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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:
19
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
exposure are 52.42 ppm, 22.36 ppm, 12.27 ppm and 5.564 ppm
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
exposure are 1258.08 ppm, 1073.28 ppm, 883.44 ppm and 534.144 ppm
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’
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII
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
4 ) R e g re s s io n e q u a tio n -
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
Table -4. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Carbosulfan (for 72 hrs).
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’
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 19.6119 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
4 ) R e g re s s io n e q u a tio n -
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
Table -5. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Carbosulfan (for 96 hrs).
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’
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII
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
4 ) R e g re s s io n e q u a tio n -
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’
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII
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'
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII
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
Table – 8. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Profenofos (for 72 hrs).
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
4 ) R e g re s s io n e q u a tio n -
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
Table – 9. Calculation of Regression equation for LC10 and LC50 values of Lamellidens marginalis exposed to Profenofos (for 96 hrs).
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
I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII
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
4 ) R e g re s s io n e q u a tio n -
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.
Fig. 1.1.2 Provisional and regression lines for Lamellidens marginalis to carbosulfan
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
Log of concentration
45
Fig. 1.1.3 Provisional and regression lines for Lamellidens marginalis to carbosulfan
for 72 hrs.
Fig. 1.1.4 Provisional and regression lines for Lamellidens marginalis to carbosulfan
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
Log of concentration
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
Log of concentration
46
Fig. 1.2.1 Provisional and regression lines for Lamellidens marginalis exposed to
profenofos for 24 hrs.
Fig. 1.2.2 Provisional and regression lines for Lamellidens marginalis exposed to
profenofos 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 1.7 1.8
Em
pir
ica
l p
rob
it
Log of concentration
Provisional line
Regression line
47
Fig. 1.2.3 Provisional and regression lines for Lamellidens marginalis exposed to
profenofos for 72 hrs.
Fig. 1.2.4 Provisional and regression lines for Lamellidens marginalis exposed to
profenofos for 96 hrs.
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
Log of concentration
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
Log of concentration
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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