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ST. ALOYSIUS INSTITUTE OF TECHNOLOGY

PRACTICAL FILEON

Environmental Engg. - II CE- (703)

SUBMITTED TO - SUBMITTED BY-

List of Experiment-

1. To study the various standards for waste water. (Theory)2. To study the sampling techniques for waste water. (Theory)3. To determine the alkalinity in water sample.4. To determine the acidity in water sample.5. Determination of Dissolved Oxygen in the water and waste water sample.6. Determination of Biological Oxygen demand of a waste water sample.7. Determination of Chemical Oxygen demand of a waste water sample.8. Bacteriological analysis of drinking water.9. Determination of various types of solids in the waste water sample.

INDEX

Sr. No.

Experiment Page No. Date of Experiment

Date of Submission

Remark/Sign

1To determine the acidity of the given sample of waste water.

2 To determine the amount of the following types of alkalinity present in the given samples:

3To determine the quantity of dissolved oxygen present in the given sample by using modified Winkler’s (Azide modification) method.

4 To determine the amount of B.O.D. exerted by the given sample.

5 To determine the Chemical Oxygen Demand (C.O.D.) for given sample.

6 Bacteriological analysis of drinking water

7 Determination of various types of solids in the waste water sample.

EXPERIMENT NO.-1

Aim : To determine the acidity of the given sample of waste water.Principle : Acidity of waste water is its quantitative capacity to neutralise a strong base to a designated pH. Strong minerals, acids, weak acids such as carbonic and acetic and hydrolysing salt such as ferric and aluminium sulphates may contribute to the measured acidity. According to the method of determination, acidity is important because acid contributes tocorrosiveness and influences certain chemical and biological processes. It is the measure of the amount of base required to neutralise a given sample to the specific pH. Hydrogen ions present in a sample as a result of dissociation or hydrolysis of solutes are neutralised by titrationwith standard alkali. The acidity thus depends upon the end point pH or indicator used. Dissolved CO2 is usually the major acidity component of unpolluted surface water. In the sample, containing only carbon dioxide-bicarbonatecarbonate,titration to pH 8.3 at 25°C corresponds to stoichiometric neutralisation of carbonic acid to carbonate. Since the colour change of phenolphthalein indicator is close to pH 8.3, this value is accepted as a standard end point for the titration of total acidity. For more complex mixture or buffered solution fixed end point of pH 3.7 and pH 8.3 are used. Thus, for standard determination of acidity of wastewater and natural water, methyl orange acidity (pH 3.7) and phenolphthalein acidity (pH 8.3) are used.Thus, in determining the acidity of the sample the volumes of standard alkali required to bring about colour change at pH 8.3 and at pH 3.7 are determined.APPARATUS1. Burette 2. Pipette3. Erlenmeyer flasks 4. Indicator solutionsREAGENTS

1. CO2 free water 2. Standard NaOH solution 0.02N 3. Methyl orange indicator solution 4. Phenolphthalein indicator solution 5. Sodium thiosulphate 0.1 N.

PROCEDURE 1. 25 mL of sample is pipetted into Erlenmeyer flask. 2. If free residual chlorine is present, 0.05 mL (1 drop) of 0.1 N thiosulphatesolution is added. 3. 2 drops of methyl orange indicator is added. 4. These contents are titrated against 0.02 N hydroxide solution. The end point is noted when colour change from orange red to yellow. 5. Then two drops of phenolphthalein indicator is added and titration continued till a pink colour just develops. The volumes of the titrant used are noted down.

OBSERVATION0.02 N NaOH × Sample (Methyl orange/phenolphthalein indicator)

Calculation

Model Viva Questions :-

Q1: What is pH?

A1: The pH of a solution is the negative logarithm of the hydrogen ion concentration. pH = log10 (H+ )

Q2: What is the effect of temperature on pH?

A2: The pH value of the solution increases with increase of temperature.

Q3: What is the pH of pure water at 250C?

A3: 7

Q4: What is effect of dilution on pH of an acidic solution?

A4: pH increases toward 7.

Q5: What does pH of a solution signify?

A5: It signifies the H3O+ ion concentration in moles per litre.

Q6: What does pH of a solution if it is acidic?

A6: pH of an acidic solution is less than 7.

Q7: What is an acid-base indicator?

A7: An acid-base indicator is an organic compound which changes its colour within certain pH range.

Q8: What is ionic product of water?

A8: Kw = [H3O+] [OH-]

Q9: What do you mean by universal indicator?

A9: It is a mixture of several indicators having different pH ranges.

Q10: Does addition of a salt having a common Ion on to weak acid change the pH of the solution?A10: Yes, the change the pH of solution increases

EXPERIMENT NO.2.Aim- To determine the amount of the following types of alkalinity present in the given samples.(a) Hydroxide alkalinity (b) Carbonate alkalinity (c) Bicarbonate alkalinity (d) Hydroxide–Carbonate alkalinity (e) Carbonate–Bicarbonate alkalinity PrincipleThe alkalinity of water is a measure of its capacity to neutralize acids. It is primarily due to salts of weak acids, although weak or strong bases may also contribute. Alkalinity is usually imparted by bicarbonate, carbonate and hydroxide. It is measured volumetrically by titration with 0.02 N sulphuric acid and is reported in terms of CaCO3 equivalent. For samples whose initial pH is above 8.3, the titration is conducted in two steps. In the first step, the titration is conducted until the pH is lowered to 8.2, the point at which phenolphthalein indicator turns from pink to colourless. This value corresponds to the points for conversion of carbonate to bicarbonate ion. The second phase of titration is conducted until the pH is lowered to 4.5, corresponds to methyl orange end point, which corresponds to the equivalence points for the conversion of bicarbonate ion to carbonic acid.Apparatus

1. Burette 2. Erlenmeyer flask 3. PipettesReagents1. Carbon dioxide free distilled water. 2. Phenolphthalein indicator. 3. Methyl orange indicator. 4. 0.1 N sodium thiosulphate solution 5. 0.02 N sulphuric acid. Procedure1. Pipette 50 mL of sample into a clean Erlenmeyer flask (V). 2. Add one drop of sodium thiosulphate solution, if residual chlorine is present. 3. Add two drops of phenolphthalein indicator; if the pH is above 8.3, colour of solution becomes pink. 4. Titrate against standard sulphuric acid in the burette, till the colour just disappears. Note down the volume(V1). 5. Then add two drops of methyl orange indicator, the colour turns yellow. 6. Again titrate against acid, until the colour turns to orange yellow. Note down the total volume (V2).


1. Why is water alkaline?

It is due to the presence of hydroxide, carbonate and bicarbonate ions.

2. What is the colour of phenolphthalein in acid and alkaline medium?

Colourless in acid and pink in alkaline.

3. What are the ions determined by phenolphthalein?

Hydroxide ion and half of carbonate ion.

4. What is the role of methyl orange indicator in determining alkalinity?

Total alkalinity is determined by using methyl orange.

5. Can OH, CO32-& HCO3-can exist together?

No, because OH- & HCO3- combine together to form carbonate ions

6- What do u mean by alkanity of water?It is defiend as the amount of standard acid in mg required to neutralizeonelitre of given water sample.

7 :Which constituent are responsible for alkalinity in water?

It is due to presence of hydroxide ion, carbonate ions and bicarbonate.

8. Why two indicators are used in determining alkalinity of water?

It is because of different Ph of water due to different ions causing alkalinity. The two indicators

give end point at different PH i.e. phenolphthalein at PH at 8-3 and methyl orange 3 to 4.5.

9; - How much alkalinity is permissible for drinking water?

It should be less than 100 ppm.

10 ;- On which factors the use of acid indicator in a titration depends ?

It depends upon the PH of the solution.

EXPERIMENT NO.3.

Aim- To determine the quantity of dissolved oxygen present in the given sample(s) by using modified Winkler’s (Azide modification) method.

PrincipleDissolved Oxygen (D.O.) levels in natural and wastewaters are dependent on the physical, chemical and biochemical activities prevailing in the water body. The analysis of D.O. is a key test in water pollution control activities and waste treatment process control.Improved by various techniques and equipment and aided by instrumentation, the Winkler (or iodometric) test remains the most precise and reliable titrimetric procedure for D.O. analysis. The test is based on the addition of divalent manganese solution, followed by strong alkali to the water sample in a glass-stoppered bottle. D.O. present in the sample rapidly oxidises in equivalent amount of the dispersed divalent manganous hydroxide precipitate to hydroxides of higher valency states. In the presence of iodide ions and upon acidification,the oxidised manganese reverts to the divalent state, with the liberation of iodine equivalent to the original D.O. content in the sample. The iodine is then titrated with a standard solution of thiosulphate.

Apparatus1. 300 mL capacity bottle with stopper 2. Burette 3. Pipettes, etc.

Reagents1. Manganous sulphate solution (MnSO4.4H2O) 2. Alkali-iodide azide reagent 3. Conc. sulphuric acid (36 N) 4. Starch indicator 5. Standard sodium thiosulphate solution (0.025N) 6. Standard potassium dichromate solution (0.025N)

Procedure1. Add 2 mL of manganous sulphate solution and 2 mL of alkali-iodide azide reagent to the 300 mL sample taken in the bottle, well below the surface of the liquid. (The pipette should be dipped inside the sample while adding the above two reagents.) 2. Stopper with care to exclude air bubbles and mix by inverting the bottle at least 15 times.

When the precipitate settles, leaving a clear supernatant above the manganese

3. hydroxide floc, shake again. 4. After 2 minutes of settling, carefully remove the stopper, immediately add 3 ml concentrated sulphuric acid by allowing the acid to run down the neck of the bottle. 5. Restopper and mix by gentle inversion until dissolution is complete. 6. Measure out 203 mL of the solution from the bottle to an Erlenmeyer flask. As 2 mL each of manganese sulphate and azide reagent have been added, the proportionate quantity of yellow solution correspondsto 200 mL of sample is

7.Titrate with 0.025 N sodium thiosulphate solution to a pale straw colour.8. Add 1–2 mL starch solution and continue the titration to the first disappearance of the blue colour and note down the volume of sodium thiosulphate solution added (V), which gives directly the D.O. in mg/L.

Model Viva Questions :-1.What method is used to determine dissolved oxygen?Iodometric method.2.What is Winkler’s reagent?Manganese sulphate, alkali iodide and concentrated sulphuric acid.3.What is the amount of dissolved oxygen present in pure water?7 - 9 mg/litre.4. What is the importance of dissolved oxygen?It is the fundamental requirement for survival of all aquatic livingorganisms.5.Why estimation of dissolved oxygen is of great significance?Decrease in dissolved oxygen in water leads to decay and death of aquatics and causes foul odour.6.What is the role of KI used in estimation?KI liberates iodine. Amount of iodine liberated is equal to dissolvedoxygen present.7.Complete the reactionMnSO4+ OH → Mn(OH)2

– white precipitate8.What is Winkler’s method?Estimating dissolved oxygen using manganese sulphate, alkali iodideand concentrated sulphuric acid.

EXPERIMEMT NO.4.

Aim- To determine the amount of B.O.D. exerted by the given sample(s).

Principle -The Biochemical Oxygen Demand (B.O.D.) of sewage or of polluted water is the amount of oxygen required for the biological decomposition of dissolved organic matter to occur under aerobic condition and at the standardized time and temperature. Usually, the time is taken as 5 days and the temperature 20°C as per the global standard. The B.O.D. test is among the most important method in sanitary analysis to determine the polluting power, or strength of sewage, industrial wastes or polluted water. It serves as a measure of the amount of clean diluting water required for the successful disposal of sewage by dilution. The test has its widest application in measuring waste loading to treatment plants and in evaluating the efficiency of such treatment systems. The test consists in taking the given sample in suitable concentrations in dilute water in B.O.D. bottles. Two bottles are taken for each concentration and three concentrations are used for each sample. One set of bottles is incubated in a B.O.D. incubator for 5 days at 20°C; the dissolved oxygen (initial) content (D1) in the other set of bottles will be determined immediately. At the end of 5 days, the dissolved oxygen content (D2) in the incubated set of bottles is determined.

Then, mg/L B.O.D. = (D1 – D2 )/P.

where, P = decimal fraction of sample used.D1 = dissolved oxygen of diluted sample (mg/L), immediately after preparation.D2 = dissolved oxygen of diluted sample (mg/L), at the end of 5 days incubation.

Among the three values of B.O.D. obtained for a sample select that dilution showing the residual dissolved oxygen of at least 1 mg/L and a depletion of at least 2 mg/L. If two or more dilutions are showing the same condition then select the B.O.D. value obtained by that dilution in which the maximum dissolved oxygen depletion is obtained.

THEORY- Biochemical Oxygen Demand (BOD) -The amount of oxygen required by the bacteria while stabilizing decomposable organic matter under aerobic conditions. Decomposable means that organic matter can serve as food for the bacteria and energy is derived from its oxidation.

Biochemical oxygen demand is a measure of the quantity of oxygen used by microorganisms (e.g., aerobic bacteria) in the oxidation of organic matter.

Natural sources of organic matter include plant decay and leaf fall. However, plant growth and decay may be unnaturally accelerated when nutrients and sunlight are overly abundant due to human influence.

Urban runoff carries pet wastes from streets and sidewalks; nutrients from lawn fertilizers; leaves, grass clippings, and paper from residential areas, which increase oxygen demand.

Oxygen consumed in the decomposition process robs other aquatic organisms of the oxygen they need to live. Organisms that are more tolerant of lower dissolved oxygen levels may replace a diversity of more sensitive organisms.

BOD Level (in ppm) Water Quality

1 – 2 Very Good-not much organic waste present

3 – 5 Moderately clean

6 – 9 Somewhat polluted

10+ Very pollute

Apparatus -

1. B.O.D. bottles 300mL capacity 2. B.O.D. incubator 3. Burette 4. Pipette 5. Air compressor 6. Measuring cylinder etc.

Reagents -

1. Distilled water 2. Phosphate buffer solution 3. Magnesium sulphate solution 4. Calcium chloride solution 5. Ferric chloride solution 6. Acid and alkali solution 7. Seeding 8. Sodium sulphite solution 9. Reagents required for the determination of D.O.

Procedure - 1. Place the desired volume of distilled water in a 5 litre flask (usually about 3 litres of distilled water will be needed for each sample). 2. Add 1mL each of phosphate buffer, magnesium sulphate solution, calcium chloride solution and ferric chloride solution for every litre of distilled water. 3. Seed the sample with 1–2 mL of settled domestic sewage. 4. Saturate the dilution water in the flask by aerating with a supply of clean compressed air for at least 30 minutes. 5. Highly alkaline or acidic samples should be neutralised to pH 7. 6. Destroy the chlorine residual in the sample by keeping the sample exposed to air for 1 to 2 hours or by adding a few mL of sodium sulphite solution.7. Take the sample in the required concentrations. The following concentrations are suggested:

Strong industrial waste: 0.1, 0.5 and 1 per cent Raw and settled sewage: 1.0, 2.5 and 5 per cent Oxidised effluents: 5, 12.5 and 25

per cent Polluted river water: 25, 50 and 100 per cent 8. Add the required quantity of sample (calculate for 650 mL dilution water the required quantity of sample for a particular concentration) into a 1000 mL measuring cylinder. Add the dilution water up to the 650 ml mark. 9. Mix the contents in the measuring cylinder. 10. Add this solution into two B.O.D. bottles, one for incubation and the other for determination of initial dissolved oxygen in the mixture. 11. Prepare in the same manner for other concentrations and for all the other samples. 12. Lastly fill the dilution water alone into two B.O.D. bottles. Keep one for incubation and the other for determination of initial dissolved oxygen. 13. Place the set of bottles to be incubated in a B.O.D. incubator for 5 days at 20°C. Care should be taken to maintain the water seal over the bottles throughout the period of incubation. 14. Determine the initial dissolved oxygen contents in the other set of bottles and note down the results. 15. Determine the dissolved oxygen content in the incubated bottles at the end of 5 days and note down the results. 16. Calculate the B.O.D. of the given sample.

Note: The procedure for determining the dissolved oxygen content is same as described in the experiment under “Determination of dissolved oxygen”.


1. Why is it important to have a water tight seal on my BOD bottles?

-As a precaution against drawing air into the BOD bottle during the 5-day incubation, you must have a water tight seal.

Having a water tight seal will prevent the following:-

Possible contamination A change in the volume of solution in the bottle The bottle stopper from becoming stuck

2. What type of BOD bottles should I use?

-To ensure a water tight seal, InterLab Supply recommends using Wheaton’s 300-mL BOD bottles. Wheaton’s BOD bottles have a flared mouth and use tapered ground-glass stoppers which are the bottle features recommended in The Standard Methods for Examination of Water and Wastewater.

3. Why is it important to run the BOD test?

-Most importantly, the BOD test is required by law. The BOD test is used to determine how much oxygen is being used by aerobic microorganisms in the water to decompose organic matter. If these aerobic bacteria use too much of the dissolved oxygen in the water, there will not be enough left over for the fish, insects, and other organisms that rely on the oxygen to live. In this situation, the rich diversity of life in a healthy river is reduced to a low diversity (but sometimes high volume) of pollution-tolerant organisms.

4. Why is it important to read my 5-day DO±6 hours of incubation?

-You want to keep a tight time fram when you read your final DO. The 21st edition of Standard methods now suggests that reading the final DO ± 6 hours will yield the most accurate test results.

5.hat temperature should I store my BOD bottles during the 5-day incubation period?

-Standard Methods states your bottles should be stored at 20 ± 1°C throughout the 5-day incubation period. Remember, temperature directly affects the metabolic rate of the bacteria. Incubating your bottles at 20 ± 1°C provides the perfect "controlled" environment for the bacteria to reproduce.

6. Why is it important to store my BOD bottles in the dark during the 5-day incubation period?

-Excluding all light prevents the possibility of photosynthetic (algae) production of DO.

7.Why is it important not to have any bubbles present in any the BOD bottles?

-You do not want to add additional oxygen to your samples. Therefore, no visible buttles should be present during preparation, or any time during the test.

EXPERIMENT NO. 5

Aim - To determine the Chemical Oxygen Demand (C.O.D.) for given sample.

Principle -

Potassium dichromate is a powerful oxidising agent in acidic medium and is obtained in high state of purity.

The reaction involved is:CnHaOb + cCr2O72– + 8cH+ = nCO2 + (a+8c)/2 H2O + 2cCr3+

where, c = 2/3n + a/6 – b/3

C.O.D. results are reported in terms of mg of oxygen. N/8 or 0.125 N solution of oxidising agent is used in the determination. Normality double the strength is used. This allows the use of larger samples. Thus, each ml of 0.25 N solution dichromate is equivalent to 2 mg of oxygen. An excess of oxidising agent is added, the excess is determined by another reducing agent such as ferrous ammonium sulphate. An indicator ferroin is used in titrating the excess dichromate against ferrous ammonium sulphate. Blanks are used also treated and titrated to get the correct value of C.O.D.

Apparatus - 1. Reflux apparatus 2. Burettes 3. Pipettes

Reagents - 1. Standard potassium dichromate solution 0.25N. 2. Sulphuric acid reagent. 3. Standard ferrous ammonium sulphate. 4. Ferroin indicator solution. 5. Mercuric sulphate. 6. Sulphuric acid crystals.

Procedure - 1. Place 50.0 mL of sample in a 500 mL refluxing flask. 2. Add 1g mercuric sulphate and a few glass beads. 3. Add sulphuric acid to dissolve the mercuric sulphate and cool. 4. Add 25.0 ml 0.25 N potassium dichromate solution and mix well. 5.Attach the flask to the condenser and start the cooling water.

5. Add the remaining acid reagent (70 mL) through the open end of condenser and mix well. 6. Apply heat and reflux for 5 hours. 7. Cool and wash down the condenser with distilled water. 8. Dilute the mixture to about twice its volume and cool to room temperature. 9. Titrate the excess dichromate with standard ferrous ammonium sulphate using ferroin indicator (2 to 3 drops). 10. The colour change from blue green to reddish indicates the end point. 11. Reflux in the same manner a blank consisting of distilled water of equal volume as that of the sample.


1) Why COD values are always higher then BOD values?

Biochemical oxygen demand (BOD) is a measure of the amount of oxygen that bacteria will consume while decomposing organic matter under aerobic conditions or biochemical oxygen demand only measures the amount of oxygen consumed by microbial oxidation and is most relevant to waters rich in organic matter Whereas Chemical oxygen demand (COD) does not differentiate between biologically available and inert organic matter, and it is a measure of the total quantity of oxygen required to oxidize all organic material into carbon dioxide and water. COD values are always greater than BOD values, but COD measurements can be made in a few hours while BOD measurements take five days.

2.What would be the role of Ag2SO4 in COD determination?

Silver Sulphate catalyses the reaction and also assists in the oxidation of the nitrogen compounds. The secondary catalyst, Silver Sulfate (AgSO4) assists oxidization of straight-chain hydrocarbons such diesel fuel and motor oil. The reaction between chloride and silver sulfate creates silver chloride (AgCl). The reduction of silver sulfate correspondingly reduces the activity needed to oxidize straight chain hydrocarbons, a negative interferent one would think. Chemical Oxygen Demand

3. Write the NEQS for COD.

National Environmental Quality Standards for Municipal and Liquid Industrial Effluents (mg/L, Unless Otherwise Defined)

Sr. No Parameter Existing

Standards

Revised StandardsInto Inland

WaterInto Sewage Treatment Into Sea

1 Chemical Oxygen Demand (COD)1 150 mg/l 150 400 400

4. Compare BOD and COD.

Chemical oxygen demand (COD) is a measure of the capacity of water to consume oxygen during the decomposition of organic matter and the oxidation of inorganic chemicals such as ammonia and nitrite. COD measurements are commonly made on samples of waste waters or of natural waters contaminated by domestic or industrial wastes. Chemical oxygen demand is measured as a standardized laboratory assay in which a closed water sample is incubated with a strong chemical oxidant under specific conditions of temperature and for a particular period of time. A commonly used oxidant in COD assays is potassium dichromate (K2Cr2O7) which is used in combination with boiling sulfuric acid (H2SO4). Because this chemical oxidant is not specific to oxygen-consuming chemicals that are organic or inorganic, both of these sources of oxygen demand are measured in a COD assay.

Chemical oxygen demand is related to biochemical oxygen demand (BOD), another standard test for assaying the oxygen-demanding strength of waste waters. However, biochemical oxygen demand only measures the amount of oxygen consumed by microbial oxidation and is most relevant to waters rich in organic matter. It is important to understand that COD and BOD do not necessarily measure the same types of oxygen consumption.

5. Write the applications of COD data to environmental Engineering?

Chemical Oxygen Demand COD test is a measure of the relative oxygen-depletion effect of a waste contaminant. It has been widely adopted as a measure of pollution effect. To determine the amount of pollution in a water stream to try to control and limit the amount of chemicals that can pollute the lakes and rivers if left in a final effluent or discharge stream. Some Municipalities want to measure the amount of chemicals in the incoming stream in order to asses surcharges as a way of measuring how much additional treatment their plant will have to do in order to get the incoming water clean. It is used in Process Control in Influent/effluent for removal efficiencies.Chemical Oxygen Demand COD is extensively used in analysis of industrial waste. It is particularly valuable in surveys designed to determine the losses of sewage. Results are obtained within short time and control measures can be taken on the same day. It is very useful in finding out the toxic condition and presence of biologically resistant organic substance.Chemical oxygen demand is a vital test for assessing the quality of effluents and waste waters prior to discharge. The Chemical Oxygen Demand (COD) test predicts the oxygen requirement of the effluent and is used for monitoring and control of discharges, and for assessing treatment plant performance. The impact of an effluent or waste water discharge on the receiving water is predicted by its oxygen demand. This is because the removal of oxygen from the natural water reduces its ability to sustain aquatic life. The COD test is therefore performed as routine in laboratories of water utilities and industrial companies. Chemical Oxygen Demand

EXPERIMENT NO. 6

Aim - Bacteriological analysis of drinking water

TheoryThe most common and widespread danger associated with drinking water is contamination, either directly or indirectly, by sewage, other wastes or human and animal excrement1. About 25 years ago, authoritative estimates indicated that each year some 500 million people are affected by water-borne or water associated disease, and as many as 10 mfflion of these die2. In a recent estimate based on WHO reports suggests that 80% of all human illnesses in the dcveloping world are caused by biological contamination3. Faecal pollution of drinking water may introduce a variety of intestinal pathogens. Their presence being related to microbial diseases and carriers present in the community, which may cause diseases from mild gastroentritis to severe and sometimes fatal dysentry, cholera or typhoid. Other organisms, naturally present in the environment and not regarded as pathogens, may also cause opportunist disease1. Ideally, drinking water should not contain any microorganisms known to be pathogenic. It should be free from bacteria indicative of pollution with excreta4. The majority of the population in developing countries is not adequately supplied with potable water, and thus obliged to use unsafe water for domestic and drinking purposes5 Pakistan, a developing country, is also facing a problem of wholesome water supply. This study was initiated to determine bacterial loads and contaminants in drinking water in and around Islamabad as water quality guidelines form a basis for judgement of the acceptability of public drinking water supplies5.

MATERIAL AND METHODS Sample collection:One hundred and twelve water samples were aseptically collected in sterilized bottles from various sources in and around Islamabad. Sampling was done from May to October, 1984. Samples were collected from wells, springs, rivers and municipal tap water supplies. All samples were immediately transported to the laboratory and processed within two hours.Sample Processing:A. pH of water: pH of all the water samples was recorded by means of a pH meter.

B. Bacteriological Analysis:

i) Presumptive test for coliformsa. Untreated water samples: Water samples were processed as described by Rand et al6. Briefly, five tubes of double strength lactose broth (containing durham tube) were inoculated with 10 ml water sample (in each tube) and two tubes of single strength with 1.0 ml and 0.1 ml respectively. After incubation at 35°C for 48 hours production of acid and the presence of gas in any of the durham tubes was considered positive. Number of the positive tubes was recorded and most probable number (MPN) was calculated according to MPN tables.

b. Treated water samples: In case of chlorinated or sand filtered water, 50 ml of double strength Mac-Conkey's broth was inoculated with 25 ml ofwáter sample and incubated at 35°C for 48 hours7. The rest of the procedure was same as for untreated water samples.

ii) Confirmatory test for faecal coil-forms:One ml from each positive tube of presumptive coliforms was inoculated in Brilliant Green Lactose bile Broth (BGLB) tube. After incubation at 44.5°C in a water bath for 24 hours; tubes with gas and turbidity were considered positive. Positive tubes were further cultured on Eosine Methylene Blue agar (EMB) for isolation of faecal coliforms7. Isolated colonies were confirmed by using biochemical tests10 as well as Systek kit No.1.

iii) Confirmatory test for faecal streptococci:Positive tubes of presumptive coliform test were subcultured in glucose broth and incubated for 2 hours at 37°C. Sodium azide (0.25 gm/500 ml) was then added and incubation carried out at 44.5°C for a further 48 hours. Positive tubes showing acid were subcultured on MacConkey agar plates and incubated at 37°C for 24 hours; The presence of small red pinpoint colonies were indicative of Streptococcus faecalls. Gram staining and the production of acid in mannitol and lactose only, but not in raffinose, confirmed their presence.

iv) Standard Plate Count (SPC):

The standard plate count was done by pour plate technique using 10 fold dilutions (upto 10-6) in ringers solution. One ml of each dilution was poured (duplicates) in empty, sterilized petridishes. About 12 to 15 ml of plate count agar (kept at 45°C in a waterbath) was added to each plate. Plates after solidification were incubated at 37°C for 24 to 48 hours. Plates showing 3 0—300 colonies were counted to determine the SPC per ml of sample tested.

v) Analysis of other enteric pathogens.

Salmonella, Shigella and vibrio spp. Fifty ml of selenite broth and same quantity of alkaline peptone water in flasks were inoculated with 25ml water (in each flask). After incubation for 16-18 hours at 37°C subcultures from the former was made on Xylose Lysine Dextrose agar (XLD) plate and on Thiosulphate Citrate Bile salt Sucrose agar (TCBS) from the later. Plates were incubated at 37°C for 24 hours. Suspected colonies were identified biochemically and serologically.Results - The water samples investigated in this study were mainly analysed for the bacteriological contamination. The pH of the water samples (112) fall between pH 7—8.3. Water samples were analysed categorized in two different groups (Table I).

Out of 57 untreated water samples, 46 (8 1%) were found positive for the presence of coliforms while out of 55 treated water samples, 21 (38.0%) were found positive for the same. The samples which were found positive for coliform presumptive test underwent confirmatory test. Out of 67 positive samples 26 were confirmed for the presence of faecal coliforms. In addition 6 water samples were found positive for Streptococcus faecalis. Load of Viable aerobic bacteria per ml of the water sample was determined, (Table II).Twenty nine (26%) specimens showed no growth on the plate count agar and were mostly from treated water samples. Other samples enumerated counts between < 10 to> 106/ml. Escherichia coli (27%) was the major pathogen among the other bacterial isolates (Table III)

followed by Streptococcus faecalis (6%) and Staphylococcus aureus (2%). Other organisms were Pseudomonas Spp. (20%), enteric bacteria (14.5%) and environmental bacteria (30%). Some of the samples contained more than one isolate. All the water samples investigated for the presence of Salmonella, Shigella and Vibrio cholerae were found negative.

Discussion An acceptable pH for drinking water is between pH 6.5 to pH 8.5, recommended by WHO as a guideline value and in the absence of a distribution system acceptable range may. be broader. AU the water samples examined in this study were in acceptable pH range.For the presumptive coliforms test, the WHO guideline for both treated and untreated water samples is 0/ 100 ml4, but in an occasional untreated water sample 3 coliform/100 ml are allowed on the condition that these would not be found in consecutive water samples. 12 The coliform group as an indicator bacteria are used to evaluate the potability of drinking water and the presence of any coliform organisms is an indication of a contaminated source, inadequate treatment or post treatment contamination13. In unpiped water supplies, sometimes upto 10 coliforms/l00 ml are allowed but they should not occur repeatedly; if occurrence is frequent and sanitary protection cannot be improved, an alternative source must be found if possible. 12 In this study 81 % of the untreated and 38% of the treated water samples were positive for MPN, showing a high contamination and risk to public health The detection of faecal (Thermotolerant) coliform organisms provide definite evidence of faecal pollution4 and they were found in 39% of the positive samples. Search for Streptococcus faecalis is not carried out routinely. Its main value is when doubt is expressed that large number of irregular types of coliforms found in a sample of water are of faecal origin. Confirmation of faecal pollution would then rely on finding £ faecaiis in the water8. Since they survive longer in water than coliform bacteria they should be referred as indicator of faecal pollution in water and shelfish. In the present study they

were found only in 6% of the water samples examined. The main value of colony counts lies in the comparison of results obtained from regular samples from the same supply so that any significant change from the normal range in a particular location can be detected. As the SPC in most of the untreated water samples (Table II) was very high, it is therefore desirable to disinfect all supplies of drinking water before distribution. Supplies derived from protected sources which are distributed without disinfection should be similar in quality to that of disinfected drinking water. Where it is impracticable to supply water to consumers through a piped distribution network and where untreated sources such as wells, bore-holes and springs which may be naturally pure must be used, considerable reliance should be placed on sanitary examination and not exclusively on the results of bacteriological examination.The high percentage of E. coil (27%) provides a definite evidence of faecal pollution in water.Staphylococcus aureus (2%) which is relatively recently accepted as indicator organisms in food and water, provides a useful indication that faecal contamination has occurred in water. Pseudomonas spp. are common inhabitant of soil and water and found in small numbers in the faeces of man and animals. These were isolated in about 20% samples and are of public health importance as some’ species cause a variety of suppurative infections in man. Enterotoxigenic strains of pseudomonas spp. alongwith other species of Enterobacter, Kiebsiella and Acinetobacter have been isolated from cases of infantile diirrhoea in Addis Ababa during surveys in 1974 and 1977.Enterotoxigenic species of proteus with other enterotoxigenic bacteria have also been reported in a study on food and water from an Ethiopian community. Environmental bacteria (Table Ill) include Alcaligenes spp., Acinetobacter spp., Yeast spp. and Bacillus spp. which are usually found in soil. Presence of such high bacterial counts and presence of faecal coliforms and other indicator organisms as Streptococcus faecaiis, Staphy - lococcus aureus indicate inadequate treatment, post treatment contamination and contaminated water sources. Therefore, everything possible should be done to prevent pollution of the drinking water, special attention being given to the safe disposal of excrement. But the significance of routes of transmission other than drinking water should not be underestimated as the provision of a safe potable water supply by itself will not necessarily prevent infection without accompanying improvement in sanitation and personal habits. Education in simple hygiene is also essential.


1.How is an SPC performed?

A sample of product is blended in an appropriate solution and aliquots of the suspension, after dilution as necessary, are applied to the medium. The inoculated plate is incubated under required conditions and after a specified time, the number of visible colonies is counted. The results are typically expressed as colony forming units (C.F.U.)/g. or /mL.

2. What is the purpose of an SPC?Obtaining an estimate of the number of microorganisms in a feed product can be used to evaluate sanitary practices during processing and handling. It can also be used to determine potential sources of contamination by testing line samples taken at successive stages of receiving, storage, processing, transport, and feeding. Selective testing for pathogens, is costly, time consuming and risky. SPC is generally a cheaper and quicker test. 3.What are the limitations?

SPC measures most microbiological growth, but does not differentiate between the naturally occurring bacteria, yeast, molds, etc. and the pathogenic or spoilage organisms.

EXPERIMENT NO. 7

Aim - Determination of various types of solids in the waste water sample.

Theory-

Solids refer to matter suspended or dissolved in water or wastewater. Solids may affect water or effluent quality in a number of ways. Water with high dissolved solids is generally of inferior palatability and may induce unfavorable physiological response (intestinal distress) in the transient consumer (i.e tourists). Highly mineralized waters are unsuitable for many industrial applications. High suspended solids content can also be detrimental to aquatic plants and animals by limiting light and deteriorating habitat.

Total solids - includes all solids present in a water sample. Determined directly by evaporating a known volume of an unfiltered water sample in a 105 oC oven.

Total dissolved solids - includes all solids present in a water sample filtered through a 1.2 m m filter. Determined by evaporating a known volume of the filtrate sample in a 105 oC oven.

Total suspended solids - includes all solids present in a sample that remain on a 1.2 m m filter. Determined by filtering a known volume of sample and placing the filter and filter container in a 105 oC oven for 24 hours to evaporate the water.

Fixed solids - solids that remain after firing a sample in a 550 oC muffle furnace. Can be performed on total, dissolved, or suspended samples to determine - total fixed solids, fixed dissolved solids, or fixed suspended solids.

Volatile solids - solids that removed by firing a sample in a 550 oC muffle furnace. Can be performed on total, dissolved, or suspended samples to determine - total volatile solids, volatile dissolved solids, or volatile suspended solids.

Procedure -

Total Solids:

Weigh an evaporation dish to the nearest 0.1 mg. Accurately measure a known volume (as much as will fit without spilling) of your sample into an evaporation dish and place in the 105 oC oven. After all of the water has evaporated, let the dish cool in a dessicator, weigh the dish plus the remaining solids and record.

Total Suspended Solids: Weigh and identify your crucible/filter. Filter as much of your sample as possible (usually 300-400 mL) through the crucible/filter combination. Place the crucible in the 105 oC oven for 24 hours. Let cool to room temperature in a dessicator. Weigh the crucible plus the solids and record. This is the total suspended solids.

Fixed and Volatile Suspended Solids: After measuring the total suspended solids, fire the crucible in a 550 oC muffle furnace for 1 hour. Weigh the crucible plus the remaining solids. This is the fixed suspended solids. The difference between the total and fixed suspended solids is the volatile suspended solids.

Calculations (See T&S figure 2.7 p.60 for a more complete discussion of the relationships between the different solids fractions. Also note that the balance weighs in units of grams, g, and the equations below require milligrams, mg.):

Total solids:

A = weight of dried residue + dish after 24 hrs at 105 oC (mg) B = weight of dish (mg)

Total suspended solids:

A = weight of crucible + filter + residue after 24 hrs at 105 oC (mg) B = weight of crucible + filter (mg)

Volatile and Fixed suspended solids:

A = weight of crucible + filter + residue after 24 hrs at 105 oC (mg) B = weight of crucible + filter + residue after 1 hr at 550 oC (mg) C = weight of crucible + filter (mg)


1.The pore size of filter paper used in filtration isAns- 0.2µm or smaller

2.The crucible used in experiment is made ofAns- porcelain

3.Total suspended solid are mostly responsible forAns- Turbidity

4.The chemical substance used in desiccators isAns – calcium chloride

5.The dissolved solids that imposed BOD areAns- Volatile solids

6.Define Total solids?Total solids - includes all solids present in a water sample. Determined directly by evaporating a known volume of an unfiltered water sample in a 105 oC oven.

7.Define Total dissolved solids?

Total dissolved solids - includes all solids present in a water sample filtered through a 1.2 m m filter. Determined by evaporating a known volume of the filtrate sample in a 105 oC oven.

8.Define Total suspended solids ?

Total suspended solids - includes all solids present in a sample that remain on a 1.2 m m filter. Determined by filtering a known volume of sample and placing the filter and filter container in a 105 oC oven for 24 hours to evaporate the water.

9.Define Fixed solids

Fixed solids - solids that remain after firing a sample in a 550 oC muffle furnace. Can be performed on total, dissolved, or suspended samples to determine - total fixed solids, fixed dissolved solids, or fixed suspended solids.

10.Define Volatile solids

Volatile solids - solids that removed by firing a sample in a 550 oC muffle furnace. Can be performed on total, dissolved, or suspended samples to determine - total volatile solids, volatile dissolved solids, or volatile suspended solids.

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