WASTEWATER
LABORATORY PROCEDURES
& CHEMISTRY
U.S. ENVIRONMENTAL PROTECTION AGENCY
REGION VII
1735 BALTIMORE
KANSAS CITY, MISSOURI - 64108
OFFICE OF INTERMEDIA PROGRAMS
MANPOWER & TRAINING PROGRAM
SURVEILLANCE & ANALYSIS DIVISION
TECHNICAL SERVICES BRANCH
JUNE 1975
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EPA 907/9-75-002
WASTEWATER
LABORATORY PROCEDURES AND CHEMISTRY
This manual has been adapted from Chapter 14 (by James Patterson)
of "Operation of Wastewater Treatment Plants - A Field Study Course."
The complete Field Study Course has been prepared for EPA by
California State University - Sacramento and is available at a nominal
charge. For information on ordering the complete course, write to:
Dr. Kenneth D. Kerri
Department of Civil Engineering
California State University
6000 Jay Street
Sacramento, California 95819
Environmental Protection Agency
Region VII
1735 Baltimore
Kansas City, Missouri 64108
Office of Intermedia Programs Surveillance & Analysis Division
Manpower & Training Program Technical Services Branch
JUNE 1975
Znwwmn^-i r- - -
v;-' . ; -Lo0bl°n Agency
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The Superintendent of Documents
classification number is:
EP.1.8: 111
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EPA REVIEW NOTICE
This manual has been reviewed by the Office of Water
Programs, EPA, and approved for publication. Approval
does not signify that the contents necessarily reflect
the views and policies of the Environmental Protection
Agency. Mention of trade names or commercial products
does not constitute endorsement or recommendation for
use by the Environmental Protection Agency or California
State University, Sacramento.
n
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TABLE OF CONTENTS
CHAPTER 14 LABORATORY PROCEDURES AND CHEMISTRY
Page
14.0 Introduction 14-1
14.00 Should You Start This Lesson Now? 14-1
14.01 Material in This Lesson 14-2
14.02 References 14-3
14.03 Acknowledgements 14-4
14.1 Glossary of Terms and Equipment 14-4
14.10 Terminology 14-4
14.11 Equipment 14-6
14.2 Safety and Hygiene 14-12
14.20 Laboratory Safety 14-12
14.21 Personal Hygiene for Wastewater
Treatment Plant Personnel 14-17
14.3 Sampling 14-21
14.30 Importance 14-21
14.31 Accuracy of Laboratory Equipment 14-21
14.32 Selection of a Good Sampling Point
to Obtain a Representative Sample . 14-22
14.33 Time of Sampling 14-23
14.34 Composition and Preservation of Samples 14-23
14.35 Sludge Sampling ' 14-25
14.36 Sampling Devices 14-26
14.37 Summary 14-27
14.4 Laboratory Work Sheet 14-30
14.5 Plant Control Tests 14-35
Test No. Title
1 Total Alkalinity 14-37
2 Biochemical Oxygen Demand or BOD 14-37
3 Carbon Dioxide (C02) in Digester Gas 14-38
4 Chemical Oxygen Demand or COD 14-43
5 Chlorine Residual 14-50
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Test No. Title Page
6 Clarity 14-59
7 Coliform Group Bacteria 14-62
8 Dissolved Oxygen or DO 14-82
I. In Water 14-82
II. In Aerator 14-90
9 Hydrogen Sulfide (H2S).- 14-103
I. In Atmosphere. . . 14-103
II. In Wastewater 14-104
10 pH 14-107
11 Settleability of Activated Sludge Solids. . 14-113
I. Settleability 14-113
II. Sludge Volume Index (SVI) 14-115
III. Sludge Density Index (SDI) 14-117
12 Settleable Solids 14-119
13 Sludge Age 14-123
14 Sludge (Digested) Dewatering
Characteristics .... 14-126
1^ Supernatant Graduate Evaluation 14-129
16 Suspended Solids 14-133
I. Gooch Crucible 14-133
II. Centrifuge 14-146
17 Temperature 14-150
I. Wastewater 14-150
II. Digester Sludge 14-154
18 Total and Volatile Solids (Sludge) 14-156
19 Turbidity (See Clarity)
20 Volatile Acids 14-164
21 Volatile Solids (See Total Solids)
14.6 Recommended General Laboratory Supplies 14-181
14.7 Additional Reading 14-185
IV
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OBJECTIVES
Chapter 14. Laboratory Procedures and Chemistry
Following completion of Chapter 14 you should be able to:
1. Work safely in a laboratory.
2. Know how to operate laboratory equipment.
3, Collect representative samples of influents to and effluents
from a treatment process as well as sample the process.
4. Prepare samples for analysis.
5. Perform plant control tests.
6. Recognize shortcomings or precautions for the plant control
tests.
7, Record laboratory results.
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CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
14.0 INTRODUCTION
14.00 Should You Start This Lesson Now?
Laboratory procedures and results are the means by which we control
the efficiency of our treatment processes and measure the effective-
ness of the processes. To operate your plant as efficiently as
possible, you must understand the laboratory procedures and relate
them to the actual operation of your plant.
This lesson has been given to you at this time mainly for reference
purposes. When you read the lessons on the treatment processes you
should begin to wonder how certain tests are performed that are
essential for proper plant operation. At this time you should refer
to this lesson for a general discussion and a description of the
laboratory procedure.
It might seem logical to you to complete this lesson first in order
to better understand the operational aspects of the treatment process
lessons. Many operators and potential operators who were interested
in this profession have taken this course. Most of them have said
that they wanted to learn about the treatment processes first and
then learn how to apply the lab procedures to plant operation. Many
potential operators experienced difficulty with the terminology when
they tried to work this chapter before completing the lessons on the
treatment processes. If you are an experienced operator arid are
anxious to learn more chemistry and to obtain a better understanding
Of lab procedures, you may decide to try this lesson first.
14-1
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Past experience has indicated that most operators prefer to use
tjiis section as a reference while studying the lessons on treat-
ment processes. You are the operator who wants to learn more
abou"t treatment plant operation, and you are encouraged to use
this material in any manner that you feel best fits ycur par-
ticular situation and professional goals. Now is the time for
you to decide whether you are going to:
1. Thumb through this lesson, proceed through the chapters .
on treatment processes, and then complete this lesson;
2. Complete the lessons on treatment processes, referring
to this lesson when interested, and then complete this
lesson;
3. Complete this lesson and then the lessons on treatment
processes; or
4. Follow your own plan.
14..01 Material in This Lesson
A few of the' lab procedures outlined, in this chapter are,.not
."Standard Methods" (4),1 but are used by many operators because
.they, are simple and easy to perform. Some of these procedures
are not accurate enough for scientific investigations, but are
satisfactory for successful plant control and operation. When
lab data must be submitted to regulatory agencies for;monitoring
and enforcement purposes, you should request the agency,to ', ,
provide you with a list of approved test procedures.
Each test section contains the following information:
1. Discussion of test.
2. What is tested?
3. Apparatus.
4. Reagents.
5. Procedures.
6. Precautions.
7. Examples.
8. Calculations.
Numbers in parentheses refer to references in Section 14.02,
14-2
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If you would like to read an introductory discussion on laboratory
equipment and analysis, the Water Pollution Control Federation has
a good publication entitled "Simplified Laboratory Procedures" (3).
Good discussions on the use of the analytical balance may be found
in "Laboratory Procedures" (1) or "Simplified Procedures" (3),
14.02 References
1, "Laboratory Procedures for Operators of Water Pollution Control
Plants" by Joe Nagano. Obtain from Secretary-Treasurer,
California Water Pollution Control Association, P.O. Box 61,
Lemon Grove, California 92045. Price $3.25 to members of CWPCA;
$4.25 to others.
2. "EPA Methods for Chemical Analysis of Water and Wastes", Ana-
lytical Quality Control Laboratory, 1014 Broadway, Cincinnati,
Ohio 45202 (October 1974)
3. "Simplified Laboratory Procedures for Wastewater Examination,"
WPCF Publication No. 18, 1968, 60 pages. $2 to WPCF members;
$3 to others.
4- "Standard Methods for Examination of Water and Wastewater,"
13th Edition, 1971, 874 pages. $16.50 to WPCF members;
$22.50 plus postage to others.
Both References 3 and 4 may be obtained by writing:
Water Pollution Control Federation
3900 Wisconsin Avenue
Washington, D.C. 20016
Order forms may be found in the Journal of the Water Pollution Control
Federation.
14-3
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14.03 Acknow1edgments
Many of the illustrated laboratory procedures were provided by
Mr. Joe Nagano, Laboratory Director, Hyperion Treatment Plant,
City of Los Angeles, California. These procedures originally
appeared in Laboratory Procedures for Operators of Water
Pollution Control Plants, prepared by Mr. Nagano and published
by the California Water Pollution Control Association. The
lists of equipment, reagents, and procedures outlined in this
chapter are similar to those listed in the references in
Section 14.02. Use of information from these references is
gratefully acknowledged.
14.1 GLOSSARY OF TERMS AND EQUIPMENT
14.10 Terminology
> Greater than.
DO > 5 mg/1, would-be
read as DO greater than
5 mg/1, ~~
< Less than.
DO < 5 mg/1, would be
read as DO less than
5 mg/1.
Aliquot (AL-li-kwot).
Portion of a sample.
Ambient Temperature (AM-bee-ent). Temperature of the surroundings.
Amperpmetric (am-PURR-o-MET-rick). A method of measurement that
records electric current flowing or generated, rather than record-
ing voltage. Amperoraetric titration is an electrometric means of
measuring concentrations of substances in water.
Anaerobic Environment (AN-air-G-bick). A condition in which "free"
or dissolved oxygen is not present.
14-4
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Blank. A bottle containing dilution water or distilled water,
but the sample being tested is not added. Identical tests are
frequently run on a sample and a blank and the differences
compared.
Buffer. A measure of the ability or capacity of a solution or
liquid to neutralize acids or bases. This is a measure of the
capacity of water or wastewater for offering a resistance to
changes in the pH.
Composite (proportional) Samples (coir,-POZ-j.t). Samples collected
at regular intervals in proportion to the existing flow and then
combined to form a sample representative, of the entire period of
flow over a given period of time.
Pistillate. In the distillation of a sample, a portion is
evaporated; the part that is condensed afterwards is the distillate.
End Point. Samples are titrated to the end point. This means
that a chemical is added, drop by drop, to a sample until a
certain color change (blue to clear, for example) occurs which
is called the end point of the titration. In addition tq a color
change, an end point may be reached by the formation of a precipi-
tate or the reaching of a specified pH, An end point iray be
detected by the use of an electronic device such as a pH meter.
Flame Polished. Sharp or broken edges of glass (such as the end
of a glass tube) are flame polished by placing the edge in a flane
and rotating it. By allowing the edge to melt slightly, it will
become smooth.
M pr^ Molar. A molar solution consists of one gram molecular
weight of a compound dissolved in enough water to make one liter
of solution. A gram molecular weight is the molecular weight of
a compound in grams. For example, the molecular weight of sulfuric
acid (P^SOiJ is 98. A 1M solution of sulfuric acid would consist
of 98 grams of H^SCV dissolved in enough distilled water to make
one liter of solution.
Molecular Weight. The molecular weight of a compound in grains is
the sum of the atomic weights of the elements in the compound. The"
molecular weight of sulfuric acid (H2SQLf) in grams is 98.
Atomic • Number Molecular
Element Weight of Atoms Weight
H 1 2 2
S 32 . 1 32
0 16 4 64_
98
14-5
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N or Normal. A normal solution contains one gram equivalent
weight of a reactant (compound) per liter of solution. The
equivalent weight of an acid is that weight of a compound which
contains one gram atom of ionizable hydrogen or its chemical
equivalent. For example, the equivalent weight of sulfuric
acid (^SOtJ is 49 (98 divided by 2 because there are two re-
placeable hydrogen.ions), A IN solution of sulfuric acid
would consist of 49 grams of F^SO^ dissolved in enough water
to make one liter.
Oxidation (ox-i-DAY-shun). Oxidation is the addition of oxygen,
removal of hydrogen, or 'the removal of electrons from an element
or compound. In wastewater treatment, organic ma'tter is oxidized
to more stable substances.
Percent Saturation. Liquids can contain in solution limited
amounts of compounds and elements. 100% saturation is the
maximum theoretical amount that can be dissolved in the solution.
If more than the maximum theoretical amount is present, the
solution is supersaturated.
o f, „. J_. Amount in Solution ,„„„
% Saturation = rr—: -r —• 1 * 100%
Maximum Theoretical
Amount in Solution
Reagent (re-A-gent). A substance which takes part in a chemical
reaction that is used to measure, detect, or examine other sub-
stances.
Representative Sample. A portion of material or water identical
in content to that in the larger body of material or water being
sampled.
Titrate. To titrate a sample, a chemical solution of known
strength is added on a drop-by-drop basis until a color change,
precipitate, or pH in the sample is observed (end point).
Titration is the process of adding the chemical solution to
completion of the reaction as signaled by the end point.
14.11 Equipment
Equipment can be better described by a photo or a sketch than
a written description; consequently, this portion of the
glossary will describe equipment in this manner. Photos of
equipment shown were provided by Van Waters § Rogers.
14-6
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ILLUSTRATIONS OF LABORATORY
60809-021 Series
Test Tube
60824-116 Series
Culture Tube
Without Lip
13912-207
Beaker
30209-025
Funnel
29140-023
Flask,
Erlenraeyer
(ER-len-MY-er)
Wide Mouth
29619-642
Flask,
Volumetric
29110-102
Flask,
Boiling
Flat Bottom
23130-049
23131-020
Condenser
29126-022
Flask,
Boiling
Round Bottom
Short Neck
29209-083
Flask,
Distilling
29415-100
Flask,
Filtering
30294-024
Funnel,
Buchner
With
Perforated Plate
14-7
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Bottle,
Reagent
23835-000
Crucible
Gooch
(GOO-ch)
Porcelain
g
y *•/
17685-005
Support, Buret
§ Buret Clamp
Bottle,
BOD
24707-2S5
Cylinder,
Graduated
23810-021
Crucible
Porcelain
25310-019
Dish,, '
Evaporating
-v-
17454-443
Buret
(bur-RET)
25313-017
Dish,
Evaporating
Shallow Form
17590-044
Buret
Automatic
14-8
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Clamp, Beaker,
Safety Tongs
217SO-009
, Dish
Safety Tongs
21792.904
Clamp, Flask,
Safety Tpngs
21611-046
Clamp, Utility
62765-029 Series
Tripod, Concentric
Ring
21770-028
Clamp, Test Tube
21877-000
Clamp Holder
17951-029
Burner, Bunsen
62730-024 Series
Triangle
Fused
14-9
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66187-004
Cone,
Irahoff
(IM-hoff)
66190-009
Cone Support
25353-248
Dish, Petri
68176-325
Color Comparison
Tubes, Nessler
25026-026
Desiccator
(DES-ick-kay-tor)
52368-022 Series
Oven, Mechanical Convection
53047-024 Series
r.Pipette ;
(P IE-pet)
Volumetric ,
53224-028 Series
Pipet, Serological
61048-033 Series
Thermometer, Dial
33976-009
Hot Plate
30632-003
Muffle Furnace, Electric
14-10
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35960-000
BOD Cabinet
34114-055
pH Meter
57980-000
Spectrophotometer
Weight = 95.5580 gm.
11274-008 Reading Scale
11274-008
Balance, Analytical
54906-001
Pump, Air Pressure $ Vacuum
60776-002
Test Paper, pH 1-11
14-11
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CHAPTER 14 LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 1 of 8 Lessons)
14.2 SAFETY AND HYGIENE, by A.E. Greenberg from California Water
Pollution Control Association Operators Laboratory Manual
14.20 Laboratory Safety
Safety is important in the laboratory as well as in the rest
of the treatment plant. Therefore, each employee working in
a laboratory should be thoroughly familiar with this section.
On questions of safety, consult your state's General Industrial
Safety Orders or similar document and Sax's "Dangerous Chemicals".2
Personnel working in a wastewater treatment plant laboratory
must realize that a number of hazardous materials and conditions
exist. _PREVENT_ ACCIDENTS.. Be alert and careful.. Be aware of
potential Dangers at all times. The major threats to you are
listed for your safety,
1. Infectious Materials
Wastewater and sludge contain millions of bacteria, some
of which are infectious and dangerous, and can cause
diseases such as tetanus, typhoid, dysentery, poliomeiytds",
and hepatitis. Personnel handling these materials should
thoroughly wash their hands with soap and water, particularly
before handling food. Do not pipette wastewater or polluted
samples by mouth. Use a rubber bulb. Though not mandatory,
inoculations by your County Health Department are recommended
for each employee.
2 See Sax, N.I,, Dangerous Properties of Industrial Materials
Third Edition, ReinholdY New York',' 1968", price $35.
14-12
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2. Corrosive Chemicals
A. Acids
(1) Examples: Sulfuric, hydrochloric, nitric, glacial
acetic, Pomerpy solutions Nos. 1 and 2, and chromic
acid cleaning solutions,
(2) Acids are extremely corrosive to human tissue, metals,
clothing, wood, cement, stone, and concrete. Use glass-
ware or polyethylene containers.
(3) In case of accidental spills, immediately dilute
large portion? of water and neutralize the acid
sodium carbonate or bicarbonate
unto,! bubbling and foaming stops.
Clean up neutralized material.
If spills occur on bench tops,
dilute, neutralize, and squeegee
into sink. If spills occur on
person, immediately wash pff
with water, If spills occur
on face (spills pf concentrated
acid), immediately flood with
large quantities of cold water.
Notify supervisor. Remember to
add acid to water, but not" reverse.
Pour and pipette carefu1ly to
prevent spilling and dropping.
Prevent contact with metals,
particularly equipment.
with
with
B. Bases
(1) Examples: Sodium hydroxide, potassium hydroxide,
ammonium hydroxide, alkaline iodide sodium azide
solution.
(2) Handle with extra care and respect, They are extremely
corrosive to skin, clothing, and leather. Use glass-"
ware and polyethylene containers,.
Ammonium hydroxide is extremely irritating to the
eyes and respiratory system. Ppur ammonium hydrpxide
under a laboratory hood with fan in operation.
14-13
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(3) In case of accident, wash with large quantities
of water and use saturated boric acid solution,
to neutralize.
Miscellaneous
(1) Chlorine gas solution avoid inhalation. Handle
in hood. Secure cover to prevent escape of vapors.
(2) Ferric salts, Ferric chloridf very corrosive to
metals. Avoid body contact and wash off imme-
diately,
(3) Strong oxddants^---avoid body contact. 1,'ash off
immediately. Use of perchloric acid by untrained
personnel must be prohibited.
3. TQXJC Materials
Avoid ingesting qr inhaling,
A. Solids: Cyanides, chromium, cadmium, and other heavy
metal compounds.
B. Liquids: Use in vented hood. Carbon tetrachlori'Je,
ammqnium hydroxide, nitric acid, bromine, chlorine
water, aniline dyes, formaldehyde, chloroform, and
carbon disulfide. Carbon tetraehloride is absorbed
into skin on contact; its vapors will damage the lungs;
and it will build up in your body to a dangerous level.
C, Gases: Use in vented hood. Hydrogen sulfide, chlorine,
ammonia, nitric, hydrochloric acid.
D. Most laboratory chemicals have toxicity warnings and
antidotes on their labels. Learn about the materials
you use, Don't breathe, eat, or drink them; and if
they come in contact with your body, quietly apply
large quantities of water to wash the substance away.
14-14
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4. Explosive or Inflammable Materials ;
A. Gases:- Acetylene, hydrogen,
B, Li quids: Carbon disulfide, benzene, ethyl ether,
petroleum ether, acetone, gasoline. , >:
Store these materials according to fire regulations^to
prevent fire hazards. If large quantities must T?e "stxired,
they should be located in a separate storage building.
Do not uss near open flame or exposed heating elements-.'
Use under a vended laboratory hood. Do not,distill t'$ dry-
ness or explosive mixtures may result. Use .face mask. Do
not throw flammable liquids into sinks. Cigarette-.discard
may cttuse fire. Do not let gas cylinders fa IT. ^"~"
5. Broken Equipment
A. Inexpensive^Iterns--Beakers and flasks should be dis-
carded/ except for minor chips which .can be flame
polished3 easily,
Bt cxpensiye Iterns--Shou1d be set aside for salvage if
possible. Discard if damaged beyond repair. ~ ?-%
" Flame Polished. Sharp or broken edges of glass (such as the
end of a glass tube) are flame polished by placing the edge
}.n^a flame and rotating it. By allowing the edge to melt
slightly, it will become smooth.
14-15
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6. Mi see 11 aneous
A. Use safety goggles or face mask
in any experiment in which there
is danger to the eyes. Never look
into the end of the test i ube during
reaction or heating.
Use care in making rubber- to- glass
connections. Lengths of glass
tilting should be supported uhile
they are being inserted into rubber.
The ends of the glass should be
flame polished, and either wetted
or coverec.' with a lubricating jelly
for ease in joining connections.
Never use grease or oil. Gloves
or grippers should be worn when
making such connections, and the
tubing should be held as close to
the e-.d being inserted as possible
to prevent bending or breaking.
Never try to fo::ce rubber tubing
or stoppers from glassware. Cut
the rubber or material off,
B. Always check labels on bottles to
make sure that the chemical selected
is correct. All chemicals and bottles
should be cleaVly 'labeled'. Never "
handle chemicals with bare hands .
Use spatula, spoon, or tongs.
C, Never work in a poorly ventiJated
area. Toxic fumes even in mild
concentrations can knock you out.
Be sure you have adequate venti-
lation before you start work in tm
laboratory.
.0. Stioking and eating should be avoifei when working with
infectious materials such as \«/astewater ano sludge. Never
use laboratory glassware for serving the food.
E. Always use the proper type of equipment for handling hot
containers, such as ^rc^ective gloves, tongs, clothing,
glasses, etc,
14-16
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F. Where cylinders of oxygen or other compressed gases
are used in the laboratory, they should be stored in
separated and ventilated sections. They should be
chained or clamped in an upright position while being
used. The protective caps should never be removed until
the cylinder is set and clamped in place, ready for
attachment of valve gage and connections. Always use
fittings approved for the cylinder being used and care-
fully follow instructions.
G. Iji working in the plant, be carefijl around:
(1) Digesters--Do not smoke.
(2) Chlprinators—B® aware of chlorine leaks. Chlorine
may be detected by its odor, or a white mist will
form near a rag soaked in ammonia.
(3) Power and BJ,ower--Wear ear plugs or ear covers if
working over one hour in engine room.
(4) Open Wastewater Tanks—Be carefulj don't fall in.
(5) Closed Wastewater Tanks—Avoid running over tank
covers by foot or vehicle.
(6) In Tanks or Ne,ar Construction—Wear hard hats.
14.21 Personal Hygiene for Wastewater Treatment Plant Personnel
Although it is highly unlikely that personnel can contract diseases
by working in wastewater treatment plants, such a possibility does
exist with certain diseases.
1, Some diseases are contracted through breaks in the skin, cuts,
or puncture wounds. In such cases the bacteria causing the
disease may be covered pver and trapped by flesh, creating a
suitable anaerobic; environment1* in which the bacteria may
thrive and spread throughput the body.
Anaerobic Environment (AN-air-0-bick). A condition in which
"free" or dissolved oxygen is not present.
14-17
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For projection against diseases contracted through breaks
in the skin, cuts, or puncture wounds/ everyone working in
or around wastewater must receive immunization from tetanus.
Immunization must be received before the infection occurs.
To prevent diseases from entering open wounds, care must be
taken to keep wounds protected either with band aids or, if
necessary, with rubber gloves or waterproof protective
clothing. ! .
2. Diseases that may be contracted through the gastrointestinal
system or through the mouth are typhoid, cholera, dysentery,
amebiases, worms, salmonella, infectious hepatitis, and
polio virus. These diseases are transmitted by the infected
wastewater materials being ingested or swallowed by careless
persons. The best protection against these diseases is
furnished by thorough c1eansing. Hands, face, and body
should be thoroughly washed wl'th soap and water, particularly
the hands, in order to prevent the transfer of any unsanitary
materials or germs to the mouth while eating. A change of
working clothes into street clothes before Leaving work is
highly recommended to prevent carrying unsanitary materials
to the employee's home. Personal hygiene, thorough cleansing,
and washing of the hands are effective means of protection.
Immunization is provided for typhoid and polio. Little is
known about infectious hepatitis except that it can be trans-
mitted by wastewater. It is frequently associated with gross
wastewater pollution.
3, Diseases that may be contracted by breathing contaminated
air include (1) tuberculosis, (2) infectious hepatitis, and
(3) San Joaquin fever. There has been no past evidence to
indicate the transmission of tuberculosis through the air
at wastewater treatment plants. However, there was one
case of tuberculosis being contracted by an employee who
fell into wastewater and, while swimming, inhaled waste-
water into his lungs. San Joaquin fever is caused by a
fungus which may be present in wastewater. However, there
is no record of operators contracting the disease while on
the job.
14-18
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bfst insurtnee against these diseases is proper personal
hygiene an4 imnnaniESfion. Your plant should have an immunization
program aga;in?t (l); tetapus, (20 typhoid, (3) polio, and (4) small-
pox (although smallpox i^ not related to wastewater) . The
imm«#ti Cation;* shwjtf be provided to protect you. Check with your
IqcaJ, Or sWte health department for recommendations regarding
In the washing of hands, the kind of soap is less important than
th$ though BSe of the soap, (Special disinfectant soaps are
not essential.)
The u«e of protective cjpthing is very important, particularly
gloves and boots. The protection of wounds and cuts is also
important. Report injuries and take care of them.
The responsibility rests upon you.
There is no absolute insurance against contraction of disease
in a wast^water treatfnent plant. However, the likelihood of
transmission is practically negligible. There appears to be no
special risk in working at treatment plants, In fact, operators
may receive a natural immunization by working in this environment.
14-19
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QUESTIONS
14.2A Why should you always use a rubb'er bulb to" -
pipette wastewater or polluted wate'r?
' f, ;
14.2B Why are inoculations against disease recommended
for people working around wastewatei4? " '
14.2C What would you do if you spilled a concentrated '
acid on your hand?
14.2D True or False: You may add acid to water, but
never water to acid.
14,2E If you are working in a', wastewater treatment
plant, why should you change your clothes b-ef-bre
going home at night?
14-20
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14.3 SAMPLING, by Joe Nagano, from California Water Pollution
Control Association Operator? Laboratory Manual ,.',.,
14.30 Importance . ...-•''',
Before any laboratory tests are performed, it is highly important
to obtain a proper, representative sample. Without a representative
sample, a test shpuld not even be attempted because the test .result
will be incorrect and meaningless. A laboratory test without, a good
sample will most likely lead to erroneous conclusion? and confusion.
The largest errors produced in laboratory tests .are usually,, caused
by imprp^er sampling, poor preservation, 'or lack of enough mixing"
diirlrif corn Ositlng^and testing. ' , T1 -^ •-•-•- — • -
14.31 Accuracy of Laboratory Equipment
Laboratory equipment, in itself, is generally quite accurate.
Analytical balances weigh to 0.1 milligram. Graduated cylinders,
pipettes, and burettes usually measure to 1% accuracy, so that the
errors introduced by these items should total less than .5%,. and
under the worst possible conditions only 10%, Under ideal conditions
let us assume that a test of raw wastewater- for suspended solids
should run about 300 mg/1. Because of the previously mentioned
equipment or apparatus variables, the value may actually range
fro|a £70 to 330 mg/1. Results in this range are reasonable for
operation. Other less obvious factors are usually present which
make it quite possible to obtain results which are 25, SO, o.r'even
100% in error, unless certain precautions are taken. Some examples
will illustrate hpw these errors are produced.
The City of Los Angeles Terminal Island Treatment Plant is a
primary treatment facility with a flow of 8 million gallons per
day. It has an aerated grit chamber, two circular 85-foot clari-
fiers of 750,000 gallon capacity, and two digesters 100 and 75 feet
in diameter. ' , "
Composite (Proportional) Samples (com-POZ-it.). Samples collected
at regular intervals in proportion,to the existing flow and;then
cpmbined to form a sample representative of the entire-,period of
flow over a given period of time, " * Y* '•.,;"? *\
14-21
-------
Monthly summary calculations based upon the suspended solids test
showed that about 8,000 pounds of suspended solids were being
captured per day during sedimentation assuming 200 mg/1 for the
influent and 100 mg/1 for the effluent. However, it also appeared
that 12,000 pounds per day of raw sludge solids were being pumped
out of the clarifier and to the digester. Obviously, if sampling
and analyses had been perfect, these weights would have balanced.
The capture should equal the removal of solids. A study was made
to determine why the variance in these values was so great. It
would seem logical to expect that the problem could be due to
(1) incorrect testing procedures, (2) poor sampling, (3) incorrect
metering of, the wastewater or sludge flow, or (4) any combination
of the three or all of them. . . .
In the first case, the, equipment was in excellent condition.
The operator was a conscientious and able employee who was
found to have carried out the laboratory procedures carefully
and who had previously run successful tests on comparative
samples. It was concluded that the equipment and test proce-
dures were Completely satisfactory.
14.32 Selection of a Good Sampling Point to Obtain . , ;>-
a.Representative Sample , .
A survey was the1* made.to determine if sampling stations were in
need of relocation. By using Imhoff cones and running settleable
solids tests along the influent channel and the aerated,grit.,
chamber, one could quickly recognize that the best mixed, and
most representative samples were to be taken from the aerated
grit chamber rather than the influent channel.
The settleable solids ran 13 ml/1 ;i.n the aerated grit chamber
against 10 ml/I in the channel. By the simple process of
determining the best sampling station, the suspended solids
value in the influent was corrected from 200 mg/1 to the more
representative 300 mg/1. Calculations, using the correct
figures,, changed the solids capture from 8,000 pounds to 12,000
pounds per day and a balance was obtained.
This study clearly illustrates the importance of selecting a
good sampling point in securing a truly representative sample.
It emphasizes the point that even though a test is accurately
performed, the result may be enjb^rely" ^rr^ph^us' and meaningless
insofar as use' for process control isT concerned, unless a good
represgrit.aHve saniple is taken. Furthermore, a good sample is
highly dependent: upon the sampling' station. Whenever possible,
14-22
-------
select a place where mixing is thorough and the wastewater quality
is uniform. As the solids concentration increases, above about
200 mg/1, mixing becomes even more significant because the waste-
water solids will tend to separate rapidly with the heavier solids
settling toward the bottom, the lighter solids in the middle, and
the floatables rising toward the surface. If, as is usual, a
one-gallon portion is taken as representative of a million-gallon
flow, the job of sample location and sampling must be taken
seriously.
14.33 Time of Sampling
Let us consider next the time and frequency of sampling. In
carrying out a testing program, particularly where personnel
and time are limited due to the press of operational responsi-
bilities, testing may necessarily be restricted to about one
test day per week. If the operator should decide to start his
tests early in the week, by taking samples early on Monday
morning he may wind up with some very odd results.
One such incident will be cited. During a test for ABS (alkyl
benzene sulfonate), samples were taken early on Monday morning
and rushed into the laboratory for testing. Due to the detention
time in the sewers, these wastewater samples actually represented
Sunday flow on the graveyard shift, the weakest wastewater obtain-
able. The ABS content was only 1 mg/1, whereas it would normally
run 8 to 10 mg/1. So the time and day of sampling is quite important,
and the samples should be taken to represent typical weekdays or
even varied from day to day within the week for a good cross-section
of the characteristics of the wastewater.
14,34 Compositing and Preservation of Samples
Since the wastewater quality changes from moment to moment and
hour to hour, the best results would be obtained by using some
sort of continuous sampler-analyzer. However, since operators
are usually the sampler-analyzer, continuous analysis would
leave little time for anything but sampling and testing. Except for
tests which cannot wait due to rapid chemical or biological changa -
of the sample, such as tests for dissolved oxygen and sulfides, a
fair compromise may be reached by taking samples throughout the
day at hourly or two-hour intervals.
When the samples are taken, they should be immediately refrigerated
to preserve them from continued bacterial decomposition. When all
of the samples have been collected for a 24-hour period, the samples
from a specific location should be combined or composited together
according to flow to form a single 24-hour composite sample.
14-23
-------
To prepare a composite sample, (1) the rate of wastewater flow
must be metered and (2) each- grab sample must then be taken
and measured out in direct proportion to the volume of flow
at that time. For example, Table I illustrates the hourly flow
and sample volume to be measured out for a 12-hour proportional
composite sample.
TABLE I
DATA COLLECTED TO PREPARE, PROPORTIONAL COMPOSITE SAMPLE
Time
6 AM
7 AM
8 Mi
9 AM
10 AM
11 AM
Flow
MGD
0.2
0.4
0.6
1.0
1.2
1.4
Factor
100
100
100
100
100
100
A sample composited
Sa£vple
20
40
60
100
120
140
in this
Vol Time
12 N
1 PM
2 PM
3 PM
4 PM
5 PM
manner would
Flow
MGD
1.5
1.2
1.0
1.0
1.0
0.9
total
Factor
100
100
100
100
100
100
1140 ml.
SampleVol
150
120
100
100
100
90
1140
Large wastewater solids should be excluded from a sample,, particu-
larly those greater than one-quarter inch in diameter,
Pur i n g compos i t in g
A very important point should be emphasized.
and at the exact moment of testing, the
remixed so that they w i 1 1 beof the s age cgnroosit . i on and _as wejLL_
T!dxeda£whenthCT_wereori^ra^ly sampled. Sometimes sue!- re^ixii
may become lax., so that all the solids are not uniformly suspended.
Lack of mixing can cause low results in samples of solids that
settle out rapidly, such as those in activated sludge or raw vjasto
water. Samples must therefore be mixed thoroughly and poured
quickly before any settling occurs. If this is not done,, errors
of 25 to 50% may easily occur. For example,, on the same mixed
liquor sample, one person may find 3,000 mg/1 suspended solids
while another person may determine that there are only 2,000 mg/1
due to poor mixing,, When such, a composite sample is tested, a
reasonably accurate measurement of the quality of the day's flow
can be made.
If a 24-hour sampling program is not possible, perhaps due to
insufficient personnel or the absence of a night shift, single
representative samples should be taken at a time when typical
characteristic qualities are present in the wastewater. The
samples should be taken in 'accordance with the detention time
14-24
-------
required for treatment. For example, this period may exist
between 10 AM and 5 PM for the sampling of raw influent. If
a sample is taken at 12 Noon, other samples should be taken
in accordance with the detention periods of the serial processes
of treatment in order to follow this slug of wastewater or plug
flow. In primary settling, if the detention time in the pri-
maries is two hours, the primary effluent should be sampled at
2 PM. If the detention time in the succeeding secondary treat-
ment process required three hours, this sample should be taken
at 5 PM.
14.35 Sludge Sampling
In sampling raw sludge and feeding a digester, a few important
points should be kept in mind as shown in the following illus-
trative table.
For raw sludge from a primary clarifier at Los Angeles' Terminal
Island Plant, the sludge solids varied considerably with pumping
time as shown by samples withdrawn every one-half minute,
TABLE II
DECREASE IN PERCENT TOTAL SOLIDS DURING PUMPING
Cumulative
Pumping Time Total Solids Solids
In Minutes Percent: Average
0.5 7.0 7.0
1.0 7.1 7.1
1.5 7.4 7.2
2.0 7.3 7.2
2.5 6.7 7.1
3.0 5.3 6.8
3.5 4.0 6.4
4.0 2.3 5.9
4.5 2.0 5.5
5.0 1.5 5.1
14-25
-------
Table II shows that the solids were heavy during the first
2.5 minutes, and thereafter rapidly became thinner and
watery. Since sludge solids should be fed to a digester
with solids as heavy as possible and a minimum of water.
the pumping should probably have been stopped at about
3 minutes. After 3 minutes, the water content did become
greater that desirable.
In sampling this sludge, the sample should be taken as a
composite by mixing small equal portions taken every 0.5
minutes during pumping. If only a single portion of sludge
is taken for the sample, there is a chance that the sludge
sample may be too thick or too thin, depending upon the
moment the sample is taken, A composite sample will pre-
vent this possibility.
It should also be emphasized again that as a sludge sample
stands, the solids and liquid separate due to gasification
and flotation or settling of the solids, and that it is
absolutely necessary to thoroughly remix the sample back
into its original form as a mixture before pouring it for
a test.
When individual samples are taken at regular intervals
in this manner, they shorld be carefully preserved to
prevent sample del ?::-•:• ,t~'.-r If bacterial action. Re-
frigeration is an ox-ellent method of preservation an-
is generally preft.-ii.ble to chemicals since chenucais aay
interfere with tests such as BOD and COD.
14.36
Automatic sampling devices are wonderful timesavers and s'loul3 be
en,ployed where possible. However,, like anything automatic,
problems of which the operator should be aware do arise in their
use. Sample lines to auto-samplers may build up growths which
may periodically slough off and contaminate the sample with a
high solids content. Very regular cleanout of the intake line
is required. Another problem occurred at Los Angeles' Hyperion
Plant when the reservoir for the automatic sampler was attacked
by sulfides. Metal sulfides flaked off and entered the sample
container producing misleading high solids results. The
reservoir was cleaned and coa.ted with coal-tar epoxy and little
further difficulty has been experienced.
14-26
-------
Manual sampling equipment includes dippers, weighted bottles,
hand-operated pumps, and cross-section samplers. Dippers con-
sist of wide-mouth corrosion resistant containers (such as
cans or jars) on long handles that collect a sample for testing.
A weighted bottle is a collection container which is lowered
to a desired depth. At this location a cord or wire removes
the bottle stopper so the bottle can be filled. Sampling pumps
allow the inlet to the suction hose to be lowered to the sampling
depth. Cross-sectional samplers are used to sample where the
wastewater and sludge may be in layers, such as in a digester or
clarifier. The sampler consists of a tube, open at both ends,
that is lowered at the sampling location. When the tube is at
the proper depth, the ends of the tube are closed and a sample
is obtained from different layers.
Many operators build their own sampler (Fig. 14.1) using the
material described below:
1. Sampling Bucket. A coffee can attached to an eight-foot
length of 1/2-inch electrical conduit or a wooden broom
handle with a 1/4-inch diameter spring in a four-inch loop.
2. Samp1ing Bottle, Plastic bottle with rubber stopper equipped
wi'tn two"3/8-1nch glass tubes, one ending near bottom of
bottle to allow sample to enter and the other ending at the
bottom of the stoppe~ to allow the air in the bottle to
escape while the sample is £.'11 ing the bottle.
For sample containers, wide-mouth plastic bottles are recommended.
Plastic bottles, though somewhat expensive initially, not cnly
greatly reduce the problem of breakage and metal contamination,
but are much safer to use. The wide- mouth bottles ease the
washing problem. For regular samples, sets of plastic bottles
bearing identification labels should be used.
14,37 Summary
1. Representative samples must be taken before any tests are
made.
2. Select a good sampling location.
3. Collect samples and preserve them by refrigeration.
4. If possible, prepare 24-hour composite samples. Mix samples
thoroughly before compositing and at the time of the test.
14-27
-------
1/2" Conduit
Length to Suit A.
1/4" Spring to Retain Sample Bottle
Coffee Can
Quart
Plastic
Bottle
/
A
-<-
f!f
Jl
Rubber Stopper V|_j j !/
Glass Tube Von
Glass Tube - Cut to
fit 1/2" clearance from
bottom of bottle
Fig. 14.1 Sampling bottle
14-28
-------
QUESTIONS
14.3A What are the largest sources of errors found in
laboratory results?
14.3B Why must a representative sample be collected?
14.3C How would you prepare a proportional composite
sample?
14-29
-------
14.4 LABORATORY WORK SHEET
All laboratory results should be recorded immediately alter a
sample has been measured. There is no standard laboratory form;
however, your plant or the agency that regulates your discharge
may have a preferred form. Figure 14.2 is a typical laboratory
work sheet (sometimes called a bench sheet) and will be referred
to throughout the chapter.
14-30
-------
PLANT
DATE
SUSPENDED SOLIDS $ DISSOLVED SOLIDS
SAMPLE
Crucib le
Ml Sample
Wt Dry § Dish
Wt Dish
Wt Dry
M Wt Dry,, gm x 1,000,000
mg/ ~ - Ml Sample
Wt. Dish § Dry
Wt Dish § Ash
Wt Volatile
o, „ i _ Wt Vol „
Wt Dry
BOD
# Blank
SAMPLE
DO Sample
Bottle #
% Sample
Blank or adj blank
DO after incubation
Depletion, 5 days
Dep %
I
i
i
i i
Nitrate N03
Sample
Graph Reading
Sett. Solids
Sample
Direct Ml/1
COD
Sample
Blank Titration
Sample Titration
Depletion
,, Dep x N FAS x 8000
mg/1 = . f.
Ml Sample
Fig. 14.2 Typical laboratory work sheet
14-31
-------
TOTAL SOLIDS
SAMPLE
Dish No.
Wt Dish
Wt Dish
Wt Wet
Wt Dish +
Wt Dish
Wt Dry
% Solids »
Wet
Dry
Wt
Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
% Volatile - Wt Vo1
Wt Dry
PH
Vol. Acid
Alkalinity as CaC03
x 100%
x 100%
Grease (Soxlet)
Sample
Ml Sample
Wt Flask + Grease
Wt Flask
Wt Grease
me/1 = Wtl-GrJe_ase» m.g_ x
Ml Sample
H2S (Gas) (Starch-Iodine)
Blank
Sample
Diff
Diff x .68
mg/1 x 43.6
* OOP
Ml
Ml
Ml
mg/1
grain/TOO cu ft
Fig. 14.2 Typical laboratory work sheet (continued)
14-32
-------
END OF LESSON 1 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
EXPLANATION OF DISCUSSION AND REVIEW QUESTIONS
Work this portion ,o£ the discussion and review questions after you
have completed answering the questions in Lesson 1. At the end of
each lesson in this chapter you will find some discussion and review
questions that you should complete before continuing.
The purpose of these questions is to indicate to you how well you
understand the material in this chapter.
14-33
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 1 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook before continuing.
1. What precautions should an operator take to protect himself
from diseases when working in a wastewater treatment plant?
2. Why should work with certain chemicals be conducted under a
ventilated laboratory hood?
3. What is meant by a representative sample?
4. How would you obtain a representative sample?
14-3:4
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 2 of 8 Lessons)
14.5 PLANT CONTROL TESTS
Tests in this section are listed in alphabetical order. Many
of the tests are conducted at primary, secondary, and advanced
wastewater treatment plants. Certain tests are commonly used
to control digester operation and activated sludge plants.
Typical plant and special plant control tests are summarized below,
A. Typical PlantL Control Tests
TEST NO. TITLE
2
4
5
6
7
8
9
10
12
16
17
Biochemical Oxygen Demand or BOD, Procedure with DC
Chemical Oxygen Demand or COD
Chlorine Residual
Clarity
Coliform Group Bacteria,
Dissolved Oxygen or DO
Hydrogen Sulfide
PH
Settleable Solids
Suspended Solit>s (Gooch Crucible)
Temperature (Wastewater)
Digester Control Tests
TEST NO.
1
3
14
15
17
20
21
TITLE
Alkalinity, Procedure with Volatile Acids
Carbon Dioxide (C02) in Digester Gas
Sludge Dewatering Characteristics
Supernatant Graduate Evaluation
Temperature (Digester Sludge)
Volatile Acids
Total and Volatile Solids (Sludge)
14-35
-------
C. Activated Sludge Control Tests
TEST NO. TITLE
8 Dissolved Oxygen (In Aerator)
11 Settleability
13 Sludge Age
11 Sludge Density Index (SDI)
11 Sludge Volume Index (SVI)
16 Suspended Solids (Centrifuge)
14-36
-------
(Total Alkalinity)
(BOD)
Total Alkalinity
The alkalinity test is located with the volatile acid test be-
cause the volatile acid/alkalinity relationship is critical in
the successful operation of sludge digesters.
2. Biochemical Oxygen Demand or BOD
The BOD test is placed with the dissolved oxygen (DO) test be-
cause to measure the rate of oxygen uptake in the BOD test, the
DO must be measured.
14-37
-------
3. Carbon Dioxide (C02) in Digester Gas
A. Discussion
Changes in the anaerobic sludge digestion process will be observed
in the gas quality and are usually noted after the volatile acids
or volatile acid/alkalinity relationship starts to increase. The
C02 content of a properly operating digester will range from 30%
to 40% by volume. If the percent is above 44%, the gas will not
burn. The easiest test procedure for determining this change is
with a C02 analyzer.
B. What is Tested?
Sample Preferred
C02 in Digester Gas 30% - 35% by Volume
METHOD A
C,, Apparatus
1. One Bunsen burner
2. Plastic tubing
3. 100 ml graduated cylinder
4. 250 ml beaker
\
D. Reagents
C02 Absorbent (KOH). Add 500 g potassium hydroxide (KOH) per liter
of water.
14-38
-------
Cco2)
E. Outline of Procedure
Clean out sampling line
by allowing gas from
sampling outlet to burn
until line is full of
gas from digester.
Gas
Outlet
Bunsen
Burner
2. Displace air in
graduated
cylinder.
3. Place graduate upside
down in beaker containinj
C02 absor-
bent.
Insert hose in graduate
and run gas for 60 seconds.
5. Remove hose from
graduate ar)d then
turn off gas.
Wait 10 minutes.
zbt
O
a
O
R .id volu:.'.c
gas rerr - •*?* .
nearest ml.,
to
PRECAUTIONS
1. Avoid any open flames near the digester.
2. Work in a well ventilated area to avoid the formation of ex-
plosive mixtures of methane gas.
3, If your gas sampling outlet is on top of your digester, turn
on outlet and vent the gas to the atmosphere for several
minutes to clear the line of old gas. Start with step 2S
displace air in graduated cylinder, NEVER ALLOW ANY SMOKI_NG_
OR FLAMES NEAR THE DIGESTER AT ANY TIME.
14-39
-------
(C02)
PROCEDURE
1. Measure total volume of a 100 ml graduate by filling it to
the top with water (approximately 125 ml) . Record this
volume.
2. Pour approximately 125 ml of C02 absorbent in a 250 ml beaker.
CAUTION: Do not get any of this chemical on your skin
or clothes. Wash immediately with running water until
slippery feeling is gone or severe burns can occur.
3. Collect a representative sample of gas from the gas dome on
the digester, a hot water heater using digester gas to heat
the sludge, or any other gas outlet. Before collecting the
sample for the test, attach one end of a gas hose to the gas
outlet and the other end to a Bunsen burner. Turn on the
gas, ignite the burner, and allow it to burn digester gas
for a sufficient length of time to insure collecting a
representative gas sample.
4. With gas running through hose from gas sampling outlet, place
hose inside inverted calibrated graduated cylinder and allow
digester gas to displace air in graduate. Turn off gas,
CAUTION: The proper mixture of digester gas and air
is explosive when exposed to a flame,
5. Place graduate full of digester gas upside down in beaker
containing C02 absorbent.
6. Insert gas hose inside upside down graduate.
7, Turn on gas, but donot blow out liquid. Run gas for at
least 60 seconds.
8. Carefully remove hose from graduate with gas still running.
9. I mme di a t e T
10. Wait for ten minutes and shake gently. If liquid continues
to rise, wait until it stops.
11. Read gas remaining in graduate to nearest ml. (Fig. 14,3)
14-40
-------
(C02)
Fig. 14.3 C02 measurement using in-
verted graduated cylinder
F. Example
Total Volume of Graduate = 126 ml
Gas Remaining in Graduate ~ 80 ml
G. Calculation
CO =
Gas
Total Volume, ml
= (126 ml - 80 ml)
_46_
126
37%
126 ml
x 100%
x 100%
x 100%
.365
126 /46.0
37 8
8 20
7 56
640
630
14-41
-------
(C02)
METHOD B
(ORSAT)
The Orsat gas analyzer can measure the concentrations of carbon
dioxide, oxygen, and methane by volume in digester gas. To
analyze digester gas by the Orsat method, follow equipment manu-
facturer's instructions. This procedure is not recommended for
the inexperienced operator.
QUESTIONS
3. A What are the dangers involved in running the
C02 in digester gas test?
3.B What is the percent COa in a digester gas if
the total volume of the graduated cylinder is
128 ml and the gas remaining in the cylinder
after the test is 73 ml?
14-42
-------
4. Chemical Oxygen Demand or COD
A. Discussion
COD is a good estimate of the first-stage oxygen demand for most
municipal wastewaters. An advantage of the COD test over the BOD
test is that you do not have to wait for five days for the results.
The COD test also is used to measure the strength of wastes that
are too toxic for the BOD test. COD is usually higher than the
BOD, but the amount will vaiy from waste to waste. The method
related here is a quick, effective measure of the strength of a
waste.
B. What is Tested?
Sample Common Range, mg/1
Influent 200 - 400
Effluent 40 - 80
Industrial Waste 200 - 4000
C. Apparatus
Two 50 ml graduated cylinders
10 ml pipette
50 ml burette
Boiling flask
Reflux condenser
Hot plate
14-43
-------
(COD).
D. Reagents
1. Standard potassium dichromate (I^C^Oy) 0.250 N, Dissolve
12.259 g dried K2Cr207 in distilled water and make up to
1 liter.
2. Surfuric acid-silver sulfate reagent. Add 22 g of silver
sulfate (Ag2SOzt) to a 9-lb bottle of concentrated sulfuric
acid (H2S<\). It takes one to two days to dissolve.
3. Standard ferrous ammonium sulfate solution, 0.25 N. Dissolve
98 g FeCNHit)2(SOit)2'6H20 in distilled water, add 20 ml
concentrated f-^SO^, cool and dilute to 1 liter. This solution
is unstable and must be standardized daily.
4. Ferroin Indicator. Dissolve 1.485 g of 1,10 phenanthroline
(Ci2H8N2«H20), together with 0.695 g ferrous sulfate crystals
(FeSOit«7H20) , in water and make up to 100 ml.
5. Silver sulfate, reagent powder,
6. Mercuric sulfate (HgSO^) analytical grade crystals.
14-44
-------
(COD)
E. Outline of F'rocedure
5. Add 30 ml
H2S(VAg2SOu
Solution
4. Add 10 ml
0.25 N K,Cr207
3. Add 2 ml
cone, H9
2. Add
20 ml
Sample
1.
,Cooling Water
Vent -
7.
Reflux Two
Hours, Cool
$ Wash Down
Add Ferroin 9,
Indicator
Titrate
to red
end point.
Reflux condenser, Friedrichs, VWR - 23157-001
Flask, boiling, flat bottom, VWR - 29113-068
PROCEDURE
1. Place 0.4 g mercuric sulfate into a 250 ml Erlenmeyer flask
with a ground glas? neck.
2. Measure 20.0 ml sample into the flask.
3. Add 2.0 ml concentrated sulf iric acid. Swirl until contents
are welL mixed.
4. Pipette 10.0 ml standard potassium dichromate solution into
the flask.
14-45
-------
(COD)
5. Carefully add 30 ml sulfuric acid-silver sulfate reagent
into the flask while swirling the flask. Use caution.
Make sure contents of the flask are thoroughly mixed before
heat is applied.
6. Add a few glass beads to reduce bumping and connect to con-
denser. The reflux mixture must be thoroughly mixed before
heat is applied. If this is not dene, local hot spots on
bottom of flask may cause mixture to be blown out of flask.
7. Prepare a blank5 by repeating above steps and by substituting
distilled water for the sample.
8. Reflux samples and blank for two hours. (If sample mixture
turns completely green, the sample was too strong. Dilute
sample with distilled water and repeat above steps substi-
tuting diluted sample.)
9. While the samples and blank are refluxing, standardize the
ferrous ammonium sulfate solution:
a. Pipette 10.0 ml standard potassium dichromate solution
into a 250 ml Erlenmeyer flask. Add about 100 ml of
water.
b. Add 30 ml concentrated HpSO^ with mixing. Let cool,
c. Add 2-3 drops ferroin indicator, titrate wiuh ferrous
ammonium sulfate (FAS) solution. Color change o^ solution
is from orange to greenish to red.
ml FAS
10 ml Ki,Cr207
Concentration Ratio, R =
mi
10. After refluxing mixture for two hours, -;ash down, condenser.
Let cool. Add distilled water to about 140 ml,
11. Titrate reflux mixtures with standard FAS.
Blank - ml FAS __
Sample - ml FAS ^
Blank. A bottle containing dilution water or distilled water,
but the sample being testec is not added. Tests are frequently
run on a sample and a b1ank and the differences compared.
14-46
-------
(COD)
F. Precautions
1. Wastewater sample should be well mixed. If large particles
are present, sample should be homogenized.
2. Flasks and condensers should be clean and free from grease
or other oxidizable materials, otherwise erratic results
would be obtained.
3. The standard ferrous ammonium sulfate solution is unstable
and should be standardized daily or each time the COD test
is performed.
4. Use extreme caution in handling concentrated H^SOt,.. Spillage
on skin or clothing should be immediately washed off and
neutralized.
5. The solution must be well mixed before it is heated. If the
acid is not completely mixed in the solution when it is heated^
the mixture could spatter and some of it will pass out the vent,
thus ruining the test.
6. Mercury sulfate is very toxic. Avoid skin contact and
breathing of this chemical.
G. Example
1. Standardization of ferrous amrucnium sulfate, FAS.
ml 0.25 N K2Cr207 = 10.0
ml FAS = 11.0
Concentration _ ml KoCr207
Ratio, R = —J-—
ICU^
11.0
2. Sample test.
Sample Taken = 20.0 ml
A = ml FAS used for blank = 10.0 ml
B = ml FAS used for sample = 3.0 ml
14-47
-------
(COD)
H. Calculation for COD
\ '
Method 1
COD, mg/1 = (A - B) x R x 100
= (10.0 - 3.0) (10/11) (100)
= 635 mg/1
Method 2 (According to Standard Methods)
COD, mg/1 . (A - B) x CjcJOOO
ml Sample
where
C = Normality of FAS
N = Normality of K2Cr207 Standard
ml KCr20?
x N
ml FAS
= 0.227
COD, mg/1 = M-i-_ I" °)i°-'-227J
= 635 mg/1
14-48
-------
QUESTIONS
4.A What does the COD test measure?
4.B What are some of the advantages of the COD test
over the BOD test?
14-49
-------
5. Chlorine Residual
A. Discussion
A chlorine residual should be maintained in a plant effluent
for disinfection purposes. The amount of residual remaining
in the treated wastewater after passing through a contact
basin or chamber may be related to the numbers of bacteria
allowed in the effluent by regulatory agencies.
Method A (lodometric) is used for samples containing waste-
water, such as plant effluents or receiving waters. Method B
(Deleted). Method C (Amperometric? Titration) gives the best
results, but the titrator is expensive.
B. What is Tested?
Common Range, mg/1
Sample (After 30 MinutesJ
Effluent 0.5 - 2.0 mg/1
C. Apparatus
METHOD A (lodometric)
1. One 250 ml graduated cylinder
2. One 10 ml measuring pipette
3. One 500 ml Erlenmeyer flask
4. Two 5 ml measuring pipettes
5. One 50 ml Buret
Amperometric (am-PURR-o-MET-rick). A method of measurement
that records electric current flowing or generated, rather
than recording voltage. Amperometric titration is an electro-
metric means of measuring concentrations of substances in water.
14-50
-------
(Chlorine Residual)
METHOD B (Orthotolidine-Arsenite or OTA)
One permanent glass color comparator
Three comparator cells
METHOD C (Amperometric Titratioi j
See Standard Methods
D. Reagents
METHOD A
1. Standard phenylarsine oxide solution, 0.00564 N. Dissolve
approximately 0.8 g phenylarsine oxide powder in 150 ml
0.3 N NaOH solution. After settling, remove upper 110 ml
of this solution into 800 ml distilled water and mix thoroughly,
Adjust pH up to between 6 and 7 with 6 N HC1 and dilute to
950 ml with distilled water. To standardize this solution
accurately measure 5 to 10 ml of freshly standardized
0.0282 N iodine solution into a flask and add 1 ml KI
solution. Titrate with phenylarsine oxide solution, using
starch solution as an indicator. Adjust to exactly 0.00564 N
and recheck against the standard iodine solution; 1.00 ml =
200 yg available chlorine. CAUTION: Toxic - avoid ingestion.
2. Potassium iodide, crystals.
3. Acetate buffer solution, pH 4.0. Dissolve 146 g anhydrous
NaC2H302, or 243 g NaC2H302 • 3H20, in 400 ml distilled water,
add 480 g concentrated acetic acid, and dilute to 1 liter
with distilled water.
4. Standard iodine titrant, O.Q282 N. Dissolve 25 g KI in a
little distilled water in a 1-liter volumetric flask, add
the proper amount of 0.1 N iodine solution exactly standard-
ized to yield a 0.0282 N solution, and dilute to 1 liter.
Store in amber bottles or in the dark, protecting the solution
from direct sunlight at all times and keeping it from all
contact with rubber.
5. Starch indicator. Make a thin paste of 6 g of potato starch
in a small quantity of distilled water. Pour this paste into
one liter of boiling, distilled water. Allow to boil for a
few minutes, then settle overnight. Remove the clear super-
natant and save; discard the rest. For preservation, add two
drops of toluene (C6H5CH3).
14-51
-------
(Chlorine Residual)
METHOD B
Deleted
METHOD C
See Standard Methods
14-52
-------
(Chlorine Residual)
E. Procedure
METHOD A
1\ Place 5.00 ml
phenylarsine
oxide solution
to Erlenmeyer
flask
2. Add excess
KI
(approx 1 g)
3. Add 4 ml
acetate buffer
solution
Add 200 ml
sample
5. Mix with
stirring
rod
6.
Add 1 ml
starch
solution
mi
Titrate until
blue color
first appears
and remains
after mixing
14-53
-------
(Chlorine Residual)
METHOD A
1. Place 5.00 ml 0.00564 N phenylarsine oxide solution in an
Erlenmeyer flask.
2. Add excess KI (approx. 1 g).
3. Add 4 ml acetate buffer solution, or enough to lower the
pH to between 3.5 and 4.2 .
4. Pour in 200 ml of sample.
5. Mix with a stirring rod.
6. Add 1 ml starch solution just before titration.
7. Titrate to the first appearance of blue color, which
remains after complete mixing.
METHOD B
1. Label the three comparator cells "A," "B," and "C." Use
0.5 ml of orthotolidine reagent in 10-ml cells, 0.75 ml in
15-ml cells, and the same ratio for other volumes of sample.
Use the same volume of arsenite solution as orthotolidine.
2. Add orthotolidine reagent to Cell A.
3. Add sample to mark on wall of Cell A. Mix quickly, and
immediately (within 5 seconds) add arsenite solution. Mix
quickly again and compare with color standards as rapidly
as possible.
Free available chlorine and
interfering colors, A = mg/1
4. Add arsenite solution to Cell B.
14-54
-------
(Chlorine Residual)
5. Add sample to mark on wall of Cell B. Mix quickly, ami
immediately add orthotolidine reagent. Mix quickly again
and compare with color standards as rapidly as possible.
Interfering colors present _ ,,
in immediate reading, B} " - - -
6. Compare with color standards again in exactly 5 minutes.
Interfering colors present _ ...
in 5 -minute reading, B2 -
7. Add orthotolidine reagent to Cell C.
8. Add sample to mark on wall of Cell C. Mix quickly and compare
with color standards in exactly 5 minutes.
Total amount of residual chlorine _ ,,
and interfering colors present, C ~ - °
F. Examples and Calculations
Method A
Titration of a 200 ml sample required 0.4 ml of 0.0282 N I.
„ 1 . „ . , , ,, (1 - ml I) 1000
Chlorine Residual, me/1 = £• • \ ..... • •.,• < ........ ,
' & Sample Volume, ml
(1 - 0.4) (1000}
200
= CO. 6) (5)
= 3.0 mg/1
NOTE: The larger the ml of I used in the titration, the smaller
the (1 - ml I) term and thus the lower the chlorine residual.
This is why this test is sometimes called the back titration
test for chlorine residual. If 1 ml of I is used in the
titration, you have titrated back to a zero chlorine residual,
14-55
-------
(Chlorine Residual)
Method B
Results from the OTA test on a plant effluent.
A = 0.5 mg/1
B! = 0.2 mg/1
B2 = 0.3 mg/1
C = 1.4 mg/1
Total Available Residual
Chlorine, mg/1
~ 2
= 1.4 mg/1 - 0.3 mg/1
= 1.1 mg/1
Free Available Residual
Chlorine, mg/1
~ l
= 0.5 mg/1 - 0.2 mg/1
= 0.3 mg/1
Combined Available
Residual Chlorine, mg/1
Total Available -
Residual Cl, mg/1
1.1 mg/1 - 0.3 mg/1
0.8 mg/1
Free Available
Residual Cl, mg/1
Total available residual chlorine consists of free available chlorine
(HOC1 and OC1~) and combined available chlorine (chloramines--compounds
formed by the reaction of chlorine with ammonia).
14-56
-------
(Chlorine Residual)
QUESTIONS
5.A Why should plant effluents be chlorinated?
5.B Discuss the important differences between the lodometric
titration, orthotolodine, and amperometric titration
methods of measuring chlorine residual.
END OF LESSON 2 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 3.
14-57
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 2 of 8 Lessons)
Chapter 14. Laboratqry Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The problem
numbering continues from Lesson 1.
5. How can you obtain a representative sample of digester gas?
6. Why is the COD test run?
7. Why should a chlorine residual be maintained in a plant effluent?
14-58
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 3 of 8 Lessons)
6. Clarity
A. Discussion
All high quality effluents should have a clarity reading taken
at high noon or some other specific time. This test is based
on how far you can see through your plant effluent under similar
conditions at the same time every day. The objective of the test
is to indicate the clearness or clarity of the plant effluent.
The test can be performed either in the lab by looking down
through the effluent in a graduated cylinder, or in the field
by looking down through the effluent in a clarifier or chlorine
contact basin. Sometimes this test is referred to as a tur-
bidity measurement, but you are interested in the clarity of
your effluent.
B. What is Tested?
Common Range
Sample QField^Test)_
Secondary Clarifiers: Poor Good
Trickling Filter 1 ft 3 ft
• Activated Sludge 3 ft 6 ft
Activated Sludge Blanket
in Secondary Clarifier 1 ft 4 ft
Chlorine Contact Basins 1 ft 5 ft
C. Apparatus
1. One clarity unit (Secchi (SECK-key) Disc) and attached
cord marked in one-foot units.
2. One 1000 ml graduated cylinder
3. Hach Turbidimeter, Model 2100 A
D. Reagents
None
14-59
-------
E. Procedures
1. Field Test. Tie end of marked nylon rope to handrail where
tests will be run, for example, in final sedimentation unit.
Always take tests at the same time each day for comparable
results. Lower disc slowly until you just lose sight of it.
Stop. Bring up slowly until just visible. Stop, Look at
the marks on the rope to see the depth of water that you can
see the disc through. Bring up disc and store. Record results,
-. Use a clean 100° ml graduate. Fill with a well-
mixed sample up to the 1000 ml mark. During every test the
same lighting conditions in the lab should be maintained.
Look down through the liquid in the cylinder and read the
last visible number etched on the side of the graduate and
record results.
Hach Turbidimeter. Follow manufacturer's instructions.
Whether you use one or each of these tests, you should run
either test at the same time every day and under similar
conditions for comparable results.
14-60
-------
(Clarity)
F. Example and Calculation
1. Each foot of depth is better clarity with Secchi disc.
2. Each 100 ml seen in depth is better clarity.
3. Turbidimeter reading indicates degree of clarity.
QUESTION
6.A What does the clarity test tell you about
the quality of effluent?
6,B What happens when you attempt to measure
clarity under different conditions, such
as lighting and clarifier loadings?
14-61
-------
7. Coliform Group Bacteria
A. Discussion
Coliform bacteria are measured to indicate the presence of
bacteria originating in the intestines of warm-blooded animals.
High coliform counts indicate the usefulness of water may have
been impaired by fecal contamination. Coliform bacteria are con-
sidered harmless, but their presence may be indicative of the
presence of disease-producing organisms that may be found with
them.
B. What is Tested?
Samole_ ' Usual Range, MPN/100 ml
Effluent:
Primary 5,000 to 1,000,000
Nonchlorinated Secondary >240,000
Chlorinated Secondary 50 to 500
Receiving Waters 1,000 to 1,000,000
C. Sampling Bottles
Polypropylene wide-mouthed bottles with 200 to 400 ml capacity are
used to collect samples. Before sterilization by autoclave, add
sodium thiosulfate (0.1 ml of a 10% solution per 4 ounce bottle) to
the bottles to neutralize any chlorine residual in the samples.
When filling bottles in the field, do not flush out sodium thio-
sulfate or contaminate sample or bottle. Fill bottles approximately
three-quarters full, maintain at 4° C with ice during transport and
start test in lab within eight hours after sampling,
D. Media Preparation
1. General Discussion
Careful media preparation is necessary to meaningful bacterio-
logical testing. Attention must be given to the quality, mixing,
and sterilization of the ingredients. The purpose of this care
is to assure that if the bacteria being tested for are indeed
present in a sample, every opportunity is presented for their
14-62
-------
(Coliform)
development and ultimate identification. Much bacteriological
identification is done by noting changes in the medium; conse-
quently, the composition of the medium must be standardized.
Much of the tedium of media preparation can be avoided by purchase
of dehydrated media (Difco, BBL, or equivalent). The operator
is advised to make use of these products; and, if only a limited
amount of testing is to be done, consider using tubed, prepared
media.
2. Glassware
All glassware must be thoroughly cleansed using a suitable detergent
and hot water (160°F), rinsed with hot water (180°F) to remove all
traces of residual detergent, and finally rinsed with distilled or
deionized water.
3. Water
Only distilled water or demineralized water which has been tested
and found free from traces of dissolved metals and bactericidal
and inhibitory compounds may be used for preparation of culture
media.
4. Buffered8 Dilution Water
Prepare a stock solution by dissolving 34 grams of KH^POt,. in
500 ml distilled water, adjusting the pH to 7.2 with IN NaOH.
Prepare dilution water by adding 1.25 ml of the stock solution
per liter of distilled water. This solution can be dispersed
into various size dilution blanks or used as a sterile rinse water
for the membrane filter test.
Buffer. A measure of the ability or capacity of a solution
or liquid to neutralize acids or bases. This is a measure
of the capacity of water or wastewater for offering a
resistance to changes in the pH.
14-63
-------
(Colifom?)
5. Co liform Test—Fermentation Tube Method
a. Lactose Broth or Lauryl Tryptose Broth
For the presumptive coliform test, dissolve the recommended
amount of the dehydrated medium in distilled water. Dispense
solution into fermentation tubes containing an inverted glass
vial. Autoclave the capped tubes at 121°C for 15 minutes.
b. Brilliant Green Bile Lactose Broth
For the confirmed coliform test, dissolve 40 grams of the
dehydrated medium in one liter of distilled water. Dispense
and sterilize as with Lactose Broth.
c. Compensation for Diluting Effect of Samples
Large volumes of samples can dilute the medium in the fermen-
tation tube. Use the concentrations listed below to compensate
for diluting effects when using lauryl tryptose broth.
No. ml Ml of sample Nominal No. grams
medium or dilution concentration dehydrated
in tube before medium per
inoculation liter
10 0.1 to 1.0 Ix 35.6
10 10 2x 71.2
20 10 1.5x 53.4
35 100 4x 137.3
6. Coliform Test-ElevatedTemperature for Fecal Coliforms
EC Broth
For the fecal coliform test, dissolve 37 grams of the dehydrated
medium in one liter of distilled water. Dispense and sterilize
as with Lactose Broth.
7. Co li form Tes t- -Memb rang Fi Iter Method
M-Endo Broth
Prepare this medium by dissolving 48 grams of the dehydrated
product in one liter of distilled water which contains 20 ml
f ethyl alcohol per liter. Heat solution to boiling only--
JO NOT AUTOCLAV^. Prepared media should be stored in a
refrigerator and used within 96 hours.
14-64
-------
(Coliform)
8. Autoclaving
Steam autoclaves are used for the sterilization of the liquid media
and associated apparatus. They sterilize (killing of all organisms)
at a relatively low temperature of 121°C within 15 minutes by
utilizing moist heat.
Components of the media, particularly sugars such as lactose, may
decompose at higher temperatures or longer heating times. For this
reason adherence to time and temperature schedules is vital.
Autoclaves operate in a manner similar to the familiar kitchen
pressure cooker:
1. Water is heated in a boiler to produce steam.
2. The steam is vented to drive out air.
3. The steam vent is closed when the air is gone.
4. Continued heat raises the pressure to 15 lbs/in2 (at this
pressure, pure steam has a temperature of 121°C) .
5. The pressure is maintained for the required time.
V.
6. The steam vent is opened and the steam is slowly vented
until atmospheric pressure is reached. (Fast venting will
cause the liquids to boil.)
7. Sterile material is removed to cool.
\
In autoclaving fermentation tubes, a vacuum is formed in the inner
tubes. As the tubes cool, the inner tubes are filled with sterile
medium. Capture of gas in this inner tube from the culture of
bacteria is the evidence of fermentation.
14-65
-------
CColiform)
E. Test for Coliform Bacteria
1. General Discussion
The test for coliform bacteria is used to measure the suitability
of a water for human use. The test is not only useful in determin-
ing the bacterial quality of a finished water, but it can be used
by the operator in the treatment plant to guide him in achieving
a desired degree of treatment,
2. Multitube Fermentation Technique
Coliform bacteria are detected in water by placing portions of
a sample of the water in lactose broth. Lactose broth is a
standard bacteriological medium containing lactose (milk) sugar
in tryptose broth. The coliform bacteria are those which will
grow in this medium at 35 °C temperature and ferment and produce
gas from the sugar within 48 hours. Thus to detect these bac-
teria the operator need only inspect fermentation tubes for gas.
In practice, multiple fermentation tubes are used in a decimal
dilution for each sample.
3. Materials Needed
1. Fifteen sterile tubes of lactose broth are needed
for each sample.
2. Use five tubes for each dilution.
3. Dilution tubes or blanks containing 9 ml or 99 ml
of sterile buffered distilled water.
4. Quantity of one and 10 ml sterile pipettes.
14-66
-------
(Coliform)
4. Technique for Inoculation and/or Dilution of Sample (Fig. 14.4)
All inoculations and dilutions of wastewater specimens must be
accurate and should be made so that no contaminants from the air,
equipment, clothes or fingers reach the specimen, either directly
or by way of the contaminated pipette.
1. Shake the specimen bottle vigorously 20 times before removing
sample volumes.
2. Into the first five lactose tubes pipette 1.0 ml of sample
directly into each tube. (It is important to realize that the
sample volume applied to the first 5 tubes will depend upon the
type of water being tested. The sample volume applied to each
tube can vary from 10 ml (or more) for high quality waters to
as low as 10 or 0.00001 ml (applied as 1ml of a diluted sample)
for raw wastewater specimens).
Note: When delivering the sample into the culture medium, deliver
sample portions of 1ml or less down into the culture tube near
the surface of the medium. Do_ not deliver small sample volumes
at the top of the tube and allow them to run down inside the tube;
too much of the sample will fail to reach the culture medium.
Note: Use 10 ml pipette for 10 ml sample portions, and 1 ml pipette
for portions of 1 ml or less. Handle sterile pipettes only near the
mouthpiece, and protect the delivery end from external contamination.
3. Pipette 1/10 ml or 0.1 ml of raw sample into each of the next
5 lactose broth tubes. This makes a 0.1 dilution.
4. To make the 0.01 dilution, place 1 ml of well mixed raw sample
into 99 ml of sterile buffered dilution water. Mix thoroughly
by shaking. This bottle will be labeled bottle A.
5. Into each of the next 5 lactose broth tubes place directly 1 ml
of the 0.01 dilution, from bottle A.
At this point you have 15 tubes inoculated and can place these
three sets of tubes in the incubator; however, your sample specimen
may show gas production in all 15 fermentation tubes.
This means your sample was not diluted enough and you have no
usable results. To obtain usable results it is recommended that the
first time a sample is analysed that 30 tubes having a range of
six dilutions be setup. In most cases this will give usable results.
6. To make a 1/1000 or 0.001 dilution add 0.1 ml from the 1/100
dilution bottle (Bottle A) directly into each tube of five more
lactose broth tubes.
14-67
-------
(Coliforw^
7. To make a 1/10000 or 0.0001 dilution take 1 ml from Bottle A
and place this 1 ml into 99 ml of sterile buffered dilution water.
Mix diluted sample thoroughly by shaking. This bottle will be
called Bottle B.
8. From the 0.0001 dilution (Bottle B) pipette 1.0 ml of sample
directly into each tube. Set 5 tubes up with this dilution.
9. To make a 1/100000 or 0.00001 dilution pipette 0.1 ml of
sample directly into each tube. Set 5 tubes up with this dilution.
The first time a sample is analysed 30 tubes of lactose broth
should be prepared. Once the appropriate dilutions are established
that give usable results for determining the MPM Index only 15 tubes
need by prepared for subsequent samples to be analysed.
14-67a
-------
COLIFORM BACTERIA TEST FIG. 14.4
1 ml TO EACH TUBE
n
O.t ml TO EACH TUBE
WATER SAMPLE
1 ml RAW SAMPLE
1 ml TO EACH TUBE
I I
f~\
0
0
u
0.1 ml TO EACH TUBE
BOTTLE A
99 ml STERILE
BUFFERED
DILUTION
WATER
p
t ml TO BCti
0.1 ml TO EACH TUBE
BOTTLE
99ml STERILE
BUFFERED
DILUTION
WATER
' & S
9 W
u
DILUTION
NO DILUTION
0.1
0.01
0.001
0.0001
1
IKCUBATE ALL TUBES AT 35° C ± 0.5°C FOR 24 HOURS
GAS IN INNER VIAL
IS A + TEST RESULT
14-68
-------
5. 24-Hour Lacotse Broth Presumptive Test
Place all inoculated lactose broth tubes in 35 C ^ 0.5°C incubator.
After 24+2 hours have elapsed, examine each tube for gas formation
in inverted vial (inner tube). Mark + on report form for all tubes
that show presence of gas. Mark - for all tubes showing no gas
formation. Save all positive tubes for confirmation test. The
negative tubes must be reincubated for an additional 24 hours.
6. 48-Hour Lactose Broth Presumptive Test
Record both positive and negative tubes at the end of 48 + 3 hours.
Save all positive tubes for confirmation test.
7. 24-Hour Brilliant Green Bile Confirmation. Test
Confirm all presumptive tubes that show gas at 24 or 48 hours.
Transfer, with the aid of a sterile 3 mm platinum wire loop,
one loop-full of the broth from the lactose tubes showing gas,
and inoculate a corresponding tube of BGB (Brilliant Green Bile)
broth by mixing the loop of broth in the BGB broth. "Discard"
all positive lactose broth tubes after transferring is completed.
Always sterilize inoculation loops and needles in flame immediately
before transfer of culture; do not lay loop down or touch it to any
nonsterile object before making the transfer. After sterilization
in a flame, allow suffic;e™.t time for cooling, in the air, to pre-
vent the heat of the loop from killing the bacterial cells being
transferred. Sterila wooden applicator sticks also are used to
transfer cultures, expecially in the field where a flame is not
available for sterilization.
After 24 hours has elapsed, inspect each of the BGB tubes for gas
formation. Those with sny amount of gas are considered positive
and are so recorded on the data sheet. Negative BTB tubes are
reincubated for an additional 24 hours.
8. _48^Hpur Brilliarit green Bile Confirmation Test
1. Examine tubes for gas at the end of the 48+3 hour period.
Record both positive and negative tubes.
2. Complete reports by decoding MPN index and recording MPN
on work sheets.
14-69
-------
9. Methods of calculations of the most probable number
Select the highest dilution with all positive tubes, before a
negative tube occurs, plus the next two dilutions, (See
Example No. 1).
14-70
-------
EXAMPLE NO. 1
m...*,... 6AS 6AS 6*S 6AS GAS
DILUTION ^ ^ ^ ^ __ RESULTS
o.
5 out of 5
0.1
GAS GAS GAS GAS GAS
5 out of 5
0.01
GAS GAS GAS GAS GAS
5 out of 5 •—i
0.001
GAS MO GAS NO GAS NO GAS GAS
2 out of 5
0.0001
NO GAS NO GAS SO GAS NO GAS MO SAS
0 ouf o! 5
0.00001
NO GAS NO GAS m GAS NO GAS NO GAS
0 out of 5
14-70a
-------
From the code 5-2-0 in the MPN Table (Table III) the MPN index
is 49. Using the following formula the # of coliform bacteria
/100 ml is determined.
MPN a constant
MPN/100 ml = Index from table x 10
Dilution giving all positive,
before a negative tube occurs
= 49 x 10 = 49000
0.01
14-70b
-------
Example number 2
Dilutions
No. of tubes positive
out of 5 tubes set up
at each dilution
0 0.1 0.01 0.001 0.0001 0.00001
0
0
Our code in example 2 is 5-0-0. Going to the MPN index (Table III)
we read the MPN Index of 23. Using the formula as in example number
1
23 x 10
MPN/100 ml = 0.001
MPN/100 ml = 230,000
MPN
or
= 230,000 per 100 ml
Example number 3
Dilutions
No. of tubes positive
out of 5 tubes set up
at each dilution
0 0.1 0.01 0.001 0.0001 0.00001
In example number 3 the code 5-1-0-1 is unreasonable because probability
would indicate that the 0.01 dilution should have 1 tube in 5 giving a
positive result. In this case it is assumed that the 0.01 dilution
should have given 1 tube in 5 as positive and the code 5-1-1 is used to
determine the MPN/100 ml.
Using the formula:
MPN/100 ml
46 x 10
0
MPN
460/100 ml
14-70C
-------
(Coliform)
TABLE III
MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
Five 1-ml
portions
0
0
0
1
1
1
2
2
3
0
0
0
0
1
1
1
2
2
2
3
3
4
0
0
0
0
1
1
1
2
2
2
Five 0. 1 ml
portions
0
1
2
0
1
2
0
1
0
0
' 1
2
3 '
0
1
2
0
1
2
0
1
0
1
1
2
3
0
1
2
0
1
2
MPN Index
(organisms
per 100 ml)
<2
2
4
2
4
6 |
4
6
6
2
4
6 ]
8
4
6
8
6
8
10
8
10
11
5
7
9 :
12
7
9
12
9
12
14
14-71
-------
(Coliform)
TABLE III (cont'd.)
MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
Five 1-ml
portions
3
3
4
0
0
0
1
1
1
1
2
2
Five 0.1 ml
portions
0
1
0
0
1
2
0
1
2
3
0
1
2 i 2
!
5 0
3
4
4
5
0
0
0
0
1
1
1
2
2
2
3
3
3
1
0
1
0
0
1
2
3
0
1
2
0
1
2
0
1
2
MPN Index
(organisms
per 100 ml)
12
14
15
8
11
13
11
14
17
20
14
17
20
17 i
21
21
24
25
13
17
21
25
17
21
26
22
26
32
27
33
39
14-72
-------
(Coliform)
TABLE III (cont'd.)
MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Five 1-ml
portions
4
4
5
5
0
0
0
0
0
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
Five 0.1 ml
portions
0
1
0
1
0
1
2
3
4
0
i
2
3
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
MPN Index
(organisms
per 100 ml)
34
40
41
48
23
31
43
58
76
33
46
63
84
49
70
94
120
148
177
79
109
141
175
I 212
253
130
172
221
278
345
426
240
348
542
920
1600
>2400
j
14-73
-------
(Coliform)
F. Test for Fecal Coliform Bacteria
1. General Discussion
Many regulatory agencies are measuring the bacteriological
quality of water using the fecal coliform test because this
test is a more reliable test for indicating the potential
presence of pathogenic organisms than is the coliform group
of organisms. The procedure described is an elevated temper-
ature test for fecal coliform bacteria.
2. Materials Needed
Equipment required for the tests are the same as those required
for the 24-Hour Lactose Broth Presumptive Test, a water bath,
and EC Broth.
3. Procedure
1. Run lactose broth or lauryl tryptose broth presumptive
test.
2. After 24 hours temporarily retain all gas-positive tubes,
3. Label a tube of EC broth to correspond with each
gas-positive tube of broth from presumptive test.
4, Transfer one loop-full of culture from each ga?-
positive culture in presumptive test to the
correspondingly labeled tube of EC broth.
5. Incubate EC broth tubes 24 t 2 hours at 44.5°C i
0.2°C in a waterbath with water depth sufficient
to come up at least as high as the top of the culture
medium in the tubes. Place in waterbath as soon as
possible after inoculation and always within 30 minutes
after inoculation.
6, After 24 hours remove the rack of EC cultures from
the waterbath, shake gently, and record gas pro-
duction for each tube. Gas in any quantity is a
positive test.
7. As soon as results are recorded, discard all tubes.
This is a 24-hour test for EC broth inoculations and
not a 48-hour test.
14-74
-------
(Coliform)
Transfer any additional 48-hour gas positive tubes
from the presumptive test to correspondingly labeled
tubes of EC broth. Incubate for 24 ± 2 hours at
44.5°C ± 0.2°C and record results on data sheet.
Codify results and determine MPN of fecal coliforms
per 100 ml of sample.
G. Membrane Filter Method
1. General Discussion
In addition to the fermentation tube test for coliform bacteria,
another test is used for these same bacteria in water analysis.
This test uses a cellulose ester filter, called a membrane filter,
the pore size of which can be manufactured to close tolerances.
Not only can the pore size be made to selectively trap bacteria
from water filtered through the membrane, but nutrients can be
diffused up through the membrane to grow these bacteria into
colonies. These colonies are recognizable as coliform because
the nutrients include fuchsin dye which peculiarly colors the
colony. Knowing the number of colonies and the volume of water
filtered, the operator can then compare the water tested with
water quality standards.
2. Materials Needed
1. One sterile membrane filter having a 0.45y pore size.
2. One sterile 47 mm Petri dish with lid.
3. One sterile funnel and support stand.
4. One sterile pad.
5. One receiving flask (side-arm, 1000 ml).
6. Vacuum pump, trap, suction or vacuum gage, connecting sections
of plastic tubing, Glass "T" hose clamp to adjust pressure by-
pass.
7. Tweezers, alcohol, Bunsen Burner, grease pencil.
14-75
-------
(Coliform)
8. Sterile buffered distilled water for rinsing made up in
100-500 ml quantities.
9. M-Endo Media.
10. Sterile pipettes—two 5 ml graduated, one 1 ml for aliquot
or one 10 ml for larger aliquot. Quantity of one ml pipettes
if dilution of sample is necessary. Also, quantity of dilution
water blanks if dilution of sample is necessary.
11. One moist incubator at 35° C temperature. Auxiliary incubator
dish with cover.
14-76
-------
(Coliform)
3. Illustration of Inoculation of Membrane Filter
Fig. I
1. Center membrane filter on
filter holder. Handle mem-
brane only on outer 3/16
inch with tweezers sterilized
before use in ethyl or methyl
alcohol and passed lightly
through a flame.
Fig. II
2. Place funnel
onto filter
holder.
Fig. Ill
3, Pour or pipette sample
aliquot into funnel.
s Avoid spattering. After
suction is applied rinse
two times with sterile
buffered distilled water.
Fig. IV
Fig. V
Remove membrane filter from
filter holder with sterile
tweezers. Place membrane
on pad. Cover with Petri
top.
Incubate in
inverted
position for
22+2 hours.
Count colonies
on membrane.
14-77
-------
(Coliform
4. Procedure for Inoculation of Membrane Filter
All filtrations and dilutions of water specimens must be accurate
and should be made so that no contaminants from the air, equipment,
clothes or fingers reach the specimen either directly or by way of
the contaminated pipette.
1. Secure tubing from pump and bypass to receiving flask. Place
palm of hand on flask opening and start pump. Adjust
suction to % atmosphere with hose clamp on pressure bypass.
Turn pump switch to OFF.
2. Set sterile filter-support-stand and funnel on receiving flask.
Loosen wrapper. Rotate funnel counter-clockwise to disengage
pin. Recover with wrapper.
3. Place Petri Dish on bench with lid up. Write identification
on lid with grease pencil.
4. Open sterile filter pad package. Light Bunsen burner.
5. Sterilize tweezers by dipping in alcohol and passing quickly
through Bensen burner.
6. Center membrane filter on filter stand with tweezers after
lifting funnel. Membrane filter with printed grid should
show grid uppermost (Fig. 1).
7. Replace funnel and lock against pin (Fig. 11).
80 Add a small amount of the sterile dilution water to funnel.
This will help check for leakage and also aid in dispersing
small volumes (Fig. Ill).
o
9, Shake sample or diluted sample. Measure proper aliquot
with sterile pipette and add to funnel.
10. Now start vacuum pump.
9
Aliquot (AL-li-kwot). Portion of sample.
14-78
-------
(Coliform)
12. After filtration of entire sample is finished, rinse two
times wit1-1 sterile buffered distilled water, pouring just
below inner lip of funnel. Allow each rinse to completely
pass through funnel before proceeding to next rinse.
13. When membrane filter appears barely moist, switch pump to
OFF.
14. Sterilize tweezers as before.
15. Remove membrane filter with tweezers after first removing
funnel as before (Fig. 1).
16. Center membrane filter on pad containing M-Endo medium
with a rolling motion to insure water seal. Inspect
membrane to insure no captured air bubbles are present.
(Fig. IV).
17. Place inverted Petri Dish in incubator for 22+2 hours.
5. Procedure for Counting Membrane Filter Colonies
1. Remove Petri Dish from incubator.
2. Remove lid from Petri Dish.
3. Place Petri Dish with filter under illuminating light. Tilt
membrane filter in base of Petri Dish so that green and
yellow-green colonies are most apparent. Direct sunlight
has too much red to facilitate counting.
4. Count individual colonies utilizing an overhead fluorescent
light. The coliform colony is characterized by a "metallic
sheen" and only those colonies showing ANY amount of this
sheen are considered to be coliforas,
5. Report total number of "coliform colonies" on work sheet.
Use the membranes that show from 20 to 80 colonies and do
not have more than 200 colonies of all types (including non-
sheen or, in other words, non-coliforms).
14-79
-------
Example:
A total of 42 colonies grew after filtering 10 ml of the undiluted
sample.
No. of colonies counted x 100 ml
Bacteria/100 ml=Volume of sample filtered
Example: =(42 colonies) (100ml) »(4.2 (100 ml) = 420 per 100 ml.
(10 ml) (100 ml) 100 ml
A total of 30 colonies grew after filtering 20 ml of a 1/100 or 0.01
diluted sample.
Bacteria/100 ml=No. of colonies counted x 100 ml
Volume of sample filtered x dilution factor
=30 x 100 ml
20 ml x 0.01
=15000 bacteria/100 ml
QUESTIONS
7.A Why should sodium thiosulfate crystals be added to
sample bottles for coliform tests before sterilization?
7.B Steam autoclaves effect sterilization (killing of all
organisms) at a relatively low temperature (p C)
within _^ minutes by utilizing moist heat.
7.C Calculate the Most Probable Number (MPN) of coliform
group bacteria from the following test results:
Dilutions 0 -1 -2 -3 -4 • -5
Readings 555120
7.D How is the number of coliforms estimated by the membrane
for filter method ?
END OF LESSON 3 of 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of the.discussion and review questions
before continuing with Lesson 4.
14-80
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 3 of 8 Lessons)
Chapter 14.' Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The
problem numbering continues from Lesson 2.
8. Why must the clarity test always be run under the
same conditions?
9. What is the purpose of the coliform group bacteria
test?
10. What does MPN mean?
14-81
-------
The following graphical method may be helpful and is added here
as an alternate:
PROCEDURE FOR USE OF GRAPH
Step 1: Locate the point on the horizontal axis which corresponds
to the coliform value obtained from the laboratory analysis.
Step 2: Draw a vertical line from the point located in Step 1 up
to the diagonal line on the graph.
Step 3: Draw a horizontal line from the point on the diagonal line
wh^ch was located in Step 2 to the vertical axis on the
left side of the graph.
Step 4: The point on the vertical axis which was located in Step 3
corresponds to the logarithm of the colifprm value.
Step 5: Repeat Steps 1 through 4 for each coliform value which was
obtained in the given time period.
Step 6: Sum all of the logarithm values obtained in Step 4.
Step 7: Divide the sum of the logarithms by the number of logarithms
summed in Step 6.
Step 8: Locate the point on the vertical axis which corresponds to
the value obtained in Step 7.
Step 9: Draw a horizontal line from the point located in Step 8 to
the diagonal line on the graph.
Step 10: Draw a vertical line from the point on, the diagonal line
which was located in Step 9, down to the horizontal axis
of the graph.
Step 11: The point on the horizontal axis which was located in Step
10 corresponds to the geometric mean coliform value.
14-81 a
-------
The following graphical method may be helpful and is added here
as an alternate:
EXAMPLE OF USE OF GRAPH
Given; The following coliform levels (in numbers per 100 ml) were
determined for eight effluent samples collected during a
1-month period: 375, 425, 78, 17, 1098, 8, 9327, and 172.
jPindj The monthly geometric mean value of th$ e,ight effluent
coliform samples.
A. The logarithm of each coliform determination i§ selected
from the graph using Steps 1 through 5 of the procedure.
Sample Coliform Determination Logarithm of Coliform Determination
1. 375 2.57
2. 425 2.63
3. 78 1.89
4. 17 1.23
5. 1,098 3.04
6. 8 0.90
7. 9,327 3.97
8. 172 2/24
Total = 18.47
B. The sum of the logarithm determinations is obtained (Step 6
of the procedure).
C. The arithmetic average of the logarithm is obtained (Step 7
of the procedure). Arithmetic average of logarithms =
Total - 18,47 = 2.31
Number of samples 8
D. The geometric mean value is selected from the graph using
Steps 8 through 11 of the procedure. Geometric mean value =
200/100 ml. ~~" '
14-81b
-------
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„„„„ _Jj. 1 Ul—U,i,_J. L srssiL^iAsi
fe^^afea^Ka&s^s^ai&aiJ^^^Ssfe^Ei^ ^^^fi^ssi^*™*^"^™^^™™™"" '•**'
S'O
O'l
3T
O
8 °
S l O 00
<->
O
0'5
jo
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 4 of 8 Lessons)
3. Dissolved Oxygen or DO and Biochemical Oxygen Demand or POD
I. IN WATER
A. Discussion
The dissolved oxygen (DO) test is, as the name inplies, the testing
procedure to determine the amount pf oxygen dissolved in samples of
water or wastewater. There are various types of tests that can be
run to obtain the amount of dissolved oxygen. This procedure is the
Sodium Azide Modification of the Winkler Method and is best suited for
relatively clean waters. Interfering substances include color,
organics, suspended solids, sulfides, chlorine, and ferrous and
ferric iron. Nitrites will not interefere with the test if fresh
azide is used.
The generalized principle is that iodine will be released in pro-
portion to the amount of dissolved oxygen present in the sample.
By using sodium thiosulfate with starch as the indicator, one can
titrate the sample and determine the amount of dissolved oxygen.
B. What is Tested?
Sample
Influent
Primary Clar. Effluent
Secondary Effluent
Oxidation Ponds
Activated Sludge--
Aeration Tank Outlet
Common Range, mg/1
Usually 0,>1 is very good.
Usually 0, Recirculated from
filters > 2 is good,
50% to 95% Saturation, 3 to
>8 is good.
1 to 25+*
>2 desirable
(> means greater than)
(* supersaturated with oxygen)
14-82
-------
(DO and BOD)
!
C. Apparatus
METHOD A (Sodium,Azide Modification of Winkler Method)
1. Buret, graduated to 0.1 ml. j
I
2. Three 300 ml glass-stoppered BOD bottles
3. Wide-mouth Erlenmeyer flask, 500 ml.
4. One 10 ml measuring pipette.
5. One 1-liter reagent bottle to collect activated sludge.
METHOD B (DO Probe)
Follow manufacturer's instructions. See Section H for Discussion,
Calibration, and Precautions.
D. Reagents
1. Manganous sulfate solution. Dissolve 480 g rcanganous sulfate
crystals (MnSO^. 4H20) in 400 to 600 nil distilled water. Filter
through filter paper., then add distilled water to the filtered
liquid to make a 1-liter volume.
2. Alkaline iodide-sodium azide solution. Dissolve 500 g sodium
hydroxide (NaOH) in 500 to 600 ml distilled water; dissolve
150 g potassium iodide (KI) in 200 to 300 ml distilled water
in a separate container. Exercise caution. Mix chemicals in
pyrex glass bottles using a magnetic stirrer. Add the chemicals
to the distilled water slowly and cautiously. Avoid breathing
the fumes and body contact with the solution. Heat is pro-
duced when the water is added, and the solution is very
caustic. Place an inverted beaker over the top of the mixing
container and allow the container to cool at room temperature.
• Mix both solutions when they ar,e cool.
Dissolve 10 g sodium azide (NaNs) in 40 ml of distilled water.
Exercise caution again. This solution is poisonous.
Add the sodium azide solution with constant stirring to the
cooled .solution of alkaline iodide; ther, add distilled water
to the mixture to make a 1-liter volume. Sod:um azide will
decompose in time and is no good after tnree months.
14-83
-------
(DO and BOD)
3. Sulfuric acid. Use concentrated reagent-grade acid .v
Handle carefully, since this material will burn hands and
clothes. Rinse affected parts with tap water to prevent
injury.
CAUTION: When working with alkaline azide and sulfuric acid,
keep a nearby water faucet running for frequent hand rinsing.
4. 0.0375 N sodium thiosulfate solution. Dissolve exactly
9.308 g sodium thiosulfate crystals (Na2S203'5H20) in
freshly boiled and cooled water and make up to 1 liter.
For preservation, add 0.4 g or 1 pellet of sodium hydroxide
(NaOH). Solutions of "thio" should be used within two weeks
to avoid loss of accuracy due to decomposition of solution.
5. Starch solution. Make a thin paste of 6 g of potato starch
in a small quantity of distilled water. Pour this paste
into one liter of boiling, distilled water, allow to boil
for a few minutes, then settle overnight. Remove the clear
supernatant and save; discard the rest. For preservation,
add two drops toluene (C6H5CH3).
6. Copper sulfate solution. Make a 10 percent solution by dis-
solving 10 grams of copper sulfate in 100 ml of water.
Sodium Azide Modification of the Winkler Method
NOTE: The sodium azide destroys nitrates which will
interfere with this test.
E. Outline of Procedure
4. Mix by
Inverting
White floe
NO no
1.
Take
300 ml
Sample
2.
Add
2 ml
MnSO^
below
surface
Add
2 ml
KI +
NaOH
below
surface
0 0
#00
0 o
Brown floe
DO present
Reddish-
Brown
Iodine
Solution
14-84
-------
(DOand BOD)
Titration of Iodine Solution:
I. Pour Bottle
Contents
into Flask.
Reddish-
Brown
Pale
Yellow /*•
Blue,
2. Titrate
3. Add 5tarch
Indicator
"ft
/ V Clear
End Point
14-85
-------
(DO and BOD)
PROCEDURE
The reagents are to be added in the quantities, order, and methods
as follows:
1. Collect a sample to be tested in 300 ml (BOU) bottle taking
special care to avoid aeration of the liquid being collected.
Fill bottle completely and add cap.
2. Remove cap and add 2 ml of manganous sulfate solution below
surface of the liquid.
3. Add /' ml of alkaline-iodide-sodium azide solution below the
surface of the liquid.
4. Replace the stopper, avoid trapping air bubbles, and shake
well by inverting the bottle several times. Repeat this
shaking after the floe has settled halfway. Allow the floe
to settle halfway a second time.
5. Acidify with 2 ml of concentrated sulfuric acid by allowing
the acid to run down the neck of the bottle above the surface
of the liquid.
6. Restopper and shake well until the precipitate has dissolved.
The solution will then be ready to titrate. Handle the
bottle carefully to avoid acid burns.
7. Pour contents of bottle into an Erlenmeyer flask.
8. If the solution is brown in color, titrate with 0.0375 N
sodium thiosulfate until the solution is pale yellow color.
Add a small quantity of starch indicator and proceed to
step 10.
9. If the solution has no brown color, or is only slightly
colored, add a small quantity of starch indicator. If
no blue color develops,1 there is zero Dissolved Oxygen.
If, -a blue color does develop, proceed to step 10.
10. Titrate to the first disappearance of the blue color. Record
the number of ml of sodium thiosulfate used.
11. The amount of oxygen dissolved in the original solution will
be equal to the number of ml of sodium thiosulfate used in
the titration provided significant interfering substances are
not present.
mg/1DO = ml sodium thiosulfate
14-86
-------
JC and 10D)
Example
The DO titration of a 300 ml sample requires 5.0 ml of 0.0375 N
Sodium Thiosulfate. Therefore, the dissolved oxygen concentra-
tion in the sample is 5 mg/1.
G. Calculation
You 'will want to find the percent saturation of DO in the
i-ffiuent of your secondary plant, ^lie DO 3.5 5.0 mg/1 and the
temperature is 20CC. At 209C, 100% DO saturation is 9.2 mg/1.
The dissolved oxygen saturation values are given in Table IV,
Note that as the temperature of water increases, the DO satura-
tion value (100% Saturation Column) decreases. Table IV gives
hVi";, DO saturation values for temperatures in °C and C'F.
iX.) Saturation, % = -SP °f S^Ple> "8/1. x, P0%
00 at :'">ra; Saturation, mg/1
H. DO Probe
Discussion
Measurement of the dissolved oxygen (DO) concentration with a
probe and electronic readout meter is a satisfactory substitute
for the Sodium Azide Modification of the Winkler Method, under
many circumstances. The probe is recommended when samples con-
t. in substances -,.-hj.ch interfere with the modified Winkler procedure,
such as sulfite, tniosulfate, polythionate, mercaptans, free
chlorine or hypochlof-te, organic substances -eadily hydrplyzed
in alkaline solutions, free iodine, intense color o^ turbidity,
»nd biological "Iocs. \ continuous record of the dissolved
14-87
-------
(DO t!nd BOD)
TABLE IV
LFFECT OP 1TMPHKATURE ON OXYGEN SATURATION
FOR A CHLORIDE CONCENTRATION OF ZERO Mg/":
°c
0
1
•>
.i.
3
4
5
6
7
8
-1
' 10
11
12
13
'14 '
15
16
17
18
' '
2U_
21
22
23
24'
i. - -
°F
32.0
33.8
35.6
37.4
39.2
41.0
42.8
44.6
46.4
48.2
50.0
51.8
53.6
55.4
57.2
60.0
61.8
63.6
65.4
67. J
68.0
69.8
71.6
73.4
75.2
77.0
mg/1 DO at
saturation
14.6
14.2
13.8
13.5
13.1
12.8
12.5
12.2
11.9
11.6
11.3
11.1
10.8
10.6
ID. 4
10.2
10.0
9. ~
9.b
9.4
9^2
9.0
8.8
8.7
8.5
8.4
14-88
-------
[Dp and 30f>i
oxygen content of aeration tanks and receiving waters may be
obtained -using a probe. In determining the BOD of sapples.,
a probe may be used to determine the DO initially and u/te,r
the five-day incubatior period of the blanks and samp.e «i. t;.:
2. Procedure
Follow manufacturer's instructions.
3. Calibration
To be assured that the DO prone reading provides the dissolved
oxygen content of the sample, the probe must be calibrated. lake
a sample that does not contain substances that interfe/e with
either the probe reading or the ipodified Winkler procedure.
Split the sample. Measure the DO in one portion of the sample
using the modified Winkler procedure and compare this result with
the DO probe reading on the other portion of the sample. Adjust
the probe reading to agree with the results from the modified
Winkler procedure.
When calibrating the probe in an aeration tank of the activated
sludge process, do not attempt to measure the dissolvec oxygen
in the aerator and then adjust the probe. The biological floes
in the aerator will interfere with the modified Winkler procedure,
and the copper sulfate-sulfamic acid procedure is not sufficiently
accurate to calibrate the probe. An aeration tank probe may be
calibrated by splitting an effluent sample, measuring the DO by
the modified Winkler procedure, and comparing results with the
probe readings. Always keep the membrane in the tip of the r>ro;>e
frpm drying because the probe can lose its accuracy until re-
conditioned.
4. Precautions
1. Periodically check the calibration of the probe.
2. Keep the membrane in the tip of the probe from drying out.
3. Dissolved inorganic salts, sucn as found in sea water, can
influence the readings from a probe.
4. Reactive compounds, such as reactive gases and sultur coru-
poxands, can interfere with the output of a probe.
.-;, Don't place the probe directly over a diffuser because you
want to measure the dissolved oxygen in the water being
treated, not the oxygen in the air supply to the aerator.
14-89
-------
(DO and BOD)
8. Dissolved Oxygen
II. IN AERATOR
Copper Sulfate-Sulfamic Acid Flocculation, page 413, 12th Edition,
1965, "Standard Methods".
A, Discussion
This modification is used for biological floes that have high
oxygen utilization rates in the activated sludge process, and
when a DO probe is not available. It is very important that
some oxygen be present in aeration tanks at all times to maintain
aerobic conditions.
This test is similar to the regular DO test except that copper
sulfate is added to kill oxygen-consuming organisms, and sulfamic
acid is added to combat nitrites before the regular Dp test is run.
NOTE;' If the results indicate a DO of less than 1 mg/j, it is
possible that''the DO in the aera'tion tank is ZERO1
When the DO in" the aeration tank' is near zero, consider-
able DO from the surrounding atmosphere can mix with the
sample when it is collected, when the inhibitor is addend,
while the solids are settling, and when the sample is
transferred to a BOD bottle for the DO test. If you use
this test, use a deep container and avoid stirring. See
article by Hughes and Reynolds JWPCF, Vol. 41, pg. 184,
January 1969, for a discussion of the shortcomings of
this test.
B. What is Tested?
Sample C ommon DO Range,
Aerator Mixed Liquor 0.1-3.0
C. Apparatus
1. One tall bottle, approximately WOO ml.
2. Regular DO apparatus.
14-90
-------
(DO
BOD)
!>. Reagt p.ts
1. Copper sulfate-sulfamic acid inhibitor solution. .^ssolve
32 g technical grade sulfamic acid O!H2S02OH) without heat
in 475 ml distilled water. Dissolve 50 g copper sulfate,
CuSOi4-5H20, in 5f'r; ml water. Mix the two solution5? together
and add 23 ml concentrated acetic acid.
2. Regular DO reagents.
Outline of Procedure
1. Add 10 ml of
inhibitor.
Jip into mixed 3. Settle
liquor sairpjc,
:.to7r;;er settle.
4. Siphon over 300 ml
of sample into
SOD : j-;t:e.
1. Add at least 10 ml of inhibitor (5 ml copper sulfate and
5 ml sulfamic acid) to any TALL bottle (1-cmart milk oottle)
fcith an approximate volume oTTdOO ml. °lace filling tube
near the bottom. An emptying tube is placed approximately
1/4 inch from the top of the bottle cork. Attach bottle to
rod or alum: nura conduit and lower into aeration tank.
2. Allow bottle to fill and then withdraw.
3. Let stand, un* •.J clear s.pernatant liquor can be siphoned into
a 300 ml " ? botx .e. Do not aerate in transfer,
4. Then run regular DO.
^4-91
-------
fOQ and HOP!
F. and G. Example and Calculations
Same as regular DO test.
QUESTIONS
R,A Calculate the percent dissolved oxygen saturation if
the receiving water DO is 7.Q rag/1 and the temperature
is 10 °C.
8.B How would you calibrate the DO probe in an aeration tan*.1'
8.C What are the limitations of the copper sulfate-suiramie acid
procedure for measuring DO in an aeration tank when the DO
in the tank is very low?
14-92
-------
(DO and BOD)
Biochemical Oxygen Demand or BOD
A. Discussion i
The BOD test gives the amount of oxygen used by microorganisms j
to utilize the substrate (food) in wastewater when placed in a j
Controlled temperature for five days. The DO (dissolved oxygen))
is measured at the beginning and recorded. After the 5-day '
jncubation period the DO is again determined. The BOD is then
calculated on the oasis of the reduction of DO and the size of
sample. This test is an estimate of the availability of food !
in the sample ffoo^ .or organisms that take up oxygen) expressed
ir. terms of oxv , . use. Results of a BOD test indicate the rate
of oxidation a.nu provide an indirect estimate of the Availability
to ov*eanistns o*1 concentration of the waste.
Samples are incubaxoa i«..>r a standard period of five days because
a vra.:tiou of the total BO'. «•.•>. M be exerted curing this period.
fhe :':t;ir,ate or total. BOD i... normally never run for plant control,
• Disadvantage of t;>e BU1 *• st is that 'che res Jits are not *.v'ail-
:-'•• ie until f: ve. days , at't^r tne sample was collected,
.<•. iVhat is Tested?
Sample Common Range, mg/1
Influent 15C >'^
Primary Effinen* 60 - 160
Secondary Efflueir 10 - 60
j.tester Supernatant 1000 - 4000-r
Industrial Wastes 100 - 3000-
C. Apparatus
300 ml BOD bottles with ground glass stoppers
:, Incubator, 20 °(
7. Pipettes, 10 ml gr; .Uct'ced, 1/32 to 1/16-inch diameter tip
*, Burette and stand
:, Erlenmeyer flask, ?nO ml
14-93
-------
(DO and BOD)
D. Reagents^
See Section U, page 14-°
-------
(DO and BOD)
1. Fill 2 BOD bottles
with BOD dilution
water.
OUTLINE OF PROCEDURE
4.
20°C
A
1 Incubate
I 5 days
I
i
Test for D.O.
3. Fill with
dilution water
I
2. Add
sample
5. Immediately test 2 § 4 for initial D.O.
6. Add
2 ml
below
surface
Test for D.O.
0.375 N
Na2S203
7. Add 2 ml
Alkaline KI
below
surface
8. Add 2 ml 9. Transfer 10. Titrate
H2SOtt Bottle Con-
tents to
14-95 Flask
-------
(DO and BOD)
E. Outline of Procedure
The test is made by measuring the oxygen used or depleted during
a 5-day period at 20°C by a measured quantity of wastewater sample
seeded into a reservoir of dilution water saturated with oxygen.
This is compared to an unseeded or blank reservoir of dilution
water by subtracting the difference and multiplying by a factor
for dilution. See outline on Page 14-107.
PROCEDURE
1. BOD bottles should be of 300 ml capacity with .ground glass
stoppers and numbers. To clean the bottles, carefully rinse
with tap water followed by distilled water.
2. Fill two bottles completely with dilution water and insert
the stopper tightly so that no air is trapped beneath the
stopper. Siphon dilution water from its container when
filling BOD bottles.
3. Set up one or more dilutions of the sample to cover the
estimated range of BOD values. From the estimated BOD,
calculate the volume of raw sample to be added to the BOD
bottle based on the fact that:
The most valid DO depletion is 4 mg/1. Therefore,
ml of sample added _ (4 mg/1) (300 ml)
per 300 ml ~ Estimated BOD, mg/1
1200
Estimated BOD, mg/1
Examples:
a. Estimated BOD = 400 mg/1
ml of sample added _ 1200
to BOD bottle ~ 400
= 3 ml
-------
(DO and BOD)
b. Estimated BOD = 200 rog/1: use 6 ml
100 mg/1: use 12 ml
20 mg/1: use 60 ml
When the BOD is unknown, select more than pne sample size.
For example, place several samples--! ml, 3 ml, 6 ml, and
12 mi—into four BOD bottles,
For samples with very high BOD values, it may be, difficult
to accurately measure small volumes or to get a truly repre-
sentative sample. In such a case, initial dilution should
first be made on the sample. A dilution of 1:10 is convenient.
4. To perform the BOD test, first fill two BOD bottles with
BOD dilution water. Nos. (1) and (2) in illustration,
Page 14-107
5. Next, for each sample to be tested, carefully measure out the
two portions of sample and place, them into two new BOD bottles,
Nos. (3) and (4). Add dilution water until the bottles are
completely filled. Insert the stoppers. Avoid entrapping air
bubbles. Be sure that there are water seals on the stoppers.
6, On bottles (2) and (4) immediately determine the initial
dissolved oxygen.
7. Incubate the remaining dilution water blank and diluted sample
at 20°C for five days. These are bottle? (1) and (3).
8. At the end of exactly five days (± 3 hours), test bottles
(1) and (3) for their dissolved oxygen by using the sodium
azide modification of the WinkTer" method or a DO probe.
At the end of five days, the oxygen content should be at
least 1 mg/1. Also, a depletion of 2 mg/1 or more is
desirable. Bottles (1) and (2) are only used to check the
dilution water quality. Their difference should be less
than 0.2 mg/1 if the quality is good and free of impurities.
14-97
-------
(DO and BOD)
F. Precautions
Since this is a bioassay (BUY-o-ass-SAY), that is, living organisms
are used for the test, environmental conditions must be quite exact.
1. The temperature of the incubator must be at 20°C. Other
temperatures will change the rate of oxygen used.
2. The dilution water should be made according to Standard
Methods for the most favorable growth rate of the bacteria.
This water must be free of copper which is often present
when copper stills are used by commercial dealers. Use all
glass or stainless steel stills.
3. The wastewater must also be free of toxic wastes, such as
hexavalent chromium.
4. If you use a cleaning solution to wash BOD bottles, be sure
to rinse the bottles several times. Cleaning agents are
toxic and if any residue remains in a BOD bottle, a BOD
test could be ruined.
5. Wastewater normally contains an ample supply of seed bacteria;
therefore seeding is usually not necessary.
G. Chlorinated Samples
It is very difficult to obtain reliable and reproducible results
from the BOD test, and a chlorinated sample is even more difficult.
For this reason, samples for BOD tests should be collected before
chlorination.
H. Example
BOD Bottle Volume = 300 ml
Sample Volume = 15 ml
Initial DO of 0 _ /n
Diluted Sample = 8'° mg/1
DO of Sample and Dilution A n ,,
After 5-day Incubation = 4'U mg/i
14-98
-------
(DO and BOD)
I. Calculations
HOD,
mg/1
Initial DO of
Diluted Sam-
pie, ng/1
DO of Diluted"')
Sample After
5-Day Incuba-
tion, mg/1
30D Bottle Vol., xil
Sample Volume, ml
f \
(8.0 mg/1 - 4.0 mg/1) H^ "*!
(4.0) (300)
15
SO mg/1
For acceptable results, the percent depletion of oxygen in the BOD
test should range from 30% to 80% depletion.
„ n , _.
% Deplete on
DO of Diluted Sample, mg/f|
-^_ DO After^ 5 Jays, mg/lj
DO of Diluted Sample, mg/1
(3.0 ing/]. - 4.0 mg/1) x 1Q{J%
8.0 mg/1
4 x 100%
= 50%
When a sample reo^ires a large volume in the BOD test and a small
amount of dilution water, or if a sample has a high DO (plant or
pond effluent), the initial JO of the mixture may be determined
as fellows.
Example:' BOD Bottle Volume
Sample Volume
Sample DO
DO of Dilution Water
DC of Sample and Dilution
After 5-Day Incubation
300 ml
60 ml
2.0 ii'g/1
8.0 mg/1
4.0 mg/1
14-99
-------
(DO and BOD
DO of Initial
Mi xture of
Dilution Water
and Sample, mg/1
m] of Sample x DO of Sample + ml of
Dilution H20 x DO of pilution HzO
BOD Bottle Volume
BOD, mg/1
60 ml x 2.0 nig/1 + 24Q ml x 8.0 mg/1
300 ml
120 + 1920
300
6.8 mg/1
6.8
300/2040.0
18CO
240.0
240.0
DO o£
Diluted
Sample,
mg/1
N
DO After
- 5 Days,
mg/1
J
^ Bottle Vol. ,^ ml
Sample \/ol., ml
= (6.8 mg/1 - 4.0 mg/1)
500 ml
'60 ml
NOTES
= (2.8),
200
60
= 14.0 mg/1
1, On effluent samples where the DO is run on the sample and the
blue bounces back on the end point titration, this indicates
nitrite interference and can cause the BOD to be higher than
actual by as much as 10% to 15% of the answer. This fact
should be considered in interpreting your results. The end
point also may waver because of decomposition of azide in an
old reagent or resuspension of sample solids. To correct a
wavering end point, try preparing a new alkaline-azide solution
or more of the old solution should be used because it may be
decomposing.
2. Researchers and equipment manufacturers are continually striving
to develop quicker and easier tests to measure BOD. If you find
a test procedure that provides you with an effective operational
control test, use it. Be sure to check with your regulatory
agencies for the procedures they require you to use in your
effluent monitoring program.
14-100
-------
QUESTIONS
8.D How would you determine the ampunt of organic
material in wastewater?
8.E How would you prepare dilutions to measure the
BOD of cannery waste having an expected BOD of
2000 mg/1?
8.F What is the BOD of a sample of wastewater if
a 2 ml sample in a 300 ml BOD bottle had an
initial DO of 7.5 mg/1 and a final DP of 3.9 mg/1?
8.G Why should samples for the BOD be collected
before chlorination?
8.H Why should opened bottles of "Thio" be used or
restandard!zed within two weeks?
END OF LESSON 4 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 5.
14-101
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 4 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The problem
numbering continues from Lesson 3.
11. What is the fbrmula'for calculating the percent
saturation of DO?
12. What precautions should be exercised when using
a DO probe?
13. What is a blank, as referred to in laboratory
procedures? .
14. What are some of the disadvantages of the BOD test?
15. What precautions should be taken when running a
BOD test?
16. Calculate the BOD of a 5 ml sample if the initial
DO of the diluted sample was 7.5 mg/1 and the DO
of diluted sample after 5-day incubation was 3.0 mg/1?
14-102
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 5 of 8 Lessons)
9,. Hydrogen Sulfide (H2S)
I. IN ATMOSPHERE
A. Discussion
The rate of concrete corrosion is often directly related to the rate
of H2S production or amount of H2S in the atmosphere. This test deals
with the time it takes a paper tape or unglazed tile to turn black.
It is a qualitative measurement of the H2S present in the sewer atmos-
phere. H2S is recognized by its characteristic odor of rotten eggs.
B. What is Tested?
Samle Common
Atmosphere in sewers, out- Not black in ,, , 0. ,
•, ^ ,- c . , -, , , = Good. 24+ hr
lets from force mains, wet 24 hours
pits, pumping stations, and
influent areas to treatment Black in less R , , ,
plants. than 1 hour '
C, Apparatus
Lead acetate paper or unglazed tile soaked in lead acetate.
D, Reagents
Saturated lead acetate solution.
E. Procedure
1. Obtain pieces of unglazed tile or use lead acetate paper. Cut
tile with hacksaw into % inch strips.
2, Soak strips in tile in lead acetate solution.
14-103
-------
LH2S)
3. Dry tile in drying oven or air dry.
4. An open manhole or any point where wastewater is exposed to the
atmosphere is a good test site. Drive a nail between metal crown
ring of manhole, concrete, or other convenient place. Tie paper
or tile with cotton string to nail and then replace it and return
in half an hour or less. If tile is not black or substantially
colored, return periodically until black, If H2S is present as
indicated by a color change, then measure flow, temperature, pH,
and BOD for further evaluation of problem.
II. IN WASTEWATER
A, Piscussion
In sewers, when there is no longer any dissolved oxygen, H2S tests
are run to determine the rate of H2S increase as the wastewater
travels to a pumping station or treatment plant. If the wastewater
is exposed to the atmosphere, H2S will be released and a typical
rotten egg odor will be detected. Anaerobic bacteria found in
wastewater can liberate H2S from the solids. When the gas leaves
the wastewater stream and comes in contact with moisture and
oxygen, sulfuric acid is formed which is very corrosive to concrete.
Not all odors in wastewater are from H2S, and there is no correlation
between H2S and other odors. The total H2S procedure is good up to
18 mg/1, and higher concentrations must be diluted before testing.
H2S production can be controlled by up-sewer aeration which reduces
US formation and also stabilizes the wastewater in the collection
svstem.
14-104
-------
(H2S)
B. What is Tested?
Sample Wastewater From Possible Results, mg/1
the Following Locations GocKl' ~ " Bad
Sewers ,1 1
Outlets from force mains .1 1
Wet pits, pumping stations .1 .5
Influents to treatment plants Preferably 0 .5
All of the above locations should be sampled, if pertinent, when
using up-stream aeration to control H2S.
C. Apparatus
1. One LaMotte-Pomeroy Sulfide Testing Kit to test:
a. Total Sulfides
b. Dissolved Sulfides
c. Hydrogen Sulfide in solution
Obtain from LaMotte Chemical Products Company. Order by Code
#4630, $27.50, FOB, Chestertown, Maryland 21620.
2. One LaMotte-Pomeroy Accessory Hydrogen Sulfide Kit for testing
H2S in air and gases (not essential). Obtain from LaMotte
Chemical Products Company. Order by Code #4632, $22.00, FOB,
Chestertown, Maryland 21620.
D. Reagents
The instructions are in the kit.
E, Procedure
The instructions are in the kit.
Note: No EPA evaluation of test kit was available at time this
manual was prepared.
14-105
-------
(H2S)
F. Example
The instructions are in the kit.
G. Calculations
The instructions are in the kit.
QUESTION
9.A Why would you measure the H2S concentration:
1. In wastewater?
2. In the atmosphere?
14-106
-------
10. p_H
A. Discussion
The intensity of the alkaline or acid strength of water is
expressed by its pH.
Mathematically, pH is the logarithm of the reciprocal of the
hydrogen ion concentration, or the negative logarithm of the
hydrogen ion concentration.
1 .
pH = log TTTTV = -log (FT)
For Example
If a wastewater has a pH of 1, then the hydrogen ion concentration
(H+) = 10'1 = 0.1.
If pH = 7, then (H+) = 1Q-7 = 0.0000001.
pH Scale
0 increasing acidity -- 7 -- increasing alkalinity 1
1 +- 2 «-
• 3 +- 4 •*- 5 •*- 6 x\ 8 •> 9 -
Neutral
6 through 8
*• 10 ->• 11 -> 12 -> 13
In a solution, both hydrogen ions (H+) and the hydroxyl ions (OH")
are always present. At a pH of 7, the concentration of both byiU-oqon
and hydroxyl ions equals 10~7 moles per liter. When the pH is loss
than 7, the concentration of hydrogen ions is greater tli.T; the hyiroxyl
ions. The hydroxyl ion concentration is greater than the hydrogen ions
in solutions with a pH greater than 7.
The pH test indicates whether a treatment process may continue to
function properly at the pH measured. Each process in the plant has
its own favorable range of pH which must be checked routinely,
Generally a pH value from 6 to 8 is acceptable for best organism
activity.
14-107
-------
fpH)
The paper tape colorimetric comparison method is explained in
this section. This is not considered a "Standard Method" but j
will give a rough indication of the pH. Most wastewater contains
many dissolved solids and buffers which tend to minimize pH changes,
There are many ranges of pH tapes available. Normally a range of
5 to 8 will cover the inplant control testing.
B. What is Tested?
Wasewater Common
Influent or Raw Wastewater (domestic) 6.8 to 8.0
Raw Sludge (domestic) 5.6 to 7.0
Digester Recirculated Sludge or
Supernatant 6.8 to 7.2
Plant Effluent Depending on
Type of Treatment 6.0 to 8.0
C. Minimum Apparatus List
1. pH Meter.
or 2. Three rolls of paper tapes (range 5 to 8) .
or 3. Colorimetric set (range 6.8 to 8.4) — permanent glass
can be used with chlorine comparator or liquid color tubes
that are less stable.
D. Reagents
(to be used with corresponding apparatus listed under Section C)
1. Buffer tablets of various pH values. Distilled water.
2 . None .
3, Brom thyml blue (for pH 6.2 to 7.6).
Phenol red (for 6,4 to 8.0).
14-108
-------
(pi I)
E, Procedures
Use the same samples used for the other tests.
METHOD A (pH Meter)
Procedure '
1. Due to the differences between the various makes and models
of pH meters commercially available, specific instructions
cannot be provided for the correct operation of all instru-
ments. In each case, follow the manufacturer's instructions
for preparing the electrodes and operating the instrument.
2. Standardize the instrument against a buffer solution with a
pH approaching that of the sample.
3. Rinse electrodes thoroughly with distilled water after re-
moval from buffer solution.
4. Place electrodes in sample and measure pH.
5. Remove electrodes from sample, rinse thoroughly with dis-
tilled water.
6. Immerse electrode ends in beaker of pH 7 buffer solution.
7. Shut off meter.
Precautions
1, To avoid faulty instrument calibration, prepare fresh buffer
solutions as needed, once per week, from commercially avail-
able buffer tablets.
2. pH meter, buffer solution, and samples should all be at the
same temperature (constant) because temperature variations
will give erroneous results,
3. Watch for erratic results arising from electrodes, faulty
connections, or fouling of electrodes with oily or precipitated
matter."
14-109
-------
(pH)
METHOD B (Paper Tape)
Procedure
1. Measure pH directly in tank or immediately after collecting
sample.
2. Tear off tape lh to 2" long. Dip half of tape in tank or
sample and quickly read results. '
3. Remove tape and compare color with colors on package, and
record pH on Laboratory Work Sheet in proper column from
which" the sample came. For example, if the sample came
from the plant influent and the color of the portion of the
tape wetted by the sample matches a color on the package
indicating a pH of 7.2, then record 7.2 on Laboratory Work
Sheet in the influent column on the pH row. (See Fig. 14.2
second page of work sheet).
This procedure applies to liquids that have solids which separate
(settle or float) easily.
METHOD B (Paper Tape-High Solids Cone, in Sample)
Procedure
The following procedure is for samples containing higher solid
concentrations such as found in the raw sludge, digester recircu-
lated sludge, digester supernatant, and digested sludge samples,
1. Obtain representative samples and identify them.
2. Allow samples to stand until some of the solids have settled
and water is visible above the solids. Sufficient water
should be above the solids to allow the tape to be dipped in
the sample and not discolored by the solids.
3. Bend the tape by making a sharp crease ^ from end. Very carp-
fully allow tape to touch liquid surface.
End of 4. Remove tape from liquid surface
bent I and compare the color with pH
~»~ I—
tape
color standard on the package.
Record on Laboratory Work Sheet,
14-110
-------
(pH)
METHOD C (Colorimetric Comparitor)
Procedure • :
i
1. Fill the three tubes or two rectangular bottles provided with
-the comparitor unit to the indicator line with a portion of
the sample being tested.
2. Add the recommended amount of indicator solution.
3. Place the tubes in the comparitor in such a way that the color
standards are opposite the tubes not containing the indicator
s olut i on.
4. Compare the colors by rotating the comparitor disk or changing
the standard color solution vials. Read the pH of the indi-
cator having the color closest to the color of the sample.
Record results on Laboratory Work Sheet.
5. Thoroughly wash and dry sample tubes when test is completed
and before returning tubes to comparitor unit for storage.
F. Precautions
a
1. Collect fresh samples and test immediately. The pH of „
sample can change rapidly due to loss of C0~ .and biological
activity. A fresh effluent sample could have a pH of 6.5
and after standing overnight the pH could be 8,0.
2. Always measure aerator pH directly in the aerator.
3. The pH of a composite sample will not accurately describe pii
conditions in your plant. A ten-minute slug of a highly acid
waste can upset plant performance for a day or longer, but
you may not notice it in a composite sample. Measure pH in
place, frequently and quickly, for best description of
environment encountered' by'"brgahisms' in' treatment' processes .
14-111
-------
QUESTIONS
10.A How would you measure the pH by the paper tape
colorimetric comparison method for:
1. Plant influent?
2. Raw sludge?
10.B What precautions should be exercised when using
a pH meter?
14-112
-------
11. Settleability of Activated Sludge Solids
I. SETTLEABILITY
A, Discussion
This test is run on mixed liquor or return sludge and plotted on
attached graph (Fig. 14.5). All pertinent information is filled
in for process control of aerators.
-p
o
_Q
a
Q)
0
0
60
10 15 20
Time, minutes
14.5 Settleability of activated sludge solids
Settleability is important in determining the ability of the solids
to separate from the liquid in the final clarifier. The activated
sludge solids should be returned to the aeration tank, and the
quality of the effluent is dependent upon the absence of solids
flowing over the effluent weir.
The suspended solids should be run on the same sample of mixed
liquor that the Settleability test is run. This will allow.you Lo
calculate the Sludge Volume Index (SVI) or the Sludge Density Index
(SDI) which are explained in other sections.
14-113
-------
(Settleability)
The 2000 ml graduate that is filled with mixed liquor in the
settleability test is supposed to indicate what will happen to
the mixed liquor in the final clarifier--the rate of sludge
settling, turbidity, color, and volume of sludge at the end of
60 minutes.
B. What is Tested?
Sample
Mixed Liquor or
Return Sludge
Working Range
Depends on desirable mixed
liquor concentration
C. Apparatus
2000 ml graduated cylinder.10
D. Reagents
None.
E. Procedure
--Mix sample and pour
into 2000 ml graduate.
Sample
2. Record settleable solids, %, at 5-minute intervals,
950-
-I l
730-
-1 L.
550 -
Time,
min. 0
J C
510-
•'. •'
$%fc2
'W%#.
u'S ; 'A?': ;M
^'s&N;
/
4
470-
10
15
20
30
60
10 Mailory Direct Reading Settleometer (a 2 liter graduated cylinder
approximately 5 inches in diameter and 7 inches high). Obtain from
Scientific Glass Apparatus Co., Inc., 735 Broad Street, Bloomfield,
New Jersey. Catalog No. JS-1035. Price $16.50 each.
14-114
-------
(Settieabllj-ty)
1. Collect a sample of mixed liquor or return sludge,
2. Carefully mix sample and pour into 2000 ml graduate. Vigorous
shaking or mixing tends to break up floe and produces slower
settling or poorer separation. '
3, Record settleable solids, %, at regular intervals. ;
F. Example and Calculation J
The percent settling rate can be compared for the various days of
the week and with other measurements—suspended solids, SVI, per-
cent sludge solids returned, aeration rate, and plant inflow. A
very slow settling mixed liquor usually requires air and solids
adjustment to encourage increased stabilization during aeration.
A very rapidly settling mixed liquor usually gives poor effluent
clarification.
II. SLUDGE VOLUME INDEX (SVI)
A. Discussion
The Sludge Volume Index (SVI) is used to indicate the condition
of sludge (aeration solids or suspended solids) for settle-
ability in a secondary or final clarifier. The SVI is the volume
in ml occupied by one gram of mixed liquor suspended solids""a*fTer"
30 minutes of settling. It is a useful test to indicate changes
in sludge characteristics. The proper SVI range for your plant
is determined at the time your final effluent is in the best con-
dition regarding solids and BOD removals and clarity.
B. What_is Tested?
Sample Preferable Range, SVI
Aerator Solids or .. ~
Suspended Solids
14-115
-------
C. Apparatus
See 11. Settleability of Activated Sludge Solids, Part I,
Settleability, and 16. Suspended Solids,
D. Reagents
None. , I
E. Procedure
See Section 11, I, on Settleability, and 16, Suspended Solids^
F. Example
30-minute settleable solids test = 360 ml or 18%.
Mixed liquor suspended solids = 1500 mg/1. - :
G. Calculations
Sludge Volume _ % Settleable Solids x 10^.^
Index, SVI Mixed Liquor Suspended' Sol id's / ing/~i
18 x 10,000
"
- 18Q° •
" 15 1
= 120
14-116
-------
(Settleability - SDI)
III. SLUDGE DENSITY INDEX (SDI)
A. Discussion
The Sludge Density Index (SDI) is used in a way similar to the SVI
to indicate the settleability of a sludge in a secondary clarifie? or
effluent. The calculation of the SDI requires the same information
as the SVI test.
SDI = mg/l °£ suspended solids in mixed liquor
ml/1 of settled" mixed liqupr solids x ID
or
SDI = 100/SVI
B. What is Tested?
Sample Preferable Range, SDI
Aerator Solids or
Suspended Solids 0.4-1.0
C. through G.
These items are not included because of their similarity to the
SVI test.
QUESTIONS
11.A Why should you run settleability tests on mixed liquor?
11.B What is the Sludge Volume Index (SVI)?
11.C Why is the SVI test run?
11.D What is the relationship between the Sludge Density
Index (SDI) and SVI?
END OF LESSON 5 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 6.
14-117
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 5 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The problem
numbering continues from Lesson 4.
17. Hydrogen sulfide is measured because it causes
18. What factors promote H2S production in sewers?
19. The pH scale runs from to , with 7 being neutral.
20. Calculate the SVI if the mixed liquor suspended solids are
2000 mg/1 and the 30-minute settleable solids test is 500 ml
or 25%.
21. Calculate the SDI if the SVI is 125.
14-118
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 6 of 8 Lessons)
12. Settleable Solids
A. Discussion
The settleable solids test is the volume of settleable solids in
one liter of sample that will settle to the bottom of an Imhoff
cone during a specific time period. The test is an indication of
the volume of solids removed by sedimentation in sedimentation
tanks, clarifiers, or ponds. The results are read directly in
milliliters from the Imhoff cone.
B. What is Tested?
Sample Common Ranges Found
12 ml/1 medium wastewater
Influent 20 ml/1 strong wastewater
8 ml/1 weak wastewater
Primary Effluent 0,1 ml/1 - 3 ml/1
Secondary Effluent Trace —0.5 ml/1
Over .5 ml/1 poor
C. Apparatus
1. Imhoff Cones.
2, Rack for holding Imhoff Cones.
3. Glass stirring rod, or wire.
14-119
-------
D. Outline of Procedure
(Settleable Solids)
Mix well and
pour 1 liter
into Irnhoff
Cone.
Settle
45 Minutes
Gently Stir
Sides
1 Liter
Read-
Sludge
Volume
Settle
15 Minuses
1.
2.
3.
4.
5.
PROCEDURE
Thoroughly mix the wastewater sample by shaking and immediately
fill an Imhoff cone to the liter mark.
Record the time of day that the cone was filled. T =
Allow the waste sample to settle for 45 minutes.
Gently spin the cone to facilitate settling of material adhering
to the side of the cone.
After one hour, record the number of milliliters of settleable
solids in the Imhoff cone. Make allowance for voids among the
settled material.
14-120
-------
(Settleable Solids)
6. Record the settleable solids as ml/1 or milliliters per
liter.
Settleable Solids, Influent = ______ ml/1
Settleable Solids, Effluent = ml/1
Settleable Solids, Removal = ml/1
E. ExajnpJLe
Samples were collected from the influent and effluent of a primary
clarifier, After one hour, the following results were recorded:
Sette able
Influent ' 12.0
Effluent 0,2
F. Calculations
1. Calculate the efficiency or percent removal of the above primary
clarifier in removing settleable solids.
% Removal _ (Inf 1. Set Sol , ml/1 - Ef f 1. Set^ Sol, ml/1) x ]OQ%
of Set Sol ~ Influent Set Sol, ml/1
12 ml/1 - 0.2 ml/1 Inn0 -12,0
= - » • • « ..—— -—- «-. .»,..»^..«. '
12 ml /I
____
= liil x 100% 12 / 11,8
12 10^3
1 00
* 98% -_£
40
2. Estimate the gallons per day of sludge pumped to a digester
from the above primary clarifier if the flow is 1 MGD (1 million
gallons per day) . In your plant, the Imhoff cone may not
measure or indicate the exact performance of your clarifier
14-121
-------
(Settleable Solids)
or sedimentation tank, but with some experience you should
be able to relate or compare your lab tests with actual
performance.
Sludge Removed by Clarifier, ml/1
= Influent Set Sol, ml/1 - Effluent Set Sol, ml/1
= 12 ml/1 - 0.2 ml/1
= 11.8 ml/1
To estimate the gpd (gallons per day) of sludge pumped to a
digester, use the following formula:
Sludge to Digester, gpd
= Total Set Sol Removed, ml/1 x 1000 x Flow, MGD
- n ° mA Y 1000 mg „ 1 M gal
— i, 1 . O . . A L -., A ,
M mg ml day
= 11,800 gpd
This value may be reduced by 30 to 75% due to compaction of
the sludge in the clarifier.
If you figure sludge removed as a percentage (1.18%), the sludge pumped
to the digester would be calculated as follows:
Sludge to Digester, gpd
Flow of 1,0(30 ,'OoTTgpct
„, , „. , 1.18% x 1,000,000 gpd
Sludge to Digester, gpd = - - * w.
= 11,800 gpd
G. Clinical Centrifuge
Settleable solids also may be measured by a small clinical centri-
fuge. A mixed sample is placed in 15 ml graduate API tubes and
spun for 15 minutes. The solid deposition in the tip of the tube
is related to plant performance for plant control. A centrifuge
also is used in Section 16, Suspended Solids, II, Centrifuge.
QUESTION
«t
12. A Estimate the volume of solids pumped to a digester
in gallons per day (gpd) if the flow is 1 MGD, the
influent settleable solids is 10 ml/1, and the eff-
luent settleable solids is 0.4 ml/1 for a primary
clarifier.
14-122
-------
13, Sliulpe Are
A. Discussion
Sludge age Is a control guide that is widely used and is a rough
indicator of the length of time a pound of solids is rraintai ncd
nudcr aeration in the system. The basis for calculating the sludge
age is weight of suspended solids in the mixed liquor in the aeration
tank divided by weight of suspended solids added per day to the
aerator.
Suspended Solids in Mixed Liquor, mg/1
Sludge Age, = x._ Aerat or Vojume^ in_ MG^ x_ _8Jj4_ Ibs / g a 1
d:iys SS in Primary Effluent, mg/1*
x Daily Flow, HGD x 8.34 Ibs/gal
Any significant additional loading placed on the aerator by the
digester supernatant liquor must be added to the above loadings by
considering the additional flow (MGD) and concentration (mg/1).
The selection of the method of determining sludge age is discussed
in Chapter 7, Activated Sludge.
Common Range, ii'g/1
Suspended solids in aerator Depends on p
and BOD or suspended solids
in primary effluent
Sludge age Conventional
2.5-6 days,
* NOTE: Sludge age is calculated by three different meUiods:
1. Suspended solids in primary effluent, mg/1
2. Suspended solids removed from primary effluent.s mg/1, cr
primary effluent, suspended solids, ing/1 - final effluent
suspended solids, mg/1
3. BOD or COD in primary effluent, mg/1
14-123
-------
(Sludge Age)
C. Apparatus
See 16, Suspended Solids Test.
D. Reagents
None.
E. Procedure
See 16, Suspended Solids Test.
F. Example
Suspended Solids in Mixed Liquor = 1500 mg/1
Aeration Tank Volume = 0.50 MG
Suspended Solids in Primary Effl. = 100 mg/1
Daily Flow =2.0 MGD
G. Calculations
Susp. Solids in Mixed Liquor, mg/1
Sludge Age, _ x Aerator Vol., MGx_ 8.34 Ibs/gal
days Susp. Solids in Primary Effl., mg/1
x Flow, MGD x 8.34 Ibs/gal
- Mixed Liquor Susp. Solids, Ibs
Primary Effluent SS, fbT/day
1500 mg/1 x 0.50 MG x 8.54 Ibs/gal
100 mg/1 x 2.0 MGD x 8.34 Ibs/gal
1500 x 0.50
100 x 2.0
7.5
2.0
= 3.75 days
14-124
-------
QUESTION
13.A Determine the sludge age in an activated sludge process
if the volume of the aeration tank is 200,000 gallons
and the suspended solids in the mixed liquor equals
2000 mg/1. The primary effluent SS is 115 mg/1, and
the average daily flow is 1.8 MGD.
14-425
-------
14. Sludge (Digested) Dewatering Characteristics
A. Discussion
The dewatering characteristics of digested sludge are very
important. The better the dewatering characteristics or
drainability of the sludge, the quicker it will dry and the
less area will be required for sludge drying beds.
B, What is Tested?
PREFERRED RANGE
Sample . Method A Method B
Digested Sludge Depends on 100-200 ml
appearance
C.
METHOD A
1000 ml graduated cylinder.
METHOD B
1. lir.hoff cone with tip removed,
2, Sand from drying bed.
3. 500 ml beaker.
D. Reagents
None.
E. Procedure
Two methods are presented in this section. Method A relies on a
visual observation and is quick and simple. The only problem is that
operators on different shifts might record the same sludge draining
characteristics differently. Method B requires 24 hours, but the
results are recorded by measuring the vplume of liquid that passed
through the sand. Method B would be indicative of what would happen
if you had sand drying beds.
14-126
-------
(Sludge Dewatering)
METHOD A
1. Add digested sludge
to 1000 ml graduate.
Sample
Container
Pour sample from graduate
back into container.
3. Watch solids
adhere to
cylinder wall;
^p
S&KI
aJ&^feAUv
1. Add sample of digested sludge to 1000 ml graduate.
2, Pour sample back into sample container. Set graduated
cylinder down.
3. Watch graduate. If solids adhere to cylinder wall and
water leaves solids in form of rivulets, this is a
good dewatering sludge on a sand drying bed (Fig, 14.6).
Fig. 14.6
Sludge on graduated
cylinder walls for
sludge dewatering
test
-------
^Sludge Uewatering)
METHOD B
1. Pour digested sludge 2. Place beaker under 3. Measure liquid
on top of sand in tip and wait 24 that has passed
Imhoff cone. hours. through the sand.
Broken Tip
1.
2.
3.
4,
5.
Broken glass Imhoff cone that has tip removed and a glass wool
plug in the end to hold the sand in the cone.
Fall halfway with sand from sand drying bed.
Fill remainder to 1 liter with digested sludge.
Place 500 ml beaker under cone tip and wait 24 hours.
Record liquid that has passed through sand in ml. If
less than 100 ml has passed through sand, you have poor
sludge drainability.
QUESTION
14.A What are the differences in the use of (1) a graduated
cylinder and (2) an Imhoff cone, filled with sand, that
has a broken tip, to neasure the dewaterJng characteristics
of digested sludge?
14-128
-------
15. Supernatcnt. Graduate evaluation
A. Discussion
The digester supernatant solids test measures the percent of
settleable solids being returned to the plant heaclworks. The
settleable solids falling to the bottom of a graduate should
not exceed the bottom 5no of the graduate in most secondary
plants. When this happens, you are imposing a load on the
primary settling tanks that they were not designed to handle.
If the solids exceed 5% you should run a suspended solids
Gooch crucible test (Section 16) on the sample and calculate
the recycle load on the plant that is originating from the
digester.
B. What is Tested?
Sample Common Values
Supernatant % Solids should be <5°
C. Apparatus
100 ml graduated cylinder.
D. Reagents
None.
14-129
-------
F;. Procedure
Fill 100 ml graduate
with supernatant.
Supernatant
Sample
100 ml Graduate
(Supernatant)
2. After 60 minutes,
read ml of solids
at bottom.
JL
10 ml
1.
2.
3.
Fill a 100 ml graduated cylinder with supernatant sample.
After 60 minutes, read the ml of solids that have settled
to the bottom.
Calculate supernatant solids, %.
Supernatant Solids, % - ml of Solids
F. Ivx a mp 1 e
Solids on bottom of cylinder, 10 ml.
G. CaIculations
Supernatant Solids, % = ml of Solids
= 10 ml
= 10% Solids (High) by Volume
14-130
-------
QUESTION
15.A Why should the results of the supernatant solids test
be less than 5% solids?
END OF LESSON 6 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of discussion and review questions before
continuing with Lesson 7.
14-131
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 6 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The
problem numbering continues from Lesson 5.
22. Calculate the efficiency or percent removal of a
primary clarifier when the influent settleable
solids are 10 ml/1 and the effluent settleable
solids are 0.3 ml/1.
23. Why does the actual volume of sludge pumped from
a clarifier not agree exactly with calculations
based on the settleable solids test? '
24. What does sludge age measure?
25. Why should the dewatering characteristics of
digested sludge be measured?
26. What happens to the plant when the supernatant
from the digester is high in solids?
14-132
-------
Ji 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 7 of 8 Lessons)
16'. Suspended Solids
I. GOOCH CRUCIBLE
A. Discussion
One of the tests run on w%astewater is to determine the amount of
material suspended within the sample. The result obtained from
the suspended solids test does not mean that all of the suspended
solids settle out in the primary clarifier or, for that matter, in
the final clarifier. Some of the particles are of such size and
weight that they will not settle without additional treatment.
Therefore, suspended solids are a combination of settleable solids
and those solids that remain in suspension.
B. What is Tested?
SampJLe Common r Ran_ges,^ mg/JL
Influent . Weak 150 - 400+ Strong
Primary Effluent Weak 60- - 150+ Strong
Secondary Effluent 10 Good - 60+ Bad
Activated Sludge Tests Depending on Type of Process
Mixed Liquor 1000 - < 5,000
Return or Waste Sludge 2000 - < 12,000
Digester Tests:
Supernatant 3000 - < 10,000
When supernatant suspended solids are greater than 10,000 mg/1,
the total solids test is usually performed.
14-133
-------
(Suspended Solids - Gooch)
1. 2.4 cm glass fiber filter.
2. No. 4 Gooch crucible.
3. Distilled water.
4. Filter flask.
5. Graduated cylinder.
6. Vacuum pump or aspirator.
7. Oven.
8. Analytical balance.
0. Outline of Procedure
The procedure is outlined on Page 14-157.
(Method with Gooch Crucible and Glass Fiber Filter)
14-134
-------
(Suspended Solids - Gooch)
'iltering Flask
Seat filter, by add-
ing distilled water
and applying vacuum.
n n n
O
5. Dry crucibles in oven
at 103CC.
4. Cool
5, Wei^h crucible.
Pour
measured
volume of
sample in
Gooch
crucible.
7. Filter out suspended
solids with vacuum.
8. Wash graduate, crucible,
and filter with distilled
water to complete solids
transfer.
n n n
a
O
[y \ vj
Drv crucibles plus
suspended solids
at 103CC,
11. iVeigh crucible
plus suspended
solids.
nrm
10. Cool.
14-135
-------
(Suspended Solids - Gooch)
t. Preparation of Gooch Crucible
1. Put a No. 4 Gooch crucible into filtering apparatus.
2. Insert 2.4 cm glass fiber filter and center it.
3. Apply suction.
4. Wash filter with 100 ml of distilled water to seat well.
5. Dry at 103°C for one hour.
6, If volatile suspended solids are to be determined, ignite
crucible in muffle furnace for one hour at 550°C.
7, Cool in desiccator.
8. Weigh and record tare weight.
1;. How to Perform the Test
1. Depending on the suspended solids content, measure out a
25, 50, or 100 ml portion of a well mixed sample into a
graduated cylinder. Use 25 ml if sample filters slowly.
Use larger volumes of sample if samples filter easily,
such as secondary effluent. Try to limit filtration time
to about 15 minutes or less.
2. Wet prepared Gooch crucible with distilled water and apply
suction.
3. Filter sample through the Gooch crucible.
4. Wash out dissolved solids on the filter with about 20 ml
of distilled water. (Use two 10 ml porLions.)
5. Dry crucible at 103°C for one hour or other specified time.
Some samples may require up to three hours to dry if the
residue is thick.
6. Cool crucible in desiccator for 20-30 minutes.
7. Weigh and record weight.
8. Total Weight = g
Tare Weight = g
Solids Weight = g
14-136
-------
{.Suspended Solids - Gooch)
Precautions
1. Check and regulate the oven temperature at 103° - 105°C.
2. Observe crucible and glass fiber for any possible leaks. A
leak will cause solids to pass through and give low results.
The glass fiber filter may become unseated and leaky when the
crucible is placed on the filter flask. The filter should be
reseated by adding distilled water to the filter in the crucible
and applying vacuum before filtering the sample.
3. ' Mix the sample thoroughly so that it is completely uniform in
suspended solids when measured into a graduated cylinder before
sample can settle out. This is especially true of samples heavy
in suspended s'o'lTdsV'such as raw wastewater and mixed liquor in
activated sludge which settle rapidly. The test can be no better
than the mix.
4. It is a good practice to prepare a number of extra Cooch crucibles
for additional tests if the need arises. If a test result appears
faulty or questionable, the test should be repeated. Check filtration
rate and clarity of water passing through the filter.
II. Example and Calculations
This section is provided to show you the detailed calculations. After
some practice, most operators use the lab work sheet as shown at the
end of the calculation-s.
CALCULATIONS FOR SUSPENDED SOLIDS TEST
(or use lab work sheet at end of calculations)
F.xamplc: Assume the following data.
Volume of sample = 50 ml.
Recorded Heights
Crucible weight 21.6329 g
Crucible plus dry solids 21.6531 g
Crucible plus ash11 21.6360 g
11 Obtained by placing the crucible plus dry solids in a muffle
furnace at 550°C for one hour. The crucible plus remaining
ash are cooled and weighed.
14-137
-------
(Suspended Solids - Gooch)
1. Compute total suspended solids.
21.6531 g Weight of Crucible plus Dry Solids, grams
~ 21.6529 g - Weight of Crucible, grams _,....,..,
= 0.0202 g = Weight of Dry Solids, grams
or
= 20.2 mg
1000 milligrams (mg) = 1 gram (g)
or
20.2 mg = 0.0202 g
Total
Suspended _ Weight of Dry Solids.max 1000
Solids, Sample Volume, ml
mg/1
__ _ 1000 ml/1 404.
= 20.2 mg x -— / —
50 ml 507 20200.
200
200
= 404 mg/1 200
2. Compute volatile or organic suspended solids.
21.6531 g Weight of Crucible plus Dry Solids, g
- 21.6360 g_ - Weight of Crucible plus Ash, g _
= 0.0171 g = Weight of Volatile Solids, g
or
= 17.1 me
Volatile
Suspended = Weight of Volatile Solids^ ^ mg^ _x 1000 ml/I
Solids, Sample Volume, nil
mg/1
17.1 mg x 1000 ml/1 342_
50 ml 507 17100
150
= 342 mg/1 21Q
200
100
100
14-138
-------
(Suspended Solids - Gooch)
3. Compute-the percent volatile solids.
Volatile = (height Volatile, mg) 100%
Solids, °6 Weight Total Dry Solids, mj
mg
100%
= 84.71
.8465
20.2 •' 17.10
16 16
940
808
1320
1212
1080
1010
4. Compute fixed or inorganic suspended solids.
21.6360 g
- 21.6529 g
= 0.0031 g
or
= 3.1 m
Weight of Crucible plus Ash,
- Weight of Crucible, g
= Weight of Fixed Solids, g
Fixed
Suspended
Solids,
nig/I
Weight of Fixed^ Solids, mg x 1000 ml/1
Sample Volume, ml
5.1 mg x 1000 ml/1
50 ml
= 62 mg/1
To check your work:
Fixed Susp. Solids
Total Susp. Solids, mg/1 - Volatile
Susp. Solids, mg/I
= 40-4 mg/1 - 342 mg/1
= 62 mg/1 (Check)
404
-342
62
14-139
-------
(Suspended Solids - C-ooch)
5. Compute the percent fixed solids,
.,. , c i-j o (Weight Fixed, ing) x 100%
f'ixed Solids, % = — •• P-- — • — • - '— •••••• — — — •— -
Weight Total, mg
100%
20.2 mg
" = 15 . 3%
The above calculations are also performed on a Laboratory Work
Sheet (Fig. 14.7) to illustrate the use of the work sheet.
CALCULATIONS- FOR OVERALL PLANT REMOVAL OF
SUSPENDED SOLIDS IN PERCENT
Example : Assume the following data.
Influent suspended solids 202 mg/1
Primary Effluent suspended solids 110 mg/1
Secondary Effluent suspended solids 52 mg/1
Final Effluent suspended solids 12 mg/1
To calculate the percent removal or treatment efficiency for a
particular process or the overall plant, use the following formula:
Removal, % = -. ."... x 100%
In
Compute percentage removed between influent and primary effluent
Removal, % = C,1?1. ,". Out) x 10Q%
In
(202 mg/1 - 110 mg/1)
202 mg/1
= - x 100%
202 -110
92
= 45,5%
14-140
-------
(Suspended'.Solids - Gooch)
Compute percentage removed between influent and secondary effluent:
Removal, % - (In " Out) x 100% '
In
- (202 mg/1 - 52 mg/1) 202
— ' "' "" • *' "'• ' • rr' • ' X lUU-o
202 mg/1 , -52
' . • 150
150 100% *74
_ „ x JLUU-8 /
202 202/ 150.00
141.4
8 60
= 74% 8 08
52
Compute percentage removed between influent and final effluent
(overall plant percentage removed):
Removal, % = dp. .-. Out? x 100% '
In
, .(202 mg/1 - 12. mg/1) y lop%
202 mg/1
» 122 x 100%
202
= 94.1% removal for the plant in suspended solids
CALCULATIONS FOR POUNDS SUSPENDED SOLIDS REMOVED PER DAY
Example: Assume the following data.
Influent suspended solids 200 mg/1
Effluent suspended solids 10 mg/1
Flow in million gallons/day 2 MGD
1 gallon of water weighs 8.34 Ibs
14-141
-------
(Suspended Solids - Gooch)
Compute pounds suspended solids removed:
The general formula for computing pounds removed is
R d (Concentration In, mg/1 - Concentration Out, mg/1)
Ibs/day x Flow, MGD x 8.34 Ib/gal
(200 mg/1 - 10 mg/1) x 2 MGD x 8.34 Ib/gal
= 190 x 2 x 8.34
= 3169 Ibs/day of suspended
solids removed by plant
8.34
380
000
6672
2502
3169.20
DERIVATION
This section is not essential to efficient plant operation, but is
provided to furnish you with a better understanding of the calcu-
lation if you are interested. For practical purposes,
1 mg/1 = 1 ppm or 1 part per million
or =1 mg/million mg, because 1 liter = 1,000,000 mg
Therefore:
Ibs _ mg M gal Ibs
day M mg day gal
= Ibs/day
14-142
-------
[Suspended Solids - Gooch)
PLANT
DATE
CLEAN WATER
SUSPENDED SOLIDS § DISSOLVED SOLIDS
SAMPLE
Crucible
Ml Sample
Wt Dry $ Dish
Wt Dish
mc/l =
S
Wt Dry
m
OOP
Ml Sample
Wt Dish § Dry
Wt Dish § Ash
Wt Volatile
vol =
Wt Dry
x 100
INFL.
#015
50
21.6531
21.6329
0.0202
404 mg/1
21.6531
21.6360
0.0171
84.73
BOD
# Blank
SAMPLE
DO Sample
Bottle #
% Sample
Blank or adj blank
DO after incubation
Depletion, 5 days
Dep %
Nitrate N03
Sample
Graph Reading
Sett. Solids
Sample
Direct Ml/1
COD
Sample
Blank Titration
Sample Titration
Depletion
DeP x N FAS
8000
Ml Sample
Fig. 14.7 Calculation of solids content
on Laboratory Work Sheet
14-143
-------
(Suspended Solids - Gooch)
TOTAL SOLIDS
SAMPLG
Dish No.
"Wt Dish $ Wet
Wt Dish
Wt Wet
Wt Dish + Dry
Wt Di sh
Wt Dry
, o-lid_ _ Wt Dry x 100%
Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
- V-latilc - Wt V01 X 10°%
Wt Dry
pH
Vol. Acid
alinity as CaC03
Grease (Soxlet)
Sample
Ml Sample
Wt Flask + Grease
Wt Flask
Wt Grease
Wt Grease, mg x 1000
Ml Sample
ing/1
H2S (Gas) (Starch-Iodine)
Blank
Sample
Diff
Diff x .68
mg/1 x 43.6
Ml
.Ml
Ml
mg/1
grain/100 cu ft
Fig. 14.7 Calculation of solids content on
Laboratory Work Sheet (continued)
14-144
-------
QUESTIONS
16.A Why does some of the suspended material in wastewater fail
to be removed by settling or flotation within one hour?
16.B Given the following data:
100 ml of sample
Crucible weight 19.3241 g
Crucible plus dry solids 19.3902 g
Crucible plus ash 19.3469 g
Compute:
a. Total suspended solids
b. Volatile suspended solids
c. Percent volatile
d. Fixed suspended solids
e. Percent fixed
16.C Compute the percent removal of suspended solids by the
primary clarifier, secondary process (removal between
primary effluent and secondary effluent), and overall
plant:
Influent suspended solids = 221 mg/1
Primary effluent SS = 159 mg/1
Final effluent SS = 33 mg/1
16.D If the data in problem 16.C is from a 1,5 MGD plant,,,
calculate the pounds of suspended solids removed:
a. By the primary unit
b. By the secondary unit
c. By the overall plant
14-145
-------
16. Suspended Solids
II. CENTRIFUGE
A. Discussion
This procedure is frequently used in plants as H. quid-; and easy
method to estimate the suspended solids concentration of the
mixed liquor in the aeration tank instead of the regular suspended
solids test. Many operators control the solids in their aerator
on the basis of centrifuge readings. Others prefer to control
solids using Fig. 14.8. Irs either case, the operator should
periodically compare centrifuge readings with values obtained
from suspended solids tests. If the solids are in a good settling
condition, a 1% centrifuge solids reading could have a suspended
solids concentration of 1000 mg/1. However, if the sludge is
feathery, a 1% centrifuge solids reading could have a suspended
solids concentration of 600 ir.g/1.
The centrifuge reading versus mg/1 suspended solids chart (Fig 14.8)
Must be developed for each plant by comparing centrifuge readings
with suspended solids determined by the regular Gooch crucible
method. The points are plotted and a line of best fit is drawn
as shown in Fig, 14.8. This line must be periodically checked by
comparing centrifuge readings with regular suspended solids tests
because of the large number of variables influencing the relation- .
ship, such as characteristics of influent waste, mixing in aerator,
and organisms in aerator. If you don't have a centrifuge or if
your solids content is over 1500 mg/1, determine suspended solids
by the regular method.
B. What is^ Tested?
Sample Common Range
Suspended Solids in Mechani- 800 - 1200 mg/1
cal Aeration Tanks
Suspended Solids in Diffused 1000 - 3000 mg/1
Aeration Tanks
C. Apparatus
1. Centrifuge.
2. Graduated centrifuge tubes, IS ml,
14-146
-------
(Suspended Solids - Centrifuge)
I). Reagents
None.
B. Procedure
1. Collect sample in regular sampling can.
2, Mix sample well and fill each centrifuge tube to the 15 ml
line with sample.
3. Place filled sample tubes in centrifuge holders.
4. Crank centrifuge at fast speed as you count slowly to 60.
Be s".re tc count and cruik at the same speed for all tests.
It is extremely important to peiform each step exactly the
sane every time.
5. Remove one tube and read the amount of suspended solids con-
centrated in the bottom of the tube. This reading will be
1/10 of ml. Results in other tubes should be compared.
6. Refer to the conversion graph to determine suspended solids
in mg/1.
NOTF.: The reason for filling tubes to the 15 ml mark is that the
graph (Fig. 14.8) is computed for samples of this size.
'•', Example
Suspended solids concentration on bottom of centrifuge tube is 0.4 ml.
G. Calculations
From Fig. 14.8, find 0.4 ml on centrifuge reading side and follow
line horizontally to line on chart. Drop downward from line on chart
to mg/1 suspended solids and read result of 900 mg/1.
If the suspended solids concentration is above or below the desired
range, then you should make the proper changes in the pumping rate
of the waste and return sludge. For details on controlling the solids
concentration, refer to Chapter 7, Activated Sludge.
14-147
-------
CO
200
400
500 600 700
Suspended Solids
a.
CO
o
i—'
H-
a-
m
n
(T>
r+
i-i
H-
H5
00
CD
-------
(Suspended Solids - Centrifuge)
Development of Fig. 14.8
To develop Fig. 14.8 take a sample from the aeration tank and
measure suspended solids and also centrifuge a portion of the
sample to obtain the centrifuge sludge reading in ml pf sludge
at the bottom of the tube. Obtain other samples of different
solids concentrations to obtain the points on the graph. Draw
a line of best fit through the points. Periodically the points
should be checked because the influent characteristics and con-
ditions in the aeration tank change.
QUESTION
16.E What is the advantage of the centrifuge test
for determining suspended solids in an aeration
tank in comparison with other methods of measuring
suspended solids?
14-149
-------
17. Temperature
I. WASTEWATER
A. Discussion
This is one of the most frequently taken tests. One of the many
uses is to calculate the percent saturation of dissolved oxygen
in the DO test. (Refer to DO Test for procedure.)
Changes of plus or minus 4°F from the average or expected value
should be investigated and the cause corrected if possible.
For example, an influent temperature drop may indicate large
volumes of cold water from infiltration. An increase in temperature
may indicate hot water discharged by industry is reaching your plant.
A temperature measurement should be taken where samples are
collected for other tests. This test is always immediately
performed on a grab sample because it changes so rapidly.
Always leave the thermometer in the liquid while reading
the temperature. Record temperature on suitable work sheet,
including time, location, and sampler's name.
B. What is Tested?
Sample Common Range
Influent12 65°F to 85°F13
Effluent12 ' 60°F to 95°F or
higher from ponds
Receiving Water12 60°F to ambient
temperature1 **
Digester (Recirculated 60°F to 100°F
Sludge before Heat Ex-
changer- -Supernatant)
12 If dissolved oxygen (DO) measurements are performed on any
samples, the temperature should be measured and recorded.
13 Depends on season, location, and temperature of water supply.
14 Ambient Temperature (AM-bee-ent). Temperature of the
surroundings.
14-150
-------
(Temperature)
C. Apparatus
1. One NBS (National Bureau of Standards) thermometer for
calibration of the other thermometers.
2. One Fahrenheit mercury-filled, 1° subdivided thermometer.
3. One Celsius (formerly called Centigrade) mercury-filled,
1° subdivided thermometer.
4. One metal case to fit each thermometer.
There are three types of thermometers and two scales.
Scales
1. Fahrenheit, marked °F.
2. Celsius, marked °C (formerly Centigrade).
Tliermome_ters
1. Total immersion. This type of thermometer must be totally
immersed when read. This will change most rapidly when
removed from the liquid to be recorded.
2. Partial immersion. This type thermometer will have a solid
line around the stem below the point where the scale starts.
3. Dial. This type,has a dial that can be easily read while
the thermometer is still immersed. Dial thermometer readings
should be checked (calibrated) against the NBS thermometer.
Some dial thermometers can be recalibrated (adjusted) to read
the correct temperature of the NBS thermometer.
13. Reagents
None.
14-151
-------
(Temperature)
E. Procedures
Use a large volume of sample, preferably at least a 2-pound coffee
can or equivalent volume. The temperature will have less chance
to change in a large volume than in a small container. Collect
sample in container and immediately measure and record temperature.
Do not touch the bottom or sides of the sample container with the
thermometer. To avoid breaking or damaging glass thermometer,
store it in a shielded metal case. Check your thermometer accuracy
against the NBS certified thermometer by measuring the temperature
of a sample with both thermometers simultaneously. Some of the
poorer quality thermometers are substantially inaccurate (off as
much as 6°F),
F. Example
To measure influent temperature, obtain sample in large coffee can,
immediately immerse thermometer in can, and record temperature when
reading becomes constant. For example, 72°F.
-------
(Temperature)
°C = 5/9 (OF - 32°)
77
= 5/9 (77° - 32°) -32_
45
= 5/9 (45°) , 5
9 < 45
= 25° 5
X:;
25"
QUESTIONS
17.A What could a change in influent temperature indicate?
17.B Why should the thermometer remain immersed in the
liquid while being read?
17.C Why should thermometers be calibrated against an
accurate NBS certified thermometer?
14-153
-------
17. Temperature -. , t- < - '- .-'
II. DIGESTER SLUDGE
A. Discussion
The rate of sludge digestion in a digester is a function of the
digester temperature. The normal temperature range in a digester
is around 95 to 98°F. The temperature of a digester should not
be changed by more than 1°F per day because then the helpful
organisms in the digester are unable to adjust to rapid temperature
changes.
B. Apparatus and Procedure
Ref-r to I., WASTEWATER.
END OF LESSON 7 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 8.
14-154
-------
DISCUSSION^ AND REVIEW QUESTIONS
(Lesson 7 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name
Date
Write the answers to these questions in your notebook.
numbering continues from Lesson 6.
The problem
27. Given the following data:
100 ml of sample
Crucible weight
Crucible plus dry solids
Crucible plus ash
Compute:
1. Total suspended solids
2. Volatile suspended solids
3. Percent volatile
28.
29.
30.
31.
19.9850 g
20,0503 g
20.0068 g
Estimate the pounds of solids removed per day by a primary
clarifier if the influent suspended solids is 220 mg/1 and
the effluent suspended solids is 120 mg/1 when the flow is
1.5 MGD.
What is the ambient temperature?
Convert a temperature reading of 50°F to °C.
Why should the temperature of a digester not be changed by
more than 1°F per day?
14-155
-------
CHAPTER 14. LABORATORY PROCEDURES AND CHEMISTRY
(Lesson 8 of 8 Lessons)
18. Total and Volatile Solids (Sludge)
A. Discussion
Total solids measure the combined amount of suspended and dissolved
materials in the sample.
This test is used for wastewater sludges or where the solids can be
expressed in percentages by weight and the weight can be measured
on an inexpensive beam balance to the nearest .01 of a gram. The
total solids are composed of two components, volatile and fixed
solids. Volatile solids are composed of organic compounds which
are of either plant or animal origin. Fixed solids are inorganic
compounds such as sand, gravel, minerals, or salts.
B. What is Tested?
COMMON RANGE, % BY WEIGHT
Sample
Raw Sludge
Raw Sludge plus Waste
Activated Sludge
Recirculated Sludge
Supernatant:
Good Quality, has
Suspended Solids
Poor Quality
Digested Sludge
to Air Dry
1
Total
6% to 9%
2% to 5%
.5% to 3%
< 1%
> 5%
3% too Thin
to < 8%
Volatile
75%
80%
75%
50%
50%
Fixed
25% ± 6%
20% ± 5%
25% ±5%
50% ± 10%
50% ±
14-156
-------
C. Apparatus
1. Evaporating dish.
2. Analytical balance.
3. Drying oven, 103° - 105°C.
4. Measuring device—graduated cylinder.
5. Muffle furnace, 550°C.
D. Outline of Procedure
v
2. Cool
1. Ignite empty dish
in muffle furnace
4. Measure out
sludge
3. Weigh
dish
5. Evaporate water at
103-105°C
\
7. Weigh dish
+ residue
6. Cool dish
+ residue
14-157
-------
(Total and Volatile Solids)
PROCEDURE
1. Dry the dish by ignition in a muffle furnace at 550°C for
one hour. Cool dish in desiccator.
2. Tare the evaporating dish to the nearest 10 milligrams, or
0.01 g on the Mettler single pan balance. Record the weight
as Tare Weight = _ _ J _ _ a ; __. _., . Sms<
3. Weigh dish plus 50 to 100 ml of well mixed sludge sample.
Record total weight to nearest 0.01 gram as Gross Weight =
gms.
4. Evaporate the sludge sample to dryness in the 103°C drying
oven.
5. Weigh the dried residue in the evaporating dish to the
nearest 10 milligrams, or 0.01 g. Record the weight as
Dry Sample and Dish = gms.
6. Compute the net weight of the residue by subtracting the
tare weight of the dish from the dry sample and dish.
E. Precautions
1. Be sure that the sample is thoroughly mixed and is representative
of the sludge being pumped.Generally, where sludge pumping is
intermittent, sludge is much heavier at the beginning and is less
dense toward the end of pumping. Take several equal portions of
sludge at regular intervals and mix for a good sample.
2. Take a large enough sample. Measuring a 50 or 100 ml sample
which is closely equal to 50 or 100 grams is recommended.
Since this material is so heterogeneous (non-uniform), it is
difficult to obtain a good representative sample with less
volume. Smaller volumes will show greater variations in answers,
due to the uneven and lumpy nature of the material.
3. Control oven temperature closely at 103° - 105°C. Some solids
are lost at any drying temperature. Close control of oven
temperature is necessary because higher temperatures increase
the losses of volatile solids in addition to the evaporated
water.
14-158
-------
Heat dish long enough to insure evaporation of water,
usually about 3-4 hours. If heat drying and weighing
are repeated, stop when the weight change becomes small
per unit of drying time. The oxidation, dehydration,
and degradation of the volatile fraction won't completely
stabilize until it is carbonized or becomes ash.
Since sludge is so non-uniform, weighing on the analytical
balance should probably be made only to the nearest 0.01
grams or 10 milligrams.
Outline of Procedure for Volatile Solids
(continue from total solids test)
1, Ignite dried solids
at 550°C
2. Cool
3, Weigh fixed solids
1.
2.
3.
4.
PROCEDURE
Determine the total solids as previously described in Section D.
Ignite the dish and residue from total solids test at 550°C for
one hour or until a white ash remains.
Cool in desiccator for about 30 minutes.
Weigh and record weight of Dish Plus Ash =
gms,
14-159
-------
(Total and Volatile Solids)
G. Example
Weight of Dish (Tare) = 20.31 g
Weight of Dish plus
Wet Solids (Gross) = 70.31 g
Weight of Dish plus
Dry Solids = 22.81 g
Weight of Dish plus Ash = 20.93 g
H. Calculations
See Laboratory Work Sheet (Fig. 14.9) or calculations shown below.
1. Find weight of sample.
Weight of Dish plus Wet Solids (Gross) = 70.31 g
Weight of Dish (Tare) = 20.31^ g
Weight of Sample = 50.00 g
2. Find weight of total solids.
Weight of Dish plus Dry Solids = 22.81 g
Weight of Dish (Tare) = 20.31 g
Weight of Total Solids = 2.50 g
3, Find % solids.
% solids = (Weight of Solids, g) 100%
Weight of Sample, g
(2.50 g) 100%
50.00 g
= 5%
4. Find weight of volatile solids.
Weight of Dish plus Dry Solids = 22.81 g
Weight of Dish plus Ash = 20.95 _g
Weight of Volatile Solids = 1.88 g
14-160
-------
5. Find % volatile solids.
% Volatile Solids = (Weight of Volatile Solids, gjj.00%
Weight of Total Solids, g
(1.88 g) 100%
2.50 g
= 76%
QUESTION
18.A What is the origin of the volatile solids found in a
digester?
18.B What is the significance of volatile solids in a
treatment plant?
19. Turbidity
See Clarity.
14-161
-------
PLANT
DATE
SUSPENDED SOLIDS § DISSOLVED SOLIDS
SAMPLE
Crucible
Ml Sample
Wt Dry § Dish
Wt Dish
" Wt Dry
-._/, _ Wt Dry, gm x 1,000,000
6 Ml Sample
Wt Dish $ Dry
Wt Dish $ Ash
Wt Volatile
% vol - -l^2i x 100%
Wt Dry
BOD
# Blank
SAMPLE
% Sample
Dep %
Nitrate N03
Sample
Graph Reading
COD
Scimple
Blank Titration
Sample Titration
Depletion
rag/1 =
le
#
e
r adj blank
r incubation
on, 5 days
Sett. Solids
Sample
Direct Ml/1
X80°
Ml Sample
Fig. 14.9 Calculation of total solids
on Laboratory Work Sheet
14-162
-------
TOTAL SOLIDS
SAMPLE
Dish No.
Wt Dish § Wet
Wt Dish
Wt Wet
Wt Dish + Dry
Wt Dish
Wt 'Dry
?- c -. • !„ _ wt Dry x i°°%
Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
* Volatile - Wt Vo1 x 100%
Wt Dry
pH
Vol. Acid
Alkalinity as CaC03
RAW
7
70.31
20.31
50.00
22.81
20.31
2.50
5.0%
22.81
20.93
1.88
76%
Grease (Soxlet)
Sample
Ml Sample
Wt Flask + Grease
Wt Flask
Wt Grease
mg/1 = Wt Grease, mg x 1000
Ml Sample
H2S (Gas) (Starch-Iodine)
Blank
Sample
Diff
Diff x .68
mg/1 x 43.6
Ml
Ml
Ml
mg/1
grain/100 cu ft
Fig. 14.9 Calculation of total solids on
Laboratory Work Sheet (continued)
14-163
-------
20. VoJLatile Acids and Total Alkalinity
A. Discussion
Volatile acids are determined on sludge samples from the digesters.
Most modern digesters have sampling pipes where you can draw a
sample from various levels of the tank. Be sure to allow the
sludge in the line to run for a few minutes in order to obtain a
representative sample of the digester contents. Samples also may
be collected from supernatant draw-off tubes, or thief holes.15
The concentrations of volatile acids and alkalinity are the first
measurable changes that take place when the process of digestion
is becoming upset. The volatile acid/alkalinity relationship can
vary from 0.1 to about 0.5 without significant changes in digester
performance. When the relationship starts to increase, this is a
warning that undesirable changes will occur unless the increase is
stopped. If the relationship increases above 0.5, the composition
of the gas produced can change very rapidly, followed by changes
in the rate of gas production, and finally pH.
In a healthy and properly functioning digester, the processes or
biological action taking place inside the digester are in equilibrium.
When fresh sludge is pumped into a digester, some of the organisru
in the digester convert this material to volatile (organic) acid.?,
In a properly operated digester, other organisms feed on the newly-
produced volatile acids and eventually convert the acids to methane
(CHiJ gas, which is burnable and carbon dioxide (C02). If too much
raw sludge is pumped to the digester or the digester is not function-
ing properly, an excess of volatile acids are produced. If excessive
amounts of volatile acids are produced, an acid environment unsuitable
for some of the organisms in the digester will develop and the digeste.
may cease to function properly unless the alkalinity increases too.
Routine volatile acids and alkalinity determinations during trie
start-up process for a new digester are a must in bringing the
Oigester to a state of satisfactory digestion,
Routine volatile acids and alkalinity determinations during digestion
are important in providing the information which will enable the
operator to determine the health of the digester.
15 Thief Hole. A digester sampling well.
14-164
-------
For digester control purposes, the volatile acid/alkalinity relation-
ship should be determined. When the volatile acid/alkalinity
relationship is from less than 0.1/1.0 to 0.5/1.0, the loading and
seed retention of the digester are under control. When the relation-
ship starts increasing and becomes greater than 0.5/1.0, the digester
is out of control and will become "stuck" unless effective corrective
action is taken.
B. What is Tested?
Sample Desirable Range
Recirculated Sludge 150 - 600 mg/1
(expect trouble if alkalinity less than
two times volatile acids]
METHOD A
(Silic Acid Method)
C. Apparatus
1. Centrifuge or filtering apparatus.
2. Two 50 ml graduated cylinders.
3. Two medicine droppers.
4. Crucibles, Gooch or fritted glass
5. Filter flask
6. Vacuum source
7. One 50 ml beaker
8. Two 5 ml pipettes
9. Buret
14-165
-------
D. Reagents
1. Silicic acid, solids, 100-mesh. Remove fines from solid
portion of acid by slurrying the acid in distilled water
and removing the supernatant after allowing settling for
15 minutes. Repeat the process several times, Dry the
washed acid solids in an oven at 103°C aid then store in
a desiccator.
2. Chloroform-butanol reagent. Mix 300 ml chloroform, 100 ml
n-butanol, and 80 ml 0.5 N H SO in separatory funnel and
allow the water and organic Iaye?s to separate. Drain off
the lower organic layer through filter paper into a dry
bottle.
3. Thymol blue indicator solution. Dissolve 80 mg thymol
blue in 100 ml absolute methanol.
4. Phenolphthalein indicator solution. Dissolve 80 mg
phenolphthalein in 100 ml absolute methanol.
5. Sulfuric acid, 10 N.
6. Standard solium hydroxide reagent, 0.02 N. Prepare in
absolute methanol from cone. NaOH stock solution in water.
14-166
-------
12. Outline of Procedure
3.
Add a
few drops
of thymol
blue.
Separate solids by
centrifuging or
filtering sample.
2. Measure 10-15 ml
of sample into
beaker.
4. Add
10 N H2S04
dropwise until
thymol
blue turns
red
Add 5 ml
acidified
sample.
7. Add 50 ml
chloroform-butanol
Place 10 g silic
acid in crucible
and apply suction,
Apply suction until
all of reagent has
entered solid acid
column.
9, Remove filter flask.
10. Add a few
drops of
phenoIphthale in
11. Titrate with
0.02N NaOH,
14-167
-------
PROCEDURE
1. Centrifuge or filter enough sludge to obtain a sample of
10 to 15 ml. This same sample and filtrate should be
used for both the volatile acids test and the total
alkalinity test.
2. Measure volume (10 to 15 inl) of sample and place in a
beaker.
3. Add a few drops of thymol blue indicator solution.
4. Add 10 N H^SO^, dropwise, until thymol blue color just turns
to red.
5. Place 10 grams of silicic acid (solid acid) in crucible
and apply suction. This will pack the acid material
and the packed material is sometimes called a column.
6. With a pipette, distribute 5.0 ml acidified sample
(from step 4) as uniformly as possible over the column.
Apply suction briefly to draw the acidified sample into
the silicic acid column. Release the vacuum as soon as
the sample enters the column.
7. Quickly add 50 ml chloroform-butanol reagent to the column.
8. Apply suction and stop just before the last of the reagent
enters the column.
9. Remove the filter flask from the crucible.
10. Add a few drops of phenolphthalein indicator solution to
the liquid in the filter flask.
11. Titrate with 0.02 N NaOH titrant in absolute methanol, taking
care to avoid aerating the sample. Nitrogen gas or C0£ - free
air delivered through a small glass tube may be used both to
mix the sample and to prevent contact with atmospheric C0~
during titration [ CC-2 - free air may be obtained by passing
air through ascarite or equivalent].
Volume of NaOH used in sample titration, a =- ml.
12. Repeat the above procedure using a blank of distilled water.
Volume of NaOH used in blank titration, b = ml.
14-168
-------
F. Precautions
1. The sludge sample must be representative of the digester.
The sample line should be allowed to run for a few minutes
before the sample is taken. The sample temperature should
be as warm as the digester itself.
2. The sample for the volatile acids test should not be taken
immediately after charging the digester with raw sludge.
Should this be done, the raw sludge may short-circuit to
the withdrawal point and result in the withdrawal of raw
sludge rather than digested sludge. Therefore, after the
raw sludge has been fed into the tank, the tank should be
well mixed by recirculation or other means before a sample
is taken.
3. If a digester is performing well with low volatile acids
and then if one sample should unexpectedly and suddenly
give a high value, say over 1000 mg/1 of volatile acids,
do not become alarmed. The high result may be caused by
a poor, nonrepresentative sample of raw sludge instead
of digested sludge. Resample and retest. The second
test may give a more typical value. When increasing
volatile acids and decreasing alkalinity are observed,
this is a definite warning of approaching control problems.
Corrective action should be taken immediately, such ^
reducing the feed rate, reseeding from another digest--
maintaining optimum temperatures, improving digester mixmg,,
decreasing sludge withdrawal rate, or cleaning the tank
of grit and scum.
14-169
-------
G. Example
bquivalent Weight of Acetic Acid, A =60 mg/ml
Volume of Sample, B = 10 ml
Normality of NaOH tit rant, N = 0.02 N
Volume of NaOH used in sample titration, a = 2.3 ml
Volume of NaOH used in blank titration, b = 0.5 ml
H. Calculation
Volatile Acids, mg/1 _ A x 1000 ml/1^ x N (a - b)
(as acetic acid) B
60 mg/ml x 100°. P1/.1. x, P.'P2 Q2'5 ml. ". P_\5
10 ml
= 216 mg/1
METHOD B
(Nonstandard Titration Method)
C. Apparatus
1. One pH meter.
2. One adjustable hot plate.
3. Two Burets and stand.
4. One 100 mi beaker.
D. Reagents
1. pH 7.0 buffer solution
2. pH 4.0 buffer solution
3. Standard acid.
I. Standard base.
14-170
-------
E. Outline of Procedure
3. Titrate with
1. Separate solids by
centrifuging or re-
moving water above
ye'.T led sample.
o ., SUlf
2. Measure
50 ml $ J°.a
, . 4.0.
place in
beaker.
Vv?j *~
uric ac
pH of
/T\
o oo| \\
V
id '
* r
A
i rnn\
If •/;••.-.
4. Note acid used
and continue
titrating to
pH 3.5 to 3.3.
*•*
.)
5, lightly boil
- r -' : for
'nutes.
6. Cool in water bath.
7. Titrate to ra of 4.0,
with 0.05 ;\ NaOH, note
buret reading, and com-
plete titration to - pi!
of 7.0.
14-171
-------
PROCEDURE
1. Buffer the pll meter at 7.0 and check pH before treatment
of sample to remove the solids. Filtration is not necessary.
Decanting (removing water above settled material) or centri-
fuging sample is satisfactory. Do not add any coagulant aids.
2. Titrate 50 ml of the sample in a 100 ml beaker to pH 4.0
with the appropriate strength sulfuric acid (depends on
alkalinity), note acid used, and continue to pH 3.5 to 3.3.
A magnetic mixer is extremely useful for this titration.
3. Carefully buffer pH meter at 4.00 while lightly boiling the
sample a minimum of three minutes. Cool in cold water bath
to original temperature.
4. Titrate sample with standard 0.050 N sodium hydroxide up to
pH 4,00, and note buret reading. Complete the titration at
pH 7,0. (If this titration consistently takes more than
10 ml of the standard hydroxide, use 0.100 N NaOH.)
5. Calculate volatile acid alkalinity (alkalinity between pH
4.0 and 7.0).
Volatile Acid _ ml 0.050 N NaOH x 2500
Alkalinity mi Sample
For a 50 ml sample the volatile acid alkalinity equals
50 x ml 0.050 N NaOH, or 100 x ml 0.100 N NaOH.
6. Calculate volatile acids.
Case 1: > 180 mg/1 volatile ncid alkalinity.
Volatile Acids = Volatile Acid Alkalinity x 1.50
Case 2: < 180 mg/1 volatile acid alkalinity,
Volatile Acids = Volatile Acid Alkalinity x l.on
Steps 1 and 2 will give the analyst the pH and total alkalinity,
two control tests normally run on digesters. The difference
between the total and the volatile acid alkalinity is bicarbonate
alkalinity. The time required for Steps 3 and 4 is about ten
minutes.
This is an acceptable method for digester control to determine
the volatile acid/alkalinity relationship, but not of sufficient
accuracy for research work.
14-172
-------
For details regarding this test see DeLallo, R., and Albertson,
O.E., Volatile Acids by Direct TitTatian^ Water Pollution Control
Federation, Vol. 33, No. 4, pp 356-365, April 1961. The procedure
is reproduced from the article.
F. Example and Calculation
Titration of pH 4.0 to 7.0 of a 50 ml sample required 8 ml of
0.05 N NaOH.
Step 5 - Calculate volatile acid alkalinity (alkalinity between
pH 470 and 7.0).
Volatile Acid _ ml 0.05 N NaOH x 2500
Alkalinity, mg/1 " ml Sample
8 ml x^ 2500
50 ml
= 400 mg/1
Step_j5_ - Calculate volatile acids.
Case 1: 400 mg/1 > ?. f':" mg/1. '.herefore,
Volatile ., , .., . . , ... ,. .. , _„
, . , ,. = Volatile Acid Alkalinity x 1.50
Acids, mg/1 J
- 400 mg/1 x 1.50
= 600 mg/1
QUESTION
20.A What is the volatile acid concentre" ' ir -" /; a
digester if a 50 ml sample required : .: oC 0.05
NaOH for a titration from a pH of 4.',' to 7.0V
14-173
-------
Total A Ika Unity
A. Discussion
Tests for total alkalinity of digesters are normally run on settled
supernatant samples. The alkalinity of the recirculated sludge is
a measure of the buffer capacity in the digester. When organic
matter in a digester is decomposed anaerobically, organic acids
are formed which could lower the pH, if buffering materials
(buffer capacity) were not present. If the pH drops too low, the
organisms in the digester could become inactive or die and the
digester becomes upset (no longer capable of decomposing organic
matter).
For digester control purposes, the volatile acid/alkalinity relation-
ship should be determined. When the volatile acid/alkalinity
relationship is from less than 0.1/1.0 to 0.5/1.0, the loading and
seed retention of the digester are under control. When the relation-
ship starts increasing and becomes greater than 0.5/1.0, the digester
is out of control and will become stuck unless effective corrective
action is taken. The pH will not be out of range as long as the
volatile acid/alkalinity relationship is low. This relationship
gives a warning before trouble starts.
All samples must be settled so that a liquid free of solids is availir:
for the test. Tests cannot be calculated correctly if solids are in
the sample.
B. What is Tested?
Sample Common Ra-.ge
Recirculated Sludge 2-10 Times Vo'atMe
14-174
-------
(Volatile Acids and Total Alkalinity)
C. Apparatus
1. Centrifuge and centrifuge tubes, or settling cylinder.
2. Graduated cylinders (25 ml and 100 ml)
3. 50 ml Buret
4. 400 ml Erlenmeyer Flask or 400 ml beaker
5. pH Meter or a methyl orange chemical color
indicator may be used (see Procedure)
D. Reagents
1. Sulfuric Acid, 0.2 N. Prepare stock solution of approximately
0.1 N by cautiously adding 2.8 ml of concentrated sulfuric
acid (H^OiO to 1 liter of distilled water. Dilute 200 ml of
the 0.1 N stock solution to 1 liter with boiled distilled
water. Standardize against 0.02 N sodium carbonate (Step 2).
2. Sodium Carbonate, 0.02 N. Dry in oven before weighing. Dis-
solve 1.06 g of anhydrous sodium carbonate (Na2CC>3) in boiled
distilled water and dilute to 1 liter with distilled water.
3. Methyl Orange Chemical Color Indicator. Dissolve 0.5 •:>
methyl orange in 1 liter of distilled water.
14-175
-------
E. Procedure
4. Titrate
Centrifuge
or settle
3. Place electrodes of
pH meter in beaker
2. Add 190 ml of
distilled water
Add 2 drops of
methyl orange
TZJT7
This proceuure is followed to measure the alkalinity of a sample
and also the alkalinity of a distilled water blank.
1. Take a clean 400 ml beaker and add 10 ml or less of clear
supernatant (in case of water or distilled water, use 200 ml
sample). Select a sample volume that will give a useable
titration volume. If the liquid will not separate from the
sludge by standing and a centrifuge is not available, use
the top portion of the sample. This same sample and filtrate
should be used for both the total alkalinity test and the
volatile acids test.
2. Add 190 ml distilled water (in case of water or distilled
water determination skip this step).
14-176
-------
(Volatile Acids and Total Alkalinity)
.">. Place the electrodes of pH meter into the 400 ml beaker
containing the sample.
4. Titrate to a pH of 4.5 with 0.02 N sulfuric acid. (In
case of a lack of pH meter, add 2 drops of methyl orange
indicator. In this case, titrate to the first permanent
change of color to a, red-orange color. Care must be
exercised in determining the change of color and your
ability to detect the change will improve with experience.)
5. The alkalinity of the distilled water should be checked
and if significant, subtracted from the calculation,
6. Calculate alkalinity.
Alkalinity of
Distilled = ml of 0.02 N H2S04 x 5*
Water, mg/1
Total Alka- _ " ml of 0.02 N H2SOk x 100* - mg/1
linity, mg/1 ~ alkalinity of distilled H20
F. Example
Results from alkalinity titrations on
1. Distilled Water 4 ml 0.02 N H2SOtt
2. Recirculated Sludge 19.8 ml 0.0? N H:,S04
G, C aIculations
Alkalinity of . ,, - ,_ ., ., „...
r. f. 1 j T, n /i = ml (rf" O.i)2 N HobU,, x 'a
Distilled H20, mg/1 z +
= 4 n'l x 5
= 20 >'!.-'!
Hlse 5 if measuring alkalinity of water or distilled wat-:>r
(200 ml sample) and 100 if measuring alkalinity of sludge
(10 ml sample).
14-177
-------
Total Alka-
linity, mg/1,
of recircu-
lated sludge
ml of 0.02 N H2SOi4 x 100 - mg/1 alka-
linity of distilled H20
19.8 ml x 100 - 20 mg/1
1980 mg/1 - 20 mg/1
1960 mg/1
QUESTIONS
20.B Why would you run a total alkalinity test on
recirculated sludge?
20.C What is meant by the buffer capacity in a
digester?
20.D If the total alkalinity in a digester is
2000 mg/1 and the volatile acids concen-
tration is 300 mg/1 per liter, what is
the volatile acid/alkalinity relationship?
14-178
-------
2 * •
See Total Solids.
END OF LESSON 8 OF 8 LESSONS
on
Laboratory Procedures and Chemistry
14-179
-------
DISCUSSION AND REVIEW QUESTIONS
(Lesson 8 of 8 Lessons)
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Write the answers to these questions in your notebook. The
problem numbering continues from Lesson 7.
32. Why are solids only weighed to the nearest 0.01 gram
when determining the total and volatile solids content
of digesters?
33. What is a thief hole?
34. What relationship is the critical control factor
in digester operation?
14-180
-------
14.6 KECOMMENI'O GENERAL LABORATORY SUPPLIES
Supplies needed in addition to apparatus listed for tests. Source:
WPCF Publication No. 18, Simplified Laboratory Procedures for Waste-
water Examination.
Quantity Description
12 Pinch clamps, medium
200 Corks, assorted
1 Cork borer set, sizes 1 through 6
1 Cork borer sharpener
2 Ib Glass tubing, 8 mm
4 Thermometers, -20° to 100°C
40 ft Rubber tubing, 1/4-in. ID, 3/32-in. wall
2 Ib Rubber stoppers, assorted (sizes 6 through 12)
1 Tripod, concentric ring, 6 in. OD
1 Latest edition, Standard Methods for the Examination oj
Water & Wastewater
2 Funnels, 50 mm
2 Funnels, 100 mm
2 pair Balance watch glasses, 3 in.
4 Beakers, Pyrex, 1000 ml
4 Beakers, Pyrex, 600 ml
6 Beakers, Pyrex, -in-; ;, i
•t Beakers, Pyrex, 250 nJ
Beakers, Pyrex, 100 ml
4 Beakers, Pyrexs SO ml
2 Bunsen burner>
2 Brushes, medium
2 Brush, B
2 Brush, A
2 Brush, Flask
2 Aprons, plastic, 42 in, length
3 Wire gauzes, 4 x 4 in.
3 Triangles, 2-1/2 in, per side
1 tube Stopcock lubricant
14-181
-------
SUPPLEMENTAL EQUIPMENT FOR THE BOD TEST
Quantity Desc ripti on
12 Flask, Erlenmeyer, 500 ml
12 Flask, Erlenmeyer, 250 ml
2 Pipettes, volumetric, 25 ml
2 Pipettes, volumetric, 10 ml
2 Pipettes, volumetric, 5 ml
2 Flasks, volumetric, graduated to contain and
deliver 1000 ml
2 Flasks, volumetric, graduated to contain and
deliver 500 ml
2 Flasks, volumetric, graduated to contain and
deliver 100 ml
6 Bottles, 32 oz
6 Bottles, 16 oz
6 Bottles, 8 oz
24 BOD bottles, with funnel opening
2 Burets, 50 ml
1 Buret clamp, double
2 Bottles, dropping, 30 ml
2 Spatulas, 75-mm blade
3 Bottles, storage, 2-1/2 gal
1 Buret support, medium
9 Ib Sulfuric acid, CP
5 Ib Sodium hydroxide pellets, CP
j'2 Bulb, rubber, pipette, 2 ml
24 Holder, rubber, stor.per
4 Flask, volumetric, w'o stopper, 100 ml
2 Ib Potassium iodide, CP
1 Ib Starch, soluble potato
1 Ib Sodium thiosulfi; te, CP
5 Ib Manganous sulfate, CP
100 g Sodium azide, CP
1 Ib Magnesium sulfate
1/4 Ib Ferric chloride
1 Ib Potassium phosphate, mono-basic
1 Ib Potassium phosphate, dibasic
14-182
-------
Quantity
Description
] Ih Sodium phosphate, dibasic heptahydrate
1/4 Ib Ammonium chloride
1 oz Potassium bi-iodate, primary standard
1 Ib Potassium dichromate
10 g Sodium diethyldithio carbamate
1 Incubator, BOD
1 Refrigerator
1 Ib Calcium chloride, 20 mesh
1
1
6 Ib
25 g
SUPPLEMENTAL EQUIPMENT FOR THE CHLORINE RESIDUAL TEST
Quantity Description
Comparator, water analysis
Disc for comparator, chlorine
Hydrochloride acid, CP
Orthotolidine dihydrochloride
SUPPLEMENTAL EQUIPMENT FOR SOLIDS ANALr ..S
Quantity Description
1 Brush, camel hair, 1 - \ a, vide
1 Balance wit,-\ cove °
] Weights, balance set, 50 g
12 Crucibles, Gooc1 , No. 4
2 Holders, cruciMe
2 Cylinder, graduated, 1000 ml
2 Cylinder, gradua* •', Snn ml
2 Cylinder, gradual ' "50 ml
4 Cylinder, graduated, 100 ml
4 Cylinder, graduated, 50 ml
2 Cylinder/ graduated, 25 ml
1 Cylinder, graduated, 10 ml
1 Desiccator, 250 mm
1 Desiccator olate
12 Dishes, r-vaporating, size 0
14-183
-------
Quantity
Description
3 Flask, filtering, 500 ml
2 Pipettes, 25 ml
6 Pipettes, 10 ir.l
2 Pipettes, 5 rcl
1 Hot plate, 660 w
2 Tongs, crucible
1 Tongs, furnace, 18 in,
8 ft Tubing, rubber, (heavy) 1/4-in. ID
2 Filter pumps
1 Clock, interval timer, 2 hr
1 Furnace, muffle
2 boxes Paper, filter, glass fiber, 2.4 cm
1 Water baths, four-hole
1 Balance, platform, triple beam
2 Bottles, washing, polyethylene, 500 ml
; 6 Pencils, wax, red
2 boxes Filter paper, 12.5 cm, Whatman No. 41
1 bottle Ink, marking, black
1 Ib Rod, glass, 6 mm
1 File, triangular, 4 in.
12 . Bulb, rubber, pipet, 2 oz
1 ' Balance desiccator
1 Oven, drying
24 2.4 cm glass fiber filter
2 Buchner funnel, size 2A
6 Tube "T", connecting, 1/4-in.
5 Ib Drierite
SUPPLEMENTAL EQUIPMENT FOR COLIFORM GROUP
BACTERIA ANALYSES
Quantity Description
1 Sterilizer or autoclave
12 3 mm wire transfer loop
24 , Pipets, measuring, 10 ml
48 Pipets, measuring, 1 ml, or quantity of disposable
sterile pipets
14-184
-------
Many equipment suppliers will furnish suggested equipment lists
upon request and indication of size of plant and tests being
performed. Lists may be obtained from:
Central Scientific Company Van Waters § Rogers
1700 Irving Park Road- Post Office Box 2062
Chicago, lllinios ' Terminal Annex
, . Los Angeles, California 90054
•14.7 ADDITIONAL READING
a. MOP 11
b. New York Manual, pages 127-148
c. Texas Manual, pages 565-587
d. Laboratory Procedures for Operators of Water Pollution Control
Plants,'- Nagano, Joe. Obtain from Secretary-Treasurer, California
Water Pollution Control Association, P.O. Box 61, Lemon Grove,
California °2045. Price $3.25 to members of the CWPCA; $4.25
to others.
e. Simplified Laboratory Procedures for Wastewater Examination,
WPCF Publication No. 18, Water Pollution Control Federation,
3900 Wisconsin Avenue, Washington, D.C. 20016. Price $2.00
to members; $4.00 to others. Indicate your member association
when ordering.
f. Standard Methods for Examination of Water and Wastewater, pro-
duced by APHA, AWWA, and WPCF, Water Pollution Control Federation,
3900 Wisconsin Avenue, Washington, D.C. 20016. Price $16.50 to
members prepaid only-; otherwise $22.50 plus postage. Indicate
your member association when ordering.
g. Chemistry for Sanitary Engineers, Sawyer, Clair N. and
McCarty, Perry L., McGraw-Hill Book Company, New York, 1967.
Price $13.50.
h. Methods for Chemioal Analysis of Water and Wastes, 1971,
Environmental Protection Agency, Water Quality Office, Analytical
Quality Control Laboratory, 1014 Broadway, Cincinnati, Ohio 45202.
For sale by Superintendent of Documents, Government Printing Office,
Washington, D.C. 20402, Stock Number 5501-0667. Price $3.00.
14-185
-------
14-186
-------
SUGGESTED ANSWERS
Chapter 14. Laboratory Procedures arid Chemistry
14.2A A bulb should always be used to pipette wastewater
or polluted water to prevent infectious materials
from entering your mouth..
14.2B Inoculations are recommended to reduce the possibility
of contracting diseases.
14.2C Immediately wash area where acid spilled with water and
neutralize the acid with sodium carbonate or bicarbonate,
14.2D True. You may add acid to water, but never reverse,
14.2E Work clothes should be changed before going home at
night to prevent carrying unsanitary materials and
diseases home which could infect you and your family.
14.3A The largest sources of errors -.found in laboratory results
are usually caused by improper sampling; poor preservation;
and lack of sufficient mixing, compositing, and testing.
IB
14.3R A representative sample must be collected or the test
results will not have any significant meaning. To
efficiently operate a wastewater treatment plant, the
operator must rely on test results to indicate to him
what is happening.
14.3C A proportional composite sample may be prepared by collect-
ing a sample every hour. The size of this sample is
proportional to the flow when the sample is .collected.
All of these proportional samples are mixed together to
produce a proportional composite sample. If an equal
volume of sample was collected each hour and mixed, this
would be simply a composite sample.
3.A The dangers encountered in running t-he CC>2 on digester
gas include:
1. Digester gas contains methane, which is
explosive when mixed with air.
2. The C02 gas absorbent is hafmful to your skin.
14-187
-------
» - (Total Volume, ml - Gas Remaining, ml) x 100%
3.B a LU2 - Total Volume, ml
(128 ml - 73 ml) x 100% 128
128 ml - 75
55
= !§ x 100%
4.A The COD test is a measure of the strength of a waste
in terms of its chemical oxygen demand. It is a good
estimate of the first-stage oxygen demand. (Either
answer is acceptable.)
4.B The advantage of the COD test over the BOD test is
that you don't have to wait five days for the results.
5. A Plant effluents should be chlorinated for disinfection
purposes to protect the bacteriological quality of the
receiving waters.
5.B The idometric method gives good results with samples
containing wastewater, such as plant effluent or re-
ceiving waters. Orthotolidine will give satisfactory
results if used within 20 minutes of the application
of chlorine; however, the entire chlorine demand may
not yet have been satisfied. Amperometric titration
gives satisfactory results, but the equipment is ex-
pensive.
6.A The clarity test indicates the relative change of depth
you can see down in the final clarifier or contact basin
This reflects a visual comparison of color, solids, and
turbidity from one test to the next. OR_ Indication of
quality of effluent.
6.B When clarity is measured under different conditions the
results can not be compared. You won't be able to tell
whether your plant performance is improving, staying the
same, or deteriorating.
14-188
-------
7,A Sodium thiosulfate crystals should be added to sample
bottles for coliform bacteria tests before sterilization
to neutralize any chlorine that may be present when the
sample is collected. Care must be taken not to wash the
bottles out when a sample is collected.
7.B 121°C within 15 minutes.
7.C Dilutions -2 -3 -,4 -5
Readings 5 __1 2_ 0
MPN = 63,000/100 ml
7.D The number of coliforms is estimated by counting the
number of colonies grown on the membrane filter.
8.A DO Saturation, % = DO of Sample, mg/1 x 100%,
DO at Saturation, mg/1
(7.9 mg/1) 100% .699
11.3 mg/1
= 70%
8.B To calibrate the DO probe in an aeration tank, a
sample of effluent can be collected and split. The
DO of the effluent is measured by the modified Winkler
procedure, and the probe DO reading is adjusted to agree
with the Winkler results.
8.C When the DO in the aeration tank is very low, the
copper sulfate-sulfamic acid procedure can give high
results. The results are high because oxygen enters
the sample from the air when the sample is collected,
when the copper sulfate-sulfamic acid inhibitor is
added, while the solids are settling, and when the
sample is transferred to a BOD bottle for the DO test.
8.D BOD test or volatile solids test.
14-189
-------
8.F. To prepare dilutions for a cannery waste with an expected
BOD of 2000 mg/1, take 10 ml of sample and add 90 ml of
dilution water to obtain a new sample with an estimated
BOD of 200 mg/1 (10 to 1 dilution);
BOD Dilution, ml «*
1200
EstimateTd BOD/ mg/l
1200
8.F
BOD,
mg/1
6 ml
Initial DO of
Diluted
pie, mg/1
pO of Diluted
Sample After
BOD Bottle Vol., ml
Sample Volume/ mJ
* (7.5 mg/1 - 3.9 mg/1)
500ml
* (3.6 mg/1) (130)
* 540 mg/1
8.G Samples for the BOD test should be collected before chlori
nation because chlorine interferes with the organisms in
the test. It is difficult to obtain accurate results with
dechlorinated samples,
8.H A solution of sodium thipsulfate at 0.0375 N is very weak
and unstable and will not remain accurate over two weeks.
9. A (1)
You wpuld measure thp fyS in the wastewater to know
the strength of H2S and an indication of the corrosion
taking place on the concrete.
(2) HaS in the atmosphere produces a rotten egg odor. It
is indicative of anaerobic decomposition of organics
in wastewater which occurs in the absence of oxygen.
10. A (1) To measure plant influent pH with a paper tape,
collect representative sample, mix sample with a
clean stirring rod, and dip tape in sample while
it is still moving. Compare tape color with pack-
age color and record resu 1 ts ,
(2) To measure raw sludge pH with a paper tape first
allow raw sludge sample to settle. Dip tape in
liquid at top, compare resulting color, and record
results.
pH of both samples should be measured in place or as soon
as possible.
14-190
-------
10. B Precautions to be exercised when usiftg a pH wet^r include: >
(1) Prepare fresh .buffer solution weekly for
calibration purposes.-.
(2) pH meter, samples, and buffer solutions should
all be at the same" temperature.
(3) Watch for erratic results arising from faulty*
operation of pH meter or fouling of electrodes
with interfering matter.
11. A Settleability tests should be run on the mixed liquor to
determine the settling characteristics of the sludge floe
at regular intervals for 60 minutes. The results -are used
in the SVI and SDI determinations.
11. B The SVI is the volume in ml occupied by one gram of mixed
liquor suspended solids, after 30 minutes, of settling.
ll.C The SVI test is used to indicate changes in sludge
characteristics.
11. D Sludge Density Index (SDI) = 100/SVI . - -
Sludge , '••.", ••-.'•••
12. A, t;° 1)1" :...= ;(Tot,al Vet ''So f. Removed, ml/1) (1000) (Flow, MGD)
-
gpd = (10 ml/1 - 0.4 ml/1) (1000 nig/ml) (1 M Gal/day)
9.6-,ml
M mg
-= 9600 gpd,
1000 ms
ml
Cal
day
This value may be reduced by 30 to 75% due to
compaction of the sludge•in -the clarifier.
13.A The sludge age of.a 200,000 gallon aeration tank that has
2000 mg/1 mixed liquor suspended solids, a primary effluent
of 115 mg/1 SS, and an Average.flow of 1.8 MGD:
'-,''•' • *" -
Sludge , ' Vol'of Aeration' Tank ' •"'" ,. , onnn /^
Age, - ;: >2MG .. - ^-Sus Solids, 2000 mg/1
days Flow, MGD,, .,1..,8 :x. Primary Effl','T15 rag/1
0.2 MG x 2000 mg/1 ' •
~ 1.8-MGD'X 115 mg/1
= 1.93
14-191
-------
14.A (1) Results from the graduated cylinder are available
immediately, but different operators may interpret
the results differently.
(2) Results are not available until the next day, but
different operators will record the same result.
15.A If the supernatant solids test is greater than 5%,
the supernatant could be placing a heavy solids load on
the plant and the appropriate operational adjustments
should be made.
16.A The specific gravity is very near that of H20 and is not
light enough to float nor heavy enough to settle.
16.B Solids calculations will be shown in detail here to illus-
trate the computational approach and the units involved.
After you understand this approach, use of the laboratory
work sheet on the following pages is more convenient.
a; Total Suspended Solids
Volume of Sample, ml = 100 ml
Weight of Dried Sample 5 Dish, grams = 19.3902 g
Weight of Dish (Tare Weight), grams = 19.3241^g
Dry Weight = 0.0661 g
or = 66.1 mg
Total
Suspended _ Weight of Solids^ m^jx J-OOO ml/1
Solids, Volume of Sample, ml
mg/1
66.1 mg x 1000 ml/1
100 ml
= 661 mg/1
b. Volatile Suspended Solids
Weight of Dried Sample § Dish, grams = 19,3902 g
Weight of Ash PT Dish, grams = 19.3469 g
Weight Volatile, grains = 0.0433 g
or 43.3 mg
14-192
-------
Volatile
Suspended _ Weight of Volatile, mg x 1000 ml
Solids, Volume of Sample, ml
mg/1
(43.3 mg) (10_0p| ml/1)
100 ml
= 433 mg/1
c. Percent Volatile Solids
o ,, ,.,..-, c TJ Weight Volatile, mg x 100%
% Volatile Solids = ~~-~e~————...-. ..>\,..A... ,—_
Weight Dry, nig
x 100%
661 mg 661 433.0
6
= 65.5% 36 40
33 05
" 3 350
3 305
d. Fixed Solids
Total Suspended Solids, mg/1 = 661 mg/1
Volatile Suspended Solids, rng/1 = 433 mg/1
Fixed Solids, mg/1 = 228 mg/1
e. Percent Fixed Solids
Total Solids, % = 100.00%
Volatile Solids, % = _65_._50%
Fixed Solids, % = 34.5 %
or
o ,-• j Fixed, mg „
% Fixed = —> *—~ x 100%
Total, mg
= 228jng x 10(J%
661 mg
= 34.5% (Check)
14-193
-------
16. C Calculate Percent Reduction through Primary:
% Removal = -..Out) x 100% In = Infl»ent tp
jn plant or unit
Out = What is leaving
plant or unit
- C221 mg/ 1-159 wg/1)
--- __ - _ x 100,
,7 ,28
100% 221 '62.0 "
44 2
= 28% reduction through primary ^
17 63
Calculate Percent Removal by Secondary System:
o r, ! (In - Out) ,_,.0 In = 159
% Removal = - - x 100% . __,
In primary effluent
Out = 33 mg/1 SS in
final effluent
= (159 mg/1 - 33 mg/1) «,
159 mg/1
.79
, * - 159/ 126.0
= 79% removal from primary
effluent to final effluent > - • •
14 70
J4 31
Calculate Overall Plant Efficiency:
% Removal - (In. T. PPtt. x 100% In = 2^ 1>g/l SS in
In plant influent
Out = 33 mg/1 SS in
plant effluent
- 33 m/1)
221 mg/1
x 100%
= 85.5% overall plant removal
14-194
-------
16,D Calculate the pounds of solids removed per day by each unit:
Amount
Removed, = Cone. Reduction, mg/1 x Flow, MGD x 8.34 Ib/gal
Ib/day
where MGD = million gallons per day
A. Influent, mg/1 = 221 mg/1
Primary Effluent, mg/1 = 159 mg/1
Primary Removal, mg/1 = 62 mg/1
Amount Removed, ,,„ ,.,. ,. _ .,„.,-. fa -.
Ib/day (Primary) = <62 mg/1) (1'5 MGD) (8"34
= 775.6 Ibs/day
removed by primary
B. Primary Effluent, mg/1 = 159 mg/1
Final Effluent, mg/1 = 53^ mg/1
Secondary Removal, mg/1 = 126 mg/1
Amount Removed, /i-./- /i-, ri r *tr-^ rn ~,» TL / i-.
Ib/day (Secondly) = (126 ^/D (1'5 MGD) (8'34 lb/gal)
• = 1576 Ib/day
removed by secondary
C. Influent, mg/1 = 221 mg/1
Final Effluent, mg/1 = 55 mg/1
Overall Removal, mg/1 = 188 mg/1
Amount
Removed, = (188 mg/1) (1.5 MGD) (8.34 Ib/gal)
mg/1
= 2351 Ibs/day
removed by plant
or = Primary Removal, Ib/day + Secondary, Ib/day
= 775 + 1576
= 2351 (Check)
14-195
-------
16.E The advantages of the centrifuge over the regular
suspended solids test are:
(1) Speed of answer! Not as accurate as
other methods, but results are sufficiently
close.
(2) Answers very acceptable if suspended solids
concentration is below 1000 mg/1.
Disadvantage: Small plants cannot always afford
the' $500 or "more cost of the centrifuge.
17.A Changes in influent temperature could indicate a new
influent source. A drop in temperature could be caused
by cold water from infiltration, and an increase in
temperature could be caused by an industrial waste dis-
charge.
17.B The thermometer should remain immersed in the liquid
while being read for accurate results. When removed
from the liquid, the reading will change.
17.C All thermometers should be calibrated against an
accurate National Bureau of Standards thermometer
because some thermometers can be purchased that
are substantially inaccurate (off as much as 6°).
18.A Volatile solids found in a digester are organic compounds
of either plant or animal origin,
18.B Volatile solids in a treatment plant represent the waste
material that may be treated by biological processes.
20.A Volatile Acid ml 0.05 N NaOH x 2500
Alkalinity, mg/1 ml Sample
5 ml x 2500
50 ml
= 250 mg/1
Since 250 mg/1 > 180 mg/1,
Volatile Acids, .. . . ., .-,»,,,- -^ , ™
,, = Volatile Acid Alkalinity x 1.50
= 250 mg/1 x 1.50
= 375 mg/1
14-196
-------
20.B The alkalinity test is run to determine the buffer capacity
and the volatile acids/alkalinity relationship in a digester.
20.C The buffer capacity in a digester as measured by the total
alkalinity tests indicates the capacity of the digester to
resist changes in pH.
VoJLatile Acid = 500 mg/1
Alkalinity 2000 mg/1
= 0.15
14-197
-------
OBJECTIVE TEST
Chapter 14. Laboratory Procedures and Chemistry
Name Date
Please write your name and mark the correct answers on the IBM answer
sheet. There may be more than one correct answer to each question.
TRUE OR FALSE (1-10):
1. A rubber bulb should be used to pipette wastewater or polluted
water.
1. True
2. False
2. Acid may be added to water, but not the reverse.
1. True
2. False
3. Always, wear safety goggles when conducting any experiment in which
there may be danger to the eyes.
1. True
2. False
4. Smoking and eating should be avoided when working with infectious
material such as wastewater and sludge.
1. True
2. False
5. In the washing of hands after working with wastewater, the kind
of soap is less important than the thorough use of soap.
1. True
2. False
6. The pH scale may range from 0 to 14, with 7 being a neutral solution.
1. True
2. False
14-198
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7. If at all possible, samples for the BOD test should
be collected before chlorination.
1. True
2. False
8, The COD test is a measure of the chemical oxygen
demand of wastewater.
1. True
2. False
9. The BOD test is a measure of the organic content of
wastewater.
1. True
2. False
10. The answers from the total solids and suspended solids
test are always the same.
1. True
2. False
Possible definitions of the words listed below are given on the
right. For each word listed on the left, try to find its defini-
tion on the right. Mark the number of the definition in the
answer column for each word. For example, if the definition of
a word is after the number 2, mark column 2 on your answer sheet
after the word.
Word Definition
1. Surrounding
11. Aliquot 2. Capacity to resist pH change
12. Ambient 3, Portion of a sample
13. Blank 4. Inside
14. Buffer 5. Test run without sample
15. Large errors in laboratory tests may be caused by:
1. Improper sampling
2. Large samples
3. Poor preservation
4. Poor quality effluent
5. Lack of mixing during compositing
14-199
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16. The most critical factor in controlling digester
operation is the:
1. C02
2. Gas production
3. Volatile solids
4. Volatile acids/alkalinity relationship
5. pH
17. The COD test:
1. Measures the biochemical oxygen demand
2. Estimates the first-stage oxygen demand
3. Measures the carbon oxygen demand
4. Estimates the total oxygen demand
5. Provides results quicker than the BOD test
18. A clarity test on plant effluent:
1. TellsMf the effluent is safe to drink
2. Is measured by an amperemeter
3. Should always be measured at the same time
4. Should always be measured under the same light conditions
5. Is measured by a Secchi Disc
19. Coliform group bacteria are:
1. Measured by the membrane filter method
2. Measured by the multiple fermentation technique
3. Measured by the modified Winkler procedure
4. Harmful to humans
5. Indicative of the potential presence of bacteria
originating in ttie intestines of warm-blooded animals
20. The saturation concentration of dissolved oxygen in
water does not vary with temperature.
1. True
2. False
21. DO probes are commonly used to measure dissolved oxygen
in water in:
1. Aeration tanks
2. Sludge digesters
3. Manholes
4. Streams
5. BOD bottles
14-200
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22. Hydrogen sulfide:
1. Reacts with moisture and oxygen to form a substance
corrosive to concrete
2. Is sometimes written as I^S
3. Smells like rotten eggs
4. Is formed under aerobic conditions
5. Should not be controlled in the collection system.
23. Results from the settleability test of activated sludge
solids may be used to:
1. Calculate SVI
2. Calculate SDI
3. Calculate sludge age
4. Determine ability of solids to separate from liquid
in final clarifier
5. Calculate mixed liquor suspended solids.
24. Results of the settleable solids test run using Imhoff
cones may be used to:
1. Calculate the Imhoff Settling Index
2. Calculate the efficiency of a treatment process.
3. Calculate the pounds of solids pumped to the digester
4. Indicate the quality of the influent
5. Indicate the quality of the effluent
25. Precautions that must be observed in running the suspended
solids-Gooch crucible test include:
1. Collecting and testing a representative sample
2. Proper temperature level in oven at all times
3. Lack of leaks around and through the glass fiber
4. Thoroughly mixing sample before testing
5. Discarding any large chunks of material in sample
26. A chlorine residual should be maintained in a plant effluent:
1. To keep the chlorinator working
2. For disinfection purposes
3. For testing purposes
4. To protect the bacteriological quality of the receiving
waters
5. None of these
14-201
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PLANT
DATE
SUSPENDED SOLIDS & DISSOLVED SOLIDS
, SAMPLE
Crucible
Ml Sample
Wt Dry & Dish
Wt Dish
Wt Dry
/I _ Wt Dry, gm x 1 ,000,000
; Ml Sample
: ' Wt Dish & Dry
Wt.Dish & Ash
-.- . yt-V.Qla.t11e
1 Vnl - Wt Vo1 x 100
-•. ^•"rM-.vWfr.Dry x IUU
BOD
Nitrate
Sample
Graph Reading _
COD
Sample
Blank Titration
Sample Titration
Depletion
mnn Pep x N FAS x 8000
mg/1 = Ml Sample -
# Blank
'SAMPLE
DO Samole
Bottle #
% Sample
Blank or adj blank
DO after incubation
Depletion, 5 days
Dep %
Sett. Solids
Sample
Direct Ml/I
Typical Laboratory Work Sheet
-------
TOTAL SOLIDS
SAMPLE
Dish No.
Wt Dish & Wet
Wt Dish
Wt Wet
Wt Dish +
Wt Dish
Wt Dry
% Solids
Dry
Wt Dry
Wt Wet
x 100%
o1
x 100%
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
% Volatile = * n
Wt Dry
pH
Vol. Acid
Alkalinity as CaC03
Grease (Soxlet)
Sample
Ml Sample
Wt Flask + Grease
Wt Flask
Wt Grease
ma/1 = Wt Grease, mg x 1000
y Ml Sample
H2S (Gas) (Starch-Iodine)
Blank
Sample
Diff ZZ
Diff x .68
mg/1 x 43.6
Ml
Ml
Ml
mg/1
grain/100 cu ft
Typical Laboratory Work Sheet (Continued)
14-203
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
Wastewater Laboratory Procedures and Chemistry
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
1O. PROGRAM ELEMENT NO.
U. S. Environmental Protection Agency
Office of Intermedia Programs
Manpower and Training Program
Region VII, 1735 Baltimore, Kansas City, Missouri 64108
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This manual has been adapted from Chapter 14 of "Operation of Wastewater Treatment
Plants - A Field Study Course" for limited distribution in Region VII. This manual
is intended to serve as a training reference material for personnel of the Regional
^Surveillance and Analysis Program to assist them in providing assistance to treatment
plant operators who have been identified as being in need of greater skills to perform
necessary laboratory analyses.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COS AT I Field/Group
Water Pollution Control Laboratory
Analysis
Water Pollution Control
DISTRIBUTION STATEMENT
LIMITED
19. SECURITY CLASS (This Report)'
UNCLASSIFIED
21. NO. OF PAGES
240
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
14-204
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