Civl407 Labmanual09

CIVL407
UNIVERSITY OF BRITISH COLUMBIA

Department of Civil Engineering
Laboratory Exercise
Manual
www.civil.ubc.ca/home/env_lab/CIVL407_LABMANUAL09.pdf
CIVL407 ENVIRONMENTAL LABORATORY ANALYSIS
Laboratory Exercise Guide
University of BC
Department of Civil Engineering,
Environmental Engineering Laboratory, Rm 1301
6250 Applied Science Lane
Vancouver, BC, Canada, V6T 1Z4
Table of Contents
Course Description .................................................................................. 1
SCHEDULE: ................................................................................................. 1
Station/Drawer Supply List ..................................................................... 2
First Session: Laboratory Safety and Orientation ................................ 3
Laboratory Report Write-Up Guidelines ................................................ 5
Laboratory Exercises .............................................................................. 6
Laboratory 1: Solids Determination ....................................................... 7
Laboratory 2: COD, DO and BOD5 ........................................................ 11
Laboratory 3: Chloride Determination ................................................. 16
Laboratory 4: Chlorine and Chlorine Demand .................................... 23
Laboratory 5: Bacteriological Examination of Sewage

...................... 28
Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity

... 38
Laboratory 7a: Coagulation and 7b: Coagulation & Softening .......... 46

C I V L 4 0 7 E N V I R O N M E N T A L L A B O R A T O R Y A N A L Y S I S
Course Description
Testing procedures used in water quality studies and in the operation of water and
wastewater treatment plants. Prerequisite Chem 151.
SCHEDULE:
Lecture: Room MCML 160 Wednesday 1:00 pm
Laboratory CEME1301:
1) Tuesday (time to be arranged by consensus)
2) Wednesday 2:00 to 5:00 pm
3) Thursday (time to be arranged by consensus)
4) Friday (time to be arranged by consensus)
(Note: Lab Session must have at least 4 students to run)
Date 2009 Subject
Sep. 9 - 11 No Lab
Sep. 16 only Laboratory Safety & Orientation (~1 hr)
Sep. 22 - 25 #1 Solids determination
Sep. 29 – Oct 2 #2 Dissolved oxygen, COD, BOD
Oct. 6 - 9 #3 Chloride
Oct. 13 - 16 No lab - Thanksgiving week - catch up on lectures
Oct. 20 -23 #4 Chlorine demand
Oct. 27 - 30 #5 Bacteriological Examination of waste water
Nov. 3 – 6 #6 Acidity, alkalinity, hardness, colour, turbidity
Nov. 10 - 13 No lab - Remembrance Day - catch up on lectures
Nov. 17 – 20 #7a Coagulation
Nov. 24 - 27 #7b Coagulation and Softening
Dec. 1 - 4 No lab
Lecturer: Dr. Eric Hall email: ehall@civil.ubc.ca 604-822-2707

Lab instructor: Paula Parkinson email: parkin@civil.ubc.ca 604-822-4397
Teaching Assistants:
Lab TA: Alireza Abedini email: ali.abedini@yahoo.ca 604-827-5369
Marker TA: Soubhagya Pattanayak (Pattu) email: soubhagya.pattanayak@gmail.com
1
Station/Drawer Supply List

The following items will be maintained in each stations supply drawer or at station on bench
when required:
2 permanent marker pens

2 stir bars and a bar retriever
1 stirring motor (plate)
2 small buret funnels
4 30 ml beakers
4 50 ml beakers
2 100 ml beakers
2 150 ml beakers
2 250 ml beakers
2 1 L plastic beakers
2 600 ml plastic beakers
2 400 ml plastic beakers
2 10 ml grad cylinders
0-14 pH strips
Thermometer
2 Stir stick and/or spatula
Tweezers (Millipore or Gelman for membrane filters))
On benches each row:

Latex gloves- small, medium and large
Non-latex/Nitrile gloves (kept on reserve for latex sensitive persons only)
On benches as required:
Filter racks with funnels
#4 Whatman filter paper or equivalent
Vacuum flasks and hoses
8 1 L grad cylinders
8 3 L plastic beakers
2
First Session: Laboratory Safety and Orientation

1. NO Food or drink. No sandals or open-toe or heel shoes. Solid leather best.
2. Personal Protective Equipment: Lab coat (long), eye protection, gloves as required.
3. Sanitation: antibiotic soap for hand washing, disinfectant for bench surfaces.
4. Hygiene: At times you will be working with sewage. Be aware that handling your pens,
calculators, packs, etc. with contaminated gloves will contaminate those items. Putting
pens in your mouth that may have been on the bench or handled with gloves could be
hazardous. Wearing lab coats out of the lab is forbidden. Please take your lab coat away in
a plastic bag or leave in assigned drawer.
5. Safety Equipment: Eye Wash Station, Safety Shower.
6. WHMIS: Workplace Hazardous Materials Information System is a government regulation

requiring that information about hazardous substances be obtained, read and understood
before using it. MSDS: is a supplier information sheet that provides this information.
7. Spills: Wipe up all spills immediately, wash down, disinfect and dry the bench. Please ask
for assistance if chemicals are spilled.
8. Broken Glass: Be careful not to break glass, but if you do please ask for assistance. DO
NOT PUT BROKEN GLASS INTO THE REGULAR GARBAGE CONTAINER. If you cut
yourself, no matter how minor, please see TA or instructor for help.
9. Clean-up: You must leave your work station as you found it - clean and dry. All glassware
must be rinsed well (at least 5 times with tap water) and put on paper towels at your station
or on drying racks, if there is room. Do not put lids and small items onto racks that may slip
through to the filthy drip tray. Remember to empty and rinse burets as well. Pipettes must
be placed with tips pointing up into the pipet basket supplied.
10. Pipettes/ graduated cylinders/ beakers/ flasks: Information:

a. Volumetric pipettes are the most accurate way to measure volume. They are
available in 0.5, 1.0, 2.0...up to 10.0 ml, 15, 20, 25, 50 and 100 ml. These should
be used whenever standard solutions are measured or when upmost accuracy is
required.
b. Most pipettes are calibrated ―to deliver‖ or TD and should never be blown out.
They are calibrated by weighing the volume of distilled water that will flow from
them by gravity, with the tip against the side of the receiving vessel. A small
amount of liquid always remains in the tip and must not be blown out.
c. Some pipettes may be ―to deliver with Blow-Out‖. They are calibrated the same
way as TD except that the remaining drop is blown into the receiving vessel.
They are usually identified by a double etched or coloured band at the top of the
pipette.
d. Graduated or ―measuring‖ pipettes are not as accurate as volumetric pipettes
and the one having the maximum capacity closest to the volume required is the
3
most accurate one to select. Some are blow-out, some are not meant to drain
completely - pay attention to the markings.
e. Graduated cylinders: Not as accurate as pipettes, especially at smaller volumes.
They are useful for measuring samples 20 mls - 1000 mls. Use the size of
cylinder CLOSEST TO THE VOLUME YOU WISH to measure for best accuracy.
Ie If you want to measure 20 mls, do not use a 1 L graduated cylinder. Cylinders
are available: 5 mls, 10 mls, 25 mls, 50 mls, 100 mls, 250 mls, 500 mls, 1, 2 & 4
L
f. Beakers: are cylindrical in shape with straight sides and flat bottoms; markings
are not accurate at all - merely approximations. Used to hold/transfer
approximate volumes of samples, chemicals or for titrations when accuracy is not
required.
g. Flasks: Volumetric flasks are very accurate and should be used when diluting
standards (together with volumetric pipettes). Erlenmeyer or conical flask
markings are not accurate. They are containers best suited for ―swirling‖ liquid ie
to mix reagents with samples in a colourimetric determination (Lab 4 or 6).
11. NEVER mouth pipette. Pipette bulbs or pumps must be used and great care must be
taken not to contaminate bulbs. A demonstration will be given. If liquid should enter a bulb
or pump, please rinse out immediately and set aside to dry. Do not squish bulbs in the
direction of another person.
12. Gas/air/vacuum taps: Do not touch these unless instructed to do so. Undetected gas
leaks can be deadly. Even the force of air from the air taps can be dangerous because it
can contain grit or water.
13. Cup sinks: Please do not use these sinks for the disposal or delivery of liquids. Taps can
be unpredictable in force and are close to eye level. Chemical residues can be splashed in
your face.
14. Stir bars: Please remove from containers before pouring contents down the drain.
15. Glassware/supplies: Each station has a drawer where some glassware and other useful
tools will be kept. Additional supplies are available in the supply room (around the corner).
Pipettes are in drawers indicated.
16. Chemicals will be put out as required for each lab and should be used sparingly, not
wastefully as quantities are limited. Make sure that you label beakers and flasks to avoid
confusion and waste.
4
Laboratory Report Write-Up Guidelines
Guidelines for laboratory report write up are also given in the CIVL407 Manual Course Notes
and summarized here. The short report is intended to be a concise yet complete laboratory
report whereas the long report is more elaborate. The mark allocation for some sections for
both reporting format is indicated in parenthesis in the table below. Please note that the
marking outline is intended only as a guideline to assist in report write-up and that marks would
also be allocated for overall presentation and clarity of the report.
REPORT ELEMENTS SHORT REPORT LONG REPORT

LABS 1 TO 6 LAB 7
Title page Required Required
Objective Required Required
Materials and Method Details are not required. Required
State that the procedures Include a brief description of
were in accordance with the procedure. Sufficient detail
CIVL 407 Lab Manual should be provided so that what
and note any deviations was done could be understood
to the manual’s and the experiment could be
procedure. repeated from the procedure
reported.
Observations and Results Required [1] Required [2]
Sample Calculations Required [2] Required [2]
Discussion Brief discussion required Required [5]
Including sources of error [2]
Conclusions Required Required [2]
Questions and Answers Required [4] Required [6]
Other Students’ Data and - Required [1]
Results
References Required Required
Presented in an
acceptable format.
Total Mark [10] [20]
5
Laboratory Exercises
#1 Solids determination
#2 Dissolved oxygen, COD, BOD
#3 Chloride
#4 Chlorine demand
#5 Bacteriological Examination of waste water
#6 Acidity, alkalinity, hardness, colour, turbidity
#7a Coagulation
#7b Coagulation and Softening
6
Laboratory 1: Solids Determination
WARNING OBJECT:
-to become familiar with a number of the most common sewage
You will be handling treatment plant tests and to illustrate some of the difficulties in performing
sewage samples which
these tests; to measure sewage treatment plant efficiency in removing
may contain
pathogenic bacteria.
residue.
Use pipette bulbs, wipe
up all spills and wash MATERIALS:
with disinfectant, keep -Imhoff cones, samples of raw sewage, sludge, final effluent, oven
hands and pencils muffle furnace, desiccators, balance, evaporating dishes, tin dishes,
away from your mouth graduated cylinders, distilled water bottles.
and wash hands
frequently. A. SETTLEABLE SOLIDS
1. Mix the samples thoroughly and fill individually marked Imhoff cones to
Wear PPE at all times.
the 1 litre mark. A 1 L graduated cylinder may be used in place of the
Imhoff cone for the sludge sample, if necessary.
2. Settle for 45 minutes, gently dislodge any solids that have clung to the
sides using a stirring rod, settle for 15 minutes longer, and record the
volume of settleable solids. If large pockets of liquid form between the
particles of settled matter, subtract an estimated volume from the
measured volume of matter. Do not include any surface floating material as
settled solids.
(Note: Sludge Volume Index is the volume occupied by 1 gram of sludge after
30 minutes of settling. This is a different test to the one we are doing using
Imhoff cones and should not be confused.)
B. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550oC

1. Obtain the tare weight of a properly prepared porcelain evaporating dish (i.e. one that has
been washed, dried, pre-fired and desiccated - done for you, prior to session).
2. Mix the sample in the carboy thoroughly, subsample enough for Part B and Part C into a
beaker and then, immediately after stirring the beaker, rapidly pour sample into a 50 ml
graduated cylinder to approximately equal 35-40 mls (if using 50 ml dishes), recording the
exact volume in your table. [Notes: to allow for rinsing and to avoid spillage when
transferring dish to oven, don't pour more than 40 mls of sample]. For the smaller dish,
meant for high solids sample (aerobic sludge), use a 10 ml grad. cylinder and pour ~10
mls.
3. Pour the sample into the dish.[Note: it may be harder to wash out solids if you allow them
to settle before pouring into the dish - so be quick.] Using very small amounts of distilled
water, rinse the cylinder, adding the ―rinsings" to the dish.
4. Make sure that you have recorded all the pertinent details: Dish #, tare weight, sample
source and sample volume. Put the dish in the 103-105°C oven for drying. Do not write on
crucibles as high temperatures during the next steps will burn writing off. Use indelible
markings on dishes.
7
5. After drying overnight, the dish will be transferred to a desiccator. Obtain the gross weight
of the dish [Note: it should be room temperature before weighing.]. Subtract the tare
weight to obtain the total solids weight and calculate the mg/L value.
6. The dish containing the dried total solids can now be placed in a muffle furnace or, if
instructed, on a cart in preparation for staff to do so when all are ready. The samples will
be fired at 550°C for one hour. The furnace will then be allowed to cool significantly
before dishes are removed to a desiccator. Once dishes are at room temperature they
can be re-weighed. The loss in weight represents the volatile solids lost from the originally
measured sample volume. Conversely, the solids remaining represent the fixed solids.
Calculate the mg/L volatile and fixed solids.
C. SUSPENDED SOLIDS - TOTAL AND VOLATILE
1. Obtain the tare weights of three aluminum dishes each containing a glass fibre filter
(already pre-washed, dried and fired).
2. Using the sample poured out for Part B (for consistency), stir the beaker contents and
then rapidly (so that it doesn't settle) measure a volume of sample into the appropriate
graduated cylinder. [Hint: pour out small portions (say 25 mls at a time for Effl, 10 mls for
Raw & 5 mls for sludge) of well-mixed sample and filter entire portion before pouring
another portion; when filtering slows significantly do not add more. Record the total
volume filtered.] Rinse the graduated cylinder with small amounts of distilled water and
add to filter. Suggested portions: 100-200 ml of final effluent, 25-50 mls of raw sewage
and 5-10 ml of sludge using graduated cylinders to measure - not beakers.
3. Carefully remove filter from filtration apparatus and transfer it back to the aluminum dish.
Pinch sides of dish in a bit to protect the filter from oven drafts. Place the aluminum dish
into the 103°C oven to dry for at least one hour (or leave drying overnight).
4. Transfer dish to a desiccator, cool and weigh. DO NOT DISCARD! The gain in weight
represents the total suspended solids of the sample. Calculate as the mg/L total
suspended solids.
5. The aluminum dish and filter (from #4) must now be fired in a muffle furnace at 550°C for
at least 30 minutes (or until a constant weight is achieved). For the time being, place the
dishes on a cart designated by the instructor. Lab personnel will perform the timed-firing
after the class. After firing the dishes will be moved to a desiccator. They must be allowed
to cool completely before re-weighing them to determine the loss on ignition or volatile
suspended solids. Please return tomorrow to obtain final weight.
6. Calculate the mg/L total volatile solids and total fixed suspended solids.
7. From the above determinations it will now be possible to obtain a value for the dissolved
solids present in the sample. Enter all data in the tables on following page.
Calculate the percent solids reduction through the treatment plant.
% reduction in settleable matter _________

% reduction in total solids _________
% reduction in volatile solids _________
% reduction in fixed solids _________
% reduction in suspended solids _________
% reduction volatile suspended solids _________
% reduction fixed suspended solids _________
% reduction dissolved solids _________
8
Lab 1 Questions
1. What major types of solids are removed in primary treatment? in secondary

treatment?
2. If sludge is used in Part A, compute the sludge volume index (SVI). Discuss the
meaning and significance of this index.
3. What two techniques can be used to overcome or correct for the loss of material from
filter pads when firing at 550oC?
4. Raw sewage sludge goes through an anaerobic digestion process, where the volatile
solids are reduced from 65% to 40%. If all of the volatile solids reduced is given off as gas
and if the specific gravity of the volatile solids is 1.3 and fixed solids 2.50, what is the
percentage reduction in solids volume?
5. Why must residue samples be brought to ambient temperature before weighing?
6. Are your values for percent reduction about what you would expect for this type of
plant? Discuss. (Primary treatment typically removes about 60 percent of total suspended
solids and about 35 percent of BOD5; dissolved impurities are not removed. It is usually used
as a first step before secondary treatment. Secondary treatment typically removes more than
85 percent of both suspended solids and BOD5.)
9
DATA AND RESULTS

Raw Sewage Aerobic Sludge Final Effluent
A. Settleable Matter
After 1 hour (mL/L)
B. Total Solids
Dish marking (do not use ink - use permanent mark on dish)
Sample volume (mL)
Gross weight - oven dry (G)
Dish tare weight (G)
Total solids (G)
Total solids (mg/L)
Gross weight - fired (G)
Loss on ignition (G)
Volatile solids (mg/L)
Fixed solids (mg/L)
Percentage fixed residue
C. Suspended Solids
Tin dish marking (do not use ink - use embossed mark)
Sample volume (ml)
Gross weight - oven dry (G)
Tin dish with filter tare weight (G)
Total suspended solids (G)
Total suspended solids (mg/L)
Gross weight - fired (G)
Loss on ignition (G)
Volatile suspended solids (mg/L)
Fixed suspended solids (mg/L)
Dissolved solids (mg/L)
10
Laboratory 2: COD, DO and BOD5
OBJECT: -to familiarize you with the commonest tests run for assessing
treatment plant efficiency and pollution loading to receiving waters; to illustrate
the shortcomings of these tests; and to demonstrate the use of dissolved
WARNING oxygen probes.
You will be using
concentrated acid Part A: COD - Closed Reflux, Colourimetric Method
and other corrosive
and toxic NOTE: Open reflux method is the most accurate means of determining COD
chemicals, as well because a large sample size is used (20 mls). The closed reflux method is
as handling more economical but because a small sample volume (2 mls) is used,
samples probably homogenization of samples containing suspended solids may be necessary in
containing order to get a representative sample and to improve reproducibility. Digests
pathogenic from either method can be titrated or read colourimetrically.
bacteria. Wipe up
all spills
immediately, Materials: Raw sewage and final effluent samples, vials with pre-measured
wash/rinse bench reagents, Hach block digester, spectrophotometer, potassium hydrogen
tops with phthalate standards: 800, 400, 200, 100 and 50 mg/L as COD, pipettes, etc.
water/disinfectant.
NEVER pour water Procedure:
into acid. Keep 1. At your station you will find four vials with pre-measured solutions
fingers, pencils, etc containing potassium dichromate and sulfuric acid (prepared according to
out of your mouth. standard methods, except without mercuric sulfate). Put identifying labels
Always use and on each tube i.e. your initials, Raw Sewage (Infl) and/or Effluent (Effl) in the
store pipettes with
upper 1/4 of the tube using permanent markers or on the top of lid.
the top higher than
the tip, otherwise
2. Pipette 2 mls of sample (using volumetric pipettes unless particulate is
reagents will run to present, in which case, use wide mouth graduated pipettes) in duplicate,
the bulb end and into the appropriate tubes and screw cap on snugly. If sample is known to
make pipetting be outside the standard range (above 800 mg/L), a smaller aliquot (making
difficult and volume up to 2 mls with distilled water) or 2 mls of a pre-diluted sample can
contamination be used. Make sure sample is well mixed with the acid contents of the vial.
likely. [Note: it should look homogenous and be careful it will be very hot!]
Wash hands 3. Take vials to fumehood and place in block digester - noting the location of
thoroughly before your well-labeled, well-mixed samples in the 25-position digester.
leaving lab. 4. Standards have been digested for you, before the lab, and are at the
spectrophotometer, ready to read. Allow samples to digest for 30 minutes
(normally this would be 2 hours). Remove the vials (put in beaker to carry)
and allow them to cool to room temperature (use cold water to speed up if
necessary).
5. The instructor will demonstrate the use of the spectrophotometer, which is set at 600 nm to read
absorbance of light by the sample. The vials should be clean and dry on the outside. Read your
samples and the set of COD standards: 800, 400, 200, 100, 50 and 0 COD as mg O2 /L.
6. Prepare calibration curve, absorbance versus concentration and calculate the COD in samples.
If less than two mls of sample or diluted sample was used, a volumetric correction must be
made to the value obtained from the curve.
11
DO NOT DISCARD CONTENTS OF VIALS. LEAVE IN BEAKER AT YOUR STATION.
Part B: DISSOLVED OXYGEN (DO)
Materials: Raw sewage and final effluent samples, dilution water, alkaline-iodide-azide reagent,
manganous sulfate, concentrated sulphuric acid, starch indicator, 0.025 N sodium thiosulphate,
thermometers, BOD bottles, 10-ml graduated pipettes, burette stand with burettes, 250-ml
graduated cylinder, 500-ml Erlenmeyer flasks.
Note: You are going to measure DO on four different liquids using two different methods. The
four liquids are cold tap water, aerated dilution water, raw sewage and final effluent.
You will use a chemical titration (Winkler - the azide modification of the iodometric method) and a
dissolved oxygen probe and meter. The probe has been previously calibrated.
DISSOLVED OXYGEN PROBE
1. Fill four BOD bottles with the four samples (cold tap water, aerated dilution water, raw sewage
or influent and final effluent), right to the top and place stopper on bottle. (Fill carefully, taking
care not to entrain additional air. For the cold tap water sample, submerge the hose in the
bottle and allow the water to overflow for 15-30 seconds or until water is cold).
2. Determine DO, using the DO probe and meter, for each of the four samples. Instructions on the
use of the probe will be given in the lab. Record the temperature of each sample using the DO
meter. You can save these bottles for the Winkler method.
WINKLER TITRATION (Azide modification)
1. Fill a BOD bottle with each of the four samples (or use ones saved from A - which may have to
be topped up to exclude air). Stopper the bottles without trapping any air bubbles (lift stopper to
let bubbles out or add a little extra sample if air bubbles are seen inside).
2. Place the bottles in the sink and one at a time remove the stopper, add 1 (one) ml of
manganous sulfate reagent using plastic dropper provided (keeping the dropper tip just below
the surface) and quickly replace the lid. To the same bottle, add 1 ml of alkaline-iodide-azide
solution, again quickly replacing the lid. Carefully tip the liquid out of the space around the
stopper (caution -it may be corrosive) and invert several times, as demonstrated, to thoroughly
mix contents. Set bottle back in sink. Repeat the same procedure with the other three samples.
3. Allow the floc to settle to about 1/3 the bottle volume, invert several times again to remix and
allow floc to settle again. When the floc has settled the second time to about 1/3 bottle volume,
remove the stopper, add 1 ml of concentrated H2SO4 and quickly re-stopper. Tip out the liquid
from the area around the stopper and invert several times to mix thoroughly. The chemical floc
should completely dissolve. (Note: biological solids originally present in the sample will not
dissolve.) Repeat for other three samples.
4. Transfer 201 ml of each sample bottle (do one at a time) to a 500 ml conical flask. Use the
specially marked volumetric flask to dispense the 201 ml.
5. Titrate this volume with 0.025 N Na2S2O3 until only a faint yellow or "straw" colour remains.
Note: if solution already seems pale yellow add starch right away. Add enough starch solution to
give a strong blue colour (one dropper full) and continue to titrate, drop-wise, until the first
complete disappearance of the blue colour. Neglect any reappearance of the blue colour.
Record the volume of titre. (Hint: 1 ml of titrant = 1 mg/L DO. If you measured a very low DO
concentration with the meter, then the same sample will require little or no titre and may not
even turn blue when starch is added - think about it!)
12
6. Complete all four titrations.

7. Determine the percent saturation for each sample.
Dissolved Oxygen Data
Sample/Data Raw Sewage Final Effluent Tap Water Dil’n Water
Bottle #
Sample Volume (mls)
Initial Buret reading
Final Buret reading
Net Titre (mls)
DO conc. (mg/L)
DO conc. (Probe)
Temp (oC) (thermometer)
Saturation concentration
Percent saturation
DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD

For a quick and easy determination of the percent saturation value for dissolved oxygen at a given
temperature, use the saturation chart above. Pair up the mg/l of dissolved oxygen you measured
and the temperature of the water in degrees C. Draw a straight line between the water temperature
and the mg/l of dissolved oxygen. The percent saturation is the value where the line intercepts the
saturation scale. Note that this nomogram assumes that the water is at sea level. The saturation
value can also vary slightly depending on barometric pressure with lower values expected when a
storm front moves through as compared to bright and sunny "high pressure" days.
13
BIOCHEMICAL OXYGEN DEMAND

Note: Because of the logistics of organization some groups may have 6 day BOD instead of the
usual 5 day. Be sure to note this in your lab write-up.
Materials: Raw sewage and final effluent samples, dilution water, BOD bottles, 10-ml graduated
pipettes, graduated cylinders. Dissolved oxygen meter and probe.
1. Using the volumes and samples provided by the instructor, carefully measure the samples (Raw
and Effluent) into BOD bottles. Two dilutions will be made for each sample and each dilution will
be in duplicate for a total of eight bottles plus one bottle for the blank (mentioned in Step 4). Be
sure to record the bottle numbers and sample volumes on the following table - do not write on
the bottles and do not use the numbers on the lids.
2. Carefully top up the BOD bottles with DILUTION WATER, to bring the levels above the ground
glass neck of the bottle. Try not to aerate the contents by keeping the filling tube below the
surface of the liquid in the bottle or carefully running the water down the side of the bottle. Fill
one BOD bottle with DILUTION WATER alone. Because you want to be sure that the dilution
water has no BOD, be careful not to contaminate it with sample. The blank is used to check that
the dilution water has negligible BOD.
3. Measure the Initial DO in all 9 bottles using a probe and meter. Record the meter number and
use same one for final measurements.
4. Stopper the BOD bottles, being sure to exclude any air bubbles that might be trapped under the
stopper by adding a little dilution water if necessary. All bottles should have liquid in the well
around the stopper - if not add some dilution water (or distilled water) to the well. Place plastic
caps over all bottles. These caps are designed to prevent the evaporation of the liquid around
the stopper, thereby maintaining a water seal for each bottle.
5. Place BOD sets in the 20o C incubator for five days (or 6 for Tues. groups). Note: Each group
should have 9 BOD bottles in the incubator.
6. After five (or six) days, remove bottles and measure the final DO, preferably using the same
meter and probe used to get IDO reading. Calculate the 5-day (or 6-day) BOD, showing sample
calculations. There should be negligible BOD in the Dilution water, but if there is a significant
BOD then it will have to be subtracted before the dilution factor is applied in the calculation. If the
dilution water BOD is greater than 1 ppm, it may indicate the dilution water had been
contaminated.
BIOCHEMICAL OXYGEN DEMAND (BOD) DATA

Sample source: _____________________
Raw Sewage Bottle number (not lid #) _____ _____ _____ _____
Sample volume (ml) _____ _____ _____ _____
Initial DO (mg/L) _____ _____ _____ _____
Final DO (mg/L) _____ _____ _____ _____
Final Effluent Bottle number (not lid #) _____ _____ _____ _____
Sample volume (ml) _____ _____ _____ _____
Initial DO (mg/L) _____ _____ _____ _____
Final DO (mg/L) _____ _____ _____ _____
Dilution water Bottle number (not lid #) _____

Sample volume (ml) _____
Initial DO (mg/L) _____
Final DO (mg/L) _____
14
BOD5 Calculation
When dilution water is not seeded (our water is not seeded for this exercise):
When seeded water is used:
where: D1 = DO of diluted sample immediately after preparation, mg/L

D2 = DO of diluted sample after 5 d incubation at 20oC, mg/L.
P = decimal volumetric fraction of sample used
B1 = DO of seed control before incubation, mg/L
B2 = DO of seed control after incubation, mg/L, and
f = ratio of seed water in diluted sample to seed in seed control=
(% seed in diluted sample) / (%seed in seed control).
Summary
Raw Sewage, Diln. 1 ________ ________
Raw Sewage, Diln. 2 ________ ________ Average ________
Final Effluent, Diln. 1 ________ ________
Final Effluent, Diln. 2 ________ ________ Average ________
Percentage BOD reduction through treatment plant ___________
Questions
1. At what relative times should influent and effluent samples be taken from a sewage treatment
plant in order to determine true % removal efficiency?
2. Give two reasons why samples for dissolved oxygen should be ―fixed‖ in the field, if at all
possible.
3. Calculate theoretical oxygen demand for 300 mg/L of methyl alcohol.
CH3OH + ³/2 O2 → CO2 + 2H2O
4. Show on a graph the approximate dissolved oxygen sag curves in a river receiving two wastes.
Both wastes have equal BOD5 temperature and volume. One waste has a K value of 0.17, the
other 0.25.
5. If an industrial waste has a COD of 450 mg/L, what would you expect its I) BOD5 and ii) BODL to
be.
6. What interference does the Azide Modification of the Winkler Dissolved Oxygen method
overcome and why is it the most common method used for domestic sewage?
7. Why is a blank measured in the COD test? (Refer to Standard Methods, closed reflux,
colourimetric.)
8. Why is your COD result different from the BOD result?
15
Laboratory 3: Chloride Determination
OBJECT: -to acquaint you with the use of specific ion electrodes, known
addition and calibration techniques and colourimetric and potentiometric
WARNING
titrations; to illustrate the differences in operation and accuracy between
You will be using instrumental methods and wet chemical methods.
concentrated
acid and other MATERIALS:
very dangerous
chemicals. -millivolt meter, chloride specific ion electrode, reference electrode (double
Mercuric junction), magnetic stirrer, beakers, buffer, standards, semi-log graph paper;
thiocyanate is
spectrophotometer, 125 ml Erlenmeyer flasks, graduated cylinders; pH meter
very toxic. Use
personal
with double-junction electrode and silver billet electrode, burettes, conc.
protective HNO3, standard chloride solution (0.5 mg/L), standard AgNO3 solution,
equipment: volumetric pipettes; standard Hg(NO3)2, indicator-acidifier reagent, indicator,
gloves, goggles NaHCO3, beakers; sample.
or glasses and
lab coats. If Note and consider carefully: Different methods of analysis have different
chemicals are optimum ranges. You will be told the approximate concentration in the
spilled, alert sample and it is up to you to make sure that the sample will fit within the
instructor/TA specified ranges. That means you will probably have to dilute the sample by
immediately. the most accurate means available.
I. CHLORIDE - SPECIFIC ION ELECTRODE
1. Standards of 1000, 100 and 10 ppm (mg/L) have been prepared for you and are at the meter.
Unless already done by a previous group, pour out 100 mls of each standard into separate,
labelled beakers. Add 2 mls of IONIC STRENGTH ADJUSTMENT (ISA) buffer to each using the
plastic dropper attached to the chemical (filled to the mark).
2. Unless already done by a previous group (values written on the board), place the lowest
standard on the stirrer and lower the electrodes into it. Turn on stirrer and while stirring record
the millivolt (mv) reading when the reading appears to be stable. As probe tends to drift for
some time, it may be expedient to select a ―standard time to read‖ (i.e. 2 minutes) and use it for
all standards and samples. Repeat for rest of standards, rinsing and blotting dry the electrodes
between each.
3. Measure 100 mls of sample, added 2 mls of ISA, stir and obtain a reading for it.
4. Using semi-log paper or software equivalent, plot the millivolt readings (linear axis) against the
concentration of the standards (log axis) to produce a calibration curve. Determine the
concentration of chloride in the sample.
5. Method of Standard Addition: Add 5 mls of the stock (4 mg/ml) chloride solution (Vs) to the
sample measured in Step 3 (E1) and take a new reading after value is stable (E2 ).
16
Calculate the chloride concentration, where:

E1 = potential of sample (mV)
E2 = potential of sample plus stock addition
E = potential change = E2 - E1
Vo = volume of sample
Vs = volume of stock solution added
A = mg Cl- added to the sample
S = "slope" = change in potential over 10 fold change in concentration of standard (use
data from Step 2).
mg Cl- in sample = __________________________
mg/L Cl- = _______________________
II. CHLORIDE - BY COLOURIMETRIC METHOD
The principle of this method is based on the following formula:

2Cl- + Hg(SCN)2 ➔ HgCl2 + 2SCN-
SCN- + Fe+++ ➔ Fe(SCN)++ (Reddish brown)
1. Using a 25 ml graduated cylinder, measure 25 mls of ―zero‖ standard or ―blank‖ (halide-free

distilled water), the three standards (2.0, 4.0 and 8.0 ppm), and the sample (diluted if necessary)
into separate and labelled 125 ml Erlenmeyer flasks (5 in total).
2. Marking the time (T=0), add 5 ml of the ―combined reagent‖ (containing ferric alum and mercuric
thiocyanate solutions) to the blank first, using the dispenser provided. (Use extreme caution as
this is a corrosive & toxic chemical.) Mix thoroughly by gently swirling the flask.
3. Wait two or three minutes and then add the reagent to the first (lowest) standard; after a few
more minutes, add the reagent to the second standard and so on for the rest of the standards and
sample (which may need to have been diluted). [Note: this delay between additions is to allow
you time to measure absorbance of each at the 10 minute time required - they can’t all be at 10
minutes at once.]
4. After 10 minutes has passed (since the combined reagent was added to the blank), use a plastic
dropper to transfer blank into a spectrophotometer cuvette (do not fill more than 3/4), discard and
refill at least twice to rinse cuvette. Insert cuvette into the spectrophotometer (wavelength must
be set at 463 nm) and press ―zero‖ or ―ref set‖ (depending on model) to make the meter read
zero. (Alternatively, zero instrument on distilled water and obtain an absorbance value for the
blank, which then must be subtracted from the standards and sample.)
5. Every two or three minutes thereafter you will rinse the cuvette with the next standard or sample,
fill, insert in the spectrometer and record the absorbance displayed on the meter. (No further
adjustment is required after the blank or distilled water has been set to zero.)
6. Prepare a calibration curve by plotting the absorbance readings versus the concentration of
chloride in the standards. Determine the chloride concentration in the sample from the calibration
curve. If sample is above the highest standard, it should be rerun from the start after diluting.
17
III. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION
The principle of this method is as follows: Cl- ions in the sample combine with added mercuric ions
forming a soluble but virtually undissociate-able complex. After all Cl- ions have been bound, the
excess mercuric ions combine with diphenylcarbazone (DPC) to produce a violet colour (the end-
point).
Hg++ + 2Cl- ➔ HgCl2 and Hg++(excess) + DPC ➔ violet colour
1. Pour 100 ml of sample (or a portion diluted such that the concentration is less than 100 mg/L)
into a 250 ml beaker..
2. Add 1 ml (use pipette to measure accurately) of indicator-acidifier reagent to sample. The colour
of the solution should be green-blue at this point. (Light green indicates a pH of less than 2.0;
pure blue, a pH of greater than 3.8. For most samples, the 1 ml of the indicator-acidifier reagent
will adjust the pH to 2.5 ± 0.1.
3. Titrate with 0.014N Hg(NO3)2 to a definite purple end-point. Near the end-point, the solution
should be green-blue and will turn to pure blue (no green) within a few drops of the purple end-
point.
4. Determine the blank by titrating 100 ml of distilled water containing 10 mg of NaHCO 3 which
serves to provide some alkalinity to the blank (to make it similar to the sample) and provides a
correction for alkalinity and reagents - if one is necessary. Don’t forget to add the indicator. The
titre from this step must be subtracted from the titre for the sample.
Calculate the chloride concentration:
where: A = ml titrant for sample

B = ml titrant for blank
N = normality of titrant
IV. CHLORIDE BY POTENTIOMETRIC TITRATION
Note: There are only two stations set up for this method and it takes time to complete therefore it
may not be possible for every group to do both standard and sample. Reorganize groups to ensure
that every group has both sets of data needed to complete the lab write-up. A reference set of data
will be provided for comparison.
Part One: Standard titration

1. Place 10.0 ml (use volumetric pipette) of standard chloride solution in a 250 ml beaker, dilute to
about 100 ml with distilled water, and add 2.0 ml of concentrated HNO3 (use plastic dropper).
Nitric acid is in the sink and is very corrosive.
2. Immerse stir bar, place beaker on stir plate and lower electrodes into the solution. Take care that
the stirrer will not hit the electrodes.
3. Start the stirrer and set the millivolt reading to zero or record initial mv reading [Note: Some
meters cannot be set to zero].
4. Begin titrating, in increments, with standard AgNO3, recording the volume and millivolt reading
for each increment. At the start, large increments (1-2 mls) may be added; then, as the end-point
of the reaction is approached (△ mV/ml increases), smaller increments (0.1 to 0.2 ml or drop-
wise) should be added, until past the end-point when larger increments can be used again (△
18
mV/ml decreases). Continue the titration about 3 ml past the end-point. The end-point occurs at
the greatest mV change per unit addition of AgNO3.
5. Plot a differential titration curve to determine the exact end-point.
6. Calculate the Normality of the AgNO3 using the following equation:
N A g N 3O 1 0.0 × 0.0 1 4 1

m lo f A g N 3O
Part Two: Sample
1. 1Measure 100 ml of sample or portion made up to 100 ml, if dilution is necessary into a
250 ml beaker (use a graduated cylinder to measure).
2. 2Add 2 ml of HNO3 and proceed as described in Part One. (If pH of the sample were
quite high it would be necessary to first adjust the pH to about 4 and then add 2 ml
HNO3. It is not, in this case.)
3. Plot three curves for this titration:
a. Millivolt reading versus ml of titrant.
b. Millivolts per ml versus ml of titrant.
c. Millivolts per ml per ml versus ml of titrant.
In plotting b and c, which are merely first and second derivatives, be careful to use the
average value of ml of titrant on the abscissa.
4. Calculate the chloride concentration in the sample using the following equation:
Where: A = ml AgNO3
B = ml Blank
N = normality of titrant (AgNO3)
D = ml of sample
ml mg/L Cl-
From graph a ╶────────╴ ╶────────╴

b ╶────────╴ ╶────────╴
c ╶────────╴ ╶────────
19
Chloride - Potentiometric Titration

RAW DATA FIRST DERIVATIVE SECOND DERIVATIVE
A B C D E F G H I J
Volume Meter Ave. Ave. of ∆(∆E/∆Vol)
AgNO3 Reading (E) ∆(Vol) ∆(E) (AgNO3) ∆(E)/∆(Vol) ∆(∆E/∆Vol) ∆[Ave.(Vol)] Ave.(Vol) /ml
Added (mV) Volume
(Vol) (ml) (ml) (mV) (ml) (mV/ml) (mV/ml) (ml) (ml) (mV/ml/ml)
20
RAW DATA FIRST DERIVATIVE SECOND DERIVATIVE

A B C D E F G H I J
Volume Meter ave
AgNO3 Reading ∆(Vol) ∆(E) (AgNO3) ∆(E)/∆(Vol) ∆(∆E/∆Vol) ∆[Ave(Vol)] Ave of ∆(∆E/∆Vol)
Added (E) Volume Ave(Vol) /ml
(Vol) (ml) (mV) (ml) (mV) (ml) (mV/ml) (mV/ml) (ml) (ml) (mV/ml/ml)
21
INTERFERENCES IN CHLORIDE DETERMINATIONS
POTENTIOMETRIC METHOD
■ Iodide, fluoride and bromide titrate as chloride.
■ Other interferences: ferricyanide, chromate, dichromate, ferric ion.
■ Pretreatment of environmental samples usually required.
MERCURIC NITRATE TITRATION METHOD
■ Iodide, fluoride and bromide titrate as chloride.
■ Chromate, ferric, and sulfite ions interfere when concentration greater than 10 mg/L.
■ Colour may obscure or interfere with end-point determination.
■ Sample pH may need adjustment beyond the capacity of indicator-acidifier reagent.
■ Environmental samples usually require complex pre-treatment.
COLOURIMETRIC METHOD
■ Bromide, iodide, fluoride, cyanide, thiosulphate, hydrazine, and nitrite interfere.
■ Colour in the sample may interfere in the absorbance measurement.
■ Environmental samples usually require pretreatment.
SPECIFIC ION ELECTRODE
■ All halides interfere (the electrode is a halide electrode).
■ High levels of ions which form very insoluble salts of silver may deposit a layer of salt on the
electrode membrane, causing electrode malfunction. Also, strongly reducing solutions may form a
surface layer of silver.
■ Mercury must be absent from samples.
■ In practice, specific ion electrodes are rarely usable in environmental samples.
KNOWN (STANDARD) ADDITION TECHNIQUE
■ The known addition techniques will only correct for certain types of interferences. In the above
tests, known addition cannot correct for any ions that test as chloride ion. It can only correct for
substances that enhance or suppress the test response proportional to the amount of chloride ion
present.
PRETREATMENT TECHNIQUES
■ There is no "standard" pre-treatment technique. Each sample type requires development of an
appropriate method which, in itself, requires knowledge of the compounds in the sample which are
likely to interfere. This is a complex, time-consuming process requiring a sound knowledge of chemistry
and extensive experience working with environmental samples.
Lab 3 Questions
1. In the Mohr method for chlorides (described in Sawyer and McCarty) what would the equilibrium
concentration of silver ions be (in mg/L) when the chloride concentration has been reduced to 0.3
mg/L?
2. Outline the advantages and disadvantages of all the methods used in this lab to measure chloride
3. From your chloride results, can you see any advantage in using the first or second derivative curve?
Discuss.
22
Laboratory 4: Chlorine and Chlorine Demand
WARNING OBJECT: to acquaint you with disinfection of water supplies and to

Glacial acetic acid is very demonstrate techniques for the determination of chlorine residuals; to
corrosive. Bleach/chlorine illustrate the concepts of free and combined chlorine residuals, chlorine
solutions are strong demand and break-point chlorination.
oxidants. Personal
protective equipment Materials: 600 ml beakers, 250 ml Erlenmeyer flasks, 250 ml volumetric
required at all times when flasks, pipettes, burettes and stands, 100 ml graduated cylinder, bleach
working in the lab are: eye or hypochlorite solution, 0.025 N sodium thiosulphate, chlorine free
protection, gloves and lab water, (concentrated) glacial acetic acid, potassium iodide (KI) crystals,
coats. When weighing starch indicator, phosphate buffer solution, N,N-diethyl-p-
chemicals at the balance,
phenylenediamine (DPD) indicator solution, standard ferrous ammonium
clean up all spills with a
sulphate (FAS) solution.
brush into a container and
wash crystals down the
drain. Wipe up all liquid REFERENCE:
spills immediately. Standard Methods for the Examination of Water and Wastewater
Methods: 2350 B, C; 4500-Cl F.
I. PREPARATION OF STOCK CHLORINE SOLUTION

(Iodometric method - total chlorine residual)
1. Prepare a stock solution of strong chlorine water by adding 5 mls of bleach solution (sodium
hypochlorite) to about 200 mls of water in a 250 ml volumetric flask. Make up to the 250 ml and invert
flask several times to mix. Rinse a burette and fill it with this stock chlorine solution.
2. Take a 250 ml Erlenmeyer flask and add about 1 gram of potassium iodide, about 50 ml of chlorine-
free water (distilled water will do), about 5 ml of glacial acetic acid (use a graduated cylinder), and
exactly 10 ml of 0.025 N sodium thiosulphate solution (use a volumetric pipette).
3. Mix well (on stir plate with stir bar), add 1 ml of starch solution, mix again. Titrate rapidly with the
stock chlorine solution (in burette) until the appearance of a constant purplish-blue colour.
4. From the amount of chlorine solution used, calculate the chlorine concentration in the stock solution
(See Data and Results section for formula).
23
IIa. Breakpoint Chlorination using Free and Combined Chlorine Residuals Values
1. Set up a numbered row of six 600 ml beakers each containing 500 mls of the sample provided (it
has been prepared for you, using Ammonium chloride and Glutamic acid). See note IIb. 1before
proceeding.
2. Chlorine dosing procedure: Allow at least 5 minutes of time to elapse between application of chlorine
to successive beakers, in order to allow for time to titrate the free and combined chlorine residuals,
refill the burette, etc. once the 15 and 60 minute contact duration has been reached. Add the
dosages given by the instructor (see board): Time (T) = 0 add dosage to beaker #1; T=5 add next
dosage to beaker #2; T=10 dose beaker #3; T=15 dose beaker #4, and so on.
3. After 15 minutes of contact time, and again after 1 hour of contact time determine the free and
combined chlorine (see below) in 100 ml samples taken from each of the beakers at the appropriate
time spacing (15 minutes and 60 minutes after the addition of the chlorine dose to each beaker).
Note that immediately after applying the dose to Beaker # 4 you will have to start titrating a sample of
Beaker #1. Coordinate yourselves well for this!
IIb. Determination of Free and Combined Chlorine by the DPD Method
1. Place 5 ml of phosphate buffer solution and 5 ml of DPD solution in a 250 ml conical (Erlenmeyer)
flask and swirl to mix. These 6 flasks can be prepared at once to make them ready for the next
step (best to do so ahead of the dosing process).
2. Add 100 ml of sample or diluted* sample using a 100 ml graduated cylinder and mix again.
(*NOTE: If total chlorine is likely to exceed 5 mg/L use a smaller sample and dilute to a total
volume of 100 mls with chlorine-free water. It is likely that the top one or two doses will require
dilution.) Mix usual volumes of buffer reagent and DPD indicator solution with distilled water before
adding sufficient sample to bring total volume to 110ml or the test will not work. (i.e. 5 ml + 5 ml +
50 ml chlorine-free water + 50 mls of sample will give a 1:1 dilution; 5 ml + 5 ml + 75 ml water + 25
ml of sample will give a 1:4 dilution)
a. Free chlorine: titrate rapidly with standard FAS (Ferrous ammonium sulfate) titrant until the
red colour is discharged. Record the burette reading (mls) = Reading A. Note: if no initial red
colour do not add FAS, volume =0.
b. Monochloramine: Add two drops of KI solution (to the same sample) and mix. Continue
titrating until red colour (if any) is discharged once again (Reading B). Go to c.
c. Dichloramine: To the same sample add about 1 g KI and mix to dissolve. Let stir for 2 min
(or until completely dissolved) and then continue titrating until red colour (if any) is
discharged (Reading C). For dichloramine concentrations greater than 1 mg/L, let stand 2
min more if colour drifts back (indicating incomplete reaction) continue titrating to colourless.
3. Alternatively: (Not to be done in this lab.) Free and combined chlorine or total chlorine: To obtain
total chlorine in one reading, add full amount of KI at the start (ie 2 drops KI solution and 1 g of KI
crystals) to a fresh sample, mix to dissolve, let stand for two minutes and titrate until the red colour
(if present) is discharged. Let stand 2 minutes more; if colour drifts back (indicating an incomplete
reaction), titrate to colourless again. Record the burette reading (Reading C).
Total chlorine (Free plus Combined) = Reading C

Free residual chlorine = Reading A
Combined residual chlorine (dichloramine plus monochloramine) = Reading C - A
Monochloramine= B-A
Dichloramine= C-B
24
DATA AND RESULTS
I. Standardization of chlorine stock solution:

Volume of sodium thiosulphate used = ______ mls (A)
Normality of sodium thiosulphate used = ______ (N)
Volume of stock chlorine solution = ______ mls (B)
Cl concentration of stock solution = = _________mg/L Cl as Cl2*
*To determine bleach concentration as percent you must multiply first by your dilution (i.e. 5/250) then
convert mg/L to g/100 mls = %. Guaranteed analysis is normally printed on the container and is usually
5-6%.
II. FAS titrant concentration is 1 ml = 100 µg Cl as Cl2
1. Determine the concentration of total, free and combined residual chlorine for each applied
chlorine dose.
2. Make a plot of the three residual chlorine components against the applied chlorine dosage.
Make a separate graph for the 15 minute and 1 hour contact time.
3. Discuss your results as a function of the residual forms of chlorine present at different chlorine
dosages and at different contact times.
4. How would you expect the forms of residual chlorine and their speed of formation to change as
a function of 1) temperature and 2) pH?
Lab 5 Questions
1. Why is it important to determine chlorine residuals in domestic water supplies?
2. Given: HOCl ⇄ H+ + OCl-
Keqv = 2.7 x 10-8 at 25o C
% HOCl = 27
What is the pH of the solution?
3. Under what conditions is the practice of break point chlorination necessary?
4. The rate of kill of bacteria by chlorination follows first-order reaction kinetics.
a. If this is true, what percentage of bacteria would be killed in 10 minutes at a chlorine

residual of 0.5 mg/L if 50% are killed in 1½ minutes at this concentration?
b. If a sewage sample contains 10 x 106 coliforms/100 ml, how many coliforms would
remain after a chlorine contact time of 10 minutes? 20 minutes?
25
15 MINUTE CONTACT TIME
BEAKER NUMBER 1 2 3 4 5 6
Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl2)
Applied Cl dosage (mg/L Cl as Cl2)
Time of Cl addition
Time of Residual measurement
Volume of FAS to endpoint A
Volume of FAS to endpoint B (includes A)
Volume of FAS to endpoint C (includes A & B)
Free chlorine (mg/L Cl as Cl2) (A)
Monochloramine (mg/L Cl as Cl2) (B-A)
Dichloramine (mg/L Cl as Cl2) (C-B)
Combined residual chlorine (mg/L Cl as Cl2) (C-A)
Total chlorine (mg/L Cl as Cl2)
60 MINUTE CONTACT TIME
26
BEAKER NUMBER 1 2 3 4 5 6
Vol. of Cl Soln. (mls) (Stock=_____mg/L Cl as Cl2)
Applied Cl Dosage (mg/L)
Time of Cl addition
Time of residual measurement
Vol. of FAS to endpoint A (mls)
Vol. of FAS to endpoint B (mls) (includes A)
Vol. of FAS to endpoint C (mls) (includes A+B)
Free chlorine (mg/L Cl as Cl2) (A)
Monochloramine (mg/L Cl as Cl2) (B-A)
Dichloramine (mg/L Cl as Cl2) (C-B)
Combined residual chlorine (mg/L Cl as Cl2) (C-A)
Total chlorine (mg/L Cl as Cl2)
27
Laboratory 5: Bacteriological Examination of Sewage
OBJECT:
WARNING -to demonstrate different methods for the
As you are handling determination of the coliform group of organisms and to
and pipetting samples, acquaint you with bacteriological procedures. Students will
possibly containing only do sections marked ➼
pathogenic organisms,
please observe the MATERIALS:
following rules: Wear Multiple tube method: dilution tubes, lauryl tryptose broth tubes,
gloves, lab coats, eye Brilliant Green Lactose Broth tubes, EC tubes. (Demonstration
protection; DO NOT only)
MOUTH PIPETTE;
wipe up all spills and
disinfect the area of Membrane filter method: dilution tubes, sterile pipettes, sterile
the spill. Keep hands, dilution water, filtering apparatus, membrane filters, LES Endo
pencils, etc., away agar plates, 35oC incubator.
from your mouth. Wipe
lab benches with Heterotrophic plate count: dilution tubes, sterile pipets, agar tubes,
disinfectant before and plates, 35oC incubator, Quebec colony counter, HACH P/A Broth
after each lab. Wash with MUG. Demonstration only
hands with soap
before leaving. Do not SAMPLE: You will do either a raw sewage sample or a final
wear lab coat out of
effluent sample, not both. Get raw data from another group and
lab - leave it here or
take away in a plastic include in your report.
bag to launder.
Five different procedures will be studied - because of logistics it is
not possible for you to perform the lab as previously written.
Therefore, you will do those marked by ➼ only
Multiple-tube Fermentation: Serial dilutions of sample are incubated in lauryl tryptose broth at 35oC
for 48 hours (swirl after 24 hrs). Gas formation indicates the possible presence of coliform organisms.
Another series of tubes are inoculated from these positive lauryl tryptose tubes. Brilliant green lactose
bile (BGLB) tubes are inoculated and incubated at 35oC for 48 hours (confirmed test for total coliforms)
and EC tubes are inoculated and incubated at 44.7oC for 24 hours (confirmed test for faecal coliforms).
Bacterial numbers are calculated from the numbers of positive tubes. Hach’s Most Probable Number
Method 8368, A-1 Medium, is USEPA-accepted for testing non-potable waters. It is used as a faster
alternative to the LTB/EC procedure for enumerating faecal coliforms in water. Note: these methods will
be demonstrated but data must be recorded.
Methods:
Reading BGLB and EC tubes:
1. All tubes have been examined for gas production. Formation of gas in any amount in the
inverted vial of the BGLB broth fermentation tube at any time within 48 hr ±3 hr constitutes a
28
positive confirmed phase. Calculate the MPN value from the number of positives using the
Table 1. Express as Total coliforms.
2. ➼Copy results from the board for the demonstration set.
3. ➼Report positive EC broths incubated at the elevated temperature (44.5oC) as a positive
completed test response for both Total and faecal coliform. Parallel positive BGLB broth
cultures with negative EC broth cultures indicate the presence of non-faecal coliforms.
Completed Phase LES Endo agar Plates: A sample will be available to look at.
1. Using aseptic technique, as demonstrated, streak one LES Endo agar plate from each tube
of BGLB broth showing gas from the highest dilution and the second highest dilution. Streak
plates in a manner to insure presence of some discrete colonies. Flame loop between
second and third quadrants to improve colony isolation.
2. Incubate plates (inverted) at 35oC for 24 ± 2 hr. Prepare Gram Stain slides for examination
under a microscope.
3. Microscope Slides (Completed Phase of Multiple-Tube Fermentation): Single colonies
arising from single cells are prepared and stained for microscope examination. (Single
colonies are obtained by streaking LES Endo agar plates with an inoculum from positive
BGLB or EC broth tubes and incubating for 24 hours.) Refer to the internet or ―Bacteriology
for Sanitary Engineers‖ D.D. Mara 1974 or other for detailed instructions on the Gram Stain
procedure.
4. ➼A Gram Stain slide has been prepared and can be viewed under the microscope
afterwards or the next day when you come back to count colonies.
➼ Membrane Filter Technique: Serial dilutions of sample are collected on membrane filters
and placed on M-Endo media. Colonies that are pink to dark red with a golden green metallic
sheen are counted after incubation 20-22 hrs at 35oC.
Method:
1. Dilution tubes. Aseptically prepare one set of six dilution tubes as follows and
demonstrated by the instructor.
a. Aseptically transfer exactly 1 ml of the sample to a tube containing exactly 9 ml of
sterile diluent to make a 1:10 dilution of the sample.
b. Discard the pipette into a cylinder for disinfection.
c. Using a fresh pipette mix the contents of the 1:10 dilution tube by withdrawing about
1 ml from the bottom of the tube and ejecting it at the top: repeat several times.
d. With the same pipette used in step c transfer 1 ml of the 1:10 dilution to a second
tube of sterile diluent to make a 1:100 dilution. Discard the pipette and with a fresh
pipette mix the 1:100 dilution as in step c.
e. Make a 1:1000 dilution from the 1:100 dilution and higher dilutions similarly until you
reach the 6th tube (1:1000000)
2. Filtering. The filtration apparatus has been sterilized by autoclaving. Mount apparatus on
suction flask with valve sideways to block suction. Using sterile forceps, place a sterile
membrane filter (grid side up) onto the filtration apparatus. Pipette about 10 ml of sterile
dilution water over the surface of the filter with the suction valve still closed. Pour the full
volume of dilution tube # 5 (for Effluent) or #6 (for Raw Sewage) onto the filter, open the
valve slowly and allow all the liquid to be pulled through. Close valve and rinse the funnel
with three aliquots (portions) of sterile dilution water opening and closing valve after each
aliquot.
29
3. When filtering is complete and the valve is closed, remove filter using flame- sterilized
forceps (allow forceps to cool or they will damage the filter). Place the filter on a small LES
Endo agar plate carefully, from one edge, with a motion that excludes air as it comes in
contact with the agar. Do not push down on the filter except on the outside edge and use
only sterile forceps - applying gentle pressure.
4. Repeat for the rest of the dilution tubes ending at tube #1 for Effluent or #2 for Raw Sewage
(5 dishes in total). Incubate inverted Petrie dishes for 20-22 hr at 35oC. Be sure dishes are
labelled with your name and dilutions on the bottom of the plate (so that you don’t block view
when counting colonies).
5. Counting. After 20-22 hr, choose appropriate dilutions for counting. Use dishes with 20 to
80 coliform colonies and not more than 200 of all types. The typical coliform colony has a
pink to dark-red colour with a golden green metallic surface sheen. The sheen area may
vary in size from a small pinhead to complete coverage of the colony surface. The sheen
area may vary in size from a small pinhead to complete coverage of the colony surface.
Atypical coliform colonies can be dark red or nucleated without sheen. Colonies that lack
sheen may be pink, red, white, or colourless and are considered to be non-coliforms. The
total count of colonies (coliform and non-coliform) on Endo-type medium has no consistent
relationship to the total number of bacteria present in the original sample. A high count of
non-coliform colonies may interfere with the maximum development of coliforms. Verification
of both types of colonies is advisable as some typical sheen colonies may be non-coliform
and some atypical may be coliform. Verification is usually done by a test for lactose
fermentation. Gram staining technique can be also be used. In general, coliform bacteria are
Gram-negative (pink) rods (sometimes coccus-like).
6. -Note: ―typical‖ colonies count as coliforms in this procedure, therefore the results table
should specify #coliform colonies/100 ml.
➼ C. Heterotrophic Plate Count - Pour Plate Method: Serial dilutions of samples mixed with
agar and incubated for 48 hrs at 35oC. The number of colonies counted directly.
1. Dilution tubes. Prepare one set of six dilution tubes as described in Section previous
section.
2. Inoculating. Using a sterile pipette, transfer 1 ml of sample from dilution tube # 6 (most
dilute for Raw sewage) or #5 (for Effluent) to an empty (large) Petrie dish, removing the
cover just enough to insert pipette tip and dispense sample, then immediately replace the
cover.
3. Repeat with dilutions 5 to 2 (for Raw) and 4-1 (for Effluent).
4. Take 5 tubes of melted agar from the incubator. Let them cool down from the 60oC that they
are kept at but not enough to solidify. Test temperature on your wrist - too hot for a baby
= too hot for a bacterium. Slide the cover of a Petrie dish back just far enough to pour the
agar, pour and replace cover. Gently swirl the dish, on the bench, to mix the sample with the
agar. Repeat for the rest of the dilutions. Work quickly or the agar will solidify in the tube
before you get a chance to pour it.
5. Allow the agar to solidify in the dish before inverting, marking (on bottom edge of the dish so
as not to impede counting) and incubating for 48 hr at 35oC.
6. Counting. After 48 hr incubation, select the appropriate dilution to count. Ideally, use the
plate(s) that will give from 30 to 300 colonies/plate. The aim of using several dilutions is to
have at least one dilution giving colony counts between these limits. Use the Quebec colony
counter to aid in counting and a grease pencil to mark off colonies counted. A hand counter
is also available. Compute bacterial density and report as ―colony-forming units‖ (CFU) per
30
ml. Do not report as ―too numerous to count‖ if all plates exceed 300 colonies. Instead, if
fewer than 10/cm2, count colonies in 13 squares (of the colony counter) of the most dilute
plate having representative colony distribution. Preferably select seven consecutive squares
horizontally and six consecutive squares vertically. Multiply the number by 5 to compute
estimated colonies per plate (66 cm2 ). If more than 10/cm2, count four representative
squares, average and multiply by 57 (for plastic Petrie dish).
D. Presence/Absence Testing: HACH technique to simultaneously screen for total coliform

and E.coli, using a ready-to-use P/A Broth with MUG. MUG produces a fluorogenic product
when hydrolyzed by an enzyme specific to E.coli. The broth will turn yellow indicating total
coliform and fluoresce in the presence of E.COLI in 4 to 24 hours.
1. Carefully place each of the previously prepared and incubated bottles of media under
the UV light in the light box. Do not tilt more than 45o to avoid leakage of highly infectious
bacteria.
2. Make notes for each sample: sterile dilution water, tap water and sewage, recording the
colour, clarity and fluorescence qualities.
3. Report as presence of Total Coliform, E.Coli or negative.
➼Microscopic examination. Please allow instructor to set up the microscope for viewing as it
is difficult for the novice to succeed. Only the fine focus knob should be used. If you are not able
to see well enough to describe bacteria, as requested, refer to resources in books and on the
internet to describe what you expected to see.
Procedure:
1. Use the low power objective to locate a suitable group of bacteria for examination. (Note: Oil
should not be used for lower power objectives)
2. Rotate the objective out of the way and place one drop of immersion oil on the slide directly under
the lens (where bacteria were located). Lower the oil immersion objective (100x) until it just
touches the oil drop. While looking through the eyepiece, turn the fine focus knob until the image
comes into focus. Be careful not to lower the objective too far, grinding it into the slide, breaking
the slide and possibly damaging the lens.
3. In general, coliform are Gram-negative (pink), nonsporing, usually motile, short, plump rods,
sometimes coccus-like and 0.5 - 3 µm in size. Cells occur singly, in pairs, or in short chains. Note:
in the staining process: before decolourisation all organisms appear Gram positive (blue), after
decolourisation Gram negative organisms are no longer visible. After application of the
counterstain, Gram negative organisms are visualized (pink).
4. Description of bacteria: Describe what you see on the plates: colonial morphology of each type of
colony seen ie surface (shiny, dull, metallic, rough, smooth, concave), transparency, colour,
odour; and on the slide: typical or atypical, gram positive or negative, cocci or rods, length to
width ratio, cell groupings (single, pairs chains).
Schedule Summary
Day 1.
- Record positives in all Multiple Tube Fermentation inoculations: LTB, BGLB and EC tubes -
counts will be provided on the board.
- Prepare dilutions for Heterotrophic Plate Count and Membrane Filter technique.
- Prepare and incubate Heterotrophic (Pour) Plates using dilutions above.
- Membrane filter prepared dilutions, place on LES Endo agar dishes and incubate.
- Record Presence/absence testing results for Hach Prepared Media bottles.
- Examine prepared slides, if ready and/or if time permits.
31
Day 2.
- Come in and count colonies on LES Endo Membrane Filter dishes after 24 ± 2 hours.
- Examine prepared slides, if ready and/or if time permits.
Day 3.
Come in and count Heterotrophic Plate colonies after 48 ± 2 hours. Plates reaching 48 hours on
a weekend (Sunday) will be put in cold room until Monday to prevent overgrowth.
Bacteriological Examination Data Dilution table

Tube # 1 2 3 4 5 6
Volume diln
water 9 9 9 9 9 9
Mls of sample
pipetted 1 1 1 1 1 1
Mls of
sample/ml 1 0.1 0.01 0.001 0.0001 0.00001
pipetted
Volume left in
tube after 9 9 9 9 9 10
serial dilution
Multiple Tube Fermentation: 1 ml of each dilution tube (in the series above) was added to 5 replicas for
each dilution of LTB media. Indicate the number of positives for each dilution. A loop was used to inoculate LTB
positives into BGLB and EC media tubes.
Tube # 1 2 3 4 5 6
Mls of sample/diln Infl

tube
Effl
LTB 24 hr (+ or -) Infl
Effl
BGLB 48 hr (+ or -) Infl
Effl
EC 24 hr (+ or -) Infl
Effl
AI 24 hr (+ or -) Infl
Effl
From MPN Index table find MPN Index per 100 ml. Multiply the MPN index by the dilution factor from the series
used when dilutions other than 1/1, 1/ 10 and 1/100 are used.
32
Heterotrophic Plate Count - pour plate method, 35oC/48 hr. (1 ml is taken from each dilution
tube. Calculate the amount of original sample is in that ml.)
Tube # 1 2 3 4 5 6
Mls of sample Infl
Effl
Plate count Infl
Effl
*CFU/ml Infl
Effl
*CFU= colony-forming units
Membrane Filter Technique – 35oC/24 hr. The entire contents of each dilution tube is used.
Calculate the amount of original sample there is in each tube (note tube volume).
Tube # 1 2 3 4 5 6
Mls of sample Infl
Effl
Coliform Infl
Colonies
Effl
#Colonies/100 Infl
mls
Effl
Additional notes
Presence/Absence (P/A) Testing:
 Media chosen may be Presence/Absence broth (P/A) or Lauryl Tryptose broth with Bromcresol
Purple broth (LT/BCP) to indicate acid formation. Both media meet USEPA guidelines for testing of
total coliforms in drinking water. They are in concentrated form and merely require the addition of
100 ml of sample (or diluted sample) to the media and incubation. If zero total coliform is the
maximum contamination goal, it is not necessary to enumerate.
 With MUG reagent added to P/A Broth, you can simultaneously detect total coliforms and E.coli. A
long-wave UV light is required to detect fluorescence. MUG (4-methylumbelliferyl-β-D-glucuronide)
can be used to detect E.coli because it produces a fluorogenic product when hydrolyzed by
glucuronidase, an enzyme specific to E.coli. E.coli will fluoresce as early as 4-24 hours.
Estimation of Bacterial Density - Most Probably Number (MPN)

When more than three dilutions are used in a decimal series of dilutions, use the results from only three
of these in computing the MPN. To select the three dilutions to be used in determining the MPN index,
choose the highest dilution that gives positive results in all five portions tested (no lower dilution giving
33
any negative results) and the two next succeeding higher dilutions. Use the results at these three
volumes in computing the MPN index. In the examples given below, the significant dilution results are in
boldface. The number in the numerator represents positive tubes; that in the denominator, the total
tubes planted; the combination of positives simply represents the total number of positive tubes per
dilution:
Example 1 0.1 0.01 0.001 Combination MPN Index

ml ml ml ml of positives /100 ml
a 5/5 5/5 2/5 0/5 5-2-0 5000
b 5/5 4/5 2/5 0/5 5-4-2 2200
c 0/5 1/5 0/5 0/5 0-1-0 20
Further information can be found in the laboratory library (do not remove without permission):
Standard Methods for the Examination of Water and Wastewater -Part 9000 Microbiological Examination
 HACH publications:-Catalogue listing testing methods with descriptions.

o -The Use of Indicator Organisms to Assess Public Water Safety
o - http://www.hach.com/ Information Central/Learning Library/Microbiological Testing
 ASTM: Bacterial Indicators/ Health Hazards Associated with Water (STP635)
 D.D. Mara: Bacteriology for Sanitary Engineers
 Microbiology 321: -Manual of Microbiological Techniques
 Website: There are many websites with information including:
http://www.microbelibrary.org/microbelibrary/files/ccImages/Articleimages/keen/Gramstainkeen.htm
Lab 5 Questions: Discussion/Questions:
1. What are the sources of error in these procedures?
2. Compare MPN, MF results. Do they agree? Why or why not?
3. What is a heterotroph? Compare MPN/MF with Heterotrophic Plate Count. Do they agree? Why
or why not? What is expected?
4. Were the reductions through the sewage treatment plant as expected?
5. Compare the advantages and disadvantages of the multiple tube fermentation and membrane
filter techniques.
6. What are the important pathogens transmitted by water?
7. In handling wastewaters in the laboratory, what care must be taken to protect laboratory
personnel from pathogen infection?
34
Table 2 - MPN Index and 95% Confidence Limits for Various Combinations of Positive Results
when 5 Tubes are used per Dilution (10 mL, 1.0 mL, 0.1 mL)
Comb. MPN 95% Confidence Comb. MPN 95% Confidence
Of Index/ Limits Of Index/ Limits
Positives 100 ml Lower Upper Positive 100 ml Upper Lower
s
0-0-0 <2 - - 4-2-0 22 9 56
0-0-1 2 1 1 4-2-1 26 12 65
0-1-0 2 1 10 4-3-0 27 12 67
0-2-0 4 1 13 4-3-1 33 15 77
1-0-0 2 1 11 4-4-0 34 16 80
1-0-1 4 1 15 5-0-0 23 9 86
1-1-0 4 1 15 5-0-1 30 10 110
1-1-1 6 2 18 5-0-2 40 20 140
1-2-0 6 2 18 5-1-0 30 10 120
2-0-0 4 1 17 5-1-1 50 20 150
2-0-1 7 2 20 5-1-2 60 30 180
2-1-1 9 3 24 5-2-0 50 20 170
2-2-0 9 3 25 5-2-1 70 30 210
2-3-0 12 5 29 5-2-2 90 40 250
3-0-0 8 3 24 5-3-0 80 30 250
3-0-1 11 4 29 5-3-1 110 40 300
3-1-0 11 4 29 5-3-2 140 60 360
3-1-1 14 6 35 5-3-3 170 80 410
3-2-0 14 6 35 5-4-0 130 50 390
3-2-1 17 7 40 5-4-1 170 70 480
4-0-0 13 5 38 5-4-2 220 100 580
4-0-1 17 7 45 5-4-3 280 120 690
4-1-0 17 7 46 5-4-4 350 160 820
4-1-1 21 9 55 5-5-0 240 100 940
4-1-2 26 12 63 5-5-1 300 100 1300
5-5-2 500 200 2000
5-5-3 900 300 2900
5-5-4 1600 600 5300
5-5-5 1600 -— ----
35
Gram-negative cocci
Diplococci: usually characteristic of Neiseria spp., such
as N. meningitidis
In addition, Moraxella spp. and Acinetobacter spp.are
often diplococcal in morphology. Acinetobacter can be
pleomorphic, and sometimes appear as Gram-positive
cocci.
Coccobacilli: usually characteristic of Acinetobacter

spp., which can be either Gram-positive or Gram-
negative, and is often Gram-variable.
Gram-negative bacilli
Thin rods: usually characteristic of enterobacteriaceae,
such as E. Coli
36
Coccobacilli: usually characteristic of Haemophilus

spp., such as H. influenzae
Curved:usually characteristic of Vibrio spp.;

Campylobactor spp., such as
V. cholerae C. jejuni
Thin needle shape: usually characteristic of

Fusobacterium spp.
37
Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity
OBJECT: to familiarize you with the most common water treatment tests;
to acquaint you with the use of pH metres, pH test paper and other water
WARNING quality assessment equipment.
Please use caution
when handling all
chemicals, particularly
those labelled with MATERIALS: pH meter, pH test paper, magnetic stirrer, bromphenol blue,
specific hazards. phenolphthalein, bromcresol green, metacresol purple, and other
indicators, standard acid, standard base, burettes and stand, graduated
Use care when using cylinders, beakers, flasks, colour comparator, turbidimeters, EDTA,
lab equipment. buffer solution, hardness indicators, 1 N NaOH, water samples.
Probes, metres and
calibrated glassware
are available in limited
quantities and are very
expensive to replace. I. DETERMINATION OF pH
1. Check the pH of a small aliquot of the sample by wetting a pH
Always wear personal test paper and comparing the colour combinations to those shown
protective equipment.. on the case. (Take care not to contaminate paper in case.)
2. Calibrate the pH meter by following the procedure outlined on the
instruction sheet beside the meter unless told otherwise. Be sure
that buffers are being stirred when you perform the calibration and
that when you change buffers you thoroughly rinse the probe into
a waste beaker and blot dry so that you do not contaminate other buffers or your samples.
Normally, the two buffers that are chosen for calibration will bracket your sample ie if sample is
less than 7, then use buffers 4 and 7 to calibrate; if above 7, then use buffers 7 and 10.
3. After calibration is complete, rinse & dry probe, place a beaker containing the sample and stir-
bar on the stir-plate, lower probe, turn on stirrer and obtain a stable reading.(Do not allow the
stir bar to hit the probe.) Record value in following table.
4. Rinse probe when you are finished and leave immersed in pH 7 buffer for storage.
II. DETERMINATION OF ALKALINITY

1. Rinse and fill a labelled burette with standard acid solution.
2. Measure 100 ml of water sample into a clean, sample-rinsed graduated cylinder and transfer to
an Erlenmeyer flask. In order to remove any free residual chlorine (which interferes with the
indicator colour response), add 1 drop of 0.1 N sodium thiosulphate to the sample. Add a stir-
bar.
3. Add a full dropper of phenolphthalein (P) indicator and if the solution stays pink or red, titrate
until just colourless. Record burette reading (P). (If solution is clear after addition of P...think
what that means!)
4. Add a dropper full of bromcresol green (BG= MO) to this same water sample and continue the
titration until the colour changes from blue to yellow (at pH 4.5).
5. Record the burette reading in the following table. (BG or MO)
6. If sample is highly alkaline (titration goes over one burette full) dilute the sample and titrate
again. Apply dilution factor to obtain final result.
38
III. DETERMINATION OF ACIDITY
1. Rinse and fill another burette with standard base solution and label it.
2. Measure 100 ml of sample (with minimum agitation or exposure to air) using a graduated
cylinder and transfer carefully to an Erlenmeyer flask. Add 1 drop of sodium thiosulphate
solution.
3. Add a full dropper of bromphenol blue indicator and titrate until the colour changes from yellow
to blue ("MO" Acidity). (If the solution is already blue after indicator....?)
4. Measure out a new sample and add 1 drop of sodium thiosulphate and a full dropper of
phenolphthalein. Titrate to the first uniform pink colour (colour stays). Record the volume of
titrant required in the following table.
IV. TOTAL HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD
1. Rinse and fill a burette with standard EDTA solution and record the initial burette reading.
2. Measure out 25 mls of sample using a graduated cylinder and dilute to 50 ml with distilled water.
Pour into a 250 ml Erlenmeyer flask. Add stir-bar.
3. Add 1-2 ml of buffer solution using dropper bottle (3 full droppers) and one aliquot of Total
Hardness Indicator using the special dispenser on the chemical bottle. The buffer elevates the
pH to 10. A pH much above 10 promotes CaCO3 precipitate.
4. Titrate slowly with EDTA, while stirring, until the reddish tinge disappears from the solution and
a pure blue remains. Add the last few drops at 3-5 second intervals so that you do not
"overshoot" the end-point. A paper towel placed under the flask will facilitate this determination.
Complete the titration within 5 minutes to minimize the tendency toward CaCO3 precipitation. If
a ppt does form within the 5 minutes, it may be necessary to repeat the titration with the sample
diluted 1 to 1 (again) with distilled water. Alternatively, if the approximate hardness is known (by
unsuccessful attempts), add 90% of the titrant to the sample before adjusting the pH with buffer.
5. Record the final burette reading in the following table.
V. CALCIUM HARDNESS DETERMINATION - EDTA TITRIMETRIC METHOD

1. Refill the burette with EDTA titrant and note the initial reading.
2. Measure out 50 ml of sample into an Erlenmeyer flask and add 2.0 ml of 1 N NaOH solution
(use 3 droppers full), or a volume sufficient to produce a pH of 13-14 (designed to precipitate
the magnesium as a hydroxide). Add a stir-bar and stir to mix.
3. Add one aliquot of the Calcium Hardness Indicator and titrate with EDTA slowly, with continuous
stirring to a pure blue end-point.
4. Record the final burette reading in the following table.
VI. COLOUR - TRUE AND APPARENT

PART A: TRUE COLOUR
1. Filter about 50 mls of sample through provided filter paper, using a filter funnel and stand.
2. Pour the supernatant into one of the colour comparator cells to the etched mark and fill the other
with distilled water. Place the glass "plunger" lid on top, making sure that the top is dry and the
bottom is completely submerged in the liquid within the cell.
3. Turn the numbered dial until the colour on the dial matches the colour of the sample. Hint: Try to
match hue or tone rather than specific colour intensity. The dial reading is colour in A.P.H.A.
units. If colour appears stronger than 100 units, dilute the sample to bring it into the correct
range.
39
PART B: APPARENT COLOUR

1. Use the technique outlined in Part A, except on an unfiltered sample, to determine the
apparent colour (Removes the influence of turbidity on the reading).
2. Record all colour data in the following table.
VII. TURBIDITY - BY HACH, HELLIGE AND JACKSON CANDLE METHODS

PART A: Hach Turbidimeter
1. Place the sealed standard into holder and place light shield cap over standard. Make
sure that the cell riser is placed in the cell holder assembly before the standards in the 0-
100 and 0-1000 FTU ranges. Adjust the calibration knob to the correct reading.
2. Fill the sample cell with undiluted sample and read the turbidity. It may be necessary to
recalibrate using the appropriate standard range ie if the sample is below 10, calibrate
using the 10 FTU standard. If the sample is above 1000, it will be necessary to dilute
using filtered sample water not distilled water (which might dissolve some of the turbidity
in the sample).
3. Rinse out the sample cell and record your reading in the following table. DO NOT TURN
THE TURBIDIMETER OFF.
PART B: Hellige Turbidimeter

1. Carefully fill the 20 mm viewing depth tube to the mark with sample. Lower the plunger
into the liquid making sure no bubbles are trapped under the plunger and the top is dry.
Wipe the outside of the tube to dry it and place it in the turbidimeter (in the circular
groove of the mirror unit). Note that the rectangular door mirror is closed, that the filter
selector shows "none" and that bulb B is in use. Close the door and switch on the light.
2. Immediately balance the light intensity of the central spot with the surrounding
observation field by turning the dial on the right-hand side of the instrument. Take the
reading where the dark central part first merges with the surrounding field. If you take too
long, the turbidity may start to settle and condense on the bottom of the tube.
3. Read the scale on the dial, record the number and refer to the appropriate graph to
determine the turbidity as mg/L SiO2.
PART C: Jackson Candle Turbidity - this will be shown to class but not individually
performed.
1. Be sure that the calibrated tubes are clean before adding samples to the tubes. DO NOT
PLACE AN EMPTY TUBE IN THE TUBE HOLDER WITH THE CANDLE LIT - adding
liquid to a hot tube will cause it to shatter. Try to be quick and not burn the candles more
than a few minutes at a time. Pinch all excess charred wick with fingers before lighting or
soot may be deposited on the glass tubes, obscuring vision.
2. Pour sample into the tube in small increments until you can no longer distinguish the
shape of the candle flame. Remove about 10 mls of sample by pipette and slowly
dispense this aliquot back into the tube until the shape of the flame is no longer
distinguishable. (This allows you to make a more gradual and accurate approach to the
end-point.)
3. Remove the glass tube from the holder and read the turbidity at the meniscus of the
sample. Note that there are several different scales on the side of the tube. Record value
in following table as J.T.U.s.
40
Alkalinity relationships
Result of Hydroxide Carbonate Bicarbonate

Titration Alkalinity Alkalinity Concentration
as CaCO3 as CaCO3 as CaCO3
P=0 0 0 T
P<1/2T 0 2P T-2P
P=1/2T 0 2P 0
P>1/2T 2P-T 2(T-P) 0
P=T T 0 0
Where: P = Phenolphthalein alkalinity; T = Total alkalinity.

See Standard Methods for the Examination of Water and Wastewater: Method: 2320 A & B
41
DATA
Sample 1 Sample 2
pH - Initial pH by test paper __________ ___________
Initial pH by pH meter __________ ___________
ALKALINITY- Sample volume __________

P. Alkalinity end-point reading (mls) __________
"M.O." Alkalinity end-point (mls) __________
Initial burette reading __________
Net P. Alkalinity titre (mls) __________
Net "M.O." Alkalinity titre (mls) __________
ACIDITY- Sample volume __________ ___________

"M.O." Acidity end-point reading (mls) __________ ___________
Initial burette reading __________ ___________
P. Acidity end-point reading (mls) __________ ___________
Initial burette reading __________ ___________
Net "M.O." titre (mls) __________ ___________
Net Total acidity titre (mls) __________ ___________
Net CO2 Acidity titre (mls) __________ ___________
TOTAL Hardness Sample volume (mls) __________

Final burette reading (mls) __________
Initial burette reading (mls) __________
Net titre (mls) __________
Total Hardness as mg/L CaCO3 __________
CALCIUM Sample volume (mls) __________

Final burette reading (mls) __________
Initial burette reading (mls) __________
Net titre (mls) __________
Calcium as mg Ca++/L __________
Calcium hardness as mg CaCO3/L __________
42
Calculations
Calcium
A × B × 4 00 .8
m gC a/L 
m lo fs a m p le

B = mg CaCO3 equivalent to 1.00 ml EDTA titrant at the calcium indicator endpoint = 1.0
Calcium Hardness
m gC a C 3O/L  A × B × 1 00 0
m lo f s a m p l e

Total Hardness
A × B × 1 00 0
m gC a C3O/L 
m lo f s a m p l e

Alkalinity
A × N × 5 0,0 0 0
m gC a C3O/L 
m lo f s a m p l e
where: A= ml standard acid used

N= normality of standard acid
Acidity
A × N × 5 0,0 0 0
m gC a C3O/L 
m lo f s a m p l e
where: A= ml standard base used

N= normality of standard base
43
RESULTS
Sample 1
P. Alkalinity as CaCO3 (mg/L) ______________________________________
"M.O." Alkalinity as CaCO3 (mg/L) ______________________________________
OH- Alkalinity as CaCO3 (mg/L) ______________________________________
CO3-2 Alkalinity as CaCO3 (mg/L) _____________________________________
HCO3- Alkalinity as CaCO3 (mg/L) ______________________________________
OH- Alkalinity as OH- ion (mg/L) ______________________________________
CO3-2 Alkalinity as CO3-2 ion (mg/L) ______________________________________
HCO3- Alkalinity as HCO3- ion (mg/L) ______________________________________
"M.O." Acidity as CaCO3 (mg/L) ______________________________________
Total Acidity as CaCO3 (mg/L) ______________________________________
Free carbon dioxide as CO2 (mg/L) ______________________________________
Total Acidity as CO2 (mg/L) ______________________________________
Ca Hardness as mg Ca+2 (mg/L) ______________________________________
Ca Hardness as CaCO3 (mg/L) ______________________________________
Mg Hardness as CaCO3 (mg/L) ______________________________________
Mg Hardness as Mg+2 (mg/L) ______________________________________
Carbonate Hardness as CaCO3 (mg/L) ______________________________________
Non-carbonate Hardness as CaCO3 (mg/L) ______________________________________
Excess alkalinity as CaCO3 (mg/L) ______________________________________
Colour - True APHA Colour Units _______________NTU_______________
Apparent APHA Colour Units _______________NTU_______________
Turbidity - Hach - N.T.U. ______________________________________
- Hellige - mg/L SiO2 ____________
- Jackson Candle - J.T.U. ____________
44
Lab 6 Questions
1. Is this water suitable for a domestic water supply? Why?
2. What errors can occur in the calcium hardness determination? Why
3. How do the Hellige, Hach and Jackson Candle turbidities compare?
4. What sanitary significance does colour have?
5. A water has the following analysis:
Na+ 15 mg/L HCO3- 70 mg/L Mg+2 18 mg/L

K+ 33 mg/L Cl- 42 mg/L
Ca+2 12 mg/L SO4-2 8 mg/L
What is the carbonate, non-carbonate and total hardness of this water in mg/L as CaCO3? in
meq/L? Is the analysis complete? Why?
6. From equations (17-12) and (17-13) in Sawyer and McCarty, calculate the carbonate alkalinity in
mg/L as CaCO3 for the water sample analyzed during this lab, given K2 = 4.7 x 10-11 and Kw = 10-14.
Does this agree with the results from Part II of this lab? Explain.
ity 
5 0,0 0 0A l k a li n [H ] K w
 5 00 0 0 [H 
]
CO
3 (a s m g ) 
/L C a C3O

[H ]
1
2K2
Note: You should bring a copy of this lab to the next two sessions as you will need the
same instructions to perform the same tests in the Coagulation and Softening Labs.
45
Laboratory 7a: Coagulation and 7b: Coagulation & Softening
OBJECT:
-to illustrate the principles of coagulation and water
softening. This is a two-step experiment, to be conducted over two lab
Attention periods. The first step will be to analyse the raw water sample and the
supernate from a number of alum dosages (to be announced in the
This lab will require class) using the techniques learned in Lab. No.6. The optimum alum
patience and dosage for coagulation of the sample will then be chosen for the next
cooperation from all of session (7b). In part 7b, several lime and soda ash dosages will be
you as we have only selected for determining the efficiency of water softening. The supernate
two jar testers with 6
from the various coagulation and softening treatments will then be
paddle sites each.
Please try to analyzed.
coordinate your work
with the rest of the
class. You should
have with you the part MATERIALS:
of Exercise 7 that -Jar Test Apparatus, 1000 ml beakers, alum solution
gives instructions for (Al2(SO4)3*18H2O), soda ash solution (Na2CO3) and lime (Ca(OH)2) or
the various tests. sodium hydroxide (NaOH), and all of the materials from Lab. No. 7
Caution (except Jackson Candle and Hellige Turbidimeter).
Wear personal
protective equipment
at all times. Corrosive
chemicals in use.
46
Part A: COAGULATION USING ALUM - work in 2 teams to assess a total of 6 dosages for each team
plus a raw water sample.
1. Measure out six 1000 ml aliquots of sample (using graduated cylinder to measure, not a beaker)
and transfer to 1000 ml beakers.
2. Place beakers on the jar tester apparatus. Make sure they are centred.
3. Dispense Alum from a burette into small beakers or plastic weigh boats and add to beakers on jar
tester as close to simultaneously as possible, use a small amount of distilled water to rinse the
small beaker or weigh boat into the large beaker and then start the stirrer. Stir at 100 rpm for 1
minute, then reduce stirring rate to about 20 rpm for 10 minutes (this is just fast enough to keep the
floc suspended but not so fast that the floc is sheared). Observe and make notes describing floc
formation and note the time of appearance.
4. Stop stirring after 10 minutes. Lift & secure paddles out of beakers. Record relative floc sizes (pin-
point, small, medium, large); clarity of supernatant liquid (very clear, clear, hazy, cloudy); and
settling characteristics of the floc (rapid, moderate, slow).
5. After the flocs have settled (allow 15 min), decant the supernatant liquid carefully into another set of
beakers (try to do one continuous pour so that you do not stir up what has settled).
6. Each person of each team will be assigned one or more tests and will do all the samples for those
tests. Analyze these supernates plus a sample of raw water for pH, alkalinity (―P‖ and Total), acidity
(―MO‖ and Total), hardness (total and calcium), true colour (using the Hach Spectrophotometer set
to the Colour 465 nm method) and turbidity (using Hach turbidimeter). Remember that there is only
800-900 mls to do all the tests, so do not waste sample! Stir decanted samples before removing
portions for tests, at least until turbidity measurement has been done so as not to corrupt the
measurement.
7. Complete the coagulation summary sheet during the lab period and fill in the table on the board. In
the lecture, you will discuss the data and select the appropriate alum dosage to be used by all
groups in the water softening step next week. You will also determine the optimum lime and soda
ash dosages to soften this water and select a range of dosages surrounding this optimum for
investigation next week based on the analysis of the raw sample.
47
Raw Data - each team is responsible for their own raw data for 6 treatments.
Raw Alum
DATA Sampl mg/L mg/L mg/L mg/L mg/L
e mg/L
pH
Alkalinity-sample vol ml
Net titre to pH 8.3 ―P‖
Net titre to pH 4.5 ―TOT‖
Acidity - sample vol ml
Net titre to pH 3.7 ―MO‖
Net titre to pH 8.53―TOT‖
Total Hardness sample vol
Net titre
Ca Hardness - sample vol
Net titre
48
Calculated Data Summary – put these numbers on the board as well. Each team is responsible for data for 6 dosages.
Raw Alum Raw

DATA Sam mg/L mg/L mg/L mg/L mg/L Sam mg/L mg/L mg/L mg/L mg/L mg/L
ple mg/L ple
pH
Floc size
Clarity
Floc settling rate
Alkalinity (mg/L CaCO3) ―P‖
Alkalinity (mg/L CaCO3) ―TOT‖
Acidity (mg/L CaCO3) ―MO‖
Acidity (mg/L CaCO3) ―TOT‖
Total Hardness (mg/L CaCO3)
Ca Hardness (mg/L CaCO3)
True Colour (PtCo colour)
Turbidity (N.T.U.)
(unfiltered!)
49
Part B: Coagulation & Softening - work in 2 teams to assess a total of 12 dosage combinations, 6 for
each team plus a raw water sample.
1. Measure out six 1000 ml aliquots of sample (using a graduated cylinder to measure, not a
beaker) and transfer to 1000 ml beakers.
2. Place beakers on the jar tester apparatus. Make sure it is centred.
3. Dispense selected dosage of Alum to sample using either the 100 mg/mL or the 10 mg/mL
solution (select to minimize volume added). If you use the 100 mg/mL solution you could
dispense using a burette into a small plastic weighing dish. The dish could then be ―dunked‖ into
the sample quickly and easily. Stir at 100 rpm for one minute after all beakers have been dosed,
then reduce stirring rate to about 20 rpm for 10 minutes
4. Stop stirring after 10 minutes. Lift paddles out of beakers and secure.
5. After the flocs have settled (allow 15 min), decant exactly 800 mls of the supernatant liquid
carefully into a graduated cylinder and transfer into a clean set of beakers (try to do one
continuous pour so that you do not stir up what has settled).
6. Position beakers on apparatus and prepare your 6 lime and soda ash dosages. Lime (Ca(CO)3)
is in the balance room. Calculate how much to weigh out based on volume of 800 mls. Weigh
out 6 portions into labelled weigh dishes. Before adding to the jar tester beakers make a slurry
by adding a bit of distilled water and stirring with a stirring rod. Position in front of beakers.
Measure out appropriate volumes of 100 mg/mL soda ash (Na2CO3) into small beakers or
weigh dishes and position these in front of designated beakers. Make sure jar tester beakers
are labelled i.e. 150/250, 150/300, etc. for lime and soda ash dosages that apply.
7. In a coordinated effort, add both doses to beakers as quickly as possible and then start stirring
at 100 rpm for 1 minute then decrease to 20 rpm for 10 minutes.
8. After that 10 minutes stop stirring and raise paddles. Let settle for 15 minutes and carefully
decant as much as possible into clean beakers. Note that you now have only 600-700 mls to do
all the tests on so don’t waste it!
Lab 7 Questions
A. Coagulation
1. Analyze the response of colour and turbidity removal as a function of alum dose.
2. How does the consumption of alkalinity by alum compare with theoretical calculation based
on balanced equations? How much extra soda ash should you add to compensate for the
alkalinity consumption by 70 mg/L alum?
B. Softening
1. Based on your chemical analysis of the raw water, show by calculation the theoretical
amount of lime and soda ash required for excess lime/soda ash softening.
2. Compare your experimental softening results to the theoretical results based on solubility
equations or mass balance equations.
3. Compare removal of hardness components under the different doses - use graphical
presentation.
4. What treatment would you recommend to prepare this water for human consumption? Why?
50
Raw Data – Each team is responsible for raw data for 6 doses plus raw sample
Lime/NaOH mg/L ______ ______ ______ ______ ______ ______

Soda Ash mg/L Raw ______ ______ ______ ______ ______ ______
Sample
Alum mg/L ______ ______ ______ ______ ______ ______
___ _ _ _ _ _
______ ______ ______ ______ ______ ______
pH _ _ _ _ _ _
Alkalinity-sample vol ml
Net titre to pH 8.3
Net (Total) titre pH 4.5
Acidity - sample vol ml
Net titre to pH 3.7
Net (Total) titre pH 8.3
Total Hardness sample vol
Net titre
Ca Hardness - sample vol
Net titre
51
Calculated Data Summary – put these numbers on the board as well. Each team is responsible for data for 6 dosages + raw sample.
Lime/NaOH mg/L Raw _____ _____ _____ _____ _____ _____ Raw _____ _____ _____ _____ _____ _____
Soda Ash mg/L Sample _____ _____ _____ _____ _____ _____ Sample _____ _____ _____ _____ _____ _____
Alum mg/L _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____ _____
pH
P- Alkalinity -mg/L CaCO3
*Total Alkalinity-mg/L CaCO3
―MO‖ Acidity -mg/L CaCO3
*Total Acidity -mg/L CaCO3
Total Hardness -mg/L CaCO3
Ca Hardness -mg/L CaCO3
Mg Hardness -mg/L CaCO3
**Carbonate Hard. -mg/L CaCO3
**Non-Carb Hard -mg/L CaCO3
**Excess Alk. -mg/L CaCO3
True Colour (PtCo colour units)
Turbidity (N.T.U.)
Note:* Total Alkalinity must include Phenolphthalein Alkalinity just as Total Acidity must include ―MO‖ Acidity.
** By Calculation
52
Notes on methods:
Alkalinity: Use the station that is set up with a pH meter and acid titrant (0.02N). Rinse probe before
inserting in 100 mls of sample in a beaker; positioned probe in sample under the burette which must be
able to drip unimpeded into the sample. Allow the reading to stabilize and record pH before starting to
titrate. You may add indicator (phenolphthalein for 8.3 and bromcresol green for 4.5) if you want but
you will be relying on the pH meter to indicate the endpoint: (down) to pH 8.3 for the first endpoint and
down to 4.5 for the second endpoint. Record the volumes for each and calculate ―P‖ Alkalinity and Total
Alkalinity. Put numbers on the board.
Acidity: Use the station that is set up with a pH meter and base titrant (0.02N). Rinse probe before
inserting in 100 mls of sample in a beaker; positioned probe in sample under the burette which must be
able to drip unimpeded into the sample. Allow the reading to stabilize and record pH before starting to
titrate. You may add indicator (bromphenol blue for 3.7 and phenolphthalein for 8.3) if you want but you
will be relying on the pH meter to indicate the endpoint: (up) to pH 3.7 for the first endpoint and up to
pH 8.3 for the second. If you add the indicator for this test you will have to pour two samples (one for
each indicator) therefore, if the pH is above 3.7 record 0 for the first endpoint and just add the second
indicator. However, you will be relying on the pH meter to indicate the endpoints regardless of the
colour. Record the volumes, calculate ―MO‖ acidity and Total Acidity and put numbers on the board.
Total Hardness: Use the station set up for this. Use 25 mls of sample, add 25 mls of water, 2 mls of
Total Hardness Buffer using a plastic graduated droppers (2 x 1 ml) and the white crucibles provided.
Add one or two aliquots from the special dispenser marked Total Hardness or use 2 or 3 of the little
packets marked ManVer. Whichever you use make sure that the colour at the start is pronounced and
titrate fairly quickly until purple and slower to a final pure blue colour. Calculate and put numbers on the
board.
Ca Hardness: Use the station set up for this. Use 25 mls of sample (instead of 50 mls), add 25 mls of
water, 2 mls of 1 N NaOH using a plastic graduated dropper (2 x 1 ml) and the white crucibles provided.
Add one or two aliquots from the special dispenser marked Calcium Hardness or use 2 or 3 of the little
packets marked CalVer. Whichever you use make sure that the colour at the start is pronounced and
titrate fairly quickly until purple and slower to a final pure blue colour. Calculate and put the numbers on
the board.
Colour: There are two Spectrophotometers available, one for each team. It should be set for Colour at
465 nm and units are PtCo colour units. Zero the instrument using distilled water. Filter a portion of
sample to measure True Colour. Put values on the board.
Turbidity: There is only one Hach Turbidimeter. If it is off put the distilled water vial in the meter, close
the lid and turn the power on. It should zero on the water. Use the other vial to measure each of your
samples - with the lid closed. Put values on the board.
53
Notes on calculating dosages to use for softening (based on hypothetical results for a 70 mg/L Alum
dose of a sample with the following values)
1) Calculate meq/L
pH 6.46
CO2 = Total acidity = 27 mg/L x 27/50 = 0.54 milli-equivalents/L (meq/L)
―MO‖ Acidity = 0 mg/L
CO2 = Total acidity = 27 mg/L x 27/50 = 0.54 meq/L
―P‖ Alkalinity = 0 mg/L
Total Alkalinity = 57 mg/L x 57/50 = 1.14 meq/L
Total Hardness = 358 mg/L = 358/50 = 7.16 meq/L
Ca Hardness = 204 mg/L = 204/50 = 4.08 meq/L
2) Calculate forms of Hardness:

Magnesium hardness = T.Hardness- Ca Hardness = 7.16-4.08 = 3.08 meq/L
Carbonate Hardness = Total Alkalinity = 1.14 meq/L
Non-Carbonate Hardness = T.Hardness - Carbonate Hardness = 7.16-1.14= 6.02
3) Calculate optimum lime addition:

a) remove CO2 = 0.54 meq/L
b) convert HCO-3 to CO3 = 1.14 meq/L
c) precipitate magnesium = 3.08 meq/L
d) raise ph (use 1 meq/L) = 1.00 meq/L
Total = 5.76 meq/L
Equivalent weight of lime (Ca(OH)2) = 74/2=37 5.76 x 37 = 213 mg/L
4) Remove non-carbonate hardness: Equivalent weight of Na2CO3=106/2 = 53; 6.02 x 53 = 319 mg/L
Choose various dosage combinations based on 200 Lime and 300 Soda Ash as optimum
54

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CIVL407

UNIVERSITY OF BRITISH COLUMBIA

Laboratory Exercise Guide

Station/Drawer Supply List ..................................................................... 2

First Session: Laboratory Safety and Orientation ................................ 3

Laboratory Report Write-Up Guidelines ................................................ 5

Laboratory Exercises .............................................................................. 6

Laboratory 1: Solids Determination ....................................................... 7

Laboratory 2: COD, DO and BOD5 ........................................................ 11

Laboratory 3: Chloride Determination ................................................. 16

Laboratory 4: Chlorine and Chlorine Demand .................................... 23

Laboratory 5: Bacteriological Examination of Sewage

Laboratory 6: Alkalinity, Acidity, pH, Hardness, Colour, Turbidity

Laboratory 7a: Coagulation and 7b: Coagulation & Softening .......... 46

Date 2009 Subject

Lecturer: Dr. Eric Hall email: ehall@civil.ubc.ca 604-822-2707

Station/Drawer Supply List

2 permanent marker pens

On benches each row:

First Session: Laboratory Safety and Orientation

5. Safety Equipment: Eye Wash Station, Safety Shower.

6. WHMIS: Workplace Hazardous Materials Information System is a government regulation

10. Pipettes/ graduated cylinders/ beakers/ flasks: Information:

Laboratory Report Write-Up Guidelines

REPORT ELEMENTS SHORT REPORT LONG REPORT

#2 Dissolved oxygen, COD, BOD

#5 Bacteriological Examination of waste water

#6 Acidity, alkalinity, hardness, colour, turbidity

#7b Coagulation and Softening

Laboratory 1: Solids Determination

B. TOTAL SOLIDS and TOTAL VOLATILE AND FIXED SOLIDS AT 550oC

C. SUSPENDED SOLIDS - TOTAL AND VOLATILE

Calculate the percent solids reduction through the treatment plant.

% reduction in settleable matter _________

1. What major types of solids are removed in primary treatment? in secondary

5. Why must residue samples be brought to ambient temperature before weighing?

DATA AND RESULTS

Laboratory 2: COD, DO and BOD5

DO NOT DISCARD CONTENTS OF VIALS. LEAVE IN BEAKER AT YOUR STATION.

Part B: DISSOLVED OXYGEN (DO)

DISSOLVED OXYGEN PROBE

WINKLER TITRATION (Azide modification)

6. Complete all four titrations.

Dissolved Oxygen Data

Sample/Data Raw Sewage Final Effluent Tap Water Dil’n Water

DETERMINING PERCENT SATURATION THE "QUICK AND EASY" METHOD

BIOCHEMICAL OXYGEN DEMAND

BIOCHEMICAL OXYGEN DEMAND (BOD) DATA

Dilution water Bottle number (not lid #) _____

When seeded water is used:

where: D1 = DO of diluted sample immediately after preparation, mg/L

Percentage BOD reduction through treatment plant ___________

CH3OH + ³/2 O2 → CO2 + 2H2O

Laboratory 3: Chloride Determination

I. CHLORIDE - SPECIFIC ION ELECTRODE

Calculate the chloride concentration, where:

mg Cl- in sample = __________________________

mg/L Cl- = _______________________

II. CHLORIDE - BY COLOURIMETRIC METHOD

The principle of this method is based on the following formula:

1. Using a 25 ml graduated cylinder, measure 25 mls of ―zero‖ standard or ―blank‖ (halide-free

III. MERCURIC NITRATE METHOD FOR CHLORIDE DETERMINATION

Hg++ + 2Cl- ➔ HgCl2 and Hg++(excess) + DPC ➔ violet colour