Bhatye estuary is the richest source of aquatic flora and fauna with. characteristic estuarine environment. This estuary is very wide but shallow,

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2 Bhatye estuary is the richest source of aquatic flora and fauna with characteristic estuarine environment. This estuary is very wide but shallow, and a major part of it gets exposed at low tide. Extensive clam beds are found in this estuary. The clam beds are rich at various places and the fishing is done throughout the year; high fishing is usually carried out from November to May because of the availability of large sized clams during this period. During monsoon, large amount of freshwater influx occurs in the estuary, resulting in considerable salinity fluctuations. Clams are delicious, low cost and easily available. The Clam species like Katelysia opima, Meretrix meretrix and Paphia laterisulca are abundantly found in Bhatye estuary, at Ratnagiri throughout the year and hence, clam fishery becomes bread and butter of many fishermen as well as people engaged in collection of bivalve shells. Shells are used for lime production. There are eight lime producing cottage industries along the bank of Bhatye estuary. Bhatye estuary is the richest source of aquatic flora and fauna with characteristic estuarine environment. Clams are susceptible to pollutants in water and can accumulate toxic substances in their body which get biomagnified. As Bhatye estuary happens to be rich in clam fauna, and as they dominate in the food chain, hence can be considered as suitable models for pesticide studies in aquatic environment. Use of various pesticides increases the agricultural production and eradicates the vector born diseases. But extensive and indiscriminate use of pesticides is likely to find their ways through runoff to nearby water bodies, where they produce cumulative and deleterious effects not only on the fish but also on all food web organisms inhabiting there. Thus, destruction of one link of the food chain can cause major changes within an ecosystem.

3 Cypermethrin is one of the commonly used insecticides for controlling the pests of rice, mango, cereal, maize, cotton, etc. It is mainly used against mango hoppers, aphids, thrips, white flies, termites, turf insects, etc. So it has been a subject of biological interest due to its hazardous properties. The toxicity of any substance depends upon its concentration and physical and chemical nature under which it is tested (Eisler, 1969). Reaction and survival of the aquatic animals under toxic conditions depends upon toxicity and concentration of the toxicants and the temperature, salinity, ph, dissolved oxygen of water, etc. Temperature conditions in estuaries depend upon bathymetry of the environment, water exchange between the estuary and sea, and seasonal variation (Borego and Borego, 1982). Temperature is a very important physical parameter because it influences the biota in a water body by affecting activities such as behaviour, reproduction and metabolism. It is necessary to study the temperature variation over the given animal habitat, while studying a water body and animal s eco-physiological and toxicological aspects. In the present study, it was found that during the summer and monsoon, temperature was higher as compared to winter. An influx of fresh water through rivers and the intrusion of sea water through the lower reaches have profound influence on distribution of temperature in backwater systems (Pillai et al. 1975). It was observed that temperature, oxygen saturation, salinity are interrelated parameters. The temperature is directly proportional to salinity and inversely proportional to dissolved oxygen, it affects oxygen concentration in water. Due to increase in temperature, there is a decrease in the solubility of oxygen and intensification of the BOD. Therefore, the effect of

4 a temperature change on chemical toxicity impart may be mediated through the availability of dissolved oxygen of the water (Cairns et al. 1975). It was observed that, lethal value of the clams was low during summer as compared to monsoon and winter. Temperature effects on chemical toxicity are complex, because temperature alone may be lethal and toxicants may alter lethal thermal limits in presently unpredictable ways. Although, it is tempting to generalize that, the temperature rise always increases toxicity, changes detoxification and excretory rates and may also serve to reduce the effects on toxin uptake in aquatic organisms (Holden, 1973). Temperature and dissolved oxygen content are, due to metabolic processes, vital factors in water for the organism. In general, increase in temperature reduces the survival of clams. Threshold concentration of poison was reported to be changed with temperature variations (Lioyd and Herbert, 1962; Brown and Jordan, 1967). Environmental temperature has a great influence on the survival of the animal. Akarte and Mule (1985), while studying the pesticide toxicity to fresh water bivalve, Lamellidens corrianus, also found that, in summer the animals are sensitive to pesticide stress due to high temperature and low oxygen content of water. Akarte (1987), observed similar results while working on Lebaycid toxicity to fresh water bivalve, L. corrianus, L. marginalis, and I. careuleus. The temperature has marked effect on mortality. In general, toxicity of a substance increases with increase in water temperature (Johansson, 1973 and Ciaccio, 1971). Temperature appeared to be the major environmental variable influencing the metabolism of fish and hence it is expected to have a significant impact on the toxicity of pesticides. Temperature changes were associated with behaviour of different species of

5 fish exposed to sub-lethal levels of toxicants thus indicating stress at high temperatures and reduction in feeding rates. It is due to increased permeability of the cells for the entry of toxicants or inhibition of digestive enzymes (Ghosh and Konar, 1973) Salinity is the key trigger of other environmental characteristics. It depends on certain factors as local precipitation, water influences, mixing of fresh water with sea water and evaporation. Hence salinity is a more labile parameter than those of other estuarine parameters. In the present study, seasonal fluctuations were quite marked. Low salinity was observed during monsoon due to heavy rainfall in the Basin of Kajali River, which causes dilution. Similar observations were also made by other workers ( Morton et al., 1985; His et al., 1989; Gouda, 1996). During winter and summer months, salinity was quite high (32.6 and 36.8 mg/l respectively.) Due to cessation of monsoon floods, the neartic water was found to penetrate into the estuary along the bottom during high tide periods, it is known as recovery phase. During summer (April and May, 2009), recovery phase was rapid and high value of salinity (36.8 mg/l) was obtained. A fluctuation of salinity, its interrelationship and salinity tolerance of benthic estuarine animals was studied by many workers. ( Morton et al., 1985; His et al., 1989; Gouda, 1996; Kumbhar, 2001 and Cherry, 2003). Scanty information is available on the effects of salinity on chemical toxicity. Salinity was seen to affect Cypermethrin toxicity to Katelysia opima in the present study. It might be due to interrelation existing between salinity, temperature and oxygen saturation. A variation in ph values was observed during the study period. During the present study, the ph values were fluctuated from 8.0 to 9.8 with an

6 average of 8.9 (Summer), from 7.5 to 8.5 with an average of 8.0 (Monsoon) and from 8.1 to 8.7 with an average of 8.4 (Winter). The water was alkaline with maximum fluctuation in summer (April-May) and the minimum during winter (Dec-Jan). The observations are in agreement with earlier studies by Khabade et al., (2000). In summer, the ph increases due to increase in photosynthetic activity (Gupta, 1986 and Sastry, et. al, 1991) and the declined value of this parameter could be due to influence of fresh water influx during monsoon (Mishra et al., 1989). The variations in ph did not show any significant seasonal pattern, however, the ph values were low during monsoon due to the heavy influx of fresh water, similar observations are noted by Chandran and Rammurthy (1984) in Vellar estuary (Tamilnadu) and Kumbhar (2001) in Bhatye estuary, Ratnagiri. Dissolved oxygen (DO) sometimes referred as the measure of pulse of an aquatic ecosystem. Oxygen in water is regulated by atmospheric dissolution as well as rate of photosynthesis and community respiration. The values of oxygen saturation varied seasonally in the estuarine waters. In general, during monsoon saturation was better than those of summer and winter. During summer, minimum saturation of oxygen was observed. The decreased level of DO during summer in the present study could be attributed to the decreased oxygen holding capacity of water at high temperature (Sachidananda murthy et al., 2006). During monsoon, minimum and maximum values of dissolved oxygen were 4.42 and 5.80 ml/l, respectively. During summer, salinity exceeds and reaches at its peak (38.2mg/L) that minimize saturation of oxygen. During winter, minimum and maximum values of dissolved oxygen were 3.76 and 5.48 mg/l, respectively. Wide fluctuations

7 in oxygen contents were observed during the present study. The observed higher values of dissolved oxygen during monsoon may be due to the influx of fresh water. In addition to the freshwater runoff in the estuary, tidal ingression might also be playing a major role in enhancing the dissolved oxygen content of water. Oxygen concentration is influenced by temperature. Many substances are toxic when dissolved oxygen of water is reduced (Cairns et al., 1975). Water receiving toxic and oxygen depleting waste shows significant increase in the effective toxicity due to reduced oxygen demand. From the available information on the interaction of physico-chemical factors with seasons and chemical toxicity in the estuarine clams, it is observed that, dissolved oxygen has vital interaction with water temperature and chemical toxicity. Based on the present study it is evident that water temperature has an inverse relationship with dissolved oxygen (DO). In summer, at higher temperature rate of oxidation of organic matter in water increases and as oxygen is consumed in the process, the level of latter comes down. Secondly, at higher temperature, the water has a lesser oxygen holding capacity and O 2 is lost to atmosphere (Mishra et al., 2007). The results are in line with earlier studies on estuarine environment by Rao and Valsaraj (1984) and Maruthanyagam and Subramaniyan (1999). Oxygen concentration is influenced by temperature. Temperature affects processes important to the dissolved oxygen level in water such as the solubility of O 2 and rate of oxidation of organic matter. (Ronald et al., 1999) Many workers have studied the pyrethroid toxicity in many fishes and bivalves. Kumar et al. (2009), showed the acute toxicity of Cypermethrin for Channa punctatus with LC 50 value as 0.4mg/l. Saha (2003) reported 72hr

8 LC 50 of aqueous and acetone solublized Cypermethrin to Heterpneustes fossilis as 0.67 and 1.27 µg/l, respectively. Aydin et al. (2005) determined 48hr and 96hr LC 50 values of Cypermethrin for Common carp embryos as and 0.809µg/L. Pesticides are considered to be serious contaminants of aquatic ecosystem due to their inherent toxic nature at low concentration and high rate of bioaccumulation (Baby and Menon, 1986). The toxicity of any substance depends upon its concentration and physical and chemical condition under which it is tested (Eisler, 1970). Reaction and survival of aquatic animals under toxic conditions depends upon toxicity and concentration of toxicants, the temperature, salinity, dissolved oxygen and ph of water etc. It also depends upon the type and time of exposure to the toxicant as well as the physiological status of animal. Salinity is another important factor. During monsoon, salinity is very low, although salinity was not at optimum range; clams survived well. It might be due to shifting of clams from aerobic to anaerobic respiration. ph was not found to be directly affecting toxicity to estuarine clams. From the available information on the interaction of temperature, dissolved oxygen, ph, salinity of estuarine water and chemical toxicity in the aquatic animals, it was observed that, temperature and salinity are most likely to affect the response to acute concentrations. Threshold concentrations usually, are less affected and in some cases they are not affected at all. The degree of temperature influence and its direction depends upon the species and the type of toxicant (Holden, 1973, Mc Kim et al., 1973 and Brungs et al., 1977).

9 In the present study, it was found that toxicity depends upon certain water parameters, season and physiological status of the clam. When K. opima was subjected to different concentrations of Cypermethrin in different season. LC 0 and LC 50 concentrations were found to be 1.11 and 1.58ppm (summer); 1.11 and 1.67ppm (monsoon) and 1.86 and 2.79 ppm (winter), respectively. Observed LC 0 and LC 50 values for Cypermethrin suggests that, the toxicity is largely seasonal. This is due to labile estuarine water parameters like temperature and salinity. During summer, average temperature was 32 C. High temperature reduces dissolved oxygen content and leads to hypoxia, hence during summer, range of tolerance was very low, and LC 50 values were less as compared to values for monsoon and winter. Cairns et al., (1975) claimed that, fresh water fishes at low temperature die at a much slower rate when these are exposed to various toxicants. They studied the sensitivity of the Bluegill sunfish, Leponis macrochirus to toxic chemicals with temperature variations and opined that; slow rate of mortality at low temperature was due to physiological processes like reduced metabolic, respiratory and cardiac rates. Surwase et al. (2009) studied the effect of temperature on behaviour of L. marginalis and found that, the mortality was more in summer than monsoon and winter. During bioassay test, behaviour of clams from control and experimental group was recorded. Behaviour reflects the survival of aquatic animals and integration of many biochemical and physiological processes. Therefore, behaviour is an important area to examine when investigating the effect of toxicants on aquatic animals. Clams can isolate their tissues from the stressful external environment by closing their valves. Such adaptive behavior

10 in bivalves has been shown to be affected by environmental variables like temperature, ph, dissolved oxygen ( Akaberali and Davenport, 1982). The results of the present study are similar to these findings. The exposure of clams to Cypermethrin for a longer period shows physical adaptations against Cypermethrin. During monsoon, valve closure period was comparatively longer. It was observed in the control group as well as acute and chronic groups of K. opima. It was due to low salinity (average salinity 4.6 mg/l) that clams can close their shell valve for a considerable period (Ranade, 1970). During summer and monsoon, high temperature minimizes dissolved oxygen and hence toxicity is mediated through temperature. It has also been shown that, the toxicity of pollutant like heavy metals and pesticides increase with water temperature (Mane and Muley, 1984) to overcome these unfavourable situations, clams start engulfing more and more water through their siphons to acquire more dissolved oxygen. Clams of control, lethal and sub-lethal groups showed minimum valve closure period in summer as compared to monsoon. Clams from sub-lethal and lethal groups showed immediate valve closure after addition of Cypermethrin, but after a few hours they start opening the valves. The valve closure act was similar in all seasons in experimental groups of clams. Clams treated with a lethal concentration close their shell valves for prolonged duration in all seasons. Muley et al. (1987) while studying the acute toxicity of Metasystox on the intertidal bivalve mollusks, Donax cuneatus found that most of clams opened the shell valves and extended the foot at low concentrations (0.004 to ppm) but this was slow and late at high concentrations ( to 0.008ppm). It was also observed that, there was no protrusion of mantle edges in concentrations from

11 0.004 to ppm. Simon (1983) reported that, clam closed the shell valve when stimulated by an obnoxious toxic compound or by any change in the environment. Clams of lethal and sub-lethal group showed excreta up to initial 12 hours, while, control group of clams showed excreta up to 48 to 60 hours, depending upon season. Excreta were less in experimental groups than control groups, again, it was less during monsoon. This may be due to less feeding and discontinuous valve closure. Mucilage secretion was observed in lethal group of clams after 12 hours of acute exposure during the three seasons, as compared to control and sub lethal groups. It was probably due to mucus secretion from mucus glands of mantle and gill to keep the body free from contaminated water. Mucus which is secreted as an induced action on exposure to Cypermethrin probably forms protective layer on gill filaments and mantle, resulted in the reduction of gaseous exchange between the blood and water. All the changes result in depletion of oxygen and carbon dioxide accumulation leading to suffocation and death of clams. Lioyd (1965) suggested that, it is the cellular damage of the gills that cause respiratory distress and not the mucus coagulation. In this context, it is reasonable to assume that, gill damage along with mucus deposition in the gills resulted due to the exposure to Cypermethrin which lead to decreased efficiency for gas exchange and breakdown of vital functions of clam and finally leads to death. Heavy exudation of mucus over the body and, body dyspigmentation are attributed to dysfunction of the pituitary gland under toxic stress, causing the changes in the number and areas of mucus gland and chromatophores (Pandey et al.,1990). The above cited behavioural abnormalities of the fish, and death

12 implies that, toxic effect is mediated through the disturbed nervous and cellular enzyme system affecting the respiratory function and nervous system, which involves the controlling of almost all the vital activities. In the present study, the failure in the rhythmic shell valve closing, mucus secretion, failure to respond to the external stimulant and permanent wide opening of the shell valve in the clams exposed to different concentrations of Cypermethrin gives an insight into dysfunction. The behavioural responses of the mollusc varied in accordance with the test concentration of pesticides. Relatively reduced activity was exhibited during the early hours of exposure at all concentrations. The siphons were extended and food searching movements contributed but eventually the clams appeared to have been paralyzed as they could not retract their siphons even when mechanically stimulated. The pumping activity of the clams was also affected by the pesticide. When compared to controls, the surviving molluscs displayed excessive mucus secretion, sluggishness, gapping of shell valves and permanently extended siphons. Respiration is a measure of animal s overall energy demand under particular conditions, at which the measurements are made. The process of respiration is sensitive to a whole array of environmental as well as biological variables. Oxygen consumption is a useful measure to assess the sub-lethal effects of xenobiotics, as energy processes serve as an indicator of overall physiological state (Sigmon, 1979). The fluctuation in the estuarine environment where clams are found, are therefore bound to influence the oxygen consumption in clams. It is well known fact that the rate of oxygen consumption is used as an authentic tool for understanding the physiological

13 state of metabolic activities of an organism (Kulkarni et al.,1983). Measurement of the rate of oxygen consumption is an important parameter to access the toxicant stress on aquatic organism since it is also an index of energy expenditure to fulfill the demands due to environmental and biological alterations. Many investigators have shown that, filtration rates and oxygen consumption in bivalves are the best indication of their response to various pollutants (Capuzzo and Sasner, 1977; Mathew and Menon, 1992 and Abel, 1998). In the present study, it was found that, control group of clams showed fluctuations in the rate of oxygen consumption in all the three seasons. Decrease in oxygen consumption after pesticide stress was observed in Corbicula regularis (Lomte and Jadhav, 1982). Mali and Ambhore (2003), studied the impact of copper sulphate on the oxygen consumption in freshwater female crab, Barytephsa guerini and reported a significant decrease in the rate of oxygen consumption. In case of aquatic animals, there is no escape from toxicants and maximum part that is continuously exposed to pesticide is the respiratory surface i. e. Gill surface. High activity of gills and continuous exposure to a toxicant causes severe damage to gill surfaces and reduces the oxygen uptake capacity of respiratory organs (Roberts, 1979). The salinity and temperature data (Table 1) shows that, during monsoon when river water enters in the estuary, salinity decreases due to freshwater influx. It has been observed that, the rate of water filtration and metabolic activity decreases during monsoon because for the majority of the period valves of clams remain closed in low salinity. In monsoon and winter,

14 oxygen consumption was less than in summer. It may be due to presence of optimum range of water parameters and physiological status of clams at that time. During summer, clams exhibited a higher degree of oxygen consumption. It may be due to depleted oxygen content in water and Cypermethrin induced stress. Isono Ryosuke et al., (1998) reported that, in Japanese little neck clam, Ruditapes philippinarium, the oxygen consumption rate decreased markedly above 40 C. Clams suffer from thermal stress above 25 C and have significant mortality at around 34 o C within a few days and no heat resistance over 40 o C. Haeure et al., (1998) observed enhanced oxygen consumption rate in flat oyster, Ostrea edulis during summer. Young and Shim-Ieong-Her (1998), observed increased rate of oxygen consumption during summer in the Antarctic clam, Laternula elliptica. Similar observations were made by Ranade (1973), in case of the clams from Ratnagiri water. Clams close their shell valves for a considerable period to combat with unfavourable estuarine parameters especially salinity. Similar observations were noted by Ranade (1973), in Meretrix meretrix and Katelysia opima from Ratnagiri water. Thus, variance in physico-chemical factors affects the rate of oxygen consumption in estuarine animals (Sigmon 1979; Chandran and Ramamoorthy, 1984; Gouda and Panigrahy, 1993). Mane and Dhamne (1980) reported adaptations of clam P. laterisulca in Kalbadevi river (Ratnagiri) under such conditions. It might be possible that clams may be switching from aerobic to anaerobic respiration. As compared to summer and winter, in monsoon, the control group of clams showed decreased oxygen uptake. As a control group of clams, LC 50 group showed decreased oxygen uptake in three different seasons. The

15 observed decrease is attributed to variation in the volume of water ventilated through the gills, caused by the intermittent closure and opening of the shell valves. Here main factor responsible for decreased oxygen uptake was coagulation of mucus on gills due to Cypermethrin exposure. Coagulation of mucus causes reduction in effective transfer of oxygen to internal tissues, adversely affects the absorption of oxygen from the ambient medium. Brown and Newell (1972), have suggested that, reduced oxygen uptake may be due to reduced energy requirement caused by suppression of ciliary activity. Salakni (1965, 1968), attributed this to valve closure and termination of filtering. The reduced ciliary activity in turn results in mucus coagulation on gills. The reduced filtration rate in Mussel under stress was reported (Equifanio and Srna, 1975; Murthy, 1982; Mathew and Menon, 1987; Sujata et al. 1995). They found that endosulfan induced estuarine clams Villorita cyprinoides recorded a greater reduction in the percentage deviation of oxygen consumption, possibly due to hypoxia induced by the pollutants. The stiff suppression in the rate of oxygen consumption was probably due to the reduced efficiency of gills. In the present study, considerable mucus secretion was found in lethal concentration during three different seasons. These fluctuations in the oxygen uptake rate in estuarine clams exposed to different concentration of Cypermethrin at different seasons are due to variations in gill ventilation rate coupled with the concentration of pollutant in water and the efficiency of assimilation of oxygen via gills and also the length of time during which the shell valve is closed (Murthy, 1986; Mohan et al., 1986; Rajelaxmi and Mohandas, 1998). Tendulkar and Kulkarni (1998) found that Gafrarium diverticulum showed suppression of oxygen consumption and rate of filtration

16 after 96 hours Benzene and gear oil WSF exposure. Muley and Mane (1987) reported reduction in oxygen consumption in LC 50 group of fresh water Lamellibranch marginalis from Godavari river. Baby and Menon (1986), Kapoor and Lomte (1987), Prakasam et al.(1989), Muley(1991), Savant and Amte (1992) and Masarrat Sultana and Lomte (1998) reported similar observations in their respective animals at different sub-lethal concentrations of heavy metal and or pesticides. In the present study, during summer K. opima of control and sub-lethal group showed comparatively elevated oxygen uptake than other season. It was due to depleted dissolved oxygen content in estuarine water ( ml/L). Correlation between other parameters and oxygen content in estuarine water is discussed earlier. To cope with this hypoxia, clams tried to filter more water. Higher oxygen consumption rate exhibited by the clams of LC 0 group points out to a higher energy demand. As compared to the control group, clams treated with LC 0 concentration showed a slight elevation in oxygen consumption after initial 12 hours exposure. This was found in all the three seasons. These findings relates with the findings of many workers. Mathew and Menon(1983), reported that oxygen consumption increased in P. viridis exposed to low concentrations of Ag, while concentrations above 0.01ppm, oxygen consumption sharply decreased. Muley and Mane (1987), observed that pesticide induced increased oxygen uptake in L. marginalis. In the present study it was found that, control group of clams showed negligible fluctuations. Lethal (LC 50 ) group of clams showed significant decrease while in sub-lethal concentration (LC 0 ) group showed a significant increase in oxygen uptake in monsoon and in summer. significant increase in oxygen uptake was

17 observed up to 48 and 60 hours in summer and winter, respectively. These results were similar to Rathod and Shembekar, (2009). They observed that the rate of oxygen consumption was found to be increased initially upto 48 hours and then decreased up to end of the experiment. The decrement may be due to the respiratory distress as a consequence of the impairment of oxidative metabolism (Prashanth et al., 2003). The studies on biochemical changes enable to define the dose response relationship, threshold limit value and reversible, irreversible pollutant effect. In addition the biochemical indices of toxicity derived after a relatively short exposure time may be useful in predicting the appropriate threshold concentration of chronic effects (Christensen et al., 1977). Aquatic invertebrates have the capacity to concentrate the pesticides. There must be metabolic strategies to utilize or sequester these pesticides, depending upon their toxic potential. It is quite evident that the frequency of changes in the composition of the biochemical constituents of animal varied not only with environmental changes, but also with the seasons (Masurekar and Pai, 1977; Somvanshi 1983). Various biochemical changes under stress conditions have a great significance, since they catalyze and control the formation of various intermediates which are indispensable to all the normal physiological processes. The stress induced chemical changes are described as secondary responses of fish. In clams, glycogen, proteins and lipids act as source of energy but amongst them, glycogen is the most prime source of energy for carrying out various activities (Nikalaje et al., 2011). In the present study seasonal changes in glycogen content in different organs of Katelysia opima were recorded. It varies according to tissue,

18 season and physiological status of clam. In control group of K. opima, glycogen content was present in ascending order of gill < hepatopancreas < male gonad < foot < mantle < female gonad. Female gonad acts as depository of glycogen. Seasonal changes were prominent in control group of clams. Female gonad exhibited its peak during all the three seasons. In control group of K. opima. Gills and female gonad showed maximum glycogen during summer and minimum level during winter. Mantle exhibited maximum level during monsoon and minimum level during summer while foot and male gonad showed maximum level during winter and minimum during summer, and hepatopancreas showed maximum level during summer whereas minimum level during monsoon. The glycogen content in gill was at its peak during summer. It might be due to high energy requirement during summer. To combat with hypoxic condition, gill acquired high amount of glycogen content. In K. opima, male gonad and female gonad exhibited its peak during winter and summer and low during summer and winter, respectively. In general it shows variance with the breeding behaviour and development of the gonad. During the active gametogenesis, glycogen content was at a high level and it was low during the spawning period. Similar observations are cited by Nagbhushanam and Talikhedkar (1977) in wedge clam Donax cuneatus. Results obtained in the present study are in a good agreement with earlier workers Masumoto and Hibino (1973), Dhavale and Masurekar (1985); and George and Mathew (1996) When clams were exposed to LC 0 concentration of acute test, significant increase in glycogen content was observed in hepatopancreas in all seasons while overall considerable decrease was found in rest of the body

19 organs, mantle being the exception. Mantle showed significant increase in glycogen content in winter. Clams of LC 50 group showed increase in glycogen content only in hepatopancreas while there was considerable decrease in all other organs in all three seasons. Clams treated with chronic concentration, showed significant increase in glycogen content in gill and the other organs showed decrease in glycogen content in all three seasons. In hepatopancreas the glycogen content was seen to increase during summer and monsoon, with a decrease in winter. As compared to control group of clams, gill showed, 70.92, and 51.49% increase in glycogen content in summer, monsoon and winter, respectively. Hepatopancreas showed 36.99, 59.55% increase in glycogen content in summer and monsoon. 3.47% significant decrease in glycogen content was observed in hepatopancreas in winter. In general, there was significant increase in glycogen content in hepatopancreas in clams of LC 0 and chronic group. There was considerable decrease in glycogen content of foot, male gonad and female gonad during acute and chronic exposure. Mantle exhibited decrease in glycogen content in summer and monsoon. The observations point out that probably in most of the tissues there was switch over to anaerobic metabolism in response to Cypermethrin toxicity. It is important to mention here that the previous studies on rate of respiration in clams also showed consistency in decrease in respiratory rate which was more pronounced in LC 50 group as compared to LC 0 group on exposure to Cypermethrin. Decline in respiratory rates may be due to depletion in glycogen content. The results of the present study showed decline in glycogen content in different organs during three different seasons. These results can be correlated with the findings of Ansel, (1972); Ansari, et al.,

20 (1981); Dhavale and Masurekar, (1985); Radhakrishnan and Bassappa, (1986); Jana and Bandopadhya, (1987). A decline in glycogen in various tissues may be due to stress resulting in breakdown of tissue glycogen. Nasreen et al. (1994), also observed marked depletion in the glycogen content in all the tissues due to phenol exposure in Channa punctatus. Similar results were observed by Shakoori et al., (1996) Das et al.,(1999); Shobha et al., (2007) who reported that decrease in glycogen in muscle, gills, liver, Heart and kidney of Catla catla, when exposed to cadmium chloride and stated that glycogen reserves are being used to meet the stress through glycolysis via hexose monophosphate pathway. Toxic effects of various pesticides on carbohydrate metabolism have been investigated by many workers (Shrivastava and Shrivasata 1995, Tilak et al., 2003). The decrease in glycogen content was reported by several workers in different animals by the pesticide toxicity. ( Nagpure and Zambare, 2003; Kumar and Saradamani, 2004; Tilak et al., 2005 and Laxmi Prasad, 2009). Overall decrement of glycogen level in different body organs might be due to the prevalence of hypoxic conditions. At the tissue level, in acute exposure glycogen content decreased considerably in all the tissues except hepatopancreas and in chronic exposure, gill and hepatopancreas showed increase in glycogen content in all the three seasons, indicating greater utilization of these product for metabolic purposes. Glycogen reserves in various tissues of clams are readily utilized on exposure to Cypermethrin, which signify their rapid utilization during imposed physiological stress, perhaps through glycolytic pathway so that animal can meet their immediate energy requirement. In bivalves, the

21 glycogen provide the sugar for nucleic acid synthesis (Byne, 1976 ) and the conversion of the glycogen into fatty acid, triglycerides reserves via trios phosphate entry in the glycolytic sequence and to the production of pentose sugar for the lipogenesis are well documented (Gobott, 1976). Hepatopancreas and gills are more modified in their biochemical composition than muscles. Gita and Yeragi (1998), reported the decrease in glycogen content of muscle in the pesticide treated Scylla serrata. Gills and hepatopancreas accumulate higher levels of Cypermethrin than other organs. The significant increase in glycogen level in gill and hepatopancreas indicate that, they accumulate Cypermethrin and combat with Cypermethrin level. To meet the energy requirement of gill and hepatopancreas, there was increased mantle, foot and gonad glycogenolysis. Decrease in glycogen content has been observed in muscles and liver of fish following Cypermethrin treatment (Sheela and Muniandi, 1992). Similar findings were reported by Reddy and Bhagyalakshmi (1994) in crab, Scylla serrata, Kaviraj and Das (1994) in fish and other aquatic organisms and Tilak et al. (2009) in fresh water edible fish. Protein plays vital role in spawning and other metabolic activities. It is the main organic nutrient used to build up different body tissues. In the present study, it was observed that protein content changes according to season, physiological status of the clam and artificial environmental stress. Control group of clam exhibited high protein content in mantle, foot, male and female gonad during summer and foot and mantle exhibited low protein content in monsoon. Hepatopancreas, male and female gonad showed low protein content in winter. It may be due to spawning period. Gill is an exceptional case in which, the protein content was high in winter and low in

22 monsoon. Nagbhushnam and Talikhedkar (1977) found that in D. cuneatus, protein content remain relatively high throughout the year except a decline during the breeding period and at the time when the clams were in fully matured condition. Durve and Bal (1961), while studying biochemical composition of C. gryphoides observed high protein values throughout the year. Further, they stated that period of low value coincided with the spawning season. Protein content in the mantle, hepatopancreas and adductor muscles of bivalve L. marginalis was decreased due to mercury toxicity (Patil, 1998). Also, Mule and Lomate (1994) observed decrease in protein concentration in mantle, foot and whole body due to mercury toxicity in gastropod, Thiara tuberculata. Effect of tannery effluent on the biochemical constituents like total free amino acids, total protein, glycogen and lipids in different tissues like liver and muscles of Cyprinus carpio were studied by Jitender Kumar et al. (2004). The work suggested that protein and other constituents were decreased on the dose of the effluent and duration of exposure period. In gastropods, protein content was decreased due to the herbicide toxicity (Chaudhary et al. 1998). During this type of stress condition the protein synthesis and interconversion of amino acids, glucose and fatty acids to liberate energy get affected (Mane et al.,1986). Molluscicide copper sulphate induced toxicity in a freshwater pulmonate, L. luteola was studied by Mathur and Menon (1994) in mantle, foot and hepatopancreas. Ramana Rao and Ramamurthy (1981) studied the effect of heavy metals on terrestrial snail, Zootecus insularis. In the present study, it was observed that, increase or decrease in protein content depends upon spawning season, environmental stress,

23 concentration of toxicants and particular tissue. The clam exhibited significant increase in protein content of gill in LC 0 and LC 50 group of clams in all season while foot, male gonad and female gonad exhibited considerable decrease in protein content. Whereas, in LC 0, mantle showed high protein content in winter and hepatopancreas exhibited high protein content in monsoon. In LC 50 group, mantle exhibited high protein content in monsoon and hepatopancreas exhibited high protein content in monsoon and winter. In chronic group of clams, significant decrease in protein content was observed in all the tissues except gill and hepatopancreas. This is because according to Umminger (1970), protein is the source of energy during chronic conditions of stress. Number of studies has reported a decline in different organs of fishes treated with pesticides (Ram and Sathyanasan, 1984; Ganguly et al., 1997; Ramani, 2001). A reduction in protein content after the exposure of Cypermethrin may be due to reduced protein synthesis. The reduced protein content may also suggest increased proteolysis and it is also possible that utilization of degraded products for metabolic processes might have increased the pesticidal stress. The protein level decreases in all the organs except gill in LC 0 and LC 50 group. Decrese in protein level may be due to increased proteolytic activity or might be due to anaerobic conditions produced by Cypermethrin. Decrease in fish protein content was due to pesticidal stress or utilization of proteins in glyconeogenesis for energy production (Chaudhari and Kulkarni (1998). In the present study, there was slight increase in protein content of gill in LC 0 and LC 50 group during all three seasons. Muley et al.(1996) reported

24 increased level of protein content in gill, muscles and kidney of Tilapia mosambica exposed to 0.016ppb of endosulfan for 168 hours. Such increase in protein level may be assumed to be due to the heavy metal induced new proteins which may have some harmful effects on animal system. An increase in protein content of various tissues which could reflect stimulated protein synthesis of detoxification enzymes. Toxic level of cypermethrin in the present study showed an alteration in tissue protein suggesting disturbance in physiological activity. The importance of lipids in energy metabolism of bivalves has been reported for some species such as Tapes sp. (Beninger, 1984) and Patinopecten vessoensis (Takashi and Mori, 1971). In adult bivalves, lipids are mainly stored in gonads. It constitutes the component of reproductive material (Gabbot, 1976; Davis and Watson, 1983). Nagbhushnam et al. (1973) observed that in Wedge clam, D. cuneatus, female oysters contain lipid twice as much as males during gametogenesis. It is interesting to note that Suryanarayan and Nair (1976) studied the seasonal variations in biochemical constituents of C. radiata and found that there is no very evident correlation between lipid content and breeding season. During the study, it was observed that in control group, high lipid content was observed in mantle, gill, hepatopancreas, male gonad and female gonad in monsoon. In summer, low lipid content was observed in gill, hepatopancreas, male gonad and female gonad. The mantle showed high lipid content in monsoon and low lipid content in winter. The foot exhibited low lipid content in monsoon and high lipid content in summer. As compared to control, LC 0 group and LC 50 group showed significant decrease in lipid

25 content in foot, male gonad and female gonad. In gill and hepatopancreas, the increase in lipid content was observed in LC 0 whereas LC 50 group showed decrease in lipid content in all the three seasons. The mantle showed significant decrease in lipid content in summer and monsoon and increase in lipid content in LC 0 and decrease in lipid content in LC 50 group in winter. The chronic group exhibited decrease in lipid content in all tissue in all three seasons mantle being the exception in winter. A physiological change that takes place when organisms are exposed to lethal and sub-lethal levels of cypermethrin stress include rate of feeding as well as respiration and excretion. The net result could be a change in energy available for growth and reproduction. When the clams were subjected to LC 0 concentration, hepatopancreas and gill showed marked increase in lipids during all seasons. And all other organs exhibited overall decrease in lipid content in all seasons except mantle in winter. LC 50 group of clam showed overall decrease in lipid content in all target organs in different seasons. Clams treated with chronic concentration showed overall decrease of lipid content in all organs in three different seasons except mantle in winter. The decrease in lipid levels in above mentioned body parts in LC 0 and LC 50 groups perhaps due to an increase in lipolysis to meet higher energy demand to overcome the toxicant stress. The observed increase in lipids in some body parts is probably related to the anoxic endogenous oxidation process providing required energy for survival under Cypermethrin stress with simultaneous generation of acetyl Co-A. While studying the effects of pesticides on fresh water bivalves Swami et al.(1983), suggested that, the flux of carbohydrates through the Kreb s cycle can be controlled by the relative

26 channeling of glucose-6-phosphate through pentose phosphate pathway and generated acetyl Co-A in the pathway of lipid biosynthesis. The two metabolic pathways are complementary to each other as one provides NADPH required for lipogenesis over the other. All the effects that are observed due to the Cypermethrin exposure on the different tissues are extremely important for experimental group of clams from the energetic point of view. Any particular alteration of cell may indicate the presence of disease or the toxic substance. Brown (1968) suggested that there is clear correlation between pathological condition of cell or tissues and its affected functions. The extent of damage induced by the toxicant to a particular organ can also be judged at a cellular level. Pathological and biochemical disturbances in aquatic organisms like mollusc due to pesticide toxicity are well documented (Waykar and Lomte, 2002; 2004). Histopathological changes are mostly confined to organs directly involved in their metabolism and detoxification (Rashatwar and ilyas, 1994). In aquatic vertebrates and invertebrates gills are the organs directly exposed and the most susceptible to environmental variations hence they are target tissues for different pollutants. Gills have been identified as the primary target organ of Cypermethrin accumulation in K. Opima. These are the primary route for the entry of pesticide (Mallatt, 1985; Richmonds and Dutta, 1989). Nimmo et al. (1977) was first to report the pathological blackening of gill filaments in shrimps exposed to cadmium. Ghare and Muthekar (1979) reported blackening of gill filaments of Macrobrachium kistnensis and Cardina species on exposure to copper sulphate. In the present study, the light microscopic finding of the gill includes pathological blackening of the gill

27 filament, shrinkage of gill lamellae, lifting of epithelial lining and degeneration of tissue. The lifting of epithelial lining and its degeneration may be attributed to the active Cypermethrin accumulation. Lethal concentrations of Cypermethrin have devastating effects on gills which result in the death of the estuarine clams. It was found that, in LC 50 and in chronic group, gill is severely damaged than LC 0 group in all the three seasons. But the damage was more sever in summer. The epithelial lining may provide protection against the pollutant uptake by increasing the diffusion distance. But this lifting of epithelium, as also observed during this study in clams, formed a nontissue gap, which would result in inadequate gas exchange. Shrinkage of the respiratory area and enlargement of the blood/water barrier of the gill in the sub-lethal concentration of nickel has been reported by Randol and Shelton, (1968). Shrinkage of gill lamellae resulted in the restriction of the flow of water through the gill sieve for respiration. Riji John and Jayabalan (2007) have observed hyperplasia, lamellar fusion, curling and bulging of tips of primary gill lamellae, exudation of erythrocytes, when the fish Cyprinus carpio was exposed to sub-lethal concentration of endosulfan. The mantle or pallium is an important molluscan organ. Each pallial lobe is well marked in three regions: proximal, middle or distal and marginal region. In the present study, it was found that, in LC 50 and in chronic group, mantle is severely damaged than LC 0 group in all the three seasons. Epithelial cells, connective tissue, longitudinal and radial muscles are damaged significantly. Again, damage to mantle cellular architecture was severe in monsoon. In mantle tissue, Cypermethrin concentration may increase directly from endocytosis or from transportation of pesticide by amoebocytes. Due to

28 its high capacity of pesticide accumulation, damage was severe. The mantle is primary organ in shell production (Galtsoff, 1964). Hepatopancreas is very important target organ. The above results revealed that the structural changes occurred due to cypermethrin in all the three seasons. The disconnection of digestive and secretary cells with basement membrane, infiltration of amoebocytes in to tubules, accumulation of haemocytes, vacuolization in the cytoplasm of digestive cells and karyolysis or necrosis were the common features of cypermethrin (acute and chronic ) exposure. High vacuolation of digestive cell is known to be one of the manifestations of stress response which is indicative of increased lysosomal number. The cells of digestible tubule show atrophy and thinning of epithelium. There is tendency to generalize such changes to stressor effects mainly by genobiotic or prolonged starvation (Pipe et al. 1985). Structural assay of cellular damage have shown that, there was enlargement of cells of digestive tubules resulting in bulbous epithelial structure or total atrophy resulting in thinning. The basement membrane of each tubule was ruptured at some places. Kumbhar (2001) observed similar type of histological alterations in the hepatopancreas of K. opima after acute and chronic exposure to Cadmium. Enlargement in cells results in overall increase in volume, which is due to the formation of enlarged or giant lysosomes. This subsequently leads to atrophy of digestive cells. The functional aspects being confined to autophagic activity. Severe degeneration of epithelial cells of digestive diverticulum has been observed (Auffrett, 1988). The presence of completely damaged cells of digestive tubule is an indication of atrophy which would eventually lead to sloughing off of cells. In the present study, the tissue

29 damage was observed in LC 50 and chronic group than control and LC 0 group. The severe damage was observed in monsoon. The study of histology of male gonad showed that, in summer, the follicles were more affected in LC 0, LC 50 and chronic groups. On the other hand, in monsoon and winter, LC 0 group of K. opima showed enhanced spermatogenesis to give rise to sperms, which were also released, but in LC 50 groups, spermatogenesis was diminished and only a little quantity of sperms were produced. Cypermethrin caused severe damage to the male gonads, particularly in summer. The overall effect of Cypermethrin was more at chronic group than LC 0 and LC 50 group. This was probably because of rapid penetration of Cypermethrin deep into gonad tissue. Muley (1985), while studying the effect of pesticides on the gastropod, V. bengalensis, observed growth retardation and damage to germ and sex cells. Prabhupatkar (2004) observed similar type of histological alterations in Female gonad of Meretrix meretrix after acute exposure to Cypermethrin. These observations are in good agreement with present study. Comparing the effects of Cypermethrin on female gonads of clams, it was observed that, effects were more prominent in summer, followed by monsoon and winter. Follicular shrinkage and distortion was observed in LC 0, LC 50 and chronic groups. In LC 50 and chronic group, vacuoles in cytoplasm and degeneration of oocytes were observed. Muley (1985) observed similar changes in both LC 0 and LC 50 groups of V. bengalensis after acute exposure to folithion and lebaycid. Prabhupatkar (2004) observed similar type of histological alterations in female gonad of Meretrix meretrix after acute exposure to Cypermethrin.