Chapter 1 Condition Index, Behavioural Study and Oxygen Consumption

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

1.1 General Introduction Class Bivalvia of phylum Mollusca includes soft-bodied forms which are enclosed within a bivalve shell. Molluscs form the second largest group of animals after insects in the whole animal kingdom. They are highly adaptive and occupy all possible habitats except aerial. Most family members of this class are mussels, clams, oysters etc. Lamellidens are the most common freshwater mussels found in India, inhabiting the surfaces layers of the muddy beds of the lakes, rivers and streams. They form one of the largest groups in terms of biomass amongst the filter feeding organisms in many freshwater ecosystems (Mickael, 1995). They are important calcium and carbon accumulators; they link primary producers (bacteria and phytoplankton) with higher organisms in aquatic food-chains. The freshwater mussel plays a vital role in freshwater environment. They are often described as sessile, primary consumers which can be used to assess different routes of contaminant exposure (Farris, 2007). The freshwater mussels, Unionid mussels in particular, are more sensitive to chemical exposure and a variety of other environmental stressors, as compared to other organismal groups (Surwase, 2009). They need to be considered as a part of whole freshwater community. Mussels are filter-feeding sedentary species, especially prone to the accumulation and concentration of contaminants. As filter feeders, their vulnerability to different contaminants, chemicals and other pollutants in aquatic habitats seem to be high. Freshwater bivalves are highly sensitive to toxic contaminants. Exposure assessment is essential to understanding the potential effects of contaminants to non-target animal populations. Recent surveys have indicated declining diversity and density of mussels because of different contaminants in aquatic ecosystems. Mussels readily bio accumulate both organic (Moore M, 1985) and metal pollutants (Vairengo et.al., 1985) thereby rendering them more accurate in situ bio-indicators of pollution than fish (Cajaraville et. al., 2000).

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Human beings also consume mussels and therefore, any pollutant which can accumulate in mussel tissues has the potential to enter higher tropic level organisms via the food chain. Mussels are sessile, which make them useful as bio-indicators since they stay in one place and their health status yields information relevant to a particular location (Dondero et.al., 2006). Mussels’ lifestyle is stationary, filter feeding, inhabiting the benthic environment where pollutants usually end up and accumulate. Therefore, a mussel provides many advantages as a model to assess toxic effect of environmental pollutants in organisms (Al Amri et. al., 2012). The impact of aquatic pollutants is thought to be significantly different in various organs/tissues along with their own biological function variability. For example, gill tissue and digestive gland of mussels play important roles in food collection, absorption and digestion. Chronic exposure of mussels to pollutants in water and sediments may ultimately impair their nutrient absorption ability, compromise their growth and reproduction (Smital et. al., 2004). Owing to high filtering capacity of mussels, gill tissues are not only continuously in contact with pollutants in waters, but they may also concentrate pollutants contained therein due to the remarkably high volume of water that they filter (Au, 2004). Pollution is an alarming problem that the mankind is facing today. We unknowingly do a lot of activities that terribly ruin the nature. Water contamination is one of the severe factors that affect the aquatic ecosystem. Aquatic environment is continuously being contaminated with metals and toxic chemicals from industrial, agricultural and domestic activities. Heavy metal contamination interferes with ecological balances of an ecosystem and produces devastating effects on environment quality (Farombi et. al., 2007). The freshwater mussels play a vital role in food chain and today, sadly, are among the most threatened aquatic species in the world. One of the major issues implicated in this decline is water pollution. Fresh water mussel populations have suffered a lot because of habitat disturbances & commercial demands. They need to be considered as a part of whole freshwater community, particularly because they link up various aquatic food chains involving the edible fish, which form an important resource of protein for the human population. Since mussels exist in direct contact with contaminated aquatic

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption sediments and are exposed to water-borne contaminants, they represent interesting specimens in eco-toxicological studies. Therefore, mussels have been used extensively as sensitive bio-indicators for aquatic pollutants (Livingstone et.al., 1992) they represent interesting specimens in eco-toxicological studies since they exist in direct contact with contaminated aquatic sediments and they are exposed to water-borne contaminants.

1.2 Aim and objectives of the present study There are several advantages of mussels, as they occur in most of the water bodies and they have stationary, filter feeding life style, inhabiting benthic environment, where different contaminants accumulate. Mussels readily bio accumulate both organic (Moore, 1985) and metal (Viarengo, 1985) pollutants. The present work is proposed to study biological effects of contaminants like cupric chloride dihydrate, PAH (Anthracene) individual and combined exposure of both together for seven (T1) and fourteen days (T2) on mussels.

The objectives of the present study: 1. Estimation of level of metals in water & in tissues of bivalves, collected from different reservoirs of water 2. Assessment of health of animals at the time of collection from their natural reservoirs 3. Analyses of acute toxicity of contaminants such as - Cupric chloride di-hydrate (A), Anthracene (B) and combined effect of mixture (C=A+B) on Lamellidens corrianus in laboratory 4. Periodic observations of behavioural changes on exposure to A, B and C 5. Determination of rate of oxygen consumption 6. Determination of neurotoxicity by analysis of AChE activity 7. Determination of alterations in biomarkers of oxidative stress, such as MDA, SOD, CAT, GST and GR 8. Assessment of histopathological changes in gills after exposure to different contaminants 9. Estimation of Metallothionein content upon exposure to cupric chloride di-hydrate

(Cucl2.2H20) in bivalves

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

10. Quantification of metal bioaccumulation in soft tissues of bivalve 11. Assessment of genotoxicity in the gill cells by using comet assay 12. Detection of chromosomal damage with the help of micronuclei formation, MN test 13. Assessment of recovery potential of animals following the A, B and C exposures

1.3 Selection of experimental animal The present study deals with the investigation and assessment of toxicity of cupric chloride dihydrate and anthracene individual and their combined effects of both together on non-target fresh water bivalve Lamellidens corrianus in laboratory, this species selected for the present study because it fulfils most of the criteria for a standard test species (Fishes, mollusks, and crustaceans) as described by Adelman and Smith (1976). i. Test species should be capable of being maintained in the laboratory, with healthy conditions for at least one month, ii. It must be available throughout the year with proper and required size, iii. It should be easy in handling, collection and transportation, iv. It should be available in sufficient numbers for repeating the toxicity tests, v. It must have a constant response to toxicants tested under similar controlled conditions of exposure. It has been observed that bivalves are the most suitable and are easily harvestable from the water bodies, they are widely used as ecological indicators because they are sensitive to ecological stress and being filter feeders they accumulate contaminants from water bodies which makes them highly suitable as test organism. Since L. corrianus fulfils above criteria and in environmental conditions it gets constantly exposed to toxic contaminants directly or indirectly through food chain, hence it is considered as an ideal test species in toxicological studies.

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

The taxonomic classification of L. corrianus is as under,

1.3.1 Systematic Position

Phylum: Mollusca

Class: Bivalvia

Subclass: Paleoheterodonta

Order: Trigoinoida

Superfamily: Unionoidea

Family: Unionidae

Subfamily: Ambleminae

Tribe: Amblemini

Genus: Lamellidens

Species: corrianus

Figure 1.1: Lamellidens corrianus

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

1.3.2 Collection and Maintenance in the laboratory The freshwater mussels L. corrianus, were collected from the reservoirs around Pune and Ozar Tal. Junner Dist. Pune (shell- length 7-9 cm). This species is found at the bottom of ponds, rivers, lakes and other fresh water bodies. They are mostly found buried in mud in stagnant and slow running water hence can be easily collected. The water from the site of collection was examined to ascertain any traces of metal contamination. Physicochemical analysis of reservoir water was done using standard methods. The common physiochemical characteristics of the water from collection site are given in the table 1.1. Table 1.1: Physicochemical characteristic of water Sr. Parameters (mg/l) Nandegaon Nandegaon No. (N 18°33' E 73°42') (N 18°33' E 73°42') 1. Temperature 29.7 29.4 2. pH 9.29 9.5 3. Conductivity 164 138 4. TDS 115 123 5. Salts 81 72 6. Chlorophyll (µg/l) 0.94 0.344 7. T. Phosphorus 2.44 4.756 8. T. K. Nitrogen 0.78 0.98 9. Total hardness 152 228 10. Dissolved Oxygen 7.0 7.6 11. COD 39.5 49.4 12. BOD 12.7 18 13. Nitrate 8.0 4.725

1.3.3. Condition index (CI) Introduction: Condition indices (CIs) have been used to measure the well-being of mussels (Narkko et.al.). It theoretically integrates the effect of changing trophic conditions over time, the good relationship between condition index and habitat characteristic provides a scientific framework for its use as a good ecological indicator of aquaculture intensity (Filgueira et. al., 2012). Mussel ‘health’ condition index is used as an economic indicator for determination of the quality of marketed product (Orban et. al., 2002).

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Materials and Methods: Mussels were collected from collection sites. After collection, at the laboratory five mussels were cleaned and dissected on the same day and wet weighed. Whole soft tissues and shells of mussels were kept into drying oven at 60ºC and then weighed after 96 hrs in order to determine their dry weights. The condition index was calculated according to the prescribed formula given by (Walne, 1976). Condition index= Dry flesh weight (g)/Dry shell weight (g) x 100 This index is widely used because it is easily standardized and it has more universal application than the other condition indices (Lucas and Beninger, 1985). Therefore, this condition index facilitates the methodology standardization and it is essential for environment monitoring programs. Result: Average condition index of mussels collected from field was 12.89, which showed that animals were healthy in their natural reservoir. Table 1.2: Condition index of field collected L. corrianus

SR. Dry Flesh weight Condition index Dry shell weight (SW) No. (FW) (FW/SW X 100) 1 2.17 16.53 12.42 2 2.22 20.7 9.53 3 3.39 18.18 13.13 4 1.79 19.84 10.72 5 2.19 17.64 18.65

1.3.4 Acclimatization: The freshwater mussels L. corrianus, were collected from collection sites, after collection the mussels were cleaned to remove mud, algal mass and debris. Then brought to the laboratory, acclimated in fiberglass tanks containing aerated, re-circulating water at a density of around 2 lit of water per mussel for 14 days. The temperature of the water was maintained at 24±1ºC and the water was renewed on a daily basis after every 24 hours and any dead mussels were removed and discarded immediately. The animals were fed daily ad libitum with algal suspensions of spirulina according to the method of

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Amanullah et al. (2010). Mussels were acclimatized to laboratory condition for fifteen days in de-chlorinated tap water prior to experimentation, only good conditioned mussels were used for exposure experiments.

1.4 Selection of toxicants and exposures

1.4.1. Cupric chloride dihydrate (CuCl2.2H2O): Copper is widely distributed heavy metal, its persistence in the environment results in toxicity. Copper in trace quantity is essential for metabolic processes but when it is present in excess quantity, it results in toxicity (Davenport and Redpath, 1984). The main source of Cu is various industrial effluents. Copper is extensively used as biocide within antifouling paints. It can bio-accumulate within the soft tissues of mollusks and induce corresponding enzymatic responses. It has thus become an established fact that the accumulation and effects of metals on biota depend rather on their bioavailability than on their total concentration in water. Taking into account the environmental concerns caused by excess Cu, it is important to assess the impact of Cu on the health of organisms so as to protect the aquatic ecosystems. Based on LC50 of cupric chloride dihydrate in this test species, sub lethal concentration (LC1-0.1ppm) was considered as suitable dose for further experiments.

1.4.2. PAH (Anthracene- C14H10): Polycyclic aromatic hydrocarbons (PAHs) are persistent chemicals of water and their concentrations could range from undetectable to up to milligrams per litre in industrial effluents and in heavily-polluted areas. When PAHs enter aquatic systems, they deteriorate the environment. These PAHs are able to readily accumulate in aquatic biota, in concentrations that are depended merely upon species ability to bio-transform them. Since there is a wide variation in physiological responses, which determine PAHs bioavailability and toxic potency, a lot of studies have been focused on their effects on aquatic species, including mussel. Sixteen PAH compounds are considered as priority pollutants because of carcinogenic and mutagenic effects of their metabolites on aquatic organisms. Bivalves readily accumulate hydrocarbons such as PAH and polychlorinated biphenyls (PCB) in the environment and have been widely used as 'bio accumulators' in

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption environmental monitoring programs. PAHs constituents, such as anthracene, are effectively toxic for aquatic organisms, including bivalve mollusks. Based on literature, sub lethal concentration of anthracene (0.5 ppm) was considered as suitable dose for exposure.

1.4.3. Combined exposure (CuCl2.2H2O + Anthracene): For combined exposure, mixture of 0.1 ppm of cupric chloride dihydrate and 0.5ppm of anthracene was selected. In this, the effect of combined exposure of cupric chloride dihydrate (0.1 ppm) and of Anthracene (0.5 ppm) together was studied. After collections of the animals from the collection sites, animals of uniform shell length (8-9 cm) were selected for exposure experiments, specimen from the same site was used for each and every single set of experiment. Animals were cleaned to remove mud, debris and faecal matters. Then the cleaned animals were acclimatized in the laboratory conditions for fourteen days, contamination by excretory product, faecal matter and metabolism was checked by daily renewal of water. Animals were divided in following four groups with six animals in each group to study the effects of the selected contaminants (Toxicants).

Group I (Control): Animals in de-chlorinated tap water served as control group. For PAH exposure, first control set was maintained in water and second control set was maintained insolvent (acetone 0.05% v/v) Group II (A):

Animals were exposed to 0.1 ppm of CuCl2.2H2O (LC1) for 7 days (T1) and 14 days (T2). After exposure, animals of each group (T1 and T2) were transferred to water without CuCl2.2H2O for 4 days for assessment of recovery (R1 and R2) Group III (B): Animals were exposed to 0.5 ppm of anthracene up to 7 days (T1) and 14 days (T2) Animals after exposure were transferred to water without anthracene up to 4 days for assessment of recovery (R1 and R2)

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Group IV (C): Animals were exposed to combined exposure of 0.1 ppm of Cupric Chloride dihydrate + 0.5 ppm of anthracene for up to 7 days (T1) and 14 days (T2) Animals after exposure were transferred to water without any toxicant up to 4 days for assessment of recovery (R1 and R2) Animals from each group were sacrificed to collect the tissues for biochemical estimations, genotoxicity assessment and histopathological studies.

1.5 Behavioral responses 1.5.1 Introduction: There is positive correlation between animal behaviour and environment fluctuations. Various biotic and abiotic factors play an important role in changing behavioural activities of the animals. Animal behaviour is a particular way in which an organism responds to environmental stimulations; especially a response which can be observed (Webster’s 2005). Chemical pollution is becoming a serious problem worldwide, particularly in India. Most of the agricultural, industrial and other regular activities dump untreated chemical wastes with toxic metals in water bodies which lead to bioaccumulation of toxic substances and stress full environment. Stress full environment may cause various alarming reactions in aquatic ecosystems and organisms (Madhyastha, 1996). The toxicants cause stress on the organisms; the behavioural changes are the immediate response to the toxicant and as such are indicators of possible stress.

1.5.2. Materials and Methods: For the observations of behavioural changes, the animals were exposed for 7 days (T1) and 14 days (T2) to:- A) Cupric chloride dihydrate (0.1 ppm) B) Anthracene (0.5 ppm) and C) Combined exposure (A+B)

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

4 days of recovery period (R1 and R2) was given after each exposure. Behavioural changes in control as well as exposed animals were closely observed and recorded regularly.

1.5.3. Results : The following results depict the comparison in behaviour of animals kept in the toxicant-free fresh water versus those kept in toxicant-exposed water. The behaviour of bivalves subjected to toxicant-exposed water was distinctly atypical to those subjected to toxicant-free water.

I) Behavioural observations in control animals: Freshly collected bivalves were placed in containers with normal fresh water; initially bivalves closed their shell valves and imprisoned themselves inside the shell. After some time, they opened their shell valves and slowly extended out their foot. After opening the shell valve pallial edges were extended out, the siphon was protruded outside the shell valves. Excreta accumulated normally. There was no excess mucus secretion. With the gentle mechanical stimulus, bivalves retracted the extended organs and closed shell valves immediately.

II) Behavioural observations after exposures:- After the exposures of A) cupric chloride dihydrate (0.1 ppm), B) anthracene (0.5ppm) and C) combined (A+B) for 7 days (T1) and 14 days (T2) followed by 4 days of recovery (R1and R2), the common behavioural observations of L. corrianus were as under - On immersion in toxicant mixed water, the bivalves immediately retracted the foot in the shell and closed the shell valves and then bivalves slightly began to open the shell valve after some period. The gentle stimulus with glass rod made the extended organs to retract and bivalve closed the shell valves immediately (Duration of retraction for extended organs was 10-12 seconds). The shell valves were closed mostly till next day.

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

After 24 hours, open shell valves were observed, pallial edges, foot and siphons were extended out, the gentle mechanical stimulus made the extended organs to retract and the shell valves closed immediately (Duration: 20-22 seconds). The closed shell valves were observed up to 24 - 26 hours, thereafter valves opened intermittently and foot was extended out. After 48 hours, extended foot, pallial edges and siphon were observed. Gentle mechanical stimulus resulted in retraction of foot and closing of valves. Duration of response was increased up to 50-55 seconds. On the 7th day of exposure slightly swollen foot was observed. Bivalves slowly closed the shell valves after gentle stimulus (Duration: 70-75 second). Mucus secretion was also seen during earlier exposure duration (7 days). The secretion of mucus was observed to be higher after 14 days of exposures as compared to 7 days of exposures to cupric chloride dihydrate (0.1 ppm), anthracene (0.5ppm) and combined treatment. After 14 days of exposure for A, B and C the foot was swollen. The mantle edges remained at the shell border, while siphons protruded out of the shell. On the 14th day of all the exposures, bivalves appeared inactive, swollen foot along with the pallial edges and siphon was outside with excess mucus. Gentle mechanical stimulus made the extended organs to retract slowly as the swollen foot was trapped in the shell valves; duration of response being 2 to 3 minutes. It indicated that toxicant exposure caused slow and prolonged body activities.

1.5.4 Discussion: Behavioural changes are immediate responses to environmental toxicants; significant alterations in the behaviour pattern of the organisms resulted from environmental pollution and toxicant exposure (Surwase, 2009). Townsend et al. (2014) reported variations in the behavioural activities of sediment dwelling Tellinid bivalve Macomona liliana, following exposure to contaminated sediment, the chronic, slow level stressors disturb the behavioural activities, slow and prolonged body activities were observed. The rate of feeding, movement also gets affected. The prolonged exposure also results in reduction in fitness or health condition of organisms or it may not recover and potentially lead to death.

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Behavioural alterations due to bio-accumulations of heavy metals (copper, lead and zinc) were observed in bivalves Macomona liliana. It was reported that 10 days of heavy metal exposure resulted in metal bio-accumulation which affect the feeding behaviour, for filter feeder organisms ingestion is a dominant route of metal exposure, it may affect feeding habits (Fukunaga and Anderson, 2011). EL-Shenawy (2003) reported the variations in shell valve opening, foot and siphon protruding with heavy metal exposure in clam Ruditapes decussates. Variability in metabolic and respiration rates was also observed. Toxicity potential of distillery effluent on the behaviour of freshwater bivalve Parreysia favidens was studied; it was observed that distillery effluent was very toxic. After exposure, bivalve showed avoiding response by withdrawing their body parts and closing their shell valves immediately. Excess secretion of mucus may be due to hyper activity of mucus gland as protective response, it reflected the toxicity stress (Shandilya et al. 2010). The change in the behavioural activities of animal can be taken as sensitive indicator of environmental toxicity stress. Behavioural changes due to acute toxicity of copper in freshwater snail Bellamya bengalensis, was reported by Kamble and Kamble (2014). The various physiological as well as morphological changes were recorded in copper sulphate exposed snail, foot movements and tentacular movements were reduced, quick response to pin touch or other stimulus was not found, mucus secretion was much more. At the end snails became immotile and lost their movements. Mucus secreting cells serve in excretory and defense mechanisms, the excess secretion of mucus was reported in common mussel Mytilus edulis due to toxic effects of zinc, the zinc exposure resulted in an acute inflammatory reaction in gill which may lead to copious production of mucus (Hietanen et al. 1988). Pillay (2013) reported mucus secretion responses in mussel Perna perna, exposed to various sub lethal copper concentrations, difference in mucus production rates were examined at different copper concentrations exposure, the results revealed that increasing copper concentrations exposure had significant impact on mucus production. In mussel, mucus production is one of the essential detoxification mechanisms. Mucus is a complex carbohydrate which is essential for maintaining homeostasis in molluscs; it helps in

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption feeding by selection and entanglement of food particles from water. It plays an important role in depuration against environmental contaminants; it may be seen as the first line of defense against foreign toxicants around the gills, which are continuously in contact with water, thus use of mucus production is an essential biomarker recommended for toxicological studies. In bivalve, mucus secreted by gill cells comes first in contact with water and acts as barrier to prevent diffusion of toxicants in to soft tissues. The exposure of high concentrations of Zn and Fe resulted in excess production of mucus, it indicated important role of mucus in metal depuration in deep sea hydrothermal mussel Bathymodiolus azaricus (Kadar, 2007). Motile animals can protect themselves by running away from polluted or toxicant contaminated area, but bivalves are sedentary unable to run away they avoid toxic effects by producing mucus. Mucus production might help to reduce exposure of body surface to toxicant or by withdrawing body parts in to the shell valves. Shell closing mechanism might act as protective phenomenon against the toxicant exposure (Nagarthnamma and Ramamurthi, 1982).

1.6 Effect of Cupric Chloride dihydrate and Anthracene on oxygen consumption (OC)

1.6.1 Introduction: In aquatic organisms many, physiological processes like respiration, metabolism, growth, reproduction, development etc. have been reported to be affected by different contaminants accumulated in water bodies. In mussels, gills are the major sites of environmental interactions. Gill maintains steady water current, filter the water, collect and process of food particles simultaneously. Thus a gill plays an important role in the respiration and bioaccumulation (Rozenn et. al., 2009). The respiration is considered as one of the important parameter for understanding the variations in physiological processes in the mussels, since many features of aerobic metabolism can be also studied by measuring of the rate of oxygen consumption. The rate of oxygen consumption shows a common metabolic index which helps to determine the physiological state of mussel under various ecological circumstances (Zotin and

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

Ozernyuk, 2004). Oxygen consumption in mussel depends on various internal as well as external factors. Internal factors such as age, body weight etc and external factors such as temperature, seasons, food and feeding conditions, water saturations with oxygen, contaminants in water affect the rate of oxygen consumption (Famme, 1980; Hamburger et. al.,1983; Sukhotin, 1988). Oxygen consumption (OC) is a very sensitive physiological process, therefore alteration in the respiratory activity is considered as an indicator of toxicity stress in mussels exposed to heavy metals. (Sarkar, 1999). Billinski and Jones, (1973) had suggested that decreased oxygen consumption resulted due to disturbances in oxidative and phosphorylative processes. Lomte and Jadhav (1982) also showed that copper sulphate, mercuric chloride causes decrease in oxygen consumption in L. corrianus. Metal contaminants can increase or decrease the rate of oxygen consumption. They act as respiratory depressants in perna indica (Krishnakumar et. al., 1990).

1.6.2. Materials and Methods: The freshwater mussels, L. corrianus were collected from the reservoirs around Pune (shell- length 7-9 cm). After acclimatization in laboratory conditions, the next step was to estimate the oxygen consumption. A series of 1L volume capacity rectangular glass jars were filled with water. As a precaution to avoid air bubbles, a thick layer of coconut oil was spread on water surface to prevent atmospheric contact. Then, a single animal was exposed separately in separate jar for cupric chloride di-hydrate, PAH (anthracene) and combined treatment. Three individual sets for experimental (one each for A, B and C) and control were arranged simultaneously.

Determination of oxygen Content in the water sample: Before exposure to the contaminants, initial oxygen content of water was determined by collecting the sample in a narrow mouthed glass stoppered sample bottle of known volume by Winkler’s method (Annon, 1984). Oxygen consumed by the mussel was calculated by finding out the difference between the initial and final oxygen content in the animal chambers. Same method of determination of oxygen content was followed

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption for each exposure. The dissolved oxygen content in the control and treated water samples was calculated.

1.6.3. Results A) Exposure to cupric chloride dihydrate: Alterations in the oxygen consumption (OC) of fresh water mussel Lamellidens corrianus, exposed to cupric chloride dihydrate (0.1ppm) is represented in figure 1.2 and 1.3, for 7 days (T1) and 14 days (T2) of exposure followed by 4 days of recovery (4 DR : R1 and R2) for both the exposures. The results revealed that as compared to control, acceleration in the respiration rate was observed. A significant (p<0.05) increase (132%) was noted after 7 days of cupric chloride dihydrate (0.1ppm) exposure (T1) (figure 1.2). After four days of recovery period (R1), the significant rate of respiration recorded was (106%). The reduction in the rate of respiration was observed after extended duration of exposure (T2) (figure 1.3), the significant reduction was (69%) and after 4 days of recovery (R1) it was (56%).

B) Exposure to Anthracene: Bivalves were exposed to anthracene (0.5ppm) for 7 days (T1) and 14 days(T2) and after both exposure durations, they were allowed to cure in toxicant free fresh water for 4 days (4DR : R1 and R2). In both cases oxygen consumption was recorded, the results revealed that (Figure 1.2) in case of 7 days of exposure (T1) oxygen consumption was increased to some extent (121%). After four days of recovery (R1) the rate of respiration was (96%). As duration of anthracene (0.5ppm) exposure was extended for 14 days (T2), the significant reduction in oxygen consumption (55%) was noted (figure 1.3), followed by (34%) recovery after four days (R2).

C) Combined exposure: In this case animals were exposed to mixture of cupric chloride dihydrate (0.1ppm) and anthracene (0.5ppm) for 7 days (T1) and 14 days (T2) of exposure and after

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption both exposures animals were allowed to recover for 4 days (R1) in toxicant free fresh water. The results depicted increase in oxygen consumption after T1 exposure (113%), however after four days of recovery (R1) bivalve did not recover significantly (104%). After extended duration (T2) of combined effect, bivalve showed significant decrease (figure 1.2) in oxygen consumption (46%), after 4 days they recovered (R2) to some extent (37%).

control 7 DT 4DR 0.8 a 0.7

0.6 b

0.5

0.4

mg/lit/hr 0.3

0.2

0.1

0.0 A B C

Figure 1.2: Alterations in the rate oxygen consumption (OC) of L. corrianus after 7 days (T1) of exposure to (A) - cupric chloride dihydrate (0.1 ppm), (B) - Anthracene (0.5 ppm) and (C) - combined exposure followed by 4 days of recovery (R1). a There are significant differences ( p< 0.05) between the control and treated groups, b There are significant differences (p< 0.05) between the treated and 4 day recovery groups.

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

0.7 control 14 DT 4DR

0.6

0.5

0.4 a mg/lit/hr 0.3 b a 0.2 a

0.1

0.0 A B C

Figure 1.3: Alterations in the rate of oxygen consumption (OC) of L. corrianus after 14 days (T2) of exposure to (A) - cupric chloride dihydrate (0.1 ppm), (B) – Anthracene (0.5 ppm) and (C) -combined exposure followed by 4 days of recovery (R2). a There are significant differences ( p< 0.05) between the control and treated groups, b There are significant differences (p< 0.05) between the treated and 4 day recovery groups

1.6.4. Discussion: Oxygen taken up during the process of respiration is essential in the body. In aquatic organisms like mussels, rate of oxygen consumption is an important index for indicating the metabolic state as their whole body is continuously bathed by the surrounding water. When any metal contaminant or organic contaminant enters the water body and bio accumulates in the aquatic organisms, it primarily affects the metabolism which is related with the oxygen consumption. Thus respiration is a very sensitive physiological process and any change in the respiratory activity has been used as an indicator of stress in the animals exposed to toxicants (Sarkar, 1999). The present study showed that there was an increase in the rate of oxygen consumption when the bivalves were exposed to (A) cupric chloride dihydrate, (B) anthracene and (C) combined effects of both for 7 days (T1), but there was decline in the rate of oxygen consumption after the fourteen days of exposure (T2).

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

As bivalves were exposed to cupric chloride dihydrate, initially oxygen consumption increased, similar results were observed when L. marginalis was exposed to sub lethal concentration of heavy metal, oxygen consumption rate was high on the 10th day of exposure and there after it was decreased gradually (Mohanraj and Hameed, 1991). Similar findings were reported by Krishnakumar et al. (1990), in Perna viridis, when exposed to copper and mercury, initially animal showed increased oxygen consumption. When duration of cupric chloride dihydrate exposure was extended for 14 days the significant reduction in oxygen consumption was observed, the prolonged exposure and toxicity of heavy metals lead to the oxygen depletion in the mantle cavity of bivalve, therefore the oxygen consumption decreased, this is supported by the findings of Moon and Pitchard (1970) in case of Mytilus californianus, oxygen depletion in the mantle cavity was rapid with the contaminant exposure as bivalve closed the shell valve. Valve closure is an avoidance behaviour which leads to depletion of the oxygen in the mantle cavity; this was in accordance with the observations of Brand and Taylor (1974) in case of bivalve Arcticais landica. Akberali and Earnshaw (1982), reported that the prolonged exposure to copper, lead and zinc caused damage in the gill at cellular level affecting the respiration which led to reduction in oxygen consumption. Similarly, effects of chronic copper exposure on oxygen consumption in the green mussel Perna viridis was investigated, the result showed that an increase in the copper body burden caused decrease in oxygen consumption by 12.8% and 24.8% (Sze and Lee, 2000). Minakshi and Mahajan (2013) studied the effects of Thiamethoxam on oxygen consumption of the freshwater bivalve, L. marginalis, it was observed that the rate of oxygen consumption was significantly decreased after every 24 to 96 hours as compared to control, it was observed that the toxicant affect the respiratory metabolism and decrease the rate of oxygen consumption. In bivalves gill plays a key role in the exchange of gases, all the metabolic pathways also depend upon the normal functioning of the gills, the various toxic

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption contaminants damage to gill tissues and result in respiratory stress (Magare and Patil, 2000) Mahajan and Zambare (2007) studied the effects of heavy metal nickel on the oxygen consumption of fresh water bivalve Corbicula striatella and reported that heavy metals caused disruption of normal gill structure and resulted in decrease of the oxygen consumption rate (OCR). The decrease in OCR of the fresh water fish Amblypharyngodon mola was observed due to heavy metal exposure, the result showed that heavy metal exposure may lead to excess secretion of mucous, this mucous forms a thin film over the gills, which reduces the efficiency of oxygen uptake of the fish (Shelke and Wani, 2005). Stefanoni and Abessa (2011) evaluated the effects of Linear Alkyl benzene sulphonate (LAS) on the mussel Perna perna the result showed that there was slight increase in the respirationrate, the increase in the oxygen consumption rate (OCR) may be related to metabolic alteration caused by LAS. Cheney et. al., (2001) reported diverse patterns of alterations in the respiratory rate of the freshwater mussel Elliptio complanata exposed to polycyclic aromatic hydrocarbons like chrysene, anthracene and naphthalene, the results revealed that PAH had diverse effects on the metabolic activity, it also stimulated heart rate which led to alterations in the oxygen consumption. Polycyclic aromatic hydrocarbons caused long term effects on the physiological metabolism, when Pacific oyster- Crassostrea gigaswere exposed to different concentrations of polycyclic aromatic hydrocarbons (anthracene, fluoranthene, fluorene, phenanthrene and pyrene) the results revealed that the rate of respiration increased as exposure level increased, it suggested that oyster could spend more energy to eliminate toxins from the body and adapted themselves to toxicity stress, it indicated the close relation between oxygen consumption and tissue burden of organic toxicants (Jeong and Cho, 2007). Zebra mussels significantly accumulate hydrophobic contaminants like PCB and PAH which lead to two fold increase in oxygen consumption, it showed that greater oxygen demand was linked with contaminant uptake which alters the metabolic activities (Bruner et. al., 1994).

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption

The long term exposure to copper salts resulted in the high level metal bioaccumulation in the gill tissues; it also caused histopathological damage to the gill epithelium which altered the rate of respiration and oxygen uptake. This may be the probable reason for the reduction in the oxygen consumption. The present results were in confirmation with the previous studies by Kumarasamy and Karthikeyan (1999), they reported histopathological damage to the gill epithelium and affected respiration in estuarine bivalve M. casta.

1.7 Conclusions Condition index showed that animals were healthy in their natural habitat at the time of collection. Behavioural changes are indicators of possible toxicity stress. Extending duration of exposure up to 14 days led to excess uptake of toxicant, slow retraction of extended organs. Delayed shell closure responsiveness showed decline in neuromuscular control; slow and prolonged body activities represented lethargic conditions in bivalves. Excess mucus secreted by L. corrianus after the toxicant exposure played an important role in overcoming toxicity stress and complemented its preventive role in the toxicity. It is clear that toxicant exposure has an impact on behavioural changes and hence, behavioural changes can be used as effective biomarker for assessment of stress. The results depicted that alteration in the rate of respiration are exposure duration dependent. Initially, in case of 7 days (T1) of exposure for all exposure conditions (A, B and C) acceleration in the rate of oxygen consumption was observed, it might be because of the activated behavioural and respiratory response of bivalve upon the toxicant exposure. Bivalve could spend more energy to eliminate toxins from body and for adopting toxicity stress, oxygen consumption was increased initially. The decreased rate of respiration was observed upon prolonged exposure for 14 days (T2) in all exposure conditions (A, B & C). The extended duration of exposure of (A)-cupric chloride dihydrate (0.1ppm), (B)-anthracene (0.5ppm) individually and (C)- combined attributed to the toxic effects on the gill surface, it led to inactivity and closing of shell valves which caused depletion in the rate of oxygen consumption. Thus

Chapter 1: Condition Index, Behavioural Study and Oxygen Consumption measurement of respiration rate could be useful parameter for detection of toxicity stress in bivalves.