STUDY ON DRINKING WATER QUALITY ANALYSIS FOR PHYSIOCHEMICAL AND MICROBIAL PARAMETERS

Dhanush N. Begur ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 10 July 2020 Final Accepted: 14 August 2020 Published: September 2020 Study aims to analyze the physicochemical and microbial parameters of water samples collected from places in and around Mangalore, Karnataka, India and accesses the objectives for physicochemical parameters of samples ,for biological parameters and to compare and ensure safe water quality standards for human consumption with ISI(International standard Institute)specification for drinking water (portable water). There are 3 parts, first part consists of Introduction to the research , second consists of methods and methodology used for various tests and third consists of the final results obtained with discussions and conclusion to end with.

Water microbiology is concerned with the microorganisms that live in water, or can be transported from one habitat to another by water. Water can support the growth of many types of microorganisms. This can be advantageous. For example, the growth of some bacteria in contaminated water can help digest the poisons from the water. However, the presence of other disease-causing microbes in water is unhealthy and even life threatening. For example, bacteria that live in the intestinal tracts of humans and other warm-blooded animals, such as E. coli, Salmonella, Shigella, and Vibrio, can contaminate water if feces enter the water. Contamination of drinking water with a type of Escherichia coli known as O157:H7 can be fatal. The contamination of the municipal water supply of Walkerton, Ontario, Canada in the summer of 2000 by strain O157:H7 sickened 2,000 people and killed seven people (Chapelle et al., 2000).The intestinal tract of warm-blooded animals also contains viruses that can contaminate water and cause disease. Examples include rotavirus, enteroviruses, and coxsackievirus. Another group of microbes of concern in water microbiology are protozoa. The two protozoa of the most concern are Giardia and Cryptosporidium. They live normally in the intestinal tract of animals such as beaver and deer. Giardia and Cryptosporidium form dormant and hardy forms called cysts during their life cycles. The cyst forms are resistant to chlorine, which is the most popular form of drinking water disinfection, and can pass through the filters used in many water treatmentplants. If ingested ISSN: 2320-5407 Int. J. Adv. Res. 8(09), 453-465 454 in drinking water they can cause debilitating and prolonged diarrhoea in humans, and can be life threatening to those people with impaired immune systems. Cryptosporidium contamination of the drinking water of Milwaukee, Wisconsin with in 1993 sickened more than 400,000 people and killed 47 people.
Many microorganisms are found naturally in freshwater. These include bacteria, cyanobacteria, protozoa, algae, and tiny animals such as rotifers. These can be important in the food chain that forms the basis of life in the water. For example, the microbes called cyanobacteria can convert the energy of the sun into the energy it needs to live. The plentiful numbers of these organisms in turn are used as food for other life. The algae that thrive in water are also an important food source for other forms of life.
The region of a water body near the shoreline (the littoral zone) is well lighted, shallow, and warmer than other regions of the water. Photosynthetic algae and bacteria that use light as energy thrive in this zone. Further away from the shore is the limnetic zone. Photosynthetic microbes also live here. As the water deepens, temperatures become colder and the oxygen concentration and light in the water decrease. Now, microbes that require oxygen do not thrive. Instead, purple and green sulfur bacteria, which can grow without oxygen, dominate. Finally, at the bottom of fresh waters (the benthic zone), few microbes survive. Bacteria that can survive in the absence of oxygen and sunlight, such as methane producing bacteria, thrive (Chapelle et al., 2000).
Water can also be an ideal means of transporting microorganisms from one place to another. For example, the water that is carried in the hulls of ships to stabilize the vessels during their ocean voyages is now known to be a means of transporting microorganisms around the globe. One of these organisms, a bacterium called Vibrio cholerae, causes life threatening diarrhoea in humans.
Drinking water is usually treated to minimize the risk of microbial contamination. The importance of drinking water treatment has been known for centuries. For example, in pre-Christian times the storage of drinking water in jugs made of metal was practiced. Now, the anti-bacterial effect of some metals is known. Similarly, the boiling of drinking water, as a means of protection of water has long been known.
Chemicals such as chlorine or chlorine derivatives has been a popular means of killing bacteria such as Escherichia coli in water since the early decades of the twentieth century. Other bacteria-killing treatments that are increasingly becoming popular include the use of a gas called ozone and the disabling of the microbe's genetic material by the use of ultraviolet light. Microbes can also be physically excluded from the water by passing the water through a filter. Modern filters have holes in them that are so tiny that even particles as miniscule as viruses can be trapped (Madigan et al., 2000).
An important aspect of water microbiology, particularly for drinking water, is the testing of the water to ensure that it is safe to drink. Water quality testing can be done in several ways. One popular test measures the turbidity of the water. Turbidity gives an indication of the amount of suspended material in the water. Typically, if material such as soil is present in the water then microorganisms will also be present. The presence of particles even as small as bacteria and viruses can decrease the clarity of the water. Turbidity is a quick way of indicating if water quality is deteriorating, and so if action should be taken to correct the water problem.
In many countries, water microbiology is also the subject of legislation. Regulations specify how often water sources are sampled, how the sampling is done, how the analysis will be performed, what microbes are detected, and the acceptable limits for the target microorganisms in the water sample. Testing for microbes that cause disease (i.e., Salmonella typhymurium and Vibrio cholerae) can be expensive and, if the bacteria are present in low numbers, they may escape detection. Instead, other more numerous bacteria provide an indication of faecal pollutionof the water. Escherichia coli (E. coli) have been used as an indicator of faecal pollution for decades. The bacterium is present in the intestinal tract in huge numbers, and is more numerous than the disease-causing bacteria and viruses. The chances of detecting E. coli are better than detecting the actual disease causing microorganisms. Escherichia coli also had the advantage of not being capable of growing and reproducing in the water (except in the warm and food-laden waters of tropical countries). Thus, the presence of the bacterium in water is indicative of recent faecal pollution. Finally, Escherichia coli can be detected easily and inexpensively (Madigan et al., 2000).

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The importance of the provision of a wholesome supply of drinking water has been recognised since at least the times of the Romans with major advances being made during the nineteenth century. The provision of safe drinking water is one of the most important steps that can be taken to improve the health of a community by preventing the spread of water-borne disease. The maintenance of a sufficient supply of wholesome drinking water is a complex undertaking in which individuals from many disciplines have a role.

Legislation and water quality standards for public water supplies
The new European Directive for drinking water prescribes standards for the quality of drinking water, water offered for sale in bottles or containers and water for use in food production undertakings. The Directive specifies two types of parameter values, namely mandatory and non-mandatory. Mandatory standards, covering 28 microbiological and chemical parameters for mains water, are essential for health and the environment, and have to be met by specified dates. Non-mandatory indicator values, covering 20 further microbiological, chemical and physical parameters are prescribed for monitoring purposes. Any contravention of an indicator value must be investigated, but remedial action need be taken only where there is a risk to public health. The Water Industry Act, the Water Supply (Water Quality) Regulations and the Private Water Supplies Regulations transpose the Directive into English law. The Act places a duty on water companies to supply only water that is wholesome at the time of supply. The Act also creates a criminal offence of supplying water that is unfit for human consumption. Wholesomeness is defined by reference to the prescribed concentrations or values and other requirements. Prescribed concentrations or values are specified for microbiological, chemical and physical parameters. National legislation includes some standards and requirements in addition to those required by the Directive. All water covered by the regulations must be microbiologically wholesome. Prescribed concentrations or values for microbiological parameters rely on well proven indicator organisms, such as coliform bacteria, Escherichia coli, Enterococci, Clostridium perfringens, and colony counts. In addition to meeting standards, water must not contain any micro-organism (other than a parameter) or parasite at a concentration which would constitute a potential danger to human health

Materials And Methods:-Materials
The chemicals used in the study were of analytical grade procured from HiMedia and Merck and Co. And Sigma Aldrich

Sample collection:
Samples were collected in sterile containers from above spots. Collection of water samples were made for the study of physical, chemical parameters. The temperature and pH were noted on the spot and remaining factors were analysed in the laboratory without lapse of much time.

Analysis of Physicochemical parameters Colour
Pure water has no colour. The presence of some acids, weeds or industrial effluents may cause colour change of drinking water.

Determination of temperature
Temperature is noted at the time of sampling with a thermometer (Trideviand Goel, 1986).
Determination of pH by electrometric method (Trideviand Goel, 1986). Principle: The basic principle of electrometric pH measurement is determination of the activity of the hydrogen ion by potentiometric measurements using a standard hydrogen electrode usually glass electrode is used. The electromotive force (emf) produced in the glass electrode system varies linearly with pH

Procedure:
The electrode was thoroughly washed with distilled water and then with the sample ↓ The electrode was dipped in the sample and reading was noted down (Trivedi and Goel, 1986) Procedure:

Determination of Carbon dioxide
Take 100 ml of the sample in a conical flask ↓ Add 3-4 drops of phenolphthalein indicator, if the colour changes pink free CO2 is absent ↓ Note down the burette reading, suing the formula. If it remains colourless then iot is titrated against 0.05N NaOH till pink colour appears.
Where, A-Volume of NaOH added B-N-normality of NaOH

Hardness of water
Principle EDTA (Ethylenediamine tetra acetic acid) forms colorless stable complexes with Ca2+ and Mg2+ ions present in water at pH 9-10. To maintain the pH of the solution at 9-10, buffer solution (NH 4 Cl + NH 4 OH) is used. Erichrome Black-T (E.B.T) is used as an indicator. The sample of hard water must be treated with buffer solution and EBT indicator which forms unstable, wine-red coloured complex with Ca2+ and Mg2+ present in water. (https://www.cutm.ac.in/pdf/Env%20Engg%20Lab%20Manual.pdf) Procedure: 50ml of sample was taken in a conical flask ↓ 2ml of ammonia buffer solution was added ↓ The above solution was titrated aganist 0.02N EDTA solution using Eriochrome black-T as indicator ↓ The burette rerading was noted when the colour changes from red to blue ↓ Hardness can be calculated using the formula (Tridevi and Goel, 1986) Hardness mg/l = Alkalinity (Trivedi and Goel, 1986) Procedure: Take 100ml of the sample in a conical flask ↓ Add 2-3 drops of phenolphthalein indicator, if the remains colourless, alkalinity is absent ↓ If contents turns pink, titrate with 0.1 N HCl till colour disappears Note down the burette reading for phenolphthalein alkalinity ↓ Now add 2 drops of methyl orange indicator and titrate and titrate further till the colour changes from pale yellow to orange/pink

Analysis of Biological parameters Dissolved oxygen Principle
It is based on oxidation of potassium iodide. The liberated iodine is titrated against standard hypo solution using starch as a final indicator. Since oxygen in water is in molecular state and not capable to react with KI, an oxygen carrier manganese hydroxide is used to bring about the reaction between KI and O 2 .Manganous hydroxide is produced by the action of potassium hydroxide and manganous sulphate.

MPN (Most Probable Number) test Principle
Since human fecal pathogens vary in kind (viruses, bacteria, protozoa) and in number, it would be impossible to test each water sample for each pathogen. Instead, it is much easier to test for the presence of non-pathogenic intestinal organisms such as E. coli. E. coli is a normal inhabitant of the intestinal tract and is not normally found in fresh water. Therefore, if it is detected in water, it can be assumed that there has been faecal contamination of the water.

Standard water analysis Presumptive test
In the presumptive test, a series of lactose broth tubes are inoculated with measured amounts of the water sample to be tested. The series of tubes may consist of three or four groups of three, five or more tubes. The more tubes utilized, the more sensitive the test. Gas production in any one of the tubes is presumptive evidence of the presence of coliforms. The most probable number (MPN) of coliforms in 100 ml of the water sample can be estimated by the number of positive tubes.

The Confirmed Test
In order to confirm the presence of coliforms, it is necessary to inoculate EMB (eosin methylene blue) agar plates from a positive presumptive tube. The methylene blue in EMB agar inhibits Gram positive organisms and allows the Gram negative coliforms to grow. Coliforms produce colonies with dark centres. E. coli and E. aerogenes can be distinguished from one another by the size and colour of the colonies. E. coli colonies are small and have a green metallic sheen, whereas E. aerogenes forms large pinkish colonies.

The Completed Test
The completed test is made using the organisms which grew on the confirmed test media. These organisms are used to inoculate a nutrient agar slant and a tube of lactose broth. After 24 hours at 37°C, the lactose broth is checked for the production of gas, and a Gram stain is made from organisms on the nutrient agar slant. If the organism is a Gram negative, non-spore-forming rod and produces gas in the lactose tube, then it is positive that coliforms are present in the water sample.
460 BOD bottle full of dilution water for 5 days at 20ºC for 3 days at 27ºC. DO uptake of dilution water should not be more than 0.2mg/L and preferable not more than 0.1mg/L. 5. Determine BOD of the seeding material. This is seed control. From the value of seed control determine seed DO uptake. The DO uptake of seeded dilution water should be between 0.6mg/L and 1mg/L Sample preparation: 1. Neutralize the sample to pH 7, if it is highly acidic or alkaline. 2. The sample should be free from residual chlorine. If it contains residual chlorine remove it by using Na2S2O3 solution as described below. 3. Take 50mL of the sample and acidify with addition of 10mL 1 + 1 acetic acid. Add about 1g Kl. Titrate with 0.025N Na 2 S 2 O 3 , using starch indicator. Calculate the volume of Na 2 S 2 O 3 required per Litre of the sample and accordingly add to the sample to be tested for BOD. 4. Certain industrial wastes contain toxic metals, e.g. planting wastes. Such samples often require special study and treatment. 5. Bring samples to 20 ± 1ºC before making dilutions 6. If nitrification inhibition is desired, add 3mg 2-chloro-6-(trichloromethyl) pyridine (TCMP) to each 300mL bottle before capping or add sufficient amount to the dilution water to make a final concentration of 30mg/L. Note the use of nitrogen inhibition in reporting results. 7. Samples having high DO contents, DO ≥ 9mg/L should be treated to reduce the DO content to saturation at 20ºC. Agitate or aerate with clean, filtered compressed air. 8. Dilution of sample: Dilutions that result in a residual DO of at least 1mg/L and DO uptakes of at least 2mg/L produce reliable results. Make several dilutions of the pretreated sample so as to obtain about 50% depletion of DO or DO uptake of 2mg/L. Prepare dilutions as follows: 9. Siphon out half the required volume of seeded dilution water in a graduated cylinder or volumetric flask without entraining air. Add the desired quantity of mixed sample and dilute to the appropriate volume by siphoning dilution water. Mix well with plunger type mixing rod to avoid entraining air.
Sample processing: 1. Siphon the diluted or undiluted sample in three labeled bottles and stopper immediately. 2. Keep 1 bottle for determination of the initial DO and incubate 2 bottles at 20ºC for 3days. See that the bottles have a water seal. 3. Prepare a blank in triplicate by siphoning plain dilution water (without seed) to measure the O2 consumption in dilution water. d. Also prepare a seed blank in triplicate to measures BOD of seed for correction of actual BOD. 4. Determine DO in a BOD test can in the blank on initial day and end of incubation period by Winkler method as described for DO measurement. 5. DO estimation in a BOD test can also be done by membrane electrodes. A DO probe with a stirrer is used to determine initial and final DO after incubation in BOD samples. The semi permeable membrane provided in the DO probe acts as a diffusion barrier against impurities between sensing element and sample

Calculations
Calculate BOD of the sample as follows: a. When dilution water is not seeded BOD as O2 mg/L = (D1 -D2) x 100 / % dilution b. When dilution is seeded BOD O2 mg/L = [(D1 -D2) -(B1 -B2)] x 100 / % dilution c. When material is added to sample or to seed control BOD O2 mg/L = [(D1 -D2) -(B1' x B2'] x F x 100/ % dilution where, D1 = DO of sample immediately after preparation, mg/L D2 = DO of sample after incubation period, mg/L B1 = DO of blank (seeded dilution water) before incubation, mg/L B2 = DO of blank (seeded dilution water) after incubation, mg/L F = ratio of seed in diluted sample to seed in seed control (Vol. Of seed in diluted sample / Vol. of seed in seed control) B1'= DO of seed control before incubation, mg/L B2'= DO of seed control after incubation, mg/L In calculations, do not make corrections for DO uptake in dilution water. (https://www.cutm.ac.in/pdf/Env%20Engg%20Lab%20Manual.pdf)

Chemical Oxygen Demand (COD) of the water sample Principle
COD is the measure of oxygen consumed during the oxidation of the oxidisable organic matter by a strong oxidising agent. Potassium dichromate in the presence of sulphuric acid is generally used as an oxidising agent. The excess of potassium dichromate was titrated against sodium thiosulphate using starch as an indicator. The amount of potassium dichromate used is proportional to the oxidisable organic matter present in the sample.
Procedure 50ml each of the sample were taken in three conical flask ↓ Simultaneously three distilled water blank standards were also taken ↓ 5ml of K 2 Cr 2 O 7 solution was added to each of the six flasks ↓ Flasks were kept in the water bath at 100ºC for 1hr ↓ Samples were allowed to cool for 10mins ↓ 5ml of potassium iodide was added to each of the flasks ↓ 10ml of H2SO4 was added to all the flasks ↓ The contents of each flasks was titrated against 0.1M sodium thiosulpahte until the appearance of pale yellow colour ↓ 1ml of starch solution was added, for the appearance of the blue colour ↓ Again it was titrated against the titrant until the blur colour disappears completely (Tridevi and Goel, 1986) COD of sample in mg/L =   fig. 4.1).

Standard plate count
Among the samples tested for safe human consumption, the well water sample had higher number of colonies i.e., 86 CFU/mL in 10 -3 dilution and 47 CFU/mL in 10 -5 dilution respectively. The least number of colonies were seen in bore well water (

Discussion:-
Life is possible because of water; hence use of water of good quality makes healthy life. However, physicochemical and biological properties of vary with sources of, types of water and geographical regions. There are certain chemicals present in water in higher or lower amount than the required ones similarly, there are number of pathogenic water borne protozoa, fungi, bacteria (Dubey et al., 2008).
Water microbiology is concerned with the microorganisms that live in water, or can be transported from one habitat to another by water. Water can support the growth of many types of microorganisms. This can be advantageous. For example, the growth of some bacteria in contaminated water can help digest the poisons from the water. However, the presence of other disease-causing microbes in water is unhealthy and even life threatening (Chapelle et al., 2000).
Drinking water is usually treated to minimize the risk of microbial contamination. The importance of drinking water treatment has been known for centuries. An important aspect of water microbiology, particularly for drinking water, is the testing of the water to ensure that it is safe to drink. Water quality testing can be done in several ways. In many countries, water microbiology is also the subject of legislation. Regulations specify how often water sources are sampled, how the sampling is done, how the analysis will be performed, what microbes are detected, and the acceptable limits for the target microorganisms in the water sample (Madigan et al., 2000).
Surface water and ground water samples were taken for various physico chemical parameters and biological parameters. The samples were collected from 4 different places in and around Mangalore using sterilized glass bottles in their respective environments and the bottles were brought back to laboratory for further analysis Under aseptic conditions selected physico chemical parameters and biological parameters were being tested. The results obtained were compared with the ISI (Indian Standard Institute) specifications and the samples were evaluated for drinking water standards.
In the present study, the physicochemical parameters tested were in the range specified for drinking water standards. Along with this test; samples were serially diluted and tested for spread plate method using PDA and Nutrient agar. Samples were also analyses for MPN that indicates the presence of faecal contamination. However, in this study the values obtained in the MPN as well as SPC method indicates that the bacterial count in the well water is slightly higher than the other sources obtained. Therefore, regular methods such as boiling and purification of water before consumption should be suggested.