Physico-chemical and Microbiological Characterization of Recycled , Open and Eutrophicated Lake Water Grabs and Assessment of its Effects on A Vertebrate System Zebra Fish ( Danio rerio ) : A Case Study of Mallathahalli Lake , Bangalore , India

Bangalore is a metropolitan and capital city of the state of Karnataka in India, it is considered to be the fastest growing Information technology and Biotechnology hub of the world. In contrast with the same the city is subjected to continuous development through anthropogenic activities where in it has lost most of its wetlands like lakes and the existing water bodies are exploited to the highest order the resultant of which is seen through the spread of diseases and loss of existing natural aquatic bodies and loss of ecosystem. In the present study grab water samples were collected from three distinct sampling stations of Mallathahalli Lake, Bangalore, India and assessed for various physical and chemical parameters betwixt December 2014 and February 2015; Microbiological evaluation was also extrapolated for the lake betwixt the same period. The average values recorded for the physical parameters like pH and temperature for Recycled, Open and Eutrophicated lake water was found to be within the limit. The average values recorded for the chemical parameters from the three sampling stations were found to be within the standards set by World Health Organization (WHO) for drinking water quality. The DO, BOD and COD values were found to be varying in the three different sampling stations implicating organic pollution. Bacteriological examination of water indicated high microbial activity and proved water to be unfit for human consumption. The effect of the water quality was supported by heart beat count and acetyl choline esterase activity which was established in zebra fish a vertebrate model. Zebra fish analysis concluded recycled water to be of better quality when compared to eutrophicated and open water system.

Wetlands play an important role in survival of life forms on planet earth and they are a pivotal source of water.Water is one of the basic amenities for survival of fauna and flora; lakes, estuaries, rivers etc form an integral part of wetland ecosystem.Lentic ecosystems especially lakes are the most productive ecosystems of the biosphere and they are constantly deteriorated by chemical pollution and anthropogenic activities; this has lead to loss of ecosystem and reduced socioeconomic development (Constanza 1997, Rapport et al., 1998).The lakes occupied about 4.8% of geographical area of Bangalore and served as the major water source for drinking and irrigation (Ramachandra et al., 2001), The wetlands account for drinking water, fish culture, replenish ground water and also reduce natural calamities (Gurunathan 2006), Human activities has led to loss of wetlands the resultant of which is discontinuation of drainage networks in the city of Bangalore (Deepa et al., 1998) and the surviving lakes are reduced to cesspools due to continuous exploitation and dumping of waste (Ramachandra 2007).High pH, TDS, Sulphates and Phosphates has altered the nutrient concentration of the lakes in Bangalore and reduced the dissolved oxygen levels in the lakes leading to fish mortality (Zutshi et al., 2008).Fish death and sewage disposal has increased the microbial population resulting in diseases and infections.According to APHA (1992) pathogenic organisms transmitted through water are Salmonella, Klebsiella, E.coli, Shigella etc. Microbial population cause spoilage of fish after its death and also results in its decomposition (Nandini 1995).Water clogging inside water distribution system results in formation of bio-films and may result in spread of disease (Lee et al., 2010).The chemical pollutants and microbial population has largely deteriorated the water quality which in turn has influenced the composition, distribution and abundance of macro invertebrates and vertebrates (Zutshi et al., 2007), unplanned development of the city, industrial and domestic wastes, autochthonous wastes mainly due to plants have a direct effect on the water quality, influencing the light penetration and efficacy of the lakes (Ramachandra 2009).Ecological succession and growth of water weeds is the result of eutrophication observed in ibalur lake, Bangalore (Raj et al., 2014).Mallathahalli lake water was hard water type and can be used for irrigation (Ravikumar et al., 2013).Wetland ecosystems like lakes in Bangalore are largely degraded by pollutants and urbanization, with this ideology the objective of the study was to assess Mallathahalli Lake for physico-chemical characters and microbial characters and to evaluate its effects on zebra fish (Danio rerio) a vertebrate model.

Sampling station-Mallathahalli Lake
Mallathahalli Lake is a natural fresh water lake which is positioned at latitude of 12.9603 and a longitude of 77.4967, it is situated about 14 kilometres from the Kempegowda bus terminus and categorised under the south ward of urban Bangalore, India.The lake has a periphery of about 3.5 kilometres with a catchment area of 625 hectares; the lake was recouped in recent years for recreational purpose by bunds and fencing.Million litres per Day -Sewage Treatment Plant (MLD-STP) was set up to treat sewage water.The lake is bordered by a crematorium to the south, areca plantation to the west, a sewage treatment plant to the north and it is encroached by houses to the east.Three sampling stations were identified to collect water samples.Sample A-Recycler was a point near MLD-STP, at this point sewage water was recycled and ingested into the lake, Sample B-Boat camp was the second sampling station and this area was of the open water system thereby was well aerated and received good sunlight.Sample C-Plant was the site where in plants was found growing along with algal forms indicating high degree of eutrophication.Sampling method: Physico chemical and Microbial characterization BOD bottles and well cleaned plastic bottles were used to collect water samples from the sampling stations betwixt December 2014 and February 2015.Dissolved oxygen was fixed at the site, pH and temperature was determined at the sampling stations.Standard procedures prescribed in NEERI lab manual by Trivedi and Goel (1986) and APHA (1998) were used to judge the parameters like DO, BOD, COD, Sulphates, Calcium, Alkalinity, Ammonical nitrogen, Turbidity, Sodium and Potassium.Standard methods prescribed in lab manual of Aneja (2003) was followed to determine microbial parameters like most probable number (MPN) and total microbial counts.

Zebra fish (Danio rerio) examination
Fully developed breeding stock of zebra fish were procured form local aquarium vendors and grown for a week in small glass tanks fitted with a sieve at the bottom.The tanks were well aerated and lit.The fishes were fed with flakes twice a day and were allowed to fertilize.Spawning happened early in the morning and the eggs were trapped in the sieve which was further transferred to petriplates containing reverse osmosis water and the lake water samples to establish morphological changes and heart beat count of the embryos to correlate their growth and development.

Assay for Acetyl choline esterase activity
Titrimetric method for acetylcholine esterase activity (Guruprasad et al., 2014) was followed.Adult male fishes which were grown in the lake water samples was subjected to euthanization to dissect out the brain cells, the brain cells were homogenized with phosphate buffer in a pre-chilled mortar using the pestle.The homogenate was centrifuged with phosphate buffer at 4° C and the supernatant served to be the enzyme source.The supernatant was diluted to 20X strength for further assay.The diluted enzyme solution was mixed with acetylcholine iodide and titrated with sodium hydroxide with phenolphthalein as the indicator.

RESULTS AND DISCUSSION
The lake water was analysed in three different sampling stations betwixt December 2014 and February 2015.The data for various physical and chemical parameters measured are tabulated.The data for Sample A -Recycler over the three sampling seasons is recorded in table 1. Similarly the data for the sample B-Boat camp and sample C-Plant are recorded in table 2 and 3 respectively.The microbial parameters which were examined are also recorded and indicated in table 5 and 6 respectively.The zebra fish analysis which was carried out to determine the effects on a biotic system are tabulated in table 7 and 8.
The pH of the lake water was found to be 7.31, the average pH value of the Recycler, Boat camp and Plant was found to be 7.53, 7.26 and 7.13 respectively.High pH imparts a bitter taste to the water and may also corrode water distribution system.A pH range of 6-9 is expected for survival of aquatic life failing which there is slow deterioration of life forms (Alabaster et al., 1980).Temperature of lake water is dependent on the dissolved oxygen (Salah 2014); higher temperature will result in chemical reactions.The mean values of temperature were found to be 22.33 samples A and B and 22.66 for sample C respectively.Dissolved oxygen is supposed to be 5mg/L for ecological sustenance (Ramachandra et al., 2001) below which it may lead to stress in survival (Sisodiya et al., 2006).The DO values for day 1 and day 5 observed is depicted in figure 5. Sample C-Plant showed a very low DO level.Highest DO

Fig. 5 .Fig. 1 .
Fig. 5. indicating the values of Dissolved oxygen (Expressed in mg/L) for the grab samples Recycler, Boat Camp and Plant for the month of December 2014, January 2015 and February 2015

Table 1 .
Indicating the Physico-Chemical parameters for the grab sample A -Recycler for the month of December 2014, January and February 2015

Table 2 .
indicating the Physico-Chemical parameters for the grab sample B -Boat Camp for the month of December 2014, January and February 2015

Table 3 .
Indicating the Physico-Chemical parameters for the grab sample C -Plant for the month of December 2014, January and February 2015

Table 4 .
Indicating the average values of Physico-Chemical parameters for the grab samples A-Recycler, B-Boat Camp and C-Plant the month of December 2014, January and February 2015

Table 5 .
Indicating the Most Probable Number results for the grab samples (Recycler, Boat Camp and Plant) for the month of December 2014, January and February 2015

Table 6 .
Indicating the colony characters and the gram characteristics of isolated microorganisms

Table 7 .
Indicating the Heart Beat count recorded in Zebra fish (Danio rerio) for the samples Recycler, Boat Camp and Plant

Table 8 .
Indicating the Acetyl choline esterase activity recorded in Zebra fish (Danio rerio) for the samples Recycler, Boat Camp and Plant lake which was found to be within the standard.Alkalinity is said to increase with pH and chemical pollutants, the lake water showed a value of 136.88 mg/L.The average values of the sampling zones are indicated in table 1, 2 and 3 respectively.The lake water was found to be within the acceptable range and proved to be good.Clay, silt, organic pollution, phytoplankton may lead to turbidity of water (kishore et al., 2005).1.5NTU,31 NTU, and 6 NTU are the values of the three sampling stations as indicated in table 4.This indicates Sample B to be highly turbid.Ground water contains high sodium depositions when compared to surface waters(WHO 1979).47.5mg/l, 71.03 mg/l and 96 mg/l were the observed values of sodium in the sampling points Recycler, Boat camp and Plant respectively, these values clearly depict that the