produce and had (and still has) a low human toxicity, but is

extremely active toward almost all insects. As a tool in antimalarial

campaigns, it was extremely efficient. By the end of World War II it was

used to combat insect-transmitted diseases and agricultural and household

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pests like flies and bedbugs. The production reached the maximum in

1963 with 8.13-107 kg in the U.S. alone.

In the 1960s, it was discovered that DDT was preventing many fish

eating birds from reproducing, which was serious threats to biodiversity.

Rachel Carson wrote the best selling book “Silent Spring” about

biological magnification. DDT is now banned in at least 86 countries, but

it is still used in some developing nations to prevent malaria and other

tropical diseases by killing mosquitoes and other diseases carrying

insects. Bans and restrictions of DDT usage have since reduced the

production volume of this first and efficient modern pesticide. Today an

international treaty has been signed to restrict its use to very few

applications in vector control. DDT is therefore not very important as a

commercial product anymore. There are no patent protections. Because of

environmental problems, its usefulness is limited. Furthermore, insect

resistance to DDT would in any case have restricted its usefulness.

Since introduction of synthetic organochlorine and

organophosphorous insecticides in the 1940’s there has been a rapid

increase in the use of chemicals of high biological activity for the pest

control. Globally 4.6 million tons of chemical pesticides are annually

sprayed into the environment. There are currently about 500 pesticides

with mass applications, of which organochlorined pesticides, some

herbicides and the pesticides containing mercury, arsenic and lead are

highly poisonous to the environment. The Pesticide Manual from 1979

(C. Worthing, 6th edition, British Crop Protection Council) presents 543

active ingredients. Approximately 100 of these are organophosphorus

insecticides and 25 are carbamates used against insects. The issue of The

Pesticide Manual from 2000 (T. Tomlin, 12th edition, British Crop

Protection Council, 49 Downing St., Farnham, Surrey GU9 7PH, U.K.,

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www.bcpc.org) describes 812 pesticides and lists 598 that are superseded.

Today’s 890 synthetic chemicals are approved as pesticides throughout

the world and the number of marketed products is estimated to be 20,700.

Organophosphorus insecticides are still the biggest group of insecticides

with, according to The Pesticide Manual, about 67 active ingredients on

the market, but the pyrethroids are increasing in importance, with 41

active ingredients. The steroid demethylation inhibitors (DMIs) constitute

the main group of fungicides (Breidbach and Kutsch, 1995).

Photosynthesis inhibitors (triazines 16, ureas 17, and other minor groups)

and the auxin-mimicking aryloxyalkanoic acids (Bakke, 1978) are still

very popular as herbicides, but many extremely potent inhibitors of

amino acid synthesis e.g., the sulfonylureas (Bloomquist, 1996) are

becoming more important. Lead arsenate, mercury salts, and some

organic mercury compounds, zinc arsenate, cyanide salts, nicotine,

nitrocresol, and sodium chlorate were sold with few restrictions. Very

few of these early pesticides are now regarded as safe. The world had a

very strong need for safe and efficient pesticides.

In view of the world’s limited croplands and growing population

(Zhang et al., 2006; Zhang, 2008), and mans attempt to modernize the

methods of agriculture to increase the food production, pesticides have

gained importance for the very effective control of agricultural crops and

domestic animals (Zhang et al., 2007, 2008c; Zhang, 2009). Pesticide

consumption in India has increased from 2353 MT in 1955 to 40,672 MT

in 2005 for technical grade chemical pesticides. In March, 2005, 186

technical grade pesticides were registered in the country for use under

section 9(3) of Insecticides Act, 1968 (Directorate of Plant Protection and

Quarantine, Govt. of India). Indian pesticide industry has achieved the

status of second largest basic pesticide manufacturer in Asia after Japan.

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Interestingly, India’s consumption of pesticides per hectare is low (0.5

kg/ha) when compared with world averages like those of Korea (6.60

kg/ha) and Japan (12.0 kg/ha). According to the pesticide industry

statistics, India spends only $3/ha on pesticides compared to $24/ha spent

by the Philippines, $255/ha by South Korea and $633/ha by Japan (TERI,

2000). However, the contamination of food products in the country is

alarming. About 20% of Indian food products contain pesticide residues

above tolerance level compared to only 2% globally (TERI, 2000). This

is primarily due to their non-judicious use in certain areas/states, lack of

awareness and inadequate information dissemination amongst the

farming community. Pesticide usage for cultivation of food crops

amongst different states of India indicates a mixed pattern. The per

hectare pesticide usage is highest in Punjab (923 g/ ha) as compared to

other agriculturally advanced states like Haryana (843 g/ha), Andhra

Pradesh (548 g/ha), Tamil Nadu (410 g/ha), Karnataka (216 g/ha) and

Gujarat (47 g/ha) (Agnihotri, 2000).

These applied pesticides are transported into the nation’s waters via

runoff from rainstorm events, atmospheric deposition, and spray drift.

However pesticides have become to pose a major problem by their

indiscriminate use. Only 1% of the sprayed pesticides are effective, 99%

of pesticides applied are released to non-target soils, water bodies and

atmosphere, and finally absorbed by almost every organism (Zhang et al.,

2011). Some pesticides such as soil fumigants and nematocides are

applied directly into soil to control pests and plant diseases presented in

soil. The transport, persistence or degradation of pesticides in soil

depends on their chemical properties as well as physical, chemical and

biological properties of the soil. All these factors affect sorption/

desorption, volatilization, degradation, uptake by plants, run-off, and

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leaching of pesticides. Sorption is the most important interaction between

soil and pesticides and limits degradation as well as transport in soil.

Pesticides bound to soil organic matter or clay particles are less mobile,

bio available but also less accessible to microbial degradation and thus

more persistent (PAN, 2010). Soil organic matter is the most important

factor influencing sorption and leaching of pesticides in soil. Addition of

organic matter to soil can enhance sorption and reduce risk to water

pollution. It has been demonstrated that amount and composition of

organic matter had large impact on pesticides sorption. For example soil

rich on humus content are more chemically reactive with pesticides than

non-humified soil (Farenhorst, 2006). Persistence of pesticides in soil can

vary from few hours to many years in case of OC pesticides. Despite OC

pesticides were banned or restricted in many countries, they are still

detecting in soils (Shegunova, et al., 2007; Toan et al., 2007; Li et al.,

2008; Hildebrandt et al., 2009; Jiang et al., 2009; Ferencz and Balog

2010). Pesticides from soil percolate in to the water bodies.

Insecticides applied to crops and in urban areas do not just

disappear. It's true that these pesticides break down after a given time, but

some of these pesticides are very persistent and remain in the

environment for long periods. Persistence is a good quality for some

pesticides because it means that it remains effective in killing pests for a

long time. However, this attribute means that pesticides are around long

enough to enter water sources under some conditions. This also means

that pesticides entering water may remain toxic longer. Rainfall and

irrigation can wash pesticides from sites of application into the water

system. These pesticides can accumulate in invertebrates and fish; and

pass through the food chain to birds, mammals, and even human.

Insecticides cause serious ecotoxicological problems mainly due to their

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persistence and high toxicity. The use of Lindane is now a day prohibited

in most countries but this organochlorine persists in soils and may reach

the marine environment through erosion processes (Hamza-Chaffai et al.,

1998).

The pesticides even when applied in restricted areas are washed

and carried away by rains and floods to larger water bodies like ponds

and rivers (Das, 1989). The work of Holden (1972), Miles (1978) and

several such workers has clearly established that the pesticide residues are

transported to the aquatic environment. Pesticide pollution alters the

physicochemical properties of water (Richardson, 1988). Heavy

contamination of pesticides in water, in turn, leads to oxygen depletion

and cases of poisoning and mass mortality of fishes and other aquatic

organisms are not uncommon. Pesticides and related chemicals destroy

the delicate balance between species that characterizes a functioning

ecosystem (Zaheer Khan and Francis, 2005). The fresh water organisms

are particularly susceptible to these pollutants, since their habitats are

confirmed and escape from such circumscribed and polluted habitats is

impossible. A major environmental impact has been the widespread

mortality of fish and invertebrates due to the contamination of aquatic

systems by pesticides. Most of the fish in Europe’s Rhine river were

killed by the discharge of pesticides, and at one time fish populations in

the great lakes became very low due to pesticide contamination. An

estimated 50,000 kg of dead fish was reported in 1985 in the Miranda

river in South America as a result of pesticide exposure (Alho and vieira,

1997). Pesticides are considered to be one of main reasons for demise of

the commercial fishery in the Azov Sea. In the late 1980s pesticide input

to this sea was about 100 000 t/yr. and consisted primarily of compounds

from the organophosphate group (Semenov et al., 1998). It was examined

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that about 13% of fish kills in England and Wales and about 25% in

Scotland in 1967, were due to pesticides (Anon, 1970). Yunus and Linn

(1971) noted a great loss of important food fishes after a spraying

program was initiated to increase rice production in Malaysia.

Undesirable effects caused by pesticides to the aquatic organisms and

their hazards are elegantly reviewed by many workers (Ponogi et al.,

2000; Livingstone, 2001; Mastumoto et al., 2006) either directly or

indirectly. Snails, fishes and bivalves may frequently encounter these

pesticides (Thelin and Gianessi 2000a, 2000b; Burkepile et al., 2000;

Dutta et al., 2006).

Estimates are that nearly one-half of the groundwater and well

water in the United States is or has the potential to be contaminated

(Holmes et al., 1988; USGS, 1996). EPA (1990) reported that 10% of

community wells and 4% of rural domestic wells have detectable levels

of at least one pesticide of the 127 pesticides tested in a national survey.

The bald eagle continues to be threatened by the use of several

pesticides, including the organophosphate insecticides terbufos, fonofos,

and phorate; warfarin, an anticoagulant rodenticide; and the insecticide

carbofuran. The FWS has been urging the EPA to cancel all forms of

carbofuran since the early 1990s because of its extreme toxicity to

wildlife. According to the FWS, illegal use of carbofuran and other

highly toxic chemicals for predator control has killed a number of bald

eagles. The National Wildlife Health Research Center has diagnosed over

one hundred cases of pesticide poisonings in bald eagles in the past

fifteen years (Litmans and Miller, 2004).

Human pesticide poisonings and illnesses are clearly the highest

price paid for all pesticide use. The total number of pesticide poisonings

in the United States is estimated to be 300 000/year (EPA, 1992).

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Worldwide, the application of 3 million metric tons of pesticides results

in more than 26 million cases of non-fatal pesticide poisonings (Richter,

2002). Of all the pesticide poisonings, about 3 million cases are

hospitalized and there are approximately 220,000 fatalities and about

750,000 chronic illnesses every year (Hart and Pimentel, 2002). The use

of pesticides carries severe risks to human health, the environment,

biodiversity, food security and income of small-scale farmers and

agricultural workers. These problems are particularly severe in

developing countries. In India, the first report of poisoning due to

pesticides was from Kerala in 1958, where over 100 people died after

consuming wheat flour contaminated with parathion (Karunakaran,

1958). The Poison Information Centre in NIOH, Ahmadabad reported

that OP compounds were responsible for the maximum number of

poisoning (73 %) among all agricultural pesticides (Dewan and Saiyed,

1998). One incidence occurred in Bhopal, India, where more than 5,000

deaths resulted from exposure to accidental emissions of methyl

isocyanate from a pesticide factory. In the United States, there are 67

thousands human pesticide poisonings per year. In China, there are 0.5

million human pesticide poisonings with 0.1 million deaths per year

(Zhang et al., 2011).

Ecobichon (1991) also states that as many as 25,000 cases of

pesticide-related illnesses occur annually among agricultural workers in

California. Although California is at the top of the list of pesticide

consumption, there are more poisonings in the developing nations. In the

tropical countries more insecticides than herbicides are used. Insecticides

are usually more toxic, and because of the hot weather, it is very

unpleasant to wear protective clothes.

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Toxic effects of pesticides in the aquatic organisms are exquisitely

discussed by many workers. Radhakrishnan et al., (1986) reported the

presence of 11 chlorine based pesticides in the significant levels in

mussels. Bhide (1987) studied the toxic effects of certain pesticides on

behavior, mortality and development of the fresh water snail, Pila

globosa. Keem and Lee (1988); Muley and Mane (1988); Doherty (1990)

and Han and Hung (1990) have discussed the effects of pollutants in the

bivalves. Menon (1992) studied the toxic effects in bivalve to metal

mixture. Pardeshi (1992) studied pesticide toxicity to Lymnaea

accuminata. Serrano et al., (1995) studied the toxicity and

bioaccumulation of organophosphorous pesticides in molluscs. Sing et

al., (1996) investigated the toxicity of organophosphorous pesticides to

the bioenergetics of a fresh water snail, Indoplanorbis exustus. Impact of

pesticides on the freshwater bivalve, Parreysia cylindrica was studied by

Waykar (1998) and Chaudhari (1999). Noor Alam and Sadhu (2001)

reported the toxicity of kedett 36 (monocrotophos 36% SL) to a common

paddy field fish, Channa striatus. Srivastava and Sigh (2001) evaluated

toxicity of alphamethrin diamethoat and carbaryl pesticides to the fresh

water snail, Lymnaea accuminata. Gurushankara et al., (2003) studied the

toxicity of malathion insecticide in tadpole and adults of Rana

limnocharis. Rabia (2009) studied the acute toxicity of alpha-

cypermethrin on adult Nile tilapia (Oreochromis niloticus L.).

Many pesticides belonging to different groups are available in the

market, out of them Carbosulfan and Profenofos are most commonly used

pesticides which are selected for the present study. The toxicity

evaluation of these pesticides was carried out on the fresh water bivalves,

Lamellidens marginalis which is commonly found in the fresh water

bodies.

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However, to our knowledge, none of these studies focused on the

suitability of the mixture toxicity concepts under environmental realistic

conditions. In fact, the main purpose of these studies was to assess the

potential ecological impact of realistic exposure scenarios on and to

evaluate standards to be protective for the aquatic environment.

Carbosulfan:

Carbosulfan is the common name of the insecticide Marshal 25%

EC. It belongs to the carbamate chemical class. Chemically it is 2, 3-

dihydro-2, 2-dimethylbenzofuran-7-yl (dibutylaminothio)

methylcarbamate. It is a systemic insecticide with contact and stomach

action through cholinesterase inhibition. Its activity is due to in vivo

cleavage of the N-S bond, resulting in conversion to carbofuran.

Carbosulfan formulated as Marshal 25% EC was evaluated by Ensaf

S.Idris in seasons 2003-05 for the control of aphids, Aphis gossypii, in

potato (El-Habieb, 2005).

It is a broad spectrum insecticide, nematicide, miticide, effective

against pests and mites with stomach and contact action. Carbosulfan is

closely related to its main metabolite carbofuran, a major pesticide in its

own right. It is used to control many kinds of insects including: cotton

aphid, American bollworn, black cutworms, stalk borers, cabbage aphids,

cabbage caterpillars, rice fulgorids and thrips. Carbosulfan is safe to crops

and effective to both pests and larva by good systemic properties, low

residue and long-term effect.

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Most of our knowledge, carbamates are restricted either to

vertebrates or to insects and there is little information on molluscs.

Ebenso et al., (2005) observed effect of carbamate molluscicide on

African giant snail, Limicolaria aurora. Boran et al., (2007) studied the

acute toxicity of cabamates (Carbaryl, Methiocarb and carbosulfan) to the

rainbow trout, Oncorhynchus mykiss and guppy, Poecilia reticulata.

Various investigators studied the toxicity of carbamates in aquatic

organisms (Jadhav et al., 1996; Waykar and Lomte, 2001; Radwan et al.,

2008).

Profenofos:

Profenofos is the common name of the insecticide curacron 50%

E.C. It belongs to the organophosphate chemical class. Chemically it is:

(RS)-O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate

(C11H15BrClO3PS). Profenofos is a pesticide of thiophosphate series. It is

a wide-spectrum insecticide with easy biodegradation and a high

bioactivity for antiloxic pests. It can be used to control pests in cotton,

fruit trees and vegetables with an excellent effect on cotton.It are non-

systemic insecticide and acaricide with contact and stomach action

exhibits a translaminar effect. Have ovicidal properties. It is used for

control of insects and mites on cotton, maize, sugar beet, soya beans,

potatoes, vegetables, tobacco, and other crops. In Korea it is used for pest

control such as white fly, rocket and plantlouse.

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Recently, organophosphorous pesticides, carbamates, pyrethroids

and triazines have largely replaced the organochlorine compounds in

agricultural activities. The organophosphorous pesticides chlorpyrifos,

phosphorothioate,Chlorfenvinphos, phosphorothioate, methidathion and

phosphorothioate are widely used in the countries of the European Union

(UNED 1991) and have been detected at µg/L level in surface water of

the Spanish Mediterranian coasts (Hernandez et al., 1996). Recent studies

proved the risks of these organophosphorous pesticides due to their short

and long term effects on the survival and accumulation ability in the

tissues of aquatic organisms (Serrano et al., 1995; Van den Brink et al.,

1995). The organophosphate pesticides modify the activity of several

enzymes (Mohiyuddin et al., 2010; Joseph and Raj, 2011).

The organophosphates are found in environment with enough

frequency (Ballesteros and Parrado, 2004) to constitute an ecological risk.

Their concentration in water sources (Barcelo et al., 1990; Konstantinou

et al., 2006), in air (Tuduri et al., 2006) and food (Bai et al., 2006; Darko

and Akoto, 2008) can vary between a few ppb to ppm levels. There are

some studies dealing with the ecotoxicology of organophosphates

(Burkepile et al., 1999; Zhang et al., 2008) but few provide data about the

hazards of the degradation products (Kim et al., 2006; Kralj et al., 2007;

Virag et al., 2007). Maheshwari et al., (2001) reported that

organophosphate pesticide (Triazophos) was more toxic than other

insecticides. Joseph and Raj (2010) studied the toxicity of Curacron

(profenofos) in Cyprinus carpio.

The insecticides of the organophosphorus and carbamate classes

are widely used and highly effective pest control agents. Although there

are agents within these two classes that have other pesticidal uses, such as

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fungicidal or herbicidal applications, it is the insecticides (which also

have utility as nematocides, acaricides, and helminthicides) that display

the greatest neurotoxic properties. Any agent designed to kill pests is of

potential danger to nontarget organisms, such as humans, if the molecular

target for the pesticide also exists as an important entity in the nontarget

organism. Such a common molecular target exists for the

organophosphorus and carbamate insecticides. The members of these two

insecticidal classes are inhibitors of acetylcholinesterase (AChE). The

inhibition of AChE mediates most, if not all, of the clinical signs of

toxicity during an acute intoxication. Because of the environmental and

metabolic ability of these two classes of agrochemicals, they were

important replacements for the persistent and bioaccumulative

organochlorine insecticides, which were the predominant agricultural

chemicals in the 1950s and 1960s. The use of the organophosphorus

insecticides (less accurately but more commonly called

organophosphates: OPs) and the carbamates has been an important

component in the control of insects in agriculture, buildings, home

gardens, and public health since the 1950s. While attempts have been

made by the agrochemical industry to improve the pest vs nontarget

organism selectivity, and these attempts have frequently been very

effective, it remains a fact that some of the agents with high-use patterns

are still moderately or highly toxic to mammals (Cruz and White, 1995;

Olivera, 1997). Because of their intense use, it is inevitable that human

exposures will occur, and, despite important safety precautions being in

place, some of these exposures are likely to be high level and life

threatening during accidents.

Therefore, toxicity testing is an essential component of evaluating

water pollution. It is a study of change in ecological degradation,

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reflecting environmental pollution. Toxicants like heavy metal ions and

most of pesticides are non-degradable, resulting into bioaccumulation

within the ecosystem and its biotic components.

The organism used to determine the toxicity are called biotests and

the individual indices showing the change in the biochemical and

physiological process used for determination of degree of poisoning are

called tests. As per as the toxicity test is concerned, bioassay method is

very important. According to Sprague, (1973) “bioassay is a test in which

the quality or strength of the material is determined by the reaction of

living organisms to it.” Though in the bioassay tests, impact of pesticide

on the organism is assessed, it is very important to improve and

standardize the bioassay methodology to obtain accurate toxicity data.

The purpose of toxicity tests is to produce data concerning the

adverse effect of an agent on test organisms. The most common type of

toxicity test with aquatic animals is the acute mortality test, which is

usually conducted to obtain information about a median lethal

concentration (LC50). The data produced by the test generally consists of

the percentages of organisms that are killed by different concentrations of

a toxicant after specified length of exposure like 24, 48, 72 and 96 hours.

LC50 is concentration in which 50 % of the experimental animals

survive. Estimation of LC50 by interpolation involves plotting of data in a

graph with concentration on X- axis, while percentage survival on Y-

axis. A straight line is drawn between two points representating survival

at two successive concentrations that were lethal to more and less than

half of the total number of test animals exposed to the toxicant. The

concentration at which this line crosses the 50 % survival line is the LC50

value (Litchfield and Wicoxon, 1949).

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In order to assess the potential effect of pollutants on aquatic biota,

the toxicity tests are must in pollution study. The physical and chemical

tests are not enough to assess the potential effects of pollutants on aquatic

flora and fauna. The susceptibility of different aquatic organisms varies

species to species. Therefore toxicity tests are useful to solve certain

queries such as -

1) To decide the doses for the screening of detoxifying drugs.

2) To evaluate short term and long term effects of toxicants to aquatic

biota.

In toxicology, different terminologies such as acute, sub acute,

chronic, lethal, sub lethal, short term or long term toxicity test, etc have

been used to study the pattern of response in the organisms. There are two

categories of toxic effects i.e. acute and chronic (Alderdice, 1967). In

acute toxicity a large dose of poison (usually lethal) is administered for a

short duration, and in chronic toxicity a small dose (may be either sub-

lethal or lethal) of poison is administered over a long duration. The

bioassay is frequently used inter-changeably. Toxicity results are

expressed as LC50 (lethal concentration) values. It is the calculated

concentration of toxicant in water that produces 50% mortality of test

organisms during specific period. From LC50 values safe concentration or

tolerable concentration of a toxicant can be determined. This is necessary

for the study of physiological responses to the toxicant in organism.

In present investigation, the toxicity tests of the pesticides

carbosulfan and profenofos in fresh water bivalve, Lamellidens

marginalis are carried out.

Toxicity tests have been found useful for providing answers to the

following human curiosities:

Suitability of an environmental condition for aquatic life.

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Relative toxicity of different toxicants to test species.

Relative sensitivity of species to different toxicants.

Permissible discharge rates of effluents.

Permissible concentration of pollutants for sustainable aquatic life.

Toxicity of pollutants to common species of food chain.

Sub-lethal effect(s) of a toxicant on a particular phase of species’ life

cycle.

Short and long term effects of toxicants to aquatic fauna.

Effectiveness of water treatment methods.

To decide the proper dose of toxicant for pest control.

To throw more light on ramifications of toxicity, selection of

toxicity tests is the most important aspect. These tests are classified

according to:

1) Method of exposure to test solution (static, renewal, flow through and

re-circulation) (Alabaster, 1969; Torzwell, 1971; Sanborn, 1974;

Leenwarngh, 1980; Van Wijngoarden et al., 1993).

2) Duration (short, intermediate and long term).

3) Purpose (monitoring odour, taste, growth rate, and relative toxicity/

relative sensitivity as a function of effluent quality.

Among these tests, short-term tests are exploratory and useful for

routine monitoring the efficiency of treatment methods for the quality of

discharge. They determine LC50 and give quick assessment of relative

toxicity of different toxicants to different species. These tests are also

useful for estimating a toxicant concentration to be used in studying

intermediate and long-term impact. They permit determination of

(i) Sub-lethal effect(s) on behavioral, anatomical, histological,

biochemical and teratological aspects,

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(ii) Maximum acceptable/allowable toxicant concentration (MATC)

and

(iii) Application factor (AF).

Though very little conceptual change has been made to the

theoretical frame-work in the early acute toxicity tests (Doudoroff, 1977),

many workers have optimized parameters such as selection of test

species, characteristics of diluent water, acclimatization, reporting of

results, computation of data etc (Torzwell, 1971; National Academy of

Sciences, 1973; Doudoroff, 1977; Buikema et al., 1982).

Acute toxicity:

Acute toxicity tests are carried out to (a) detect sensitive species of

an eco-system (Penny and Adams, 1953), with respect to pollutants, (b)

assess the degree of damage to target organ(s) and (c) correlate it with

consequent behavioral and physiological disorder(s). Brown and Parsons

(1978) listed twelve basic types of investigations in toxicity, most of them

being carried out in laboratory. Main aim of these toxicity tests is for (1)

preliminary screening of chemicals for assessing the extent of risk to an

aquatic organism(s) and (2) identifying the chemicals causing death of

test species so that their de-pollution measures receive concerted

attention. Rehwoldt et al., (1973); Eisler (1977) have studied that acute

toxicity tests to throw light on the lethal effects of industrial pollutants on

aquatic life. Their ultimate objective has been to define a concentration at

which a test pollutant is capable of producing a tangible response, usually

deleterious to the test population under controlled conditions of exposure.

Criteria for the selection of experimental model:

Adelman and Smith (1976) and APHA, AWWA and WPCF (1985)

have defined the following criteria for the selection of test species:

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a) They should occur, or be closely related to species, which occur, in

the waste receiving water being tested.

b) They should be capable of being held in the laboratory in healthy

condition for at least one month.

c) They should be sufficient in number for repeating the tests for

statistically meaningful conclusions.

d) They should represent an important tropic level or economic

resource in the eco-system of the receiving water.

Based on these criteria, test organisms (crustaceans, mollusks, fishes

etc) are standardized for toxicity evaluation by the developed countries.

In these bioassays, (a) experimental organisms are exposed to a

series of suspected concentrations of a toxicant, (b) exposure period is

generally 24, 48, 72 and 96 hrs, (c) experimental conditions are

adequately controlled, mimicking those which are found in nature and (d)

an observation on acute toxicity is expressed in terms of LC50, indicating

50% mortality, at a particular concentration of a toxicant or a median

tolerance limit (TLM) (McLeese et al., 1982).

Acute toxicity bioassays are conducted in meeting at least one of

the six following objectives (Carter, 1962; Murthy, 1986). They qualify

to be (a) scientifically meaningful, (b) legally desirable/defensible, (c)

simple to carry out reproducibly for an independent validation, (d) cost-

effective for repeating a number of times, (c) ecologically significant in

predicting an acute toxicity capability of a toxicant and (e) considered to

have the greatest utility.

Chronic toxicity:

Bioassay for this type of toxicity demonstrates the effects of long-

term exposure of test species to a toxicant at a concentration much lower

than lethal level. Its objective is to determine MATC (Maximum

20

Acceptable Toxicant Concentration) and AF (Application Factor) of a

toxicant in an effluent (Mishra and Tiwari, 1998). These tests are of two

types: (i) partial life cycle and (ii) complete life cycle.

These tests are designed to evaluate a concentration of a toxicant

that will interfere with (1) oxygen uptake, (2) physiological/biological

responses, (3) food consumption / utilization, (4) growth and

development, (5) gross sign(s) of intoxication and (6) reproductive

potential of an aquatic organism, besides providing a more sensitive

measure of toxicity than provided by an acute toxicity test.

Chronic effect depends mainly on persistence of pollutant(s),

period of exposure and capacity of an organism to degrade/eliminate

accumulated pollutants (Christensen et al., 1977).

One of the victims among non target organisms is mollusks which

are commercially important to man. Representatives of class bivalvia are

very important for evaluating the levels of pollution of given area because

the group comprises sedentary filter feeders which can accumulate

xenobiotics from the environments. They are suspension feeders in the

primary stages of food chain and influence the organization and

functioning of ecosystem (Mane et al., 1986). Bivalves are extensively

used in monitoring programmes in the marine environment due to their

ability to concentrate pollutants to several orders of magnitude above

ambient levels in sea water, commonly, the levels of contaminants

accumulated in the tissues has been used to indicate the degree of

chemical contamination in the environment (Madfa and Abdel-Moati,

1998).

Mussels found on the riverbed are sold in clusters in markets.

Present investigation has undertaken to find LC50 values of the pesticides

21

profenofos and carbosulfan for Lamellidens marginallis as a pre-requisite

to conducting acute and chronic toxicity tests for further study.

22

MATERIALS AND METHODS

Medium sized fresh water bivalves, Lamellidens marginalis (50-55

mm in shell length) used in the present study, were collected from Girna

dam area, situated at 200, 28

1,58

0 N latitude and 74

0,43

1, 13

11E longitude

and forty four Km away from Chalisgaon. The bivalves were cleaned and

kept in plastic troughs containing dechlorinated water for 2-3 days to

acclimatize to the laboratory conditions. Overcrowding was avoided by

keeping small number of bivalves in different troughs. Water from the

troughs was changed every day. The animals were not feed during the

experimentation.

The analysis of the physico-chemical properties like water

temperature, pH, dissolved oxygen (Wrinkler’s method), free CO2

carbonates, bicarbonates and total alkalinity of water used in the bioassay

tests were estimated according to APHA,1980 (Table -1).

The medium sized active acclimatized bivalves were selected for

evaluation of toxicity. Ten bivalves each were exposed to 10 to 15

different concentrations of each pesticide in 10 litres of water in plastic

troughs. Series of static bioassay tests were conducted under laboratory

conditions. The concentration of pesticides, profenofos (50%E.C., an

organophosphate) and carbosulfan (25% E.C., a Carbamate) were

maintained by changing the polluted water in troughs after every 24

hours. The resulting mortality was recorded in the range of 10% to100%

for each concentration for the duration of 24, 48, 72 and 96 hours, as

acute exposure. Parallel controls were also maintained without the

pollutant. Animal behavior is also observed.

Cm = Control mortality.

It was observed that there was no mortality in control group of

bivalve. The mortality data obtained in experimental animals for each

dose was calculated by Finney’s formula.

23

P =r

n × 100

Where,

P = percent mortality,

r = mortality observed,

n = number of animals exposed in batch.

The mortality data thus obtained was put into probit/log

concentration transformation, so as to plot probit regression lines. These

regression lines were plotted for the purpose of calculating the required

concentration of pesticides to produce 50% mortality and 20% mortality.

The standard error of the log LC50 (Variance ‘V’ of the calculated log

LC50) and ƒ 2

(Chi-square) value and Fiducial limits to pesticides were

calculated from regression equation. The lethal dose and safe

concentration of pesticides were calculated from the above data.

Calculation of regression line:

To plot a well studied straight line between log concentration and

probit kill, the method described by Finney (1964) and simplified by

Busvine (1971) was followed. To trace a regression equation and to plot a

regression line the steps carried out are given below:

The regression equation was calculated for the bivalve,

Lamellidens marginalis when exposed for 24, 48, 72 and 96 hours to

pesticides profenofos and carbosulfan.

1. In the first column of the table serial numbers of trough were entered.

2. In column No. II the concentration of the pesticides in ppm was

entered.

3. In the IIIrd

column, headed ‘x’ the log of respective concentration to

the base 10was entered.

24

4. In the IVth

column, headed ‘n’ the number of animals taken in a batch

was noted.

5. In column No. V, observed mortality for 24, 48, 72 and 96 hours was

recorded.

6. The percent mortality (P) entered in column No.VI was calculated by

formula,

P =r

n × 100

If the mortality occurred in the control set of animals, then by

using the Abbots formula the corrected mortality was calculated and

entered in the VIth

column.

P =Om − Cm

100 − Cm × 100

7. The empirical probit value was read from Table 1 (Transformation of

percentage to probits) from Finney’s book, and recorded in column

No. VII.

8. The empirical probits were plotted against ‘x’ (log of concentration of

pesticides).The provisional straight line was drawn to suit the

maximum points, judging its position by eye.

9. The expected probit (Y) values were read from the provisional line of

the graph for values of ‘x’ and tabulated in column VIII with two

places of decimals.

10. From the column e = 00 of table II in Finney’s book, the weighing

co-efficient (w) for y was read and entered in column No. IX.

11. Each weighing coefficient (w) was multiplied by ‘n’ (number of

bivalves exposed per batch) from column II and then product W

were listed in column X.

25

12. From table No. IV (Finney’s book) the working probit (y) value was

read corresponding to each ‘y’ and ‘p’ and listed in column No. XI.

13. Then for each row, the value of W and ‘x’ as well as W and ‘y’ were

multiplied and the products Wx and Wy were listed in the column

XII and XIII.

14. The products of column X, XII and XIII were summed up at the foot

of each representative column and were abbreviated as SW, SWX

and SWY respectively.

15. In column XIV, XV and XVI the products of W multiplied by x 2, W

multiplied by y2

and W multiplied by x and y were entered

respectively. The summations of the products of column XIV, XV

and XVI are SWx2 and SWy

2 and SWxy respectively and they were

entered at the foot of each column.

16. Then the values for x and y were calculated by using the following

equations.

x =SWx

SW y =

SWy

SW

17. Now the value of the estimated regression co-efficient ‘b’ was

calculated by the following equation.

b =SWy − x . SWy

SWx2 − x . SWx

18. The regression equation may now be written as

Y = y + b X − x

19. From the regression equation value of ‘y’ corresponding to the

original values of ‘x’ were calculated and entered in column XVII

as improved expected probit y’. These values of improved

26

expected probit (y') should not differ by more than 0.2 as compared

to the expected probits (y) in column VIII. However, if there is no

discrepancy the value of ‘y’ were taken as improved expected

probit y' and the whole cycle of calculation from VIII was

repeated.

20. The regression line was then plotted between log concentration (x)

and improved expected probit y'.

Calculation of LC10 and LC50:

For the calculation of LC10 and LC50 values of the pesticides from

regression equation, y = 3.7184 and y = 5 (values from Finney's table

no.1) were kept to calculate x values. Antilogs of the x values are the

LC10 and LC50 of the pesticides in ppm. The LC10 and LC50 values for 24,

48, 72 and 96 hours which were calculated for the pesticides carbosulfan

and profenofos are given in table.10.

Calculation of accuracy of the LC50:

The variance ‘v’ of the calculated log LC50 was calculated by

the expression.

V =1

b2

1

SW+

(m − X )2

SWx2 −(SWx)2

SW

Where, V =Variance (The Standard error of LC50)

Calculation of Chi-square (ƒ2 ) values:

The value of ƒ2 (Chi-square) was calculated to test the homogeneity of

the data. This is given by the expression.

27

ƒ2 = (SWy

2 -x . SWy) – b (SWxy - x . SWy)

The value of ƒ2 was compared with the table of the statistics for n-2

degree of freedom (where ‘n’ is the number of experiments) should this

value be higher than the figure of ƒ2

for the 5% level, there is indication

of heterogeneity.

Calculation of Fiducial limits:-

The Fiducial limits m1 and m2 with 95 % confidence were

calculated from the variance (v) by the following formula:

M1 = m − 1.96 V

and

M1 = m + 1.96 V

Where M = calculated log LC50 Value

V = variance (standard error of LC50)

Calculation of lethal dose:-

The lethal dose calculated due to its importance from agricultural

point of view. The lethal dose was calculated by the following formula:

Lethal dose = LC50 value × time of exposure.

Calculation of safe concentration:-

Hart et al., (1945) have proposed a formula for calculation of safe

concentration of toxicants for animals.

C =48 hrs TLM × 0.3

S2

28

Where C = safe concentration and

S =24 hrs TLM

48 hrs TLM=

24 hrs LC50

48 hrs LC50

Where TLM = median tolerance limit, or known as LC50 value,

which is the concentration at which 50% of the test bivalves were killed

in particular time period.

29

OBSERVATIONS AND RESULTS

The physico-chemical characteristics of the water used for holding

bivalve and as diluents were examined and are presented in table 1.

Acute toxicity tests were carried out in the laboratory upto 96 hours

duration for two pesticides, profenofos and carbosulfan. The LC10 and

LC50 values were calculated for 24, 48, 72 and 96 hours by the method

described by Finney (1964) and simplified by Busvine (1971). The results

obtained after toxicity evaluation of pesticides to Lamellidens marginallis

are summarized in table 2 to 10.

The LC10 and LC50 values for pesticides are shown in table 10. The

LC10 values for 24, 48, 72 and 96 hours exposure to profenofos are 30.09

ppm, 6.702 ppm, 4.585 and 2.211 ppm respectively. The LC10 values for

24, 48, 72 and 96 hours exposure to carbosulfan are 25.29 ppm, 10.07

ppm, 4.467 ppm and 1.629 ppm respectively. The data obtained indicates

that carbosulfan is more toxic than profenofos and Lamellidens

marginalis showed more sensitivity to carbosulfan.

The LC50 values for 24, 48, 72 and 96 hours exposure were

calculated. LC50 values for profenophos at 24, 48, 72 and 96 hours

exposure are 60.60 ppm, 26.46 ppm, 11.65 ppm and 6.191 ppm

respectively. LC50 values for carbosulfan at 24, 48, 72 and 96 hours


respectively. LC50 values for carbosulfan are less indicating the high

toxicity of pesticide.

The calculated accuracy for the log LC50 values are summarized in

the table 10 under the column Variance ‘V’. The Variance values of LC50

for profenofos are 0.002126, 0.0073628, 0.003612 and 0.004548 for 24,

48, 72 and 96 hours respectively. The Variance values of LC50 for

carbosulfan for 24, 48, 72 and 96 hours are 0.0017845, 0.016275,

0.0042193 and 0.005151 respectively.

30

The Fiducial limits for log LC50 values are cited in table 10 under

the column Fiducial limit M1 and M2. The 95% confidence of LC50 values

(Fiducial limit) to pesticides are M1 (minimum limit) and M2 (maximum

limits). The minimum fiducial limits for 95% confidence at 24, 48, 72

and 96 hours values in ppm of profenofos are 1.692127, 1.25452,

0.948599 and 0.65962 and maximum fiducial limits are 1.872873,

1.59088, 1.184200 and 0.92398 respectively. The minimum fiducial

limits for 95% confidence at 24, 48, 72 and 96 hours in ppm of

carbosulfan are 1.63671, 1.09936, 0.961386 and 0.60473 and maximum

fiducial limits are 1.80229, 1.59944, 1.216014 and 0.88607 respectively.

The safe concentrations of the pesticides profenofos and

carbosulfan are calculated and summarized in table 10 under column of

safe concentration ‘C’. The safe concentrations for profenofos and

carbosulfan are 1.513372 and 1.22060 ppm respectively. Carbosulfan is

most toxic and profenofos is least toxic among the two pesticides.

Lethal doses for the pesticides are entered in the table 10 under the

column ‘lethal dose’. For the immediate 100% mortality of the bivalve,

Lamellidens marginallis, the lethal dose was calculated. The lethal dose

for the pesticide profenofos at 24, 48, 72 and 96 hours exposure are

1454.40 ppm, 1270.08 ppm, 838.80 ppm and 594.336 ppm respectively.

The lethal dose for the pesticide carbosulfan at 24, 48, 72 and 96 hours


respectively.

The order of toxicity in increasing manner is profenofos <

carbosulfan. The ƒ2 values for the pesticides, profenofos and carbosulfan

are calculated and given in table 10 under the column the column ƒ2

value. The ƒ2 values for profenofos at 24, 48, 72 and 96 hours exposure

are 0.185322, 0.457804, 0.157022 and 0.628979 respectively. The ƒ2

31

values for carbosulfan at 24, 48, 72 and 96 hours exposure are 0.335019,

0.367902, 0.26072 and 1.19483 respectively..

The chi-squares ƒ2 values are summarized in the table 10 under the

column ƒ2. These values are used to test the homogeneity of data. The

chi-square test for heterogeneity showed that there is no significant

difference between the observed and the calculated values.

32

ANIMAL BEHAVIOUR

A) Behaviour of bivalves in control groups:

1) The bivalves when immersed in water retracted their body inside

the shell and closed the shell valves.

2) After few minutes, they extended foot and opened the shell valves.

3) The bivalves opened the shell valves, extended pallial edges as

well as the siphons out of the valves. The gentle mechanical

stimulus made the extended organs to retract in the shell valves

immediately.

4) Excreta were accumulated every time and comparatively little

mucus was secreted.

5) Dead bivalves had opened shell valves with foot retracted in the

valves. Gentle mechanical stimulus had no effect.

B) After exposure to various pesticides, the behaviour of the bivalves

was as follows:

1) At the time of immersion, the bivalves immediately retraced the

foot in the shell and closed the valves.

2) After some period, the bivalves slightly opened the shell valves and

protruded the foot slightly along with the pallial edges and siphon

out side the shell valves.

3) The bivalves which initially opened the shell valves trapped the

slightly swollen foot between the shell valves and remained closed

to avoid further penetration of pesticide inside the body.

4) Copious mucus secretion was seen for carbosulfan while little

mucus was secreted in profenofos.

5) Swelling of foot was observed for profenofos, while it shrinked in

carbosulfan. With increase in exposure time restricted movements

were observed.

33

6) The bivalves opened the shell valves and extended the swollen foot

out side the shell valves. Mechanical stimulus made these bivalves

to retract the foot slowly in the shell valves. The mantle edges

remained at the border of shell valves with siphons protruded

outside the shell valves.

7) The time of siphon opening was also reduced with the time of

exposure to the pesticides.

8) Eggs and embryos at various stages of development were released

and were encapsulated in gelatinous mass.

9) The bivalves when died widely opened the shell valves with foot

shrunken (carbosulfan) and swollen (profenofos). Gentle

mechanical stimulus had no effect.

34

Table-1. Physico-chemical characteristics of water used during

toxicity tests of pesticides for Lamellidens marginallis

Sr. No. Parameters Values

1 pH 7.22 ± 0.1699

2 Air temperature 28oC ± 2.2173

oC

3 Water temperature 24oC ± 2.2173

oC

4 Dissolved oxygen 1.612 ± 0.720

5 Bicarbonates 60 ± 10

6 Total alkalinity

(Bromocresol indicator) 71.666 ± 10.40

7 Acidity (phenolphthalein) 4.526 ± 2.6792

8 Salinity 0.20478

9 Chlorides 113.44

10 T.D.S. 40

All parameters are expressed in mg/l except pH and temperature.

35

Table -2. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Carbosulfan (for 24 hrs).

Sr.

No.

Conc.

Of

Pesti-

cide

ppm

Log Of Conc.

to base

10 'x'

No. of

animals

exposed

'n'

Mortality

for 24hrs

'r'

Percentage

Mortality

P=(100 X r)

n

%

Empirical

Probit

‘ X’

Expected

Probit

'Y'

Weighing

Coeffi-

cient

'w'

Weight

W= nw

Working

Probit

'y'

Wx Wy Wx² Wy² Wxy

Improved

Expected

Probit

Y'

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII

1. 30 1.4771 10 2 20 4.1584 4.1 0.47144 4.7144 4.160 6.96364 19.6119 10.28599 81.58552 28.96874 4.0184

2.

40

1.6021 10 3 30 4.4756 4.6 0.60052 6.0052 4.479 9.62093 26.89729 15.41369 120.4730 43.09215 4.5245

3.

50 1.6990 10 4 40 4.7467 4.9 0.63431 6.3431 4.748 10.77693 30.11704 18.3100 142.9957 51.16885 4.9169

4.

60 1.7782 10 6 60 5.2533 5.2 0.62742 6.2742 5.253 11.15678 32.95837 19.83899 173.1303 58.60658 5.2376

5.

70 1.8451 10 7 70 5.5244 5.5 0.58099 5.8099 5.524 10.71985 32.09389 19.77919 177.2866 59.21643 5.5085

6.

80 1.9031 10 8 80 5.8416 5.8 0.50260 5.0260 5.841 9.56498 29.35687 18.20311 171.4735 55.86905 5.7433

SW =

34.1728

SWx=

58.8031

Swy =

171.035

SWx² =

101.8310

SWy² =

866.9400

Swxy =

296.9218

2 - .

S W x 5 8 .8 0 3 1

1 ) = = = 1 .7 2 0 7 6

S W 3 4 .1 7 2 8

S W y 1 7 1 .0 3 5

2 ) = = = 5 .0 0 5 0 0

S W 3 4 .1 7 2 8

3 ) b = x S W x

x

y

S W xy - x . S W y 2 9 6 .9 2 1 8 -1 .7 2 0 7 6 × 1 7 1 .0 3 5= = 4 .0 4 9 1 5

S W x 1 0 1 .8 3 1 0 - 1 .7 2 0 7 6 × 5 8 .8 0 3 1

4 ) R e g re s s io n e q u a tio n -

Y = y + b (X - x )

= 5 .0 0 5 0 0 + 4 .0 4 9 1 5 (X - 1 .7 2 0 7 6 )

= 4 .0 4 9 1 5 X - 1 .9 6 2 6 1

36

Table -3. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Carbosulfan (for 48hrs).

Sr. No.

Conc.

Of Pesti cide

ppm

Log Of

Conc. To Base

10

'x'

No. of animals

exposed

n

Mortality for 48 hrs

r

Percentage Mortality

P=(100 X r)

n

%

Empirical Probit

X

Expected Probit

Y

Weighing Coeffi-

cient

w

Weight

W= nw

working Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved Expected

Probit

Y’


1. 10 1.0000 10 1 10 3.7184 3.7 0.33589 3.3589 3.719 3.35890 12.49175 3.35890 46.45681 12.49715 3.7070

2. 15 1.1761 10 3 30 4.4756 4.4 0.55788 5.5788

4.477 6.56122 24.97629 7.716659 111.8188 29.37461 4.3587

3. 20 1.3010 10 4 40 4.7467 4.9 0.63431 6.3431 4.748 8.25237 30.11704 10.73634 142.9957 39.18227 4.8208

4. 25 1.3979 10 5 50 5.0000 5.2 0.62742 6.2742 4.997 8.77070 31.35218 12.26057 156.6668 43.82721 5.1794

5. 30 1.4771 10 7 70 5.5244 5.5 0.58099 5.8099 5.524 8.58180 32.09389 12.67616 177.2866 47.40588 5.4725

6. 35 1.5441 10 8 80 5.8416 5.8 0.5026 5.0260 5.841 7.76064 29.35687 11.98321 171.4735 45.32994 5.7204

SW =

32.3909

SWx = 43.28563

SWy = 160.3880

SWx2 = 58.73183

SWy2 = 806.6982

SWxy = 217.6170

S W x 4 3 .2 8 5 6 3

1 ) x = = = 1 .3 3 6 3 5

S W 3 2 .3 9 0 9

S W y 1 6 0 .3 8 8 0 2

2 ) y = = = 4 .9 5 1 6 4

S W 3 2 .3 9 0 9

S W x y - x . S W y 2 1 7 .6 1 7 0 6 -1 .3 3 6 3 5 × 1 6 0 .3 8 8 0 23 ) b = = = 3 .7 0 0 3 8

2 5 8 .7 3 1 8 3 - 1 .3 3 6 3 5 × 4 3 .2 8 5 6 3S W x - x . S W x


Y = y + b (X - x )

= 4 .9 5 1 6 4 + 3 .7 0 0 3 8 (X - 1 .3 3 6 3 5 )

= 3 .7 0 0 3 8 X + 0 .0 0 6 6 4

37


Sr. No.

Conc.

Of

Pestici-

de

ppm

Log Of Conc.

To Base

10 'x'

No. of

animals

exposed

n

Mortality for 24

hrs

r

Percentage

Mortality

P=(100 X r)

n

%

Empirical

Probit

X

Expected

Probit

Y

Weighing

Coeffi-

cient

w

Weight

W= nw

working

Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved

Expected

Probit

Y’


1.

6 0.7782 10 2 20 4.1584 4.1 0.47144 4.7144 4.160 3.66874 19.6119 2.85501 81.58552 15.26198

4.0929

2.

10 1.0000 10 4 40 4.7467 4.8 0.62742 6.2742 4.747 6.2742 29.78363 6.27420 141.3829 29.78363

4.7409

3.

14 1.1461 10 5 50 5.0000 5.2 0.62742 6.2742 4.997 7.19086 31.35218 8.24144 156.6668 35.93273 5.1678

4.

18 1.2553 10 7 70 5.5244 5.5 0.58099 5.8099 5.524 7.29316 32.09389 9.15511 177.2866 40.28746 5.4869

5.

22 1.3424 10 8 80 5.8416 5.8 0.5026 5.0260 5.841 6.74690 29.35687 9.05704 171.4735 39.40866 5.7413

SW =

28.0987

SWx =

31.17388

SWy =

142.1985

SWx2 =

35.58282

SWy2 =

728.3953

SWxy =

160.6745

2 - .

S W x 3 1 .1 7 3 8 8

1 ) = = = 1 .1 0 9 4 4

S W 2 8 .0 9 8 7

S W y 1 4 2 .1 9 8 5

2 ) = = = 5 .0 6 0 6 8

S W 2 8 .0 9 8 7

x3 ) b =

x S W x

x

y

S W xy - . S W y 1 6 0 .6 7 4 5 - 1 .1 0 9 4 4 × 1 4 2 .1 9 8 5 = = 2 .9 2 1 7 8

S W x 3 5 .5 8 2 8 2 - 1 .1 0 9 4 4 × 3 1 .1 7 3 8 8


Y = y + b (X - x )

= 5 .0 6 0 6 8 + 2 .9 2 1 7 8 (X - 1 .1 0 9 4 4 )

= 2 .9 2 1 7 8 X + 1 .8 1 9 1 4

38


Sr.No.

Conc.

Of

Pestic

ide

ppm

Log Of Conc.

To

Base 10 'x'

No. of

animals

exposed

n

Mortality

for 24hrs

r

Percentage

Mortality

P=(100 X r)

n

%

Empirical

Probit

X

Expected

Probit

Y

Weighing Coeffi-

cient

w

Weight

W= nw

working Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved Expected

Probit

Y’


1.

2 0.3010 10

2 20 4.1584 4.1 0.47144 4.7144 4.160 1.41903 19.6119 0.42712 81.58552 5.90318 3.9319

2.

4 0.6021 10 3 30 4.4756 4.6 0.60052 6.0052 4.479 3.61573 26.89729 2.17703 120.4730 16.19486 4.6555

3. 6 0.7782 10 5 50 5.0000 5.0 0.63662 6.3662 5.000 4.95417 31.83100 3.85534 159.1550 24.77088 5.0787

4. 8 0.9031 10 6 60 5.2533 5.2 0.62742 6.2742 5.253 5.66623 32.95837 5.11717 173.1303 29.76471 5.3789

5. 10 1.0000 10 7 70 5.5244 5.5 0.58099 5.8099 5.524 5.8099 32.09389 5.8099 177.2866 32.09389 5.6119

6.

12 1.0792 10 9 90 6.2816 5.6 0.55788 5.5788 6.123 6.02064 34.15899 6.49747 209.1555 36.86438 5.8022

SW =

34.7487

SWx = 27.48571

SWy = 177.5514

SWx2 = 23.88405

SWy2 = 920.7860

SWxy = 145.5919

2 - .

S W x 2 7 .4 8 5 7 1

1 ) = = = 0 .7 9 0 9 9

S W 3 4 .7 4 8 7

S W y 1 7 7 .5 5 1 4

2 ) = = = 5 .1 0 9 5 8

S W 3 4 .7 4 8 7

x3 ) b =

x S W x

x

y

S W xy - . S W y 1 4 5 .5 9 1 9 - 0 .7 9 0 9 9 × 1 7 7 .5 5 1 4 = = 2 .4 0 3 2 7

S W x 2 3 .8 8 4 0 5 - 0 .7 9 0 9 9 × 2 7 .4 8 5 7 1


Y = y + b (X - x )

= 5 .1 0 9 5 8 + 2 .4 0 3 2 7 (X - 0 .7 9 0 9 9 )

= 2 .4 0 3 2 7 X + 3 .2 0 8 6 2

39

Table – 6. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Profenofos (for 24 hrs).

Sr.

No.

Conc.

Of Pesti-

cide

ppm

Log Of

Conc.

to base

10

'x'

No. of

animals exposed

n

Mortality

for 24hrs

r

Percentage Mortality

P=(100 X r)

n

%

Empirical

Probit

X

Expected

Probit

Y

Weighing

Coeffi-cient

w

Weight

W= nw

Working

Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved

Expected Probit

Y’


1

30 1.4771 10 1 10 3.7184 3.7 0.33589 3.3589 3.719 4.96143 12.49175 7.32853 46.45681 18.45156

3.7129

2 45 1.6532 10 3 30 4.4756 4.4 0.55788 5.5788 4.477 9.22287 24.97629 15.24725 111.8188 41.29080

4.4551

3

60 1.7782 10 5 50 5.0000 5.0 0.63662 6.3662 5.000 11.3203 31.831 20.12989 159.1550 56.60188

4.9820

4 75 1.8751 10 6 60 5.2533 5.4 0.60052 6.0052 5.250 11.2603 31.5273 21.11428 165.5183 59.11684

5.3903

5

90 1.9542 10 8 80 5.8416 5.7 0.53159 5.3159 5.834 10.3883 31.01296 20.30088 180.9296 60.60553

5.7219

SW =

26.625

SWx =

47.15335

SWy =

131.8392

SWx2 =

84.12073

SWy2 =

663.8785

SWxy =

236.0665

S W x 4 7 .1 5 3 3 5

1 ) x = = = 1 .7 7 1 0 1

S W 2 6 .6 2 5

S W y 1 3 1 .8 3 9 2

2 ) y = = = 4 .9 5 1 7 0

S W 2 6 .6 2 5

S W x y - x . S W y 2 3 6 .0 6 6 5 - 1 .7 7 1 0 1 × 1 3 1 .8 3 9 23 ) b = = = 4 .2 1 4 5 8

2 8 4 .1 2 0 7 3 - 1 .7 7 1 0 1 × 4 7 .1 5 3 3 5S W x - x . S W x

4 )

R e g re s s io n e q u a tio n -

Y = y + b (X - x )

= 4 .9 5 1 7 0 + 4 .2 1 4 5 8 (X - 1 .7 7 1 0 1 )

= 4 .2 1 4 5 8 X - 2 .5 1 2 4

40

Table -7..Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Profenofos (for 48hrs).

Sr no.

Cocn. Of

Pesti-cide

ppm.

Log of conc.

to

base

10

'X'

No. of animals

exposed

'n'

Mortality for 48

Hrs.

'r'

Percentage mortality

P=(100 X r)

n

%

Empirical probit

Expected probit

'Y'

Weighing Coeffi

cient

'w'

Weight

W = nw

Working probit

'y'

Wx

Wy

Wx2

Wy2

Wxy

improved expected

probit

y'


1 10 1.0000 10 2 20 4.1584 4.1 0.47144 4.7144 4.160 4.7144 19.6119 4.7144 81.585521 19.6119 4.0918

2 20 1.3010 10 4 40 4.7467 4.7 0.61609 6.1609 4.747 8.01533 29.2458 10.4279 138.82978 38.04878 4.7384

3 30 1.4771 10 5 50 5.0000 5.0 0.63662 6.3662 5.000 9.40351 31.831 13.8899 159.15500 47.01757 5.1167

4 40 1.6020 10 6 60 5.2533 5.2 0.62742 6.2742 5.253 10.0513 32.9584 16.1021 173.13033 52.79931 5.3851

5 50 1.6990 10 8 80 5.8416 5.4 0.60052 6.0052 5.793 10.2028 34.7881 17.3346 201.52760 59.10502 5.5934

SW=

29.5209

SWx=

42.3873

Swy=

148.435

SWx2=

62.469

SWy2=

754.22823

Swxy=

216.5826

41


Sr.

No.

Conc.

Of Pesti-

Cide

ppm

Log Of

Conc.

to base 10

'x'

No. of

animals exposed

n

Mortality

for 72 hrs

r

Percentage

Mortality P=(100 X r)

n

%

Empirical

Probit

X

Expected

Probit

Y

Weighing

Coeffi-cient

w

Weight

W= nw

working

Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved

Expected Probit

Y’

I II III IV

V VI VII VIII IX X XI XII XIII XIV XV XVI XVII

1.

6 0.7782 10 2 20 4.1584 4.1 0.47144 4.7144 4.160 3.66874 19.61190 2.85501 81.58552 15.26198 4.0880

2. 10 1.0000 10 4 40 4.7467 4.8 0.62742 6.2742 4.747 6.27420 29.78363 6.27420 141.3829 29.78363 4.7899

3.

14 1.1461 10 6 60 5.2533 5.2 0.62742 6.2742 5.253 7.19086 32.95837 8.24144 173.1303 37.77359 5.2523

4. 18 1.2553 10 7 70 5.5244 5.6 0.55788 5.5788 5.523 7.00306 30.81171 8.79095 170.1731 38.67794 5.5979

5.

22 1.3424 10 8 80 5.8416 5.8 0.50260 5.0260 5.841 6.74690 29.35687 9.05704 171.4735 39.40866 5.8735

6. 26 1.4150 10 9 90 6.2816 6.0 0.43863 4.3863 6.242 6.20661 27.37928 8.78236 170.9015 38.74169 6.1033

SW =

32.2539

SWx =

37.09039

SWy =

169.9018

SWx2 =

44.0010

SWy2 =

908.6468

SWxy =

199.6475

S W x 3 7 .0 9 0 3 9

1 ) x = = = 1 .1 4 9 9 5

S W 3 2 .2 5 3 9

S W y 1 6 9 .9 0 1 8

2 ) y = = = 5 .2 6 7 6 3

S W 3 2 .2 5 3 9

S W x y - x . S W y 1 9 9 .6 4 7 5 - 1 .1 4 9 9 5 × 1 6 9 .9 0 1 83 ) b = = = 3 .1 6 4 7 2

2 4 4 .0 0 1 0 - 1 .1 4 9 9 5 × 3 7 .0 9 0 3 9S W x - x . S W x


Y = y + b (X - x )

= 5 .2 6 7 6 3 + 3 .1 6 4 7 2 (X - 1 .1 4 9 9 5 )

= 3 .1 6 4 7 2 X + 1 .6 2 5 2 6

42


Sr.

No.

Conc.Of Pest-

Icide

ppm

Log Of Conc.

to base

10

'x'

No. of

animals

exposed

n

Mortality

for 96 hrs

r

Percentage

Mortality

P=(100 X r) n

%

Empirical

Probit

X

Expected

Probit

Y

Weighing

Coeffi-

Cient

w

Weight

W= nw

working

Probit

y

Wx

Wy

Wx²

Wy²

Wxy

Improved

Expected

Probit

Y’s


1. 2 0.3010 10 1 10 3.7184 3.7 0.33589 3.3589 3.719 1.01102 12.49175 0.30432 46.45681 3.76001 3.5935

2. 4 0.6021 10 3 30 4.4756 4.4 0.55788 5.5788 4.477 3.35899 24.97629 2.02245 111.8188 15.03822 4.4564

3. 6 0.7782 10 4 40 4.7467 4.9 0.63431 6.3431 4.748 4.93620 30.11704 3.84135 142.9957 23.43708 4.9610

4. 8 0.9031 10 6 60 5.2533 5.2 0.62742 6.2742 5.253 5.66623 32.95837 5.11717 173.1303 29.76471 5.3189

5.

10 1.0000 10 8 80 5.8416 5.5 0.58099 5.8099 5.808 5.80990 33.74390 5.80990 195.9846 33.74390 5.5966

SW =

27.3649

SWx = 20.78235

SWy = 134.2873

SWx2 = 17.09519

SWy2 = 670.3863

SWxy = 105.7439

S W x 2 0 .7 8 2 3 5

1 ) x = = = 0 .7 5 9 4 5

S W 2 7 .3 6 4 9

S W y 1 3 4 .2 8 7 3

2 ) y = = = 4 .9 0 7 2 8

S W 2 7 .3 6 4 9

S W x y - x . S W y 1 0 5 .7 4 3 9 - 0 .7 5 9 4 5 × 1 3 4 .2 8 7 33 ) b = = = 2 .8 6 5 3 2

2 1 7 .0 9 5 1 9 - 0 .7 5 9 4 5 × 2 0 .7 8 2 3 5S W x - x . S W x


Y = y + b (X - x )

= 4 .9 0 7 2 8 + 2 .8 6 5 3 2 (X - 0 .7 5 9 4 5 )

= 2 .8 6 5 3 2 X + 2 .7 3 1 2 1

43

Table-10. Relative toxicity of different pesticides against Lamellidens marginalis.

Sr.

No.

Name of

Pesticide

Time

of

Expo-

sure

Regression equation

+ b ( X - )

LC50

Values

ppm

LC10

Values

ppm

Variance

‘V’

Fiducial limits Lethal

dose ppm

Safe

Concen-

tration

‘C’

ƒ2

Values M1 M2

1. Carbosulfan

24

Y = 4.04915X-1.96261 52.42 25.29 0.0017845 1.63671 1.80229 1258.08

1.22060

0.335019

48

Y = 3.70038X-0.00664 22.36 10.07 0.0162750 1.09936 1.59944 1073.28 0.367902

72

Y = 2.92178X+1.81914 12.27 4.467 0.0042193 0.961386 1.21601 883.44 0.26072

96

Y = 2.40327X+3.20862 5.564 1.629 0.005151 0.60473 0.88607 534.144 1.19483

2. Profenofos

24

Y = 4.21458X- 2.51240 60.60 30.09 0.002126 1.692127 1.87287 1454.40

1.513372

0.185322

48

Y = 2.14831X+1.93350 26.46 6.702 0.0073628 1.25452 1.59088 1270.08 0.457804

72

Y = 3.16472X+1.62526 11.65 4.585 0.0036123 0.948599 1.18420 838.80 0.157022

96

Y = 2.86532X+2.73121 6.191 2.211 0.004548 0.65962 0.92398 594.336 0.628979

44

Fig. 1.1.1 Provisional and regression lines for Lamellidens marginalis to carbosulfan

for 24 hrs.


for 48 hrs.

3.5

4

4.5

5

5.5

6

1.4 1.5 1.6 1.7 1.8 1.9 2

Em

pir

ica

l p

rob

it

Log of concentration

3.5

4

4.5

5

5.5

6

0.9 1 1.1 1.2 1.3 1.4 1.5 1.6

Em

pir

ica

l p

rob

it


45


for 72 hrs.


for 96 hrs.

3.5

4

4.5

5

5.5

6

0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5

Em

pir

ica

l p

rob

it


3.5

4

4.5

5

5.5

6

6.5

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Em

pir

ica

l p

rob

it


46

Fig. 1.2.1 Provisional and regression lines for Lamellidens marginalis exposed to

profenofos for 24 hrs.



3.5

4

4.5

5

5.5

6

1.4 1.5 1.6 1.7 1.8 1.9 2

Em

pir

ica

l p

rob

it


3.5

4

4.5

5

5.5

6

0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

Em

pir

ica

l p

rob

it


Provisional line

Regression line

47





3.5

4

4.5

5

5.5

6

6.5

0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5

Em

pir

ica

l p

rob

it


3.5

4

4.5

5

5.5

6

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1

Em

pir

ica

l p

rob

it


48

Fig. 1.3.1 Comparison of safe concentration, LC10 and LC50 values of carbosulfan.

Fig. 1.3.2 Comparison of safe concentration, LC10 and LC50 values of profenofos.

0

10

20

30

40

50

60

24 48 72 96

Co

nc.

in

pp

m

Time of exposure

Safe conc.

LC 10

LC50

0

10

20

30

40

50

60

70

24 48 72 96

Co

nc.

in

pp

m

Time of exposure

Safe conc.

LC 10

LC50

49

DISCUSSION

The role of pesticides as agrochemicals in the promotion of our

economy is very important. These chemical substances of organic and

inorganic nature have brought benefits to mankind by destroying the

insect pests of various crops, resulting increased food production and

controlling the vectors. The extensive use of pesticides in the agriculture

resulted into its residues remain in the fields (Bhide et al., 2006).

Agricultural runoff from such fields leads to water pollution and causes

hazards to several non target organisms such as aquatic invertebrates and

vertebrates (Swarnakumari and Tilak, 2010). Moore and Waring (2001)

reported even low levels of pesticide in the aquatic environment causes

significant long-term effect on reproductive functions of animals.

Undesirable effect caused by pesticides to the aquatic organisms and their

hazards are elegantly reviewed by many workers (Brock et al., 2000;

Sarkar et al., 2003; Relyea, 2005; Pennati et al., 2006)

Behavior affects the survival of aquatic invertebrates and reflects

the integration of many biochemical and physiological processes.

Therefore, behavior is an important area to examine when investigating

the effect of toxicants on aquatic invertebrates. Pollutant at very low

concentration might be perceived by an organism’s sensory system. If the

stimulant is recognized harmful, avoidance may follow. Motile organisms

can protect themselves by running away from the polluted area. In those

forms with little or no mobility organism such as mollusc avoidance may

take the form of reducing exposure of external body surface through

mucus production (Concetta and Pia, 2005), or taking body in to shell or

by closure of siphons when exposure to polluted condition persist. Shell

closing mechanism might be the protective device against the toxicant

and provides good tolerance in the molluscs (Nagaratnamma and

Ramamurthi, 1982). Chaudhari (1988) reported many behavioral changes

50

in pesticide exposed snail, Bellamya bengalensis like sudden withdrawal

of foot inside the shell, closing of operculum and copious mucus

secretion. The extruded mucus forms a protective barrier preventing

direct contact between the toxin and the epithelia of the skin or digestive

tract, so reducing the toxicity of the chemicals (Triebskon and Ebert,

1989). Mucus secretion was also observed in Corbicula striatella on

exposure to pesticides (Jadhav, 1993) and in Parreysia favidens against

heavy metal exposure (Bhamre et al., 1996).

In the present study various behavioral changes are observed

during the pesticide exposure to the freshwater bivalve, Lamellidens

marginalis. Copious mucus secretion was seen after carbosulfan

exposure, while little mucus was secreted after profenofos exposure.

Swelling of foot was observed for profenofos, while foot shows shrinkage

in carbosulfan. With increase in exposure time restricted movements were

observed in bivalves. The bivalves opened the shell valves and extended

the swollen foot outside the shell valves. Mechanical stimulus made these

bivalves to retract the foot slowly in the shell valves. The mantle edges

remained at the border of shell valves with siphons protruded outside the

shell valves. The time of siphon opening was also reduced with the time

of exposure to the pesticides. Eggs and embryos at various stages of

development were released and were encapsulated in gelatinous mass.

Similar behavioral changes are observed by many workers while studying

toxicity of different pesticides on bivalves (Chaudhari 1988; Jadhav et al.,

1996; Waykar and Lomte, 2001).

The toxicity tests are necessary in pollution study because chemical

and physical tests are not sufficient to access the potential effect of

pollutants, on aquatic biota. Different kinds of organisms are not equally

susceptible to the same toxicant. The toxicity tests are useful for various

purposes as (i) To study the suitability of environment to organisms, (ii)

51

to determine favorable and unfavorable concentrations of pollutants in the

environment to the organism and (iii) To determine safe concentration of

pollutants to organisms etc. Chronic exposure to toxicant alters the

biochemical composition and physiological processes of the organisms.

Prior to the study of biochemical and physiological changes in the

organism, it is very essential to evaluate the LC50 concentration of the

toxicant. These tests provide quickest and reliable information about the

toxicity of the toxicant, however it is not exactly equivalent to field

results (Sanders, 1969).

In present investigation, acute toxicity tests were carried out in the

laboratory up to 96 hours duration for two pesticides, profenofos and

carbosulfan. The regression equations for 24 48, 72 and 96 hours were

obtained and are summarized in table no. 10. The LC10 and LC50 values,

the Fudicial limits, lethal dose, safe concentration and chi-square values

were calculated for 24, 48, 72 and 96 hours and obtained results are

presented in the table no. 10 and figure nos. 1.1.1 to 1.2.4. The data

obtained indicates that the rate of mortality of fresh water bivalve,

Lamellidens marginalis was increased with increasing concentration and

the time of exposure to profenofos and carbosulfan i.e. mortality rate is

directly proportional to the time of exposure and concentration of the

pesticides. This is consistent with earlier reports (Waykar and Lomte,

2001; Galloway, 2002; Shingadia and sakthivel, 2003; Jarrad et al., 2004;

Boran et al., 2007). Since the LC50 value of carbosulfan for 96 hours is

less (5.564 ppm) than that of profenofos (6.191 ppm), it is further

concluded that, fresh water bivalve, Lamellidens marginalis is more

susceptible to carbamate pesticide (carbosulfan) than organophosphate

pesticide (profenofos). It might be due to greater residual property of

carbosulfan than that of profenofos in the fresh water bivalve. Similar

observations have also been reported by various workers using different

52

toxicants and different test animals. Prasad Rao et al., (1994) reported

increased rate of mortality with increase of concentration and period of

exposure to endosulfan in snail, Lymnaea luteola. Deshmukh (1995)

evaluated toxicity of Parreysia corrugata to heavy metals and reported

toxicity increased with period of exposure and concentration. Masarrat

(1995) found increased rate of mortality in Lamellidens marginalis to

increased concentration of heavy metals. Fernandez et al., (1996)

reported that survival rate decreases with increase in pollutant

concentration. Waykar and Lomte (2001) reported that toxicity of

pesticides increases with increase in time of exposure in fresh water

bivalve, Parreysia cylindrical. Wright, (2001) and Saha et al., (2002)

reported that the differences may also be associated with water quality

parameters and the purity of the chemical.

The susceptibility of animals varies from pollutant to pollutant.

According to Pickering and Henderson (1968) pesticide induced mortality

patterns of aquatic organism are dependent on various factors like age,

sex and animal weight. It is also dependent on the stages of development

and periods of exposure (Macek et al., 1969). The physical factors also

influence the toxicity of the aquatic pollutants (Sprague, 1973). Eisler

(1970) found that in static bioassays, temperature, salinity and pH

influences the toxicity of pesticides. The toxicity of any compound

depends on many factors, such as the chemical and physical form of the

compound, route of administration, dose and duration of exposure, time

elapsed after exposure, dietary level of the interacting elements,

physiological conditions, nutritional status, age and sex of the exposed

individuals (Haratym-Maj, 2002; Khan et al., 2009; Aslam, et al., 2009).

Mortality decreases with decrease in concentration of the pesticides

(Sohahil Ahemad and Muhammad Farhan, 2006).

53

Pratap et al., (1979) suggested that pollutants in the water brings

changes in physico-chemical properties like change in pH of water,

asphyxiation owing to oxygen depletion and prolonged sub lethal effect

of precipitate suspension in water columns. These changes can be the

cause of mortality in the organisms exposed to effluents. A number of

environmental stress causing factors such as temperature, oxygen,

salinity, pH, and pollutants alter the metabolic rate.

In the aquatic animals gills are the important organs of respiration.

Damage to the gills by different pesticides has been reported by number

of workers (Lomte and Waykar, 1998; Tilak et al., 2001; Waykar and

Lomte, 2002; Shukla et al., 2004; Tilak et al., 2005; Nagrajan and

Kumar, 2006; Swarnakumari and Tilak, 2010 and Waykar and Tambe,

2011). Number of workers has reported that pesticide stress affects the

respiratory physiology and decreased the rate of oxygen consumption in

bivalves. A reduction in oxygen consumption is observed when the

bivalves were exposed to the toxicant and the mortality is due to effect of

metabolism of energy synthesis (Fugare et al., 2002; Tilak and Swarna

Kumari, 2009). Waykar and Lomte (2003) reported decreased rate of

oxygen consumption in fresh water bivalve, Parreysia cylindrical after

exposure to pesticides. Sontakke (1992), Jadhav, (1993) and Fugare, et

al., (2002) reported decreased in oxygen consumption in bivalve after

exposure to toxicants. It seems therefore the anoxia may be an important

factor causing death in organisms exposed to pollutants (Skidmore, 1964;

Burton et al., 1972).

Another factor causing death may be subtle effect of pollutant on

the osmoregulatory mechanism of the animal. It is well known that the

gills are involved in ionic regulation (Hughes and morgan, 1973; Evans,

1975) and hence impairment of gill may affect osmoregulation (Sultana

and Lomte, 1998).

54

Organophosphate and carbamate pesticides show low

environmental persistence but display high acute toxicity. In general these

compounds inhibit the acetylcholinesterase (AChE) participating in nerve

impulse transmission (Shengye et al., 2004). Acetylcholinesterase is the

key enzyme of the nervous system. The inhibition causes an

accumulation of acetylcholine in synapses with disruption of the nerve

function, which can result in death (Tucker and Thompson, 1987).

Organophosphates inhibit the function of acetylcholinesterase which is

irreversible. Since the principle site of action of Ops is the nervous

system, it causes variety of toxic effects. These effects have been studied

in many animals including human in laboratories as well as in their

natural habitat, due to either accidental or intentional exposures (Mineau,

1991; Ecobichon and Joy, 1994).

Many investigators have reported the toxicity of pesticides to

different species of animals. Sensitivity of the two crustaceans Dapnia

magna and Gammarus lacustris to the organochlorine and

organophosphate compounds was studied by Gaufin et al., (1965). Butler

(1966) reported that organophosphate compounds were much less toxic to

oysters than chlorinated hydrocarbons. Gupta et al., (1979) reported that

aldrin was highly toxic than ethyl parathion to the fishes. Bhagyalaxmi

(1981) reported that sumithion is more toxic than methyl parathion and

malathion to crab, Oziotelphusa senex. Nair and Nair (1982) studied

toxicity of five organophosphorous pesticides to Alithrophus typus and

found folithion is most toxic as compared to dimecron. Patil, (1999)

reported that the LC50 value of monocrotophos for 96 hours exposure of

bivalve, Corbicula striatella is 22.7007 ppm, for delfin it is11.3857 ppm

and for fenvalorate it is 8.1275 ppm. Lata et al. (2001) recorded LC50

values for carbaryl and carbofuran exposed to catfish Clarias batrachus

at 24, 48, 72 and 96 hours. It was between 16.27 to 2.75 ppm for carbaryl

55

and between 1.47 to 3.84 ppm for carbofuran. Bhatnagar et al., (2003)

studied the toxicity effects of organophosphorous and organochlorine

pesticides on common carp, Cyprinus carpio. Mishra and Bohidar (2005)

reported the percentage of mortality of catfish, Heteropneustes fossilis

after exposure to various concentrations of organophosphate insecticide,

methyl parathion for 24, 48, 72 and 96 hours and noted as 10.40, 9.60,

7.20 and 6.60 mg/L respectively.

Weatherby, (1879) studied the acute toxicity of endosulfan to three

freshwater snails, Melanoides tuberculata (Muller, 1774), Thiara

granifera (Lamarck, 1822) and Planorbella duryi. Mule and Lomte

(1993) observed monocrotophos toxicity to Thiara tuberculata. Bhamre

et al., (1996) studied the acute toxicity of heavy metals to freshwater

bivalve, Parreysia favidens. They concluded that as the period of

exposure increases, the rate of mortality also increases and the pesticide

proves to be more toxic even in lower concentrations when the exposure

is prolonged. Mahajan (2005) observed toxicity of mercury, arsenic and

lead on fresh water bivalve, Bellamya bengalensis. Radwan et al., (2008)

reported that pesticide methomyl has more toxic effect on snail E.

vermiculata than methiocarb. Bhosale (2009) reported that cisplatin is

more toxic than 5-flurouracil exposed to Corbicula striatella. Many other

workers had studied toxicity and behavioral changes in fresh water fishes

(Venkata Rathnamma et al., 2008, Charjan et al., 2008; Krishnamurthy

and David, 2010). Ramesh et al., (2009) observed behavioral responses

of the fresh water fish Cyprinus carpio (Linnaeus) after sublethal

exposure to chlorpyrifos. Logaswamy et al., (2010) reported that the

pesticide malathion has more toxic effects on fish as compared to

quinalphos.

Data on LC50 values on exposure to different pesticides is useful in

the final evaluation of the pollution of aquatic environment by pesticides.

56

Moreover, the ultimate objective of toxicity tests is prediction of

acceptable concentration of a pesticide in the environment. The LC50

values can be used for acute and chronic treatment to evaluate the

morphological and physiological damage to the tissues of the molluscs.

This becomes pertinent in the light of the fact that the species forms

staple diet and has commercial value. To sustain its normal population,

pollution from agricultural activities needs proper control and

management.

The present investigation on the toxicity evaluation of fresh water

bivalve, Lamellidens marginalis clearly indicates that any pollutant

present in the aquatic environment is harmful to animal as it directly or

indirectly affects the individual. The mode of action of these pollutants

and LC50 values were different. Among the pesticides carbosulfan was

found to be more toxic than profenofos. According to the toxicity of these

pesticides to the bivalve they can be arranged as carbosulfan >

profenofos. The based on the present findings, the safe concentrations for

carbosulfan and profenofos are 1.22060 and 1.51337 respectively,

suggested its minimum use and help to a greater extent in controlling the

water pollution.

57

SUMMARY

The chapter entitled “Toxicity Evaluation” deals with sources of

pollution, with further explanation on the nature of pollutants,

whose biological effects have immersed the science of toxicity.

In present study, toxic effect of pesticides carbosulfan and

profenofos on freshwater bivalve, Lamellidens marginalis was

studied for 24, 48, 72 and 96 hours.

Through toxicity tests, LC10, LC50 values, lethal concentration, safe

concentration, Fiducial limit etc have been evaluated; calculation

of % mortality is described. Regression line and regression equation

has been calculated.

An attempt has been made to simplify this intractable process for a

biologist to understand, since it alone provides the basis for the

calculation of LC10, LC50, chi square values for reliability of data,

Fiducial limits, lethal and safe concentration of pollutants etc.

All these results are summarized in tabulated and graphical form.

This is followed by discussion of results in the light of observations

made earlier by several distinguished researchers on the toxicity of

pesticides.

Carbosulfan pesticide was found to be more toxic than profenofos.

In the present study it was observed that the rate of mortality of fresh

water bivalve, Lamellidens marginalis, was increased with

increasing concentration and the time of exposure to pesticide

Carbosulfan and profenofos i.e. mortality rate is directly

proportional to the time of exposure and concentration of the

pesticides.

58

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