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First Edition (Revised August 2008)

HANDBOOK
LABORATORY MANUAL FOR
ENVIRONMENTAL SCIENCE

Editors

1st Edition: Ahmad Naim Ahmad Yahaya


Nadia Razali,
st
1 Revision: Robert Thomas Bachmann, Elmy Nahida Othman

Publisher
UniKL MICET

1
Table of Content Page

Experiment 1 .................................................................................................................. 3
Determination of Total Solids Dried, Total Dissolved Solids, Total Suspended Solids
and Fixed and Volatile Solids Ignited (APHA / ASTM method 2540B-E). ................. 3
Experiment 2 ................................................................................................................ 11
Determination of pH, Temperature, Colour and Turbidity (APHA / ASTM methods
4500-H+, 2120 and 2130). ............................................................................................ 11
Experiment 3 ............................................................................................................... 22
Determination of Total Kjeldahl Nitrogen (TKN) (APHA / ASTM 4500-Norg)............... 22
Experiment 4 ................................................................................................................ 27
Determination of Phosphate (APHA / ASTM 4500-P) ............................................... 27
Experiment 5 ................................................................................................................ 31
Determination of COD in Wastewater (Vial Method) ................................................. 31
Experiment 6 ................................................................................................................ 39
Determination of Chemical Oxygen Demand (COD) Open Reflux Method (APHA /
ASTM 5220 B) ............................................................................................................ 39
Experiment 7 ................................................................................................................ 42
Determination of BOD in Wastewater (METHOD No. 405.1) ................................... 42
Experiment 8 ................................................................................................................ 52
Determination of Dissolved Oxygen (DO) and 5-Day Biological Oxygen Demand
(BOD5) (APHA / ASTM 5210 B) ................................................................................ 52
Experiment 9 ................................................................................................................ 56
Correlation of Biological Oxygen Demand (BOD) and Chemical Oxygen Demand
(COD) .......................................................................................................................... 56
Experiment 10 .............................................................................................................. 59
Determination of Ammonia (NH3) (APHA / ASTM 4500-NH3) ................................ 59
Experiment 11 .............................................................................................................. 63
Determination of Heavy Metals: Lead (Pb) and Arsenic (As) by the Direct Air-
Acetylene Flame Method (APHA / ASTM 3111 B) ................................................... 63
Experiment 12 .............................................................................................................. 66
Microbiological Analysis - Standard Presumptive Total Coliform Fermentation
Technique (APHA / ASTM 9221 B) ........................................................................... 66
Experiment 13 .............................................................................................................. 72
Oil and Grease Analysis (APHA / ASTM 5520B) ...................................................... 72

2
Experiment 1
Determination of Total Solids Dried, Total Dissolved
Solids, Total Suspended Solids and Fixed and Volatile
Solids Ignited (APHA / ASTM method 2540B-E).
Date of Revision:

Objective
To determine the total solid dried (TSD), total dissolved solid (TDS) and
total suspended solid and fixed and volatile solids Ignited (FVSI) in a
wastewater samples.

Total Solids Dried (TSD) at 103 105oC (Method 2540 B)

1. General Discussion

a. Principle: A well-mixed sample is evaporated in a weighed dish and dried to


constant weight in an oven at 103 to 105C. The increase in weight over that
of the empty dish represents the total solids. The results may not represent the
weight of actual dissolved and suspended solids in wastewater samples (see
above).

b. Interferences: Highly mineralized water with a significant concentration of


calcium, magnesium, chloride, and/or sulphate may be hygroscopic and
require prolonged drying, proper desiccation, and rapid weighing. Exclude
large, floating particles or submerged agglomerates of nonhomogeneous
materials from the sample if it is determined that their inclusion is not desired
in the final result. Disperse visible floating oil and grease with a blender before
withdrawing a sample portion for analysis. Because excessive residue in the
dish may form a water-trapping crust. Limit sample to no more than 200 mg
residue.

2. Apparatus

a. Evaporating dishes: Dishes of 100 mL capacity made of one of the following


materials:

1) Porcelain. 90 mm diam.
2) Platinum-Generally satisfactory for all purposes.
3) High-silica glass (Vycor. Product of Corning Glass Works. N. Y or equivalent)

b. Muffle furnace for operation at 550C.


c. Steam bath.
d. Desiccator, provided with a desiccant containing a color indicator of moisture
concentration or an instrumental indicator.
e. Drying oven, for operation at 103 to 105C.
f. Analytical balance, capable of weighing to 0.1 mg.

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g. Magnetic stirrer with TEE stirring bar.
h. Wide-bore pipets (Kimble Nos. 37005 or 37034B or equivalent).

3. Procedures

a) Preparation of evaporating dish: Since total dried (i.e. TSD) and volatile solids (i.e.
FVSI) are to be measured ignite clean evaporating dish at 550C for 1 h in a muffle
furnace. If only TSD are to be measured, heat clean dish to between 103 to 105C
for 1 hr. Store and cool dish in desiccators until needed. Weigh immediately before
use.

b) Sample analysis: Choose a sample volume that will yield a residue between 2.5 and
200 mg. Pipet a measured volume of well-mixed sample, during mixing, to a
preweighed dish. For homogeneous samples, pipet from the approximate midpoint
of the container but not in the vortex. Choose a point both mid-depth and midway
between wall and vortex. Evaporate to dryness in a drying oven (103-105C). Stir
sample with a magnetic stirrer during transfer. If necessary, add successive sample
portions to the same dish after evaporation to increase the yield (between 2.5 and
200 mg). CAUTION: When evaporating in a drying oven, lower temperature to
approximately 2C below boiling to prevent splattering. Dry evaporated sample for
at least 1 h in an oven at 103 to 105C, cool dish in desiccators to balance
temperature, and weigh. Repeat cycle of drying, cooling, desiccating, and weighing
until a constant weight is obtained, or until weight change is less than 4 % of
previous weight or 0.5 mg, whichever is less. When weighing dried sample, be alert
to change in weight due to air exposure and/or sample degradation. Analyze at least
10 % of all samples in duplicate. Duplicate determinations should agree within 5 %
of their average.

4. Calculation

(A - B) x 1000
mg total solids/L
sample volume, mL

Where;
A = weight of dried residue + dish [mg], and
B = weight of dish, [mg]

5. Precision

Single-laboratory duplicate analyses of 41 samples of water and wastewater were


made with a standard deviation of differences of 6.0 mg/L.

4
Total Dissolved Solids (TDS) Dried at 180oC (Method 2540 C)

1 General Discussion

a) Principle: A well-mixed sample is filtered through a standard glass fibre filter, and
the filtrate is evaporated to dryness in a weighed dish and dried to constant weight
at 180C. The increase in dish weight represents the total dissolved solids. This
procedure may be used for drying at other temperatures.

The results may not agree with the theoretical value for solids calculated from
chemical analysis of sample (see above). Approximate methods for correlating
chemical analysis with dissolved solids are available (Sokoloff, 1933). The filtrate
from the total suspended solids determination (Method 2540D) may be used for
determination of total dissolved solids.

b) Interferences: Highly mineralized waters with a considerable calcium, magnesium,


chloride and/or sulphate content may be hygroscopic and require prolonged drying,
proper desiccation, and rapid weighing. Samples high in bicarbonate require careful
and possibly prolonged drying at 80C to insure complete conversion of bicarbonate
to carbonate. Because excessive residue in the dish may form a water-trapping crust,
limit sample to no more than 200 mg residue.

2. Apparatus

Apparatus in TSD is required and in addition:


a. Glass fibre filter disks without organic binder
b. Filtration apparatus: One of the following, suitable for the filter disk selected:
Membrane filter funnel
Gooch crucible, 25 mL to 40 mL capacity, with Gooch crucible adapter
Filtration apparatus with reservoir and coarse (40 - 60 m) fritted disk as
filter support
c. Suction flask, of sufficient capacity for sample size selected.
d. Drying oven, for operation at 180 2oC

3. Procedures

a) Preparation of glass-fibre filter disk: If pre-prepared glass fibre filter disks are used,
eliminate this step. Insert disk with wrinkled side up into filtration apparatus. Apply
vacuum and wash disk with three successive 20mL volumes of reagent-grade water.
Continue suction to remove all traces of water. Discard washings.

b) Preparation of evaporating dish: If volatile solids are to be measured, ignite cleaned


evaporating dish at 550C for 1 h in a muffle furnace. If only total dissolved solids
are to be measured, heat clean dish to 180 2C for 1 h in an oven. Store in
desiccators until needed. Weigh immediately before use.

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c) Selection of filter and sample sizes: Choose sample volume to yield between 2.5
and 200 mg dried residue- If more than 10 min are required to complete filtration,
increase filter size or decrease sample volume.

d) Sample analysis: Stir sample with a magnetic stirrer and pipette a measured volume
onto a glass-fiber filter with applied vacuum. Wash with three successive 10 mL
volumes of reagent- grade water, allowing complete drainage between washings,
and continue suction for about 3 min after filtration is complete. Transfer total
filtrate (with washings) to a weighed evaporating dish and evaporate to dryness in
a drying oven. If filtrate volume exceeds dish capacity add successive portions to
the same dish after evaporation. Dry for at least 1 h in an oven at 180 4 2C, cool
in a desiccators to balance temperature, and weigh. Repeat drying cycle of drying,
cooling, desiccating, and weighing until a constant weight is obtained or until
weight change is less than 4% of previous weight or 0.5 mg, whichever is less.
Analyse at least 10 % of all samples in duplicate. Duplicate determinations should
agree within 5% of their average. If volatile solids are to be determined, follow
procedure in FVSI (method 2540E).

4. Calculation

(A - B) x 100
mg total dissolved solids/L
sample volume, mL

where :
A weight of dried residue dish, mg, and
B weight of dish, mg

5. Precision

Single laboratory analysis of 77 samples of a known of 293mg/L were made with


made a standard deviation of differences of 21.20mg/L

6
Total Suspended Solids (TSS) Dried at 103 105oC (Method 2540 D)

1. General discussion

a. Principle: A well-mixed sample is filtered through a weighed standard glass-fibre


filter and the residue retained on the filter is dried to a constant weight at 103 to
105C. The increase in weight of the filter represents the total suspended solids. If
the suspended material clogs the filter and prolongs filtration, it may be necessary
to increase the diameter of the filter or decrease the sample volume. To obtain an
estimate of TSS, calculate the difference between total solids dried (TSD) and total
dissolved solids (TDS).

b. Interferences: See TSD. Exclude large floating particles or submerged


agglomerates of non-homogeneous materials from the sample if it is determined
that their inclusion is not desired in the final result. Because excessive residue on
the filter may form a water-entrapping crust, limit the sample size to that yielding
no more than 200 mg residue. For samples high in dissolved solids thoroughly wash
the filter to ensure removal of dissolved material. Prolonged filtration times
resulting from filter clogging may produce high results owing to increased colloidal
materials captured on the clogged filter.

2. Apparatus

Apparatus listed in section TSD is required, except for evaporating dishes, steam
bath and 180oC drying oven. In addition: Planchet, aluminium steel, 65mm diam.

3. Procedure

1. Preparation of glass-fiber filter disk: If pre-prepared glass fibre filter disks are used,
eliminate this step. Insert disk with wrinkled side up in filtration apparatus. Apply
vacuum and wash disk with three successive 20 mL portions of reagent-grade water.
Continue suction to remove all traces of water, turn off vacuum and discard wash-
ings. Remove filter from filtration apparatus and transfer to an inert Aluminium
weighing dish. If at Gooch crucible is used, remove crucible and filter combination.
Dry in an oven at 103 to 105C for 1 h. If volatile solids are to be measured, ignite
at 550C for 15 min in a muffle furnace. Cool in desiccators to balance temperature
and weigh. Repeat cycle of drying or igniting, cooling, desiccating, and weighing
until a constant weight is obtained or until weight change is less than 4 % of the
previous weighing or 0.5 mg, whichever is less. Store in desiccators until needed.

2. Selection of filter and sample sizes: See TDS. For non-homogeneous samples such
as raw wastewater, use a large filter to permit filtering a representative sample.

3. Sample analysis: Assemble filtering apparatus and filter and begin suction. Wet
filter with a small volume of reagent-grade water to seat it. Stir sample with a
magnetic stirrer at a speed to shear larger particles, if practical, to obtain a more
uniform (preferably homogeneous) particle size. Centrifugal force may separate
particles by size and density, resulting in poor precision when point of sample
withdrawal is varied. While stirring, pipet a measured volume onto the seated glass-
fiber filter. For homogeneous samples, pipet from the approximate midpoint of

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container but not in vortex. Choose a point both mid-depth and mid-way between
wall and vortex. Wash filter with three successive 10 mL volumes of reagent-grade
water, allowing complete drainage between washings, and continue suction for
about 3 min after filtration is complete. Samples with high dissolved solids may
require additional washings. Carefully remove filter from filtration apparatus and
transfer to an aluminum weighing dish as a support. Alternatively, remove the
crucible and filter combination from the crucible adapter if a Gooch crucible is used.
Dry for at least 1 h at 103 to 105C in an oven, cool in desiccators to balance
temperature, and weigh. Repeat the cycle of drying- desiccating, and weighing until
a constant weight is obtained or until the weight change is less than 4% of the
previous weight or 0.5 mg, whichever is less. Analyse at least 10 % of all samples
in duplicate. Duplicate determinations should agree within 5% of their average. If
volatile solids are to be determined, treat the residue according to method 2540 E.

4. Calculation

(A - B) x 100
mg total suspended solids/L
sample volume, mL
where;
A Weight of filter dreid residue, mg and
B weight of filter, mg

5. Precision

The standard deviation was 5.2 mg/L (coefficient of variation 33%) at 15 mg/L, 24
mg/L (10%) at 242 mg/L, and 13 mg/L (0-76%) at 1707 mg/L in studies by two
analysts of tour sets of 10 determinations each. Single-laboratory duplicate analyses
of 50 samples of water and wastewater were made with a standard deviation of
differences of 2.8 mg/L.

8
Fixed and Volatile Solids Ignited (FVSI) at 500oC (Method 2540 E)

1. General Discussion

a. Principle: The residue from Method TSD, TDS and TSS is ignited to constant
weight at 550oC. The remaining solids represet the fixed total, dissolved, or
suspended solids while the weight lost on ignition is the volatile solids. The
determination is useful in control of wastewater treatment plant operation because
offers a rough approximation of the amount of organic made present in the solid
fraction of wastewater, activated sludge, and industrial wastewater.

b. Interferences: Negative errors in the volatile solids may he produced by loss of


volatile made during drying. Determination of low concentrations of volatile solids
in the presence of high fixed solids concentrations may be subject to considerable
error. In such cases, measure for suspect volatile components by an- other test for
example, total organic carbon.

2. Apparatus

See methods 2540 B, C or D.

3. Procedure

Ignite residue produced by Method TSD, TDS or TSS to constant weight in a muffle
furnace at a temperature of 550oC. Ignite a blank glass fibre filter along with
samples. Have furnace up to temperature before inserting sample. Usually, 15 to 20
min ignitions are required for 200mg residue. However, more than one sample
and/or heavier residues may overtax the furnace and necessitate longer ignition
times. Let dish or filter disk cool partially in air until most of the heat has been
dissipated. Transfer to a desiccators for final cooling in a dry atmosphere. Do not
overload desiccators. Weigh dish or disk as soon as it has cooled to balance
temperature. Repeat cycle of igniting, cooling, desiccating and weighing until a
constant weight is obtained or until weight change is less than 4% or 0.5 mg,
whichever is less. Analyse at least 10 % of all samples in duplicate. Duplicate
determinations should agree within 5 % of their average.

4. Calculation
(A - B) x 100
mg volatile solids/L
sample volume, mL

(B - C) x 100
mg fixed solids/L
sample volume, mL

where;
A weight of residue dish before ignition, mg
B weight of residue dish or filter after ignition, mg and
C weight of dish or filter, mg

9
5. Precision

The standard deviation was 11 mg/L at 170 mg/L volatile total solids in studies by
three laboratories on four samples and 10 replicates. Bias data on actual samples
cannot be obtained.

References

Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (1998) Standard Method for the
Examination of Water and Wastewater. 20th Edition. Joint publication by APHA,
ASTM and WEF; ISBN: 0-87553-235-7.
Sokoloff, V.P. (1933) Water crystallization in total solids of water analysis. Ind. Eng.
Chem., Anal. Ed. 5: 336.

Sample

TSS Filtrate TDS TSD


(2540 D) (2540 C) (2540 B)

Residue Residue Residue

VFSI
(2540 E)

10
Experiment 2
Determination of pH, Temperature, Colour and
Turbidity (APHA / ASTM methods 4500-H+, 2120 and
2130).
Date of Revision:

OBJECTIVE

To determine the pH, temperature, colour and turbidity of (waste)water samples

pH (APHA/ASTM 4500 H)

Introduction

Measurement of pH is one of the most important and frequently used tests in water and
wastewater analysis. Practically every phase of water supply and wastewater treatment,
e.g. acid-base neutralization, water softening, precipitation, coagulation, disinfection
and corrosion control, is pH dependent. pH is used in alkalinity and carbon dioxide
measurements and many other acid-base equilibria. At a given temperature the intensity
of the acidic or basic character of a solution is indicated by pH or hydrogen ion activity.
Alkalinity and acidity are the acid- and base-neutralizing capacities of a water and
usually are expressed in milligrams CaCO3 per litre. Buffer capacity is the amount of
strong acid or base, usually expressed in moles per litre, needed to change the pH value
of a 1 L sample by 1 unit.
The pH value as defined by Sorenson (1909) is the negative logarithm to base 10 of the
hydrogen ion activity (mol/L). This term may be represented by

pH -log [H ]

Pure water is very slightly ionized and at equilibrium the ion product is

KW = [H+][OH-] Equation 1
= 1.01 x 10-14 mol2/L2 at 25C

and

[H+] = [OH-]
= 1.005 x 10-7

Because of ionic interactions in all but very dilute solutions (ionic strength <0.1), it is
necessary to use the activity of an ion and not its molar concentration. Use of the term
pH assumes that the activity of the hydrogen ion, H+, is being considered. The
approximate equivalence to molarity, [H+], can be presumed only in very dilute
solutions (ionic strength < 0.1).
A logarithmic scale is convenient for expressing a wide range of ionic activities.
Equation 1 in logarithmic form and corrected to reflect activity is:

11
(-log10 H+) + (-log10 OH-) = 14 Equation 2

or

pH + pOH = pKW

where:
pH = -log10 H+
pOH = -log10 OH-

Equation 2 states that as pH increases pOH decreases correspondingly and vice versa
because pKW is constant for a given temperature.
At 25C, pH 7.0 is neutral, the activities of the hydrogen and hydroxyl ions are equal,
and each corresponds to an approximate activity of 10-7 mol/L. The neutral point is
temperature-dependent and is pH 7.5 at 0C and pH 6.5 at 60C.
The pH value of a highly dilute solution is approximately the same as the negative
common logarithm of the hydrogen ion concentration. Natural waters usually have pH
values in the range of 4 to 9, and most are slightly basic because of the presence of
bicarbonates and carbonates of the alkali and alkaline earth metals.

1. General discussion
a) Principle: the basic principle of electrometric pH measurement is determination of
the activity of the hydrogen ions by potentiometric measurement using a standard
hydrogen electrode and a reference electrode. The hydrogen electrode consists of a
platinum electrode across which hydrogen gas is bubbled at a pressure of 101 kPa.
Because of difficulty in its use and the potential for poisoning the hydrogen
electrode, the glass electrode is commonly used. The electromotive force (emf)
produced in the glass electrode system varies linearly with pH. This linear
relationship is described by plotting the measured emf against the pH of different
buffers. Sample pH is determined by extrapolation.
Because single ion activities such as H+ cannot be measured, pH is defined
operationally on a potentiometric scale. The pH measuring instrument is calibrated
potentiometrically with an indicating (glass) electrode and a reference electrode
using NIST buffers having assigned values so that:

pHB = -log10 H+

12
The operational pH scale is used to measure sample pH and is defined as:

F E X E S
pH X pH B
2.303 RuT

Where:
pHX = potentiometrically measured sample pH
F = Faraday constant (9.649 x 104 Coulomb / mole)
EX = sample emf [V]
ES = buffer emf [V]
Ru = universal gas constant (8.314 J/mole/K)
T = absolute temperature [K]

The equation for pHX assumes that the emf of the cells containing the sample and
buffer is due solely to hydrogen ion activity unaffected by sample composition. In
practice, samples will have varying ionic species and ionic strengths, both affecting
H+ activity. This imposes an experimental limitation on pH measurement; thus, to
obtain meaningful results, the differences between EX and ES should be minimal.
Samples must be dilute aqueous solutions of simple solutes (<0.2 M).
Determination of pH cannot be made accurately in nonaqueous media, suspensions,
colloids, or high-ionic-strength solutions.

b) Interferences: The glass electrode is relatively free from interference from color,
turbidity, colloidal matter, oxidants, reductants, or high salinity, except for a sodium
error at pH > 10. Reduce this error by using special low sodium error electrodes.
pH measurements are affected by temperature in two ways: mechanical effects that
are caused by changes in the properties of the electrodes and chemical effects
caused by equilibrium changes. In the first instance, the Nernstian slope increases
with increasing temperature and electrodes take time to achieve thermal
equilibrium. This can cause long-term drift in pH. Because of chemical equilibrium
affects pH, standard pH buffers have a specified pH at indicated temperatures.
Always report temperature at which pH is measured.

Experimental Procedure

List of Glassware/apparatus

pH indicator paper
pH meter
Thermometer

List of reagent

NIST buffer solutions pH 4.00, 7.0 and 10.0

13
Procedure

1. Dip the indicator paper or test strips into the sample.


2. Compare the resulting colour with the manufacturer's standards.
3. Calibration: Calibrate the pH meter (see manufacturer's instruction). Carry out
the calibration using two standard buffer solutions (AKA 2-point calibration).
The choice of which pH buffer to use depends on the results from step 2. The
pH of the sample should be between the two buffer solutions pH values. For
example, if the pH obtained in step 2 is 5, then use the buffer solution of pH
4.62 and 7.00 to do the calibration. Carefully rinse the pH probe with distilled
water, blot dry and immerse pH probe in pre-rinse buffer solution. Once
equilibrium between pH probe and pre-rinse buffer has been established remove
pH probe from solution, blot dry and insert into calibration solution and
calibrate. Proceed likewise for second buffer.
4. Sample analysis: Carefully rinse the calibrated probe and blot dry. Insert pH
probe into sample, stir gently and wait until equilibrium between pH probe and
sample has established. For buffered samples or those of high ionic strength,
condition electrodes after cleaning by dipping them into sample for 1 min. Blot
dry, immerse in a fresh portion of the same sample and read pH. With dilute,
poorly buffered solutions, equilibrate electrodes by immersing in three or four
successive portions of the same sample. Take a fresh sample to measure pH.
5. Take the temperature of the sample(s) with a thermometer provided.

Precision and Bias

By careful use of a laboratory pH meter with good electrodes, a precision of 0.02 and
an accuracy of 0.05 pH units can be achieved. However, 0.1 pH unit represents the
limit of accuracy under normal conditions, especially for measurement of water and
poorly buffered solutions. For this reason, report pH values to the nearest 0.1 pH unit.

14
TEMPERATURE

Temperature is not used to evaluate directly either potable water or wastewater.


However, it is an important parameter because of its effect on chemical reactions and
reaction rates, aquatic life and the suitability of the water for beneficial uses. Normally,
temperature can be measured with any good mercury-filled Celsius thermometer.

15
COLOUR (APHA/ASTM 2120)

Introduction

Pure water is colourless, however water in nature is often coloured due to suspended
matter. This type of water is said to have an apparent colour. Water's true colour is when
the suspended matter is removed from the water leaving only dissolved solids behind.
To measure colour, methods involving comparison with standardized coloured
materials are often used. Colour comparison tubes containing a series of standards may
be used for direct comparison of water samples that have been filtered to remove the
apparent colour. The platinum-cobalt method of measuring colour is the standard
method. Results are expressed as in true colour units (TCUs) where one unit is
equivalent to the colour produced by 1 mg/L of platinum in the form of chiorplatinate
ions. This method is useful in measuring potable water and water in which the colours
are due to, naturally occurring materials. The colour value of water is extremely pH-
dependent and increases as the pH of the water is raised. When reporting a colour value,
specify the pH at which colour is determined.

EXPERIMENTAL PROCEDURE

List of Glassware/apparatus

Distilled water
Membrane filter 0.2 m
14 Nessler Tube (50 ml)
1 Collecting flask (1 L)
1 Glass rod
1 Glass pipet

Colour Measurement

1. Dilute the following volume of stock colour standard (prepared by the


demonstrator) with distilled water to 50mL in nessler tubes. These tubes now
contain a series of standards, which will be used for direct comparison of water
sample given by the demonstrator.

Volume of Stock Colour Standard (Unit)


Standard (mL)
0.5 5
1.0 10
1.5 15
2.0 20
2.5 25
3.0 30
3.5 35
4.0 40
4.5 45
5.0 50

16
6.0 60
7.0 70

(Protect these standards against evaporation and contamination when not in use)

Procedure

1. Observe the wastewater sample colour by filling a. matched nessler tube to the 50
mL mark with sample.
2. Compare it with the range of standards prepared.
3. Look vertically downward through the tubes toward a white surface (use a piece of
white paper).
4. Make a visual comparison of the sample with known concentrations of the prepared
standards.
5. If the colour exceeds 70 units, dilute sample with distilled water in known
proportions until the colour is within the range of the standards.
6. Measure pH of each sample with pH meter.

A x 50
Colour Uni ts
B
Where
A = estimated colour of a diluted sample
B = volume of sample taken for dilution

17
TURBIDITY (APHA/ASTM 2130)

Introduction
Turbidity is a measure of the extent to which light is either absorbed or scattered by
suspended material in water. Since absorption and scattering are influenced by both size
and surface characteristics of the suspended material, turbidity is not a direct
quantitative measurement of suspended solids. To illustrate this point, one small stone
in a glass of water will not produce any turbidity measurement. However, if this stone
is made into thousands of small particles of colloidal size, some turbidity measurement
is expected even though the mass of the solids has no changed.

Turbidity may be measured using a turbidity meter consisting of a nephelometer with a


light source for illuminating the sample and one or more photoelectric detectors with a
readout device to indicate the intensity of scattered light at right angles to the path of
the incident light. Turbidity measurements by the standard nephelometry procedure are
reported as nephelometric turbidity units (NTU).

Apparatus

Laboratory nephelometer
Sample cells

Reagents

Dilution water (filtered distilled water; filter pore size 0.1 m):
Stock of primary standard formazin suspension
Secondary standards (e.g. commercial stock suspensions of 4000 NTU formazin
certified by manufacturer; check shelf life!);

Procedure

a) General measurement techniques: Proper measurement techniques are important in


minimizing the effects of instrument variables as well as stray light and air bubbles.
Regardless of the instrument used, the measurement will be more accurate, precise
and repeatable if close attention is paid to proper measurement techniques.
Measure turbidity immediately to prevent temperature changes and particle
flocculation and sedimentation from changing sample characteristics. If
flocculation is apparent, break up aggregates by agitation. Avoid dilution whenever
possible. Particles suspended in the original sample may dissolve or otherwise
change characteristics when the temperature changes or when the sample is diluted.
Remove air or other entrained gases in the sample before measurement. Preferably
degas even if no bubbles are visible. Degas by applying a partial volume, adding a
non-foaming type surfactant, using an ultrasonic bath, or applying heat. In some
casees, two or more of these techniques may be combined for more effective bubble
removal. For example, it may be necessary to combine addition of a surfactant with
use of an ultrasonic bath for some severe conditions. Any of these techniques, if
misapplied, can alter sample turbidity; use with care. If degassing cannot be applied,
bubble formation will be minimized if the samples are maintained at the
temperature and pressure of the water before sampling.

18
Do not remove air bubbles by letting sample stand for a period of time because
during standing, turbidity-causing particulates may settle and sample temperature
may change. Both of these conditions alter sample turbidity resulting in a non-
representative measurement.
Condensation may occur on the outside surface of a sample cell when a cold sample
is being in a warm, humid environment. This interferes with turbidity measurement.
Remove all moisture from the outside of the sample cell before placing the cell in
the instrument. If fogging recurs, let sample warm slightly by letting it stand at room
temperature or by partially immersing it in a warm water bath for a short time. Make
sure samples are again well mixed.

b) Nephelometer calibration: Follow the manufacturers operating instructions. Run


at least one standard in each instrument range to be used. Make certain the
nephelometer gives stable readings in all sensitivity ranges used.

c) Measurement of turbidity: Gently agitate sample. Wait until air bubbles disappear
and pour sample into cell. When possible, pour well-mixed sample into cell and
immerse it in an ultrasonic bath for 1 to 2 s or apply vacuum degassing, causing
complete bubble release. Read turbidity directly from instrument display.

19
Interpretation of Results

Report turbidity readings as follows:

Turbidity Range Report to the


[NTU] nearest NTU
0 1.0 0.05
1 - 10 0.1
10 - 40 1
40 - 100 5
100 - 400 10
400 - 1000 50
>1000 100

When comparing water treatment efficiencies, do not estimate turbidity more closely
than specified above. Uncertainties and discrepancies in turbidity measurements make
it unlikely that results can be duplicate to greater precision than specified.

References

Clesceri, L.S., Greenberg, A.E. and Eaton, A.D. (1998) Standard Method for the
Examination of Water and Wastewater. 20th Edition. Joint publication by APHA,
ASTM and WEF; ISBN: 0-87553-235-7.
Sorenson, S. (1909) Uber die Messung und die Bedeutung der Wasserstoffion
Konzentration bei Enzymatischen Prozessen. Biochem. Z. 21: 131.

20
Report;

1. Fill up the given table


2.
pH
Temperature Colour Turbidity
Sample ID Indicator pH meter [C] (Units) [NTU]
paper

3. Calculate of colour content in water sample.

4. Define pH, colour, temperature and turbidity in your own words.

5. Name the physical water quality parameters of concerns to environmental


engineers.

6. What are the sources of temperature increase in water bodies?

21
Experiment 3
Determination of Total Kjeldahl Nitrogen (TKN) (APHA /
ASTM 4500-Norg)
Date of Revision:

Objective
To determine the ammonium nitrogen and organic nitrogen (TKN) of a waste water
sample

1. Selection Method

The major factor that influences the selection of a macro- or micro-kjeldahl method
to determine organic nitrogen is the concentration of organic nitrogen- The macro-
kjeldahl method is applicable for samples containing either low or high concen-
trations of organic nitrogen but requires a relatively large sample volume for low
concentrations. In the semi-micro-kjeldahl method, which is applicable to samples
containing high concentrations of organic nitrogen, the sample volume should be
chosen to contain organic plus ammonia nitrogen (kjeldahl nitrogen) in the range of
0.2 to 2 mg.

2. Storage of Samples

The most reliable results are obtained on fresh samples. If an immediate analysis is
not possible preserve samples by acidifying to pH 1.5 to 2.0 with concentrated
H2SO4, and storing at 4C. Do not use HgCl2, because it will interfere with ammonia
removal.

3. Interferences

i. Nitrate: During digestion, nitrate in excess of 10 mg/L can oxidize a portion of


the ammonia released from the digested organic nitrogen, producing N2O and
resulting in a negative interference. When sufficient organic matter in a low
state of oxidation is present, nitrate can be reduced to ammonia, resulting in a
positive interference. The conditions under which significant interferences
occur are not well defined and there is no proven way to eliminate the
interference in conjunction with the methods described herein.

ii. Inorganic salts and solids: The acid and salt content of the digestion reagent is
intended to produce a digestion temperature of about 360 to 370C. If the
sample contains a very large quantity of salt or inorganic solids that dissolve
dining digestion, the temperature may rise above 400oC, at which point
pyrolytic loss of nitrogen begins to occur. To prevent an excessive digestion
temperature, add more H2SO4 to maintain the acid-salt balance. Not all salts
cause precisely the same temperature rise, but adding of 1mL H2SO4/g salt in
the sample gives reasonable results. Add the extra acid and the digestion reagent
to both sample and reagent blank. Too much acid will lower the digestion
temperature below 360oC and result in incomplete digestion and recovery. If

22
necessary, add more sodium hydroxide-sodium thiosulfate before the final
distillation step to neutralize the excess acid. Large amounts of salt or solids
also may cause bumping during distillation. If this occurs, add more dilution
water to the samples after digestion.
iii. Organic matter: During digestion. H2SO4 organic matter to CO, and H2O. If a
large amount of organic matter is present, a large amount of acid will be
consumed, the ratio of salt to acid will increase, and the digestion temperature
will increase. If enough organic matter is present, the temperature will rise
above 400C, resulting in pyrolytic loss of nitrogen. To prevent this, add to the
digestion flask 10mL concentration H2SO4/3g COD. (For most organic
substances, 3 g COD equals about 1g organic matter). Alternately add 50mL
more of digestion reagent/g COD. Additional sodium hydroxide-sodium
thiosullate reagent may be necessary to keep the distillation pH high. Because
reagents may contain traces of ammonia, treat the reagent blank identically with
the samples.

4. Use of a Catalyst

Although it generally is desirable to avoid using mercury because of its toxicity and
the problems associated with disposal of residues, mercury is the catalyst of choice.
Only selenium is as effective as mercury, but selenium is highly toxic and there are
potential interferences associated with its use. Digestion of some samples may be
complete or nearly complete without the use of a catalyst or with the use of a less
toxic catalyst, such as copper. If copper is substituted for mercury, add 10 mL of a
solution containing 25.115 g CuSO4 to each macro-kjeldahl digestion flask with
50mL digestion reagent from which the HgO has been omitted. Use 2 mL of CuSO4
solution for the semi-micro method. If the mercury catalyst is omitted, report this
deviation and indicate, if possible the percentage recovery relative to the results for
similar samples analyzed using the mercury catalyst.

5. Total nitrogen is comprised of organic nitrogen, ammonia, nitrite and nitrate.


Kjedahl nitrogen is the sum of ammonium and those organic nitrogen compounds,
which can be converted into ammonium under Kjedahl reaction conditions. The
proportion of organic nitrogen can be calculated by subtraction of the ammonium
content from that of Kjedahl nitrogen. In this section Kjedahl nitrogen will be
determined since nitrate and nitrite generally occur in trace quantities in surface
water. Kjedahl nitrogen is determined by the following method. In the presence of
sulfuric acid, potassium sulfate, cupric sulfate catalyst, amino nitrogen of many
organic materials is converted to ammonium. Free ammonia will also be converted
to ammonium. After addition of base, the ammonia is distilled from an alkaline
medium and absorbed in boric or sulfuric acid. The ammonia may determine
colorimetrically, by ammonia-selective electrode, or by titration with a standard
mineral acid.

Experimental Procedure

1. List of equipment/glassware
i. Digestion apparatus
ii. Distillation apparatus
iii. Burette

23
iv. Retort stand

2. Chemicals
i. Sodium hydroxide 30% 5L + 3L (SCR)
ii. Boric acid 2% 5L
iii. Sulfuricacid 98% 200mL
iv. Distillad water 5L
v. Kjedahl catalyst 12 tablets

3. Preparation of reagent.
A) Sodium hydroxide (NaOH) 30%, 8L

Dissolve 2400g Sodium hydroxide in distilled water


Mark up to 8000mL with distilled water.
Shake well.

B) Boric acid 2%, 5L

Dissolve 100g Boric acid in distilled water


Mark up to5L with distilled water.
Shake well

4. Preparation of blank and sample:

i. Blank:
15mL Sulfuric acid + 2 tablets of catalyst + 100mL of distilled water.

ii. Sample:
5mLSulfuric acid + 2 tablets of catalyst + 100 ml wastewater sample

Add 10mL of sulphuric acid, concentration and heat the tube in the Buchi digestion unit
until the mixture turns green and sulphur trioxide fumes are generated. Continue heating
gently for a further half hour and then allow the flask to cool.

5. Procedures:

A) Digestion:
i. Turn on the switch of the digestion unit.
ii. Let it 10 min to warm up
iii. Place the digestion glasses into the digestion glass holder and put on the rack.
iv. Make sure the suctions are place in the right position.
v. Turn on the scrubber.

B) Distillation:

24
i. Turn on the distillation unit.
ii. Select "PREHEATING " and " START "until the time BEEP
iii. Place the vessel for the blank to the distillation unit.
iv. Select "DISTILLATION" and START".
v. When the time "BEEP", take the collected in the conical flask and titrate with
sulfuric acid 0.02m until the color change. Repeat the distillation procedure with
the entire sample vessel.

6. Calculation

(A - B) x C x 2800
Total Kjeldahl, mg/L
S
Where :
A mL of standard 0.01M H 2SO 4 solution used in titrati ng sample
B mL of standard 0.01M H 2 SO 4 solution used in titrati ng blank
C Actual molarity of 0.01M sulphuric acid solution
S mL of sample digested

Sample Volume Initial Buret Final Buret Volume of Titrant


(mL) Reading Reading (mL)
Sample 1

Sample 2

Sample 3

Sample 4

Sample 5

Blank

Calculate the concentration of nitrogen in mg NH3-N/L of sample

Question;

1. Define total nitrogen in wastewater?

2. How is total Kjedahl nitrogen content measured?

3. Why do we need to digest the sample?

4. What are significance of nitrogen analysis in water pollution control?

References

25
1. Hamner. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.
edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

26
Experiment 4
Determination of Phosphate (APHA / ASTM 4500-P)
Date of Revision:

Objectives

To introduce the different types and sources of phosphorus in water and


wastewater.
To determine the concentration of phosphates in different water samples

Introduction

Phosphorus occurs in water and wastewater as phosphates. These are classified as


orthophosphates (H2PO4- , HPO42-, PO43-), polyphosphates such as Na3(PO3)6 used in
synthetic detergents and organically bound phosphates. These different forms of
phosphates arise from a variety of sources. All polyphosphates gradually hydrolyze in
water to the stable ortho form, while decaying organic matter decomposes biologically
to release phosphate. Orthophosphates are, in turn, synthesized back into living animal
or plant tissue to form organic phosphate.

Experimental Procedure

Phosphorus analyses consist of two general steps:

a) Conversion of the phosphorus to dissolved orthophosphate and


b) Colorimetric determination of dissolved orthophosphate.

There are several procedures to determine phosphate concentration, depending on the


concentration of phosphorus in the water sample. The vanadomolybdate method is used
for phosphorus concentration in the range of 1-20mg/L, the stannous chloride method
is used for phosphorus concentration in the range of 1- 6 mg/I and the ascorbic acid
method is used for the range 0.01 - 6mg/L. However, for this laboratory session, only
the vanadomolybdate method will be carried out.

Vanadomolybdate Method

Principle:

In a dilute orthophosphate solution, ammonium molybdate reacts under acid condition


to form molydophosphoric acid. In the presence of vanadium, yellow
vanadomolydophosphoric acid is formed. The intensity of the yellow colour is
proportional to the phosphate concentration.

27
Chemicals

10mL 0.02 N H2SO4


10mL 0.02 N NaOH
2.5g Ammonium molybdate, (NH4)6Mo7O244H2O
125mg Ammonium metavanadate, NH4VO3
200mg Activated carbon
50mL Concentrated HCl
10mL 0.02N NaOH
10mL 0.02N H2SO4
21.95mg Anhydrous KH2PO4

List of glassware/apparatus

3 100mL conical flask


3 250mL beakers
1 50mL measuring cylinder
7 50mL volumetric flask
1 100mL volumetric flask
1 200mL volumetric flask
1 pH meter
2 Spectrophotometer, for use at 400 to 490nm (Use both Analog and Automatic)
1 Filtration Apparatus
1 Glass rod
1 Whatman Filter paper No. 42

*Note; All glassware must be acid washed with hot dilute HCl and rinsed with distilled
water

Preparation of Vanadate-Molybdate reagent

1. Solution A: Dissolve 2.5g ammonium molybdate in 30 mL distill water in a 100


mL conical flask

2. Solution B: Dissolve 0.125 g (or 125 mg ) ammonium metavanadate in 30 mL distill


water in a 100 mL conical flask, heat slowly and bring to boil. Cool and add 33mL
concentrated HCl. Cool solution B to room temperature, pour Solution A into
Solution B, mix, and dilute to 100 mL.

Preparation of Standard Phosphate Solution

Dissolve 21.95 mg anhydrous KH2PO4 in distill water and dilute to 100mL in a


100mL volumetric flask. The resulting concentration of phosphate will be 100mg/L
1.00mL KH2PO4 solution = 0.100mg PO43- -P.

Procedure

28
a. Sample pH adjustment:

i. Determine the pH of the 50 int water sample by using a calibrated pH probe.


ii. Read and record the pH
iii.If pH of sample is between 4-10, no adjustment is required.
iv. If pH of sample is greater than 10, add drop by drop 0.02 N H2SO4 and stir
using glass rod until the pH meter reads less than 10.
v. If pH of sample is less than 4, add drop by drop, 0.02 N NaOH until the pH
meter reads pH greater or equal to 4.
b. If water sample is coloured, the colour must be removed using the following steps:

i. Place 50mL sample in a conical flask. Add 200 mg activated carbon and
shake for 2-3 minutes
ii. By using the filtration apparatus, filter the water sample to remove the
carbon

c. Colour development in sample

1. Place 35mL sample in a 50mL volumetric flask. Label this flask as sample
2. Prepare a blank using 35mL distill water in another 50 ml, volumetric flask
Label this flask as 'blank'.
3. Add 10mL vanadate-molybdate reagent to each flask containing sample and
blank. Then dilute both flasks to 50mL with distill water.
4. After 10 minutes or more measure the absorbance of blank and sample at
wavelenght of 400 to 49 nm. The colour once developed is stable for days.

d. Preparation of calibration curve.

1. Prepare a calibration curve by using the standard phosphate solution for


concentration in the range of 1-20mg/L (1, 2, 5, 10, 15, 20 mg/L PO43- -P)
using 100mg/L Standard solution

2. To prepare standard phosphate solution of concentration 1mg/L. By using


the following formula, the amount of standard solution required could be
obtained:
x mL 3
* Standard solution of PO 3 P Con. required
Vol of flask use
where; x mL is the amount of the standard solution required

3. To produce the calibration curve, 35mL from each concentration is placed


in separate 50mL volumetric flasks. Followed by the addition of 10mL
vanadate-molybdate reagent and diluted to 50mL with distilled water.
4. After 10 minutes or more, measure the absorbance. Plot a graph of
absorbance against concentration for the standard phosphate solutions. This
should be a straight line and must pass through the origin.

29
5. Calculation from the calibration curve, read off the value of the sample in
mg/L PO43- -P. Then using the formula below, calculate the concentration of
PO43- -P in the samples.
3
3 mg/L PO 4 P x 1000
mg/L PO 4 P
mL sample

i. Information on water samples

Types of Initial pH Required pH Required Absorbance


water samples adjustment colour Units
(x/) removal (x/)

ii. Calibration curve

No. Concentration of Standard PO43--P, mg/L Absorbance Units


1.
2.
3.
4.
5.

iii. Include calibration graph in your report.

iv. Questions

a. What are the concentrations of phosphate in the water samples?

b. Why is a calibration of phosphate curve required in this experiment?

c. What is the importance of phosphate in waste water treatment?

References
1. Hammer. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.
edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

30
Experiment 5
Determination of COD in Wastewater (Vial Method)
Date of Revision:

31
32
33
34
35
36
37
38
Experiment 6
Determination of Chemical Oxygen Demand (COD)
Open Reflux Method (APHA / ASTM 5220 B)
Date of Revision:

Objective
To determine the COD of wastewater sample

Introduction

The chemical oxygen demand (COD) test is widely used as a means of measuring the
organic strength of domestic and industrial wastewater. This test allows the
measurement of a waste in terms of the total quantity of oxygen required for the
oxidation to carbon dioxide and water. It is based on the fact that all organic compounds
can be oxidized by the action of strong oxidizing agents under acid conditions.

Most types of organic matter are oxidized by a boiling mixture of chromic and sulphuric
acids. A sample is refluxed in strongly acid solution with a known excess of potassium
dichromate (K2Cr2O7). After digestion, the remaining unreduced K2Cr2O7 is titrate with
ferrous ammonium sulphate to determine the amount of K2Cr2O7, consumed and the
oxidizable matter is calculated in terms of oxygen equivalent.

Experimental Procedure

Chemicals

12.259 g K2Cr2O7
6.67 g Ag2SO4 (technical grade)
550 mL Concentrated H2SO4
1.485 g 1, 10-phenanthroline monohydrate
695 mg FeSO4 7H2O
98 g Fe(NH4)2(SO4)26H2O
1 g HgSO4

Glassware/apparatus

Reflux apparatus
Hot plate
Pipets, Class A and wide-bore.
Glass beads

Reagents Preparation

1. Standard potassium dichromate solution, 0.04167M: Dissolve 12.259 g K2Cr2O7,


previously dried at 150C for 2hr, in distilled water and dilute to 1000 mL.

39
2. Sulphuric aid reagent: Dissolve 6.67 g Ag2SO4, reagent or technical grade, crystal
or powder, with 500 mL concentrated H2SO4. Let stand 1 to 2 d to dissolve. Mix.

3. Ferroin indicator solution: Dissolve 1.485 g 1, 10-phenanthroline monohydrate and


695 mg FeSO4.7H2O in distilled water and dilute to 100 mL.
4. Standard ferrous ammonium sulphate (FAS) titrant, approximately 0.25M: Dissolve
98 g Fe(NH4)2(SO4)26H2O in distilled water. Add 20 mL concentrated H2SO4, cool,
and dilute to 1000 mL. Standardize this solution daily against standard K2Cr2O7,
solution as follows:

5. Dilute 25.00 mL standard K2Cr2O7 to about 100 mL. Add 30 mL concentration


H2SO4 and cool. Titrate with FAS titrant using 010 to 0.15 mL (2 to 3 drops) ferroin
indicator.

Volume 0.0416M K 2 Cr2 O 7 solution t itrated, mL x 0.25


Molarity pf FAS Solution
volume FAS used in titrati on, mL
6. Mercuric sulphate HgSO4, crytals or powder.

The Open Reflux Method

1. Pipet 50.00 mL of sample into a 500 mL refluxing flask.


2. Add 1 g HgSO4, several glass beads, and very slowly add 5.0 mL sulphuric
acid reagent with mixing to dissolve HgSO4. Cool while mixing to avoid
possible loss of volatile materials.
3. Add 25.00 mL 0.04167 M K2Cr2O7 solution and mix.
4. Attach flask to condenser and turn on cooling water. Add remaining sulphuric
acid reagent (70 mL) through open end of condenser.
5. Continue swirling and mixing while adding sulphuric acid reagent.

(CAUTION: Mix reflux mixture thoroughly before applying heat to prevent local
heating of flask bottom and a possible blowout of flask contents)

6. Cover open end of condenser with a small beaker to prevent foreign material
from entering refluxing mixture and reflux for 2hr. Cool and wash down
condenser with distilled water.
7. Disconnect reflux condenser and dilute mixture to about twice its volume
with distilled water. Cool to room temperature.
8. Titrate excess K2Cr2O7 with FAS, using 0.10 to 0.15 mL (2 to 3 drops) ferroin
indicator. Although the quantity of ferroin indicator is not critical, use the
same volume for all titrations. Take as the end point of the titration the first
sharp colour change from blue-green to reddish brown that persists for 1 min
or longer.
9. In the same manner, reflux and titrate a blank containing the reagents and a
volume of distilled water equal to that of sample.

40
Calculation
(A - B) x M x 8000
COD as mg O 2 / L
mL sample
where;
A mL FAS used for blank
B mL FAS used for sample
M molalarity of FAS, and
8000 milliequiv alent weig ht of oxygen x 1000mL/L

References

1. Hammer. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.


edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

Questions

1. Define COD in wastewater

2. How is COD content measured?

3. Why does the COD and BOD analysis usually give different results for the same
sample?

4. Calculate the COD of your wastewater sample.

41
Experiment 7
Determination of BOD in Wastewater (METHOD No.
405.1)
Date of Revision:

42
43
44
45
46
47
48
49
50
51
Experiment 8
Determination of Dissolved Oxygen (DO) and 5-Day
Biological Oxygen Demand (BOD5) (APHA / ASTM
5210 B)
Date of Revision:

Introduction

The biochemical oxygen demand (BOD) determination is an empirical test in which


standardized laboratory procedures are used to determine the relative oxygen
requirements of wastewaters, effluents and polluted water. The test measures the
oxygen utilized during a specified incubation period for the biochemical degradation of
organic material (carbonaceous demand) and the oxygen used to oxidize the inorganic
material such as sulfides and ferrous iron. It may also measure the oxygen used to
oxidize the reduced forms of nitrogen unless their oxidation is prevented by an
inhibitor.

Chemicals

8.5 g KH2PO4
21.75 g K2HPO4
33.4 g Na2HPO4 7H2O
1.7 and NH4Cl
1.15 g
22.5 g MgSO4 7H2O
27.5 g CaCl2
0.25 g FeCl3 6H2O
28 mL Concentrated H2SO4
40 g NaOH
3 x 3 mg 2-chloro-6-(trichloromethyl) pyridine

Apparatus

4 300mL bottles having a ground glass stopper and flared mouth


Waterbath or incubator, thermostatically controlled at 20 1oC
Dissolve Oxygen Method

Reagents Preparation

Prepare reagents in advance but discard if there is any sign of precipitation or biological
growth in the stock bottles.

1. Phosphate buffer solution: Dissolve 8.5 g KH2PO4, 21.75 g K2HPO4, 33.4 g


Na2HPO4 7H2O, and 1.7 g NH4Cl in about 500 mL distilled water and dilute to 1 L.
The pH should be 7.2 without further adjustment.

52
2. Magnesium sulfate solution: Dissolve 22.5 g MgSO4 7H2O in distilled water and
dilute to I L.

3. Calcium chloride solution: Dissolve 27.5 g CaCl2 in distilled water and dilute to 1
L.

4. Ferric chloride solution: Dissolve 0.25 g FeCl3 6H2O in distilled water and dilute
to 1 L.

5. Acid - While stirring, slowly add 28 mL concentrated sulphuric acid (H2SO4) to


distilled water. Dilute to 1 L.

6. Alkali - Dissolve 40 g sodium hydroxide (NaOH) in distilled water. Dilute to 1 L.

7. Nitrification inhibitor, 22-chloro-6-(trichloromethyl) pyridine

8. Dilution water: Use demineralised, distilled, tap, or natural water for making
sample dilutions.

Preparation of dilution water

1. Place 1 L of water in a 1 L bottle and add 1 mL each of phosphate buffer, MgSO4,


CaCl2, and FeCl3 solutions/L of water.

2. Before use bring dilution water temperature to 20 3C

3. Saturate with DO by shaking in a partially filled bottle.

Sample pretreatment

Check pH of all samples before testing.

1. For samples containing caustic alkalinity (pH > 8.5) or acidity (pH < 6.0) -
neutralize samples to pH 6.5 to 7.5 with a solution of H2SO4 or NaOH.

2. Sample temperature adjustment - Bring samples to 20 1C before making


dilutions.

3. Nitrification inhibition - add 3 mg of 2-chloro-6-(trichloromethyl) pyridine (TCMP)


to each 300 mL bottle before capping.

Sample Dilution Technique

1. Prepare dilutions either in graduated cylinders or volumetric glassware, and then


transfer to BOD bottles or prepare directly in BOD bottles.

DO and BOD Test

1. Your demonstrator will give you samples.

53
2. Samples given to you may require dilution. In the absence of prior knowledge, use
the following dilutions: 0.0 to 1.0% for strong industrial wastes, 1 to 5% for raw
and settled wastewater, 5 to 25 % for biologically treated effluent, and 25 to 100 %
for polluted river waters. Your demonstrator will tell you where the samples came
from.

3. Carry out the sample pretreatment and sample dilution (follow the above
techniques).

4. You should have 3 bottles. Fill up 2 bottles with original (or diluted, inhibited) and
diluted samples and the last one with control to the brim of the bottles.

Bottle 1 - original sample (or diluted, inhibited) (t = 0 and 5 day)


Bottle 2 - diluted sample (t = 0 and 5 days)
Bottle 3 - control sample (t = 0 and 5 days)

5. The control bottle should contain only the dilution water.

6. Determine the initial DO concentration using the DO probe (calibrate first as


instructed by the manufacturer) for the diluted samples (in duplicate) and control
sample.

7. Stopper the bottles tightly, water seal and incubate for 5 days at 20C.
After 5 days incubation determine the final DO in diluted sample and control.

Calculation

BOD 5 , mg/L [(a - b) - (c - d)]n


Where;
a DO of sample before incubator
b DO of sample after 5 days incubation
c DO of control before incubation
d DO of control after 5 days incubation
n dilution factor (if the dilution is 1 :100, n 100)

References

1. Hammer. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.


edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

54
Report

1. DO of sample before incubation

2. DO of sample after 5 days incubation

3. DO of control before incubation

4. DO of control after 5 days incubation

5. Dilution factor

Question

1. Define BOD in wastewater

2. How is BOD content measured?

3. Why do we need to know the content of oxygen in water?

4. What is the function of dilution water in the experiment?

55
Experiment 9
Correlation of Biological Oxygen Demand (BOD) and
Chemical Oxygen Demand (COD)
Date of Revision:

Objective
To determine the correlation of BOD and COD of a wastewater sample

Introduction

The COD test will give a good estimate of oxygen demand for most wastewaters. An
advantage of the COD test over the biochemical oxygen demand (BOD) test is that only
2 to 3 hours is needed for the COO analysis to be carried out as opposed to 5 days for
the BOD test.

The COD test is also used to measure the strength of wastes that are too toxic for the
BOD test. However, the COD test should be considered an independent measurement
and not a quick substitute for the DOD test.

The COD value is usually higher than the ROD, hut the amount will vary from waste
to waste. The results of the COD are higher that the corresponding BOD test for several
reasons. Many organic compounds, which are dichromate oxidizable: are not
biochemically oxidizable. Certain inorganic substances, such as sulphides, sulphites,
thiosulfates, nitrites and ferrous iron are oxidized by dichromate, creating an inorganic
COD, which is misleading when estimating the organic content of the wastewater. In
short, the COD test analyzes both biodegradable and non-degradable (refractory)
organic matter.

Since the COD will report virtually all organic compounds, it is proportional to the
BOD only for readily assimilable substances like sugars from a pharmaceutical waste.
'When considering routine plant control, the BOD is note useful test because of the long
incubation time. It is therefore useful to develop correlations between BOD and COD.

56
Experimental Procedure

Chemicals

12.259 g K2Cr2O7
6.67 g AgISO4
550 mL Concentrated H2SO4
1.485 g 1, 10-phenanthroline monohydrate
695 mg FeSO4 7H2O
98 g Fe(NH4)2(SO4)26H2O
8.5 g KH2PO4
21.75 g K2HPO4
33.4 g Na2HPO4 7H2O
1.7 g NH4Cl
22.5 g MgSO4 7H2O
27.5 g CaCl2
0.25 g FeCl3m6H2O
28 mL Concentrated H2SO4
40 g NaOH
1.15 g NH4Cl
2-chloro-6-(trichloromethyl) pyridine

Apparatus

4 300mL bottles having a ground glass stopper and flared mouth


Waterbath or incubator, thermostatically controlled at 20 1oC
Dissolve Oxygen Method
Reflux apparatus
Hot plate
Pipets, Class A and wide-bore.
Glass beads

1. Prepare all reagents as described in experiments 6 and 7 for COD and BOD
analysis respectively.

2. The demonstrator will give you a sample of wastewater, which originates from
pharmaceutical waste.

3. Filter sample first to get only the soluble organics.

4. Do at least 4 dilutions for the wastewater to carry out the COD and BOD tests as
described in experiment 6 and 7.

5. For the same diluted wastewater sample, analyze the COD and BOD of the
sample.

57
Report

COD Test 1st Dilution 2nd Dilution 3rd Dilution 4th Dilution
Volume of
FAS used for
blank(mL)
Volume of
FAS used for
sample (mL)
Molarity of
FAS (mL)

BOD Test 1st Dilution 2nd Dilution 3rd Dilution 4th Dilution
DO of sample
before
incubation
DO of sample
after 5 days
incubation
DO of control
before
incubation
DO of control
after 5 days
Dilution factor

Calculate the HOD and COD for the 4 samples above. Plot the relationship between the
BOD and COD for the pharmaceutical wastewater.

Question

1. What is the purpose of establishing the BOD/COD correlation?

2. What type of wastewater can obtain a good correlation of BOD and COD?

3. List the different between the COD and the BOD tests.

References

1. Hammer. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.


edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

58
Experiment 10
Determination of Ammonia (NH3) (APHA / ASTM 4500-
NH3)
Date of Revision:

Objective
To familiarise with the different forms and sources of ammonia in water sample
To determine the concentration of ammonia in different water samples

Related Experiment

For discussion on common forms of nitrogen and related experimental procedure,


please refer to Experiment Determination of Total Kjedahl Nitrogen

Introduction

Ammonia is a gas at temperatures and pressures normally found in natural water


systems. The gas (NH3) exists in equilibrium with the aqueous ionic form called
ammonium (NH4+).


NH3 H 2 O NH OH
The hydroxyl ion (OH-) concentration in the water, and thus the pH, controls the relative
abundance of each species.

Ammonia is used by green plants for photosynthesis. It can be formed in water by the
microbiological degradation of nitrogen-containing organic compounds. Ammonia is
also considered as a serious water pollutant because of its toxic effect to fishes. In
wastewater, the concentration of ammonia may be greater than 40 mg/L.

Principle

The test for ammonia nitrogen involves a distillation process. The analysis procedure
is based on shifting the equilibrium between the ammonium ion and free ammonia (see
equation below), and the release of gaseous ammonia along with the steam that is
produced when the water is boiled. The steam containing ammonia is condensed and is
collected in a boric acid solution. The amount of ammonia in the sample is determined
by the quantity of boric acid consumption in the collecting beaker. This may be
measured by back titration of the solution with a standard acid to determine the amount
of \borate ion produced.

acidic

NH 4 NH3 H
basic

Experimental Procedure

59
Chemicals

100 mL 0.02 N H2SO4


1 bottle Phenolphthalein indicator
10 mL 0.02 N NaOH
10 g Boric Acid
100 mg Methyl red indicator
100 mL 95 % Ethanol
50 mg Methylene blue
20 mL Phosphate buffer

List of glassware/apparatus

1 pH meter
1 TKN Apparatus
4 250mL beakers
1 burette
1 wash bottles
1 100mL measuring cylinder
2 250mL conical flask
1 500mL volumetric flask

Preparation of Reagents

Use ammonia-free water in making all reagents and dilutions

a. Mixed indicator solution

1. Dissolved 100mg methyl red indicator in 50 mL 95% ethanol.


2. Dissolved 50 mg methylene blue in 25 mL 95% ethanol
3. Combine solutions from steps 1 and 2

b. Indicating Boric Acid Solution

1. Dissolved l0g H3BO3 in ammonia-free distilled water


2. Add 5mL of mixed indicator solution
3. Dilute to 500mL in 500mL volumetric flask

c. Phosphate Buffer

1. Dissolve 8.5g KH2PO4, 21.75 g K2HPO4, 33.4 g Na2HPO4.7H20, and 1.7 g NH4C1
in about 500mL distilled water and dilute to 1 L

Procedure

60
1. Place 50 mL of water sample in a Kjeldahl flask and mark as 'Sample'

2. Add 50 mL of distilled water.

3. Add 5 drops of phenolphthalein indicator and mixed thoroughly.

4. By using a dropper, add 0.02 N NaOH until a permanent slight pink colour is
obtained which mark the endpoint of phenolphthalein.

5. Then add phosphate buffer a little at a time and stir (about 10-20 mL) until the pH
is between 7.1 and 8.0. Check the pH by using a pH meter.

6. Prepare a blank by using, 100mL of distilled water in a Kjeldahl flask. Mark the
flask as 'Blank'

7. Add 5 drops of phenolphthalein indicator and mixed thoroughly.

8. By using a dropper, add 0.02 N NaOH until a permanent slight pink colour is
obtained which mark the endpoint of phenolphthalein

9. Then add phosphate buffer, a little at a time and stir (about 10-20 mL) until the pH
is between 7.1 and 8.0. Check the pH by using a pH meter.

10. Connect the two Kjeldahl flasks containing sample and distilled water to the
Kjeldahl apparatus.

11. Refluks ollect 250 mL of the distillate in a 500 mL conical flask containing, 20 mL
boric acid solution. The tip of the delivery tube must be below the surface of the
boric acid solution to prevent the ammonia from escaping to the air.

12. Titrate the distillate with 0.02 N H2SO4 until pale bluish end point.

Calculation

(A B) x 14 x 0.02 x 1000
mg/L NH 3 N
mL sample
where;
A volume H 2 SO 4 used for titrat ion of sample
B volume H 2 SO 4 used for titrat ion of blank
14 MW N
0.02 N H 2 SO 4

Report

61
1. Titration Results

Water Sample Final volume Initial volume Volume of Amount of


of 0.02N of 0.02N 0.02N H2SO4 NH3 N
H2SO4 H2SO4 used present, mg/L
1. Blank
2.
3.
4.

2. Show one example on the calculation of NH3-N content in water sample

3. Discussions on results obtained

4. What is the importance of ammonia in water or wastewater?

5. What are the sources of ammonia in water?

6. Give the equation which shows that ammonia exists in equilibrium with the ionic
form.

7. What is the concentration of ammonia in wastewater?

8. Name the acid in which ammonia will be collected?

9. What is the endpoint of the titration using 0.02 N H2SO4

References

1. Hammer. MJ., and Hammer, J.M.Jr.,'Water and Wastewater Technology', 3rd.


edition, Prentice Hall International Editions, 1996
2. Clesceri, L.S., Greenberg, A.B. and Trussel, R.R.(1992) Standard Method for the
Examination of Water sad Wastewater 1Sth ed. American Public Health
Association (APHA).
3. Metcalf and Eddy (2003). Wastewater Engineering Treatment and Reuse. 4th
Edition. McGraw Hill.

62
Experiment 11
Determination of Heavy Metals: Lead (Pb) and Arsenic
(As) by the Direct Air-Acetylene Flame Method (APHA /
ASTM 3111 B)
Date of Revision:

Objectives

To familiarize in general the atomic absorption spectrophotometer


To determine the concentration of Lead (Pb) and Arsenic (As) in water sample
To compare the concentration of metals present in the water sample with the
values of Standard B

Introduction

Here are several types of metals that are toxic and are found in trace amounts in water
and wastewater. These metals also known as heavy metals and examples are Nickel
(Ni), cadmium (Cd), Copper (Cu), Zinc, Lead (Pb) etc. are amongst the metals
discharged in effluents by industries that uses these metals in their operational.
Examples of some of the industries that are involved with discharging metals are
petrochemicals, electroplating, heavy machinery, and battery producing industry, just
to mention a few.

Method of Determination

The concentration of an element in solution is determined by measuring the quantity of


light of specific wavelength absorbed by atoms of the elements released in a flame.
Figure 9.1 shows an atomic absorption spectrophotometer, which consist of an
atomizer-burner to convert the element in solution to free atoms in an air acetylene
flame. This is followed by a monochromator (prism and slit) to disperse and isolate the
light waves emitted, and a photomultiplier to detect and amplify the light passing
through the monochromator. The light source is a lamp with a cathode formed of the
same element being determined, since each element has characteristic wavelengths that
are readily absorbed.

63
The light passing through the sample is separated in the monochromator into its
component wavelength. The photomultiplier then receives only the isolated resonance
wavelength. The amount of energy at the characteristic wavelength absorbed in the
flame is proportional to the concentration of the element in the sample.
Experimental Procedure

Chemicals

10mL 1000mg/L Pb stock solution


10mL 1000mg/L As stock solution
10mL concentrated HCl

List of glassware/apparatus

10 100mL volumetric flask with cap


1 250mL beaker
1 Dropper
AAS (Atomic Absorption Spectrophotometer) using air
1 Acetylene flame for Pb determination
1 AAS (Atomic Absorption Spectrophotometer with graphite furnace)
And using argon hydrogen air flame for As determination

Note: This is a demo session only. Laboratory technician will perform this experiment
but you are required to prepare the standards for the calibration curve. Three
groups will share preparing the standards, blank and one water sample.

Preparation of standard 100mg/L from stock solution 1000mg/L Pb/As

1. The stock solution is first diluted to become 100mg/L by transferring 10mL of


1000mg/L into 100mL capacity volumetric flask.
2. Then add distilled water to the mark
3. The flask is then inverted several times to mix the contents properly.

Preparation of standards from 100mgL new stock solution for Pb/As

64
Calculate the amount required to obtain 0, 0.5, 1.0, 1.5 and 2.0mg/L standard solution
for Pb/As from 100mg/L new stock solution. Example calculation: To prepare
0.5mg/L Pb/As.

x
*100mg/L 0.5mg/L
100
where, x is the amount of standard required
100 is the capacity of volumetri c flask used
100mg/L is the new stock solution
0.5mg/L standard solution required to prepare
Therefore, x is 0.5mL

1. Pipette 0.5mL of 100mg/L stock solution


2. Place in 100mL volumetric flask
3. Add distilled water until the mark
4. Add 10 drops of con. HCl. Please carry out this step at the fume cupboard.
5. Mix the contents by inverting the flask several times.
6. Repeat the same steps (1- 5) for preparing 1 mg/L, 1.5 mg/L and 2.0 mg/L
standard solutions.
7. For 0 mg/L or blank, use 100mL distilled water instead and add 10 drops of HCl.
8. For the sample, place 100mL of water sample in 100mL volumetric flask and
then add 10 drops of con. HCl
9. After preparation for sample, standards and blanks has completed, the technician
will operate the AAS, plot the standard curve and measure the concentration of
Pb and As for you

References

1. Hammer, M.J., and Hammer, J.M.Jr., Water and Wastewater Technology, 3rd.
edition, Prentice Hall International Editions, 1996.

2. Clesceri, L.S., Greeberg, A.E. and Trussel, R.R.(1992) Standard Method for the
Examination of Water and Wastewater,18th ed. American Public Health
Association (APHA).

65
Experiment 12
Microbiological Analysis - Standard Presumptive Total
Coliform Fermentation Technique (APHA / ASTM 9221
B)
Date of Revision:

Objectives

To differentiate total coliforms from other bacteria


To understand the technique of dilution to extinction
To determine the MPN(Most Probable Number) of bacteria per 100mL sample

Related Experimental Topic

Introduction to bacteria in this experiment is related to the previous experiment 'Aerobic


Plate Count Method'.

Introduction

The colifom group consist of several types of bacteria belonging to the family
Enterobacteriaceae. This colifonn group is defined as all aerobic and facultative
anaerobic, Gram-negative, nonspore-forming, rod shaped bacteria that ferment lactose
with gas and acid formation within 48 hours at 35 C.

The standard test for the coliform group may be carried out by the multiple-tube
fermentation technique or by the membrane filter technique. The multiple tube method
gives less accurate quantification than counts on solid media, but is used whenever the
observation of gas production is an important part of the test. Growth of bacteria is
detected by increasing turbidity of the media and is accompanied by a change in
individual colour due to acid production. Gas production is detected by placing a small
inverted Durham tube in the media to trap any gas produced.

Experimental Procedure

Chemicals

5g Lactose
10 g Peptone
2.5 g Sodium Chloride
1g Bromocresol Purple
100 mL Ethanol
50 mL Ringers solution

66
List of glassware/apparatus

2 Pipettes (1 mL) in metallic container with cover


2 500 mL beaker
1 250 mL beaker
1 500 mL Volumetric flask
1 Measuring cyclinder
2 Pipettes aids
Dilution bottles (or fermentation tubes) with
29 caps
1 Incubator, 35 0.5 oC
1 Autoclave

Preparation of McConkey's Broth

1. Place lactose, peptone and sodium chloride in 500 mL beaker and mix with
250 mL distilled water (A)

2. In another 500mL beaker, dissolved 2.5g bilesalt in 100mL distilled water (B)

3. Mix A and B in a 500 mL volumetric flask and add 100mL distill water

4. Adjust the pH to 7.4

5. Then add 1% Bromocresol Purple in ethanol (10mL + 1L Broth)

6. Add distilled water until the final volume is 500mL

Preparation of 1 % Bromocresol purple

1. Place 1g Bromocresol Purple salt(C4H16Br2O5S) in 250mL beaker

2. Add 100mL ethanol and mix thoroughly.

Autoclave 4 dilution bottles containing 9mL Ringers solution, pipettes in metallic


container, 25 fermentation tubes containing 12mL McConkeys Broth and Durham
tubes, for 15 -20 minutes at 121oC.

67
Procedure

1. The sample is serially diluted in Ringers solution

2. Arrange fermentation tubes in rows of five each in a test tube rack.

3. Using a sterile pipette and starting with the highest dilution, sets of five tubes of
McConkeys Broth are inoculate with :

5 x 1 mL (10-4 sample dilution)


5 x 1 mL (10-3 sample dilution)
5 x 1 mL (10-2 sample dilution)
5 x 1 mL (10-1 sample dilution)
5 x 1 mL (100 or original water sample)

4. Mix water sample and McConkeys' Broth gently.

5. Incubate inoculated tubes or bottles at 35 0.5 C. After 24 2 hrs swirl each


tube or bottle gently and examine for growth, gas, and acidic reaction (shades of
yellow colour) and, if no gas or acidic reaction is evident, reincubate and re-
examine after. 48 3 hr..

6. Record presence or absence of heavy growth, gas and acidic production.

Interpretation

1. Production of gas or acidic growth in the tubes within 48hrs 3hrs constitutes a
positive presumptive reaction.

2. The absence of gas or acidic growth in the tubes within 48hrs 3hrs constitutes a
negative test.

Calculation of Most Probable Numbers (MPN)

Record the number of positive reactions for each set of tubes. Apply the following
rules:

Use only three consecutive sets of dilutions for calculation of the MPN value. Where a
series of dilutions has been used, select the smallest sample volume giving some
positive reactions with two preceding sets of dilutions, multiply the value derived from
the table by the dilution factor used to give the estimated number of organisms per
100mL

Example
In the example given below, the significant dilution results are shown in boldface. The
number in the numerator represents positive tubes; the denominator represents the total

68
number of tubes. The number of positives simply represents the total number of positive
tubes per dilution:

Combination MPN
Example 1mL 0.1mL 0.01mL 0.001mL 0.0001mL
of positives index/100mL
A 5/5 5/5 2/5 0/5 0/5 5-2-0 5000
B 5/5 4/5 2/5 0/5 0/5 5-4-2 2200
C 0/5 1/5 0/5 0/5 0/5 0-1-0 20

References

1. Hammer, M.J., and Hammer, J.M.Jr., Water and Wastewater Technology, 3rd.
edition, Prentice Hall International Editions, 1996.

2. Clesceri, L.S., Greeberg, A.E. and Trussel, R.R.(1992) Standard Method for the
Examination of Water and Wastewater,18th ed. American Public Health
Association (APHA).

Question

1. What is the function of the inverted Durham tube in the fermenting tube?

2. How many fermenting tubes are required to perform MPN

3. What are the positive reactions showing the presence of presumptive coliforms in
the fermenting tubes?

4. What is the name of the broth to culture coliforms?

5. What is the family name of coliform group?

69
Report

Individual Batch Results

Sample Dilution 1 10-1 10-2 10-3 10-4


Source No of
Positives

Combination
of positives
Factor
applied
MPN/100mL

70
Group Results

Sample Batch MPN/100mL


Raw water 1
Tap water 2
Primary effluent 3
Secondary effluent 4

Discussion and conclusions

71
Experiment 13
Oil and Grease Analysis (APHA / ASTM 5520B)
Date of Revision:

Objectives

Introduction

Experimental Procedure

Chemicals

List of glassware/apparatus

Preparation of

Procedure

Interpretation

Calculation of

References

72