LABORATORY ANALYSES IN
TREATMENT PLANT OPERATIONS
TRAINING MANUAL
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF WATER PROGRAMS
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LABORATORY ANALYSES IN
TREATMENT PLANT OPERATIONS
This course is offered for operators and other
personnel responsible for laboratory control and
management in wastewater treatment.
ENVIRONMENTAL PROTECTION AGENCY
Water Programs Operations
TRAINING PROGRAM
December 1972
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FOREWORD
These manuals are prepared for reference use of students enrolled in
scheduled training courses of the Office of Water Programs, Environmental
Protection Agency.
Vu.e to the ZA.mA.tzd ava-cZab-ttA-ty o 6 the manuals,
A.t A.A not appn.opn.A.ate to c.A.te. them cu tec.hnA.eal
n-e^enencei -tn bA.b-tA.ogn.apliA.ei on. othen fionmi o&
pubZA.catA.0 n.
Refienence-i to pnodueti and manu.6actun.eni a>te ion.
¦cZZuitnatA.on ontij, iuch nefien.encei do not -empty
pnoduct endofii ement by the O^A-ce o<5 Waten. Pn.ogn.ami,
E nvA.no nmentat Pn.0tect4.0n A genet/.
The reference outlines in this manual have been selected and developed with
a goal of providing the student with a fund of the best available current
information pertinent to the subject matter of the course. Individual
instructors may provide additional material to cover special aspects of
their own presentations
This manual will be useful to anyone who has need for information on the
subjects covered However, it should be understood that the manual will
have its greatest value as an adjunct to classroom presentations. The
inherent advantages of classroom presentation is in the give-and-take
discussions and exchange of information between and among students and
the instructional staff.
Constructive suggestions for improvement in the coverage, content, and
format of the manual are solicited and will be given full consideration.
Joseph Bahnick
Acting Chief
Direct Technical Training Branch
Division of Manpower and Training
Office of Water Programs
Environmental Protection Agency
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TRAINING PROGRAM
Through the Office of Water Program, Environmental Protection Agency conducts
programs of research, technical assistance, enforcement, and technical
training for water pollution control.
Training is available at five installations of the Agency. These are- the
National Training Center located at the Robert A. Taft Sanitary Engineering
Center in Cincinnati, Ohio, the Robert S. Kerr Water Research Center, Ada,
Oklahoma, the Southeast Water Laboratory, Athens, Georgia, the Pacific
Northwest Water Laboratory, Corvallis, Oregon, and the Hudson-Delaware
Basins Office, Edison, New Jersey.
The objectives of the Training Program are to provide specialized training in
the field of water pollution control which will lead to rapid application of new
research findings through updating of skills of technical and professional
personnel, and to train new employees recruited from other professional or
technical areas in the special skills required. Increasing attention is being
given to development of special courses providing an overview of the nature,
causes, prevention, and control of water pollution.
Scientists, engineers, and recognized authorities from other Agency programs,
from other government agencies, universities, and industry supplement the
training staff by serving as guest lecturers. Most training is conducted in the
form of short-term courses of one or two weeks' duration. Subject matter
includes selected practical features of plant operation and design, and water
quality evaluation in field and laboratory. Specialized aspects and recent
developments of sanitary engineering, chemistry, aquatic biology, microbiology,
and field and laboratory techniques not generally available elsewhere, are included.
The primary role and the responsibility of the States in the training of wastewater
treatment plant operators are recognized. Technical support of operator-training
programs of the States is available through technical consultations in the planning
and development of operator-training courses. Guest appearances of instructors
from the Environmental Protection Agency, and the loan of instructional materials
such as lesson plans and visual training aids, may be available through special
arrangement. These training aids, including reference training manuals, may be
reproduced freely by the states for their own training programs. Special categories
of training for personnel engaged in treatment plant operations may be developed
and made available to the States for their own further production and presentation.
An annual Bulletin of Courses is prepared and distributed by the Office of Water
Programs. The Bulletin includes descriptions of courses, schedules, application
blanks, and other appropriate information. Organizations and interested indi-
viduals not on the mailing list should request a copy from one of the training centers
mentioned above.
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U.S. Environmental Protection Agency
OFFICE OF WATER PROGRAMS
MANPOWER DEVELOPMENT STAFF
R. F. Guay, Director
Academic Training Branch
State and Local Operator Training
Programs
Office of Environmental Activities
Direct Technical Training Branch
National Training Center
Cincinnati, OH 45268
REGIONAL MANPOWER OFFICES
REGION I
Manpower Development Branch
Division of Air and Water Programs
424 Trapelo Road
Waltham, MA 02514
REGION II
Manpower Development and Training Offic
Air and Water Programs
26 Federal Plaza
New York, NY 10007
REGION III
Manpower Development Office
Air and Water Programs
Curtis Building
6th and Walnut Streets
Philadelphia, PA 19106
REGION IV
Manpower Development Branch
Division of Air and Water Programs
1421 Peachtree Street, NE, Fourth Floor
Atlanta, GA 30309
REGION V
Manpower Development Branch
Office of Air and Water Programs
1 N. Wacker Drive
Chicago, IL 60606
REGION VI
Manpower Development Branch
Air and Water Programs Division
1600 Patterson
Dallas, TX 75201
REGION VII
Manpower Development Branch
Air and Water Programs
1735 Baltimore
Kansas City, MO 64108
REGION VIII
Manpower Development Branch
Air and Water Division
1860 Lincoln Street - 9th Floor
Denver, CO 80203
REGION IX
Manpower Development Branch
Air and Water Division
100 California Street
San Francisco, CA 94111
REGION X
Manpower and Training Branch
Division of Air and Water Programs
1200 Sixth Avenue - Mail Stop 345
Seattle, WA 98101
7. 11. 72
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CONTENTS
Title or Description Outline Number
The Operator's Responsibilities and the Analyst's
Part in Them 1
Principles of Record Keeping 2
Sampling in Wastewater Treatment Operations 3
Laboratory Housekeeping 4
Variations in Test Results 5
Analytical Reactions - Standard Solutions 6
The Aquatic Environment Part I: The Physical Nature
of Water and Waters 7
Collection and Handling of Samples for Bacteriological
Examination 8
Bacteriological Indicators of Water Pollution 9
Examination of Water for Coliform and Fecal Streptococcus
Groups 10
Use of Tables of Most Probable Numbers 11
Membrane Filter Equipment and Its Preparation for
Laboratory 12
Membrane Filter Laboratory and Field Procedures 13
Dissolved Oxygen Determination (DO) - I
Winkler Iodometric Titration and Azide Modification 14
Laboratory Procedure for Dissolved Oxygen
Winkler1-Azide Procedure 15
Dissolved Oxygen Determination - II
Electronic Measurements 16
Biochemical Oxygen Demand Test Procedures 17
Biochemical Oxygen Demand Test Dilution Technique 18
BOD Determination - Reaerated Bottle Probe Technique 19
Effect of Some Variables on the BOD Test 20
105.12.72
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2
CONTENTS
Title or Description Outline Number
Chemical Oxygen Demand and COD/BOD Relationships 21
Laboratory Procedure for Routine Level Chemical Oxygen
Demand 22
Total Carbon Analysis 23
Acidity, Alkalinity, pH and Buffers 24
Alkalinity and Relationships among the Various Types
of Alkalinities 25
Laboratory Procedure for Total Alkalinity 26
Laboratory Procedure for Total Acidity 27
Determination of Chloride in Water Supplies 28
Determination of Sulfate in Water Supplies 29
Sources and Analysis of Organic Nitrogen 30
Ammonia, Nitrites and Nitrates 31
Laboratory Procedure for Nitrate Nitrogen Modified
Brucine Method 32
Determination of Phosphorus in the A quatic Environment 33
Laboratory Procedure for Phosphorus 34
Solids Relations in Polluted Water 35
Determination of Suspended Solids 36
Laboratory Procedure for the Determination of Total Solids 37
Laboratory Procedure for Nonfilterable (Suspended) Solids 38
Laboratory Procedure for Filterable (Dissolved) Solids 39
Laboratory Procedure for Volatile Solids 40
The Determination of Oil and Grease 41
The Basis for Chlorination of Wastewaters 42
Chlorine Determinations and Their Interpretation 43
Powers Data Sheet No. 397
Glossary - Wastewater Treatment Technology
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I INTRODUCTION
Wastewater Treatment Plant Operation involves the combined
efforts of process and equipment knowledge, diverse perform-
ance skills related to acquiring information, and using this
information effectively within the framework of situation, time,
place and human events to enhance the reuse potential of our
water resources. An operator is many things, conversely, it
requires many individuals contributing to the dynamics of
effective operations.
Contents of Section I
Outline Number
The Operator's Responsibilities and the
Analyst's Part in Them
1
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THE OPERATOR'S RESPONSIBILITIES AND THE
ANALYST'S PART IN THEM
I INTRODUCTION
A Considered as a whole, most wastewater
treatment plants serve rather small popu-
lations. Most operating staffs consist of
less than five operators, often only two.
B Analysts usually have some responsibilities
as operators. Not infrequently the plant
superintendent and shift operator share
the laboratory work and record keeping.
C In all plants, regardless of size and com-
plexities, meaningful analyses and records
are essential and vital to effective opera-
tional control, for planning and for com-
munication with others both within the plant
and outside interests.
D Todays higher goals for water resource
management require refinements and
intensification of process control. In-
creasingly complex and sophisticated
analytical procedures will be required in
an ever increasing percentage of plants.
II Working and living with others requires
assumption of broad and varied aspects of
responsibility - often automatically.
A Teamwork Requires
1 Coordination and direction
2 Cooperation of each member
3 Subordination of individual to total
objectives
4 Sharing in rewards of success
5 Sharing in burdens of disappointment
or failure.
B Responsibility
1 Implies understanding between
a One performing a task or duty and
b One assigning it
2 Proper relationship requires under-
standing of
a Nature of the obligation
b Conditions governing its execution
3 Must be accepted willingly - not
grudgingly
ID RESPONSIBILITIES OF THE OPERATOR
At any given plant the responsibilities and
obligations of the operating staff change
markedly as facilities age, undergo modi-
fication, and as performance requirements
are elevated.
A New Plant Breakin
1 Become familiar with
a Each element of the plant
b Treatment objectives
c Characteristics of receiving waters
2 Identify deficiencies
3 Recommend changes and modifications
to
a Correct deficiencies
b Add needed improvements
PC. 13. 10. 67
1-1
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The Operator's Responsibilities and the Analyst's Part in Them
4 Acquaint designer with reasons for
recommendations
5 Determine waste characteristics
6 Establish sampling and testing program
7 Develop operating schedules
8 Plan training program as personnel
are assigned to plant
9 Prepare initial budget
10 Confer with industries on
a Waste problems
b Restrictions and limitations
c Pre treatment
11 Suggest values for sewer use ordinance
B Routine Operation
The operator and analyst must look con-
stantly for ways to strengthen, refine and
modify methods to improve plant
performance.
1 Establish effective process control by
a Application of test results and
other observations
b Adjustments in operational and
maintenance practices
2 Identify adverse effects of industrial
wastes on
a Plant processes
b Receiving stream
3 Correct adverse effects of industrial
wastes
4 Keep useful meaningful records
a Physical
b Chemical
c Bacteriological - biological
5 Identify deficiencies in facilities
6 Provide officials with information for
a Budgeting and accounting
b Overall management
c Planning
7 Provide subordinates with
a Good working conditions
b Training opportunities
c Incentives
8 Prepare for plant improvements
a Enlargements
b Replacements
c Higher degree of treatment
9 Establish and maintain good public
relations
10 Comm»nicate effectively with
a Other members of staff
b Responsible local officials
c Local industries
d Selected groups (schools, service
clubs)
e General public
f Regulatory officials
g Other operators
1-2
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The Operator's Responsibilities and the Analyst's Part in Them
IV KEEPING INFORMED
The operator and the analyst are professionals
or should be. To be successful they must
keep abreast of effective methods and prac-
tices of others in their field and of new
developments as they are reported.
A Selective Reading of Technical Literature
1 Journals
2 Manufacturer's bulletins
3 Texts
B Attend Technical Meetings
1 Water Pollution Control Associations
2 Professional societies
C Participate in Training Courses
1 In plant
2 Water Pollution Control Association
3 Regulatory agencies
D Consult with Regulatory Agency
Personnel
V DIVIDENDS OF PROFESSIONALISM
Many dividends accrue from top level per-
formance, some tangible, others not easily
defined.
A Tangihle
1 Top performance of facilities
2 Complete, accurate, useful records
3 Salary benefits
4 Advancement
B Less Tangible
1 Respect and appreciation of
a Associates
b The boss
c The public
2 Leadership opportunities
3 Helping others
4 Passing Operator Certification Exam
5 Self esteem
E Visit with Other Operators and Analysts
This outline was prepared by D. M. Pierce,
1 At meetings Chief, Wastewater Section, Division of
Engineering, Michigan State Department of
2 At their plants and yours Health, Lansing, Michigan.
1-3
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II BASIC LABORATORY OPERATIONS
The team effort among individuals requires an effective
chain of events before, during, and after the action for
guidance on what to do, how to do it, and informing others
with respect to the action taken. This section considers
certain elements essential for information procurement,
validity and availability.
Contents of Section II
Outline Number
Principles of Record Keeping
2
Sampling in Wastewater Treatment
Operations
3
Laboratory Housekeeping
4
Variations in Test Results
5
Analytical Reactions - Standard Solutions
6
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PRINCIPLES OF RECORD KEEPING
I Records may not be used extensively by
those who prepare them, hence, they may
be regarded as an unessential evil. They
need to be reviewed for functional use to
limit pyramiding but its never quite certain
whether a given item will be needed. The
only common feature is the frustration
associated with the lack of an item that is
being sought.
A All functional records have one common
objective - communication among those
who have and those who need information.
1 Records may convey information from
those who do the work to those who
direct it.
2 Budget considerations are based upon
past records and future plans.
3 Stock records are a means to determine
what must be on hand to give a reason-
able probability of having a given item
when and where it is needed.
4 Performance records indicate:
a Operation efficiency
b Compliance or non-compliance with
objectives
c Help to identify and enumerate
problem areas.
5 Records may be used to provide a key
for optimization or selection of pro-
cesses, methods, individuals.
6 The record is an indication of good and
bad decisions and a source of satisfac-
tion for a job well done.
7 It may be used to create awareness and
recognition among the people served.
B Reports are of interest to a variety of
personnel of different backgrounds.
1 Supervisors are interested in what is
being done in terms of the performance
of operations, individuals, machines,
finance, etc., for planning and con-
trolling performance.
2 The public is interested in what they
are getting for their investment.
3 Plant employees are interested in their
particular contribution to the whole
operation in performance or improvement.
a The report may be a summary for
comparison of one plant in relation
to that of other plants of a similar
nature.
4 Governmental planning agencies or
administrators are interested in com-
parisons among the what is and what
ought to be done.
5 Apprentices or students seek reports
for guidance in their development.
6 Engineering problems involving selec-
tion, consultation, design, are generally
based upon reports of full-scale or
development scale operations.
C Records and reports may be classified
roughly according to the source or type
of information noted, purpose, and in-
tended contact.
1 Sensory impressions of color, odor,
appearance, and feel, convey real and
understandable concepts to the public
and to interested parties. These may
not be scientifically precise but the
impact may be tremendous.
PC. 2. 10. 67
2-1
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Principles of Record Keeping
2 Reports may contain strictly physical
measurements of length, width, flow,
volume, rate, time in relation to events.
3 The laboratory record may be expected
to contain sample identifications,
numeration of direct results and tech-
niques applied whether for biology,
chemistry or microbiological disciplines.
These are integrated into derived in-
formation including that necessary to
recheck calculations, observations on
possible interference and related
effects or conclusions.
4 The engineering report may include
conclusions and recommendations based
upon reports of the physical features,
economics, history, scientific data,
etc., for planning, design, etc.
5 The supervisors annual report may
contain performance items including
total, maximum, minimum, budget
review, major difficulties, special
developments during the year and future
plans.
6 The report is likely to represent a single
concept item giving pertinent observa-
tions and integration along one subject
or cover multiple subject areas m rela-
tion to some pertinent characteristic.
7 Any report is intended for some contact
or audience - it should be written and
structured to communicate accordingly.
a The log book for personal reference
or guidance should contain a detailed
list of work done, observed items,
techniques applied, dates, calcula-
tions and conclusions at the time.
b A report to the supervisor should
delete minor details but convey
what was done, briefly describe
methods and present results and
recommendations.
c Reports to the administrator should
stress the subject matter according
to broad aspects, giving pertinent
information and conclusions in brief,
concise, language with a minimum
of technical jargon.
d Public information releases should
contam brief single concept items
indicating some phase of the work
that is important to them, what was
done about it, or needs to be done
to contribute to their welfare.
Technical language is acceptable
if it can be explained briefly in
terms meaningful to the public.
Candor, briefness, and enthusiasm
are essential to create and hold
public trust.
II The annual operating report is a recurrent
obligation that describes your efforts to meet
a public trust and should reflect your capa-
bilities in management, scheduling, technical
and public information.
A Information should be assembled promptly
month by month to be reacfy for compilation
into an annual shortly after the closing
month.
B Review the annual report general plan with
your administrative board and solicit their
enthusiasm and backing for an informative
and interesting production. Obtain a com-
mitment on budgeting, possible use of
outside services, and personnel. Present
an outline of the annual.
C Assemble information and accessories for
production in accordance with previous
commitments. Work up layout and pro-
duction facilities and roll the presses.
An annual report is ancient history if it
is released a year later.
D It should include in an attractive format:
1 A Title Page including identification of
the plant, the city, Administrative
Board or Commission with names of
principal officials and that it is a report
of operations for the stated interval.
This preferably should be associated
with a letter of transmittal to the mayor
or other principal designated official.
2-2
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^r^cigleg ,of Record Keeping
2 Provide a brief resume of major prob-
lems or ,developmentscwi£hWllli£ period
covered.; iVplume oripounds removed'^
per dax.or\per:yearnn (.comparison td
previous;load ls.atgood start1.1'1-Mentibn
progress of new construction or'plariQr
mng .with principab.difficillties^mehtib'ned
in terms oft personn^vaequipment^ i°ncd
loading induptnaliwastes7utotarrs'ewer
extension-.puThis should' be :iri "'Th'l§
is a thumbnail' ske£chvakeep?it;that Wny3,
but it mayobe.thevOnly.partfre'adti'1' way»
f o ¦ .1 . Oil" •; <\\id.
3 Present a diagram of the plant showing
operations and, flow pattern 1 ^rpi.rctQre98
increase.interest»> Review"ipur^b"^e,res
plant and receiving water.' our no ,
> , .i;_ im i x
4 Present an organization chart with prin-
cipal officials and back> up isupport'if1 in
possible outlining, responsibilities"^
each major slot. of
5 Present a resume of plant history amjj
changes in facilities; objective'si com-
munity, or receivings water.,
i " ' c
6 Present a resume of future plans, or-
progress of construction.
7 Review area or sewer extensions.,
8 Review special services, delegated
or not, that may be of interest to,
plant-community relations.
9 Present a summary chart of flow,
strength, solids received/removed by
month with plant efficiencies indicated.
10
10
tovfere8. Stress improvement^ gr
roveron. btress impm u,
plans for improvement. If obiectionr
Rbl^VondftTons0 oecurred what happened?
mStccanor0il jaeihg done about' it? "
"'hat can pr is Beihg aart' iKmu
ssFfeat his to:
BeviJw te&il iflfl
oDmen
developmen
13
13
a
a
iStnfe rrlCc°Q»^°^
6r immediate attention.
or immediate aitenuon.
Present charted details, oxu.
Present cnarted uei aTL xrrr
^tft\CAecn^yv
(foVveerr^l 0oPpee^ifi°J^.
bb Ccposs\0if^r^0A^y^^y ^
general. ter^fpj^M^afcipn, bonded
fe'f, °orr
Cc »
¦4 ftfi- Wk I?®&
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SAMPLING IN WASTEWATER TREATMENT OPERATIONS
I INTRODUCTION
A A sample is defined as a representative
part, aliqiDt or single item from a larger
group or whole to be presented for inspec-
tion or testing for quality, also, a portion
of a statistical population whose character-
istics are studied to gain information about
the whole.
B A sample representing materials at a given
time, place and flow may be called a "catch"
or "grab" sample. A series of grab samples
obtained over a specified period of time from
the sample flow becomes a "composite"
sample when all individual samples are
combined into one. If the individual grab
sample volumes are adjusted in proportion
to flow at the time they were obtained and
before combining them the resulting mixture
is a "proportional composite. "
C Sampling requires careful observance of
procedures to be meaningful.
1 The scale up factor from the sample to
the whole commonly is very large.
Behavior of a 1 ml sample of the incoming
flow in a BOD dilution may be used to
estimate loading, a 0. 001 ml sample may
be used for estimation of membrane
filter coliform group organisms for many
million gallons of flo,v.
2 The sampler is obligated to insure that
his sample does represent the material
sampled and is protected from inadvertent
changes. Proper attention to detail and
identification is imperative.
3 Grab samples of flow into and out of process
are likely to give misleading information
if process detention time has been ignored.
If both are obtained at about the same
time a mid morning influent sample may
represent peak loading while the effluent
sample represents what entered the plant
at minimum load period.
D Changes are the principal reason for sampling
and testing for evaluation of treatment
performance.
1 Loading may be expected to change by
the hour, day, season and population
activity both in characteristics and
concentration. The long term trend
is upward.
2 Water use and requirements change.
3 Physical facilities tend to change with
time and use.
4 Operating personnel, capabilities, numbers
and motivation change with budgets,
organization and public interest.
E Sampling and testing are used to-
1 Estimate work done or remaining to
be done.
2 Identify problems.
3 Evaluate problems and response for
planning, process techniques or
equipment selection, and operational
competence purposes.
4 To evaluate compliance with requirements.
F Sampling is the initial step m a complex
series such as inspection testing interpre-
tation and active response to information
gathered. Many factors affect situation
dynamics such as objectives, probabilities,
situations, time, money, items to be
sampled and particular needs. Manpower
capabilities, skills, background, motivation
and bias have a large effect upon usefulness
of information gathered.
1 There are no fixed rules of sampling
applicable to every type of material,
item sought situation or purpose.
2 Sampling must be coordinated among those
who have or can secure and those who
need information. Each must be aware of
and respond consistently to site selections,
scheduling, techniques and communication
to be effective.
SE. MAN. rr. 3. 8. 72
3-1
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Sampling in Wastewater Treatment Operations
3 Integrity and reliability of all personnel
concerned are essential. Any lapse in
consistency results in loss of control
time money with concurrent suspicions
with respect to validity of subsequent
actions.
G This outline reviews selected factors
pertinent to sampling. It is not intended
to be complete because situations and needs
differ. References (1, 2, 3) cite specifics
for each type of item to be analyzed. Each
regulatory agency selects certain items from
standard compendia that are important to
their interest and to those in their jurisdiction.
This outline stresses considerations or judg-
ment factors. Authorities specify procedures.
II SAMPLING OBJECTIVES:
Samples are obtained because someone
needs precise information about some
material, situation or action. Constraints
and requirements for sampling and testing
depends upon who needs information, what
kind, and why. Cost commonly modifies
the action.
A Routine Record Sampling Reports:
The major wastewater treatment plant
sampling effort commonly is associated
with periodic reports to supervisors or
regulatory agencies. Sites, frequency,
techniques and handling usually are
prescribed specifically for the situation
by management and/or consultants
according to requirements of the regula-
tory agency. Requirements vary depending
upon:
1 Discharge or receiving water quality
requirements.
2 Size of the facility.
3 Discharge location in relation to
critical reuse of the receiving water.
4 Manpower budget and complexity of
treatment.
5 Load characteristics, variation, and
their relationships to design loading.
6 Public interest and activity
7 Enforcement
B Process Control Sampling1
Control sampling is intended to estimate
what is likely to happen in plant performance
rather than what has happened as in routine
operating reports. Both record and control
sampling and testing are used for performance
control. The emphasis for control sampling
and testing is to recover information in
time for corrective action before little
problems become big problems.
1 Control sampling usually is used to
characterize in-process variables
with respect to stage of completion,
environmental condition or rate of
change.
2 Control sampling and testing are used
to provide rapid estimates of process
variables that are relateable to treatment
performance and to suggest corrective
action when atypical values are evident.
Useability is more important than high
precision, and accuracy.
3 Control information includes sensory
impressions, meter readings, biological,
chemical or physical criteria. The
sampler is expected to note any unusual
conditions that may be useful for inter-
pretation of results.
4 Control applications, nature, extent of
sampling and testing vary with the
treatment and requirements and with
staff personnel customs, capabilities,
background, motivation and working
budget.
C Sampling for Enforcement Purposes:
Much more attention to detail is required
for legally acceptable sampling for courtroom
procedures than for most other sampling
operations.
3-2
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Sampling in Wastewater Treatment Operations
1 Professionally and legally accepted
methods by recognized capable trained
personnel are mandatory.
2 Documentation is essential for the history
of the sampling operation and subsequent
handling. A signed record of time,
place, conditions and sample identification
number must be logged. The sample
requires sealing and tagging for identifi-
cation and record of who did what and
when. Storage arid transfer history by
responsible individuals is essential.
3 Precise identification procedures, and
protection from tampering are main
sampling considerations. Any fact of
sample history is subject to recall by
the court.
4 Careful review of procedures and legal
guidance with respect to specifics of
the given jurisdiction are important to
enhance possibilities for information
acceptance by the court.
D Complaint Follow-up-
This usually means a special purpose
inspection and possible sampling to evaluate
the complainant's claim(s) and to guide
subsequent action. The complaint may
refer to routine or nonroutine characteristics
in the collection system treatment plant
or receiving water. Prompt and knowledgeable
handling m an honest and diplomatic manner
including feedback of communication to the
complainant through channels are good
public relations.
E Treatment Plant Design Data
Much of the information essential for design
purposes is available from previous records.
Sampling and testing to check routine data
and to evaluate unusual or difficulty treatable
wastewater mixtures are essential to provide
a versatile and reliable facility capable of
meeting foreseeable treatment requirements.
1 Loading amount and characteristics
including anticipated new contributions
and their effects are pertinent.
2 Selection of new processes or new
equipment and their performance
under conditions similar to that
anticipated commonly require
sampling and testing.
3 Sampling and testing to meet upgraded
performance requirements with
supplementary or alternate treatment
are indicated.
F Research and Development-
Investigation of new schemes, recom-
binations of established operations,
upgrading treatment with respect to
conventional removals or the removal
new or different contaminants require
a more comprehensive sampling and
testing effort than any subsequent
application of the same nature. Establish-
ment of guidelines for methodology and
interpretation are essential for the
different situation.
1 Sampling techniques and frequency
for that situation may be different
depending on feed or process
variability.
2 New interferences may be encountered
that require different handling techniques
in sampling or testing.
3 Sampling and testing are intended to
minimize surprises that may reduce
cost effectiveness during subsequent
applications.
Ill SAMPLING PRECAUTIONS BASED UPON
ITEMS TO BE SAMPLED
Biological, chemical or physical criteria
have their own characteristics and tendencies
to change. The sampler should apply
suitable techniques to obtain a representative
portion of the intended .material and to
minimize changes before the assay.
A Rapidly reacting chemicals may li/drolyze,
oxidize, form addition substitution or
complexed products different from those
3-3
-------
Sampling in Wa stewater Tr eatment Ope rations
existing at the time of sampling. High
rates of component reaction in the sample
always complicates sampling precautions
and may require the sampler to become
an analyst.
1 Storage at low temeratures is common
practice for decreasing sample reaction
rates. Freezing may be advisable.
2 Biological changes may be reduced by
adjusting the sample pH to unfavorably
high or low values, or by addition of
toxic chemicals.
3 Sample component hydrolysis such as
formation of free fatty acids and ammonia
from more complex materials is difficult
to control. Low temperatures reduce
the rate but added preservatives may
increase hydrolysis. Prompt determination
of unmodified fresh sample material is
the best choice in these situations.
4 Chemical addition to convert the item
sought into a less reactive derivative
may be used effectively such as using
zinc salts to enhance possibilities for
recovery of sample hydrogen sulfide.
5 Any modification of sample components
by addition or other treatment must not
interfere with subsequent determinations
or alter the quantities of the items sought.
The sampler is obligated to use accepted
sample modifications only and to clearly
identify treatment provided 'or guidance
in testing and interpretation.
B Gas - liquid sampling presents troublesome
situations because gas solubility varies
with pressure. If the gas is highly reaotive
such as chlorine, HCN, CCL, ®2* °r
NH the problems are magnified
0
1 Sampling profiles from top to bottom in
tanks means that each 2 feet of depth is
associated with a pressure increase of
about 1 p.s.i. Gases such as carbon
dioxide are much more soluble with
increasing depth, CO also tends to be
produced in active soxids suspensions
or deposits. The pH in treatment, plant
systems changes rapidly with COg,
HCOg and CO^3 dynamics. Any
pressure reduction or turbulence at
reduced pressure enhances loss of
and concurrent rise in pH of the
displaced sample. Electronic sensors
and in place measurement of pH permits
rapid readout without pressure change
effects upon results.
2 Dissolved oxygen solubility varies with
temperature and pressure. It also tends
to be rapidly used in active systems and
may be depleted m deposit vicinities. An
oxygen deficient sample rapidly increases
m oxygen content upon contact with air.
In-place measurement by electronic
sensors avoids possible changes due to
pressure and reaction.
3 Sampling controls including pressure,
chemical fixation, protection from
exposure etc., are possible. Chemical
fixation such as acid treatment to retain
ammonia as ammonium hydrogen sulfate,
alkali treatment to retain HCN as sodium
cyanide and others have a long history of
common use. Elaborate apparatus are
available for DO sampling to flush the
sample bottle and to minimize subsequent
exposure to the air. The apparatus tends
to become too elaborate for common use
if pressure control is built in.
4 The sampler has oecome accustomed to
measurement of temperature in place
and now because he knows that it will
change. The treatment plant sampler
will perform more effectively if he
learns to use additional remote sensors
where possible particularly where
dissolved gase3 are concerned.
C Sampling Suspended Materials:
The sampler faces real problems in
sampling water containing a conglomerate
of operationally defined suspended solids.
He is expected to manage representative
sampling of a non~ homogenous mixture
containing items such as organisms, detritus,
fibers, grease and chunks of inorganic or
organic residues with a wide range of sizes,
densities, shapes, and behavior.
3-4
-------
Sampling in Wastewater Treatment Operations
1 To be considered as suspended (particu-
late) material, the mixture must be
dispersed in water and tend to move
with it. Dissolved materials and rocks
are excluded but the suspended material
invariably includes dissolved materials,
greases, oils, organisms etc. that are
mechanically entrapped, adsurbed or
otherwise associated with the solids.
Suspended solids usually have an
additional arbitrary limitation based
upon size or ease of separation from
water. For example, it is called "grit"
if it separates from water suspension
within about one minute at a flow velocity
of about 1 ft/sec. Chunks, chips and sticks
are intended to be removed from the
sample.
2 Mixing of the main body of material or
flow is essential to disperse components
adequately for representative sampling.
This requires particular care for raw
wastewaters because many contained items
tend to slide along the bottom or float at
usual flow velocities. Mixing may be
performed by a pump, drop box, baffling
or other mixing device and the sample
site located accordingly.
3 Clearing and cleanup of manual or automatic
sample lines and devices are essential.
Flushing the lines to obtain free flow
before sampling is particularly important
for sludge lines because or grease, fibers,
sticks etc. in the flow th.it may partially
plug lines.
4 Frequency of sampling depends upon
variability if the item tested, requirements
of the needed infofmation, monsy and means
to da it. Sludge characteristics may be
so variable that it is advisable to sample
at 5 minute intervals and composite the
series to effectively estimate material
p .imped.
5 Clarified or stabilized wastewaters are
easier to sample because the particulates
remaining tend to be more uniformly
dispersed. The stabilized wastewaters are
easier to sample because the particulates
remaining tend to be more uniformly
dispersed. The stabilized sample
has been equalized and treated in
process to decrease the rate of change.
6 Solids have a tendency to change.
Dissolved solids may be converted
to suspended solids by growth,
complexation, sorp+ion or precipitation.
Fine particulates tend to become larger
particulates by coagulation and agglom-
eration. Conversely large particulates
may be dissolved or dispersed by mixing
hydrolysis etc. Cold storage is the usual
means of decreasing change rate on
samples for solids classification and
quantitation. Settling rates should be
tested promptly on fresh samples.
D Sampling for Pathogen Determinations
Health hazards related to the likely presence
of disease producing organisms in wastewater
are a major concern. The sampler is con-
cerned with specific routines characteristic of
the particular test organism group, water
mass to be sampled and use to be made of
the acquired information.
1 Clean sampling containers are required
in all sampling routines, in this case,
they must also be sterile. Special
precautions are required to protect the
sample from mtrod action of extraneous
organisms introduced during the sampling
operation or subsequent handling.
2 Samples may be stored or transported
to the test site under low temperature
conditions. No other treatment affecting
growth or death rate of the sample
organisms is permitted.
3 Time lapse between sampling and deter-
mination is critical. Best results are
obtained for holding times of less than
six hoars.
E Other Aquatic Organism Sampling:
Relationships of families of organisms
and habitats lead to inferences about
conditions depending upon the number
and variety of significant organisms
3-5
-------
Sampling in Wastewater Treatment Operations
in the system. The sampler may be
concerned with identification, relative
numbers, varieties, predominant groups,
selectivity, nutritional status, net growth,
environmental conditions, or activities of
a biological, chemical or physical nature.
Usually treatment plant objectives limit
concern to environmental conditions, growth
rates, selectivity or activities that are
pertinent to treatment plant conditions and
can be observed or measured effectively.
1 Biomonitoring, oxygen use rates, growth
rates and dynamics of organism or
substrate selection necessarily require
fresh samples under favorable environ-
mental conditions.
2 Net changes, accumulated cell mass and
residue accumulation may be preserved
and tested later.
3 Frequent and varied sampling and testing
of environmental conditions are essential
to permit interpretations of behavior.
Toxicity information on a test material
lacks validity if later information suggests
that oxygen starvation may have been a
factor.
4 Direct observation on fresh process
samples of selected indicator organisms
commonly found under favorable perform-
ance conditions are useful. Sudden
disappearance or inactivity of the usual
critters suggest drastic changes in
conditions or process.
IV SUMMARY:
A The sampler is obligated to follow prescribed
sampling sites, methodology, scheduling and
routing of samples agreed upon for his
situation.
B Sampling integrity is essential. The sample
should be clearly identified and represent the
material to be 3ampled at the time and place
designated. Any unusual conditions on site
that may affect results should be noted in
the sample record. The sample should be
protected from tampering. Preservatives,
storage conditions, and routine or special
handling should be as specified and part
of the record.
C Sampling information in this outline is
deliberately selective and incomplete
for guidance considerations. References
cited include much more detail concerning
sampling and sample handling depending
on sampled material, determinations to
be made, and use of the information. It
must be noted that any "Standard" methods
compendium includes professionally recog-
nized and recommended procedures including
certain alternates and options. These do
not become legally recognized methods until
accepted by statute or by the designated
statutory authorities, rules and regulations
for their jurisdiction. It is necessary to
determine which of the possible options are
acceptable within a given jurisdiction.
REFERENCES:
1 Standard Methods. Water and Wastewater
13th Edition 1971 APHA, AWWA, WPCF.
2 1971 Annual Book of ASTM S^anda^ds, Part
23 Water: Atmospheric Analysis. Ameri-
can Society for Testing and Materials.
3 Methods for Chemical Analysis of Water and
Wastes 1971. USSPA, Office of Water
Quality, Analytical Quality Control
Laboratory.
4 Rules and Regulations of the Water Quality
Enforcement Agency(s) including yoar
area of operations.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, WPO, EPA, Cincinnati, Ohio 45263
3-6
-------
LABORATORY HOUSEKEEPING
I Housekeeping in the general sense refers
to the management of a house and home and
the provision of equipment and services for
general welfare.
A Laboratory housekeeping places a much
greater emphasis upon organization of
facilities, equipment, and utensils into
an effective service operation.
1 Glassware must be effectively cleaned
as required and stored for use in an
orderly and accessible manner.
2 Equipment should be maintained in
effective working order, calibrated
regularly, and operated with suitable
respect for specific capabilities and
limitations.
3 Facilities should be organized in line
with conventional work loads.
4 Solutions and reagents should be pre-
cisely identified, stored in an orderly
fashion considering stability or con-
tamination, and replaced as necessary.
5 Schedule the operational routine with
requirements so that first things are
first.
6 Locate unit or allied operations so that
they can be performed with a minimum
of travel, lost time, or relocation of
needed items.
7 Schedule cleanup regularly to be ready
for analytical operations when samples
arrive.
8 Organize record operations for prompt
and easy accessibility when required.
II Facilities and equipment arrangement may
represent the difference between orderly and
hectic performance.
A Sinks, incubators, cold storage, benches,
wash-up, hoods, etc., are likely to be
fixed.
1 The problem is to arrange movable
items around these to best advantage.
2 Equipment for the BOD test should be
centered near a sink and suitable drain-
board with items used frequently stored
in the vicinity. Operations such as
filtration or microbial tests should be
suitably arranged in separate locations.
3 A laboratory cart saves time by pro-
viding a moveable platform for samples
and a place for temporary storage of
glassware to or from the 30b.
4 Electronic instruments such as the pH
meter, balance, spectrophotometers
should be located away from major
wet operations with line voltage control
if necessary.
5 Some bench space should be reserved
for non-routine operations.
6 Storage of glassware should be pro-
vided near the main line of traffic with
most commonly used items in the
favored spots for access. Supplies
should be maintained adequate to avoid
washup stops during determination
sessions.
7 Chemicals preferably should be stored
near the balance table in closed cabinets
except for special precaution items such
as volatile of flammable items.
PC. 4. 10. 67
4-1
-------
Laboratory Housekeeping
8 Prepared reagents or solutions used
in determinations should be stored
near the point of use.
9 Each item should have its place and be
returned to that location after use.
10 An inventory control of some sort is
essential to maintain chemicals, solu-
tions and glassware ready for use when
it is needed. The analyst will know or
can determine requirements for routine
use. Be sure that it is there beforehand.
This outline was prepared by F.J. Ludzack,
Chemist, National Training Center, Water
Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268,
4-2
-------
VARIATIONS IN TEST RESULTS
I INTRODUCTION
'.n any type of analytical work, the analyst
should ask the question "how well am I doing?"
or "am I getting the right results?" It is not
enough that these questions should be continu-
ally asked, but they should also be continu-
ally answered. The use of an organized
approach to answering these questions is
'<_-own as a quality control procedure. It will
the purpose of this discussion to point out
some of these procedures which can be
applied in the chemistry laboratory.
II FUNDAMENTAL CONCEPTS
--i Accuracy
For results to be accurate, the analysis
used must give values close to the true
value (see Figure 1). Errors which in-
troduce bias into results cause them to be
inaccurate. Such errors can be given an
'assignable cause" and are known as
d^tsrminate errors. There are three
fvpes of determinate errors.
1 Equipment errors
An error of this type could be the use
of a buret which is incorrectly
calibrated.
2 Method error
The most common type of error is that
associated with the presence of inter-
ferences in the sample. A low or high
yield factor for a desired constituent
may need to be developed.
3 Personal errors
These errors are attributable to indi-
vidual mistakes which are consistently
made by an analyst (e. g., not rinsing
a buret with a standard solution prior
to titration).
IMPRECISE AND INACCURATE
vX
X*
PRECISE BUT INACCURATt
ACCURATE BUT IMPRECISE
PRECISE AND ACCURATE
Figure 1. PRECISION AND ACCURACY
CH.12.4. 67
5-1
-------
Variations in Test Results
B Precision
Precision is the degree of agreement
among results obtained by repeated meas-
urements on a single sample under a given
set of conditions. In other words, it is a
measure of the degree to which results
"check. " Even when all determinate
errors are eliminated, every replicate
analysis will not give the same value.
Errors which introduce such variation
into results are due to "chance causes"
(e. g., variation in reagent addition, in-
strument response, inadvertent contamina-
tion of sample or glassware, etc.). These
errors are known as indeterminate or
random errors.
HI EVALUATION OF ACCURACY
A Spiked Samples
1 Samples which can be determined before
and after the addition of a known con-
stituent (in the concentration range of
interest) provide a way to detect deter-
minate errors. Spiked samples should
be representative and resemble actual
conditions as closely as possible. The
quantitation of bias can then be obtained
with the following measures.
Table 1
Sample: Effluent
Determination: Nitrate (Modified Brucine)
1. 55 mg/l
1.58
1.60
1.47
1.35
1 Calculation of mean error
^ 7.55 r < It
X = —-— = 1. 51 mg/l
2 Calculation of relative error
Relative Error =» ^ n O.Tfo
1« ol)
2 In some cases, it is impossible to
spike a sample so that it resembles
actual conditions (e. g., BOD and
pesticide samples). Youden^) provides
excellent techniques for detecting bias
in this situation.
a Mean error - the difference between B
the mean of the data and the true
result.
b Relative error - the mean error of
a set of data expressed as a per-
centage of the true result.
Independt "t Method
Analysis of a sample for a desired con-
stituent by two or more methods that are
entirely different in principle (gravimetric
and volumetric) may aid in the estimation
of determinate error.
EXAMPLE: An analyst determines
the nitrate content of the effluent IV EVALUATION OF PRECISION
from his sewage treatment plant to
be 0.5 mg/l. He then adds 1 mg/l A measure of the degree of agreement among
of standard nitrate solution to the results can be obtained by analyzing a single
sample. Table 1 shows the replicate sample repeatedly under a given set of
results obtained on the spiked conditions,
sample.
5-2
-------
Variations in Test Results
A Range
The range of the replicate results (differ-
ence between the lowest and the highest
value) provides a measure of indeterminate
variations.
B Standard Deviation
1 Since these indeterminate variations
conform to the laws of statistical dis-
tributions the standard deviation of the
replicates also can be used as a measure.
2 (X - X )2
1
(1)
n - 1
Xi = value of single result
X = average of results on same sample
n = number of results
Example: Table 2 contains a set of
5-day BOD results obtained on a syn-
thetic sample containing 150 mg/l of
glucose and 150 mg/l glutamic acid.
(Note: A 1% dilution was used in the
actual test). The results are those
submitted by laboratories participating
in a collaborative study. Calculate
the standard deviation of these results
X = 192 mg/l
n = 36.0
2(Xi - X)2 = 58,200
158,200'
S 'J 35
s = 41 mg/l
2 For a small number of replicates
(n < 10), the range can be used to
estimate the standard deviation (see
Table 3).
Table 2*
SAMPLE
DETERMINATION
150 mg/l glucose + 150mg/l glutamic acid (1% dilution)
5-day Biochemical Oxygen Demand
X
x -X
(X - X)2
X
X - X
(X -X)2
1
i
1
l
1
1
100 mg /1
-92 mg/l
8464 (mg/l)2
198 mg/l
6 mg/l
36 (mg/l)2
117
-75
5625
199
7
49
125
-67
4489
200
8
64
132
-60
3600
200
8
64
142
-50
2500
204
12
144
147
-45
2025
210
18
326
153
-39
1521
211
19
361
160
-32
1024
212
20
400
165
-27
729
215
23
529
165
-27
729
223
31
961
167
-25
625
224
32
1024
173
-19
361
227
35
1225
178
-14
196
229
37
1369
189
- 3
9
238
46
2116
190
- 2
4
247
55
3025
196
4
16
250
58
3.144
196
4
16
259
67
4489
197
5
25
274
82
6724
~Data taken from Water. Oxygen Demand Report
-------
Variations in Test Results
R_
"n
= s
(2)
Z(X1 - X)2
Example: Table 4 contains a set of
replicate nitrate results. Calculate
the standard deviation of these results.
a Use of formal (1)
X = 0.72 mg/1
n ~ 5
. 0134
0134
s = . 058 mg/l
b Use of formula (2)
R = . 14 mg/l
dN = 2.33
. 14 mg/l
S = 2.33
s = . 060 mg/l
Table 3*
Factors Used to Estimate the Standard
Deviation from Range
Size of
sample (n)
dN
Si
2
1. 13
.887
3
1. 69
. 391
4
2. 06
. 486
5
2. 33
. 430
6
2. 53
. 395
7
2.70
. 370
8
2. 85
. 351
9
2. 97
. 337
10
00
o
CO
. 325
*Natrella, Experimental Statistics, pp. 2-6.
Table 4
Sample: Ohio River Water
Determination: Nitrate (Modified Brucine)
X - X (X. - x)2
l l
0. 65 mg/l N
-.07
.0049
0.68
-. 04
.0016
0.70
-. 02
.0004
0.76
+. 04
. 0016
0.79
+. 07
.0049
C Relative Standard Deviation
The standard deviation expressed as a
percentage of the mean is known as the
relative standard deviation.
Example. Table 2
s = 41 mg/l
X = 192 mg/l
Relative Standard Deviation °
41 X 100
192
21%
V CONCLUSION
The concepts of precision and accuracy and
their evaluation have been presented.
REFERENCES
1 Allan, Douglas H. Statistical Quality Con-
trol. Reinhold Publishing Corporation.
New York. 1959.
2 American Society for Testing Materials.
ASTM Manual on Quality Control of
Materials. Special Technical Publica-
tion 15-C. 1951,
3 Bauer, E. L. A Statistical Manual for
Chemists. Academic Press. New
York. 1960.
5-4
-------
Variations in Test Results
4 Bennett, Carl A., and Franklin, Norman
L. Statistical Analysis in Chemistry
and the Chemical Industry. John Wiley
& Sons, Inc., New York. 1954.
5 Natrella, M.G. Experimental Statistics.
National Bureau of Standards Handbook
91. U.S. Department of Commerce,
1963.
6 Youden, W.J. The Collaborative Test.
J. AOAC. 46:55-62. January 1963.
7 Youden, W.J. The Sample, the Procedure,
and the Laboratory. Anal. Chem.
32:23-37A. December 1960.
This outline was prepared by B. A. Punghorst,
Chemist, formerly with FWPCA Training
Activities, SEC.
5-5
-------
ANALYTICAL REACTIONS - STANDARD SOLUTIONS
I INTRODUCTION
A A standard solution is one whose com-
position and concentration are known
to a high degree of accuracy. In chem-
ical analyses, it is used to determine
the concentration of a particular com-
ponent in a sample.
B These chemical analyses very frequently
involve an acid-base reaction or an
oxidation-reduction reaction.
C Three important terms connected with
these two types of chemical reactions
are- mole, equivalent weight, and
normality. These terms will be de-
fined in following sections.
II A NA LYTICAL CHEMICAL REA CTIONS
A As stated above, m a chemical analysis
a volume of standard solution is brought
into contact with a volume of sample
in order to determine the concentration
of some particular component of the
sample.
B For a given volume of sample, it is
necessary to use a definite amount of
the standard solution--too much or too
little would give erroneous results.
C For example, one cannot simply mix
together random volumes of sodium
hydroxide and hydrochloric acid solu-
tions and expect the only two substances
produced to be sodium chloride and
water.
D Unless the concentrations of the two
reagents are known and the amounts
measured accurately, excess sodium
hydroxide or hydrochloric acid will
also remain at the end of the reaction.
E The reason for these limitations is
that when molecules react with
one another, they do so in definite ratio.
Unless the number of molecules of each
reactant is known, there will always be
an excess of one of the r e a ct an ts re-
maining at the end of the chemical
reaction. As mentioned before, this
leads to erroneous analytical results.
F Because of their size, it is not possible
to count out numbers of molecules.
However, the number of molecules in
a quantity of a chemical may be found
by determining its weight and consulting
a table which lists the weights of the
atoms making up the chemical,
G For example, sodium hydroxide has the
formula NaOH. It can also be stated
that a molecule of sodium hydroxide
consists of one sodium atom, one hy-
drogen atom and one oxygen atom.
One sodium atom weighs 23 atomic
mass.units (amu). An oxygen atom
weighs 16 amu; and a hydrogen atom
weighs 1 amu.
A molecule of sodium hydroxide, there-
fore, weighs 40 amu; all amu values
have been rounded off. Forty amu is
the molecular weight of sodium hydroxide.
H A mole of any chemical is a number of
grams numerically equal to the molec-
ular weight of that chemical. One mole
of sodium hydroxide, therefore, con-
tains 40 grams (40 g).
I Similarly, the atomic w e ight of a
chlorine atom is 35 amu; that of a hy-
drogen atom is 1 amu, and the molec-
ular weight of the hydrogen chloride
molecule is 36 amu. One mole of hy-
drogen chloride weighs 36 g.
J Synonyms for mole are: mol, gram
mol, gram mole, and gram molecular
weight.
K Tables listing atomic weights of the
elements can be found in virtually all
PC. 17, a. 12.71
6-1
-------
Analytical Reactions - Standard Solutions
of the texts used for high school and
first year college chemistry courses.
L The remaining two terms mentioned in
IC (equivalent weight and normality)
will be considered as they are related to
acid-base and oxidation-reduction reac-
tions.
Ill ACID-BASE REACTIONS
A Recall that an acid is a substance which
donates a hydrogen ion, or proton, (H+)
in a chemical reaction and a base is a
substance which donates a hydroxide ion
(OH") in a chemical reaction. Reactions
involving these two ions are termed
acid-base reactions.
B In t h e case o f sodium hydroxide, the
molecular weight is 40 amu and one
mole of sodium hydroxide weighs 40 g.
One hydroxide ion is contained in the
sodium hydroxide molecule.
1 For a base, the number of grams
in a mole divided by the number of
hydroxide ions equals a quantity
called the equivalent weight. Ex-
amples are given below. All amu
values have been rounded off.
a Base - potassium hydroxide KOH
Atoms Number Wt/atom Total
K 1 39 amu 39 amu
O 1 16 16
Hill
56 amu
One mole of KOH = 56 g
Number of hydroxide ions = 1
Equivalent weight of KOH = 56 g
b Base - magnesium hydroxide
Mg(OH)2
Atoms Number Wt/atom Total
Mg 1 24 amu 24 amu
O 2 16 32
H 2 1 2
58 amu
One mole of Mg(OH>2 = 58 g
Number of hydroxide ions = 2
Equivalent weight of Mg(OH>2 = 29 g
2 For an acid, the number of grams
in a mole divided by the number of
hydrogen ions is the equivalent
weight. Examples are given below.
All amu values have been rounded
off.
a Acid - nitric acid HNO3
Atoms Number Wt/atom Total
H 1 1 amu 1 amu
N 1 14 14
O 3 16 48
63 amu
One mole of HNO3 3 63 g
Number of hydrogen 10ns = 1
Equivalent weight of HNO3 = 63 g
b Acid - sulfuric acid H2SO4
Atoms Number Wt/atom Total
H 2 1 amu 2 amu
S 1 32 32
O 4 16 j>4_
98 amu
One mole of H2SO4 = 98 g
Number of hydrogen ions 3 2
Equivalent weight of H2SO4 ° 49 g
C Normality is a method of expressing
solution concentrations. If one equiv-
alent weight of a chemical is dissolved
in a solvent and the volume brought to
one liter (1), the concentration of the
soluticr 1 s one normal (N).
1 The equivalent weight of KOH was
calculated to be 56 g. This amount
of the solid dissolved in water and
diluted to a liter would give a 1 N
solution.
2 The equivalent weight of HNO3 was
found to be 63 g. This quantity of
acid diluted to a liter would give a
1 N solution.
IV OXIDATION-REDUCTION REACTIONS
A The concepts of mole, equivalent weight
and normality, as described in previous
sections, apply also to oxidation-
reduction reactions.
6-2
-------
Analytical reactions - Standard Solutions
B One definition of an oxidation is that it
involves an increase in the oxidation
state (charge) of an atom.
For example: FeC^ —¦ FeCl3. In this
conversion the Fe has been oxidized
from +2 to +3.
It should have a high equivalent
weight so as to minimize any
errors in the analysis.
It should be readily available at a
reasonable cost.
D
A reduction is a decrease in the oxida-
tion state (charge) of an atom.
For example: KMnO^ —~ MnC^. In this
conversion the Mn has been reduced
from +7 to +4.
The equivalent weight of an oxidizing
or reducing agent is calculated by
dividing the number of grams in a mole
of the reagent by the change in charge
involved.
VI STORAGE OF STANDARD SOLUTIONS
A Standard solutions should be prepared
using high quality distilled water.
B Care should be taken to insure the
cleanliness of the glass or plastic
bottle used for storage.
C Some solutions may decompose on ex-
posure to light and should be stored in
dark bottles.
For example:
agent).
Atoms
Fe
CI
FeCl2 (used as a reducing D The stopper or cap should fit tightly.
Number
1
2
Wt/atom
56 anxu
70
Total
56 amu
70
126 amu
One mole of FeCl2 = 126 g
Change in charge = 1
Equivalent weight of FeCl2 = 126 g
The concentration of a liter of solution
which contains 126 g of FeCl2 is IN.
V PRIMARY STANDARDS
A A reagent of known purity is called a
primary standard. Primary standard
grade chemicals are available from
chemical supply houses and the National
Bureau of Standards. An accurately
measured quantity of a primary stand-
ard is used for the preparation of
standard solutions.
B Other requirements of a primary stand-
are are-
1 It must be stable at 105° C (the
temperature used for drying).
2 It should not be reactive with com-
ponents of the air, such as O2 and
CO2.
VII CALCULATIONS
A The basic formula used in volumetric
analysis is
(1 XN) of standard solution =
(1 XN) of sample
In a typical analysis, three of the four
quantities are known, or found, and
—j . , (1 XN) of standard
N of sample - —-r1 \
1 of sample
B This formula can be rearranged to give
g =1 XN Xequivalent weight
where g and equivalent weight (ew) re-
fer to the component being analyzed
and 1 and N to the standard solution.
C For example: How many g of NaOH are
present in a sample if 100.0 ml of 0.2N
HC1 are required for its titration'
g = 1 XN Xequivalent weight =
100.0 ml
1000.0 ml/1
X0. 2 X
40.0
1
= 0.8
6-3
-------
Analytical Reactions - Standard Solutions
REFERENCES
Hamilton, S.B. and Simpson, S.G.
Quantitative Chemical Analysis
11th edition. The MacMilIan Co.
New York. 1958.
Ayres, G. H.
Analysis.
New York.
Quantitative Chemical
Harper and Brothers,
1958.
2 Blaedel, W.J. and Meloche, V.W.
Elementary Quantitative Analysis:
Theory and Practice. Row,
Peterson and Co., N. Y. 1957.
This outline was prepared by Charles R.
Feldmann, Chemist, National Training
Center, MDS, OWP, EPA,. Cincinnati,
Ohio 45268.
6-4
-------
Ill THE PHYSICAL NATURE OF WATER
Water has many properties which are
unusual for liquids, upon which depend
most of the familiar aspects of the
world about us.
Contents of Section III
Outline Number
The Aquatic Environ- 7
ment Part 1:
The Physical Nature
of Water and Waters
-------
THE AQUATIC ENVIRONMENT
Part 1¦ The Nature and Behavior of Water
I INTRODUCTION
The earth is physically divisible into the
lithosphere or land masses, and the
hydrosphere which includes the oceans,
lakes, streams, and subterranean waters.
A Upon the hydrosphere are based a number
of sciences which represent different
approaches. Hydrology is the general
science of water itself with its various
special fields such as hydiograph\,
hydraulics, etc. These in turn merge
into physical chemistry and chemistry.
B Limnology and oceanography combine
aspects of all of these, and deal not only
with the physical liquid water and its
various naturally occurring solutions and
forms, but also with living organisms
and the infinite interactions that occur
between them and their environment.
C Water quality management, including
pollution control, thus looks to all
branches of aquatic science in efforts
to coordinate and improve man's
relationship with his aquatic environment.
II SOME FACTS ABOUT WATER
A Water is the onl\ abundant liquid on our
planet. It has many properties most
unusual for liquids, upon which depend
most of the lamiliar aspects ol the world
about us as we know it.
TABLE 1
UNIQUE PROPERTIES OF WATER
Property
Highest heat capacity (specific heat) of any
solid or liquid (except NH^)
Highest latent heat of fusion (except NH^)
Significance
Stabilizes temperatures of organisms and
geographical regions
Thermostatic effect at freezing point
Highest heat of evaporation of any substance
Important in heat and water transfer of
atmosphere
The only substance that has its maximum
density as a liquid (4°C)
Fresh and brackish waters have maximum
density above freezing point. This is
important in vertical circulation pattern
in lakes.
Highest surface tension of any liquid
Controls surface and drop phenomena,
important m cellular physiology
Dissolves more substances m greater
quantity than any other liquid
Makes complex biological system possible.
Important for transportation of materials
in solution.
Pure water has the highest di-electric
constant of any liquid
Leads to high dissociation of inorganic
substances in solution
Very little electrolytic dissociation
Neutral, yet contains both H+ and OH ions
Relatively transparent Absorbs much energy in infra red and ultra
violet ranges, but little in visible range.
Hence "colorless"
7-1
-------
The Aquatic Environment
B Physical Factors of Significance
1 Water substance
Water is not simply "HgO" but in
reality is a mixture of some 33
different substances involving three
isotopes each of hydrogen and oxygen
(ordinary hydrogen H1, deuterium H ,
and tritium H , ordinary oxygen O1®,
oxygen 17, and oxygen 18) plus 15 known
types of ions. The molecules of a
water mass tend to associate themselves
as polymers rather than to remain as
discrete units. (See Figure 1)
2 Density
Temperature and density Ice.
Water is the only known substance
in which the solid state will float
on the liquid state. (See Table 2)
SUBSTANCE OF WATER
Figure 1
7-2
-------
The Aquatic Environment
TABLE 2
EFFECTS OF TEMPERATURE ON DENSITY
OF PURE WATER AND ICE*
Temperature (° C) Density
Water
Ice **
-10
.99815
.9397
- 8
.99869
.9360
- 6
.99912
.9020
- 4
.99945
.9277
- 2
.99970
. 9229
0
.99987
.9168
2
.99997
4
1.00000
6
.00997
8
.00988
10
.00973
* Tabular values for density, etc., represent
statistical estimates by various workers
rather than absolute values, due to the
variability of water.
** Regular ice is known as "ice I". Four or
more other "forms" of ice are known to
exist (ice II, ice HI, etc.), having densities
at 1 atm. pressure ranging from 1.1595
to 1. 67. These are of extremely restricted
occurrence and may be ignored in most
routine operations.
This ensures that ice usually
forms on top of a body of water
and tends to insulate the remain-
ing water mass from further loss
of heat. Did ice sink, there
could be little or no carryover of
aquatic life from season to season
in the higher latitudes. Frazil or
needle ice forms colloidally at a
few thousandths of a degree
below 0° C. It is adhesive and
may build up on submerged objects
as "anchor ice", but it is still
typical ice.
1) Seasonal increase in solar
radiation annually warms
surface waters in summer
while other factors result in
winter cooling. The density
differences resulting estab-
lish two classic layers, the
epilimmon or surface layer,
and the hypolimnion or lower
layer, and in between is the
thermoclme or shear-plane.
2) While for certain theoretical
purposes a thermoclme is
defined as a zone in which the
temperature changes one
degree centigrade for each
meter of depth, in practice,
any transitional layer between
two relatively stable masses
of water of different temper-
atures (and probably other
qualities too) may be regarded
as a thermoclme.
3) Obviously the greater the
temperature differences
between epilimmon and
hypolimnion and the sharper
the gradient in the thermoclme,
the more stable will the
situation be.
4) From information given above,
it should be evident that while
the temperature of the
hypolimnion rarely drops
much below 4° C, the
epilimmon may range from
0° C upward.
5) It should also be emphasized
that when epilimmon and
hypolimnion achieve the same
temperature, stratification no
longer exists, and the entire
body of water behaves
hydrologically as a unit, and
tends to assume uniform
chemical and physical
characteristics. Such periods
are called overturns and
7-3
-------
The Aquatic Environment
usually result in considerable
water quality changes of
physical, chemical, and
biological significance.
6) When stratification is present,
however, each layer behaves
relatively independently, and
considerable quality differences
may develop.
7) Thermal stratification as
described above has no
reference to the size of the
water mass; it is found in
oceans and puddles.
8) The relative densities of the
various isotopes of water also
influence its molecular com-
position. For example, the
lighter O^g tends to go off
first in the process of
evaporation, leading to the
relative enrichment of air by
Oig and the enrichment of
water by and O^g. This
can lead to a measurably
higher content in warmer
climates Also, the temper-
ature of water in past geologic
ages can be closely estimated
from the ratio of O^g in the
carbonate of mollusc shells.
b Dissolved and/or suspended solids
may also affect the density of
natural waters.
TABLE 3
EFFECTS OF DISSOLVED SOLIDS
ON DENSITY
Dissolved Solids
Density
(Grams per liter)
(at 40 C)
0
1.00000
1
1.00085
2
1.00169
3
1.00251
10
1.00818
35 (mean for sea water) 1. 02822
c Density caused stratification
1) Density differences produce
stratification which may be
permanent, transient, or
seasonal.
2) Permanent stratification
exists for example where
there is a heavy mass of
brine in the deeper areas of
a basin which does not respond
to seasonal or other changing
conditions.
3) Transient stratification may
occur with the recurrent
influx of tidal water in an
estuary for example, or the
occasional influx of cold
muddy water into a deep lake
or reservoir.
4) Seasonal stratification involves
the annual establishment of
the epilimnion, hypolimnion,
and thermocline as described
above. The spring and fall
overturns of such waters
materially affect biological
productivity.
5) Density stratification is not
limited to two-layered systems;
three, four, or even more
layers may be encountered in
larger bodies of water.
d A "plunge line" may develop at
the mouth of a stream. Heavier
water flowing into a lake or
reservoir plunges below the
lighter water mass of the epiliminium
to flow along at a lower level. Such
a line is usually marked by an
accumulation of floating debris.
7-4
-------
The Aquatic Environment
The viscosity of water is greater at
lower temperatures (see Table 4).
This is important not only in situations
involving the control of flowing water
as in a sand filter, but also since
overcoming resistance to flow gen-
erates heat, it is significant in the
heating of water by internal friction
from wave and current action.
Laving organisms more easily support
themselves in the more viscous
(and also denser) cold waters of the
arctic than in the less viscous warm
tropical waters.
TABLE 4
VISCOSITY OF WATER (In millipoises at 1 atm)
Dissolved solids in g/L
Temp. ° C
0
5
10
30
-10
26.0
- 5
21.4
0
17.94
18. 1
18.24
18.7
5
15.19
15.3
15.5
16.0
10
13.10
13.2
13.4
13. 8
30
8.00
8. 1
8.2
8.6
100
2.84
3 Surface tension has biological as well
as physical significance. Organisms
whose body surfaces cannot be wet by
water can either ride on the surface filn
or in some instances may be "trapped"
on the surface film and be unable to
re-enter the water.
4 Incident solar radiation is the prime
source of energy for virtually all
organic and most inorganic processes
on earth. For the earth as a whole,
the total amount (of energy) received
annually must exactly balance that
lost by reflection and radiation into
space if climatic and related con-
ditions are to remain relatively
constant over geologic time.
a For a given body of water,
immediate sources of energy
include in addition to solar
irradiation: terrestrial heat,
transformation of kinetic energy
(wave and current action) to heat,
chemical and biochemical
reactions, convection from the
atmosphere, and condensation of
water vapor.
b The proportion of light reflected
depends on the angle of incidence,
the temperature, color, and other
qualities of the water. In general,
as the depth increases arithmet-
ically, the light tends to decrease
geometrically. Blues, greens,
and yellows tend to penetrate most
deeply while ultra violet, violets,
and orange-reds are most quickly
absorbed. On the order of 90%
of the total illumination which
penetrates the surface film is
absorbed in the first 10 meters of
even the clearest water, thus
tending to warm the upper layers.
5 Water movements
a Waves or rhythmic movement
The best known are traveling
waves caused by wind. These are
effective only against objects near
the surface. They have little
effect on the movement of large
masses of water.
7-5
-------
The Aquatic Environment
Standing waves or seiches occur
in lakes, estuaries, and other
enclosed bodies of water, but are
seldom large enough to be
observed. An "internal wave or
seich" is an oscillation in a
submersed mass of water such
as a hypolimnion, accompanied
by compensating oscillation in the
overlying water such that no
significant change in surface level
is detected. Shifts in submerged
water masses of this type can have
severe effects on the biota and
also on human water uses where
withdrawals are confined to a given
depth. Descriptions and analyses
of many other types and sub-types
of waves and wave-like movements
may be found in the literature.
b Tides
Tides are the longest waves known
in the ocean, and are evident along
the coast by the rhythmic rise and
fall of the water. While part and
parcel of the same phenomenon, it
is often convement to refer to the
rise and fall of the water level as
"tide", and to the accompanying
currents as "tidal currents".
Tides are basically caused by the
attraction of the sun and moon on
water masses, large and small;
however, it is only in the oceans
and certain of the larger lakes that
true tidal action has been demonstrated.
The patterns of tidal action are
enormously complicated by local
topography, interaction with seiches,
and other factors. The literature
on tides is very large.
c Currents (except tidal currents)
are steady a rhythmic water
movements which have had major
study only in oceanography although
they are best known from rivers
and streams. They primarily are
concerned with the translocation of
water masses. They may be
generated internally by virtue of
density changes, or externally by
wind or terrestrial topography.
Turbulence phenomena or eddy
currents are largely responsible for
lateral mixing in a current. These
are of far more importance in the
economy of a body of water than
mere laminar flow.
d Coriolis force is a result of inter-
action between the rotation of the
earth, and the movement of masses
or bodies on the earth. The net
result is a slight tendency for moving
objects to veer to the right in the
northern hemisphere, and to the
left in the southern hemisphere.
While the result in fresh waters is
usually negligible, it may be con-
siderable in marine waters. For
example, other factors permitting,
there is a tendency in estuarios for
fresh waters to move toward the
ocean faster along the right bank,
while salt tidal waters tend to
intrude farther inland along the
left bank. Effects are even more
dramatic in the open oceans.
e Langmuir circulation (or L. spirals)
is the interlocking rotation of
somewhat cylindrical masses of
surface water under the influence
of wind action. The axes of the
cylinders are parallel to the
direction of the wmd.
To somewhat oversimplify the
concept, a series of adjoining cells
might be thought of as chains of
interlocking gears in which at every
other contact the teeth are rising
while at the alternate contacts, they
are sinking (Figure 2).
The result is elongated masses of
waste rising or sinking together.
This produces the familiar "wind
rows" of foam, flotsam and jetsam,
or plankton often seen streaking
7-6
-------
The Aquatic Environment
windblown lakes or oceans. Certain
zoo-plankton struggling to maintain
a position near the surface of ten
collect in the down current between
two Langmuir cells, causing such
an area to be called the "red dance",
while the clear upwelling water
between is the "blue dance".
This phenomenon may be important
in water or plankton sampling on
a windy day.
6 The pH of pure water has been deter-
mined between 5. 7 and 7. 01 by various
workers. The latter value is most
widely accepted at the present time.
Natural waters of course vary widely
according to circumstances.
C The elements of hydrology mentioned
above represent a selection of some of
the more conspicuous physical factors
involved in working with water quality.
Other items not specifically mentioned
include: molecular structure of waters,
interaction of water and radiation,
internal pressure, acoustical charac-
teristics, pressure-volume-temperature
relationships, refractivity, luminescence,
color, dielectrical characteristics and
phenomena, solubility, action and inter-
actions of gases, liquids and solids,
water vapor, ices, phenomena of
hydrostatics and hydrodynamics in general.
REFERENCES
1 Buswell, A.M. and Rodebush, W.H.
Water. Sci. Am. April 1956.
2 Dorsey, N. Ernest. Properties of
Ordinary Water - Substance.
Remhold Publ. Corp. New York,
pp. 1-673. 1940.
3 Hutcheson, George E. A Treatise on
Limnology. John Wiley Company.
1957.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Office of Water Programs, EPA, Cincinnati,
OH 45268.
7-7
-------
MICROBIOLOGICAL ASPECTS OF WASTEWATER TREATMENT
Organisms in water may be implicated in purification of
it, existence in it, or in making it hazardous for man or
animal. This section considers the background and
techniques for evaluating water with respect ot pathogen
carriage.
Contents of Section IV
Outline Number
Collection & Handling of
Samples for Bacteriological
Examination
8
Bacteriological Indicators of
Water Pollution
9
Examination of Water for
Coliform and Fecal Strepto-
coccus Groups
10
Use of Tables of Most
Probable Numbers
11
Membrane Filter Equipment and
Its Preparation for Laboratory
12
Membrane Filter Laboratory
and Field Procedures
13
-------
COLLECTION AND HANDLING OF SAMPLES FOR
BACTERIOLOGICAL EXAMINATION
I INTRODUCTION
The first step in the examination of a water
supply for bacteriological examination is
careful collection and handling of samples.
Information from bacteriological tests is
useful in evaluating water purification,
bacteriological potability, waste disposal,
and industrial supply. Topics covered
include: representative site selection,
frequency, number, size of samples,
satisfactory sample bottles, techniques of
sampling, labeling, and transport.
II SELECTION OF SAMPLING LOCATIONS
The basis for locating sampling points is
collection of representative samples.
A Take samples for potability testing from
the distribution system through taps.
Choose representative points covering
the entire system. The tap itself should
be clean and connected directly into the
system. Avoid leaky faucets because of
the danger of washing in extraneous
bacteria. Wells with pumps may be
considered similar to distribution systems
B Grab samples from streams are frequently
collected for control data or application of
regulatory requirements. A grab sample
can be taken in the stream near the surface.
C For intensive stream studies on source
and extent of pollution, representative
samples are taken by considering site,
method and time of sampling. The
sampling sites may be a compromise
between physical limitations of the
Keep in mind that averaging does not
remove all variation but only minimizes
sharp fluctuations. Downstream sites
sampling may not need to be so frequent.
Samples maybe collected 1/4, 1/2 and
3/4 of the stream width at each site or
other distances, depending on survey
objectives. Often only one sample in the
channel of the stream is collected'
Samples are usually taken near the surface
D Samples from lakes or reservoirs are
frequently collected at the drawoff and
usually about the same depth and may be
collected over this entire surface.
E Collect samples of bathing beach water
at locations and times where the most
bathers swim.
IE NUMBER, FREQUENCY AND SIZE
OF SAMPLES
A For determining sampling frequency for
drinking water, consult the USPHS
Standards.
1 The total number, frequency, and site
are established by agreement with
either state of PHS authorities.
2 The minimum number depends upon the
number of users. Figure 1 indicates
that the smaller populations call for
relatively more samples than larger
ones. The numbers on the left of the
graph refer to actual users and not the
population shown by census
laboratory, detection of pollution peaks,
and frequency of sample collection in
certain types of surveys. First, decide
how many samples are needed to be
processed in a day. Second, decide
whether to measure cycles of immediate
pollution or more average pollution.
Sites for measuring cyclic pollution are
immediately below the pollution source.
Sampling is frequent, for example, every
three hours.
A site designed to measure more average
conditions is far enough downstream for
a complete mixing of pollution and water.
W.BA. sa. Id. 7. 70
3 In the event that coliform limits of the
standard are exceeded, daily samples
must be taken at the same site.
Examinations should continue until two
consecutive samples show coliform
level is satisfactory. Such samples
are to be considered as special samples
and shall not be included in the total
number of samples examined.
4 Sampling programs described above
represent a minimum number which
may be increased by reviewing
authority.
8-1
-------
Collection and Handling of Samples for Bacteriological Examination
B For stream investigations the type of use Residual chlorine tests are
study governs frequency of sampling. necessary to check neutralization of
chlorine in the sample.
C Collect swimming pool samples when use
is heavy. The high chlorine level rapidly D Lake beaches may be sampled as required
reduces the count when the pool is not in depending on the water uses.
MINIMUM NUMBER OF SAMPLES PER MONTH
1000
100
oc
100
1000
10,000
FIGURE 1
8-2
-------
Collection and Handling of Samples for Bacteriological Examination
E Salt water or estuarine beaches are
sampled as needed with frequency
depending on use.
F Size of samples depends upon examination
anticipated. Generally 100 ml is the
minimum size.
IV BOTTLES FOR WATER SAMPLES
A The sample bottles should have capacity
for at least 100 ml of sample, plus an
air space. The bottle and cap must be of
bacteriological inert materials. Resistant
glass or heat resistant plastic are
acceptable. At the National Training
Center, wide mouth ground-glass
stoppered bottles (Figure 2) are used.
All bottles must be properly washed and
sterilized. Protect the top of the bottles
and cap from contamination by paper or
metal foil hoods. Both glass and heat
I
I
resistant plastic bottles may be
sterilized in an autoclave. Hold plastic
at 121°C for at least 10 minutes. Hot
air sterilization, 1 hour at 170°C, may
be used for glass bottles.
B Add sodium thiosulfate to bottles intended
for halogenated water samples. A quantity
of 0 1 ml of a 10% solution provides 100
mg per liter concentration in a 100 ml
sample. This level shows no effect upon
viability or growth
C Supply catalogs list wide mouth ground
glass stoppered bottles of borosilicate
resistance glass, specially for water
samples.
V TECHNIQUE OF SAMPLE COLLECTION
Follow aseptic technique as nearly as
possible. Nothing but sample water must
touch the inside of the bottle or cap. To
avoid loss of sodium thiosulfate, fill the
bottle directly and do not rinse. Always
remember to leave an air space.
A In sampling from a distribution system,
first run the faucet wide open until the
service line is cleared. A time of 3-5
minutes generally is sufficient. Reduce
the flow and fill the sample bottle without
splashing. Some authorities stress
flaming the tap before collection. A
chlorine determination is often made on
the site.
B The bottle may be dipped into some waters
by hand. Avoid introduction of bacteria
from the human hand and from surface
debris. Some suggestions follow:
Hold the bottle near the base with one
hand and with the other remove the hood
and cap. Push the bottle rapidly into the
water mouth down and tilt up towards the
current to fill. A depth of about 6 inches
is satisfactory. When there is no current
move the bottle through the water
horizontally and away from the hand. Lift
the bottle from the water, spill a small
amount of sample to provide an air space,
and return the uncontaminated cap
v '
FIGURE 2
8-3
-------
Collection and Handling of Samples for Bacteriological Examination
Samples may be dipped from swimming
pools. Determine residual chlorine on
the pool water at the site. Test the
sample at the laboratory to check chlorine
neutralization by the thiosulfate.
Sample bathing beach water by wading out
to the two foot depth and dipping the
sample up from about 6 inches below the
surface. Use the procedure described in
V. B.
Wells with pumps are similar to
distribution systems. With a hand pumped
well, waste water for about five minutes
before taking the sample. Sample a well
without a pump by lowering a sterile
bottle attached to a weight. A device which
opens the bottle underneath the water
will avoid contamination by surface debris.
Various types of sampling devices are
available where the sample point is
inaccessible or depth samples are desired.
The general problem is to put a sample
bottle in place, open it, close it, and
return it to the surface. No bacteria but
those in the sample must enter the bottle.
The J - Z sampler described by Z obeli
in 1941, was designed for deep sea
sampling but is useful elsewhere (Figure)
3). It has a metal frame, breaking
device for a glass tube, and sample
bottle. The heavy metal messenger
strikes the lever arm which breaks
the glass tubing at a file mark. A
bent rubber tube straightens and the
water is drawn in several inches from
the apparatus. Either glass or collapsible
rubber bottles are sample containers.
Commercial adaptations are available.
Note the vane and lever mechanism on
the New York State Conservation
Department's sampler in Figure 4.
When the apparatus is at proper depth
the suspending line is given a sharp
pull. Water inertia against the vane
raises the stopper and water pours
into the bottle. Sufficient sample is
collected prior to the detachment of
the stopper from the vane arm allowing
a closure of the sample bottle.
The New York State Conservation
Department's sampler is useful for
shallow depths and requires nothing
besides glass stoppered sample bottles.
FIGURE 3
Reproduced with permission of the Journal
of Marine Research 4:3, 173-188 (1941) by
the Department of Health, Education and
Welfare.
8-4
-------
Collection and Handling of Samples for Bacteriological Examination
FIGURE 4
B While a sanitary survey is an indispensable
part of the evaluation of a water supply, its
discussion is not within the scope of this
lecture. The sample collector could supply
much information if desired
VII SHIPPING CONDITIONS
A The examination should commence as soon
as possible, preferably within one hour.
A maximum elapsed time between collection
and examination is 30 hours for potable
water samples and 8 hours for other
water samples (collection 6 hours and
laboratory procedures 2 hours).
Standard Methods (13th Edition)
recommends icing of samples between
collection and testing.
VIII PHOTOGRAPHS
A photograph is a sample in that it is evidence
representing water quality. Sample collectors
and field engineers may carry cameras to
record what they see. Pictures help the
general public and legal courts to better
understand laboratory data.
3 A commercial sampler is available
which is an evacuated sealed tube with
a capillary tip. When a lever on the
support rack breaks the tip, the tube
fills. Other samplers exist with a
lever for pulling the stopper, while
another uses an electromagnet.
VI DATA RECORDING
A Information generally includes: date, time
of collection, temperature of water, location
of sampling point, and name of the sample
collector. Codes are often used. The
location description must be exact enough
to guide another person to the site.
Reference to bridges, roads, distance to
the nearest town may help. Use of the
surveyors' description and maps are
recommended. Mark identification on the
bottles or on securely fastened tags.
Gummed tags may soak off and are
inadvisable.
REFERENCES
1 APHA, AWWA, WPCF, Standard Methods
for the Examination of Water and
Wastewater. (12 Ed.) 1965.
2 Prescott, S. C., Winslow, C. E.A. , and
McCrady, M. H. Water Bacteriology.
6th Ed., 368 pp. John Wiley and Sons,
Inc., New York. 1946.
3 Haney, P. D., and Schmidt, J.
Representative Sampling and Analytical
Methods in Stream Studies. Oxygen
Relationships in Streams, Technical
Report W58-2 pp. 133-42. U. S.
Department of Health, Education and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering Center,
Cincinnati, Ohio. 1958.
4 Velz, C. J. Sampling for Effective
Evaluation of Stream Pollution. Sewage
and Industrial Wastes, 22-666-84. 1950.
8-5
-------
Collection and Handling of Samples for Bacteriological Examination
5 Bathing Water Quality and Health HI
Coastal Water. 134 pp. U. &
Department of Health, Education, and
Welfare, Public Health Service, Robert
A. Taft Sanitary Engineering,
Cincinnati, Ohio. 1961.
6 Zobell, C. E. Apparatus for Collecting
Water Samples from Different Depths
for Bacteriological Analysis. Journal
of Marine Research, 4:3:173-88. 1941
This outline was originally prepared by
A. G. Jose, former Microbiologist FWPCA
Training Activities, SEC and updated by
the Training Staff, National Training Center,
DTTB, MDS, WPO, EPA, Cincinnati, OH
45268.
8-6
-------
BACTERIOLOGICAL INDICATORS OF WATER POLLUTION
Part 1. General Concepts
I INTRODUCTION
A Bacterial Indication of Pollution
1 In the broadest sense, a bacterial
indicator of pollution is any organism
which, by its presence, would demon-
strate that pollution has occurred, and
often suggest the source of the pollution.
2 In a more restrictive sense, bacterial
indicators of pollution are associated
primarily with demonstration of con-
tamination of water, originating from
excreta of warm-blooded animals
(including man, domestic and wild
animals, and birds).
B Implications of Pollution of Intestinal
Origin
1 Intestinal wastes from warm-blooded
animals regularly include a wide
variety of genera and species of
bacteria. Among these the coliform
group may be listed, and species of
the genera Streptococcus, Lactobacillus,
Staphylococcus, Proteus, Pseudomonas,
certain spore-forming bacteria, and
others.
2 In addition, many kinds of pathogenic
bacteria and other microorganisms
may be released in wastes on an inter-
mittent basis, varying with the geo-
graphic area, state of community
health, nature and degree of waste
treatment, and other factors. These
may include the following-
a Bacteria- Species of Salmonella,
Shigella, Leptospira, Brucella,
Mycobacterium, and Vibrio comma.
b Viruses- A wide variety, including
that of infectious hepatitis, Polio-
viruses, Coxsackie virus, ECHO
viruses (enteric cytopathogenic
human orphan -- "viruses in search
of a disease"), and unspecified
viruses postulated to account for
outbreaks of diarrheal and upper
respiratory diseases of unknown
etiology, apparently infective by
the water-borne route.
c Protozoa: Endamoeba histolytica
3 As routinely practiced, bacterial
evidence of water pollution is a test
for the presence and numbers of
bacteria in wastes which, by their
presence, indicate that intestinal
pollution has occurred. In this con-
text, indicator groups discussed in
subsequent parts of this outline are
as follows.
a Coliform group and certain sub-
groupings
b Fecal streptococci and certain
sub groupings
c Miscellaneous indicators of pollution
4 Evidence of water contamination by
intestinal wastes of warm-blooded
animals is regarded as evidence of
health hazard in the water being tested.
II PROPERTIES OF AN IDEAL INDICATOR
OF POLLUTION
A An "ideal" bacterial indicator of pollution
should:
1 Be applicable in all types of water
W.BA. 48f. 9. 71
9-1
-------
Bacteriological Indicators of Water Pollution
2 Always be present in water when
pathogenic bacterial constituents of
fecal contamination are present.
Ramifications of this include --
a Its density should have some direct
relationship to the degree of fecal
pollution.
b It should have greater survival time
in water than enteric pathogens.
c It should disappear rapidly from
water following the disappearance
of pathogens, either through natural
or man-made processes.
d It always should be absent in a
bacteriologically safe water.
3 Lend itself to routine quantitative
testing procedures without interference
or confusion of results due to extra-
neous bacteria
4 Be harmless to man and other animals
B In all probability, an "ideal" bacterial
indicator does not exist. The discussion
of bacterial indicators of pollution in the
following parts of this outline include
consideration of the merits and limitations
of each group, with their applications in
evaluating bacterial quality of water.
in APPLICATIONS OF TESTS FOR
POLLUTION INDICATORS
A Tests for Compliance with Bacterial
Water Quality Standards
1 Potability tests on drinking water to
meet Interstate Quarantine or other
standards of regulatory agencies.
2 Determination of bacterial quality of
environmental water for which quality
standards may exist, such as shellfish
waters, recreational waters, water
resources for municipal or other
supplies.
3 Tests for compliance with established
standards in cases involving the pro-
tection or prosecution of municipalities,
industries, etc.
B Treatment Plant Process Control
1 Water treatment plants
2 Wastewater treatment plants
C Water Quality Surveys
1 Determination of intestinal pollution
in surface water to determine type and
extent of treatment required for com-
pliance with standards
2 Tracing sources of pollution
3 Determination of effects on bacterial
flora, due to addition of organic or
other wastes
D Special Studies, such as
1 Tracing sources of intestinal pathogens
m epidemiological investigations
2 Investigations of problems due to the
Sphaerotilus group
3 Investigations of bacterial interference
to certain industrial processes, with
respect to such organisms as Pseudo-
monas, Achromobacter, or others
IV SANITARY SURVEY
The laboratory bacteriologist is not alone in
evaluation of indication of water pollution of
intestinal origin. On-site study (Sanitary
Survey) of the aquatic environment and
adjacent areas, by a qualified person, is a
necessary collateral study with the laboratory
work and frequently will reveal information
regarding potential bacteriological hazard
which may or may not be demonstrated
through laboratory findings from a single
sample or short series of samples..
9-2
-------
Bacteriological Indicators of Water Pollution
Part 2. The Coliform Group and Its Constituents
I ORIGINS AND DEFINITION
A Background
1 In 1885, Escherich, a pioneer bacteri-
ologist, recovered certain bacteria from
human feces, which he found in such
numbers and consistency as to lead him
to term these organisms "the charac-
teristic organism of human feces. "
He named these organisms Bacterium
coli-commune and B. lactis aerogenes.
In 1895, another bacteriologist,
Migula, renamed _B. coli commune as
Escherichia coli, which today is the
official name for the type species.
2 Later work has substantiated much of
the original concept of Escherich, but
has shown that the above species are
in fact a heterogeneous complex of
bacterial species and species variants.
a This heterogeneous group occurs not
only in human feces but representatives
also are to be found in many environ-
mental media, including sewage,
surface freshwaters of all categories,
in and on soils, vegetation, etc.
b The group may be subdivided into
various categories on the basis of
numerous biochemical and other
differential tests that may be applied.
B Composition of the Coliform Group
1 Current definition
As defined in "Standard Methods for the
Examination of Water and Wastewater"
(12th ed): "The coliform group includes
all of the aerobic and facultative
anaerobic. Gram-negative, nonspore-
forming rod-shaped bacteria which
ferment lactose with gas formation
within 48 hours at 35° C. "
2 The term "coliforms" or "coliform
group" is an inclusive one, including
the following bacteria which may
meet the definition above
a Escherichia coli, E. aurescens,
E. freundii, E. intermedia
b Aerobacter aerogenes, _A. cloacae
c Biochemical intermediates between
the genera Escherichia and Aero-
bacter
3 There is no provision in the definition
of coliform bacteria for "atypical" or
"aberrant" coliform strains.
a An individual strain of any of the
above species may fail to meet one
of the criteria of the coliform group.
b Such an organism, by definition, is
not a member of the coliform
group, even though a taxonomic
bacteriologist may be perfectly
correct in classifying the strain in
one of the above species.
H SUBDIVISION OF COLIFORMS INTO
"FECAL" AND "NONFECAL"
CATEGORIES
A Need
Single-test differentiations between
coliforms of "fecal" origin and those of
"nonfecal" origin are based on the
assumption that typical E, coli and
closely related strains are of fecal
origin while A. aerogenes and its close
relatives are not of direct fecal origin.
(The latter assumption is not fully borne
out by investigations at this Center.
See Table 1, IMViC Type --++). A
number of single differential tests have
been proposed to differentiate between
"fecal" and "nonfecal" coliforms.
9-3
-------
Bacteriological Indicators of Water Pollution
Without discussion of their relative merits,
several may be cited here:
B Types of Single-Test Differentiation
1 Determination of gas ratio
Fermentation of glucose by E. coli
results in gas production, with
hydrogen and carbon dioxide being
produced m equal amounts.
Fermentation of glucose by A.
aerogenes results in generation of
twice as much carbon dioxide as
hydrogen.
Further studies suggested absolute
correlation between Hg/COg ratios
and the terminal pH resulting from
glucose fermentation. This led to the
substitution of the methyl red test.
2 Methyl red test
Glucose fermentation by E. coli
typically results in a culture medium
having terminal pH in the range 4.2-
4. 6 (red color a positive test with the
addition of methyl red indicator).
A. aerogenes typically results in a
culture medium having pH 5. 6 or
greater (yellow color, a negative test).
3 Indole
When tryptophane, an amino acid, is
incorporated in a nutrient broth,
typical E. coli strains are capable of
producing indole (positive test) among
the end products, whereas A. aerogenes
does not (negative test).
In reviewing technical literature, the
worker should be alert to the method
used to detect indole formation, as the
results may be greatly influenced by
the analytical procedure.
4 Voges-Proskauer test (acetylmethyl
carbinol test)
The test is for detection of acetylmethyl
carbinol, a derivative of 2, 3, butylene-
glycol, as a result of glucose
fermentation in the presence of
peptone. A. aerogenes produces
this end product (positive test)
whereas E. coli gives a negative test,
a Experience with coliform cultures
giving a positive test has shown a
loss of this ability with storage on
laboratory media for 6 months to
2iyears, in 20 - 25% of cultures
(105 out of 458 cultures).
b Some workers consider that all
coliform bacteria produce acetyl-
methyl carbinol in glucose metab-
olism. These workers regard
acetylmethyl carbinol-negative
cultures as those which have
enzyme systems capable of further
degradation of acetylmethyl
carbinol to other end products
which do not give a positive test
with the analytical procedure.
Cultures giving a positive test for
acetylmethyl carbinol lack this
enzyme system.
c This reasoning leads to a hypothesis
(not experimentally proven) that the
change of reaction noted in certain
cultures in 4.a above is due to the
activation of a latent enzyme system.
5 Citrate utilization
Cultures of E. coli are unable to use
the carbon of citrates (negative test)
in their metabolism, whereas cultures
of _A. aerogenes are capable of using
the carbon of citrates in their metab-
olism (positive test).
Some workers (using Simmons Citrate
Agar) incorporate a pH indicator
(brom thymol blue) in the culture
medium in order to demonstrate the
typical alkaline reaction (pH 8.4 - 9. 0)
resulting with citrate utilization.
6 Elevated temperature (Eijkman) test
a The test is based on evidence that
E. coli and other coliforms of fecal
9-4
-------
Bacteriological Indicators of Water Pollution
origin are capable of growing and
fermenting carbohydrates (glucose
or lactose) at temperatures signif-
icantly higher than the body tem-
perature of warm-blooded animals.
Organisms not associated with direct
fecal origin would give a negative
test result, through their inability
to grow at the elevated temperature.
b While many media and techniques
have been proposed, EC Broth, a
medium developed by Perry and
Hajna, used as a confirmatory
medium for 24 hours at 44. 5 ±
0.2oc are the current recommended
medium and method of choice.
While the "EC" terminology of the
medium suggests "Ej. coli" the
worker should not regard this as a
specific procedure for isolation of
_E. coli.
c A similar medium, Boric Acid
Lactose Broth, has been developed
by Levine and his associates. This
medium gives results virtually
identical with those obtained from
EC Broth, but requires 48 hours of
incubation.
d Elevation temperature tests require
incubation in a water bath. Standard
Methods 13th Ed. requires this
*• temperature to be 44. 5 + 0. 2°C.
Various workers have urged use of
temperatures ranging between
43.0°C and 46. 0°C. Most of these
recommendations have provided a
tolerance of + 0. 5° C from the rec-
ommended levels. However, some
workers, notably in the Shellfish
Program of the Public Health Service,
stipulate a temperature of 44. 5 ±
0.2OC. This requires use of a water
bath with forced circulation to main-
tain this close tolerance. This
tolerance range has been instituted
in the 13th Edition of Standard Methods
and the laboratory worker should
conform to these new limits.
e The reliability of elevated temper-
ature tests is influenced by the
time required for the newly-
inoculated cultures to reach the
designated incubation temperature.
Critical workers insist on place-
ment of the cultures in the water
bath within 30 minutes, at most,
after inoculation.
7 Other tests
Numerous other tests for differentiation
between coliforms of fecal vs. nonfecal
origin have been proposed. Current
studies suggest little promise for the
following tests in this application:
uric acid test, cellobiose fermentation,
gelatin liquefaction, production of
hydrogen sulfide, sucrose fermentation,
and others.
C IMViC Classification
1 In 1938, Parr reported on a review of
a literature survey on biochemical tests
used to differentiate between coliforms
of fecal vs. nonfecal origin. A summary
follows:
No. of times
Test used for dif-
ferentiation
Voges-Proskauer 22
reaction
Methyl red test 20
Citrate utilization 20
Indole test 15
Uric acid test 6
Cellobiose fermentation 4
Gelatin liquefaction 3
Eijkman test 2
Hydrogen sulfide 1
production
Sucrose fermentation 1
a-Methyl-d-glucoside 1
fermentation
9-5
-------
Bacteriological Indicators of Water Pollution
2 Based on this summary and on his own
studies, Parr recommended utilization
of a combination of tests, the indole,
methyl red, Voges-Proskauer, and the
citrate utilization tests for this differ-
entiation. This series of reactions is
designated by the mnemonic "IMViC".
Using this scheme, any coliform culture
can be described by an "IMViC Code"
according to the reactions for each
culture. Thus, a typical culture of
E. coli would have a code ++-and a
typical A. aerogenes culture would
have a code
3 Groupings of coliforms into fecal,
non-fecal, and intermediate groups,
as shown in "Standard Methods for the
Examination of Water and Wastewater"
are shown at the bottom of this page.
D Need for Study of Multiple Cultures
All the systems used for differentiation
between coliforms of fecal vs. those of
nonfecal origin require isolation and study
of numerous pure cultures. Many workers
prefer to study at least 100 cultures from
any environmental source before attempting
to categorize the probable source of the
coliforms.
in NATURAL DISTRIBUTION OF COLIFORM
BA CTERIA
A Sources of Background Information
Details of the voluminous background of
technical information on coliform bacteria
recovered from one or more environ-
mental media are beyond the scope of
this discussion. References 2, 3, and 4
of this outline are suggested routes of
entry for workers seeking to explore this
topic.
B Recent Studies on Coliform Distribution
1 Since 1960, workers at this Center
have engaged in a continuing study of
the natural distribution of coliform
bacteria and an evaluation of pro-
cedures for differentiation between
coliforms of fecal vs. probable non-
fecal origin. Results of this work
have special significance because:
a Rigid uniformity of laboratory
methods have been applied through-
out the series of studies
b Studies are based on massive
numbers of cultures, far beyond
any similar studies heretofore
reported
Groupings of Coliforms into Fecal, Nonfecal and Intermediate Groups
Organism
Indole
Methyl
red
Voges-
Proskauer
Citrate
E.
coli. Variety I
+
+
-
-
Variety II
-
+
—
E.
freundii
(Intermediates)
Variety I
-
+
-
±
Variety II
+
+
-
+
A.
aerogenes
Variety I
-
-
+
+
Variety n
+
-
+
+
9-6
-------
Bacteriological Indicators of Water Pollution
c A wider variety of environmental and
biological sources is being studied
than in any previous series of reports.
d All studies are based on freshly
recovered pure culture isolates
from the designated sources.
e All studies are based on cultures
recovered from the widest feasible
geographic range, collected at all
seasons of the year. It is believed
that no more representative series
of studies has been made or is in
progress.
2 Distribution of coliform types
Table 1 shows the consolidated results
of coliform distributions from various
biological and environmental sources.
a The results of these studies show a
high order of correlation between
known or probable fecal origin and
the typical EI. coli IMViC code
(++--). On the other hand, human
feces also includes
numbers of A. aerogenes and other
IMViC types, which some regard as
"nonfecal" segments of the coliform
group. (Figure 1)
b The majority of coliforms attributable
to excretal origin tend to be limited
to a relatively small number of the
possible IMViC codes, on the other
hand, coliform bacteria recovered
from undisturbed soil, vegetation,
and insect life represent a wider
range of IMViC codes than fecal
sources, without clear dominance of
any one type. (Figure 2)
c The most prominant IMViC code
from nonfecal sources is the inter-
mediate type, which accounts
for almost half the coliform cultures
recovered from soils, and a high
percentage of those recovered from
vegetation and from insects. It
would appear that if any coliform
segment could be termed a "soil
type" it would be IMViC code -+-+.
d It should not be surprising that
cultures of typical E. coli are
recovered in relatively smaller
numbers from sources judged,
on the basis of sanitary survey,
to be unpolluted. There is no
known way to exclude the influence
of limited fecal pollution from small
animals and birds in such environ-
ments
e The distribution of coliform types
from human sources should be
regarded as a representative value
for large numbers of sources.
Investigations have shown that there
can be large differences in the
distribution of IMViC types from
person to person, or even from an
individual.
3 Differentiation between coliforms of
fecal vs. nonfecal origin
Table 2 is a summary of findings of
SEC workers, based on a number of
different criteria for differentiating
between coliforms of fecal origin and
those from other sources.
a IMViC type ++-- is a measurement
of E_. coli, Vairety I, and appears
to give reasonably good correlation
between known or highly probable
fecal origin and doubtful fecal origin.
b The combination of IMViC types,
++--, + , and gives
improved identification of probable
fecal origin, and appears also to
exclude most of the coliforms not
found in excreta of warm-blooded
animals in large numbers.
c While the indole, methyl red,
Voges Proskauer, and citrate
utilization tests, each used alone,
appear to give useful answers when
applied only to samples of known
pollution from fecal sources, the
interpretation is not as clear when
applied to coliforms from sources
believed to be remote from direct
fecal pollution.
9-7
-------
Table 1. COLIFORM DISTRIBUTION BY IMViC TYPES AND ELEVATED TEMPERATURE
TEST FROM ENVIRONMENTAL AND BIOLOGICAL SOURCES
IMViC
type
Vegetation
Insects
Soil
Fecal sources
Poultry
Undisturbed
Polluted
Hun-
lan
Lives
tock
No.
strains
% of
total
No. |
strains !
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
% of
total
No.
strains
% of
total
++- -
128
10. 6
134
12. 4
131
5. 6
536
80. 6
3932
87. 2
2237
95. 6
1857
97. 9
--++
237
19. 7
113
10. 4
443
18. 8
13
2. 0
245
5. 4
0
<0. 1
1
0. 1
23
1. 9
0
<0. 1
78
3. 3
1
0. 2
99
2. 2
14
0. 6
20
1. 1
+++-
2
0 2
0
<0. 1
7
0. 3
0
<0. 1
106
2. 4
59
2. 5
0
<0. 1
-+- +
168
14. 0
332
30. 6
1131
48. 1
87
13. 0
50
1. 1
1
<0. 1
5
0. 3
++- +
116
9. 6
118
10. 9
87
3. 7
22
3. 3
35
0. 8
27
1. 2
11
0. 6
-+++
32
2. 7
28
2. 6
181
7. 7
5
0. 7
21
0. 5
0
<0. 1
0
<0. 1
++++
291
24. 2
254
23. 4
159
6. 8
0
<0. 1
6
0. 1
0
<0. 1
0
<0. 1
+-++
88
7. 3
46
4. 2
67
2. 9
0
<0. 1
14
0. 2
0
<0. 1
0
<0. 1
+
87
7. 2
42
3. 9
4
0. 2
1
0. 2
2
<0. 1
0
<0. 1
0
<0. 1
-++-
5
0. 4
0
<0. 1
1
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
19
1. 6
0
<0. 1
53
2. 3
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
+-+-
2
0. 2
0
<0. 1
6
0. 3
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
+-- +
5
0. 4
8
0. 7
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
0
<0. 1
+
0
<0. 1
9
0. 8
0
<0. 1
0
<0. 1
2
<0. 1
0
<0. 1
2
<0. 1
Total
1203
1084
2348
665
4512
2339
1896
No. EC +
169*
162*
216
551
4349
2309
1765
% EC +
14. 1*
14. 9*
9. 2
82. 9
96. 4
98. 7
93. 0
¦
*120 of these *129 of these
were were —,
15 —H-f 2 7 - H—h(
11 -+— 5 ++-+
-------
Bacteriological Indicators of Water Pollution 1-9
HUMAN
EC ® 96.4
BALB ® 94.7
-- I -+- +
LIVESTOCK
EC ® 98.7
BAIB 0 98.6
SUMMARY
Type
Percent positive
—
9f 8
-+—
1.5
^
0 1
— + -t
2.8
EC®
96 3
BALB
95 3
POULTRY
EC ® 93 0
BALB ® 92.5
FIGURE 1
COLIFORMS
67 Soil Samples
(Geltli eicli. el .il.)
- + - -f I
I ++++ 1/
I—ttti Undisturbed
1 Soil
Polluted
Soil
FIGURE 2
9
-------
Bacteriological Indicators of Water Pollution
Table 2. COMPARISON OF COLIFORM STRAINS ISOLATED FROM WARM-BLOODED ANIMAL
FECES, FROM UNPOLLUTED SOILS AND POLLUTED SOILS WITH USE OF THE
IMViC REACTIONS AND THE ELEVATED TEMPERATURE TEST IN EC MEDIUM
AT 44.50 C(+0.5°) (12th ed. 1965, Standard Methods for the Examination of Water
and Wastewater)
Test
Warm-blooded
animal feces
Soil.
Unpolluted
Soil
Polluted
Vege-
tation
Insects
+ + - -
91. 8%
5. 6%
80. 6%
10. 6%
12.4%
+ + - -,
+ and
- + —
93. 3%
8. 9%
80. 7%
12. 5%
13. 2%
Indole positive
94. 0%
19. 4%
82. 7%
52. 5%
52. 4%
Methyl red positive
96. 9%
75. 6%
97. 9%
63. 6%
79. 9%
Voges-Proskauer positive
5 1%
40. 7%
97. 3%
56. 3%
40. 6%
Citrate utilizers
3. 6%
88. 2%
19. 2%
85 1%
86. 7%
Elevated temperature (EC)
positive
96. 4%
9. 2%
82. 9%
14. 1%
14. 9%
Number of cultures
studied
8, 747
2, 348
665
1, 203
1, 084
Total Pure Cultures Studied 14,047
d The elevated temperature test gives IV EVALUATION OF COLIFORMS AS
excellent correlation with samples POLLUTION INDICATORS
of known or highly probable fecal
origin. The presence of smaller, A The Coliform Group as a Whole
but demonstrable, percentages of
such organisms in environmental
sources not interpreted as being
polluted could be attributed largely
to the warm-blooded wildlife in the
area, including birds, rodents, and
other small mammals.
e The elevated temperature test yields
results equal to those obtained from
the total IMViC code. It has marked
advantages in speed, ease and
simplicity of performance, and yields
quantitative results for each water
sample. Therefore, it is regarded
as the method of choice for differ-
entiation between coliforms of
probable direct fecal origin and those
which may have become established
in the bacterial flora of the aquatic
or terrestrial habitat.
1 Merits
a The absence of coliform bacteria is
evidence of a bacteriologically safe
water.
b The density of coliforms is roughly
proportional to the amount of
excretal pollution present.
c If pathogenic bacteria of intestinal
origin are present, coliform
bacteria also are present, in much
greater numbers.
d Coliforms are always present in the
intestines of humans and other warm-
blooded animals, and are eliminated
in large numbers in fecal wastes.
9-10
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Bacteriological Indicators of Water Pollution
e Coliforms are more persistent in
the aquatic environment than are
pathogenic bacteria of intestinal
origin.
f Coliforms are generally harmless
to humans and can be determined
quantitatively by routine laboratory
procedures.
2 Limitations
a Some of the constituents of the
coliform group have a wide environ-
mental distribution in addition to
their occurrence in the intestines
of warm-blooded animals.
b Some strains of the coliform group
may multiply in certain polluted
waters ("aftergrowth"), of high
nutritive values thereby adding to
the difficulty of evaluating a pollution
situation in the aquatic environment.
Members of the A. aerogenes section
of the coliform are commonly
involved in this kind of problem.
c Because of occasional aftergrowth
problems, the age of the pollution
may be difficult to evaluate under
some circumstances.
d Tests for coliforms are subject to
interferences due to other kinds of
bacteria. False negative results
sometimes occur when species of
Pseudomonas are present. False
positive results sometimes occur
when two or more kinds of non-
conforms produce gas from lactose,
when neither can do so alone
(synergism).
B The Fecal Coliform Component of the
Coliform Group (as determined by elevated
temperature test)
1 Merits
a The majority (over 95% of the coli-
form bacteria from intestines of
warm-blooded animals grow at the
elevated temperature.
b These organisms are of relatively
infrequent occurrence except in
association with fecal pollution,
c Survival of the fecal coliform group
is shorter in environmental waters
than for the coliform group as
whole. It follows, then, that high
densities of fecal coliforms is
indicative of relatively recent
pollution.
d Fecal coliforms generally do not
multiply outside the intestines of
warm-blooded animals. In certain
high-carbohydrate wastes, such as
from the sugar beet refineries,
exceptions have been noted.
2 Limitations
a Feces from warm-blooded animals
include some (though proportionately
low) numbers of coliforms which do
not yield a positive fecal coliform
test when the elevated temperature
test is used as the criterion of
differentiation. These organisms
are E. coli varieties by present
taxonomic classification.
b There is at present no established
and consistent correlation between
ratios of total coliforms/fecal
coliforms in interpreting sanitary
quality of environmental waters.
In domestic sewage, the fecal
coliform density commonly is
greater than 90% of the total
coliform density. In environmental
waters relatively free from recent
pollution, the fecal coliform density
may range from 10-30% of the total
coliforms. There are, however,
too many variables relating to
water-borne wastes and surface
water runoff to permit sweeping
generalization on the numerical
relationships between fecal- and
total coliforms.
c At this time, evaluations are
underway regarding the survival
9-11
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Bacteriological Indicators of Water Pollution
of fecal coliforms in polluted waters
compared with that of enteric
pathogenic bacteria. In recent
pollution studies, species of
Salmonella have been found in the
presence of 220 fecal coliforms per
100 ml (Spino), and 110 fecal
coliforms per 100 ml (Brezenski,
Raritan Bay Project).
V APPLICATIONS OF COLIFORM TESTS
A Current Status in Official Tests
1 The coliform group is designated, in
"Standard Methods for the Examination
of Water and Wastewater" (13th ed.,
1971), through the Completed Test
MPN procedure as the official test
for bacteriological potability of water.
The Confirmed Test MPN procedure
is accepted where it has been demon-
strated, through comparative tests,
to yield results equivalent to the
Completed Test. The membrane filter
method also is accepted for examination
of waters subject to interstate regulation.
2 The 12th edition of Standard Methods has
introduced a standard test for fecal
coliform bacteria. It is emphasized
that this is to be used in pollution
studies, and does not apply to the
evaluation of water for potability.
This procedure has been carried to
the 13th Edition.
B Applications
1 Tests for the coliform group as a
whole are used in official tests to
comply with interstate drinking water
standards, state standards for shell-
fish waters, and in most, if not all,
cases where bacterial standards of
water quality have been established
for such use as in recreational or
bathing waters, water supplies, or
industrial supplies. Laboratory
personnel should be aware of possible
implementation of the fecal coliform
group as the official test for recreational
and bathing waters.
2 The fecal coliform test has application
in water quality surveys, as an adjunct
to determination of total coliform
density. The fecal coliform test is
being used increasingly in all water
quality surveys.
3 It is emphasized that no responsible
worker advocates substitution of a
fecal coliform test for total coliforms
in evaluating drinking water quality.
9-12
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Bacteriological Indicators of Water Pollution
Part 3. The Fecal Streptococci
I DEFINITIONS
A A fecal streptococcus as used in this
discussion refers to any streptococcus
commonly found in significant numbers
in the feces of human or other warm-
blooded animals. Other terms used are
"enterococci" or "Group D streptococci. "
B Enterococci are characterized by specific
taxonomic biochemistry. Serological
procedures differentiate the Group D
streptococci from the various groups.
Although they overlap, the three groups,
fecal streptococcus, enterococcus, and
Group D streptococcus, are not synonymous.
Because our emphasis is on indicators of
unsanitary origin, fecal streptococcus is
the more appropriate term and will include
the enterococcus as well as other groups.
C A rigid definition of the fecal streptococcus
group is not possible with our present
knowledge. The British Ministry of Health
(1956) defines the organisms as "Gram-
positive" cocci, generally occurring in
pairs or short chains, growing in the
presence of bile salt, usually capable of
development at 45° C, producing acid but
not gas in mannitol and lactose, failing to
attack raffinose, failing to reduce nitrate
to nitrite, producing acid in litmus milk
and precipitating the casein in the form of
a loose, but solid curd, and exhibiting a
greater resistance to heat, to alkaline
conditions and to high concentrations of
salt than most vegetative bacteria. "
However, it is pointed out that
"streptococci departing in one or more
particulars from the type species cannot
be disregarded in water. "
D Some workers consider that growth at
45° C and multiplication in 40% bile are
the most significant indications of fecal
origin of streptococci.
Ej
1 The first group, the Pyogenic Group,
not growing at 10° C or 45° C,
pathogenic for man and animals,
causes total lysing of red blood cells,
giving them the common name of beta
hemolytic streptococci. These orga-
nisms are not considered as belonging
to the fecal streptococci.
2 A second group, or Viridans group,
not growing at 10° C, growth at 45° C,
is an intermediate group, some
members resembling group 1 and others.
3 The third group, the Enterococcus
Group, growing at 10° C and 45o C,
and typically exhibit vigorous bio-
chemical reactions. Members of
these groups (2 and 3) are considered
to be fecal streptococci.
4 The fourth, the Lactic Group, growing
at 10° C and not at 45° C, is a common
harmless inhabitant of plants and dairy
products and bear no relationship to
the definition of a fecal streptococcus.
Examples of specific strains or species
representative of each group are-
a Streptococcus pyogenes, cause of
scarlet fever
b Streptococcus bovis, from cow
manure
c Streptococcus fecalis, from human
feces
d Streptococcus lactis, from cheese
F For special purposes, strains of individual
fecal streptocci are identified by streptocci
are identified by biochemical tests. The
techniques are discussed elsewhere.
Strains or species likely to be fecal
streptococci are- Streptococcus fecalis,
S. fecalis variety liquefaciens, S. fecalis
variety zymogenes, S. durans, S. bovis,
S, equinus, S. salivarius, and S. fecalis
biotypes usually growing at 10° C and 45° C.
9-13
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Bacteriological Indicators of Water Pollution
H HISTORY
From their discovery to the present time the
fecal streptococci appear characteristic of
fecal pollution, being consistently present in
both the feces of all warm-blooded animals
and in the environment associated with animal
discharges. As early as 1910 fecal strepto-
cocci were proposed as indicators to the
Metropolitan Water Board of London.
However, little progress resulted in the
United States until improved methods of
detection and enumeration appeared after
World War II.
Renewed interest in the group as indicators
began with the introduction of azide dextrose
broth in 1950, (Mallmann & Seligmann, 1950),
The method which is in the current edition
of Standard Methods appeared soon after.
(Litsky, et al. 1955).
With the advent of improved methods for
detection and enumeration of fecal strep-
tococci, a significant body of technical
literature has appeared.
IE SIGNIFICANCE OF FECAL STREPTO-
COCCAL INDICATORS
A The presence of fecal streptococci
indicates the presence of warm-blooded
animal pollution. Many types of wild life
may harbor fecal streptococci. One study
showed 71% of various mammalian species
yielding enterococci. Similarly, reptiles
showed 85% incidence. However, the
common rodents yielded few and sporadic
isolations. Wild birds also showed few
individuals with enterococci. (Mundt 1963),
Insects may be only accidental mechanical
carriers of fecal streptococci. However,
Geldreich, et al (1964) found high numbers
in some insects.
B Evidence indicates that fecal streptococci
do not occur in pure water or virgin soil.
In one study, Medrek and Litsky (I960)
looked specifically for occurrence of
enterococci in undisturbed or virgin soil.
Modern methods showed 8 out of 369
samples with enterococci. With only one
exception, the presence of enterococci
in a given soil sample was associated
with the presence of coliforms. The
presence of indicators in a given soil
sample is considered indicative of chance
pollution of the sampling area by animal
or bird droppings or drainage.
C Fecal streptococci do not multiply in
water. A recent report is illustrative
(Morris and Weaver, 1954). Duplicate
samples were taken of 52 polluted
Kentucky wells and examined before and
after storage for 24 hours. While the
coliform numbers increased, decreased
or changed little, the streptococcal
numbers never increased.
Other experiments on a variety of
Louisiana surface water gave same
results for streptococcal numbers.
Fecal streptococci enter a water, survive
for a period of time and die without
multiply mg.
D On the basis of laboratory data, the fecal
streptococci could be expected to be more
resistant to electrolytes like salt. If true,
the group would have special value for
salt water investigation. Further research
is needed to determine this point.
E The fecal streptococci are not considered
pathogenic. However, such strains as
S. fecalis have been known to occur with
endocarditis and other human infections.
S. fecalis var zymogenes, a beta
hemolytic streptococcus normally
inhabiting the human intestine, is also
nonpathogenic.
The fecal streptococci are alleged causes
of food-borne infections. However, the
circumstantial evidence has not been
supported by intensive studies.
IV LIMITATIONS
A Knowledge of relative survival times of
fecal streptococci and pathogens is needed.
Comparable results with many types of
pathogens under many environmental
conditions are needed. Insufficient time
9-14
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Bacteriological Indicators of Water Pollution
has elapsed since adequate media have
been developed for evidence to accumulate.
The coliforms have over 60 years of
accumulated validation for their use.
The fecal streptococci have had only about
15years of serious consideration. A few
comparisons exist, however.
When sewage is added to the soil, the
coliforms persist for long periods. Fecal
streptococci disappear rapidly. Typhoid
bacilli, added experimentally with sewage,
persist less time than the fecal strepto-
cocci. However, mineral-organic content
of the receiving soil has much influence
on persistence so that only relative rates
may be determined. (Mailman and Litsky,
1950).
B Although the enterococci are considered
as having their origin, in the intestines
of warm-blooded animals, other sources
may exist. The resistance and tolerance
of these streptococci, together with their
low minimum and high maximum limits of
growth may not only enable them to survive
but also to grow under diverse conditions
of nature. Reports have appeared that
growth occurs in food products, fresh
plants and silage. For example, some
investigations (Mundt, 1962) show that
fecal streptococci of the S. fecalis and
the S. liquefaciens types occur commonly
on plants. However, Geldreich et al,
1963 concluded after an exhaustive study
of vegetation that indicator contribution
was negligible.
V FECAL COLIFORM-FECAL
STREPTOCOCCUS RELATIONSHIPS
Because coliforms may derive from non-
fecal sources, the fecal coliforms are a more
reliable standard of comparison with strep-
tococcal densities. Even so, variation in
known ratios is marked.
A Variation may arise from different
methods, different species, different
individuals, and different geographic
locations.
Table 3. ESTIMATED PER CAPITA CONTRIBUTION OF INDICATOR MICROORGANISMS
FROM SOME ANIMALS*
Average indicator Average contribution
density per gram per capita per 24 hr
of feces
Animals
Avg wt of
Feces/24 hr,
wet wt, g
Fecal
coliform,
million
Fecal
streptococci,
million
Fecal
coliform,
million
Fecal
streptococci,
million
Ratio
FC/FS
Man
150
13.0
3.0
2,000
450
4.4
Duck
336
33. 0
54.0
11, 000
18, 000
0.6
Sheep
1, 130
o
CO
1-i
38.0
18, 000
43, 000
0.4
Chicken
182
1.3
3.4
240
620
0.4
Cow
23, 600
0.23
1.3
5,400
31, 000
0.2
Turkey
448
0.29
2.8
130
1, 300
0. 1
Pig
2, 700
3.3
84.0
8,900
230, 000
0.04
•^Publication WP-20-3, P. 102
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Bacteriological Indicators of Water Pollution
B Species differences are the main cause of
different fecal coliform-fecal streptococci
ratios. Table 3 compares fecal strep-
tococcus and fecal coliform counts for
different species. Even though individuals
vary widely, masses of individuals in a
species have characteristic proportion
of indicators. In intestinal wastes of
human origin, the ratio of number of fecal
coliforms to number of fecal streptococci
tends to be considerably greater than two-
to-one. In comparison, when such ratios
are determined for intestinal wastes from
non-human animal sources, the values
tend to be markedly less than one-to-one.
Current interpretative use of this ratio
is as follows:
Domestic Wastes
(Predominately Human) - > 4. 0
Animal Wastes -<0.7
C Determination of specific strains of fecal
streptococci sometimes is useful.
Besides ratio of indicators, the specific
types of fecal streptococci are more
typical of some animals. Humans carry
80% or over of S. fecalis group, and
characteristically the S. salivarius-mitis
group. Cows and horses carry a
majority of S. bovis and S. equinus groups.
Other farm animals have mixtures of
appreciable numbers of several groups.
Fowl, however, resemble humans in their
streptococcal flora. The S. salivarius
group, besides being uniquely human,
dies rapidly in water, giving an index of
recentness of pollution.
VI INTERPRETATION
The presence of fecal streptococci in
untreated water indicates the presence of
fecal pollution by warm-blooded animals.
In samples where the source and significance
of the coliform group has been questioned,
the presence of the streptococcus group
should be interpreted as indicating at least
a portion of the coliform group were derived
from fecal sources.
Because of the uncertainties in die-off rates,
the absence of fecal streptococci does not
necessarily mean that the water is
bacteriologically safe.
9-16
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Bacteriological Indicators of Water Pollution
Part 4. Other Bacterial Indicators of Pollution
I TOTAL BACTERIAL COUNTS
A Historical
1 The early studies of Robert Koch led
him to develop tentative standards of
water quality based on a limitation of
not more than 100 bacterial colonies
per ml on a gelatin plating medium
incubated 3 days at 20° C.
2 Later developments led to inoculation
of samples on duplicate plating media,
with one set incubated at 37° C and the
other at 20° C.
a Results were used to develop a ratio
between the 370 c counts and the
20O C counts.
b Waters having a predominant count
at 370C were regarded as being of
probable sanitary significance,
while those giving predominant
counts at 20° C were considered to
be of probable soil origin, or
natural inhabitants of the water
being examined.
B Groups Tested
There is no such thing as "total" bacterial
count in terms of a laboratory determination.
1 Direct microscopic counts do not
differentiate between living and dead
cells.
2 Plate counting methods enumerate only
the bacteria which are capable of using
the culture medium provided, under the
temperature and other growth conditions
used as a standard procedure. No one
culture medium and set of growth
conditions can provide, simultaneously,
an acceptable environment for all the
heterogeneous, often conflicting,
requirements of the total range of
bacteria which may be recovered from
waters.
C Utilization of Total Counts
1 Total bacterial counts, using plating
methods, are useful for:
a Detection of changes in the bacterial
composition of a water source
b Process control procedures in
treatment plant operations
c Determination of sanitary conditions
in plant equipment or distributional
systems
2 Serious limitations in total bacterial
counts exist because
a No information is given regarding
possible or probable fecal origin
of bacterial changes. Large numbers
of bacteria can sometimes be
cultivated from waters known to be
free of fecal pollution.
b No information of any kind is given
about the species of bacteria
cultivated.
c There is no differentiation between
harmless or potentially dangerous
forms.
3 Status of total counts
a There is no total bacterial count
standard for any of the following:
Interstate Quarantine Drinking
Water Standards
PHS regulations for water
potability (as shown in
"Standard Methods" Public
Health Service Drinking
Water Standards of 1962.)
b The most widely used current
application of total bacterial counts
in water bacteriology today is in
9-17
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Bacteriological Indicators of Water Pollution
water treatment plants, where some
workers use standard plate counts
for process control and for deter-
mination of the bacterial quality of
distribution systems and equipment.
c Total bacterial counts are not used
in PHS water quality studies, though
extensively used until the 1940's.
B Spore-Forming Bacteria (Clostridium
perfringens, or C. welchii)
1 Distribution
This is one of the most widely distributed
species of bacteria. It is regularly
present in the intestinal tract of warm-
blooded animals.
2 Nature of organism
C. perfringens is a Gram-positive,
spore-forming rod. The spores cause
a distinct swelling of the cell when
formed. The organism is extremely
active in fermentation, of carbohydrates,
and produces the well-known "stormy
fermentation" of milk.
3 Status
The organism, when present, indicates
that pollution has occurred at some
time. However, because of the ex-
tremely extended viability of the spores,
it is impossible to obtain even an
approximation of the recency of pollution
based only on the presence of
C. perfringens.
The presence of the organism does not
necessarily indicate an unsafe water.
C Tests for Pathogenic Bacteria of Intestinal
Origin
1 Groups considered include Salmonella
sp. Shigella sp. Vibrio comma,
Mycobacterium sp. Pasteurella sp,
Leptospira sp, and others.
2 Merits of direct tests
Demonstration of any pathogenic
species would demonstrate an
unsatisfactory water quality, hazardous
to persons consuming or coming into
contact with that water.
3 Limitations
a There is no available routine pro-
cedure for detection of the full
range of pathogenic bacteria cited
above.
b Quantitative methods are not avail-
able for routine application to any
of the above.
c The intermittent release of these
pathogens makes it impossible to
regard water as safe, even in the
absence of pathogens.
d After detection, the public already
would have been exposed to the
organism, thus, there is no built-in
margin of safety, as exists with
tests for the coliform group.
4 Applications
a In tracing the source of pathogenic
bacteria in epidemiological investi-
gations
b In special research projects
c In water quality studies concerned
with enforcement actions against
pollution, increasing attention is
being given to the demonstration of
enteric pathogenic bacteria in the
presence of the bacterial indicators
of pollution.
D Miscellaneous Indicators
It is beyond this discussion to explore the
total range of microbiological indicators
of pollution that have been proposed and
9-18
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Bacteriological Indicators of Water Pollution
investigated to some extent. Mention can
be made, however, of consideration of
tests for the following:
1 Bacteriophages specific for any of a
number of kinds of bacteria
2 Serological procedures for detection
of coliforms and other indicators, a
certain amount of recent attention has
been given to applications of fluorescent
antibodies in such tests
3 Tests for Pseudomonas aeruginosa
4 Tests for viruses, which may persist
in waters even longer than members
of the coliform group.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater, 12th ed.,
APHA, AWWA, WPCF. Published by
American Public Health Association,
1790 Broadway, New York, N.Y. 1965.
2 Prescott, S. C., Winslow, C.E.A., and
McCrady, M. Water Bacteriology.
John Wiley & Sons, Inc. 1946.
3 Parr, L.W. Coliform Intermediates in
Human Feces. Jour. Bact. 361.
1938.
4 Clark, H.F. and Kabler, P.W. The
Physiology of the Coliform Group.
Proceedings of the Rudolfs Research
Conference on Principles and Appli-
cations in Aquatic Microbiology. 1963.
5 Geldreich, E.E., Bordner, R.H., Huff,
C.B., Clark, H.F. , and Kabler. P.W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals.
JWPCF. 34.295-301. 1962.
6 Geldreich et al. The Fecal Coli-Aerogenes
Flora of Soils from Various Geographic
Areas. Journal of Applied Bacteriology
25-87-93. 1962.
7 Geldreich, E.E., Kenner, B.A., and
Kabler, P.W. Occurrence of
Coliforms, Fecal Coliforms, and
Streptococci on Vegetation and Insects.
Applied Microbiology. 12:63-69. 1964.
8 Kabler, P.W., Clark, H.F., and
Geldreich, E.E. Sanitary Significance
of Coliform and Fecal Coliform
Organisms in Surface Water. Public
Health Reports. 79:58-60. 1964.
9 Clark, H.F. and Kabler, P.W.
Re-evaluation of the Significance of the
Coliform Bacteria. Journal AWWA.
56-931-936. 1964.
10 Kenner, B. S., Clark, H.F., and
Kabler, P.W. Fecal Streptococci,
n. Quantification in Feces. Am. J.
Public Health. 50:1553-59. 1960.
11 Litsky, W., Mailman, W.L., and Fifield,
C. W. Comparison of MPN of
Escherichia coli and Enterococci in
River Water. Am. Jour. Public Health.
451949. 1955.
12 Medrek, T. F. and Litsky, W.
Comparative Incidence of Coliform
Bacteria and Enterococci in
Undisturbed Soil. Applied Micro-
biology. 8:60-63, 1960.
13 Mailman, W.L., and Litsky, W.
Survival of Selected Enteric Organisms
in Various Types of Soil. Am. J.
Public Health. 41:38-44. 1950.
14 Mailman, W.L., and Seligman, E.B., Jr.
A Comparative Study of Media for
Detection of Streptococci in Water and
Sewage. Am. J. Public Health.
40-286-89. 1950.
15 Ministry of Health (London). The
Bacterial Examination of Water Supplies.
Reports on Public Health and Medical
Subjects. 71:34.
9-19
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Bacteriological Indicators of Water Pollution
16 Morris, W. and Weaver, R.H.
Streptococci as Indices of Pollution
in Well Water. Applied Microbiology.
2:282-285. 1954.
18 Geldreich, E.E. Sanitary Significance
of Fecal Coliforms m the Environment.
U.S. Department of the Interior.
FWPCA Publ. WP-20-3. 1966.
17 Mundt, J.O., Coggin, J.H., Jr., and
Johnson, L. F. Growth of
Streptococcus fecalis var. liquefaciens
on Plants, Applied Microbiology.
10:552-555. 1962.
This outline was prepared by H. L. Jeter,
Director, National Training Center, OWP,
EPA, Cincinnati, OH 45268.
9-20
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EXAMINATION OF WATER FOR COLIFORM AND
FECAL STREPTOCOCCUS GROUPS
(Multiple Dilution Tube (MPN) Methods)
I INTRODUCTION
The subject matter of this outline is contained
m three parts, as follows:
A Part 1
1 Fundamental aspects of multiple dilution
tube ("most probable numbers") tests,
both from a qualitative and a quantitative
viewpoint.
2 Laboratory bench records.
3 Useful techniques in multiple dilution
tube methods.
4 Standard supplies, equipment, and
media in multiple dilution tube tests.
B Part 2
Detailed, day-by-day, procedures in tests
for the coliform group and subgroups
within the coliform group.
C Part 3
Detailed, day-by-day, procedures in tests
for members of the fecal streptococci.
D Application of Tests to Routine Examinations
The following considerations (Table 1) apply
to the selection of the Presumptive Test,
the Confirmed Test, and the Completed
Test. Termination of testing at the
Presumptive Test level is not practiced
by laboratories of this agency. It must
be realized that the Presumptive Test alone
has limited use when water quality is to
be determined.
TABLE 1
Examination Terminated at -
Type of Receiving
Water
Presumptive
Test
Confirmed Test
Completed Test
Sewage Receiving
Treatment Plant - Raw
Applicable
Applicable
Applicable
Applicable
Important where results
are to be used for control
of raw or finished water.
Application to a statis-
tically valid number of
. samples from the
Confirmed Test to estab-
lish its validity m
determining the sanitary
quality.
Chlorinated
Not Done
Applicable
Bathing
Not Done
Applicable
Drinking
Not Done
Applicable
Other Information
Applicable in all
cases where Pre-
sumptive Test alone
is unreliable.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Environmental Protection Agency."
W.BA.3L. 10.71
10-1
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MPN Methods
H BASIS OF MULTIPLE TUBE TESTS
A Qualitative Aspects
1 For purely qualitative aspects of testing
for indicator organisms, it is convenient
to consider the tests applied to one
sample portion, inocujated into a tube
of culture medium, and the follow-up
examinations and tests on results of the
original inoculation. Results of testing
procedures are definite: positive
(presence of the organism-group is
demonstrated) or negative (presence of
the organism-group is not demonstrated.)
2 Test procedures are based on certain
fundamental assumptions:
a First, even if only one living cell of
the test organism is present in the
sample, it will be able to grow when
introduced into the primary inoculation
medium,
b Second, growth of the test organism
in the culture medium will produce
a result which indicates presence of
the test organism, and,
c Third, extraneous organisms will
not grow, or if they do grow, they
will not limit growth of the test
organism, nor will they produce
growth effects that will be confused
with those of the bacterial group for
which the test is designed.
3 Meeting these assumptions usually
makes it necessary to conduct the tests
in a series of stages (for example, the
Presumptive, Confirmed, and Completed
Test stages, respectively, of standard
tests for the coliform group).
4 Features of a full, multi-stage test
a First stage: The culture medium
usually serves primarily as an
enrichment medium for the group
tested. A good first-stage growth
medium should support growth of all
the living cells of the group tested,
and it should include provision for
indicating the presence of the test
organism being studied. A first-
stage medium may include some
component which inhibits growth
of extraneous bacteria, but this
feature never should be included
if it also inhibits growth of any
cells of the group for which the
test is designed. The Presumptive
Test for the coliform group is a
good example. The medium
supports growth, presumably, of
all living cells of the coliform
group, the culture container has a
fermentation vial for demonstration
of gas production resulting from
lactose fermentation by coliform
bacteria, if present, and sodium
lauryl sulfate may be included in
one of the approved media for
suppression of growth of certain
noncoliform bacteria. This
additive apparently has no adverse
effect on growth of members of the
coliform group in the concentration
used. If the result of the first-stage
test is negative, the study of the
culture is terminated, and the result
is recorded as a negative test. No
further study is made of negative
tests. If the result of the first-
stage test is positive, the culture
may be subjected to further study
to verify the findings of the first
stage.
b Second stage: A transfer is made
from positive cultures of the first-
stage test to a second culture medium.
This test stage emphasizes provision
to reduce confusion of results due to
growth effects of extraneous bacteria,
commonly achieved by addition of
selective inhibitory agents. (The
Confirmed Test for coliforms meets
these requirements. Lactose and
fermentation vials are provided for
demonstration of coliforms in the
medium. Brilliant green dye and
bile salts are included as inhibitory
agents which tend to suppress growth
of practically all kinds of noncoliform
bacteria, but do not suppress growth
of coliform bacteria when used as
directed).
10-2
-------
MPN Methods
If result of the second-stage test is
negative, the study of the culture is
terminated, and the result is re-
corded as a negative test. A negative
test here means that the positive
results of the first-stage test were
"false positive, " due to one or more
kinds of extraneous bacteria. A
positive second-stage test is partial
vertificatioi>of the positive results
obtained in the first-stage test, the
culture may be subjected to final
identification through application of
still further testing procedures. In
routine practice, most sample exami-
nations are terminated at the end of
the second stage, on the assumption
that the result would be positive if
carried to the third, and final ,
stage. This practice should be
followed only if adequate testing is
done to demonstrate that the assump-
tion is valid. Some workers recom-
mend continuing at least 5% of all
sample examinations to the third
stage to demonstrate the reliability
of the second-stage results.
B Quantitative Aspects of Tests
1 These methods for determining bacterial
numbers are based on the assumption
that the bacteria can be separated from
one another (by shaking or other means)
resulting in a suspension of individual
bacterial cells, uniformly distributed
through the original sample when the
primary inoculation is made.
2 Multiple dilution tube tests for quantita-
tive determinations apply a Most Probable
Number (MPN) technique. In this pro-
cedure one or more measured portions
of each of a stipulated series of de-
creasing sample volumes is inoculated
in(o the first-stage culture medium.
Through decreasing the sample incre-
ments, eventually a volume is reached
where only one cell is introduced into
some tubes, and no cells are introduced
into other tubes. Each of the several
tubes of sample-inoculated first-stage
medium is tested independently,
according to the principles previously
described, in the qualitative aspects
of testing procedures.
3 The combination of positive and
negative results is used in an application
of probability mathematics to secure
a single MPN value for the sample.
4 To obtain MPN values, the following
conditions must be met:
a The testing procedure must result
in one or more tubes in which the
test organism is demonstrated to
be present; and
b The testing procedure must result
in one or more tubes in which the
test organism is not demonstrated
to be present.
5 The MPN value for a given sample is
obtained through the use of MPN Tables.
It is emphasized that the precision of
an individual MPN value is not great
when compared with most physical or
chemical determinations.
6 Standard practice in water pollution
surveys conducted by this organization,
is to plant five tubes in each of a series
of sample increments, in sample
volumes decreasing at decimal intervals.
For example, in testing known polluted
waters, the mitial sample inoculations
might consist of 5 tubes each in volumes
of 0.1, 0.01,0.001, and 0.0001 ml,
respectively This series of sample
volumes will yield determinate results
from a low of 200 to a high of 1, 600, 000
organisms per 100 ml.
10-3
-------
MPN METHODS
in LABORATORY BENCH RECORDS
A Features of a Good Bench Record Sheet
1 Provides complete identification of the
sample.
2 Provides for full, day-by-day informa-
tion about all tests performed on the
sample.
3 Provides easy step-by-step record
applicable to any portion of the sample.
4 Provides for recording of the quantitative
result which will be transcribed to sub-
sequent reports.
5 Minimizes the amount of writing by the
analyst.
6 Identifies the analyst(s)
B There is no such thing as "standard"
bench sheet for multiple tube tests; there
are many versions of bench sheets. Some
are prescribed by administrative authority
(such as the Office of a State Sanitary
Engineer), others are devised by laboratory
or project personnel to meet specific needs.
It is not the purpose of this discussion to
recommend an "ideal" bench form; however,
the form used in this training course
manual is essentially similar to that used
in certain research laboratories of this
organization. The student enrolled in the
course for which this manual is written
should make himself thoroughly familiar
with the bench sheet and its proper use.
See Figure 1.
IV NOTES ABOUT WORKING PROCEDURES
IN THE LABORATORY
A Each bacteriological examination of water
by multiple dilution tube methods requires
a considerable amount of manipulation;
much is quite repetitious. Laboratory
workers must develop and maintain good
routine working habits, with constant
alertness to guard against lapses into
careless, slip-shod laboratory procedures
and "short cuts" which only can lead to
lowered quality of laboratory work.
The student reader is urged to review the
form for laboratory surveys (PHS-875,
Rev. 1966) used by Public Health Service
personnel charged with responsibility for
accreditation of laboratories for examination
of water under Interstate Quarantine
regulations.
B Specific attention is brought to the following
by no means exhaustive, critical aspects of
laboratory procedures in multiple dilution
tube tests:
1 Original sample
a Follow prescribed care and handling
procedures before testing.
b Maintain absolute identification of
sample at all stages in testing.
c Vigorously shake samples (and
sample dilutions) before planting
in culture media.
2 Sample measurement into primary
culture medium
a Sample portions must be measured
accurately mto the culture medium
for reliable quantitative tests to l?e
made. Standard Methods prescribes
that calibration errors should not
exceed + 2. 5%.
10-4
-------
BACTERIOLOGY BENCH SHEET
Project Ohif /jww
Multiple Dilution Tube Tests
Collection Data
Date pi
Tempe
\AiM-
rature
Other Observations
Sample Station
_Time £ 50 By
_/_°c PH
Bench Number of Sample
Analyst
Analytical Record
^
Test started at BfM
t
ml
"imple
Coliform Test
Fecal
coli-
form
24
Fecal
Remarks
LTB
BGLB
EMB
LSTB
Gram
stain
A-D
EVA
24
48
24
48
24
24
48
24
48
24
48
to
~ ><
N>/i ^
/.D
1
1
1
i
1
i
1
i
i
i
|
i
i
1
1
1
OJ
1
I
1
1
1
,
dD/
1
a. on/
L
Coliform MPN/lOO ml
Confirmed:
Completed:
Fecal Coliform MPN:
Figure l. SAMPLE BENCH SHEET
Fecal Streptococcus MPN/lOO ml
A-D - EVA:
10-5
-------
MPN Methods
Suggested sample measuring practices
are as follows: Mohr measuring
pipets are recommended. 10 ml
samples are delivered at the top of
the culture tube, using 10 ml pipets.
1. 0 ml samples are delivered down
into the culture tube, near the sur-
face of the medium, and "touched
off" at the side of the tube when the
desired amount of sample has been
delivered. 1. 0 ml or 2. 0 ml pipets
are used for measurement of this
volume. 0. 1 ml samples are
delivered in the same manner as 1. 0
ml samples, using great care that
the sample actually gets into the
culture medium. Only 1. 0 ml pipets
are used for this sample volume.
After delivery of all sample incre-
ments into the culture tubes, the
entire rack of culture tubes may be
shaken gently to carry down any of
the sample adhering to the wall of
the tube above the medium.
Workers should demonstrate by actual
tests that the pipets and the technique
in use actually delivers the rated volumes
within the prescribed limits of error.
b Volumes as small as 0.1 ml routinely
can be delivered directly from the
sample with suitable pipets. Lesser
sample volumes first should be diluted,
with subsequent delivery of suitable
volumes of diluted sample into the
culture medium. A diagrammatic
scheme for making dilutions is shown
in Figure 2.
b Gas in any quantity is a positive test.
It is necessary to work in conditions
of suitable lighting for easy recog-
nition of the extremely small amounts
of gas inside the tops of some
fermentation vials.
4 Reading of liquid culture tubes for
growth as indication of a positive test
requires good lighting. Growth is
shown by amy amount of increased
turbidity or opalescence in the culture
medium, with or without deposit of
sediment at the bottom of the tube.
5 Transfer of cultures with inoculation
loops and needles
a Always sterilize inoculation loops
and needles in flame immediately
before transfer of culture; do not
lay it down or touch it to any non-
sterile object before making the
transfer.
b After sterilization, allow sufficient
time for cooling, in the air, to avoid
heat-killing bacterial cells on the
hot wire.
c Loops should be 3 mm in inside
diameter, with a capability of holding
a drop of water or culture.
For routine standard transfers
requiring transfer of 3 loopsful of
culture, many workers form three
3-mm loops on the same length of
wire.
3 Reading of culture tubes for gas
production
a On removal from the incubator,
shake culture rack gently, to
encourage release of gas which
may be supersaturated in the culture
medium.
6 As an alternative to use of standard
inoculation loops, the use of
"applicator sticks" have now been
sanctioned by the 13th Edition of
Standard Methods.
10-6
-------
MPN Methods
Figure 2. PREPARATION OF DILUTIONS
2 4
Dilution Ratios 1:10 1:10
Water
Sample
1 ml
99 ml
blank
1 ml
Delivery volume 10 ml
0. 1 ml
1 ml
lml
0. lml
Tubes
Petri Dishes or Culture Tubes
99 ml
blank
1 ml
0. 1 ml
-1 -2 -3 -4 -5
Actual volume 10ml lml 10 ml 10 ml 10 ml 10 ml 10 ml
of sample in tube
The applicator sticks are dry heat
sterilized (autoclave sterilization is
not acceptable because of possible
release of phenols if the wood is
steamed) and are used on a single-
service basis. Thus, for every culture
tube transferred, a new applicator
stick is used.
This use of applicator sticks is
particularly attractive in field
situations where it is inconvenient or
impossible to provide a gas burner
suitable for sterilization of the
inoculation loop. In addition, use of
applicator sticks is favored in
laboratories where room temperatures
are significantly elevated by use of
gas burners.
7 Streaking cultures on agar surfaces
a All streak-inoculations should be
made without breaking the surface
of fhe agar. Learn to use a light
touch with the needle, however,
many inoculation needles are so
sharp that they are virtually useless
in this respect. When the needle is
platinum or platinum-iridium wire,
it sometimes is beneficial to fuse
the working tip into a small sphere.
This can be done by momentary
insertion of a well-insulated (against
electricity) wire into a carbon arc,
or some other extremely hot environ-
ment. The sphere should not be more
than twice the diameter of the wire
from which it is formed, otherwise
it will be entirely too heat-retentive
to be useful.
10-7
-------
MPN Methods
When the needle is nichrome
resistance wire, it cannot be heat-
fused; the writer prefers to bend
the terminal 1/16 - 1/8" of the wire
at a slight angle to the overall axis
of the needle. The side of the
terminal bent portion of the needle
then is used for inoculation of agar
surfaces.
b When streaking for colony isolation,
avoid using too much inoculum. The
streaking pattern is somewhat
variable according to individual
preference. The procedure favored
by the writer is shown in the
accompanying figures. Note
particularly that when going from
any one stage of the streaking to the
next, the inoculation needle is heat-
sterilized.
8 Preparation of cultures for Gram
stain
a The Gram stain always should be
made from a culture grown on a
nutrient agar surface (nutrient agar
slants are used here) or from nutrient
broth.
b The culture should be young, and
should be actively growing. Many
workers doubt the validity of the
Gram stain made on a culture more
than 24 hours old.
c Prepare a thin smear for the staining
procedure. Most beginning workers
tend to use too much bacterial sus-
pension in preparing the dried smear
for staining. The amount of bacteria
should be so small that the dried film
is barely visible to the naked eye.
V EQUIPMENT AND SUPPLIES
Consolidated lists of equipment, supplies,
and culture media required for all multiple
dilution tube tests described in this outline
are shown in Table 2.. Quantitative infor-
mation is not presented; this is variable —
according to the extent of the testing pro-
cedure, the number of dilutions used, and
the number of replicate tubes per dilution.
It is noted that requirements for alternate
procedures are fully listed and choices are
made in accordance to laboratory preference.
10-8
-------
MPN Methods
Flame-sterilize an inoculation needle and air-cool.
Dip the tip of the inoculation needle into the bac-
terial culture being studied.
Streak the inoculation needle tip lightly back and
forth over half the agar surface, as m (1), avoid-
ing scratching or breaking the agar surface.
Flame-sterilize the inoculation needle and air-cool.
Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (1) into one-
half the unstreaked area of the agar.
Flame-sterilize the inoculation needle and air-cool.
Turn the Petri dish one-quarter-turn and streak the
inoculation needle tip lightly back and forth over one-
half the agar surface, working from area (2) into
area (3), the remaining unstreaked area.
Flame-sterilize the inoculation needle and set it aside.
Close the culture container and incubate as prescribed.
Figure 3. A SUGGESTED PROCEDURE FOR COLONY ISOLATION BY A
STREAK-PLATE TECHNIQUE
AREA 1 (Heavy inoculum]
-AREA 2
(Moderate growth]
el®
AREA 3 (Isolated colonies)
APPEARANCE OF STREAK • PLATE
AFTER INCUBATION INTERVAL
10-9
-------
MPN Methods
TABLE 2. APPARATUS AND SUPPLIES FOR STANDARD
FERMENTATION TUBE TESTS
Description of Item
Total Coliform Group
Fecal Coliformr
Presumptive
Test
Confirmed
Test
Completed
Test
(BALB)
(EC broth)
Lauryl tryptose broth or Lactose
broth 20 ml amounts of 1 5 X
concentration medium, In 25 X ISO mm
culture tubes with Inverted fermen-
tation vials, suitable caps
X
Lauryl tryptose broth or Lactose
broth 10 ml amounts of single
strength medium In 20 X 150 mm
culture tubes with inverted fermen-
totloa vials suitable caps
X
X
Brilliant green lactose bile broth 2%
in 10 ml amounts, single strength,
tn 20 X ISO mm culture tubes with
Invorted fermentation viols,
suitable caps
X
X
Eosin methylene blue agar, poured
in 100 X 15 mm Petri dishes
X
X
Endo Agar, poured in LOO X 15 mm
dlahes
Nutrient agar slant, scr*w cap tube
Boric scld lactoso broth, 10 ml
amounts of single strength medium
in fermentation tubes
X
X
X
EC Broth, 10 ml amounts of single
strength medium in fermentation
tubes
X
Formate riclnoleate broth
(provisional)
X
Culture tube racks, 10 X5 openings,
each opening to accept 25 mm dia-
meter tubes
X
X
X
X
X
Pipettes, 10 ml, Mohr type, sterile.
In suitable cons
X
Pipettes, 2 ml (optional), Morh type,
sterile, in suitable cans
X
Pipettes, 1 (pi Mohr type, sterile
in metal suitable cans
X
Standard buffered dilution water,
sterile, 99-ml amounts in screw-
capped bottles
X
Gas burner, Bunsen type
X
X
A
X
Inoculation loop, loop 3mm dia-
meter of nichrome or platlnum-
irldium wire, 26 B & S gauge, in
suitable holder (or sterile applicator
stick)
X
X
X
X
Inoculotion needle, nichrome, or
platlnum-iridlum wire, 26 B & S
gauge, in suitable holder
X
X
Incubator adjusted to 35 ~ 0 50 c
X
X
X
Waterbath Incubator, adjusted to
43 ! 0 2°C
X
Waterbath incubator, adjusted to
44 5 + 0 2°C
X
Class microscopic slides, 1" X3"
X
Slide rocks (optional)
X
Cram*staln solutions, complete set
X
Compound microscope, oil immer*
sion lens, Abbe' condebser
X
Basket fqr discarded cultures
X
X
X
X
X
Container for discarded pipottes
X
10-10
-------
Part 2
DETAILED TESTING PROCEDURES FOR MEMBERS OF THE
COLIFORM GROUP BY MULTIPLE DILUTION TUBE METHODS
I SCOPE
A Tests Described
1 Presumptive Test
2 Confirmed Test
3 Completed Test
4 Fecal Coliform Test
B Form of Presentation
The Presumptive, Confirmed, and
Completed Tests are presented as total,
independent procedures. It is recognized
that this form of presentation is somewhat
repetitious, inasmuch as the Presumptive
Test is preliminary to the Confirmed
Test, and both the Presumptive Test and
the Confirmed Test are preliminary to the
Completed Test for total coliforms.
In using these procedures, the worker
must know at the outset what is to be the
atage at which the test is to be ended, and
the details of the procedures throughout,
in order to prevent the possibility of
discarding gas-positive tubes before
proper transfer procedures have been
followed.
Thus, if the worker knows that the test will
be ended at the Confirmed Test, he will
turn at once to Section HI, TESTING TO
THE CONFIRMED TEST STAGE, and will
ignore Sections II and IV.
The Fecal Coliform Test is described
separately, in Section V, as an
adjunct to the Confirmed Test and to the
Completed Test.
H TESTING TO PRESUMPTIVE TEST
STAGE
A First-Day Procedures
1 Prepare a laboratory data sheet for
the sample. Record the following
information: assigned laboratory
number, source of sample, date and
time of collection, temperature of the
source, name of sample collector,
date and time of receipt of sample in
the laboratory. Also show the date
and time of starting tests in the
laboratory, name(s) of worker(s) per-
forming the laboratory tests, and the
sample volumes planted.
2 Label the tubes of lauryl tryptose broth
required for the initial planting of the
sample ("fable 3). The label should
bear three identifying marks. The
upper number is the identification of
the workers) performing the test
(applicable to personnel in training
courses), the number immediately
below is the assigned laboratory num-
ber, corresponding with the laboratory
record sheet. The lower number is the
code to designate the sample volume
and which tube of a replicate series is
indicated.
NOTE: Be sure to use tubes containing
the correct concentrations of culture medium
for the inoculum/tube volumes. (See the
chapter on media and solutions for multiple
dilution tube methods or refer to the current
edition of Standard Methods for Water and
Wastewater).
W.BA. 3L. 10. 71
10-11
-------
MPN Methods
Table 3. SUGGESTED LABELING SCHEME FOR ORIGINAL CULTURES AND
SUBCULTURES IN MULTIPLE DILUTION TUBE TESTS
Tube
1
Tube
2
Tube
3
Tube
4
Tube
5
Sample volume
represented
Bench number
312
312
312
312
312
Tubes with 10 ml
Volume & tube
A
B
C
D
E
of sample
Bench number
312
312
312
312
312
Tubes with 1 ml
Volume & tube
a
b
c
d
e
of sample
Bench number
312
312
312
312
312
Tubes with 0.1 ml
Volume & tube
a
c
d
e
of sample
Bench number
312
312
312
312
312
Tubes with 0.01 ml
Volume & tube
la
lb
lc
Id
le
of sample
Bench number
312
312
312
312
312
Tubes with 0.001 ml
Volume & tube
2a
2b
2c
2d
2e
of sample
Typical Example
RB
312
A
101
Lab Worker
^Identification
Bench Number
Sample Volume
Tube of Culture Medium
The labeling of cultures can be reduced by labeling only the first tube of
each series of identical sample volumes in the initial planting of the sample.
All subcultures from initial plantings should be labeled completely.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an arc of one foot
within seven seconds and withdraw the
sample portion at once.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
sample.
a Use a 10 ml pipet for 10 ml sample
portions, and 1 ml pipets for portions
of 1 ml or less. Handle sterile pipets
only near the mouthpiece, and protect
the delivery end from external con-
tamination. Do not remove the cotton
plug in the mouthpiece as this is
intended to protect the user from
ingesting any sample.
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample; otherwise sample running
down the outside of the pipet will
makt: .neasurements inaccurate.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in the
incubator at 35° + 0. 5°C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake gently. If gas is about to appear
in the fermentation vials, the shaking
will speed the process.
10-12
-------
MPN Methods
2 Examine each tube carefully Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY
QUANTITY IS A POSITIVE TEST.
3 Discard all gas-positive tubes of lauryl
tryptose broth, and return all the gas-
negative tubes to the 35°C incubator
for an additional 24+2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Be sure to record all results under the
48-hour LTB column on the data sheet.
Discard all tubes. The Presumptive
Test is concluded at this point, and
Presumptive coliforms per 100 ml can
be computed according to the methods
described elsewhere in this manual.
HI TESTING TO CONFIRMED TEST STAGE
Note that the description starts with the
sample inoculation and includes the
Presumptive Test ,stage. The Confirmed
Test preferred in Laboratories of this agency is
accomplished by means of the brilliant
green lactose bile broth (BGLB) and the
acceptable alternate tests are mentioned
in HI F. In addition, the Fecal Coliform
Test is included as an optional adjunct to
the procedure
A First-Day Procedures
1 Prepare a laboratory data sheet for the
sample. Record the following infor-
mation: assigned laboratory number,
source of sample, date and time of
collection, temperature of the source,
name of sample collector, date and
time of receipt of sample in the
laboratory Also show the date and
time of starting tests in the laboratory,
name(s) of worker(s) performing the
laboratory tests, and the sample
volumes planted.
2 Label the tutyes of lauryl tryptose broth
required for the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identification of the worker(s)
performing the test (applicable to
personnel in training courses), the
number immediately below is the
assigned laboratory number, corres-
ponding with the laboratory record
sheet. The lower number is the code
to designate the sample volume and
which tube of a replicate series is
indicated.
NOTE: If 10-ml samples are being
planted, it is necessary to use tubes
containing the correct concentration
of culture medium. This has pre-
viously been noted in n A-2.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
sample.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-ml pipets for portions
of 1 ml or less. Handle sterile pipets
only near the mouthpiece, and protect
the delivery end from external con-
tamination. Do not remove the cotton plug
in the mouthpiece as this is intended,
to protect the user from ingesting
any sample.
10-13
-------
MPN Methods
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample, otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver sample
portions of 1 ml 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.
d Prepare preliminary dilutions of
samples for portions of 0. 01 ml or
less before delivery into the culture
medium. See Table 1 for preparation
of dilutions. NOTE: Always deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much
as 30 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 35° + 0.5°C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose
broth cultures from the incubator, and
shake gently If gas is about to appear
in the fermentation vials, the shaking
will speed the process.
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a
positive (+) test and each tube not
showing gas as a negative (-) test.
GAS IN ANY QUANTITY IS A POSITIVE
TEST.
3 Retain all gas-positive tubes of lauryl
tryptose broth culture in their place
in the rack, and proceed.
4 Select the gas-positive tubes of lauryl
tryptose broth culture for Confirmed
Test procedures. Confirmed Test
procedures may not be required for all
gas-positive cultures. If, after 24-hours
of incubation, all five replicate cultures
are gas-positive for two or more con-
secutive sample volumes, then select
tHe set of five cultures representing
the smallest volume of sample in which
all tubes were gas-positive. Apply
Confirmed Test procedures to all these
cultures and to any other gas-positive
cultures representing smaller volumes
of sample, in which some tubes were
gas-positive and some were gas-negative.
5 Label one tube of brilliant green lactose
bile borth (BGLB) to correspond with
each tube of lauryl tryptose broth
selected for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop transfer one loopful of
culture from each gas-positive tube to
the corresponding tube of BGLB, Place
each newly inoculated culture into BGLB
in the position of the original gas-positive
tube.
7 After making the transfers, the rack
should contain some 24-hour gas-
negative tubes of lauryl tryptose broth
and the newly inoculated BGLB.
8 If the Fecal Coliform Test is included
in the testing procedures, consult
Section V of this part of the outline of
testing procedures.
10-14
-------
MPN Methods
9 Incubate the 24-hour gas-negative
BGLB tubes and any newly-inoculated
tubes of BGLB an additional 24 + 2
hours at 35° + 0. 5°C.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose
bile broth (BGLB). Be sure to record
results from LTB under the 4 8-hour
LTB column and the BGLB results under
the 24-hour column of the data sheet.
3 Label tubes of BGLB to-correspond with
all (if any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
loopful of culture from each gas-positive
LTB culture to the correspondingly-
labeled tube of BGLB. NOTE: AU
tubes of LTB culture which were
negative at 24 hours and became
positive at 48 hours are.to be transferred.
The option described above for 24-hour
cultures does not apply at 48 hours.
hours old^and some may be 48 hours
old. Remove such cultures from the
incubator, examine each tube for gas
production, and record results on the
data-sheet.
2' Be sure to record the results of 24-hour
' BGLB cultures in the "24" column under
BGLB and the 48-hour results under the
"48" column of the data sheet
3 Return any 24-hour gas-negative cultures
for incubation 24 + 2 hours at 35 +
0.5°C.
4 Discard all gas-positive BGLB cultures
and all 48-hour gas-negative cultures
from BGLB.,
5 It is possible that all cultural work and
results for the Confirmed Test have
been finished at this point. If so, codify
results and determine Confirmed Test
coliforms per 100 ml as described in
the outline on use of MPN Tables.
E 96-hour Procedures
4 If the Fecal Coliform Test is included
in the testing procedure, consult
Section V of the part of the outline
of testing procedures.
5 Incubate the 24-hour gas-negative
BGLB tubes and any newly-inoculated
tubes of BGLB 24+2 hours at 35° +
0.5OC.
6 Discard all tubes of LTB and all 24-hour
gas-positive BGLB cultures.
D 72-hour Procedures
1 If any cultures remain to be examined,
all will be BGLB. Some may be 24
At most only a few 48-hour cultures in
BGLB may be present. Read and record
gas production of such cultures in the "48"
column under BGLB on the data sheet.
Codify results and determine Confirmed
Test coliforms per 100 ml.
F Streak-plate methods for the Confirmed
Test, using eosin methylene blue agar or
Endo agar plates, are accepted procedures
in Standard Methods. The worker who
prefers to use one of these media in
preference to BGLB (also approved in
Standard Methods) is advised to refer to
the current edition of "Standard Methods-
for the Examination of Water and Waste-
water" for procedures.
10-15
-------
MPN Methods
IV TESTING TO COMPLETED TEST STAGE
(Note that this description starts with the
sample inoculation and proceeds through the
Presumptive and the Confirmed Test stages.
In addition, the Fecal Coliform Test is
referred to as an optional adjunct to the
procedure.)
A First-Day Procedures
1 Prepare a laboratory data sheet for the
sample. Record the following information:
assigned laboratory number, source of
sample, date and time of collection,
temperature of the source, name of
sample collector, date and time of
receipt of sample in the laboratory.
Also show the date and time of starting
tests in the laboratory, name(s) of
worker(s) performing the laboratory
tests, and the sample volumes planted.
2 Label the tubes of lauryl tryptose broth
required for the initial planting of the
sample. The label should bear three
identifying marks. The upper number
is the identification of the worker(s)
performing the test (applicable to
personnel in training courses), ,
the number immediately below is the-
assigned laboratory number, corres-
ponding with the laboratory record
sheet. The lower number is the code
to designate the sample volume and
which tube of a replicate series is
indicated. Guidance on labeling for
laboratory data number and identification
of individual tubes is described else-
where in this outline.
NOTE: If 10-ml samples are being
plated, it is necessary to use tubes
containing the correct concentration
of culture medium. This has previously
been noted elsewhere in this outline"
and referral is made to tables.
3 Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.
4 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
5 Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the
sample.
a Use a 10-ml pipet for 10 ml sample
portions, and 1-ml pipets for portions
of 1 n.l or less. Handle sterile
pipets only near the mouthpiece,
and protect the delivery end from
external contamination. Do not remove
the cotton plug in the mouthpiece
as this is intended to protect the
user from ingesting any sample.
b When using the pipet to withdraw
sample portions, do not dip the
pipet more than 1/2 inch into the
sample, otherwise sample running
down the outside of the pipet will
make measurements inaccurate.
c When delivering the sample into the
culture medium, deliver sample
portions of 1 ml or less down into
10*16
-------
MPN Methods
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.
d Prepare preliminary dilutions of
samples for portions of 0. 01 ml or
less before delivery into the culture
medium. See Table 2 for preparation
of dilutions. NOTE: Always deliver
diluted sample portions into the
culture medium as soon as possible
after preparation. The interval
between preparation of dilution and
introduction of sample into the
medium never should be as much as
30 minutes.
6 After measuring all portions of the
sample into their respective tubes of
medium, gently shake the rack of
inoculated tubes to insure good mixing
of sample with the culture medium.
Avoid vigorous shaking, as air bubbles
may be shaken into the fermentation
vials and thereby invalidate the test.
7 Place the rack of inoculated tubes in
the incubator at 35° + 0. 5°C for 24 +
2 hours.
B 24-hour Procedures
1 Remove the rack of lauryl tryptose broth
cultures from the incubator, and shake
gently. If gas is about to appear in the
fermentation vials, the shaking will
speed the process.
2 Examine each tube carefully. Record,
in the column "24" under LST on the
laboratory data sheet, each tube showing
gas in the fermentation vial as a positive
(+) test and each tube not showing gas
as a negative (-) test. GAS IN ANY
QUANTITY IS A POSITIVE TEST.
3 Retain all gas-positive tubes of lauryl
tryptose broth culture in their place in
the rack, and proceed.
4 Select the gas-positive tubes of lauryl
tryptose broth culture for the Confirmed
Test procedures. Confirmed Test
procedures may not be required for
all gas-positive cultures. If, after
24-hours of incubation, all five
replicate cultures are gas-positive for
two or more consecutive sample
volumes, then select the set of five
cultures representing the smallest
volume of sample in which all tubes
were gas-positive. Apply Confirmed
Test procedures to all these cultures
and to any other gas-positive cultures
representing smaller volumes of
sample, in which some tubes were
gas-positive and some were gas-
negative.
5 Label one tube of brilliant green lactose
bile broth (BGLB) to correspond with
each tube of lauryl tryptose broth
selected for Confirmed Test procedures.
6 Gently shake the rack of Presumptive
Test cultures. With a flame-sterilized
inoculation loop transfer one loopful of
culture from each gas-positive tube to
the corresponding tube of BGLB Place
each newly inoculated culture into
BGLB in the position of the original
gas-positive tube
7 If the Fecal Coliform Test is included
in the testing procedure, consult
Section V of this outline for details of
the testing procedure.
8 After making the transfer, the rack
should contain some 24-hour gas-
negative tubes of lauryl tryptose borth
and the newly inoculated BGLB.
Incubate the rack of cultures at 35° C
+ 0. 5° C for 24 + 2 hours.
C 48-hour Procedures
1 Remove the rack of culture tubes from
the incubator, read and record gas
production for each tube.
2 Some tubes will be lauryl tryptose broth
and some will be brilliant green lactose
10-17
-------
MFN Methods
bile broth (BGLB). Be sure to record
results from LTB under the 48-hour
LTB column and the BGLB results
under the 24-hour column of the data
sheet.
3 Label tubes of BGLB to correspond with
all (if any) 48-hour gas-positive cultures
in lauryl tryptose broth. Transfer one
loopful of culture from each gas-positive
LTB culture to the correspondingly-
labeled tube of BGLB. NOTE: AH tubes
of LTB culture which were negative at
24 hours and became positive at 48 hours
are to be transferred. The Option
described above for 24-hour LTB
cultures does not apply at 48 hours.
4 Incubate the 24-hour gas-negative BGLB
tubes and any newly-inoculated tubes of
BGLB 24 + 2 hours at 35° + 0. 50C.
Retain all 24-hour gas-positive cultures
in BGLB for further test procedures.
5 Label a Petri dish preparation of eosin
methylene blue agar (EMB agar) to
correspond with each gas-positive
culture in BGLB.
6 Prepare a streak plate for colony
isolation from each gas-positive culture
in BGLB on the correspondingly-labeled
EMB agar plate.
Incubate the EMB agar plates 24 + 2
hours at 35° + 0. 5° C.
D 72-hour Procedures
1 Remove the cultures from the incubator.
Some may be on BGLB; several EMB
agar plates also can be expected.
2 Examine and record gas production
results for any cultures in BGLB.
3 Retain any gas-positive BGLB cultures
and prepare streak plate inoculations
for colony isolation in EMB agar.
Incubate the EMB agar plates 24 +
2 hours at 35 + 0.5° C. Discard the
gas-positive BGLB cultures after
transfer.
4 Reincubate any gas-negative BGLB
cultures 24 + 2 hours at 35° + 0. 5°C.
5 Discard all 48-hour gas-negative BGLB
cultures.
6 Examine the EMB agar plates for the
type of colonies developed thereon.
Well-isolated colonies having a dark
center (when viewed from the lower
side, held toward a light) are termed
"nucleated or fisheye" colonies, and
are regarded as "typical" coliform
colonies. A surface sheen may or may
not be present on "typical" colonies.
Colonies which are pink or opaque but
are not nucleated are regarded as
"atypical colonies. " Other colony
types are considered "noncoliform. "
Read and record results as + for
"typical" (nucleated) colonies + for
"atypical" (non-nucleated pink or
opaque colonies), and - for other types
of colonies which might develop.
7 With plates bearing "typical" colonies,
select at least one well-isolated colony
and transfer it to a correspondingly-
labeled tube of lactose broth and to an
agar slant. As a second choice, select
at least two "atypical" colonies (if
typical colonies are not present) and
transfer them to labeled tubes of
lactose broth and to agar slants. As a
third cL-j'ce, in the absence of typical
or atypical coliform-like colonies,
select at least two well-isolated
colonies representative of those
appearing on the EMB plate, and trans-
fer them to lactose broth and to agar
slants.
8 Incubate all cultures transfered from
EMB agar plates 24+2 hours at 35 +
0. 5° C.
E 96-hour Procedures
1 Subcultures from the samples being
studied may include: 48-hour tubes
of BGLB, EMB agar plates, lactose
broth tubes, and agar slant cultures.
10-18
-------
MPN Methods
2 If any 48-hour tubes of BGLB are
present, read and record gas production
in the "48" column under BGLB. From
any gas-positive BGLB cultures pre-
pare streak plate inoculations for colony
isolation on EMB agar. Discard all
tubes of BGLB, and incubate EMB agar
plates 24 + 2 hours at 35 + 0.5°C.
3 If any EMB plates are present, examine
and record results in the "EMB" column
of the data sheet. Make transfers to
agar slants and to lactose broth from
all EMB agar plate cultures. In
decreasing order of preference, transfer
at least one typical colony, or at least
two atypical colonies, or at least two
colonies representative of those on the
plate.
4 Examine and record results from the
lactose broth cultures.
5 Prepare a Gram-stained smear from
each of the agar slant cultures, as
follows:
NOTE: Always prepare Gram stain
from an actively growing culture,
preferably about 18 hours old, and
never more than 24 hours old. Failure
to observe this precaution often results
in irregular staining reactions.
a Thoroughly clean a glass slide to
free it of any trace of oily film.
b Place one drop of distilled water on
the slide.
c Use the inoculation needle to suspend
a tiny amount of growth from the
nutrient agar slant culture in the
drop of water.
d Mix the thin suspension of cells with
the tip of the inoculation needle, and
allow the water to evaporate.
e "Fix" the smear by gently warming
the slide over a flame.
f Stain the smear by flooding it for 1
minute with crystal violet solution.
g Flush the excess crystal violet
solution off in gently running water,
and gently blot dry with filter
paper or with other clean absorbent
paper.
h Flood the smear with Lugol's
iodine for 1 minute.
i Wash the slide in gently running
water and blot dry with filter paper.
j Decolorize the smear with 95%
alcohol solution with gentle
agitation for 10-30 seconds,
depending upon extent of removal
of crystal violet dye, then blot dry.
k Counterstain for 10 seconds with
safranin solution, then wash in
running water and blot dry.
1 Examine the slide under the
microscope, using the oil
immersion lens. Coliform
bacteria are Gram-negative,
nonspore-forming, rod-shaped
cells, occurring singly, in pairs,
or rarely in short chains.
m If typical coliform staining reaction
and morphology are observed,
record + in the appropriate space
under the "Gram Stain" column of
the data sheet. If typical morphology
and staining reaction are not
observed, then mark it + or -, and
make suitable comment in the
"remarks" column at the right-hand
side of the data sheet.
n If spore-forming bacteria are
observed, it will be necessary to
repurify the culture from which
the observations were made.
Consult the instructor, or refer
to Standard Methods, for procedures.
At this point, it is possible that all
cultural work for the Completed Test
has been finished. If so, codify results
and determine Completed Test coliforms
per 100 ml.
10-19
-------
MPN Methods
F 120-hour Procedures and following:
1 Any procedures to be undertaken from
this point are "straggler" cultures on
media already described, and requiring
step-by-step methodology already given
in detail. Such cultures may be on:
EMB plates, agar slants, or lactose
broth. The same time-and-temperature
of incubation required for earlier studies
applies to the "stragglers" as do the
observations, staining reactions, and
interpretation of results. On con-
clusion of all cultural procedures,
codify results and determine Completed
Test coliforms per 100 ml.
V FECAL COLIFORM TEST
A General Information
1 The procedure described is an elevated
temperature test for fecal coliform
bacteria.
2 Equipment required for the tests are
those required for the Presumptive
Test of Standard Methods, a water-bath
incubator, and the appropriate culture
media.
B Fecal Coliform Test with EC Broth
1 Sample: The test is applied to gas-
positive tubes from the Standard
Methods Presumptive Test (lauryl
tryptose broth), in parallel with
Confirmed Test procedures.
2 24-hour Operations. Initial procedures
are the planting procedures described
for the Standard Methods Presumptive
Coliform test.
a After reading and recording gas-
production on lauryl tryptose broth,
temporarily retain all gas-positive
tubes.
b Label a tube of EC broth to corre-
spond with each gas-positive tube
of lauryl tryptose broth. The option
regarding transfer of only a limited
number of tubes to the Confirmed
Test sometimes can be applied here.
However, the worker is urged to
avoid exercise of this option until
he has assured the applicability of
the option by preliminary testb on
the sample source.
c Transfer one loopful of culture from
each gas-positive culture in lauryl
tryptose broth to the correspondingly
labeled tube of EC broth.
d Incubate EC broth tubes 24 + 2 hours
at*44. 5 + 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.
3 48-hour operations
a Remove the rack of EC cultures
from the waterbath, shake gently,
and record gas production for each
tube. Gas in any quantity is a
positive test.
b As soon as results are recorded,
discard all tubes. (This is a 24-
hour test for EC broth inoculations
and i.u. a 48-hour test.)
c Transfer any additional 48-hour
gas-positive tubes of lauryl tryptose
broth to correspondingly labeled
tubes of EC broth Incubate 24 +
2 hours at 44.5 + 0. 2°C.
4 72-hour operations
a Read and record gas production for
each tube. Discard all cultures.
b Codify results and determine fecal
coliform count per 100 ml of sample.
10-20
-------
MPN Methods
TESTS FOR COUFORM GROUP
SAMPLE
LACTOSE OR LAURYl TRrPTOSE BROTH
FERMENTATION TUBES (SERIAL DILUTION)
T
GAS POSITIVE
(24 HR ± 2 HR )
I
•i"
GAS NEGATIVE
I
lactose «iaurti nrrrost
WOTN ARE MTEROMNGfABlf MEDU
AMD ARE MCUBATED AT 35 OEG C±
OJ DIG C
gas positives nnes (any amount
of gas) constitute a positive
PRESUMPTIVE TEST
TOTAL INCUBATION TIME FOR LACTOSE
OR 1TB iS 43 HRS ± 3 HRS
GAS POSITIVE
GAS NEGATIVE
COLIFORM GROUP ABSENT
L.
CONFIRMATORY BROTH
BRILLIANT GREEN LACTOSE BU
ALTERNATE
CONFIRMED
EMB PLATES
LNDO AGAR
PIATES
GAS POSITIVE
GAS NEGATIVE
COUFORM GROUP
CONFIRMED
COLIFORM GROUP
NOT CO NF KM ED
EMB PLATES
NUTRIENT AGAR SLANT
LACTOSE BROTH TUBE
GRAM + AND
RODS AND/OR
SPOREFORMERS
FORMATE
RICIMOLEATE
BROTH
GRAM NEGATIVE
RODS
NO SPORES
GAS POSITIVE
GAS POSITIVE
1/
COLFORM GROUP PRESENT
COMPUTED TEST
GAS NEGATIVE
I
COLIFORM GROUP ABSENT
TRANSFER TO EMB PLATE
AND REPEAT PROCESS
HCUBATE BGLB TUBES FOR 43 HRS
t 3 HRS AT 35 DEG C* OJ DEC C
INCUBATE EMB OR ENDOAGAR
PLATES FOR 24 HRS ± 2 HRS AT
35 DEG Ct OS DEG C
GAS NEGATIVE
COUFORM GROUP ABSENT
10-21
-------
Part 3
LABORATORY METHODS FOR FECAL STREPTOCOCCUS
(Day-By-Day Procedures)
I GENERAL INFORMATION
A The same sampling and holding procedures
apply as for the coliform test.
B The number of fecal streptococci in water
generally is lower than the number of
coliform bacteria. It is good practice
in multiple dilution tube tests to start the
sample planting series with one sample
increment larger than for the coliform
test. For example: If a sample planting
series of 1.0, 0.1, 0.01, and 0. 001 ml
is planned for the coliform test, it is
suggested that a series of 10, 1.0, 0.1,
and 0.01 ml be planted for the fecal
streptococcus test.
C Equipment required for the test is the same
as required for the Standard Methods
Presumptive and Confirmed Tests, except
for the differences in culture media.
n STANDARD METHODS (Tentative)
PROCEDURES
A First-Day Operations
1 Prepare the sample data sheet and
labeled tubes of azide dextrose broth
in the same manner as for the
Presumptive Test. NOTE: If 10-ml
samples are included in the series, be
sure to use a special concentration
(ordinarily double-strength) of azide
dextrose broth for these sample
portions.
2 Shake the sample vigorously, approxi-
mately 25 times, in an up-and-down
motion.
3 Measure the predetermined sample
volumes into the labeled tubes of azide
dextrose broth, using the sample
measurement and delivery techniques
used for the Presumptive Test.
4 Shake the rack of tubes of inoculated
culture media, to insure good mixing
of sample with medium.
5 Place the rack of inoculated tubes in
the incubator at 35° + 0. 5°C for 24 +
2 hours.
B 24-hour Operations
1 Remove the rack of tubes from the
incubator. Read and record the results
from each tube. Growth is a positive
test with this test. Evidence of growth
consists either of turbidity of the
medium, a "button" of sediment at the
bottom of the culture tube, or both.
2 Label a tube of ethyl violet azide broth
to correspond with each positive culture
of azide dextrose broth. It may be
permissible to use the same confirmatory
option as described for the coliform
Confirmed Test, in this outline.
3 Shake the rack of cultures gently, to
resuspend any living cells which have
settled out to the bottom of the culture
tubes
4 Transfer three loopfuls of culture from
each growth-positive tube of azide
dextrose broth to the correspondingly
labeled tube of ethyl violet azide broth.
5 As transfers are made, place the newly
inoculated tubes of ethyl violet azide
broth in the positions in the rack
formerly occupied by the growth-
positive tubes of azide dextrose broth.
Discard the tubes of azide dextrose
broth culture.
6 Return the rack, containing 24-hour
growth-negative azide dextrose broth
tubes and newly-inoculated tubes of
ethyl violet azide broth, to the incubator.
Incubate 24 + 2 hours at 35° + 0. 5° C.
10-22
-------
MPN Methods
C 48-hour Operations
1 Remove the rack of tubes from the
incubator. Read and report results.
Growth, either in azide dextrose broth
or in ethyl violet azide broth, is a
positive test. Be sure to report the
results of the azide dextrose broth
medium under the "48" column for that
medium and the results of the ethyl
violet azide broth cultures under the
"24" column for that medium.
2 Any 48-hour growth-positive cultures
of azide dextrose broth are to be
transferred (three loopfulls) to ethyl
violet azide broth. Discard all 48-hour
growth-negative tubes of azide dextrose
broth and all 24-hour growth-positive
tubes of ethyl violet azide broth
3 Incubate the 24-hour growth-negative
and the newly-inoculated tubes of ethyl
violet azide broth 24 + 2 hours at 35°
+ 0.5OC.
2 Codify results and determine fecal
streptococci per 100 ml.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater (13th Ed).
Prepared and published jointly by
American Public Health Association,
American Water Works Association,
and Water Pollution Control
Federation. w 1971.
2 Geldreich, E.E., Clark, H.F., Kabler,
P.W., Huff, C.B. and Bordner, R.H.
The Coliform Group. II. Reactions
in EC Medium at 450 C. Appl.
Microbiol. 8:347-348. 1958.
3 Geldreich, E.E., Bordner, R.H., Huff,
C.B., Clark, H.F. and Kabler, P.W.
Type Distribution of Coliform Bacteria
in the Feces of Warm-Blooded Animals.
J. Water Pollution Control Federation.
34:295-301. 1962.
D 72-hour Operations
4 Recommend Proc. for the Bacteriological
1 Read and report growth results of all Examination of Sea Water and Shellfish
tubes of ethyl violet azide broth. 3rd Edition, American Public Health
Association. 1962.
2 Discard all growth-positive cultures
and all 48-hour growth-negative
cultures.
3 Reincubate any 24-hour growth-negative
cultures in ethyl violet azide broth 24
+ 2 hours at 35° + 0.5°C.
E 96-hour Operations
1 Read and report growth results of any
remaining tubes of ethyl violet azide
broth.
This outline was prepared by H. L. Jeter,
Director, National Training Center,
Water Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
10-23
-------
USE OF TABLES OF MOST PROBABLE NUMBERS
Part 1
I INTRODUCTION
A Using probability mathematics, it is
possible to estimate the number of bacteria
producing the observed result for any com-
bination of positive and negative results
in dilution tube tests. Because the
computations are so repetitious and time-
consuming, it is common laboratory
practice to use Tables of Most Probable
Numbers. These tables are orderly
arrangements of the possible cultural
results obtainable from inoculating various
sample increments in differential culture
media. Each possible combination of
positive and negative tube results is
accompanied by the result (MPN) of the
calculated estimate and the 95% confidence
limits of the MPN.
B The Tables of Most Probable Numbers used
in the currents 13th)1 edition of Standard
Methods for the Examination of Water and
Wastewater were developed by Swaroop.^)
Previous editions of Standard Methods have
used the tables prepared by HoskLns. '
1 Most of the tables are based on using
3 sample volumes in decreasing decimal
increments. Thus, the systems are
based on using volumes of 10 ml, 1. 0 ml,
and 0.1 ml, etc. Other quantity
relationships can be used, such as
50 ml, 10 ml, and 1. 0 ml in a table.
Tables of Most Probable Numbers can
be prepared for any desired series of
sample increments.
2 In addition, tables can be devised for
different numbers of replicate
inoculations of individual sample
volumes. For example, the MPN
Table most commonly used in the
laboratories of this agency is based
on five replicate 10 ml portions, five
1. 0 ml portions, and five 0.1 ml 'portions
A separate table is required for another
combination of sample volumes, con-
sisting of five replicate 10 ml portions,
one 1. 0 ml portion, and one 0.1 ml
portion. This is popular m bacteri-
ological potability tests on water.
MPN Tables can be prepared for any
desired combinations of replicates of
the sample increments used in a
dilution tube series.
3 An approximation of the MPN values
shown in the Tables can be obtained
by a simple calculation, developed by
Thomas. (3) The formula and
application of this calculation is shown
on a later page of this chapter.
C The method of using a Table of Most
Probable Numbers is described here,
based on the table for five 10 ml portions,
five 1.0 portions, and five 0. 1 portions.
The principles apply equally to the other
tables presented in the current edition of
Standard Methods for the Examination of
Water and Wastewater.
II DETERMINING THE MOST PROBABLE
NUMBER
A Codifying Results of the Dilution Tube
Series
If five 10 ml portions, five 1. 0 ml portions,
and five 0.1 ml portions are inoculated
initially, and positive results are secured
from five of the 10 ml portions, three of
the 1. 0 ml portions and none of the 0.1 ml
portions, then the coded result of the test
is 5-3-0. The code can be looked up in
the MPN Table, and the MPN per 100 ml
is recorded directly. If more than the
above three sample volumes are to be
considered, then the determination of the
coded result may be more complex. The
examples described in Table 1 are useful
guides for selection of the significant series
of three sample volumes.
W.BA. 42g. 10.7J
11-1
-------
Use of Tables of Most Probable Numbers
Table 1. EXAMPLES OF CODED RESULTS
No. ml sample per tube -~ 100
No. tubes per sample vol. 5
10
5
1. 0
5
0. 1
5
0. 01
5
0.001
5
Code
See Below
No. tubes in sample giving
5
4
1
5-4-1
positive results in test
5
4
0
0
0
5-4-0
(1)
4
1
0
0
0
4-1-0
(2)
5
5
4
1
1
0
5-4-2
(3)
5
5
5
4
5-5-4
(4)
5
5
5
5
5-5-5
(5)
0
0
0
0
0-0-0
(6)
0
1
0
0
0-1-0
(7)
1
0
0
0
1-0-0
(8)
Discussion of examples:
1 When all the inoculated tubes of more
than one of the decimal series give
positive results, then it is customary
to select the smallest sample volume
(here, 10 ml) in which all tubes gave
positive results. The results of this
volume and the next lesser volumes
are used to determine the coded result.
2 When none of the sample volumes give
positive results in all increments of
the series, then the results obtained
are used to designate the code. Note
that it is not permissible to assume
that if the next larger increment had
been inoculated, all tubes probably
would have given positive results and
therefore assign a 5-4-1 code to the
results.
3 Here the results are spread through
four of the sample volumes. In such
cases, the number of positive tubes in
the smallest sample volume is added
to the number of tubes in the third
sample volume (counting down from the
smallest sample volume in which all
tubes gave positive results).
4 Here it is necessary to use the 5-5-4
code, because inoculations were not
made of 0.001 ml sample volumes,
and it is not permissible to assume
that if such sample volumes had been
inoculated, they would have given
negative results, or any other arbitrarily-
designated result.
5 This is an indeterminate result. Many
MPN tables do not give a value for such
a result. If the table used does not
have the code, then look up the result
for code 5-5-4, and report the result
"greater than" the value shown for the
5-5-4 code. The first number of the
5-5-4 code is based on the 1. 0 ml
sample volume.
6 Like (5), this is an indeterminate
result. If the code does not appear in
the table being used, then look up the
result for code 1-0-0, and report the
MPN as "less than" the value shown
for the 1-0-0 code.
7 The current edition of Standard
Methods stipulates this type of code
designation when unusual results
such as this occur.
11-2
-------
Use of Tables of Most Probable Numbers
8 Note the difference from (7) above.
Inoculations of 100 ml portions were
not made, and it cannot be assumed
that the result would have called for
code 0-1-0.
B Computing and Recording the MPN
When the dilution tube results have been
codified, they are read and recorded from
the appropriate MPN Table.
1 If, as in the first four of the examples
shown under (A) the first number
in the coded result represents a 10 ml
sample volume, then the MPN per 100 ml
is read and recorded directly from the
appropriate column in the table.
2 On the other hand, if the first number
in the coded result represents a sample
volume other than 10 ml, then a
calculation is required to give the
corrected MPN. For example (4) under (A)
above, the first "5" of the 5-5-4 code
represents a sample volume of 1. 0 ml.
Look up the 5-5-4 code as if the 1. 0 ml
volume actually were 10 ml, as if the
0.1 ml volume actually were 1. 0 ml
and as if the 0. 01 ml volume actually
were 0. 1 ml. The MPN obtained (1600)
then is multiplied by a factor of 10 to
give the corrected value. A simple
formula for this type of correction is
shown on a later page of this chapter.
Table 2. Approximate Confidence Limits for Bacterial Densities as
Per Cent of MPN as Determined from Various Numbers of Tubes
in Three Decimal.Dilutions*
Number of tubes 50% 75% 80% 90% 95%
in each dilution Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper
1
33
186
18
340
15
402
10
637
6. 5
955
2
47
160
31
246
27
276
20
383
15
511
3
53
150
38
215
34
237
26
311
21
395
5
64
139
49
182
46
196
37
241
31
289
10
76
127
63
152
60
160
52
184
46
208
*The interpretation of these figures is as follows: When MPN estimates are
made on the basis of dilution tests using one tube in each of three decimal
dilutions, you will be right 50% of the time if you say that the true bacterial
density is between 33% and 186% of the MPN. If you had used 5 tubes in each
dilution you could reduce this interval to from 64% to 139% of the MPN and still
be right 50% of the time. If a greater certainty were desired, say 95%, you
would have to widen this interval to from 31% to 289%.
m PRECISION OF THE MPN VALUE
A The current edition of Standard Methods
shows for each MPN value, the 95%
confidence limits for that value. This
draws attention to the fact that a given
MPN value is not a precise measurement,
but an estimate. The 95% confidence
limits means that the observer will be
correct 95% of the time when he considers
that the actual number of cells producing
the observed combination of positive and
negative tubes was somewhere between
the stated upper limit and the stated
lower limit.
B The greater the number of replicates of
each sample volume in a dilution series,
the greater the precision (in other words,
the narrower the limits of the 95%
confidence range) of the test. The
precision of results, based on numbers
of tubes inoculated per sample volume,
is shown in Table 2.
(4)
C Woodward and other workers have
studied the precision of the MPN in
detail. Such reports should be studied
by those desiring further information
regarding the precision of 'the MPN test.
11-3
-------
Use of Tables of Most Probable Numbers
IV OCCURRENCE OF IMPROBABLE TUBE
RESULTS
A Many of the theoretically possible tube
results are omitted from the MPN Table.
For example, codes 0-0-3, 0-0-4, and
0-0-5 are not included as well as many
others. These are omitted, because, in
the opinion of the authors of the tables,
the probability of occurrence of such
results is so low as to exclude them from
practical consideration.
B The frequency of occurrence of various
code results is shown m the Table 2 both
on a theoretical basis and on the basis of
actual laboratory experience.
C From the MPN tables, it can be inferred
that the codes omitted from the MPN
Table can be expected to occur up to 1%
of the time. If, in reviewing laboratory
data, the theoretically unlikely codes
occur appreciably more than 1% of the
time, there is an indication for inquiry
into the causes. Such results can occur (1)
as a consequence of faulty laboratory
procedures, or (2) as a result of
extraneous influences in the samples.
D The current edition of Standard Methods
does not include MPN values for many
rare combinations listed in previous
editions. By pruning out those codes listed
as Group IV in Table 3, the table has been
considerably condensed. Table 4 suggests
maximum permissible numbers of samples
for various numbers of samples tested.
Table 3
FIVE-TUBE AND THREE-TUBE CODES THAT
INCLUDE 99 PER CENT OF ALL RESULTS
Group
Theoretically Ex-
pected Percentage
of Results
Theoretically Ex-
pected Cumulative
Percentage
Observed Percentage
of 360 Samples
Five-Tube-Test
Class 1 codes
550, 551, 552, 553,
67 5
67. 5
66 0
554, 500, 510, 520,
530, 540, 100, 200,
300, 400
Class 2 codes
23 1
511, 521, 531, 541,
23. 6
91 1
542, 110, 210, 310,
410, 420
Class 3 codes
501, 010, 532, 320,
7 9
69 0
7. 5
522, 220, 543, 430,
120, 533, 330, 502,
020, 544, 440, 301,
401, 431, 201, 411,
101, 311, 421, 211,
001
Improbable codes
1 0
100 0
1 4
Three-Tube Test
Class 1 codes
330, 331, 332, 300,
310, 320, 100, 200
81 5
81. 5
81
7
Class 2 codes
321, 311, 301, 210,
110, 010.
14.9
96. 4
14
1
Class 3 codes
322, 220, 201, 101
312, 120
2 7
99. 1
3
7
Improbable codes
0.9
100. 0
0
6
11-4
-------
Use of Tables of Most Probable Numbers
Table 4
MAXIMUM PERMISSIBLE NUMBERS
OF IMPROBABLE CODES FOR VARIOUS
NUMBERS OF SAMPLES TESTED
Example: From a sample of water, 5 out
of five 0. 01 - ml portions, 2 out of five
0. 001 - ml portions, and 0 out of five
0. 0001 - ml portions, gave positive
reactions.
Number of Maximum Number
Samples of Improbable Codes
1
_
15
1
16
-
45
2
46
-
83
3
84
-
130
4
131
-
180
5
181
-
233
6
234
-
290
7
291
-
350
8
351
-
413
9
414
-
477
10
478
-
543
11
E Table 5 is from International Standards
for Drinking-Water, published by the World
Health Organization, Geneva (1958). The
last three values, not shown in the WHO
publication, are from Woodward, "How
Probable is the Most Probable Number. "(4)
From the code 5-2-0 in the MPN table,
the MPN index is 49
it 49 + ki \ x7T7rr = 49' 000
(from table) 0.01
MPN Index = 49, 000
A simple approximation of the most
probable number may be obtained from
the following formula (after Thomas):
MPN/100 ml =
No. of Positive Tubes X 100
No. of ml in negative tubes) X(No. of ml
in all tubes)
Example: From a sample of water, 5 out
of five 10 - ml portions, 2 out of five
1.0 ml portions, and 0 out of five 0.1 ml
portions gave positive results.
F Several theoretically possible combinations
of positive tube results are omitted in
Table 5- These combinations are omitted
because the statistical probability of
occurrence of any of the missing results
is less than 1%. If such theoretically
unlikely tube combinations occur in more
than 1% of samples, there is need for
review of the laboratory procedures and
of the nature of the samples being tested.
When the series of decimal dilutions is
other than 10, 1. 0 and 0. 1 ml, use the
MPN in Table 5%according to the following
formula:
MPN 10
(from table) Largest quantity tested
7 y mo
MPN/100 ml = 50.22
¦J (3. 5) X(55. 5)
MPN/100 ml = 50
Note that the MPN obtained from the table
on the preceding pages with these tube
results is 49. "Most probable numbers
computed by the above formula deviate
from values given by the usual methods
by amounts which ordinarily are
insignificant. The formula is not
restricted as to the number of tubes
and dilutions used " (Thomas)
= MPN/100 ml
11-5
-------
Use of Tables of Most Probable Numbers
Table 5. MPN AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE 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
MPN Index
(organisms
per 100 ml)
Confidence Limits
(95%)
Five 10-ml
portions
Five 1-ml
portions
Five 0. 1-ml
portions
Lower limit
Upper limit
0
0
0
<2
0
0
1
2
<0.5
7
~0
0
2
4
<0.5
11
0
1
0
2
<0. 5
7
*0
1
1
4
<0. 5
11
*0
1
2
6
<0. 5
15
0
2
0
4
<0.5
11
~0
2
1
6
<0. 5
15
~0
3
0
6
<0. 5
15
1
0
0
2
<0.5
7
1
0
1
4
<0. 5
11
*1
0
2
6
<0. 5
15
~ 1
0
3
8
1
IB
1
1
0
4
<0. 5
11
1
1
1
6
<0.5
15
*1
1
2
8
1
IB
1
2
0
6
<0. 5
15
*1
2
1
8
1
IB
~ 1
2
2
10
2
23
• 1
3
0
8
1
18
*1
3
1
10
2
23
~ 1
4
0
11
2
25
2
0
0
5
<0.5
13
2
0
1
7
1
17
*2
0
2
9
2
21
»2
0
3
12
3
28
2
1
0
7
1
17
2
1
1
9
2
21
~2
1
2
12
3
28
2
2
0
9
2
21
*2
2
1
12
3
28
~2
2
2
14
4
34
2
3
0
12
3
28
~2
3
1
14
4
34
*2
4
0
IS
4
37
3
0
0
8
1
18
3
0
1
11
2
25
*3
0
2
13
3
31
3
1
0
11
2
25
3
1
1
14
4
34
*3
1
2
17
5
46
~ 3
1
3
20
6
60
3
2
0
14
4
34
3
2
1
17
5
48
*3
2
2
20
6
60
3
3
0
17
5
46
*3
3
1
21
7
63
*3
4
0
21
7
63
*3
4
1
24
8
72
~Omitted from 12th Edition Standard Methods for Water and Wastewater
11-6
-------
Use of Tables of Most Probable Numbers
Table 5. (Cont'd) MPN AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE 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 1-ml
portions
Five 0. 1-ml
portions
MPN Index
(organisms
per 100 ml)
Confidence Limits
95%
Lower limit
0
1
2
3
0
1
2
0
1
2
0
1
2
0
1
0
1
0
1
2
3
4
0
1
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
25
13
17
21
25
17
21
26
22
26
32
27
33
39
34
40
41
48
23
31
43
58
76
33
46
63
84
49
70
94
120
148
177
79
109
141
175
212
253
130
172
221
278
345
426
240
348
542
920
1600
>2400
3
5
7
8
5
7
9
7
9
11
9
11
13
12
14
14
16
7
11
15
19
24
11
16
21
28
17
23
28
33
38
44
25
31
37
44
53
77
35
43
57
90
117
145
68
118
180
210
350
800
~Omitted from 12th Edition Standard Methods for Water and Wastewater
11-7
-------
Use of Tables of Most Probable Numbers
Table 6. MPN AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE RESULTS IN A PLANTING
SERIES OF FIVE 10-ml PORTIONS OF SAMPLE
No. of Positive Tubes Out of:
Five 10 -ml Tubes
MPN per
100 ml
Limits of MPN
Lower
Upper
0
2. 2
0
6.0
1
2. 2
0. 1
12. 6
2
5 1
0.5
19.2
3
9. 2
1. 6
29.4
4
16 0
3. 3
52.9
5
> 16
8.0
Table 7. MPN AND 95% CONFIDENCE LIMITS FOR VARIOUS
COMBINATIONS OF POSITIVE RESULTS IN A PLANTING SERIES OF
FIVE 10-ml, ONE 1-ml, AND ONE 0 1-ml PORTIONS OF SAMPLE
No of Positive Tubes Out of-
MPN
per
100 ml
Limits of MPN
Five 10-ml
Tubes
One 1-ml
Tube
One 0. 1-ml
Tube
Lower
Upper
0
0
0
<2
5. 9
0
1
0
2
0 050
13
1
0
0
2
2
0.050
13
1
1
0
4.
4
0.52
14
2
0
0
5
0.54
19
2
1
0
7.
6
1. 5
19
3
0
0
8.
8
1. 6
29
3
1
0
12
3. 1
30
4
0
0
15
3. 3
46
4
0
1
20
5. 9
48
4
1
0
21
6.0
53
5
0
0
38
6.4
330
5
0
1
96
12
370
5
1
0
240
12
3700
5
1
1
>240
88
11-8
-------
Use of Tables of Most Probable Numbers
IV TABLES OF MOST PROBABLE
NUMBERS
These tables consist of the MPN indices and
95% confidence limits, within which the
actual number of organisms can lie, for
various combinations of positive and
negative tubes. Three MPN tables are
presented. Table 5 is based on five 10 ml
five 1. 0 ml and five 0.1 ml sample portions.
.Table 6 is based on five 10 ml sample por-
tions; and Table 7 is based on five 10 ml,
one 1 ml and one 0.1 ml sample portion.
3 Thomas, H. Al, Jr. Bacterial
Densities from Fermentation Tubes.
J.A.W.W.A. 34:572. 1942.
4 Woodward, R. L. How Probable is the
Most Probable Number? J. A. W. W. A.
49:1060. 1958.
5 Standard Methods for the Examination of
Water and Wastewater. 13th Edition.
Prepared and Published Jointly by
American Public Association.
American Water Works Association,
and Water Pollution Control Federation.
REFERENCES
1 Swaroop, S. Numerical Estimation of
B. coli by Dilution Method. Indian J.
Med. Research. 26:353. 1938.
2 Hoskins, J. K. Most Probable Numbers
for Evaluation of Coli-Aerogenes Tests
by Fermentation Tube Method. Public
Health Reports. 49:393. 1934.
This outline was prepared by H. L. Jeter,
Director, National Training Center,
Water Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
11-9
-------
MEMBRANE FILTER EQUIPMENT AND ITS
PREPARATION FOR LABORATORY USE
I Some equipment and supplies used in the
bacteriological examination of water with
membrane filters are specific for the method
Other items are standard in most well-
equipped bacteriological laboratories and
are readily adapted to membrane filter work.
This chapter describes needed equipment and
methods for its preparation for laboratory
use Where more than one kind of item is
available or acceptable for a given function,
sufficient descriptive information is pro-
vided to aid the worker in selecting the one
best suited to his own needs
n EQUIPMENT FOR SAMPLE FILTRATION
AND INCUBATION
A Filter Holding Unit
1 The filter holding unit is a device for
supporting the membrane filter and for
holding the sample until it passes
through the filter. During filtration
the sample passes through a circular
area, usually about 35 mm in diameter,
in the center of the filter The outer
part of the filter disk is clamped
between the two essential components
of the filter holding unit (See Plate 1)
a The lower element, called the filter
base, or receptacle, supports the
-membrane filter on a plate about
50 mm in diameter The central
part of this plate is a porous disk
to allow free passage of liquids.
The outer part of the plate is a
smooth nonporous surface. The
lower element includes fittings for
mounting the unit in a suction flask
or other container suitable for
filtration with vacuum
b The upper element, usually called
the funnel, holds the sample until
it is drawn through the filter. Its
lower portion is a flat ring that rests
on the outer part of the membrane
filter disk, directly over the non-
porous part of the filter support
plate.
c The assembled filter holding unit is
joined by a locking ring or by one
or more clamps
Characteristics of filter holding units
should include-
a The design of filter holding units
should provide for filtration with
vacuum.
(A) ASSEMBLED FILTER
HOLDING UNIT
IB) UPPER ELEMENT
LOCKING RING
(C) LOWER ELEMENT
PLATE 1
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
OUT and the Environmental Protection Agency.
W. BA. mem 60k. 11.71
12-1
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
b Filter holding units may be made
of glass, porcelain, plastic, non-
corrosive metal, or other impervious
material.
c Filter holding units should be made
of bacteriologically inert materials.
d All surfaces of the filter holding
assembly in contact with the water
sample prior to its passage through
the membrane filter should be
uniformly smooth and free from
corrugations, seams, or other sur-
face irregularities that could become
lodging places for bacteria.
e Filter holding units should be easily
sterilized by routine methods.
f The filter holding unit should be
easily and quickly assembled and
disassembled in routine operational
use.
g Filter holding units should be durable
and inexpensive Maintenance should
be simple.
3 Several forms of filter holding units
have been developed for use with
aqueous suspensions.
a SS 47 Membrane Filter Holder
(Plate 2, Figure 1)
Conical-shape funnel with a 500 ml
capacity. The base section includes
a wirescreen membrane support.
Funnel and base section are evenly
joined by a locking ring mechanism.
This assembly is designed to hold a
47 mm diameter membrane firmly in
place allowing an effective filter area
of approximately 9. 6 square centi-
meters. The entire filter unit is
made of stainless steel with the
funnel interior having a mirror-like
finish
b The Millipore Pyrex Filter Holder
(Plate 2, Figure 2)
The unit is made of pyrex glass with
coarse grade fitted support in base
for filter The upper element of
early models of glass filter holders
had a capacity of 1 liter. Currently
available units are supplied with
upper elements having 300 ml
capacity The assembled filter
holder is joined with a spring
clamp which engages on flat sur-
faces encircling the upper and
lower elements.
c Millipore Standard Hydrosol Filter
Holder (Plate 2, Figure 3)
Most components of this unit are
made of stainless metal. The
porous membrane support plate is
fine-mesh stainless steel screening
The upper element is a straight-
sided cylinder 4 to 5 inches in
diameter, constricted to a narrow
cylinder at the bottom, to fit the
lower element Capacity of the
funnel element is about 1 liter
The assembled filter holding unit is
joined by a bayonet joint and locking
ring. Accessories may be obtained
for collection of small amounts of
filtrate and for anhydrous sterilization
of the filter holding assembly.
d Gelman "Parabella Vacuum Funnel"
(PlA+e 2, Figure 5)
The unit is made of spun stainless
steel The locking ring is a bayonet-
type fitting, and is spring-loaded.
The funnel element has a 1-liter
capacity
e The Sabro Membrane Filter Holder
(Plate 2, Figure 4)
The unit is mostly of stainless steel
construction. The lower element is
a combination vacuum chamber,
filtrate receiver, and filter support-
ing element It consists of a stainless
steel cup with a metal cover The
cover is fitted with a rubber gasket
permitting airtight fit of the cover
into the top of the cup. A porous
sintered stainless steel membrane
support disk is mounted in the
center of the cover At the side
12-2
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
of the beaker is a valve to which a
pumping device can be fitted. The
upper element is a stainless steel
funnel with about 500 ml capacity.
The assembled filter holding unit is
joined by a locking ring at the base
of the upper element. This engages
on three spring clamps on the covering
plate of the lower element.
Millipore "Sterifil" filter unit
(Plate 2, Figure 6)
A funnel and flask unit of poly-
carbonate with filter base and
support of polypropylene. Manu-
facturers tables should be referred
to regarding chemicals which may
be present in the sample and their
effect on the holder and flask
elements. This unit can be safely
sterilized under steam pressure.
FILTER HOLDING UNITS
FOR AQUEOUS SUSPENSIONS
FIG. I
FIG. 3
FIG. 4
FIG. 2
FIG. 6
FIG. 5
PLATE 2
4 Care and maintenance of filter holding
units
a Filter holding units should be kept
clean and free of accumulated
foreign deposits.
b Metal filter holding units should be
protected from scratches or other
physical damage which could result
in formation of surface irregularities.
The surfaces in contact with mem-
brane filters should receive
particular care to avoid formation
of shreds of metal or other
irregularities which could cause
physical damage to the extremely
delicate filters.
c Some filter holding units have
rubber components. The rubber
parts may in time become worn,
hardened, or cracked, necessitating
replacement of the rubber part
involved
d The locking rings used in some
kinds of filter holders have two or
more small wheels or rollers
which engage on parts of the filter
holding assembly Occasional
adjustment or cleaning is necessary
to insure that the wheels turn freely
and function properly On some
units, the wheels are plastic, and
are not intended to turn. When
worn flat, they should be loosened,
turned a partial turn, and tightened
again.
B Membrane Filters and Absorbent
Pads
1 The desired properties of membrane
filters have been discussed elsewhere
Typical examples, commercially
available include:
a Millipore Filters, Type HA, white,
grid-marked, 47 mm in diameter
b S & S Type B-9, white, black-grid
mark, 47 mm diameter
12-3
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
c Oxoid cellulose acetate membrane
filters, 4.7 cm, grid-marked
2 An absorbent pad for nutrient is a
paper filter disk, usually the same
diameter as the membrane filter.
Absorbent pads must be free of
soluble chemical substances which
could interfere with bacterial
growth. They should be of such
thickness that they will retain 1.8-
2. 2 ml of liquid culture medium.
During incubation of cultures on
membrane filters an absorbent pad
saturated with liquid culture medium
is the substrate for each filter.
Absorbent pads are supplied with
the purchase of membrane filters.
Additional absorbent pads may be
purchased separately. Sterilization
in an autoclave is recommended for
absorbent pads.
C Vacuum
Water can be filtered through a membrane
filter by gravity alone, but the filtration
rate would be too slow to be practical.
For routine laboratory practice, two
convenient methods are available for
obtaining vacuum to hasten sample filtration.
1 An electric vacuum pump may be used
connected to a filtration apparatus
mounted in a suction flask. The pump
need not be a high-efficiency type. For
protection of the pump, a water trap
should be included in the system,
between the filtration apparatus and
the vacuum pump
2 A water pump, the so-called "aspirator"
gives a satisfactory vacuum, provided
there is reasonably high water pressure.
3 In emergency, a rubber suction bulb, a
hand pump, or a syringe, may be used
for vacuum. It will be necessary to
include some form of valve system to
prevent return flow of air.
D Culture Containers (Plate 3)
Most membrane filter cultures are
incubated in individual containers.
Almost any form of culture container
is acceptable if it is made of impervious
bacteriologically inert material. The
culture container should, or course, be
large enough to permit the membrane
filters to lie perfectly flat. The following
are widely used:
•1 Glass petri dishes
Conventional borosilicate glass culture
dishes are widely used in laboratory
applications of membrane filters. For
routine work, 60 mm X 15 mm petri
dishes are recommended. The common
100 mm X 15 mm petri dishes are
acceptable, but are subject to difficulties.
HYDRATOR
SUITABLE TYPES OF FORCEPS
ALCOHOL JAR
WITH
FORCEPS
PLASTIC
GLASS
TYPES OF CULTURE CONTAINERS
PLATE 3
12-4
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
2 Plastic petri dishes
Plastic containers have been developed
for use with membrane filter cultures.
Their cost is fairly low, and single-
service use feasible. They cannot be
heat-sterilized, but are supplied
sterile. They must be free from
soluble toxic substances. They can
either be loose-fitting or of a tight lid
to base friction fit.
E Other Equipment and Supplies Associated
with Sample Filtration
1 Suction flask (Plate 4)
a Most types of filter holding apparatus
are fitted in a conventional suction
flask for sample filtration. While
other sizes may be used, the 1-liter
size is most satisfactory.
b The suction flask can be connected
to the vacuum facility with thick-
walled rubber tubing. Latex rubber
tubing, 3/16" inside diameter, with
wall thickness 3/32", is suggested.
This tubing does not collapse under
vacuum, yet it is readily closed with
a pinch clamp.
c A pinch clamp on the rubber tubing
is a convenient means of cutting off
the vacuum from the suction flask
during intervals when samples are
not actually being filtered. It is
most convenient to have the vacuum
facility in continuous operation during
sample filtration work.
d In laboratories conducting a high
volume of filtration work, the
suction flask may be dispensed.
Filter-holding manifolds are available
to receive up to three filtration units.
The filtrate water is collected in a
trap (in series with the vacuum
source) which is periodically emptied.
e Another arrangement can be made
for dispensing with the suction flask.
In this case, the receptacle element
of the filtration unit is mounted in
the bench top. Instead of using a
suction flask, the lower element of
the filter holding unit has a dual
connection with the vacuum source
and with the laboratory drain. A
solenoid-operated valve is used to
determine whether the vacuum system
or the drain line is in series with
the filtration unit.
2 Ring stand with split rmg (Optional)
(See Plate 4)
SUCTION FLASK RING STAND WITH SPLIT RING
PLATE 4
When the filter holding unit is
disassembled after sample filtration,
the worker's hands must be free to
manipulate the membrane filter.
Upon disassembly of the filter holding
unit, many workers place the funnel
element, inverted, on the laboratory
bench. Some workers, to prevent
bacterial contamination, prefer a rack
or a support to keep the funnel element
from any possible source of contam-
ination. A split ring on a ring stand is
a convenient rack for this purpose.
3 Graduated cylinders
In laboratory practice, 100 ml graduated
borosilicate glass cylinders are
satisfactory for measurement of
samples greater than 20 ml.
12-5
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
4 Pipettes and cans
a Graduated Mohr pipettes are needed
for many procedures, such as
measurement of small samples, and
for preparing and dispensing culture
media. Pipettes should be available
in 1 ml and 10 ml sizes.
b Holding cans may be round or square
but must not be made of copper.
I Aluminum or stainless steel are
'acceptable.
5 Alcohol jar with forceps (Plate 3)
a All manipulation of membrane filters
is with sterile forceps. For steri-
lization, forceps are kept with their
tips immersed in ethanol or methanol.
When forceps are to be used, they
are removed from the container and
the alcohol is burned off.
b Forceps may be straight or curved.
They should be designed to permit
easy handling of filters without
damage. Some forceps have corru-
gations on their gripping tips. It is
recommended that such corrugations
be filed off for membrane filter work.
6 A gas burner or alcohol burner is needed
to ignite the alcohol prior to use of
forceps.
7 Dilution water
The buffered distilled water described
m "Standard Methods for the Examination
of Water and Wastewater" for bacterio-
logical examination of water is used
in membrane filter methods. Dilution
water is conveniently used in 99 + 2 ml
amounts stored in standard dilution
bottles. Some workers prefer to use
9.0 + 0.2 ml dilution blanks.
8 Culture medium
Bacteriological culture media used with
membrane filter techniques are dis-
cussed at length in another part of this
manual.
F Incubation Facilities
1 Requirements
a Temperature
For cultivation of a given kind of
bacteria, the same temperature
requirements apply with membrane
filter methods as with any other
method for cultivating the bacteria
in question. For example, incu-
bation temperature for coliform
tests on membrane filters should
be 35° C + 0.5° C.
b Humidity
Membrane filter cultures must be
incubated in an atmosphere main-
tained at or very near to 100%
relative humidity Failure to
maintain high humidity during
incubation results in growth failure,
or at best, in small or poorly
differentiated colonies.
2 The temperature and humidity require-
ments can be satisfied in any of
several types of equipment.
a A conventional mcubator may be
used. With large walk-in
inc.-'::tors, it is extremely difficult
to maintain satisfactory humidity.
With most conventional incubators,
membrane filter cultures can be
incubated in tightly closed con-
tainers, such as plastic petri dishes.
In such containers, required
humidity conditions are established
with evaporation of some of the
culture medium. Because the
volume of air in a tightly closed
container is small, this results in
negligible change in the culture
medium. If glass petri dishes or
other loosely fitting containers are
used, the containers should be
placed in a tightly closed container,
with wet paper or cloth inside to
obtain the required humidity con-
ditions. A vegetable crisper, such
as used in most home refrigerators.
12-6
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
is useful for the purpose. (See
Plate 3)
b A covered water bath maintaining
44. 5°C + 0.2 C is necessary for
the fecal coliform test and this
will necessitate the use of
a water-bath having forced
circulation of water.
m STERILIZATION OF MEMBRANE FILTER
EQUIPMENT AND SUPPLIES
A Filter Holding Unit
1 When is sterilization necessary'
a The filter holding unit should be
sterile at the beginning of each
filtration series. A filtration series
is considered to be interrupted if
there is an interval of 30 minutes
or longer between sample filtrations.
After such interruption any further
sample filtration is treated as a
new filtration series and requires
a sterile filter holding unit.
b It is not necessary to sterilize the
filter holding unit between successive
filtrations, or between successive
samples, of a filtration series.
After each filtration the funnel walls
are flushed with sterile water to
free them of bacterial contamination.
If properly done, the flushing pro-
cedure will remove bacteria
remaining on the funnel walls and
prevent contamination of later
samples.
2 Methods for sterilization of filter
holding unit
a Sterilization in the autoclave is
preferred. Wrap the funnel and
receptacle separately in Kraft paper
and sterilize in the autoclave 15
minutes at 121 °C. At the end of
the 15 minutes holding period in the
autoclave, release the steam
pressure rapidly, to encourage
drying of the filter holding unit.
b The unit may be sterilized by
holding it 30 minutes in a flowing
steam sterilizer.
c The unit may be immersed 2 to 10
minutes in boiling water. This
method is recommended for
emergency or field use.
d Some units (Millipore Stainless Unit)
are available with accessories per-
mitting anhydrous sterilization with
formaldehyde. The method consists
of introduction of methanol into a
wick or porous plate in the sterili-
zation accessory, assembly of the
filter holding unit for formaldehyde
sterilization, ignition of the
methanol, and closure of the unit.
The methanol is incompletely
oxidized in the closed container,
resulting in the generation of
formaldehyde, which is bactericidal.
The filter holding unit is kept closed
for at least 15 minutes before use.
e Ultraviolet lamp sterilizers are
convenient to use. A device now
commercially available for ultra-
violet sterilization of membrane
filter funnel units.
B Sterilization of Membrane Filters and
Absorbent Pads
1 Membrane filters
a Membranes are supplied in units
of 10 in kraft envelopes, or in
packages of 100 membranes. They
may be sterilized conveniently in
the packets of 10, but should be
repackaged if supplied in units of
100. Large packages of filters can
be distributed in standard 100 mm
X15 mm petri dishes, or they can
be wrapped in kraft paper packets
for sterilization.
12-7
-------
Membrane Filter Equipment and its Preparation for Laboratory Use
b Sterilization in the autoclave is
preferred. Ten minutes at 121° C
or, preferably, at 116°C is
recommended. After sterilization
the steam pressure is released as
rapidly as possible, and the filters
are removed from the autoclave and
dried at room temperature. Avoid
excessive exposure to steam.
c In emergency, membrane filters may
be sterilized by immersion in boiling
distilled water for 10 minutes. The
filters should first be separated from
absorbent pads and paper separators
which usually are included in the
package. The boiling water method
is not recommended for general
practice, as the membranes tend to
adhere to each other and must be
separated from one another with
forceps.
2 Absorbent pads for nutrient
a Unsterile absorbent pads can be
wrapped in kraft paper or stacked
loosely in petri dishes, and auto-
claved with membrane filters (ten
minutes or longer at 121°C or 116°C).
b After sterilization absorbent pads
for nutrient should be dried before
use.
C Glassware
1 Sterilization at 170° C for not less than
1 hour is preferred for most glassware
(pipettes, graduated cylinders, glass
petri dishes). Pipettes can be sterilized
in aluminum or stainless steel cans, or
they may be wrapped individually in
paper. The opening of graduated
cylinders should be covered with paper
or metal foil prior to sterilization.
Glassware with rubber fittings must not
be sterilized at 170OC, as the rubber
will be damaged.
2 Sterilization m the autoclave, 15
minutes at 121°C, is satisfactory,
and preferred by many workers. When
sterilizing pipettes it is important to
exhaust the steam pressure rapidly
and vent the containers momentarily.
This allows the vapor to leave the can
and prevents wet pipettes.
D Culture Containers
1 Glass petri dishes
a Petri dishes may be sterilized in
aluminum or stainless steel cans,
or wrapped in kraft paper or metal
foil. They can be wrapped
individually or, more conveniently,
in rolls of up to 10 dishes.
b Preferably, sterilize glass petri
dishes at 170° C for at least 1 hour.
c Alternately, they may be sterilized
in the autoclave, 15 minutes at
121°C. After sterilization steam
pressure should be released rapidly
to facilitate drying of the dishes.
Other suggested methods for
sterilization of plastic dishes
include exposure to ethylene oxide
vapor (0. 5 ml ethylene oxide per
liter of container volume), or
exposure to ultraviolet light.
Ethylene oxide is a dangerous
chemical being both toxic and
explosive, and it should be used
only '"hen more convenient and
safer methods are not available.
2 Plastic culture containers
a Because of the thermo-labile
characteristics of the plastic,
these containers cannot be heat
sterilized. Manufacturers supply
these in a sterile condition.
b For practical purposes, plastic
dishes may be sterilized by
immersion in a 70% solution of
ethanol in water, for at least 30
minutes. Dishes must be allowed
to drain and dry before use, as
ethanol will influence the perform-
ance of culture media.
This outline was prepared by H. L. Jeter,
Director, National Training Center, MDS,
WPQ EPA, Cincinnati, OH 45268.
12-8
-------
MEMBRANE FILTER LABORATORY AND FIELD PROCEDURES
I BASIC PROCEDURES
A Introduction
Successful application of membrane filter
methods requires development of good
routine operational practices. The
detailed basic procedures described in
this Section are applicable to all mem-
brane filter methods in water bacteriology
for filtration, incubation, colony counting,
and reporting of results. In addition,
equipment and supplies used in all mem-
brane filter procedures are described here
and not repeated elsewhere in such detail.
Workers using membrane filter methods
for the first time are urged to become
thoroughly familiar with these basic
procedures and precautions.
B General Supplies and Equipment List
Table 1 is a check list of materials.
C "Sterilizing" Media
Set tubes in a boiling waterbath for 10
minutes. This method suffices for
medium in tubes up to 25 X150 mm.
Frequent agitation improves dissolving
of the medium.
Alternately, coliform media can be
directly heated on a hotplate to the first
bubble of boiling. Stir the medium
frequently if direct heat is used, to avoid
charring the medium.
Do not autoclave.
D General Laboratory Procedures with
Membrane Filters
1 Prepare data sheet
Minimum data required are: sample
identification, test performed including
media and methods, sample filtration
volumes, and the bench numbers
assigned to individual membrane filters.
2 Disinfect the laboratory bench surface.
Use a suitable disinfectant solution and
allow the surface to dry before
proceeding.
3 Set out sterile culture containers m an
orderly arrangement.
4 Label the culture containers.
Numbers correspond with the filter
numbers shown on the data sheet.
5 Place one sterile absorbent pad* in
each culture container, unless an agar
medium is being used.
Use sterile forceps for all manipulations
of absorbent pads and membrane filters.
Forceps sterility is maintained by
storing the working tips in about 1 inch
of methanol or ethanol. Because the
alcohol deteriorates the filter, dissipate
it by burning before using the forceps.
Avoid heating the forceps in the burner
as hot metal chars the filter.
~When an agar medium is used, absorbent pads are not used. The amount of medium should be
sufficient to make a layer approximately 1/8" deep in the culture container. In the 50 mm
plastic culture containers this corresponds to approximately 6-8 ml of culture medium.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
Office of Water Programs, Environmental Protection Agency.
W. BA.mem. 81i. 11.71
13-1
-------
Membrane Filter Laboratory and Field Procedures
Table 1. EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)
Standard Tests
Nonstandard Teste
Item
M - Endo
Broth
Half-round glass paper weights for
colony counting,with lower half of a
2-oz metal ointment box
Hand tally, single unit acceptable,
hand or desk type
Stereoscopic (dissection) microscope,
magnification of 10X or 15X, prefer-
able binocular wide field type
Bacteriological inoculating needle
Wire racks for culture tubes,
10 openings by five openings pre-
ferred, dimensions overall approxi-
mately 6" X 12"
Phenol Red Lactose Broth in 16 X
150 mm fermentation tubes with
metal caps, 10 ml per tube
Eosin Methylene Blue Agar
(Levine) in petri plates, prepared
ready for use
Nutrient agar slants, in screw
capped tubes, 16 X126 mm
Cram stain solutions, 4 solutions
per complete set
Microscope, compound, binocular,
with oil immersion lens, micro-
scope lamp and immersion oil
Microscope slides, new, clean,
1" X 3" size
\\ ater proof plastic bags
for fecal coliform culture
dish incubation
M-Endo medium, MF dehydrated
medium in 25 X95 mm flat bottomed
screw-capped glass vials, 1.44 g
per tube, sufficient for 30 ml of
medium
Ethanol, 95% in small bottles or
screw-capped tubes, about 20 ml
per tube
Sodium benzoate solution, 12%
aqueous, in 25 X 150 mm screw-
capped tubes, about 10 ml per tube
L. E. S. Endo Agar MF, dehydrated
M-Endo medium, 0. 36 g per 25 X
95 mm flat bottomed screw-capped
glass vial, plus 0 45 g agar, for 30 ml
Lactose Lauryl Sulfate Tryptose Broth
in 25 X 150 mm test tube without
included gas tube, about 25 ml, for
enrichment in L. E. S method
X
X
L.E.S
Coliform
Delayed
Coliform
Fecal
Coliform
X
X
X
X
X
X
rcc£\i
Sit cpipfoiius
Verified
Test
X
X
X
X
X
X
X
2
-------
Membrane Filter Laboratory and Field Procedures
Table 1. EQUIPMENT, SUPPLIES AND MEDIA
Standard Tests
Nonstandard Tests
M-Endo
L.E.S.
Delayed
Fecal
Fecal
Broth
Coliform
Coliform
Coliform
Streptococcus
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Item
Funnel unit assemblies
Ring stand, with about a 3" split ring, to
support the filtration funnel
Forceps, curved-end round tipped,
special type for MF work
Methanol, in small wide-mouthed bottles,
about 20 ml for sterilizing forceps
Suction flasks, glass, 1 liter, mouth to
fit No. 8 stopper
Rubber tubing, 2-3 feet, to connect
suction flask to vacuum services, latex
rubber 3/16" I.D. by 3/32" wall
Pinch clamps strong enough for tight
compression of rubber tubing above
Pipettes, 10 ml, graduated, Mohr type,
sterile, dispense 10 per can per working
space per day. (Resterilize daily to
meet need).
Pipettes, 1 ml, graduated, Mohr type,
sterile, dispense 24 per can per working
space per day. (Resterilize daily to
meet need).
Pipette boxes, sterile, for 1 ml and
10 ml pipettes (sterilize above pipettes
in these boxes)
Cylinders, 100 ml graduated, sterile,
(resterilize daily to meet need).
Jars, to receive used pipettes
Gas burner, Bunsen or similar
laboratory type
Wax pencils, red, suitable for writing
on glass
Sponge in dilute iodine, to wipe down the
desk tops
Membrane filters (white, grid marked,
sterile,and suitable pore size for
microbiological analysis of water)
Absorbent pads for nutrient, (47 mm in
diameter), sterile, in units of 10 pads
per package Not required if medium
contains agar
Petri dishes, disposable, plastic,
50 X12 mm, sterile
Waterbath incubator 44.5 + 0.2°C
Vegetable crispers, or cake boxes,
plastic, with tight fitting covers, for
membrane filter incubations
Fluorescent lamp, with extension cord
equipped with a simple lens of about
4X magnification
Ring stand, with clamps, utility type
3
-------
Membrane Filter Laboratory and Field Procedures
Table 1 EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)
Standard Tests
Nonstandard Tests
Item
M-Endo
Broth
L. E.S.
Coliform
Delayed
Coliform
Fecal
Coliform
Fecal
Streptococcus
Verified
Test
M-FC Broth for fecal coliform,
dehydrated medium in 25 X95 mm
flat bottomed screw-capped glass
vials, 1. 23 g per tube, sufficient
for 30 ml of culture medium
Rosolic acid, 1% solution, in
0. 2N NaOH, in 25 X 150 mm flat
bottomed screw-capped tubes,
about 5 ml per tube, freshly
prepared
M-Enterococcus Agar, dehydrated
medium in 25 X150 mm screw-
capped tubes, sufficient for 30 ml,
1. 26 g per tube
Dilution bottles, 6-oz, preferable
boro-silicate glass, with screw-
cap (or rubber stopper protected
by paper) , each containing 99 ml
of sterile phosphate buffered
distilled water
Electric hot plate surface
Beakers, 400 - 600 ml (for water-
bath in preparation of membrane
filter culture media)
Crucible tongs, to be used at
electric hot plates, for removal
of hot tubes of culture media for
boiling waterbath
X
X
X
X
X
X
X
X
X
X
X
X
4
-------
Membrane Filter Laboratory and Field Procedures
6 Deliver enough culture medium to
saturate each absorbent pad, * using
a sterile pipette.
Exact quantities cannot be stated
because pads and culture containers vary.
Sufficient medium should be applied so
that when the culture container is tipped,
a good-sized drop of culture medium .
freely drains out of the absorbent pad,.
7 Organize supplies and equipment for
convenient sample filtration. In
training courses, laboratory instructors
will suggest useful arrangements,
eventually the individual will select a
system of bench-top organization«most
suited to his own needs. The important
point m any arrangement is to have all
needed equipment and supplies con-
veniently at hand, in such a pattern as
to minimize lost time in useless motions.
10 Shake the sample thoroughly.
11 Measure sample into the funnel with
vacuum turned off.
The primary objectives here are:
1) accurate measurement of sample;
and 2) optimum distribution of colonies
on the filter after incubation. To
meet these objectives, methods of
measurement and dispensation to the
filtration assembly are varied with
different sample filtration volumes.
a With samples greater than 20 ml,
measure the sample with a sterile
graduated cylinder and pour it into
the funnel. It is important to rinse
this graduate with sterile buffered
distilled water to preclude the loss
of excessive sample volume. This
should be poured into the funnel.
8 Lay a sterile membrane filter on the
filter holder, grid-side up, centered
over the porous part of the filter
support plate.
Membrane filters are extremely
delicate and easily damaged. For
manipulation, the sterile forceps
should always grasp the outer part
of the filter disk, outside the part
of the filter through which the sample
passes.
9 Attach the funnel element to the base
of the filtration unit.
To avoid damage to the membrane
filter, locking forces should only be
applied at the locking arrangement. .
The funnel element never should be
turned or twisted while being seated
and locked to the lower element of the
filter holding unit. Filter holding units
featuring a bayonet joint and locking
ring to join the upper element to the
lower element require special care on
the part of the operator. The locking
ring should be turned sufficiently to
give a snug fit, but should not be
tightened excessively.
b With samples of 10 ml to 20 ml,
measure the sample with a sterile
10 ml or 20 ml pipette, and pipette
on a dry membrane in the filtration
assembly.
c With samples of 2 ml to 10 ml, pour
about 20 ml of sterile dilution water
into the filtration assembly, then
measure the sample into the sterile
buffered dilution water with a 10 ml
sterile pipette.
d With samples of 0. 5 to 2 ml, pour
about 20 ml of sterile dilution water
into the funnel assembly, then
measure the sample into the sterile
dilution water in the funnel with a
1 ml or a 2 ml pipette.
e If a sample of less than 0. 5 ml is to
be filtered, prepare appropriate
dilutions in sterile dilution water,
and proceed as applicable in item c
or d above.
When dilutions of samples are needed,
always make the filtrations as soon
as possible after dilution of the
sample, this never should exceed
NOTE: Mention of commercial products and manufacturers does not imply endorsement
by the Office of Water Programs, Environmental Protection Agency.
13-5
-------
Membrane Filter Laboratory and Field Procedures
30 minutes. Always shake sample
dilutions thoroughly before delivering
measured volumes.
12 Turn on the vacuum.
Open the appropriate spring clamp or
valve, and filter the sample.
After sample filtration a few droplets
of sample usually remain adhered to
the funnel walls. Unless these droplets
are removed, the bacteria contained in
them will be a source of contamination
of later samples. (In laboratory
practice the funnel unit is not routinely
sterilized between successive filtrations
of a series). The purpose of the funnel
rinse is to flush all droplets of a sample
from the funnel walls to the membrane
filter. Extensive tests have shown that
with proper rinsing technique, bacterial
retention on the funnel walls is negligible.
13 Rinse the sample through the filter.
After all the sample has passed through
the membrane filter, rinse down the
sides of the funnel walls with at least
20 ml of sterile dilution water. Repeat
the rinse twice after all the first rinse
has passed through the filter. Cut off
suction on the filtration assembly.
14 Remove the funnel element of the filter
holding unit.
If a ring stand with split ring is used,
hang the funnel element on the ring,
otherwise, place the inverted funnel
element on the inner surface of the
wrapping material. This requires
care in opening the sterilized package,
but it is effective as a protection of the
funnel ring from contamination.
15 Take the membrane filter from the
filter holder and carefully place it,
grid-side up on the medium.
Check that no air bubbles have been
trapped between the membrane filter
and the underlying absorbent pad or
agar. Relay the membrane if necessary.
16 Place in incubator after finishing
filtration series.
Invert the containers. The immediate
atmosphere of the incubating membrane
filter must be at or very near 100%
relative humidity.
17 Count colonies which have appeared
after incubating for the prescribed
time.
A stereoscopic microscope magnifying
10-15 times and careful illumination
give best counts.
For reporting results, the computation
is:
bacteria/100 ml =
No. colonies counted X100
Sample volume filtered in ml
Example:
A total of 36 colonies grew after
filtering a 10 ml sample. The
number reported is:
36 colonies
10 ml
X 100 = 360 per 100 ml
13-6
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Membrane Filter Laboratory and Field Procedures
H MF LABORATORY TESTS FOR
COLIFORM GROUP
A Standard Coliform Test (Based on M-Endo
Broth MF)
1 Culture medium
a M-Endo Broth MF Difco 0749-02
or the equivalent BBL M-Coliform
Broth 01-494
Preparation of Culture Medium
(M-Endo Broth) for Standard MF
Coliform Test
Yeast extract
1.5
g
Casitone or equivalent
5.0
g
Thiopeptone or equivalent
5.0
g
Tryptose
10.0
g
Lactose
12.5
g
Sodium desoxycholate
0.1
g
Dipotassium phosphate
4.375
g
Monopotassium phosphate
1.375
g
Sodium chloride
5.0
g
Sodium lauryl sulfate
0.05
g
Basic fuchsin (bacteriological)
1.05
g
Sodium sulfite
2.1
g
Distilled water (containing
1000 ml
20.0 ml ethanol)
This medium is available in
dehydrated form and it is rec-
ommended that the commercially
available medium be used in
preference to compounding the
medium of its individual constituents.
To prepare the medium for use,
suspend the dehydrated medium at
the rate of 48 grams per liter of
water containing ethyl alcohol at
the rate of 20 ml per liter.
As a time-saving convenience, it is
recommended that the laboratory
worker preweigh the dehydrated
medium in closed tubes for several
days, or even weeks, at one operation.
With this system, a large number
of increments of dehydrated medium
(e.g., 1.44 grams), sufficient for
some convenient (e. g., 30 ml)
volume of finished culture medium
are weighed and dispensed into
screw-capped culture tubes, and
stored until needed. .Storage should
preferably be in a darkened desin-ator.
A supply of distilled water containing
20 ml stock ethanol per liter is
maintained.
When the medium is to be used, it
is reconstituted by adding 30 ml of
the distilled water-ethanol mixture
per tube of pre-weighed dehydrated
culture medium.
b Medium is "sterilized" as directed
in I, C,
c Finished medium can be retained
up to 96 hours if kept in a cool,
dark place. Many workers prefer
to reconstitute fresh medium daily.
2 Filtration and incubation procedures
are as given m I, D.
Special instructions:
a For counting, use the wide field
binocular dissecting microscope, or
simple lens. For illumination, use
a light source perpendicular to the
plane of the membrane filter, A
small fluorescent lamp is ideal for
the purpose.
b Coliform colonies have a "metallic"
surface sheen under reflected light
which may cover the entire colony, or
it may appear only in the center. Non-
coliform colonies range from
colorless to pink, but do not have
the characteristic sheen.
c Record the colony counts on the
data sheet, and compute the coliform
count per 100 ml of sample.
13-7
-------
Membrane Filter Laboratory and Field Procedures
B Standard Coliform Tests (Based on L.E. S.
Endo Agar)
The distinction of the L. E. S. count is a
two hour enrichment incubation on LST
broth. M-Endo L.E.S. medium is used
as agar rather than the broth.
1 Preparation of culture medium
(L. E.S. Endo Agar) for L.E. S.
coliform test
a Formula from McCarthy, Delaney,
and Grasso ^)
Bacto-Yeast Extract
1.2
g
Bacto- Casitone
3.7
g
Bacto - Thiopeptone
3.7
g
Bacto-Tryptose
7.5
g
Bacto-Lactose
9.4
g
Dipotassium phosphate
3.3
g
Monopotassium phosphate
1.0
g
Sodium chloride
3.7
g
Sodium desoxycholate
0. 1
g
Sodium lauryl sulfate
0.05
g
Sodium sulfite
1.6
g
Bacto-Basic fuchsin
0. 8
g
Agar
15
g
Distilled water (containing 1000 ml
2 0 ml ethyl alcohol)
b To rehydrate the medium, suspend
51 grams in the water-ethyl alcohol
solution.
c Medium is "sterilized" as directed
in I, C,
d Pour 4-6 ml of freshly prepared Agar
into the smaller half of the container.
Allow the medium to cool and solidify.
2 Procedures for filtration and incubation
a Lay out the culture dishes in a row
or series of rows as usual. Place
these with the upper (lid) or top
side down.
b Place one sterile absorbent pad in
the larger half of each container
(lid). Use sterile forceps for all
13-8
manipulations of the pads.
(Agar occupies smaller half or
bottom).
c Using a sterile pipette, deliver
enough single strength lauryl
sulfate tryptose broth to saturate
the pad only. Excess interferes.
d Follow general procedures for
filtering in I, D. Place filters on
pad with lauryl sulfate tryptose
broth.
e Upon completion of the filtrations,
invert the culture containers and
incubate at 35° C for 1 1/2 to 2
hours.
3 2-hour procedures
a Transfer the membrane filter from
the enrichment pad in the upper half
to the agar medium in the lower
half of the container. Carefully
roll the membrane onto the agar
surface to avoid trapping air
bubbles beneath the membrane.
b Removal of the used absorbent pad
is optional.
c The container is inverted and
incubated 22 hours + 2 hours + 0.5 C.
4 Counting procedures are as in I, D.
5 L. E.S. Endo Agar may be used as a
single-stage medium (no enrichment
step) in the same manner as M-Endo
Broth, MF.
C Delayed Incubation Coliform Test
This technique is applicable in situations
where there is an excessive delay between
sample collection and plating. The procedure
is unnecessary when the interval be-
tween sample collection and plating is
within acceptable limits.
1 Preparation of culture media for
delayed incubation coliform test
a Preservative media M-Endo Broth
base
-------
Membrane Filter Laboratory and Field Procedures
To 30 ml of M-Endo Broth MF
prepared in accordance with
directions in n. A, 1 of this
outline, add 1. 0 ml of a sterile
12% aqueous solution of sodium
benzoate.
L. E. S. MF Holding Medium-
Coliform: Dissolve 12.7 grams in
1 liter of distilled water. No
heating is necessary. Final pH
7.1 + 0.1. This medium contains
sodium benzoate.
b Growth media
M-Endo Broth MF is used, prepared
as described in n, A, 1 earlier in
this outline. Alternately, L. E. S.
Endo Medium may be used.
2 General filtration followed is in I, D.
Special procedures are:
a Transfer the membrane filter from
the filtration apparatus to a pad
saturated with benzoated M-Endo
Broth.
b Close the culture dishes and hold
in a container at ambient temperature. D
This may be mailed or transported
to a central laboratory. The mailing
or transporting tube should contain
accurate transmittal data sheets which
correspond to properly labeled dishes.
Transportation time, in the case of
mailed containers, should not exceed
three days to the time of reception
by the testing laboratory.
c On receipt in the central laboratory,
unpack mailing carton, and lay out
the culture containers on the labora-
tory bench.
d Remove the tops from the culture
containers. Using sterile forceps,
remove each membrane and its
absorbent pad to the other half of
the culture container.
e With a sterile pipette or sterile
absorbent pad, remove preservative
medium from the culture container.
f Place a sterile absorbent pad in
each culture container, and deliver
enough freshly prepared M-Endo
Broth to saturate each pad.
g Using sterile forceps, transfer the
membrane to the new absorbent pad
containing M-Endo Broth. Place
the membrane carefully to avoid
entrapment of air between the
membrane and the underlying
absorbent pad. Discard the
absorbent pad containing pre-
servative medium.
h After incubation of 20 + 2 hours
at 35° C, count colonies as in the
above section A, 2.
i If L. E.S. Endo Agar is used, the
steps beginning with (e) above are
omitted; and the membrane filter is
removed from the preservative
medium and transferred to a fresh
culture container with L. E. S. Endo
Agar, incubated, and colonies
counted in the usual way.
Verified Membrane Filter Coliform Test
This procedure applies to identification
of colonies growing on Endo-type media
used for determination of total coliform
counts. Isolates from these colonies are
studied for gas production from lactose
and typical coliform morphology. In
effect, the procedure corresponds with
the Completed Test stage of the multiple
fermentation tube test for coliforms.
Procedure:
1 Select a membrane filter bearing
several well-isolated coliform-type
colonies.
2 Using sterile technique, pick all
colonies in a selected area with the
inoculation needle, making transfers
into tubes of phenol red lactose broth
(or lauryl sulfate tryptose lactose
13-9
-------
Membrane Filter Laboratory and Field Procedures
broth). Using an appropriate data
sheet record the interpretation of
each colony, using, for instance,
"C" for colonies having the typical
color and sheen of coliforms, "NC"
for colonies not conforming to
coliform colony appearance on
Endotype media.
3 Incubate the broth tubes at 35° C + 0. 5°C.
4 At 24 hours:
a Read and record the results from
the lactose broth fermentation tubes.
The following code is suggested:
Code
O No indication of acid or gas
production, either with or
without evidence of growth.
A Evidence of acid but not gas
(applies only when a pH indicator
is included in the broth medium)
G Growth with production of gas.
If pH indicator is used, use
symbol AG to show evidence of
acid. Gas in any quantity is a
positive test.
b Tubes not showing gas production are
returned to the 35° C incubator.
c Gas-positive tubes are transferred
as follows.
1) Prepare a streak inoculation on
EMB agar for colony isolation, and
using the same culture.
2) Inoculate a nutrient agar slant.
3) Incubate the EMB agar plates and
slants at 35°C + 0.5°C.
5 At 48 hours:
a Read and record results of lactose
broth tubes which were negative at
24 hours and were returned for
further incubation.
b Gas-positive cultures are subjected
to further transfers as in 4c.
Gas-negative cultures are discarded
without further study; they are
coliform- negative.
c Examine the cultures transferred
to EMB agar plates and to nutrient
agar slants, as follows:
1) Examine the EMB agar plate for
evidence of purity of culture; if
the culture represents more than
one colony type, discard the
nutrient agar culture and reisolate
each of the representative colonial
types on the EMB plate and resume
as with 4c for each isolation.
If purity of culture appears evident,
continue with c (2) below.
2) Prepare a smear and Gram stain
from each nutrient agar slant
culture. The Gram stain should
be made on a culture not more
than 24 hours old. Examine under
oil immersion for typical coliform
morphology, and record results.
6 At 72 hours:
Perform procedures described in 5c
above, and record results.
7 Coliform colonies are considered
verified if the procedures demonstrate
a pure culture of bacteria which are
gram negative nonspore-forming rods
and produce gas from lactose at 35° C
within 48 hours.
E Fecal Coliform Count (Based on M-FC
Broth Base)
The count depends upon growth on a
special medium at 44. 5 + 0. 2°C.
1 Preparation of Culture Medium
(M-FC Broth Base) for Fecal
Coliform Count
13-10
-------
Membrane Filter Laboratory and Field Procedures
a Composition
Tryptose
10.0 g
Proteose Peptone No. 3
5.0 g
Yeast extract
3.0 g
Sodium chloride
5.0 g
Lactose
12.5 g
Bile salts No. 3
1.5 g
Rosolic acid* (Allied
10. 0 ml
Chemical)
Aniline blue (Allied Chemical)
0.1 g
Distilled water
1000 ml
b To prepare the medium dissolve
37.1 grams in a liter of distilled
water which contains 10 ml of 1%
rosolic acid (prepared in 0. 2 N
NaOH).
Fresh solutions of rosolic acid give
best results. Discard solutions
which have changed from dark red
to orange,
c To sterilize, heat to boiling as
directed in I, C.
d Prepared medium may be retained
up to 4 days in the dark at 2- 8° C.
2 Special supplies
Small water proof plastic sacks capable
of being sealed against water with
capacity of 3 to 6 culture containers.
3 Filtration procedures are as given in
I. D.
4 Elevated temperature incubation
a Place fecal coliform count mem-
branes at 44.5 + 0.2°C as rapidly
as possible.
Filter megnbranes for fecal coliform
counts consecutively and immediately
place them in their culture containers.
Insert as many as six culture containers
all oriented in the same way (i.e., all
grid sides facing the same direction)
into the sacks and seal. Tear off the
perforated top, grasp the side wires,
and twirl the sack to roll the open end
inside the folds of sack. Then submerge
the sacks with culture containers in-
verted beneath the surface of a 44. 5
+ 0. 2 C waterbath.
b Incubate for 22+2 hours.
5 Counting procedures
Examine and count colonies as follows.
a Use a wide field binocular dissecting
microscope with 5 - 10X magnification.
b Low angle lighting from the side is
advantageous.
c Fecal coliform colonies are blue,
generally 1-3 mm in diameter.
d Record the colony counts on the
data sheet, and report the fecal
coliform count per 100 ml of sample.
(I, D, 17 illustrates method)
[I TESTS FOR FECAL STREPTOCOCCAL
GROUP-MEMBRANE FILTER METHOD
A 48 hour incubation period on a choice of
two different media, giving high selectivity
for fecal streptococci, are the distinctive
features of the tests.
^Prepare 1% solution of rosolic acid in 0.2 N NaOH. This dye is practically insoluble in water.
13-11
-------
Membrane Filter Laboratory and Field Procedures
A Test for Members of Fecal Streptococcal
(Tentative, Standard Methods) M-
Enterococcus Agar Medium
1 Preparation of the culture medium
a Formula (The Difco formula is shown,
but equivalent constituents from
other sources are equally acceptable).
Bacto tryptose
20.0
g
Bacto yeast extract
5. 0
g
Bacto dextrose
2.0
g
Dipotassium phosphate
4. 0
g
Sodium Azide
0.4
g
Bacto agar
10.0
g
2, 3, 5, Triphenyl
0. 1
g
tetrazolium chloride
b The medium is prepared by
rehydration at the rate of 42 grams
per 1000 ml of distilled water. It
is recommended that the medium in
dehydrated form be preweighed and
dispensed into culture containers
(about 25 X 150 mm) in quantities
sufficient for preparation of 30 ml
of culture medium (1. 26 g per tube).
c Follow I, C, for "sterilizing" medium
and dispense while hot into culture
containers. Allow plates to harden
before use.
2 List of apparatus, materials, as given
in Table 1.
3 Procedure, in general, as given in I.
Special instructions
a Incubate for 48 hours, inverted,
with 100% relative humidity, after
filtrations are completed. If the
entire incubator does not have
saturated humidity, acceptable
conditions can be secured by placing
the cultures in a tightly closed
container with wet paper, towels,
or other moist material.
b After incubation, remove the
cultures from the incubator, and
count all colonies under wide field
binocular dissecting microscope
with magnification set at 10X or
2OX. Fecal streptococcus colonies
are 0.5-2 mm in diameter, and
flat to raised smooth, and vary
from pale pink to dark red in color.
c Report enterococcus count per
100 ml of sample. This is con-
veniently computed:
No. fecal streptococci per 100 ml =
No. fecal streptococcus colonies counted
Sample filtration volume in ml
B Test for Members of Fecal Streptococcal
Group based on KF-Agar
1 Preparation of the culture medium
a Formula: (The dehydrated formula
of Bacto 0496 is shown, but
equivalent constituents from other
sources are acceptable). Formula
is in grams per liter of reconstituted
medium.
Bacto proteose peptone #3
10.0
g
Bacto yeast extract
10. 0
g
Sodium chloride freagent grade)
5. 0
g
Sodium glycerophosphate
10.0
g
Maltose (CP)
20. 0
g
Lactose (CP)
1.0
g
Sodium azide (Eastman)
0.4
g
Sodium carbonate
0.636
g
(NagCOg reagent grade)
Brom cresol purple
0. 015
g
(water soluble)
Bacto agar
20.0
g
b Reagent
2, 3, 5-Triphenyl tetrazolium
chloride reagent (TPTC)
This reagent is prepared by making
a 1% aqueous solution of the above
chemical passing it through a Seitz
filter or membrane filter. It can
13-12
-------
Membrane Filter Laboratory and Field Procedures
be kept in the refrigerator in a
screw-capped tube until used,
c The dehydrated medium described
above is prepared for laboratory
use as follows:
Suspend 7. 64 grams of the dehydrated
medium in 100 ml of distilled water
in a flask with an aluminum foil
cover.
Place the flask in a boiling water-
bath, melt the dehydrated medium,
and leave in the boiling waterbath
an addional 5 minutes.
Cool the medium to 50°-600C, add
1. 0 ml of the TPTC reagent, and
mix.
1
For membrane filter studies, pour
5-8 ml in each 50 mm glass or
plastic culture dish or enough to
make a layer approximately 1/8"
thick. Be sure to pour plates before
agar cools and solidifies.
For plate counts, pour as for standard
agar plate counts.
NOTE: Plastic dishes containing
media may be stored in a dark, cool
place up to 30 days without change
in productivity of the medium, pro-
vided that no dehydration occurs.
Plastic dishes may be incubated in
an ordinary air incubator. Glass
dishes must be incubated in an
atmosphere with saturated humidity.
2 Apparatus, and materials as given in
Table 1.
3 General procedure is as given in I.
Special instructions
a Incubate 48 hours, inverted with
100% relative humidity after
filtration.
b After incubation, remove the
cultures from the incubator, and
count colonies under wide field
binocular dissecting microscope,
with magnification set at 10X or
2QX. Fecal streptococcus colonies
are pale pink to dark wine-color.
In size they range from barely
visible to approximately 2mm in
diameter. Colorless colonies are
not counted.
c Report fecal streptococcus count
per 100 ml of sample. This is
computed as follows:
No. fecal streptococci per 100 ml =
No. fecal streptococcus colonies
Sample filtration volume in ml
C Verification of Streptococcus Colonies
1 Verification of colony identification
may be required in waters containing
large numbers of Micrococcus orga-
nisms. This has been noted
particularly with bathing waters, but
the problem is by no means limited to
such waters.
2 A verification procedure is described
in "Standard Methods for the Examination
of Water and Wastewater]' 13th ed.
(1971). The worker should use
this reference for the step-by-
step procedure.
IV PROCEDURES FOR USE OF MEMBRANE
FILTER FIELD UNITS
A Culture Media
1 The standard coliform media used with
laboratory tests are used.
2 To simplify field operations, it is
suggested that the medium be sent to
the field, preweighed, in vials or
capped culture tubes. The medium
then requires only the addition of a
suitable volume of distilled water-
ethanol prior to sterilization.
13-13
-------
Membrane Filter Laboratory and Field Procedures
3 Sterilization procedures in the field
are the same as for laboratory methods.
4 Laboratory preparation of the media,
ready for use, would be permissible
provided that the required limitations
on time and conditions of storage are
met.
B Operation of the Sabro Field Unit
1 Equipment and materials
Sabro field unit
Membrane filters
Absorbent pads for nutrient
Culture containers
M-Endo Broth MF or L.E.S. Endo
Medium
Provision for heating water (optional)
Source of electricity
2 Procedure (Based on M-Endo Broth MF)
a Connect the electric cord to the
power source and to Sabro field
unit. After about 15 minutes check
the temperature 111 the incubator
drawer. The required temperature
is 35° C (95°F). If the temperature
is too low it can be increased by
turning the thermostat adiustment
screw counterclockwise. This
screw is located at the front on the
recessed divider between the two
incubator drawers. To lower
temperature, turn the adjustment
screw clockwise.
b Review the supply of expendable
materials to be used with the unit
and secure replacements as needed
(culture containers, medium,
membrane filters, absorbent pads
for nutrient, fuel, etc.).
c Sterilize the funnel unit by one of
the following procedures.
1) Immerse the equipment 2 minutes
in boiling water. The temperature
should be at least 78° C (170OF).
2) Flame-sterilize membrane filter
holder inside and both ends of
funnel (suggested by manufacturer).
d Lay in a row all the culture con-
tainers to be used in the filtration
series, and number the containers
to correspond with numbers of the
data sheet.
e Place one sterile absorbent pad in
each culture container. Use sterile
forceps for all manipulation of
absorbent pads and filters.
f Using sterile pipette deliver enough
culture medium to saturate each
absorbent pad. The amount of
culture medium required is approxi-
mately 2 ml, but cannot be precisely
stated. Sufficient medium should be
applied, that when the culture con-
tainer is tipped, a good-sized drop
of culture medium freely drains out
of the absorbent pad.
g Using sterile forceps, place a mem-
brane filter, grid- side up, on the
MF receptacle of the funnel unit.
Place the funnel portion over the
membrane, and clamp the unit with
the spring clamp provided with the
portable kit.
h Pour the water sample into the funnel
using a sterile pipette or graduate.
i Connect the tubing of the vacuum
pump to the receptable on the base
of the filter unit and draw the sample
through the membrane. After the
sample has passed through the membrane
13-14
-------
Membrane Filter Laboratory and Field Procedures
filter, rinse down the sides of the funnel
walls with at least 20 ml of sterile dilu-
tion water. Repeat the rinse twice after
all the first rinse has passed through
the filter.
j Disassemble the funnel unit and with
sterile forceps transfer the membrane
filter grid-side up to the appropriate
culture container. The membrane
should be "rolled on" the absorbent
pad containing culture medium, to
prevent entrapment of air between
the pad and the membrane filter.
k Repeat steps g - j for additional
filtrations of the same or different
sampling volumes for the water
being tested.
1 After completion of filtration, place
the culture container in an inverted
position (with membrane position
grid- side down) in the incubator
drawers.
m After completion of the last filtration
from any one sample, resterilize the
funnel unit by one of the procedures
described in instruction 2c.
n Allow the cultures to incubate
20 - 24 hours.
o Remove the cultures from the
incubator and count coliform colonies.
C Operation of Millipore Water Testing Kit,
Bacteriological
1 Supporting supplies and equipment are
the same as for the laboratory
procedures.
2 Set the incubator voltage selector
switch to the voltage of the available
supply, turn on the unit and adjust as
necessary to establish operating
incubator temperature at 35 + 0.5°C.
3 Sterilize the funnel unit assembly by
exposure to formaldehyde or by
immersion in boiling water. If a
laboratory autoclave is available, this
is preferred.
Formaldehyde is produced by soaking
an asbestos ring (in the funnel base)
with methanol, igniting, and after a
few seconds of burning, closing the
unit by placing the stainless steel
flask over the funnel and base. This
results in incomplete combustion of
the methanol, whereby formaldehyde
is produced. Leave the unit closed
for 15 minutes to allow adequate
exposure to formaldehyde.
4 Filtration and incubation procedures
correspond with laboratory methods.
5 The unit is supplied with a booklet
containing detailed step-by-step
operational procedures. The worker
using the equipment should become
completely versed in its contents and
application.
D Counting of Colonies on Membrane Filters
1 Equipment and materials
Membrane filter cultures to be
examined
Illumination source
Simple lens, 2X to 6X magnification
Hand tally (optional)
2 Procedure
a Remove the cultures from the
incubator and arrange them in
numerical sequence.
b Set up illumination source as that
light will originate from an area
perpendicular to the plane of
membrane filters being examined.
A small fluorescent lamp is ideal
for the purpose. It is highly
desirable that a simple lens be
attached to the light source.
c Examine results. Count all coliform
and noncoliform colonies. Coliform
colonies have a "metallic" surface
sheen under reflected light, which
may cover the entire colony or may
appear only on the center
13-15
-------
Membrane filter Laboratory and Field Procedures
Noncoliform colonies range from
colorless to pink or red, but do not
have the characteristic
sheen.
metallic
Enter the colony counts in the data
sheets.
Enter the coliform count per 100 ml
of sample for each membrane having
a countable number of coliform
colonies. Computation is as follows-
No. coliform per 100 ml =
No. coliform colonies on MF
No. milliliters sample filtered
X100
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewater. APHA,
AWWA, WPCF. 12th Edition. 1965.
2 McCarthy, J.A., Delaney, J. E. and
Grasso, R.J. Measuring Coliform s
in Water. Water and Sewage Works.
1961: R-426-31, 1961.
This outline was prepared by H. L. Jeter,
Director, National Training Center, MDS,
OWP, EPA, Cincinnati, OH 4&268.
13-16
-------
V OXYGEN IMPORTANCE AND DETERMINATIONS
The dissolved oxygen content of water is a
major water quality criteria in relationship
to the nature, variety, and activity of aquatic
organisms in it and degree of stabilization of it.
Contents of Section V
Outline Number
Dissolved Oxygen Determination
(DO) - I
Winkler Iodometric Titration
and Azide Modification 14
Laboratory Procedure for
Dissolved Oxygen
Winkler-Azide Procedure 15
Dissolved Oxygen Determination -II
Electronic Measurements 16
Biochemical Oxygen Demand Test
Procedures 17
Biochemical Oxygen Demand Test
Dilution Technique 18
BOD Determination - Reaerated
Bottle Probe Technique 19
Effect of Some Variables on the
BOD Test 20
Chemical Oxygen Demand and COD/
BOD Relationships 21
Laboratory Procedure for Routine
Level Chemical Oxygen Demand 22
Total Carbon Analysis
23
-------
DISSOLVED OXYGEN DETERMINATION (DO) - I
Winkler Iodometric Titration and Azide Modification
I The Dissolved Oxygen determination is
a very important water quality criteria for
many reasons:
A Oxygen is an essential nutrient for all
living organisms. Dissolved oxygen is
essential for survival of aerobic
organisms and permits facultative
organisms to metabolize more effectively.
Many desirable varieties of macro or
micro organisms cannot survive at
dissolved oxygen concentrations below
certain minimum values. These values
vary with the type of organisms, stage
in their life history, activity, and other
factors.
B Dissolved oxygen levels may be used as
an indicator of pollution by oxygen
demanding wastes. Low DO concen-
trations are likely to be associated with
low quality waters.
C The presence of dissolved oxygen
prevents or minimizes the onset of
putrefactive decomposition and the
production of objectionable amounts of
malodorous sulfides, mercaptans,
amines, etc.
D Dissolved oxygen is essential for
terminal stabilization wastewaters.
High DO concentrations are normally
associated with good quality water.
E Dissolved oxygen changes with respect
to time, depth or section of a water
mass are useful to indicate the degree
of stability or mixing characteristics
of that situation.
F The BOD or other respirometric test
methods for water quality are commonly
based upon the difference among an
initial and final DO determination for a
given sample time interval and con-
dition. These measurements are
useful to indicate-
1 The rate of biochemical activity in
terms of oxygen demand for a
given sample and conditions.
2 The degree of acceptability
(a bioassay technique) for bio-
chemical stabilization of a given
microbiota in response to food,
inhibitory agents or test conditions.
3 The degree of instability of a
water mass on the basis of test
sample DO changes over an
extended interval of time.
4 Permissible load variations in
surface water or treatment units
in terms of DO depletion versus
time, concentration, or ratio of
food to organism mass, solids, or
volume ratios.
5 Oxygenation requirements
necessary to meet the oxygen
demand m treatment units or
surface water situations.
G The DO test is the only chemical test
included in all Water Quality Criteria,
Federal, State, Regional or local.
II FACTORS AFFECTING THE DO
CONCENTRATION IN WATER
A Physical Factors:
1 DO solubility in water for an
air/water system is limited to
about 9 mg DO/liter of water at
20°C. This amounts to about
0. 0009% as compared to 21% by
weight of oxygen in air.
2 Transfer of oxygen from air to
water is limited by the interface
area, the oxygen deficit, partial
pressure, the conditions at the
CH.O.do. 31c. 12.71
14-1
-------
Dissolved Oxygen Determination (DO) - I
interface area, mixing phenomena
and other items.
Certain factors tend to confuse
reoxygenation mechanisms of
water aeration:
a The transfer of oxygen in air
to dissolved molecular oxygen
in water has two principal
variables:
1) Area of the air-water
interface.
2) Dispersion of the oxygen-
saturated water at the
interface into the body liquid.
The first depends upon the surface
area of the air bubbles m the water
or water drops in the air, the
second depends upon the mixing
energy in the liquid. If diffusors
are placed in a line along the wall,
dead spots may develop in the core.
Different diffusor placement or
mixing energy may improve oxygen
transfer to the liquid two or threefold.
b Other variables m oxygen transfer
include
3) Oxygen deficit in the liquid.
4) Oxygen content of the gas phase.
5) Time.
If the first four variables are
favorable, the process of water
oxygenation is rapid until the liquid
approaches saturation. Much more
energy and time are required to
increase oxygen saturation from
about 95 to 100% than to increase
oxygen saturation from 0 to about
95%. For example- An oxygen-
depleted sample often will pick up
significant DO during DO testing,
changes are unlikely with a sample
containing equilibrium amounts
of DO.
c The limited solubility of oxygen
in water compared to the oxygen
content of air does not require
the interchange of a large mass of
oxygen per unit volume of water
to change DO saturation. DO
increases from zero to 50%
saturation are common in passage
over a weir.
d Aeration of dirty water is practiced
for cleanup. Aeration of clean
water results in washing the air and
transferring fine particulates and
gaseous contaminants to the liquid.
e One liter of air at room temperature
contains about 230 mg of oxygen.
A 5 gal carboy of water with 2 liters
of gas space above the liquid has
ample oxygen supply for equilibration
of DO after storage for 2 or 3 days
or shaking for 30 sec.
f Aeration tends toward evaporative
cc "''"ng. Oxygen content becomes
higher than saturation values at
the test temperature, thus
contributing to high blanks.
3 Oxygen solubility varies >vith the
temperature of the water.
Solubility at 10o C is about two
times that at 30oc. Temperature
often contributes to DO variations
much greater than anticipated by
14-2
-------
Dissolved Oxygen Determination (DO) - I
solubility. A cold water often has
much more DO than the solubility
limits at laboratory temperature.
Standing during warmup commonly
results in a loss of DO due to
oxygen diffusion from the super-
saturated sample. Samples
warmer than laboratory tempera-
ture may decrease m volume due
to the contraction of liquid as
temperature is lowered. The full
bottle at higher temperature will
be partially full after shrinkage
with air entrance around the stopper
to replace the void. Oxygen in the
air may be transferred to raise the
sample DO. For example, a
volumetric flask filled to the 1000 ml
mark at 30OC will show a water
level about 1/2 inch below the mark
when the water temperature is
rpduced to 20OC. BOD dilutions
should be adjusted to 20OC + or -
1 l/2o before filling and testing.
4 Water density varies with tem-
perature with maximum water
density at 4o C. Colder or warmer
waters tend to promote stratification
of water that interferes with
distribution of DO because the
higher density waters tend to seek
the lower levels.
5 Oxygen diffusion in a water mass is
relatively slow, hence vertical and
lateral mixing are essential to
maintain relatively uniform oxygen
concentrations in a water mass.
6 Increasing salt concentration
decreases oxygen solubility
slightly but has a larger effect
upon density stratification in a
water mass.
7 The partial pressure of the oxygen
in the gas above the water interface
controls the oxygen solubility
limits in the water. For example,
the equilibrium concentration of
oxygen in water is about 9 mg DO/1
under one atmospheric pressure of
air, about 42 mg DO/liter in
contact with pure oxygen and 0 mg
DO/liter in contact with pure
nitrogen (@ 20° C).
B Biological or Bio-Chemical Factors
1 Aquatic life requires oxygen for
respiration to meet energy
requirements for growth, repro-
duction, and motion. The net
effect is to deplete oxygen resources
in the water at a rate controlled
by the type, activity, and mass of
living materials present, the
availability of food and favor-
ability of conditions.
2 Algae, autotrophic bacteria, plants
or other organisms capable of
photosynthesis may use light
energy to synthesize cell materials
from mineralized nutrients with
oxygen released in process.
a Photosynthesis occurs only
under the influence of adequate
light intensity.
b Respiration of alga is
continuous.
c The dominant effect in terms
of oxygen assets or
liabilities of alga depends upon
algal activity, numbers and
light intensity. Gross algal
productivity contributes to
significant diurnal DO
variations.
3 High rate deoxygenation commonly
accompanies assimilation of
readily available nutrients and
conversion into cell mass or
storage products. Deoxygenation
due to cell mass respiration
commonly occurs at some lower
rate dependent upon the nature of
the organisms present, the stage
of decomposition and the degree
of predation, lysis, mixing and
regrowth. Relatively high
14-3
-------
Dissolved Oxygen Determination (DO) - I
deoxygenation rates commonly are
associated with significant growth
or regrowth of organisms.
Micro-organisms tend to flocculate
or agglomerate to form settleable
masses particularly at limiting
nutrient levels (after available
nutrients have been assimilated or
the number of organisms are large
in proportion to available food).
a Resulting benthic deposits
continue to respire as bed
loads.
b Oxygen availability is limited
because the deposit is physically
removed from the source of
surface oxygenation and algal
activity usually is more
favorable near the surface.
Stratification is likely to limit
oxygen transfer to the bed load
vicinity.
c The bed load commonly is
oxygen deficient and decomposes
by anaerobic action.
d Anaerobic action commonly is
characterized by a dominant
hydrolytic or solubilizing action
with relatively low rate growth
of organisms.
e The net effect is to produce low
molecular weight products
from® cell mass with a corre-
spondingly large fraction of
feedback of nutrients to the
overlaying waters. These
lysis products have the effect
of a high rate or immediate
oxygen demand upon mixture
with oxygen containing waters.
f Turbulence favoring mixing of
surface waters and benthic
sediments commonly are
associated with extremely
rapid depletion of DO.
Recurrent resuspension of
thin benthic deposits may
contribute to highly erratic
DO patterns.
g Long term deposition areas
commonly act like point
sources of new pollution as
a result of the feedback of
nutrients from the deposit.
Rate of reaction may be low
for old materials but a low
percentage of a large mass of
unstable material may produce
excessive oxygen demands.
Tremendous DO variations are likely
in a polluted water in reference to
depth, cross section or time of day.
More stabilized waters tend to show
decreased DO variations although it is
likely that natural deposits such as leaf
mold will produce differences related
to depth in stratified deep waters.
Ill ANA LYTICA L METHOD BA CKGROUND
The basic Winkler procedure (1888) has been
modified many times to improve its work-
ability in polluted waters. None of these
modifications have been completely
successful. The most useful modification
was proposed by Alsterberg and consists of
the addition oi c ">dium azide to control
nitrite interference during the lodometric
titration. The Azide modification of the
lodometric titration is recommended as
official by the EPA-OWP Quality Control
Committee for relatively clean waters.
A Reactions
1 The determination of DO involves
a complex series of interactions
that must be quantitative to provide
a valid DO result. The number of
sequential reactions also compli-
cates interference control. The
reactions will be presented first
followed by discussion of the
functional aspects.
14-4
-------
Dissolved Oxygen Determination (DO) - I
MnSO + 2 KOH -* Mn(OH) + K SO (a)
2 Mn(OH)2 + 02 — 2 MnO(OH>2 (b)
MnO(OH)2 + 2 H2S04— MntSO^ + 3^0 (c)
Mn(SO.)_ + 2 KI — MnSO. + K„SO„ + In (d)
4 2 4 2 4 2
l2 + 2 Na2S2°3 Na2S4°6 + 2NaI (e)
2 Reaction sequence
The series of reactions involves
five different operational steps in
converting dissolved oxygen in the
water into a form in which it can
be measured.
a O —MnO(OH) — Mn(SO ) —
I —~ Thiosulfate (thio) or
phenylarsine oxide (PAO)
titration.
b All added reagents are in excess
to improve contact possibilities
and to force the reaction toward
completion.
3 The first conversion, O
MnO(OH)2 (reactions a, 5) is an
oxygen transfer operation where
the dissolved oxygen in the water
combines with manganous
hydroxide to form an oxygenated
manganic hydroxide.
a The manganous salt can react
with oxygen only in a highly
alkaline media.
b The manganous salt and alkali
must be added separately with
addition below the surface of
the sample to minimize reaction
with atmospheric oxygen via
air bubbles or surface contact.
Reaction with sample dissolved
oxygen is intended to occur
upon mixing of the reagents and
sample after stoppering the
full bottle (care should be used
to allow entrained air bubbles
to rise to the surface before
adding reagents to prevent
high results due to including
entrained oxygen).
c Transfer of oxygen from the
dissolved state to the pre-
cipitate form involves a two
phase system of solution and
precipitate requiring effective
mixing for quantitative
transfer. Normally a gross
excess of reagents are used
to limit mixing requirements.
Mixing by rapid inversion 25
to 35 times will accomplish
the purpose. Less energy is
required by inversion 5 or 6
times, allowing the solids to
settle half way and repeat the
process. Reaction is rapid,
contact is the principal
problem in the two phase
system.
d If the alkaline floe is white,
no oxygen is present.
Acidification (reactions c and d)
changes the oxygenated manganic
hydroxide to manganic sulfate
which in turn reacts with
potassium iodide to form elemental
iodine. Under acid conditions,
oxygen cannot react directly with
the excess manganous sulfate
remaining in solution.
Iodine (reaction e) may be titrated
with sodium thiosulfate standard
solution to indicate the amount of
dissolved oxygen originally
present in the sample.
a The blue color of the starch-
lodine complex commonly is
used as an indicator. This
blue color disappears when
elemental iodine has been
reacted with an equivalent
amount of thiosulfate.
14-5
-------
Dissolved Oxygen Determination (DO) - I
b Phenylarsine oxide solutions are
more expensive to obtain but
have better keeping qualities
than thiosulfate solutions.
Occasional use, field operations
and situations where it is not
feasible to calibrate thio
solutions regularly, usually
encourage use of purchased
PAO reagents.
The same thing applies for
other sample volumes when
using an appropriate titrant
normality such as-
1) For a 200 ml sample, use
0. 025 N Thio
2) For a 100 ml sample, use
0. 0125 N Thio
For practical purposes the DO
determination scheme involves the
following operations.
a Fill a 300 ml bottle* under
conditions minimizing DO
changes. This means that the
sample bottle must be flushed
with test solution to displace
the air in the bottle with water
characteristic of the tested
sample*.
*DO test bottle volumes should
be checked - discard those
outside of the limits of 300 ml
+ or - 3 ml.
b To the filled bottle
1) Add MnSO^ reagent (2 ml)
2) Add KOH, KI, NaN„ reagent
(2 ml)
Stopper, mix by inversion,
allow to settle half way and
repeat the operation.
Highly saline test waters
commonly settle very
slowly at this stage and
may not settle to the half
way point in the time
allotted.
c To the alkaline mix (settled
about half way) add 2 ml of
sulfuric acid, stopper and mix.
d Transfer the contents of the
bottle to a 500 ml Erlenmeyer
flask and titrate with 0.0375
Normal Thiosulfate*. Each
ml of reagent used represents
1 mg of DO/liter of sample.
*EPA-OWP Method
The addition of the first two DO
reagents, (MnS04 and the KOH, KI
and NaNg solutions) displaces an
equal quantity of the sample. This
is not the case when acid is added
because the clear liquid above the
floe does not contain dissolved
oxygen as all of it should be con-
verted to the particulate MnOtOH^.
Some error is introduced by this
displacement of sample during
dosage of the first two reagents.
The error upon addition of 2 ml of
each reagent to a 300 ml sample
is — X 100 or 1. 33% loss in DO.
300
This may be corrected by an
appropriate factor or by adjust-
ment of reagent normality. It is
generally considered small in
relation to other errors in sampling,
manipulation and interference,
hei." this error may be recognized
but not corrected.
Reagent preparation and pro-
cedural details can be found in
reference 1.
IV The sequential reactions for the
Chemical DO determination provides
several situations where significant inter-
ference may occur in application on
polluted water, such as
A Sampling errors may not be strictly
designated as interference but have the
same effect of changing sample DO.
Inadequate flushing of the bottle con-
tents or exposure to air may raise the
DO of low oxygen samples or lower the
DO of supersaturated samples.
14-6
-------
Dissolved Oxygen Determination (DO) - I
B Entrained air may be trapped m a DO
bottle by
1 Rapid filling of vigorously mixed
samples without allowing the
entrained air to escape before
closing the bottle and adding DO
reagents.
2 Filling a bottle with low temperature
water holding more DO than that in
equilibrium after the samples warm
to working temperature.
3 Aeration is likely to cool the sample
permitting more DO to be introduced
than can be held at the room or
incubator temperatures.
4 Samples warmer than working
incubator temperatures will be
only partially full at equilibrium
temperatures.
Addition of DO reagents results in
reaction with dissolved or entrained
oxygen. Results for DO are invalid
if there is any evidence of gas
bubbles in the sample bottle.
C The DO reagents respond to any oxidant
or reductant in the sample capable of
reacting within the time allotted. HOC1
of H2O2 may raise the DO titration
while H2S, 4 SH may react with sample
oxygen to lower the sample titration.
The items mentioned react rapidly and
raise or lower the DO rppult promptly.
Other items such as Fe or SO„ may
or may not react completely within the
time allotted for reaction. Many
organic materials or complexes from
benthic deposits may have an effect upon
DO results that are difficult to predict.
They may have one effect during the
alkaline stage to release iodine from
K1 while favoring irreversible
absorption of iodine during the acid
stage. Degree of effect may increase
with reaction time. It is generally
inadvisable to use the lodometric
titration on samples containing large
amounts of organic contaminants or
benthic residues. It would be expected
that benthic residues would tend toward
low results because of the reduced iron
and sulfur content - they commonly
favor high results due to other factors
that react more rapidly, often giving
the same effect as in uncontrolled
nitrite interference during titration.
D Nitrite is present to some extent in
natural waters or partially oxidized
treatment plant samples. Nitrite is
associated with a cyclic reaction during
the acid stage of the DO determination
that may lead to erroneous high results.
1 These reactions may be rep-
resented as follows-
2HN02 + 2 HI -I2 + 4H20 + N^02 (a)
2HN02— + l/202+ HgO + N202 (b)
These reactions are time, mixing
and concentration dependent and
can be minimized by rapid
processing.
2 Sodium azide (NaN^) reacts with
nitrite under acid conditions to
form a combination of N,. + NgO
which effectively blocks the
cyclic reaction by converting the
HNO to noninterfering compounds
of nitrogen.
3 Sodium azide added to fresh
alkaline KI reagent is adequate to
control interference up to about
20 mg of HNOg - N/liter of sample.
The azide is unstable and grad-
ually decomposes. If resuspended
benthic sediments are not detectable
in a sample showing a returning
blue color, it is likely that the
azide has decomposed in the
alkaline KI azide reagent.
E Surfactants, color and Fe+++ may
confuse endpoint detection if present
in significant quantities.
14-7
-------
Dissolved Oxygen Determination (DO) - I
F Polluted water commonly contains
significant interferences such as C.
It is advisable to use a membrane
protected sensor of the electronic type
for DO determinations in the presence
of these types of interference.
G The order of reagent addition and prompt
completion of the DO determination is
critical. Stable waters may give valid
DO results after extended delay of
titration during the acidified stage. For
unstable water, undue delay at any stage
of processing accentuates interference
problems.
ACKNOWLEDGMENTS
This outline contains significant materials
from previous outlines by J. W. Mandia.
Review and comments by C. R. Hirth and
R. L. Booth are greatly appreciated.
REFERENCE
I
1 Methods for Chemical Analysis of
Water and Wastes, EPA-AQCL,
Cincinnati, OH, July 1971.
This outline was prepared by F. J. Ludzack,
Chemist National Training Center,
MDS, OWP, EPA, Cincinnati, OH 45268.
' n
14-8
-------
LABORATORY PROCEDURE FOR DISSOLVED OXYGEN
Winkler-Azide Procedure
I INTRODUCTION
The azide modification is used for most
sewage, effluents, and streams which con-
tain nitrate nitrogen and not more than 1 mg
of ferrous iron/1. If 1 ml 40% KF solution
is added before acidifying the sample and
there is no delay in titration, the method is
also applicable in the presence of 100-200
mg/1 ferric iron.
The "Methods for Chemical Analysis of
Water & Wastes, 1971," published by the
Analytical Quality Control Laboratory of the
EPA, recommends the Winkler-Azide
method using the full bottle technique.
E Sodium Thiosulfate Stock Solution 0.75 N
Dissolve 186.15 g NagSgO^- 5 H„0 in boiled
and cooled distilled water and dilute to
1 liter. Preserve by adding 5 ml chloroform.
F Sodium Thiosulfate Standard Titrant 0. 0375N
Dilute 50. 0 ml of stock solution to 1 liter.
Preserve by adding 5 ml of chloroform.
Standardize with potassium biiodate.
EI PROCEDURE
A Addition of Reagents
1 Manganous sulfate and alkali-iodide-
azide
H REAGENTS
A Manganous Sulfate Solution
Dissolve 480 g MnSO • 4HgO in distilled
water and dilute to 1 liter.
B Alkali-Iodide-Azide Reagent
Dissolve 500 g sodium hydroxide and 150 g
potassium iodide in distilled water and
dilute to 1 liter. To this solution add 10 g
sodium azide. NaN^ dissolved in 40 ml
distilled water.
C Sulfuric Acid, Cone.
The strength of this acid is 36 N.
D Starch Solution
Prepare an emulsion of 10 g of soluble
starch in a mortar or beaker with a small
quantity of distilled water. Pour this
emulsion into 1 liter of boiling water,
allow to boil a few minutes, and let settle
overnight. Use the clear supernate.
This solution may be preserved by the
addition of 5 ml per liter of chloroform
and storage in a 10° C refrigerator.
To a full sample bottle (300 ml + 3 ml
BOD incubation), add 2 ml manganous
sulfate solution and 2 ml alkaline-
iodide reagent with the tip of each
pipette below the surface of the sample.
2 Replace the stopper, rinse under
running water and mix by inverting
4-5 times.
3 Allow the precipitate to settle until at
least 100 ml of clear supernate have
been produced. Repeat the inverting
and settling.
4 Add 2 ml conc. sulfuric acid with the
tip of the pipette above the surface of
the sample.
5 Stopper the bottle, rinse under running
water and mix to dissolve the precipitate.
6 Pour contents of bottle into a wide mouth
500 ml Erlenmeyer flask.
B Titration and Calculation
1 Titrate with 0. 0375 N thiosulfate to a
pale straw color, add 2 ml starch
solution indicator and continue titrating
to the disappearance of the blue color.
CH.O.do. lab. 3b. 12. 71
15-1
-------
Laboratory Procedure for Dissolved Oxygen
2f' Calculation
mis titrant XO. 0375 X 8000 „ ,
300 = mg/1 ]
mis titrant X 0.0125 X80 = mg/1 DO
mis titrant X 1 = mg/1 DO
mis titrant = mg/1 DO
REFERENCE
Methods for Chemical Analysis of Water
anci Wastes, EPA-AQCL, Cincinnati,
Ohio, July 1971.
This outline was prepared by J. W. Mandia,
Chemist, formerly with National Training
Center, and revised by C. R. Feldmann,
Chemist, National Training Center,
MDS, OWP. EPA, Cincinnati, OH 45268.'
15-2
-------
I INTRODUCTION
A Electronic measurement of DO is attractive
for several reasons-
1 Electronic methods are more readily
adaptable for automated analysis, con-
tinuous recording, remote sensing or
portability.
2 Application of electronic methods with
membrane protection of sensors affords
a high degree of interference control.
3 Versatility of the electronic system
permits design for a particular measure-
ment, situation or use.
4 Many more determinations per man-
hour are possible with a minor expend-
iture of time for calibration.
B Electronic methods of analysis impose
certain restrictions upon the analyst to
insure that the response does, in fact,
indicate the item sought.
1 The ease of reading the indicator tends
to produce a false sense of security.
Frequent and careful calibrations are
essential to establish workability of the
apparatus and validity of its response.
2 The use of electronic devices requires
a greater degree of competence on the
part of the analyst Understanding of
the behavior of oxygen must be supple-
mented by an understanding of the
particular instrument and its behavior
during use.
C Definitions
1 Electrochemistry - a branch of chemistry
dealmg with relationships between
electrical and chemical changes.
or electro-
metric procedures - procedures using
the measurement of potential differences
as an indicator of reactions taking
place at an electrode or plate.
3 Reduction - any process in which one
or more electrons are added to an atom
or an ion, such as + 2e —~ 20 !
The oxygen has been reduced.
4 Oxidation - any process in which one
or more electrons are removed from
an atom or an ion, such as Zn° - 2e
—~ Zn+2. The zinc has been oxidized.
5 Oxidation - reduction reactions - in a
strictly chemical reaction, reduction
cannot occur unless an equivalent
amount of some oxidizable substance
has been oxidized. For example:
2H2 + °2 ~ 2H2°
2H£ - 4e 4H hydrogen oxidized
-2
0„ + 4e <-20 oxygen reduced
Chemical reduction of oxygen may also
be accomplished by electrons supplied
to a noble metal electrode by a battery
or other energizer.
6 Anode - an electrode at which oxidation
of some reactable substance occurs.
7 Cathode - an electrode at which
reduction of some reactable substance -
occurs. For example in I.C.3, the
reduction of oxygen occurs at the
cathode.
8 Electrochemical reaction - a reaction
involving simultaneous conversion of
chemical energy into electrical energy
or the reverse. These conversions are
DISSOLVED OXYGEN DETERMINATION - II
ELECTRONIC MEASUREMENTS
2 Electronic measurements
CH. O. do. 32a. 8.70
16-1
-------
Dissolved Oxygen Determination - II
equivalent in terms of chemical and
electrical energy and generally are
reversible
9 Electrolyte a solution, gel, or mixture
capable of conducting electrical energy
and serving as a reacting media for
chemical changes. The electrolyte
commonly contains an appropriate
concentration of selected mobile ions
to promote the desired reactions
b Polarographic (electrolytic) cell -
an electrochemical cell operated in
such a way as to produce a chemical
change from electrical energy
(See Figure 2).
10 Electrochemical cell - a device con-
sisting of an electrolyte in which 2
electrodes are immersed and connected
via an external metallic conductor
The electrodes may be in separate
compartments connected by tube con-
taining electrolyte to complete the
internal circuit
a Galvanic (or voltaic) cell - an
electrochemical cell operated in
such a way as to produce electrical
energy from a chemical change,
such as a battery (See Figure 1).
KDI'
Cathode
POLAROGRAPHIC CELL
Fiturt 2
-CD-
Anode I
Cathode
2n++
S04~
Earn
CL++I
SO4-
GALVANIC CELL
Fi(or« 1
As indicated in I. C. 10 the sign of an
electrode may change as a result of the
operating mode. The conversion by the
reactant of primary interest at a given
electrode therefore designates terminology
for that electrode and operating mode
In electronic oxygen analyzers, the
electrode at which oxygen reduction occurs
is designated the cathode.
Each cell type has characteristic advantages
and limitations. Both may be used
effectively.
1 The galvanic cell depends upon
measurement of electrical energy
produced as a result of oxygen
16-2
-------
Dissolved Oxygen Determination - II
reduction. If the oxygen content of the
sample is negligible, the measured
current is very low and indicator driving
force is negligible, therefore response
time is longer
2 The polarographic cell uses a standing
current to provide energy for oxygen
reduction. The indicator response
depends upon a change in the standing
current as a result of electrons
released during oxygen reduction.
Indicator response time therefore is
not dependent upon oxygen concentration.
3 Choice may depend upon availability,
habit, accessories, or the situation.
In each case it is necessary to use
care and judgment both in selection
and use for the objectives desired.
H ELECTRONIC MEASUREMENT OF DO
A Reduction of oxygen takes place in two
steps as shown in the following equations:
1 02 + 2H20 + 2e — H202 + 20H~
2 HgOg + 2e~ — 20H~
Both equations require electron input to
activate reduction of oxygen. The first
reaction is more important for electronic
DO measurement because it occurs at a
potential (voltage) which is below that
required to activate reduction of most
interfering components (0. 3 to 0. 8 volts
relative to the saturated calomel electrode -
SCE). Interferences that may be reduced
at or below that required for oxygen
usually are present at lower concentrations
in water or may be minimized by the use
of a selective membrane or other means.
When reduction occurs, a definite quantity
of electrical energy is produced that is
proportional to the quantity of reductant
entering the reaction. Resulting current
measurements thus are more specific for
oxygen reduction.
B Most electronic measurements of oxygen
are based upon one of two techniques for
evaluating oxygen reduction in line with
equation II A. 1. Both require activating
energy, both produce a current propor-
tional to the quantity of reacting reductant.
The techniques differ in the means of
supplying the activating potential; one
employs a source of outside energy, the
other uses spontaneous energy produced
by the electrode pair.
1 The polarographic oxygen sensor
relies upon an outside source of
potential to activate oxygen reduction
Electron gam by oxygen changes the
reference voltage.
a Traditionally, the dropping mercury
electrode (DME) has been used for
polarographic measurements. Good
results have been obtained for DO
using the DME but the difficulty of
maintaining a constant mercury drop
rate, temperature control, and
freedom from turbulence makes it
impractical for field use.
b Solid electrodes are attractive
because greater surface area
improves sensitivity. Poisoning
of the solid surface electrodes is
a recurrent problem. The use of
selective membranes over noble
metal electrodes has minimized
but not eliminated electrode con-
tamination. Feasibility has been
improved sufficiently to make this
type popular for regular use.
2 Galvanic oxygen electrodes consist of
a decomposable anode and a noble
metal cathode in a suitable electrolyte
to produce activating energy for oxygen
reduction (an air cell or battery). Lead
is commonly used as the anode because
its decomposition potential favors
spontaneous reduction of oxygen. The
process is continuous as long as lead
and oxygen are in contact in the electrolyte
and the electrical energy released at
the cathode may be dissipated by an
outside circuit. The anode may be
conserved by limiting oxygen availability.
Interrupting the outside circuit may
produce erratic behavior for a time
after reconnection. The resulting
16-3
-------
Dissolved Oxygen Determination - II
current produced by oxygen reduction
may be converted to oxygen concen-
tration by use of a sensitivity coefficient
obtained during calibration. Provision
of a pulsed or interrupted signal makes
it possible to amplify or control the
signal and adjust it for direct reading
in terms of oxygen concentration or to
compensate for temperature effects.
IE ELECTRONIC DO ANALYZER
APPLICATION FACTORS
A Polarographic or galvanic DO instruments
operate as a result of oxygen partial
pressure at the sensor surface to produce
a signal characteristic of oxygen reduced
at the cathode of some electrode pair.
This signal is conveyed to an indicating
device with or without modification for
sensitivity and temperature or other
influences depending upon the instrument
capabilities and intended use.
1 Many approaches and refinements have
been used to improve workability,
applicability, validity, stability and
control of variables. Developments
are continuing. It is possible to produce
a device capable of meeting any reasonable
situation, but situations differ.
2 Most commercial DO instruments are
designed for use under specified con-
ditions. Some are more versatile than
others. Benefits are commonly reflected
in the price. It is essential to deter-
mine the requirements of the measure-
ment situation and objectives for use.
Evaluation of a given instrument in
terms of sensitivity, response time,
portability, stability, service
characteristics, degree of automation,
and consistency are used for judgment
on a cost/benefit basis to select the
most acceptable unit
B Variables Affecting Electronic DO
Measurement
1 Temperature affects the solubility of
oxygen, the magnitude of the resulting
signal and the permeability of the
protective membrane. A curve of
oxygen solubility in water versus
increasing temperature may be concave
downward while a similar curve of
sensor response versus temperature
is concave upward. Increasing
temperature decreases oxygen solubility
and increases probe sensitivity and
membrane permeability. Thermistor
actuated compensation of probe
response based upon a linear relation-
ship or average of oxygen solubility
and electrode sensitivity is not precisely
correct as the maximum spread in
curvature occurs at about 17° C with
lower deviations from linearity above
or below that temperature. If the
instrument is calibrated at a temperature
within + or - 5°C of working temperature,
the compensated readout is likely to be
within 2% of the real value. Depending
upon probe geometry, the laboratory
sensor may require 4 to 6% correction
of signal per o c change in liquid
temperature.
2 Increasing pressure tends to increase
electrode response by compression
and contact effects upon the electrolyte,
dissolved gases and electrode surfaces.
As long as entrained gases are not
contained in the electrolyte or under
the membrane, these effects are
negligible.
Inclusion of entrained gases results in
erratic response that increases with
depth of immersion.
3 Electrode sensitivity changes occur as
a result of the nature and concentration
of contaminants at the electrode sur-
faces and possible physical chemical or
electronic side reactions produced.
These may take the form of a physical
barrier, internal short, high residual
current, or chemical changes in the
metal surface. The membrane is
intended to allow dissolved gas pene-
tration but to exclude passage of ions
or particulates. Apparently some ions
or materials producing extraneous ions
within the electrode vicinity are able
to pass in limited amounts which
16-4
-------
Dissolved Oxygen Determination - II
become significant in time. Dissolved
gases include 1) oxygen, 2) nitrogen,
3) carbon dioxide, 4) hydrogen sulfide,
and certain others. Item 4 is likely to
be a major problem. Item 3 may pro-
duce deposits in alkaline media, most
electrolytes are alkaline or tend to
become so in line with reaction n.A. 1.
The usable life of the sensor varies
with the type of electrode system,
surface area, amount of electrolyte
and type, membrane characteristics,
nature of the samples to which the
system is exposed and the length of
exposure. For example, galvanic
electrodes used in activated sludge
units showed that the time between
cleanup was 4 to 6 months for electrodes
used for intermittent daily checks of
effluent DO, continuous use in the mixed
liquor required electrode cleanup in 2
to 4 weeks. Each electrometric cell
configuration and operating mode has
its own response characteristics.
Some are more stable than others.
It is necessary to check calibration
frequency required under conditions
of use as none of them will maintain
uniform response indefinitely. Cali-
bration before and after daily use is
advisable.
4 Electrolytes may consist of solutions
or gels of ionizable materials such as
acids, alkalies or salts. Bicarbonates,
KC1 and KI are frequently used. The
electrolyte is the transfer and reaction
media, hence, it necessarily becomes
contaminated before damage to the
electrode surface may occur. Electro-
lyte concentration, nature, amount and
quality affect response time, sensitivity,
stability, and specificity of the sensor
system. Generally a small quantity of
electrolyte gives a shorter response
time and higher sensitivity but also may
be affected to a greater extent by a
given quantity of contaminating sub-
stances.
5 Membranes may consist of teflon,
polyethylene, rubber, and certain
other polymeric films. Thickness
may vary from 0.5 to 3 mils (inches X
1/1000). A thinner membrane will
decrease response time and increase
sensitivity but is less selective and
may be ruptured more easily. The
choice of material and its uniformity
affects response time, selectivity and
durability. The area of the membrane
and its permeability are directly
related to the quantity of transported
materials that may produce a signal.
The permeability of the membrane
material is related to temperature and
to residues accummulated on the
membrane surface or interior. A
cloudy membrane usually indicates
deposition and more or less loss of
signal.
6 Test media characteristics control the
interval of usable life between cleaning
and rejuvenation for any type of
electrode. More frequent cleanup is
essential in low quality waters than for
high quality waters. Reduced sulfur
compounds are among the more
troublesome contaminants. Salinity
affects the partial pressure of oxygen
at any given temperature. This effect
is small compared to most other
variables but is significant if salinity
changes by more than 500 mg/1.
7 Agitation of the sample in the vicinity
of the electrode is important because
DO is reduced at the cathode. Under
quiescent conditions a gradient in
dissolved oxygen content would be
established on the sample side of the
membrane as well as on the electrode
side, resulting in atypical response.
The sample should be agitated
sufficiently to deliver a representative
portion of the main body of the liquid
to the outer face of the membrane.
It is commonly observed that no
agitation will result m a very low or
negigible response after a short period
of time. Increasing agitation will cause
the response to rise gradually until
some minimum liquid velocity is reached
that will not cause a further increase
in response with increased mixing
energy. It is important to check
mixing velocity to reach a stable high
signal that is independent of a reasonable
change m sample mixing. Excessive
16-5
-------
Dissolved Oxygen Determination - n
mixing may create a vortex and expose
the sensing surface to air rather than
sample liquid. This should be avoided.
A linear liquid velocity of about 1 ft/sec
at the sensing surface is usually
adequate.
8 DO sensor response represents a
potential or current signal in the
milli-volt or milli-amp range in a
high resistance system. A high quality
electronic instrument is essential to
maintain a usable signal-to-noise ratio.
Some of the more common difficulties
include.
a Variable line voltage or low batteries
m amplifier power circuits.
b Substandard or unsteady amplifier
or resistor components.
c Undependable contacts or junctions
in the sensor, connecting cables, or
instrument control circuits.
d Inadequately shielded electronic
components.
e Excessive exposure to moisture,
fumes or chemicals m the wrong
places lead to stray currents,
internal shorts or other malfunction.
C Desirable Features in a Portable DO
Analyzer
1 The unit should include steady state
performance electronic and indicating
components in a convenient but sturdy
package that is small enough to carry.
2 There should be provisions for addition
of special accessories such as bottle
or field sensors, agitators, recorders,
line extensions, if needed for specific
requirements. Such additions should
be readily attachable and detachable
and maintain good working characteristics.
3 The instrument should include a
sensitivity adjustment which upon
calibration will provide for direct
reading in terms of mg of DO/liter.
4 Temperature compensation and temp-
erature readout should be incorporated.
5 Plug in contacts should be positive,
sturdy, readily cleanable and situated
to minimize contamination. Water
seals should be provided where
necessary.
6 The sensor should be suitably designed
for the purpose intended in terms of
sensitivity, response, stability, and
protection during use. It should be
easy to clean, and reassemble for use
with a minimum loss of service time.
7 Switches, connecting plugs, and con-
tacts preferably should be located on
or in the instrument box rather than
at the "wet" end of the line near the
sensor. Connecting cables should be
multiple strand to minimize separate
lines. Calibration controls should be
convenient but designed so that it is
not likely that they will be inadvertently
shifted during use.
8 Agitator accessories for bottle use
impose special problems because they
should be small, self contained, and
readily detachable but sturdy enough
to give positive agitation and electrical
continuity in a wet zone.
9 Major load batteries should be
rechargeable or readily replaceable.
Lane operation should be feasible
wherever possible
10 Service and replacement parts avail-
ability are a primary consideration.
Drawings, parts identification and
trouble shooting memos should be
incorporated with applicable operating
instructions in the instrument manual
in an informative organized form.
D Sensor and Instrument Calibration
The instrument box is likely to have some
form of check to verify electronics,
battery or other power supply conditions
for use. The sensor commonly is not
included in this check. A known reference
16-6
-------
Dissolved Oxygen Determination - II
sample used with the instrument in an
operating mode is the best available
method to compensate for sensor variables
under use conditions. It is advisable to
calibrate before and after daily use under
test conditions. Severe conditions,changes
in conditions, or possible damage call for
calibrations during the use period. The
readout scale is likely to be labeled -
calibration is the basis for this label.
The following procedure is recommended'
1 Turn the instrument on and allow it to
reach a stable condition. Perform the
recommended instrument check as
outlined in the operating manual.
2 The instrument check usually includes
an electronic zero correction. Check
each instrument against the readout
scale with the sensor immersed in an
agitated solution of sodium sulfite
containing sufficient cobalt chloride to
catalyze the reaction of sulfite and
oxygen. The indicator should stabilize
on the zero reading. If it does not, it
may be the result of residual or stray
currents, internal shorting in the
electrode, or membrane rupture.
Minor adjustments may be made using
the indicator rather than the electronic
controls. Serious imbalance requires
electrode reconditioning if the electronic
check is O. K. Sulfite must be carefully
rinsed from the sensor until the readout
stabilizes to prevent carry over to the
next sample.
3 Fill two DO bottles with replicate
samples of clarified water similar to
that to be tested. This water should
not contain significant test interferences.
4 Determine the DO in one by the azide
modification of the lodometric titration.
5 Insert a magnetic stirrer in the other
bottle or use a probe agitator. Start
agitation after insertion of the sensor
assembly and note the point of
stabilization.
a Adjust the instrument calibration
control if necessary to compare
with the titrated DO.
b If sensitivity adjustment is not
possible, note the instrument
stabilization point and designate
it as ua. A sensitivity coefficient,
(ji is equal to jjq where DO is the
titrated value for the sample on
which ua was obtained. An unknown
DO then becomes DO = — . This
factor is applicable as long as the
sensitivity does not change.
Objectives of the test program and the
type of instrument influence calibration
requirements. Precise work may
require calibration at 3 points in the
DO range of interest instead of at zero
and high range DO. One calibration
point frequently may be adequate.
Calibration of a DO sensor in air is a
quick test for possible changes in
sensor response. The difference in
oxygen content of air and of water is
too large for air calibration to be
satisfactory for precise calibration
for use in water.
IV This section reviews characteristics of
several sample laboratory instruments.
Mention of a SDecific instrument does not
imply FWQA endorsement or recommendation.
No attempt has been made to include all the
available instruments, those described are
used to indicate the approach used at one
stage of development which may or may not
represent the current available model.
A The electrode described by Carrit and
Kanwisher (1) is illustrated in Figure 3.
This electrode was an early example of
those using a membrane. The anode was
a silver - silver oxide reference cell with
a platinum disc cathode {1-3 cm diameter).
The salt bridge consisted of N/2 KC1 and
16-7
-------
Dissolved Oxygen Determination - n
KOH. The polyethylene membrane was
held in place by a retaining ring. An
applied current was used in a polarographic
mode. Temperature effects were relatively
large. Thermistor correction was studied
but not integrated with early models.
Retaining Ring
Polyethylene Film
Sliver Ring
Platinum Dlek Electrolyte Layer
Figure 3
B The Beckman oxygen electrode is another
illustration of a polarographic DO sensor
(Figure 4). It cortsists of a gold cathode,
a silver anode, an electrolytic gel con-
taining KC1, covered by a teflon membrane.
The instrument has a temperature readout
and compensating thermistor, a source
polarizing current, amplifier with signal
adjustment and a readout DO scale with
recorder contacts.
SENSOR
-/ f-
-t h
ElECTRONICS
M'll'IM
Mm-1
¦0^
urc'ioiTtt cti
M'lO
IHVf • ANODl
V
Y
010 CAlHOOl
Figure 4, THE BECKMAN OXYGEN
SENSOR
C The YSI Model 51 (3) is illustrated in
Figure 5. This is another form of
polarographic DO analyzer. The cell
consists of a silver anode coil, a gold
ring cathode and a KC1 electrolyte with
a teflon membrane The instrument has
a sensitivity adjustment, temperature and
DO readout. The model 51 A has temp-
erature compensation via manual preset
dial. A field probe and bottle probe are
available
YSI Model 51 DO Se
'O' Ring
Membrane
KCL Solution
Anode Coll
Cathode Ring
Figure 5
D The Model 54 YSI DO analyzer (4) is based
upon the same electrode configuration but
modified to include automatic temperature
compensation, DO readout, and recorder
jacks. A motorized agitator bottle probe
is available for the Model 54 (Figure 6)
Til Mod«l 34 Agitator Probi
Agitator
Mtmb
Agitator ling
16-8
-------
Dissolved Oxygen Determination - n
The Galvanic Cell Oxygen Analyzer (7, 8)
employs an indicator for proportional DO
signal but does not include thermistor
compensation or signal adjustment.
Temperature readout is provided. The
sensor includes a lead anode ring, and
a silver cathode with KOH electrolyte
(4 molar) covered by a membrane film
(Figure 7).
Precision OalvonU C • 11 Oxyp«n Prob#
Silvtr Cathodi
PolytthyUna M*mbran«
Thermistor Cobl
Rttainir
Tap*r«d Section
to Pit BOO BottUs
Ploiflc Mambrqne
Ring
F The Weston and Stack Model 300 DO
Analyzer (8) has a galvanic type sensor
with a pulsed current amplifier adjustment
to provide for signal and temperature
compensation. DO and temperature
readout is provided. The main power
supply is a rechargeable battery. The
sensor (Figure 8) consists of a lead anode
coil recessed in the electrolyte cavity
(50% KI) with a platinum cathode in the tip.
The sensor is covered with a teflon mem-
brane. Membrane retention by rubber
band or by a plastic retention ring may be
used for the bottle agitator or depth
sampler respectively. The thermistor
and agitator are mounted in a sleeve that
also provides protection for the membrane.
G The EIL Model 15 A sensor is illustrated
in Figure 9. This is a galvanic cell with
thermistor activated temperature com-
psnsation and readout. Signal adjustment
is provided. The illustration shows an
expanded scheme of the electrode which
when assembled compresses into a sensor
approximating 5/8 inch diameter and 4 inch
length exclusive of the enlargement at the
upper end. The anode consists of com-
pressed lead shot in a replaceable capsule
(later models used fine lead wire coils),
a perforated silver cathode sleeve around
the lead is covered by a membrane film.
The electrolyte is saturated potassium
bicarbonate. The large area of lead
surface, silver and membrane provides
a current response of 200 to 300 micro-
amperes in oxygen saturated water at
20° C for periods of up to 100 days use (8).
The larger electrode displacement favors
a scheme described by Eden (9) for
successive DO readings for BOD purposes.
V Table 1 summarizes major characteristics
of the sample DO analyzers described in
Section IV. It must be noted that an ingenious
analyst may adapt any one of these for special
purposes on a do-it-yourself program. The
sample instruments are mainly designed for
laboratory or portable field use. Those
designed for field monitoring purposes may
include similar designs or alternate designs
generally employing larger anode, cathode,
and electrolyte capacity to approach better
response stability with some sacrifice in
response time and sensitivity. The electronic
controls, recording, telemetering, and
accessory apparatus generally are semi-
permanent installations of a complex nature.
ACKNOWLEDGMENTS
This outline contains certain materials from
previous outlines by D. G. Ballinger,
N. C. Malof, and J.W. Mandia. Additional
information was provided by C.R. Hirth,
C.N. Shadix, D.F. Krawczyk, J. Woods,
and others.
16-9
-------
Dissolved Oxygen Determination - II
WESTON & STACK
DO PROBE
©—
0—
®—
(D-
d>-
0-
(D—
-------
Model A1SA ELECTRODE COMPONENT PARTS
Cable
Connection
Cover
A15016A
O' Ring
R524
Cable Sealing
Nut
A 15017
'O' Ring
R389
Cable
embrane— Securing
'O' Ring
R317
Lead Anode
Complete
(A15024A)
0^6=? -t
Anode
Contact
A 150140
(With Sleeve S24)
Anode
Contact
Holder
A15015A
O' Ring
R622
Membrane Securing
'O' Ring
R317
Silver Cathode
A15013A
Filler Screw
Z471
Mill
MINIMI.—
» 1 1 Mill Ml II III1—
•O' Ring
R612
O' Ring
R622
End Cap
A15011A
Note: Red wire of cable connects to Anode Contact Holder
Black wire of cable connects to Anode Contact
Membrane not shown E. I. L. part number T22
Figure 9
-------
Dissolved Oxygen Determination - II
TABLE 1
CHARACTERISTICS OF VARIOUS LABORATORY DO INSTRUMENTS
Anode
Cathode
Elec
Type
Membr
DO
Sig.
Ad]
Temp.
Comp
Temp. Rdg.
Accessories for
which designed
Carrit &
Kanwisher
silver-
silver ox.
ring
Pt
disc
KC1
KOH
N/2
pol
polyeth
no
no
Recording temp.
& signal ad], self
assembled
Beckman
Aq
ring
Au
disc
KCL
gel
pol
teflon
yes
yes
yes
recording
Yellow Springs
51
Ag
coil
Au
ring
KC1
soln
sat.
pol
teflon
yes
no*
yes
field and bottle
jprobe
Yellow Springs
54
m
ii
11
¦ II
ii
yes
yes
yes
recording field
bottle & agitator
probes
Precision
Sci
Pb
ring
silver
disc
KOH
4N
galv
polyeth
no
no
yes
Weston &
Stack
300
Pb
coil
Pt
disc
KI
40%
galv
teflon
yes
yes
yes
a git. probe
depth sampler
EIL
Pb
Ag
KHCOg
galv
teflon
yes
yes
yes
recording
Delta
75
Lead
Silver
disc
KOH
IN
galv
teflon
yes
yes
no
field bottle &
agitator probe
Delta
85
Lead
Silver
disc
KOH
IN
galv
teflon
yes
yes
yes
field bottle &
agitator probe
*Pol - Polarographic (or amperimetric)
Galv - Galvanic (or voltametric)
REFERENCES
1 Carrit, D. E and Kanwisher, J.W.
Anal. Chem 31:5. 1959.
2 Beckman Instrument Company. Bulletin
7015, A Dissolved Oxygen Primer,
Fuller-ton, CA. 1962.
3 Instructions for the YSI Model 51 Oxygen
Meter, Yellow Springs Instrument
Company, Yellow Springs, OH 45387.
4 Instructions for the YSI Model 54 Oxygen
Meter, Yellow Springs Instrument
Company, Yellow Springs, OH 45387.
5 Technical Bulletin TS-68850 Precision
Scientific Company, Chicago, IL 60647
6 Mancy, K.H., Okun, D.A and Reilley,
C.N. J. Electroanal. Chem. 4.65,
1962.
7 Instruction Bulletin, Weston and Stack
Model 300 Oxygen Analyzer. Roy F.
Weston, West Chester, PA 19380.
8 Briggs, R and Viney, M. Design and
Performance of Temperature Com-
pensated Electrodes for Oxygen
Measurements Jour, of Sci.
Instruments 41 78-83. 1964.
16-12
-------
Dissolved Oxygen Determination - II
9 Eden, R.E. BOD Determinations Using
a Dissolved Oxygen Meter. Water
Pollution Control, pp. 537-539. 1967.
10 Skoog, D.A. and West, D.M. Fundamentals
of Analytical Chemistry. Holt,
Rinehart & Winston, Inc. 1966.
11 FWPCA Methods for Chemical Analysis of
Water and Wastes. FWPCA Div. of
Water Quality Research Analytical
Quality Control Laboratory, Cincinnati,
OH. p. 65-68. November 1969.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, D^TB MDS,
Environmental Protection Agency, OWP,
Cincinnati, OH 45288 and Nate Malof, Chemist
National Field Investigations Center,
Environmental Protection Agency, WPO,
Cincinnati, OH.
16-13
-------
BIOCHEMICAL OXYGEN DEMAND TEST PROCEDURES
I OXYGEN DEMAND OF POLLUTED
WATERS
Established practice includes common use
of the BOD test as a tool for estimation of
the bio-oxidizable fraction of surface waters
or wastewaters discharged to them. Any
index including a quantity per unit time such
as the BODg is a rate expression. The
ultimate demand is more important than any
one point on the progression. The results
of a bottle test with minimum seeding and
quiescent storage are not likely to be as
high as those on the same influent in a mix-
ing situation and abundant seed of secondary
treatment or receiving waters. The BOD
is "a" fraction of total oxygen requirements.
A The particular technique used for BOD
commonly is specified by State agencies
and/or supervisors. They are required
to interpret the results as obtained by
laboratory testing. It is essential that
the tester and the interpreters have a
common understanding of what was done
and how. It is highly advisable to main-
tain a given routine until all concerned
agree upon a change.
1 Each particular routine has many un-
definable factors. The particular
routine is not as important as the con-
sistency and capability with which the
result was obtained.
2 This outline and Standard Methods^
discusses several valid approaches for
obtaining BOD results. Selection of
"method" is not mtended in this outline
or in the EPA Methods Manual^2 \
B The common 5-day incubation period for
BOD testing is a result of tradition and
cost. Initial lags are likely to be over
and some unknown fraction of the total
oxidizable mass has been satisfied after
5 days
C A series of observations over a period of
time makes it possible to estimate the
total oxidizable mass and the fraction
oxidized or remaining to be oxidized at
any given time The problem is to define
the shape of the deoxygenation pattern and
its limits A fair estimate of the shape of
the deoxygenation pattern is available by
observations at 1, 2, or 3 days, 7 days
and 14 days. Increased observations are
desirable for more valid estimates of
curve shape, rate of oxidation and total
oxidizable mass or ultimate BOD
D Increasing impoundment of surface waters
and concurrent increases in complexity
and stability of wastewater components
emphasize the importance of long-term
observation of BOD. The 5-day observation
includes most of the readily oxidizable
materials but a very small fraction of the
stable components that are the main factors
in impoundment behavior
II DIRECT METHOD
A With relatively clean surface waters, the
BOD may be determined by incubation of
the undiluted sample for the prescribed
time interval. This method is applicable
only to those waters whose BOD is less
than 8 mg/1 and assumes the sample
contains suitable organisms and accessory
nutrients for optimum biological
stabilization.
B Treated effluents, polluted surface waters,
household and industrial wastewaters
commonly require dilution to provide the
excess oxygen required for the oxygen
demand determination General guidelines
for dilution requirements for a given BOD
range in terms of the percent of sample in
BOD dilution water are
For a 5-day BOD of
5-20 mg 11, use 25 to 100% sample
For a BOD of
20-100 mg/1, use 5 to 25% sample
For a BOD of
100-500 mg/1, use 1 to 5% sample
For a BOD of
500-5000 mg/1, use 0 1 to 1 0% sample
III PROCEDURES
A Cylinder Dilution Technique
CH.O.bod. 57g. 12.71
17-1
-------
Biochemical Oxygen Demand Test Procedures
1 Using an assumed or estimated BOD
value as a guide, calculate the factors
for a range of dilutions to cover the
desired depletions. Those dilutions
ranging from a depletion of 2 mgl 1
and a residual of 1 mgl 1 are most
reliable At least three dilutions in
duplicate should be used for an
unknown sample.
2 Into a one-liter graduate cylinder (or
larger container if necessary) measure
accurately the required amount of mixed
sample to give one liter of diluted waste
Fill to the on? liter mark with dilution
water..Carefully mix The initial DO by
calculation includes IDOD (VIII) a
determined initial does not. Both are
essential to estimate significance of
IDOD. Entrapment of air bubbles during
manipulation must be avoided.
3 Siphon the mixture from the cylinder into
three 300 ml glass stoppered bottles,
filling the bottles to overflowing
4 Determine the DO concentration on one
of the bottles by the appropriate
Winkler modification and record as
"Initial DO".
5 Incubate the two remaining bottles at
20°C in complete darkness The
incubated bottles should be water-sealed
by immersion in a tray or by using a
special water-seal bottle.
6 After 5 days of incubation, or other
desired interval, determine the DO on
the bottles. Average the DO concentration
of the duplicates and report as "Final DO".
B Direct Dilution Technique
1 It may be more convenient to make the
dilution directly in sample bottles of
known capacity. A measured volume of
sample may be added (as indicated in
A-l) and the bottle filled with dilution
water to make the desired sample
concentration for incubation In this
case, the sample must be precisely
measured, the bottle carefully filled,
but not overfilled, and the bottle volumes
comparable and known. Precision is
likely to be poorer than for cylinder
dilution.
2 Continue the procedure as in A-4, 5,
and 6
C Seeded Cylinder Dilution Technique
1 Many wastewaters may be partially or
completely sterile as a result of
chlorination, effects of other toxic
chemicals, heat, unfavorable pH or
other factors detrimental to biological
activity Validity of the BOD result
depends upon the presence of organisms
capable of prompt and effective bio-
degradation and favorable conditions
during the particular test Correction
of the cause resulting in sterilization
must be corrected by adjustment,
dilution, etc., prior to reinoculation to
achieve meaningful BOD data Receiving
water, biologically treated effluents, and
soil suspensions are a good source of
organisms likely to be adapted for
stabilization of wastewaters. Untreated
wastewaters provide numerous organisms
but are likely to contain nutrients
contributing to excessive seed corrections
and may require appreciable time for
adaptation before test waste oxidation
becomes significant
2 The amount of added inoculant must be
determined by trial. The concentration
added should initiate biochemical
activity promptly but should not exert
enough oxygen demand to unduly reduce
the oxygen available for sample
requirements.
3 Estimate the sample concentration
desired in accordance with A-l and
C-2 and add the sample aliquot to the
dilution cylinder
4 Add approximately half of the required
amount of dilution water to the sample
and mix. This is necessary to assure
that the concentrated waste does not
exert a toxic effect on the seed organisms
5 Measure a suitable aliquot of seed into
the bottle or cylinder and fill with
dilution water Mix the combined sample,
seed and dilution water without excessive
air entrainment
6 Continue as in III-A steps 4, 5, and 6
IV INTERPRETATION OF RESULTS
Standard Methods^ ^ includes a calculation
section that is valid and concise. Preceding
it are details of reagent preparation and
17-2
-------
Biochemical Oxygen Demand Test Procedures
procedures for the test. These will not be
reprinted here. This section considers
certain items that may cause concern about
the validity of results unless they are care-
fully considered and controlled.
A The initial DO of the BOD test obviously
should be high. The method of attaining
a high DO can trap the analyst.
1 Aeration of dilution water is the most
commonly considered treatment.
This technique does produce a high DO
but it is a treacherous ally.
a Dirty air passing through clean
dilution water can produce clean
air and dirty water. This is a
simple air-washing operation.
Filtering the entering air stream
may remove brickbats and 2 x 4's,
but filters tend to pass organic
gases, fine aerosols, and partic-
ulates.
b A stream of air passing through
water tends to cool the water by
o -
evaporation 1 to 3 C below ambient
temperature. The cooled liquid
picks up more DO than it can hold
at ambient temperature. The
physical loss of oxygen may produce
an erroneously high depletion value
for a determined initial DO, or a
low depletion on a calculated
initial DO. Erroneous blanks are
a particular concern. The dilution
water temperature/DO shift is
critical.
2 Raising DO by allowing the sample to
equilibrate in a cotton-plugged bottle
for 2 or 3 days permits oxygenation
with minimum air volume contact.
3 Shaking a partially filled bottle for a
few seconds also oxygenates with min-
imum opportunity of gas washing con-
tamination, supersaturation, or
temperature changes.
B Seeding always is a precarious procedure
but a very necessary one at times. Often
the application of seed corrections is a
" if you do, if you don't"
situation. Hopefully, seed corrections
are small because each individual
biological situation is a "universe" of
its own.
1 Unstable seeding materials such as
fresh wastewater have "seed" organisms
characteristic of their origin and
history Saprophytes resulting in
surface water stabilization may be a
small fraction of the population. Re-
actable oxygen-demanding components
produce excessive demands upon test
oxygen resources.
2 A seed containing viable organisms at
a lower energy state because of limited
nutritional availability theoretically is
the best available seed source. An
organism population grown under
similar conditions should be most
effective for initiating biochemical
activity as soon as the nutrient situation
favors more activity. The population
should not be stored too long because
organism redistribution and die-out
become limiting. This type of seed
would most likely be found in a surface
water or a treatment plant effluent
with a history of receiving the particular
material under consideration.
3 Seed sources and amounts can only be
evaluated by trial. Different seed
sources and locations require checkout
to determine the best available material
from a standpoint of rapid initiation of
activity, low correction, and predictable
high oxygen depletion under test.
C Chlorination and BOD results fundamentally
are incompatible. Chlorination objectives
include disinfection as the number one
goal. Chlorine is notoriously non-specific
in organism effects. Chlorine acts like
an oxidant in the DO determination. Test
organisms are less suitable for activity
than they were before chlorination.
Nutrients may be less available after
chlorination. Certainly the conditions are
less suitable for biological response after
chlorination. Dechlorination is feasible
with respect to the oxidizing power of
17-3
-------
Biochemical Oxygen Demand Test Procedures
chlorine, but many organic chlorine com-
pounds that do not show strong oxidizing
action still have toxic effects on biologic
response.
Numbers are obtainable after dechlorination
and reseeding. The meaning of these
numbers is obscure. At least two states
(New York and New Jersey) specify BOD's
before chlorination only.
V PRECISION OF THE BOD TEST
A The DO test precision often has been used
to suggest precision ol the BOD result
DO precision is a relatively minor and
controllable factor contributing to BOD
results. Other factors such as organism
suitability, members, adaptation and
conditional variables are much more
difficult to control or to evaluate.
B The Analytical Reference Service report
on Water Oxygen Demand, July, 1960
(Sample type VII) included the results of
seeded samples of glucose-glutamic acid
BOD results from 34 agencies on 2, 3, 5
and 7 day inrubations.
The relative geometric standard deviation
(average) was 19% on 2% sample and 24%
on 1% sample concentrations. Rate
coefficients ranged from 0 10 to 0. 27 with
a median of 0. 16 from 21 different
laboratories that participated in rate studies
VI ALTERNATE BOD TECHNIQUES
Reaeration methods are becoming increasingly
popular in order to approach more nearly the
actual waste concentration in the receiving
water It is common to obtain "sliding" BOD
results related to the concentration of waste
in a series of dilutions of the same sample.
This may result from greater possibilities
for toxic effects at higher concentrations, or
to a different selection of organisms and change
in oxidation characteristics at low concentrations
of sample The most reliable estimate of stream
behavior is likely to be from that dilution closest
to the wastewater dilution in the receiving water.
A Reaeration can be accomplished by the
usual series techniques by dumping all of
the remaining sealed bottles into a common
container when the residual DO reaches about
1. 0 mg/ 1 After reoxygenation, the remaining
bottles are refilled and a new initial DO
determined. Subsequent dissolved oxygen
depletions are added incrementally as a
summation of the total oxygen depletion
from the start of the test If necessary,
the reaeration technique may be performed
several times but at a sacrifice of double
DO determinations for each day on which
reaeration occurs
B Special methods of reaeration have evolved
to minimize the extra manipulation for
reaeration of individual sample dilutions.
1 Elmore Method
A relatively large volume of the sample
is stored in an unsealed bottle. Small
bottles are withdrawn in sets of 5 or
more, sealed, incubated, and the DO
determined at appropriate intervals
When the DO concentration in the smaller
bottles reaches 1. 0 mg/1, a new set is
withdrawn from the large unsealed
bottle, after reoxygenation if necessary
2 Orford Method
The deoxygenation is carried out in a
large sealed jug from which samples
for DO are withdrawn at appropriate
intervals. To maintain the waste level
and a sufficient DO in the jug, additional
waste is added from a second open
container See diagram
C Excess oxygen may be provided by
oxygenation with commercial oxygen
instead of with air to increase the initial
oxygen content for incubation while limiting
the number of dilutions or reaeration steps.
When oxygen is used in place of air the
oxygen saturation in water at 20°C is about
40 mg/1 instead of 9 mg/ 1 Limited results
are available hence the analyst must verify
his technique The DO tends to decrease
as soon as the bottle is opened hence, about
35 mg/1 of oxygen content is the top of the
practical working concentration There has
been no evidence that the biota is inhibited
by the higher oxygen content with respect
to BOD progression
D Reaeration or Oxygenation Advantages and
Limitations
1 Reaeration expands the range of BOD
results obtainable directly at field
concentrations, but is not advisable for
applications when the sample BOD
exceeds 50 mg/1
17-4
-------
Biochemical Oxygen Demand Test Procedures
Dilution water problems are eliminated,
to the extent that the stream sample may-
be tested without dilution.
Incubator storage space becomes a real
problem for multiple sample routine.
VII Dissolved oxygen electrodes, polarographic
and others, are feasible for use in BOD
determinations, often making it possible to
make an estimate of DO or BOD when sample
interference prevents a valid Winkler DO
determination
Electronic probe DO makes it possible to
determine many successive DO's at different
time intervals on the same bottle with
negligible sample loss. Reaeration or
extended time series, therefore, are more
feasible.
Another outline in this series describes
response of reaerated BOD,, with electronic
DO probes.
It is the responsibility of the analyst to
evaluate
1 Applicability of the specified technique
and sample.
2 To determine requirements for mixing
and possible thermal effects while
mixing in terms of instrument response
and biochemical reaction.
3 To evaluate long-term calibration or
standardization and their effects upon
precision and accuracy of the BOD
result.
VIII IMMEDIATE DISSOLVED OXYGEN
DEMAND (IDOD)
Immediate dissolved oxygen demand includes
dissolved oxygen utilization requirements of
substances such as those in I-A-3 An
arbitrary period of 15 min. has been selected
to distinguish IDOD from BOD.
REAERATION METHODS FOR B.O.D. DETERMINATION
reservoir
A A
AAA AA
sealed bottles
ELMORE METHOD
reservoir
sealed jug
AAAAAA
d.o. samples
OR FORD METHOD
17-5
-------
Biochemical Oxygen Demand Test Procedures
A The IDOD is an apparent response as
indicated by a specified technique Since
DO titration is based upon iodine titration,
any factor that causes Io response different
from that produced by tne reaction of KI
and molecular oxygen confuses the IDOD
determination.
B IDOD Determination
1 The IDOD determination includes the
determination of DO on a sample and
dilution water separately. A waste
likely to have a significant IDOD is
unlikely to show a DO.
2 According to mixing theory, it should
be possible to calculate the DO of any
definite mixture of the sample and
dilution water from the DO of component
parts and their proportion.
(See IV. E. 1).
3 The same relative proportions of sample
and dilution water should be mixed
without air entrainment and the DO
determined after 15 minutes.
4 Any difference between the calculated
initial DO as obtained in VTII-B-2, and
the determined DO in VIII-B-3 may be
designated as IDOD.
5 Sample aeration, DO interference, and
other factors confuse results for IDOD.
C Sample Calculation of IDOD
1 Sample DO checked and shown to
be 0. 0 mg/1
Dilution water DO found to be 8. 2 mg/1
Assume a mixture of 9 parts of dilution
water and 1 part (V/V) of sample
Calculated DO =
1X0=0
9 X 8.2 = 73.8
10 parts of the mixture contain 73. 8110
or 7. 4 mg DO 11. Note that mixing has
reduced the DO concentration because
the original amount is present in a
larger package.
2 The mixture described in C-3 was held
for 15 minutes and the DO determined
was 4. 3 mg/1
IDOD = DO . - DO . . X m 1QQ,
calc detm "/c sample
used
= 7. 4 - 4 3X10
= 31 mg IDOD/1
Bibliography
1 Standard Methods, APHA-AWWA-WPCF
13th Ed. 1971.
2 Methods for Chemical Analysis of Water
and Wastes, EPA-AQCL. 1971.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, OWP, EPA, Cincinnati, OH 45268.
17-6
-------
BIOCHEMICAL OXYGEN DEMAND TEST
DILUTION TECHNIQUE
I OBJECTIVES: to prepare a sample dilution for series observation of oxygen depletion versus
time for the estimation of BOD^, rate and ultimate demand. The azide modification of the
Winkler lodometric titration of dissolved oxygen will be used. An electronic DO analyzer
will be used to check the titrimetric DO.
II PROCEDURE:
A Each group will prepare 1 dilution of either effluent or influent at an assigned concentration
to enable filling 8 standard BOD bottles. One of these will be used for electronic DO mon-
itoring by the lab instructor, the other seven are for daily student determinations. Identify
each bottle with group number, I for influent or E for effluent and the assigned concentration
in %.
B Calculate the volume of sample to be used to make up a total diluted volume of 3500 ml at
the assigned concentration. Then dilute the sample as follows:
1 Add distilled water to the dilution bottle to make up about 1/2 of the total amount of
distilled water to be used. Add 1 ml each of the four mineral supplements. (These
solutions are prepared at 4 times the concentration of Standard Methods mineral
supplement reagents so the added volume is lower). Mix the supplemented dilution
water thoroughly.
2 Carefully mix the bottle of sample to be used to a uniform suspension. If large chunks
are evident, homogenize. Rapidly pour out, with mixing, into a beaker, the approximate
amount of sample required and return to lab station. Rapidly measure out, with mixing,
the required sample aliquot to make the 3500 ml sample dilution.
Mixing, time, and precision are important. Your sample may be non-representative to
the extent of carelessness in mixing, allowing settling while adjusting measurement or
measurement errors.
Add the measured quantity to the dilution bottle.
3 Carefully add distilled water to make up 3500 ml of diluted sample.
4 Stopper and shake the dilution bottle to mix the sample and to ensure that you have an
acceptable initial DO. This step will add DO or reduce supersaturation. About 15
seconds of mixing is usually adequate.
C Filling bottles is simplified if you place the dilution bottle on the center pedestal, insert
the siphon and line up your identified bottles with stoppers removed. Have a container
larger than the bottles available to accommodate the overflow while filling them. Otherwise,
move the apparatus near a sink. Fill the siphon tube and waste some of the diluted sample
to rinse this tube. Be sure that the siphon tube is used in a manner so that it is always
full of the diluted sample. Agitate the diluted sample regularly to avoid undue collection of
solids on the bottom.
CH O.bod lab. 3.12.71
18-1
-------
Biochemical Oxygen Demand Test Dilution Technique
1 Place first bottle to be filled in the overflow container or hold over sink. Insert the
siphon delivery tip all of the way down into the bottom of the test bottle. Tilt the
bottle to cover the delivery tip with water promptly. Start the siphon flow slowly to
decrease turbulence until the delivery tip is well-covered. Fill to overflowing. Slowly
raise the delivery tip while overflowing the sample. Stop the flow with the bottle com-
pletely full. Insert the bottle stopper and turn it slightly to seat it. Fill the remaining
bottles in the same manner.
2 Each bottle will have the same DO and sample mix as the next if you have followed a
consistent procedure, mixed carefully, and have not changed technique enroute.
3 One of the eight bottles is to be retained for initial titration of DO by your group. Another
bottle is to be turned in to the lab instructor for use in performance checks on all class
dilutions. The remainder are to be placed into trays for 20° cincubation. Be certain
that all bottles are properly identified.
D Dissolved Oxygen Determination
1 You are encouraged to check the DO of your sample on the electronic analyzer. A
calibrated unit will be available with assistance. The DO analyzer will not result in
more than 1/2 ml loss of sample on probe insertion. You will be able to add DO reagents
and continue the Winkler titnmetric DO on the same bottle.
2 An initial DO value (DOq) using the Winkler method must be determined today Oh succeed-
ing days, the DO^ will be determined on the incubated samples for estimation of BOD
progression. (If reaeration is required, the technique will be demonstrated). All data
is to be recorded on the sheet provided in this manual.
The Winkler-Azide procedure as presented in outline 43 is to be used for these DO
determinations.
3 A blank value for each stock bottle of distilled water will be determined by the staff.
This outline wps prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, WPQ EPA, Cincinnati, OH 45268
18-2
-------
Biochemical Oxygen Demand Test Dilution Technique
BOD DATA SHEET
Group No. Initial Date
Buret
Readings
DO
o
Buret
Headings
DOt
ADO
Sf/ID
BOD
Buret readings - initial and final
DO - initial DO in mg/liter
o
DOt = DO at indicated incubation time ^
ADOa DO change (DOq - DOt>, a depletion
Sf = the sample content as a decimal fraction in the dilution
ID = sample identification (influent or effluent)
BOD= DOq - DOt (according to Standard Methods, 13th edition, 1971)
Sf
18-3
-------
BOD DETERMINATION - REAERATED BOTTLE PROBE TECHNIQUE
I INTRODUCTION
A Customary dilution technique has certain
limitations with respect to the BOD
determination such as a prior commit-
ment on sample dilutions to be used,
number of bottles to be included to permit
Winkler DO determinations to be made
throughout a predetermined test interval,
(with sample destruction for each DO test),
possibility for anomalous effects due to
dilution water, dilution, or inconsistent
response between test bottles.
B DO probe technique offers the advantages
of nondestructive DO testing, possibilities
of adjusting routine according to sample
behavior during test, and retaining the
sample with minor losses for long term
observation (if desired) and for supplemen-
tary tests at the end of the BOD test
interval.
Note: Use of the probe results m a loss
of about 1/2 ml of each 300 ml sample.
Use dilution water with a glass rod or
bead to bring the sample volume back to
300 ml for further testing.
C This test was performed in triplicate on
the same sample at 100% concentration
with reaeration as needed to illustrate
results obtainable.
H PROCEDURE
A Sample
Final effluent (catch sample at 1100 hours)
of the Advanced Waste Treatment Research
Activated Sludge (Unit B) Sanitary Engineer:
Center Experimental Wing (September 3, 1970)
One hundred percent concentration.
B DO Probe Calibration
The probe was calibrated daily in air
saturated tap water versus the azide
modification of the iodometric titration.
A replicate test bottle of the titrated
sample was retained for initial probe
adjustment, if necessary, and to recheck
sensor response at the end of the use
period. Zero sensor response in cobalt
treated sulfite solution was checked at
three day intervals. Electronic response
and battery condition were checked daily.
C Reaeration
1 When the DO test indicated 1. 0 mg
DO/liter or appeared likely to reach
that point prior to the next test reading
the test bottles were reaerated and a
new initial DO was obtained.
2 An adapter was inserted into the neck
of the sample bottle indicating low DO
and an empty bottle inserted on the
opposite end. The combination was
shaken vigorously, while in an inverted
position to allow the transfer of sample
to the empty bottle, immediately after
transfer the combination was reinverted,
shaken as before and sample returned
to the original bottle.
3 The sample was allowed to stand for
at least five minutes to allow entrained
air to rise (generally formed a froth
ring). The froth ring was raised into
the neck of the bottle by displacement
with about 1/2 ml of dilution water.
It usually was necessary to tilt the
bottle slightly and roll it to sweep
fine air bubbles off the shoulder of the
bottle and into the neck. After careful
reinsertion of the DO sensor these
bubbles were displaced by tipping the
assembly and discarding contents of
the BOD bottle lip containing the frothy
residue.
4 It is recognized that surface active
materials and possibly other components
would concentrate m the discarded froth.
It would be possible to limit this effect
CH. O. bod. lab. 2a. 12.71
19-1
-------
BOP Determination - Reaerated Bottle Probe Technique
by allowing a longer time for the froth
to break--say 15 to 30 minutes. This
was not attempted for this demonstration.
D No attempt was made to determine DO at
any regular time interval. Observation
time is recorded for each test on a
24-hour clock basis with zero time at
midnight.
E Results are recorded in tabular form
including the date (Column 1), time m
hours (2), sample temperature (3), hours
of incubation (4), and the triplicate sample
data. For each of the samples the
observed DO, the change since the last
observation ( ADO) and the summation of
all the DO depletions observed during
the incubated interval are recorded as
£ ADO. A bold face line in the tabulated
data in the DO column indicates DO before
reaeration, the number below the line
indicates DO after reaeration to be used
as a new initial for the next observation.
F Near the end of the fifth day of mcubation
(116 hours) one of the triplicates (no. 3)
was not reaerated to check the effect of
complete depletion of oxygen. It was
reaerated on the following day and thereafter.
G The results plotted in graphic form are
presented following the BOD tabular data.
H The results of a similar respiration test
on mixed liquor from the same activated
sludge units are tabulated to show the
effects of different types of feed on DO
depletion (respiratory activity). In this
case, time is in minutes, DO is in mg/1
and ADO/minute also is m mg/1. The
first test in Table 2 represented 100 ml
of return sludge plus plant effluent to fill
the bottle. Approximately one percent
nutrient agar solution was added by
syringe (1.0 ml) to produce the response
noted in the second series, one ml of five
percent mercuric chloride produced the
effects m series 3. A new replicate
sample (100 ml sludge) and plant influent
to fill the bottle produced the results m
series 4,
III SUMMARY
A Table 1 shows that it is possible to obtain
consistent results using a DO probe plus
reaeration for BOD technique. The
major requirement is to carefully calibrate
the DO instrument and sensor on a daily
basis. It is not recommended to reaerate
as many times as found necessary here.
It could have been diluted at any time
since the sample was available. Had
the BOD response at five days been
substantially complete only two
reaerations would have been necessary.
However, since the sample was working
into second stage BOD it proceeded to
react accordingly. The oxygen demand
of 15 mg/liter at five days increased to
30 mg/liter in seven days and reached
45 mg/liter in ten days. The sample that
was allowed to deplete on the fifth day
slowly recovered but by the twelfth day
or thereafter equaled or exceeded the
BOD of the samples with residual DO at
all times. This technique requires much
less incubator space, time and manip-
ulation with an added advantage of
retaining the sample for adjustment or
extended observation. The results do
not depend upon a preconceived guestimate
which may or may not fit the situation.
B Table 2 indicates the possibility for
estimating feed acceptability in an
activated I-'dge treatment plant. On
many occasions an inquiry from an
industrial plant, a different type of
inflow or perhaps a new plant requires
a decision regarding effects at the treat-
ment plant. A respiration test by probe
technique offers an opportunity to rate
the situation within ten minutes after
probe calibration. A test of sludge and
effluent oxygen demand gives an estimate
sludge respiratory activity now. When the
same sludge is subjected to a new feed of
an acceptable nature respiratory activity
rises as in series 2. If the feed happened
to be highly toxic as m series 3 (mercuric
chloride) respiratory activity stops- -
19-2
-------
BOD Determination - Reaerated Bottle Probe Technique
obviously a dead or severely shocked
condition. A replicate sludge (Series 4)
with the regular plant influent shows a
prompt increase in activity Oxygen
requirements, permissible load ratio,
acceptable feed to sludge ratios (return
sludge adjustment) all may be estimated
on the basis of DO tests related to overall
plant performance.
C The graphic of BOD versus time includes
the COD--an estimate of first stage
oxygen demand and the TKN for the
composite effluent sample. If the total
Kjeldahl nitrogen (TKN) is multiplied by
its oxygen equivalent (4. 57) then the COD
plus TKN times 4.57 gives an estimate
of ultimate oxygen demand (138 mg/1).
The twenty day BOD was about 55 X 100/138
or forty percent of this value. The effluent
sample obtained at 1100 hours is unlikely
to contain a significant portion of the high
load period considering the primary,
aerator and secondary clarifier detention.
Also the COD and TKN may have included
materials that were chemically oxidized
but gave a poor response to biological
oxidation The BOD gives the rapid
respiratory oxygen demand. COD plus
the oxygen equivalent of N (on the same
sample) gives a quick estimate of potential
ultimate demand.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, OWP, EPA, Cincinnati, OH 45268.
19-3
-------
BOD Determination - Reaerated Bottle Probe Technique
TABLE 1
BOD Tabulated Results - Reaerated Sample, Probe DO
9/3/70 100% Sample
Hrs.
Date
Time
Toc
Incub
DO
9/3/70
1340
24. 0
0
6.7
9/3/70
1355
24. 0
0.25
6 7
9/3/70
1620
22. 0
2.6
6 5
9/4/70
1010
20 2
20.5
4.9
8.2
9/4/70
1600
20.6
26 3
7.4
9/6/70
1340
19. 5
72
1.0
8. 1
9/8/70
0940
19. 8
116
0.6
8.6
9/9/70
1235
20. 0
143
1 1
8.2
9/10/70
1200
20 0
166
1 0
8.2
9/11/70
0800
20. 0
186
1 2
8.4
9/11/70
1400
20. 0
192
5.0
8. 1
9/12/70
2035
20. 0
222
0 5
8.2
9/13/70
1130
20 0
238
6.3
8 3
9/14/70
1200
to
o
o
262
7.0
9/15/70
1340
19.6
288
5 7
9/16/70
1200
19. 3
310
5. 1
* 9/20/70
1240
21.6
480
3. 8
Sample 1
ADO
0.2
1.6
0. 8
6.4
7.5
7 5
7.2
7.0
3. 4
7.6
1.9
1.3
1 3
0. 6
1.3
E ADO
0.2
1.8
2.6
9.0
16. 5
24 0
31.2
38 2
41.6
49 2
51 1
52. 4
53 7
54.3
55 6
Sample 2
DO
6.7
6.7
6.5
4.7
8.3
7.5
1 5
8 1
0 5
7 6
1. 1
8 2
1.2
8.6
1 1
8 3
5. 3
8 4
0.6
8.2
3. 8
8.4
6 9
6 2
5.8
4.6
ADO
0 2
1 8
0.8
6 0
7.6
6.5
7 0
7.5
3 0
7 8
4.4
1.5
0.7
0.4
1.2
£ ADO
0.2
2 0
2 8
8.8
16 4
22 9
29.9
37 4
40.4
48 2
52.6
53 1
53 8
54. 2
55 4
DO
6. 8
6 8
6.6
4. 8
7.9
6 9
1. 1
7 4
0. 4
0 0
7.6
2 0
8 0
2.0
8. 5
5.7
8. 6
0. 4
8 2
4 6
8. 3
0. 9
8. 3
4. 5
8. 2
7 2
4. 9
Sample 3
ADO
0.2.1.8
1. 0
5 8
7.0
0. 4
5.6
6 0
2. 8
8. 2
3 6
7 4
3.8
1. 0
2.3
* Incubator power off
19-4
-------
Ti
Mi
0
1
2
3
4
0
1
2
3
0
1
2
3
0
1
2
3
BOD Determination - Reaerated Bottle Probe Technique
TABLE 2
ACTIVATED SLUDGE RESPIRATION DATA DO PROBE TECHNIQUE
Temp
DO
ADO
Sample
Series 1
240
7. 2
100 ml Return Sludge & Effluent
6.6
0. 6
6.2
0. 4
5 6
0 6
24. 5
5 1
0 5
4. 7
Series 2
+ 1 ml Nutrient agar
3 8
0. 9
2.7
1 1
1. 7
1. 0
24 8
4. 7
Series 3
+ 1 ml HgCl_ Soln
4 8
Ct
4. 7
Nil
4. 7
CM
5 9
Series 4
100 ml Sludge + Influent
4 6
1. 3
3 3
1. 3
2.0
1.3
19-5
-------
GRAPHIC OF REAERATED PROBE BOD RESULTS
STARTING 9/3/70 SAMPLED 1100 HOURS
COMPOSITE SAMPLE RESULTS FOR 9/3/70
COD-69 mg/LITER
TKN-15 mg/LITER
ULTIMATE DEMAND 138 mg/LITER
23 DO DETERMINATIONS
1/2 ml DISPLACED PER
DETERMINATION
ml x 100
300 = 3.8%
SAMPLE ALLOWED TO DEPLETE
INCUBATION DAYS (hrs)
I I I I
8 (216) 10 (268) 12(312) 14
I
16
I
18
-------
EFFECT OF SOME VARIABLES ON THE BOD TEST
I TIME
—
A The common equation y^ = L (1 - 10 ) for
BOD relationship indicates time as a
variable. The rate coefficient (k^indicates
that a specific percentage of material
initially present (oxygen) will be used
during a given time unit. Each successive
unit of time has less reactant present
initially than the preceding interval, hence
a definite precentage decrease results in
successively smaller amounts of reactant
use per unit of lapsed time. Increasing
kj results in a larger percentage oxygen
use per unit of time and also increases the
change in reactant mass among successive
time intervals.
B Adney's work for the British Royal Com-
mission cited 5 days passage time from
source to the ocean as maximum for
English streams. The 8th Report (1909)
largely established BOD philosophy in-
cluding the 5-day interval. At 5 days,
initial lags generally have terminated and
a substantial fraction of the long-term
oxygen demand has been exerted. If only
one time interval can be used, 7 days
permits better scheduling. Any one time
interval is "a" fraction of the total oxygen
requirement, this is a poor reference
point if we do not know how it arrived.
For example, the percentage of
oxidizable material stabilized in terms
of oxygen use at various rate factors
are.
% oxidized
k< (log 10) in 5.days Kj (log )
0.05 42% 0.11
0. 10 67% 0.23
0. 15 84% 0. 34
0. 25 94% 0. 57
0. 50 99+% 1.15
This range (F^ =2.3 k^ is commonly en-
countered in wastewater stabilization with
the higher rates characteristic of fresh oxi-
dizable material that is readily converted.
The lower coefficients are characteristic
of cell mass at later stages of oxidation
and of low-rate reactants in general
C The oxygen utilization at specified inter-
vals of time are required to estimate kj,
and L, the estimate of oxygen use at
infinite time. It is common to observe
results at equal intervals of time but
this is not essential as long as
the time intervals are accurately known.
The initial time periods are critical as
an error of a few hours in time represents
a relatively large change in reactant mass
in a system at maximum instability. Un-
equal time periods can be plotted to define
the curve from which any given mtervals
can be selected as desired.
D Increasing impoundment of surface water
provides more time for stabilization of
relatively inert soluble or suspended
pollutants and for organism adaptation
to the situation or pollutants. Long term
BOD's are essential to indicate changes
in the pattern of oxygen demand vs. time.
It may be expected that one or more
plateaus will be evident in the BOD curve
followed by a temporary rise in rate
during second stage oxidation or thereafter.
Anaerobiosis may cause a rise in rate
coefficient after aerobic conditions are
re-established. Eventually k^ stabilizes
at very low values.
1 Rate coefficients tend to be difficult to
interpret during long term BOD's
because of progressive changes and
other factors.
a The relative error of the DO test may
be a large fraction of the incremental
DO change during low rate periods.
b Cell mass may agglomerate under
quiescent test conditions and decrease
nutrient availability.
CH. O. bod. 56d. 12.71
20-1
-------
Effect of Some Variables on the BOD Test
c It is not likely that recycled nutrients
under aerobic test conditions will o.
have as much effect as recycle from
anaerobic benthic deposits in a '
stream.
2 The BOD result tends to underestimate ^ 11
deoxygenation relative to surface water = !.*
behavior because of interchanges, ^ q"
-turbulence, biota, and boundary effects. o!
Reseeding does not occur in a sealed ^ Q
bottle but reseeding is inevitable in a J
stream or treatment unit. ^
FIGURE I
DECREES C
VARIATION IN "T WITH CHANCE IN TEMPERATURE
II TEMPERATURE
A Effect on Oxidation Rate
Temperature is one of the import^mt con-
trolling factors in any biological system.
In the BOD reaction, changes in tempera-
ture produce acceleration or depression
of the rate of oxidation. Figure 1 shows
the changes in the value of k at tempera-
tures from 0 - 25°C on a common
wastewater.
B Test Temperature
In the BOD test procedure an arbitrary
temperature is usually selected for
convenience even though a wide temperature
range exists under natural conditions.
Incubation of the test containers at 20°C
for the whole period is now accepted
practice in the U.S., 18. 5°C is preferred
in England. Camp (ASCE, SA5 91:1, Oct.
65) recommended light and dark bottle
immersion in the stream.
C Temperature Correction
When it is necessary to calculate the rate
of oxidation at a temperature other than
20°, the following relationship may be
used
. 6 (Ti - T2)
k2
where-
k^ = rate coefficient at temperature T^
kg = rate at coefficient at temperature
6 = temperature coefficient, for which
Streeter and Phelps obtained the value
1. 047. 0 changes with temperature, it
appears to be higher in the range of
5-15°C than in the range of 30 to 40°C.
The value given refers to 15-30°.
The cited temperature coefficient appears
reasonable for household wastes. It may
not apply for other wastes where developing
or seed organisms may not tolerate tem-
perature changes as readily. A given
temperature coefficient should be checked
for applicability under specified conditions.
Ill pH
A The organisms involved in biochemical
conversions apparently have an optimum
response near a pH of 7.0 providing other
environmental factors are favorable, a pH
range of about 6. 5 to 8. 3 apparently is
acceptable (Figure 2). Reactivity is likely
to be significantly lower on both sides of the
acceptable pH range but microbial adapta-
tion may extend the limits appreciably.
For example, trickling filters have operated
with better than 50% treatment efficiency
at pH 3 and 10 after adaptation.
20-2
-------
Effect of Some Variables on the BOD Test
100
o £
u >>
5
80
60
1 1
" 1 I
Figure 2
- *
• #\
X
•
. 1
i 1 i
4 6 pH 0 10
B Adjustment of Concentrated Samples
When wastes are more acid than pH 6. 5 or
more alkaline than pH 8.3, adjustment to
pH 7. 2 is advisable before reliable BOD
values can be obtained.
C Dilution Samples
Standard dilution water is buffered at pH
7.2. Sample-dilution water mixtures should
be checked to make sure that the sample
buffer capacity does not exceed the capacity
of the dilution water for pH adjustment.
B Standard Methods Dilution Water
The dilution water specified for the BOD
test approximates USGS estimates for an
average U.S. mineral content of surface
water except for added phosphate buffer.
It is assumed to provide essential mineral
nutrients for most wastewaters but cannot
be expected to meet requirements for
grossly deficient wastewater nutrients both
mineral and organic. Ruchhoft (S. W. J.
13 669, 1941) summarized committee action
leading to the present dilution water.
C Other Dilution Considerations
There is a trend toward the use of receiving
water, storage-stabilized if necessary, to
evaluate waste behavior. It is advisable
to minimize dilution and consider the
nutrient level likely in the receiving water
as most valid. Any change in the environ-
ment, such as dilution, upsets the
microbial balance and requires adaptive
changes.
IV ESSENTIA L MINERA L NUTRIENTS
A Importance
In 1932 Butterfield reported on the role of
certain minerals in the biochemical oxidation
of sewage and concluded that deficient
minerals often upset metabolic response.
In addition, he found that inadequate nitrogen
and/or phosphorus was a common cause of
low BOD results in industrial wastewaters.
(Figure 3)
Figure 3
Normal
r
Lacking N 4 P
Time in Days
Effect of Mineral Nutrients on BOD
V MICROBIOLOGICA L POPU LA TION
A Need for Complex Flora and Fauna
Butterfield, Purdy, and Theriault (Pub.
Health Rep. 393, 1931) demonstrated that
an isolated species of organisms was not
as effective in biological stabilization as
a variety of species. Figure 4 summarizes
some of their data. Bhatta and Gaudy
(ASCE, SA3, 91-63, June 1965) reinvestigated
this factor. Many studies have emphasized
the need for a mixed biota in the BOD test.
It appears that bacteria are capable of
varied activities, but all species are not
capable of synthesizing all required nutrients.
Certain bacterial species may be capable
of producing enzymes, amino acids, or
growth factors needed for their use and by
other species for optimum performance.
It has been shown that oxygen demand
becomes minimal when some limit of
bacterial population has been reached.
Predation prevents such an approach to
maximum numbers and maintains a con-
tinuing bacterial growth and recycle of
nutrients among a mixed population. The
net effect is a symbiotic relation among
mixed organisms tending to enhance the
rate of stabilization or utilization of
oxygen as in the BOD test.
20-3
-------
Effect of Some Variables on the BOD Test
B Organism Adaptation
1 Early investigations in relation to the
BOD test considered domestic wastewaters
primarily. The saprophytic organisms
involved in stabilization either were
present in adequate numbers or quickly
multiplied to attain effective populations.
2 The period of adjustment required to
shift enzyme production needed to utilize
an energy source different from that
previously utilized or to shift population
variety from that favored by one food to
that favored by another food is con-
sidered an adaptation period. Dilution,
temperature, oxygen tension, pH,
nutrient type, inhibitory substances,
light and other changes all are common
inducements for microbial adaptation.
Mutation of organisms may be encountered
during adaptation but usually is not a
factor.
3 The developments in industry and
technology have resulted in discharge
of new and more varied wastewater
constituents. Microorganisms may
adapt themselves to the use of a new
substance as an energy source providing
the energy and environment are favor-
able. The receiving stream usually shows
development of an adapted microbiota
for a new or different discharge con-
stituent within hours, days or weeks
after fairly regular discharge. The
time for adaptation depends on the nature
of the constituent, available energy,
tolerance of the organisms, and environ-
mental conditions.
C Seeding
The amount of seed and its selection must
be determined experimentally. The most
effective inoculant would be that which
would produce the maximum BOD response
with minimum lag period and negligible
seed demand. This would mean some
maximum population adapted to feed and
conditions at a minimum equilibrium energy
nutrient supply.
1 Figure 5 indicates corrected BOD
progression on a synthetic feed with
river water and stale sewage inoculants
at several concentrations. The river
water resulted in higher BOD with
negligible lag and seed correction. The
seed correction at 20% concentration
of inoculant was less than 0.3 mg.
DO/1 at 5 days. It would be possible to
use this river water as a diluent without
excessive oxygen loss to produce more
valid BOD progression for that receiving
water. The lower wastewater inoculant
concentration resulted in a definite BOD
lag. Higher wastewater concentrations
produced comparable BOD progression
earlier but resulted in high seed
corrections and lowered availability of
dissolved oxygen for the sample.
2 A good secondary treated effluent
produced results similar to river water
inoculation with higher seed corrections
per increment of applied inoculant.
Soil suspensions also are very effective
sources of seed organisms with minor
seed corrections if they are reasonably
stabilized surface soils.
3 It appears that the BOD progression
most nearly indicating receiving water
oxidation would be one based upon
receiving water dilution or inoculated
with organisms from it.
4 A new or unusual wastewater may
require adapted organisms not present
in sufficient numbers in the receiving
water. Development of an adapted seed
from soil suspensions, plant effluents
or receiving water may be necessary to
evaluate oxidation potential in a plant
or receiving water at some future time.
Enrichment culture technique is bene-
ficial where small concentrations of the
test wastewater are applied regularly
with increases in wastewater concen-
trations as BOD or respiration activity
indicates increasing tolerance and
oxidation of the test waste. Both time
and concentration limits are useful to
characterize the wastewater and its
acceptability for biological stabilization.
20-4
-------
Effect of Some Variables on the BOD Test
§
c
v
M
I
I ogend
AH forma in river water —
Mixed Dacteria K. plankton
Pure culture B Aerogenea * plankton
Mixed culture bacteria —
Pure culture B Aerogenes
0
5 6 7 8
I
3
4
10 II \2 U M
9
Time in Days
h ffet t of Biological Forma on Oxygen Depletion
Figure 4
DO DEPLETION VS SEED
CONCENTRATION 5 TYPE(2% glucose-glutamic ac)
RIVER WATER
STALE W W
0 5% STALE SEW -
0 2%
01%
1-20% RIVER WATER
DAYS
F igui i- i
5 It must be recognized that BOD
progressions are most likel} to err
on the low side. A meaningful BOD
test should seek the highest consistent
oxygen demand feasible for sample and
conditions.
D Algae
When large numbers of algae are present
in surface waters, they produce significant
changes in the oxygen content. Under the
influence of sunlight excess oxygen is
produced while a net deficit occurs in the
dark. The result is a wide variation in
surface water DO depending on sample
time.
Uhen stream samples containing algae are
incubated in the laboratory the algae
survive fot a time, then die because ol the
lack of light. Short-term BOD determina-
tions may show the influence of oxygen
production bj the algae. V\ hen the algae
die, they release the stored organic load
for recycle and increase the BOD. There-
fore, samples incubated in the dark may
not be representative of the deoxygenation
process in the stream, since the benefits
of photosynthesis are lacking. Conversely,
samples incubated m the light, under
conditions of continual photosynthesis,
will^ield low BOD values.
The influence of algae on BOD is one of
the most difficult variables to evaluate.
More research is needed to develop
satisfactory methods for the accurate
determination of BOD in the presence oi
lai ge numbers of algae. Light and dark
bottle incubations suggest the magnitude
of effects.
I\ TOMCIT\
A effect
Mnce satisfaction of the BOD is accom-
plished through the action of microorgan-
isms, the presence of toxic substances
will result in depression of the oxidation
rate. In man\ cases, toxicity will
ptoduce a lag period, until tolerant
organism activity becomes significant.
Figure 6 shows the effect of cyanide on the
BOD curve. A prominent lag period is
exhibited in the 2 ppm curve, while at
10 ppm the lag extends beyond the fifth day.
An activated sludge may be adapted to work
effectivt-lj in degradation of60mgCN/).
5
-------
Effect of Some Variables on the BOD Test
i—i—i—17-1—I—I—I—T
~ 60
2 0 ppm CN
10 ppm CN -
Table I
2 3
Time In Days
Effect of Cyanide on BOD of Domestic Sewage
(2% Sewage in Formula C Dilution Water)
Figure 6
Heavy metals have similar effects depending
on history and environment. The effects of
copper and chromium are illustrated in
Figure 7.
Waste
conc.
Depletion
5 day BOD
10%
3.51
35
5%
4.53
91
2%
CO
00
0
140
1%
1.52
152
0.5%
0. 74
148
VH NITRIFICATION
A Mechanism
The oxidation process, as exemplified by
the equation
CHROMIl
_1_
I
_1_
1 ' l_
0 i ? : i 3 0 * e
ppm
LFFECT OJ MLT A LS 0\ DOD
Figure 7
B Detection
In laboratory determinations of BOD the
absence of toxic substances including
chlorine must be established before the
results can be accepted as valid.
Comparison of BOD values for several
dilutions of the waste will indicate the
presence or absence of toxicity. In Table 1
the calculated BOD for the dilutions show
higher values in the more dilute concen-
trations. It is apparent that toxicity was
present and that the toxic effect was diluted
out at a waste concentration of 2% or less.
y = L (l-10_kS
presumably involves the oxidation of
carbonaceous matter or 1st stage oxygen
demand.
o„
C H O —
C02 + h2o
The rate coefficient is normally high, giving
nearly complete oxidation in a few days.
When nitrogenous material is present its
oxidation can be shown as
°2 - °2 -
NH3 - no2 - NO,
Nitrogen oxidation may be delayed for
several days during BOD tests unless
suitable micro-biota are initially available.
Under some circumstances these two
oxidations can proceed simultaneously and
the resultant BOD curve will be a com-
posite of the two reactions.
A
L (1-10
c
"kct ) + L (1-10
-k
where yt = the simultaneous BOD of the car-
bonaceous and nitrogenous phases or 1st and
2nd stage demands.
kc and kn = the rate coefficients appli-
cable to the carbonaceous and nitrogenous
materials respectively.
20-6
-------
Effect of Some Variables on the BOD Test
L and L = the ultimate oxygen demands
characteristic of the two phases respectively.
This is the general formula for any system
characterized by two simultaneous reactions.
Principal conditions governing simultaneous
carbon and nitrogen oxidation:
1 Presence of an effective nitrifying
culture at the beginning of the test
interval (nitrifiers grow relatively
slowly).
2 Maintenance of adequate DO, believed
to be a minimum of 0. 5 to 1.0 mg/1,
for nitrifier activity.
3 Available nitrogen - in excess of that
required for synthesis. This is believed
to require a minimum of about 7 mg/1
to support active nitrification on a
continuous basis.
4 Nitrifiers appear to be more sensitive
to toxicity than most saprophytic
organisms, hence are likely to be
inhibited more readily. This is
particularly evident during nitrite to
nitrate conversion.
B It may require 5 to 10 days to establish
nitrification if the population was not
nitrifying initially. This is the basis for
the sequential carbonaceous and nitrogenous
oxidation of sewage oxidation.
1 Effects on the BOD curve indicate a
typical pattern such as in Figure 8.
The influence of nitrification m the
production of a secondary rise in the
BOD curve is so well known that any
secondary rise may be erroneously
attributed to nitrification whether or
not nitrification was involved. Actually,
a secondary rise in the curve may be
due to any oxidation system assuming
dominance after the initial oxidation
system has been completed.
2 The nitrification phenomena occurs
simultaneously in many streams,
treated effluents or partially stabilized
samples. The designation of a secondary
BOD rise to nitrification should be
based on analysis, not curve shape.
C The extent of nitrification is conclusively
shown only by periodic analysis of
ammonia, organic, nitrite and nitrate
nitrogen. The: conversion of ammonia
and organic nitrogen to oxidized nitrogen
is a definite indication of nitrification.
D Nitrification Inhibition
Plant efficiencies from a BOD standpoint
can be erroneous because nitrification
generally is not established during the
usual incubation of influent samples but
may be a major factor in effluent
incubations. It requires about 2 times
the oxygen to convert NH^ -N to NO^ -N
as to convert C to CO^ hence this is a
major fraction of stream oxygen use.
Most secondary treated effluents are
characterized by a larger fraction of
carbon than nitrogen removal which
accentuates the problem.
Pasteurization of samples, methylene
blue, chromium, and acid treatment
followed by neutralization have been used
to inhibit nitrification for estimation of
carbonaceous BOD only. Any inhibition
of nitrification also produces a change in
the sample or its behavior and may
partially inhibit carbonaceous oxidation.
Nitrification is a factor in stream self-
.purification and treatment. It does not
appear realistic to alter it for convenience.
The most realistic approach to carbon-
aceous oxidation is the measurement of
C02 or COD.
e
e
¦
l
Figure 8
20-7
-------
Effect of Some Variables on the BOO Test
VIII EFFECT OF DILUTION
When a series of dilutions are made on a
BOD sample usually the result s vary to the
extent that only an approximate BOD value
is obtained.
Statistically the probabilities of being
jiearer the actual value goes with the
nearest two of three. The 4% value
of 8. 2 depletion/4 as a minimum
possible BOD 1% concentration gives
a BOD of at least 200.
Table 2
INTERPRETATION OF BOD DATA
Sample conc.
1
DO
Depletion
BOD
Initial
1
; 8 2
-
-
Final
1
1%
, 5 5
2 7
270
2%
! 3 3
4 9
245
4%
1 0 0
Complete
*
For example, m Table 2, 1%, 2% and 4%
concentrations of sample were used. The
4% concentration became anaerobic before
the end of 5 days. The 5-day BOD of the
1% concentration was 270 and that of the
2% concentration was 245.
Statistically one value is more reliable
than the other.
Dilution
1%
2%
Difference
DO depletion
5.5 mg/1
3. 3 mg/1
2. 2 mg/1
The difference in depletion between 1 and
2% dilutions is 2.2 mg/1. This difference
may be attributed to an additional 1% of
sample added to the original 1%. If the
difference is multiplied by the dilution
factor of 100 to obtain the BOD, the result
is 220 mg/1.
1 We now have three estimates of the
BOD on a one percent concentration
basis from the two dilutions:
a the actual 1% depletion gives 270
b 2%/2 depletion gives 245
c (2%- 1%) depletion gives 220
There is the possibility that higher
concentrations may reflect significant
toxicity while lower concentrations
tend to reflect a greater proportion of
dilution water. The toxicity problem
does not appear to be significant since
the 4% sample concentration indicated
a BOD of at least 200. The higher
BOD at 1% sample concentration may
be due to a contaminated dilution water
or to the fact that a similar number of
seed organisms had less food and
utilized certain fractions that they had
passed by when they had more choice
with the 2% sample concentration.
Data is insufficient to resolve this one.
Incubations having a depletion of at
least 2 mg DO/liter and a residual of
at least 1 mg DO/liter are indicated
to be most valid^1'. Both the 1 and
2% concentrations fit this requirement
in Table 2. An average error of
+ or -0.1 ml on the DO titration would
have a smaller relative error upon
the 2% depletion.
We have a reasonable presumption
that the sample BOD of about 230 was
a good estimate. We do not have an
unequivocal basis for so stating.
Possible variations in results with
different dilutions of a given sample
are subject to many uncertainties in
the test routine.
If some cause is known - such as a
titration eror, the inclusion of ex-
traneous substances producing high
or low response, or a definite procedural
error that rules out a valid estimate of
the sample BOD- that result should be
labeled as a lost cause or unreported.
Otherwise, report what was obtained
to the best of your ability with the
provision of uncertainty for uncon-
trollables.
20-8
-------
Effect of Some Variables on the BOD Test
A CKNOW LEDGMENT:
Certain portions of this outline contain
training material from prior outlines by
D. G. Ballinger and J. W. Mandia.
REFERENCE:
Standard Methods, APHA-AWWA-WPCF,
13th edition, 1971.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, WPq EPA, Cincinnati, OH 45268.
20-9
-------
CHEMICAL OXYGEN DEMAND AND COD/BOD RELATIONSHIPS
I DEFINITION
A The Chemical Oxygen Demand (COD) is
an estimate of the proportion of the sample
matter susceptible to oxidation by a
strong chemical oxidant. The current i
(2)
edition of Standard Methods, specifies
organic material which is generally the
situation but not necessarily applicable.
B A variety of terms have been and are used
for the test described here as COD-
1 Oxygen absorbed (OA) primarily in
British practice.
2 Oxygen consumed (OC) preferred by
some, but unpopular.
3 Chemical oxygen demand (COD) current
preference.
4 Complete oxygen demand (COD)
misnomer.
5 Dichromate oxygen demand (DOC)
earlier distinction of the current pre-
ference for COD by dichromate or a
specified analysis such as Standard
Methods.
6 Others have been and are being used.
Since 1960, terms have been generally
agreed upon within most professional
groups as indicated in I-A and B-3 and
the explanation in B-5.
C The concept of the COD is almost as old
as the BOD. Many oxidants and varia-
tions in procedure have been proposed,
but none have been completely
satisfactory.
1 Ceric sulfate has been investigated,
but in general it is not a strong
oxidant.
2 Potassium permanganate was one of
the earliest oxidants proposed and
until recently appeared in Standard
Methods (9th ed.) as a standard pro-
cedure. It is currently used m
British practice as a 4-hr. test at
room temperature.
a The results obtained with perman-
ganate were dependent upon concen-
tration of reagent, time of oxidation,
temperature, etc., so that results
were not reproducible.
3 Potassium lodate or iodic acid is an
excellent oxidant but methods employing
this reaction are time-consuming and
require a very close control.
4 A number of investigators have used
potassium dichromate under a variety
of conditions. The method proposed
by Moore at SEC is the basis of the
standard procedure. Statistical
comparisons with other methods are
described. *3'
5 Effective determination of elemental
carbon in wastewater was sought by
Buswell as a water quality criteria.
(4)
a Van Slyke* ' described a carbon
determination based on anhydrous
samples and mixed oxidizing agents
including sulfuric, chromic, iodic
and phosphoric acids to obtain a
yield comparable to the theoretical
on a wide spectrum of components.
b Van Hall, et al., ^ used a heated
combustion tube with infrared
detection to determine carbon quickly
and effectively by wet sample
injection.
6 Current development shows a trend to
instrumental methnds autorn-^ting
CH.O. oc. lOe. 12. 71
21-1
-------
Chemical Oxygen Demand and COD/BOD Relationships
conventional procedures or to seek
elemental or more specific group
determination.
II RELATIONSHIP OF THE COD TEST WITH
OTHER OXIDATION CRITERIA IS
INDICATED IN TABLE 1.
A Table 1
Test
Test
Temp. °C
Reaction
time
Oxidation
system
Variables
BOD
20
days
Biol. prod.
Enz. Oxidn.
Compound, environ-
ment, biota, time,
numbers. Metabolic
acceptability, etc.
COD
145
2 hrs.
50% H2S04
K2Cr2°7
May be cata-
lyzed
Susceptibility of
the test sample to
the specified
oxidation
IDOD
20
15'
Diss. oxyg.
Includes materials
rapidly oxidized by
direct action,
Fe+ , SH.
Van Slyke
Carbon detn.
400+
1 hr.
H3PO4
HIO3
h2so4
K2Cr207
Anhydrous
Excellent approach
to theoretical oxi-
dation for most
compounds (N-nil)
Carbon by
combustion
+IR
950
minutes
Oxygen atm.
catalyzed
Comparable to
theoretical for
carbon only.
Chlorine
Demand
20
20 min.
HOCl soln.
Good NH3 oxidn.
Variable for other
compounds.
B From Table 1 it is apparent that oxidation
is the only common item of this series of
separate tests.
1 Any relationships among COD & BOD
or any other tests are fortuitous be-
cause the conditions of test tend to give
results indicating the susceptibility of
a given sample to oxidation under
specified conditions that are different
for each test.
2 If the sample is primarily composed
of compounds that are oxidized by
both procedures (BOD and COD) a
relationship may be established.
21-2
-------
Chemical Oxygen Demand and (TOP/BOD Relationships
4 COD results are available while the
waste is in the plant, not several
days later, hence, plant control is
facilitated.
5 COD results are useful to indicate
downstream damage potential in the
form of sludge deposition.
6 The COD result plus the oxygen equiva-
lent for ammonia and organic nitrogen
is a good estimate of the ultimate BOD
for many municipal wastewaters
B Limitations
1 Results are not applicable for estimating
BOD except as a result of experimental
evidence by both methods on a given
sample type.
2 Certain compounds are not susceptible
to oxidation under COD conditions or
are too volatile to remain in the oxida-
tion flask long enough to be oxidized.
a The COD procedure may be sub-
stituted (with proper qualifications)
for BOD or the COD may be used
as an indication of the dilution
required for setting up BOD
analysis.
b If the sample is characterized by a
predominance of material that can
be chemically, but not biochemi-
cally oxidized, the COD will be
greater than the BOD. Textile
wastes, paper mill wastes, and
other wastes containing high con-
centrations of cellulose have a
high COD, low BOD.
c If the situation in item b is reversed
the BOD will be higher than the
COD. Distillery wastes or refinery
wastes may have a high BOD, low
COD, unless catalyzed by silver
sulfate.
d Any relationship established as in
2a will change in response to
sample history and environment.
The BOD tends to decrease more
rapidly than the COD. Biological
cell mass or detritus produced by
biological action has a low BOD
but a relatively high COD. The
COD/BOD ratio tends to increase
with time, treatment, or conditions
favoring stabilization.
Ill ADVANTAGES AND LIMITATIONS OF
THE COD TEST(2) AS RELATED TO BOD
A Advantages
1 Time, manipulation, and equipment
costs are lower for the COD test.
2 COD oxidation conditions are effective
for a wider spectrum of chemical
compounds.
3 COD test conditions can be standardized
more readily to give more precise
results.
Ammonia, aromatic hydrocarbons,
saturated hydrocarbons, pyridine, and
toluene are examples of materials with
a low analytical response in the COD
test.
3 Dichromate in hot 50% sulfuric acid
requires close control to maintain
safety during manipulation.
4 Oxidation of chloride to chlorine is not
closely related to BOD but may affect
COD results.
5 It is not advisable to expect precise
COD results on saline water.
IV BACKGROUND OF THE STANDARD
METHODS COD PROCEDURE
A The COD procedure^ ^ considered dichro-
mate oxidation in 33 and 50 percent sul-
furic acid. Results indicated preference
of the 50 percent acid concentration for
oxidation of sample components. This is
the basis for the present standard
procedure.
21-3
-------
Chemical Oxygen Demand and COD/BOD Relationships
B Muers^ suggested addition of silver
sulfate to catalyze oxidation of certain
low molecular weight aliphatic acids and
alcohols. The catalyst also improves
oxidation of most other organic components
to some extent but does not make the COD
test universally applicable for all chemical
pollutants.
C The unmodified COD test result (A) includes
oxidation of chloride to chlorine. Each mg
of chloride will have a COD equivalent of
0. 23 mg Chlorides must be determined
in the sample and the COD result corrected
accordingly.
1 For example, if a sample shows 300
mg of COD per liter and 200 mg CI"
per liter the corrected COD result will
be 300 -(200 x 0.23)or 300 - 46 = 254
mg COD/1 oh a chloride corrected basis.
2 Silver sulfate addition as a catalyst
tends to cause partial precipitation of
silver chloride even in the hot acid solu-
tion. Chloride corrections are ques-
tionable unless the chloride is oxidized
before addition of silver sulfate, i.e.,
reflux for 15 minutes for chloride ox-
idation, add Ag SO , and continue the
reflux or use ofHgSO^(D).
" (7)
D Dobbs and Williams proposed prior
complexation of chlorides with HgS04 to
prevent chloride oxidation during the test.
A ratio of about 10 of Hg + to 1 of CI" (wt.
basis) appears essential. The CI" must
be complexed in acid solution before addi-
tion of dichromate and silver sulfate.
1 For unexplained reasons the HgSO^
complexation does not completely
prevent chloride oxidation in the
presence of high chloride concentrations
2 Factors have been developed to provide
some estimate of error in the result
due to incomplete control of chloride
behavior. These tend to vary with the
sample and technique employed.
E It is not likely that COD results will be
precise for samples containing high
chlorides Sea water contains 18000 to
21000 mgCl"/l normally. Equivalent
chloride correction for COD exceeds
4000 mg/1. The error in chloride
determination may give negative COD
results upon application of the correction.
Incomplete control of chloride oxidation
with HgS04 may give equally confusing
results.
HgS04 appears to give precise results
for COD when chlorides do not exceed
about 2000 mg/1. Interference in-
creases with increasing chlorides at
higher levels.
F The 12th edition of Standard Methods re-
duced the amount of sample and reagents
to 40% of amounts utilized in previous
editions. There has been no change in
the relative proportions in the test This
step was taken to reduce the cost of pro-
viding expensive mercury and silver sul-
fates required. Results are comparable
as long as the proportions are identical
Smaller aliquots of sample and reagents
rei-jOire more care during manipulation
to promote precision.
G The EPA Methods for COD
1 For routine level COD (samples having
an organic carbon concentration
greater than 15 mg/liter and a chloride
concentration less than 2000 mg/liter),
the EPA specifies the procedures found
m Standard Methods ^and in ASTM^.
2 For low level COD (samples with less
than 15 mg/liter organic carbon and
chloride concentration less than 2000
mg/liter), EPA provides an analytical
procedure The difference from
the routine procedure primarily in-
volves a greater sample volume and
more dilute solutions of dichromate
and ferrous ammonium sulfate.
3 For saline samples (chloride level
exceeds 1000 mg/liter and COD is
greater than 250 mg/liter), EPA
provides an analytical procedure^
involving preparation of a standard
curve of COD versus mg/liter
chloride to correct the calculations.
Volumes and concentrations for the
sample and reagents are adjusted for
this type of determination.
21-4
-------
Chemical Oxygen Demand and COD/BOD Relationships
V The precision of the unmodified COD
result shows a standard deviation of + 4% of the
mean (3)on low chloride samples. Silver
sulfate modified COD results are likely to
show a standard deviation about twice that
without catalysis, due to questionable
chloride behavior. The determination of
chloride frequently shows a coefficient of
variation (six) of 10 to 157c, hence high
chloride samples result in COD precision
controlled more by chloride behavior than
organic oxidation.
VI REMARKS PERTINENT TO EFFECTIVE
COD DETERMINATIONS INCLUDE-
A Sample size and COD limits for 0. 25 N
reagents are approximately as given.
For 0. 025 N reagents multiply COD by
0. ]. Use the weak reagents for COD's
in the range of 5-50 mg/1, (low level).
Sample Size
20 ml
10 ml
5 ml
mg COD/1
2000
4000
8000
Wipe the upper part of the flask and
lower part of the condenser with a
wet towel before disassembly to
minimize sample contamination.
Steam out the condenser after use for
high concentration samples and periodi-
cally for regular samples. Use the
regular blank reagent mix and heat,
without use of condenser water, to
clean the apparatus of residual oxidiz-
able components
Distilled water and sulfuric acid must
be of very high quality to maintain low
blanks on the refluxed samples for the
0. 025 N oxidant
ACKNOWLEDGEMENT:
Certain portions of this outline contain
training material from prior outlines by
R. C. Kroner, R. J. Lishka, andJ.W Mandia.
REFERENCES:
B Most organic materials oxidize relatively
rapidly under COD test conditions. A
significant fraction of oxidation occurs
during the heating upon addition of acid.
The color change of dichromate after
acid addition indicates the approximate
fraction of dichromate remaining. If
the mixed sample color changes from
yellow to green after acid addition the
sample was too large. Discard without
reflux and repeat with a smaller
aliquot until the color after mixing does
not go beyond a brownish hue. The
dichromate color change is less rapid
with sample components that are slowly
oxidized under COD reaction conditions.
C Chloride concentrations should be known
for all test samples and results inter-
preted accordingly.
D Special precautions advisable for the
regular COD procedure and essential
when using 0. 025 N reagents include:
1 Keep the apparatus assembled when
not in use.
2 Plug the condenser breather tube with
glass wool to minimize dust entrance.
1 Moore, W. A., Kroner, R C and Ruchhoft,
C. C. Anal. Chem 21:953 1949.
2 Standard Methods, 13th Edition, APHA-
AWWA-WPCF, 1971.
3 Moore, W.A., Ludzack, F J and
Ruchhoft, C. C. Anal. Chem. 23:1297,
1951.
4 Van Slyke, D. D. and Folch, J.J Biol
Chem. 136:509 1940.
5 Van Hall, C. E , Safranko, J. and Stenger,
V. A., Anal Chem. 35:315 1963.
6 Muers, M. M. J. Soc. Chem. Ind. (London)
55:711 1936.
7 Dobbs, R.A. and Williams, R.T., Anal.
Chem. 35:1064 1963
8 ASTM Standards, Part 23, Water:
Atmospheric Analysis, 1970.
9 Methods for Chemical Analysis of
Water and Wastes, EPA-AQCL,
Cincinnati, OH, July 1971.
See Next Page.
21-5
-------
Chemical Oxygen Demand and COD /BOD Relationships
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, MDS,
OWP, EPA, Cincinnati, OH 45268.
21-6
-------
LABORATORY PROCEDURE FOR ROUTINE
LEVEL CHEMICAL OXYGEN DEMAND
I REAGENTS
A Standard Potassium Dichromate (0.250 N):
Dissolve 12.259 g of primary standard
grade K„Cr O , which had been dried at
103° C for two nours, in distilled water and
dilute to one liter.
B Ferrous Ammonium Sulfate (0. IN):
Dissolve 39 g of Fe (NH ) (SO ) • 6H O
in distilled water.
Carefully add 20 ml of concentrated HgSO^.
Cool and dilute to one liter.
C Ferroin Indicator:
Dissolve 1.485 g 1, 10-phenanthroline
monohydrate and 0.695 g FeSO^• 7H O in
water and dilute to 100 ml. This incucator
may be purchased already prepared.
D Concentrated Sulfuric Acid (36 N):
E Mercuric Sulfate: Analytical Grade
F Silver Sulfate: Analytical Grade
G Concentrated Sulfuric Acid - Silver Sulfate:
Dissolve 22 g of silver sulfate in a 9 lb
bottle of concentrated sulfuric acid.
(1-2 days required for dissolution)
steam emerges from the top of the con-
denser for several minutes. Cool the
mixture, carefully discard the acid, and
rinse the condenser and flask with distilled
water. In order to prevent contamination
from air-borne particles, the top of the
condenser should be lightly plugged with
glass wool during storage and use.
IE STANDARDIZATION OF FERROUS
AMMONIUM SULFATE
A Dilute 10. 0 ml of the standard
potassium dichromate to about 100 ml
with distilled water.
B Add 30 ml of concentrated H2S04 and
allow to cool.
C Add 2-3 drops of ferroin indicator and
titrate to a reddish-brown end point
with the ferrous ammonium sulfate.
Calculate the normality, N, of the
ferrous ammonium sulfate.
D Calculation
N of Fe
CH.O.oc.lab.3b. 12. 71
22-1
-------
Laboratory Procedure for 1 Routine Level Chemical Oxygen Demand -
C Add 10. 0 ml of the 0. 250 N I^Cr^
and mix.
D Carefully add 28 ml of the sulfuric acid
silver sulfate reagent.
E Add several pumice granules or glass
beads to prevent bumping, and then
swirl the mixture to insure complete
mixing.
F Reflux the mixture for two hours.
G Allow the solution to cool, wash down
the condenser with distilled water, and
bring the volume of liquid to about
100 ml with water.
H Add 2-3 drops of the ferroin indicator
and titrate the solution to a reddish-
brown end point with the ferrous
ammonium sulfate.
A blank consisting of 20 ml of distilled
water and containing all reagents is
refluxed and titrated in the same manner
as the sample.
J Calculation
mg COD/1
(A-B) NX 8X 1000
ml of sample
COD 0 chemical oxygen demand
A ¦ ml Fe (NH^ (SO^ used for blank
B 0 ml Fe (NH ) (SO )„ used for sample
N = N of Fe(NH .)_ (SOJ„
4 2 4 2
8 0 equivalent weight of oxygen
A CKNOWLEDGEMENT:
Portions of this outline were taken from
an outline prepared by R. J. Liska.
REFERENCES
1. Methods for Chemical Analysis of
Water & Wastes, EPA-AQCL, 1971.
2. Standard Methods, APHA-AWWA-
WPCF, 13th Edition, 1971.
3. ASTM Standards, Part 23, 1970.
This outline was prepared by Charles R.
Feldmann, Chemist, National Training
Center, MDS, WPC) EPA, Cincinnati,
¦OH 45268.
22-2
-------
TOTAL CARBON ANALYSIS
I INTRODUCTION
A History of Carbon Analyses
In the wake of a rapid population growth,
and the increasing heavy use of our
natural waterways, the nation, and indeed
the world, is presented with the acute
problem of increased pollutional loads on
streams, rivers and other receiving
bodies. This has resulted in a growing
awareness of the need to prevent the
pollution of streams, rivers, lakes and
even the oceans. Along with this aware-
ness has developed a desire for a more
rapid and precise method of detecting and
measuring pollution due to organic
materials.
B The Methods
In the past, two general approaches have
been used in evaluating the degree of
organic water pollution.
1 The determination of the amount of
oxygen or other oxidants required to
react with organic impurities.
2 The determination of the amount of
total carbon present in these impurities.
C Oxygen Demand Analyses
The first approach is represented by
conventional laboratory tests for deter-
mining Chemical Oxygen Demand (COD)
and Biochemical Oxygen Demand (BOD).
One of the principal disadvantages of these
tests is that they are limited primarily
to historical significance, that is, they
tell what a treatment plant had been doing,
since they require anywhere from two
hours to five days to complete. Since up
to now no faster method has been
available, traditional BOD and COD
determinations have become accepted
standards of measure in water pollution
control work even though they are
essentially ineffective for process
control purposes.
Until the introduction of the Carbonaceous
Analyzer, all methods taking the second
approach, the total carbon method of
evaluating water quality, also proved
too slow.
II THE ANALYSIS OF CARBON
A Pollution Indicator
Now the carbonaceous analyzer provides
a means to determine the total carbon
content of a dilute water sample in
approximately two minutes. With proper
sample preparation to remove inorganic
carbonates, the instrument determines
the total organic carbon content in the
sample.
B Relationship of Carbon Analysis to BOD
and COD
This quantity varies with the structure
from 27 percent for oxalic acid through
40 percent for glucose to 75 percent for
methane. The ratio of COD to mg carbon
also varies widely from 0.67 for oxalic
acid through 2.67 for glucose to 5. 33 for
methane. Representative secondary
sewage effluents have given a ratio of
COD to carbon content of between 2. 5 and
3. 5 with the general average being 3. 0.
The BOD, COD and carbon contents of
these and some other representative
compounds are summarized in the follow-
ing table.
CH. MET. 24a. 12. 71
2 3 r 1
-------
Total Carbon Analysis
Sample
Stearic Acid - C^H^gOg
Glucose
Oxalic Acid
C6H12°6
C2H2°4
Benzoic Acid - C^HgOg
Phenol - C„H.O
b b
Potassium Acid Phthalate
KHC8H4°4
Salicylic Acid - C^HgO^
Secondary Effluent, Clarified
5-Day
BOD-mg/mg
786
. 73
14
1. 38
05 to 2 1 de-
pending upon
concentration
.95
1. 25
13-*
23*
4*
COD-
mg/mg
2.91
1.07
. 18
1 97
2. 36
1 15
1 60
75*
67*
36*
% Carbon
76
40
27
69
77
47
61
21*
12*
7*
* In units of mg/l
III THE CARBON ANALYZER
A Principle of Operation
Basically the carbonaceous analyzer con-
sists of three sections - a sampling and
oxidizing system, a Beckman Model 315
Infrared Analyzer, and a strip-chart
recorder
Carbonaceous Analyzer schematic
manual or
slide
valve
injection
of umple
temp
controller
Q
if
u
Q flow
¦ M 'I
condenser
(
Beckman
infrared
analyser
oxygen carrier from cylinder
A micro sample (20-40 Ml) of the water to be
analyzed is injected into a catalytic com-
bustion tube which is enclosed by an electric
furnace thermostated at 950°C. The water
is vaporized and the carbonaceous material
is oxidized to carbon dioxide (CO2) and
steam in a carrier stream of pure oxygen.
The oxygen flow carries the steam and
CO2 out of the furnace where the steam is
condensed and the condensate removed
The CO2. oxygen and remaining water vapor
enter an mir^red analyzer sensitized to
provide a measure of CO2 The output of
the infrared analyzer is recorded on a strip
chart, after which, the curve produced can
be evaluated by comparing peak height with
a calibration curve based upon standard
solutions. Results are obtained directly in
milligrams of carbon per liter
B Application
(1)
Results show that the method is applicable
for most, if noi all, water-soluble organic
compounds -- including those that contain
sulfur, nitrogen, and volatiles.
Nonvolatile organic substances can be
differentiated from volatiles, such-as
carbon dioxide or light hydrocarbons by
23-2
-------
Total Carbon Analysis
determination of carbon both before and
after the sample solution has been blown
with an inert gas.
C Sample Preparation
The Carbonaceous Analyzer is often
referred to as a total carbon analyzer
because it provides a measure of all the
carbonaceous material in a sample, both
organic and inorganic. However, if a
measure of organic carbon alone is de-
sired, the inorganic carbon content of
the sample can be removed during sample
preparation
1 Removal of inorganic carbon
The simplest procedure for removing
inorganic carbon from the sample is
one of acidifying and blowing. A few
drops of HC1 per 100 ml of sample will
normally reduce pH to 2 or less, re-
leasing all the inorganic carbon as CO2.
Five minutes of blowing with a gas free
of CO2 sweeps out the CC>2 formed by
the inorganic carbon. Only the organic
carbon remains in the sample and may
be analyzed without the inorganic inter-
ference
2 Volatile carbonaceous material
Volatile carbonaceous material that
may be lost by blowing is accounted
for by using a dual channel carbon
analyzer. Beckman's new analyzer
has the previously detailed high
temperature (950 C) furnace plus a low
temperature (150°C) one. Using
quartz chips wetted with phosphoric
acid, the low temperature channel
senses only the CC>2 (freed by the acid)
in the original sample. The remaining
organics and water are retained in the
condenser connected to this low temper-
ature furnace. None of the organics
are oxidized by the 150°C furnace.
By injecting a sample into the low
temperature furnace, a peak repre-
senting the inorganic carbon is obtained
on the strip chart. Injecting a non-
acidified sample into the high temper-
ature furnace yields a peak representing
the total carbon. The difference
between the values determined for
the two peaks is the total organic
carbon.
3 Dilute samples
If the sample is dilute (less than 100
mg/liter carbon) and is a true solution
(no suspended particles) no further
preparation is required.
4 Samples containing solids
If the sample contains solids and/or
fibers which are to be included in the
determination, these must be reduced
in size so that they will be able to pass
through the needle which has an opening
of 170 microns (needles having larger
openings may be obtained if necessary).
In most cases, mixing the sample in a
"Waring Blender will reduce the particle
size sufficiently for sampling.
IV PROCEDURE FOR ANALYSIS
A Interferences
Water vapor resulting from vaporization of
the sample, causes a slight interference in
the method Most of the water is trapped
out by the air condenser positioned immed-
iately after the combustion furnace. How-
ever, a portion of the water vapor passes
through the system into the infrared de-
tector and appears on the strip chart as
carbon. The water blank also appears on
the standard calibration curve, and is
therefore removed from the final calcu-
lation In tests of solutions containing the
following anions: NOj, CI , SO"2, PO43,
no interference was encountered with con-
centrations up to one percent.
B Precision and Accuracy
The recovery of carbon from standard
solutions is 98.5 - 100.0 percent The
minimum detectable concentration using
the prescribed operating instructions is 1
mg/l carbon. Generally, the data are
reproducible to + 1 mg/l with a standard
deviation of 0. 7 mg/l at the 100 mg/l level
23-3
-------
Total Carbon Analysis
V APPLICATIONS
Several of the many research and industrial
applications of the Carbonaceous Analyzer
are listed below
A Determine the efficiency of various waste-
water renovation processes, both in the
laboratory and in the field.
B Compare a plant's waste outlet with its
water inlet to determine the degree of
contamination contributed
C Monitoring a waste stream to check for
product loss.
D Follow the rate of utilization of organic
nutrients by micro-organisms
E To detect organic impurities in inorganic
compounds.
VI ADVANTAGES OF CARBON ANALYZER
A Speed
B Total Carbon
Another advantage is that the measure of
carbon is a total one. The oxidizing
system of the analyzer brings about com-
plete oxidation of any form of carbon No
compound has been found to which the
method is inapplicable.
VII CONCLUSIONS
The Carbonaceous Analyzer provides a
rapid and precise measurement of organic
carbon in both liquid and air samples. It
should be found useful for many research
and industrial applications, a few of which
have been mentioned.
Because of its rapidity it may be found more
useful than the more time-consuming BOD
and COD measurements for monitoring
industrial waste streams or waste treatment
processes.
REFERENCES
1 Van Hall, C. E., Safranko, John and
Stenger, V. A. Anal Chem. 35,
315-9 1963.
2 Van Hall, C. E., and Stenger, V. A
Draft of Final Report - Phase I - Con-
tract PH 86-63-94, Analytical Research
Toward Application of the Dow Total
Carbon Determination Apparatus to the
Measurement of Water Pollution.
Van Hall, C. E. , Stenger, V. A
Beckman Reprint - R6215 Taken from
Paper Presented at the Symposium on
Water Renovation, Sponsored by the
Division of Water and Waste Chemistry
ACS m Cincinnati. Jan 14-16, 1963.
This outline was prepared by Robert T. ~~
Williams, Chief, Analytical Applications
Laboratory, AWTL, NERC, Cincinnati, OH
45268 and revised by Charles J. Moench, JY
Waste Identification and Analysis, AWTL,
NERC, Cincinnati, OH 4526€.
The Carbonaceous Analyzer's most
important advantage is its speed of
analysis One analysis can be performed
in 2-3 minutes. This speed of analysis
brings about another advantage, economy
of operation Working with dilute samples,
one man can run ten to twenty carbon
determinations per hour. This is probably
more than the number of COD or BOD
tests that can even be started, much less
completed, in the same period of time.
23-4
-------
VI NUTRIENTS AND RELATED DETERMINATIONS
Chemical indications of water quality other than oxygen demand
determinations are considered in this section. These criteria
have an influence upon nutritional aspects of the aquatic environ-
ment of a favorable or unfavorable connotation depending upon
concentration, balance, and desired uses.
Contents of Section VI
Outline Number
Acidity, Alkalinity, pH and Buffers 24
Alkalinity and Relationships Among the
Various Types of Alkalinities 25
Laboratory Procedure for Total
Alkalinity 26
Laboratory Procedure for Total
Acidity 27
Determination of Chloride in Water
Supplies 28
Determination of Sulfate in Water
Supplies 29
Sources and Analysis of Organic
Nitrogen 30
Ammonia, Nitrites and Nitrates 31
Laboratory Procedure for Nitrate
Nitrogen Modified Brucine Method 32
Determination of Phosphorus in
the Aquatic Environment 33
Laboratory Procedure for Phosphorus 34
-------
ACIDITY, ALKALINITY, pH AND BUFFERS
DEFINITIONS OF ACIDS AND BASES
C Neutrality
A Arrhenius Theory of Acids and Bases
(Developed about 1887)
1 Acid: A substance which produces,
in aqueous solution, a hydrogen ion
(proton), IT*".
2 Base- A substance which produces,
in aqueous solution, a hydroxide
ion, OH".
3 The Arrhenius theory was confined
to the use of water as a solvent.
B Bronsted and Lowry Theory of Acids
and Bases (Developed about 1923)
1 Acid A substance which donates,
m chemical reaction, a hydrogen
ion (proton).
2 Base- A substance which accepts,
in chemical reaction, a hydrogen
ion (proton).
3 Bronsted and Lowry had expanded
the acid-base conce pt into non-
aqueous media, i.e., the solvent
could, but did not have to be .water.
C Further development of acid-base theory
dealing with electron pair donation or
acceptance is unimportant.
It is possible to have present in
the water chemically equivalent
amounts of acids and bases. The
water would then be described as
being neutral; i. e., there is a
preponderance of neither acid nor
basic materials. The occurrence
of such a condition would be rare.
The term "neutralization" refers to
the combining of chemically equiv-
alent amounts of acids and bases.
The two products of neutralization
are a salt and water.
HC1
NaOH
Hydro- Sodium
chloric Hydroxide
acid
NaCl + HzO
Sodium
Chloride
(a salt)
The key word in the above definitions
is "preponderance. " It is possible to
have a condition of acidity while there
are basic materials present in the
water, as well as conversely.
m HOW A RE DEGREES OF ACIDITY AND
ALKALINITY EXPRESSED?
II DEFINITIONS OF ACIDITY,
ALKALINITY AND NEUTRALITY
A Acidity
A condition in which there is a prepon-
derance of acid materials present in
the water.
B Alkalinity
A condition in which there is a prepon-
derance of alkaline (or basic) materials
present m the water.
The pH scale is used to express various
degrees of acidity and alkalinity. Values
can range from Oto 14. These two ex-
tremes are of theoretical interest and would
never be encountered in a natural water or
in a wastewater. pH readings from 0 to
just under 7 indicate an acidic condition,
from just over 7 to 14, an alkaline condition.
Neutrality exists if the pH value is exactly
7. pH paper, or a pH meter, provides the
most convenient method of obtaining pH
readings. Some common liquids and their
pH values are listed in Table 1.
CH.ALK.3.12.71
24-1
-------
Acidity, Alkalinity, pH and Buffers
TABLE 1. pH Values of Common Liquids
Household lye 13.7
Bleach 12.7
Ammonia 11.3
Milk of magnesia 10.2
Borax 9,2
Baking soda 8. 3
Sea 'water 8. 0
Blood 7.3
Distilled water 7. 0
Milk 6.8
Corn 6.2
Boric acid 5. 0
Orange juice 4. 2
Vinegar 2.8
Lemon juice 2. 2
Battery acid 0.2
5.9 - 8.4 is the common pH range for most
natural waters.
IV HARD AND SOFT WATERS
In addition to being acidic or basic, water
can also be described as being hard or soft.
A Hard water contains large amounts of
calcium, magnesium, strontium, man-
ganese and iron ions, relative to the
amount of sodium and potassium ions
present. Hard water l s objectionable
because it forms insoluble compounds
with ordinary soap.
B Soft water contains small amounts of
calcium, magnesium, strontium, man-
ganese and iron ions, relative to the
amount of sodium and potassium ions
present. Soft water does not form in-
soluble compounds with ordinary soap.
V TITRATIONS
A The conversion of pH readings into such
quantities as milligrams (mg) of acidity,
alkalinity, or hardness, is not easily
carried out. These values are more
easily obtained by means of a titration.
B In a titration, an accurately measured
volume of sample (of unknown strength)
is combined with an accurately measured
volume of standard solution (of known
strength) in the presence of a suitable
indicator.
C The strength (called normality) of the
sample is then found using the following
expression:
milliliters (ml) of sample X normality <
(N) of sample ¦ ml of standard solu-
tion X N of standard solution.
Three of the four quantities are known,
and
N of sample ¦ ml of standard solution
X N of standard solution /ml of sample.
D In modified form, and a more specific
application of the above equation, alka-
linity is calculated in the following
manner (12th ed. Standard Methods).
mg of alkalinity as mg CaCOg/liter (1)
3 ml of standard H9SO4 X N of standard
H2SO4 X 50 X 1000/ml sample.
VI INDICATORS
The term "suitable indicator" was used
above. At the end of a titration, the pH of
the solution will not necessarily be 7. It
may be above or below 7. A suitable indi-
cator, therexore, is one which undergoes
its characteristic color change at the appro-
priate pH. Below are a few examples of
indicators and the pH range in which they
undergo their characteristic color changes.
In some cases, mixed indicators may be
used in order to obtain a sharper and more
definite color change.
Operational
Indicator
pH Range
Methyl Yellow
2.8 - 4.0
Methyl Orange
3. 1 - 4.4
Methyl Red
4.4- 6.2
Cresol Purple
7.4- 9.0
Phenolphthalein
8.0- 9.6
Alizarine Yellow
10.0 - 12.0
TABLE 2. pH Range of Indicators
24-2
-------
Acidity, Alkalinity, pH and Buffers
VII BUFFERS
A A buffer is a combination of substances
which, when dissolved in water, resists
a pH change in the water, as might be
caused by the addition of acid or alkali.
Listed below are a few chemicals
which, when combined in the proper
proportions, will tend to maintain the
pH in the indicated range.
Chemicals pH Range
AceticAcid + SodiumAcetate
3.7 -
5.6
Sodium Dihydrogen Phosphate +
Disodium Hydrogen Phosphate
5.8 -
8.0
Boric Acid + Borax
6.8 -
9.2
Borax + Sodium Hydroxide
9.2 -
11.0
TABLE 3. pH Range of Buffers
B A buffer functions by supplying ions
which will react with hydrogen ions
(acid "spill"), or with hydroxide ions
(alkali "spill").
C In many instances, the buffer is composed
of a weak acid and a salt of the weak acid;
e.g., acetic acid and sodium acetate.
1 In water, acetic acid ionizes or
"breaks down" into hydrogen ions
and acetate ions.
HC2H302 = H+ + C2H302-
(acetic acid) (hydrogen ion) (acetate ion)
(proton)
This ionization occurs to only a
slight extent, however, most of the
acetic acid remains in the form of
HC2H302; only a small amount of
hydrogen and acetate ions is formed.
2 Thus, acetic acid is said to be a
weak acid.
3 In the case of other acids, ionization
into the component ions occurs to a
large degree, and the term strong acid
is applied; e.g., hydrochloric acid.
HC1 = H+ + CI"
(hydrochloric (hydrogen ion) (chloride
acid) (proton) ion)
4 The terms "strong" and "weak" are
also applied to bases. In water
solutions, those which break down
into their component ions to a large
extent are termed "strong", and
those which do not are "weak".
Sodium hydroxide is a relatively
strong base, while ammonium
hydroxide is realtively weak.
5 Sodium acetate (a salt of acetic acid)
dissociates or "breaks down" into
sodium ions and acetate ions when
placed in water.
NaC2H302 3 Na+ + C2H3O2"
(sodium acetate) (sodium ion) (acetate ion)
This dissociation occurs to a large
extent, and practically all of the
sodium acetate is m the form of
sodium 10ns and acetate ions.
D It would be difficult and expensive to
prepare large quantities of buffers for
use in a treatment plant. However,
certain naturally occurring buffers may
be available (carbon dioxide is an ex-
ample). It dissolves in water to form
the species indicated below.
co2 + h2o » h2co3
(carbondioxide) (Water) (carbonic acid)
H2C03 = H+ + HC03"
(hydrogen ion) (hydrogen car-
(proton) bonate ion)
(bicarbonate)
The hydrogen 10ns react with hydroxide
ions which might appear in the water
as the result of an alkali "spill".
H+ + OH" = H20
(in the (hydroxide ion
buffer) "spilled")
The hydrogen carbonate ions react with
hydrogen ions which might appear in
the water as the result of an acid "spill".
H+ + HCO3- = H2C03
(hydrogen ion) (in the
(proton) buffer)
"spilled"
This buffering action will be in effect as
long as there is carbonic acid present.
24-3
-------
Acidity, Alkalinity, pH and Buffers
E Buffering action is not identical with
a process in which acid wastes are
"neutralized" with alkali wastes, or
conversely. The desired effect is
achieved in both cases, however (i. e.,
the pH is maintained within a desired
range.)
This outline was prepared by C. R. Feldmann,
Chemist, National Training Center, MDS,
WPO, EPA, Cincirnati, OH 45268
24-4
-------
ALKALINITY AND RELATIONSHIPS AMONG THE
VARIOUS TYPES OF ALKAUNITIES
I PRELIMINARY
The property of water referred to as alkalinity
is usually caused by the presence of hydroxy 1,
carbonate and bicarbonate ions. To a lesser
extent, borates, phosphates and silicates
contribute but are generally present in
negligible amounts.
The concentration and ratio of the OH , CO^
and HCOg ions may be measured by titrating
a sample to certain specified pH's or end
points which are detected either by use of a
pH meter or by color indicators. Phenol-
phthalein is used for visual detection of the
first end point, (approximately pH 8) which
indicates the neutralization of NaOH and con-
version of COg to HCOg . A number of
indicators (methyl orange, methyl purple,
brom cresol green,etc.) are used for detection
of the second end point (pH 3-5) which indicates
the complete conversion of HCO to H O and
COg. The final end point is determined by
the amount of CO^ and HCO^ originally
present in the sample. If the end points are
determined electrometrically they are taken
as the mid-point of the greatest rate of pH
change per unit volume of titrant.
II RELATIONSHIPS BETWEEN HYDROXIDE,
CARBONATE, AND BICARBONATE
A LKA UNITIES
The results obtained from phenolphthalein
and total alkalinity measurements offer a
means of classification of the principal
forms of alkalinity, if certain assumptions
are made. It must first be assumed that
interferences are absent and that bicar-
bonate and hydroxide do not exist in the
same solution. According to the system
presented in Standard Methods, 12th Edition:
A Hydroxide alkalinity is present if the
phenolphthalein alkalinity is more than
one-half the total alkalinity.
B Carbonate alkalinity is present if the
phenolphthalein alkalinity is not zero
but is less than the total alkalinity.
C Bicarbonate alkalinity is present if the
phenolphthalein alkalinity is less than
one-half the total alkalinity.
Table 1. Relationships Between Phenolphthalein Alkalinity, Total Alkalinity,
Carbonate Alkalinity, Bicarbonate Alkalinity and Hydroxide Alkalinity
Lecture
Notes
Result of
Titration
OH Alkalinity
as CaCOg
CO Alkalinity
as CaCOj
HCO Alkalinity
as CaCO
Case 1
P = T
T
O
o
Case 2
p o
O
2P
o
Case 3
p = o
O
O
T
Case 4
p > It
2P-T
2(T-P)
O
Case 5
p < £t
0
2P
T-2P
P = Phenolphthalein Alkalinity T 3 Total Alkalinity
CH.ALK.2 d. 12. 71
25-1
-------
Alkalinity and Relationships Among the Various Types of Alkalinities
Table 2. Stoichiometric Volumes of Solutions of Different Normalities
Standard Solution
H2S°4
NaOH
Na2C°3
NaHCOg
Normality
0.0200
0. 0189
0.0199
0.0125
Equivalent Volumes, ml
10.0
10.6
9.9
16.0
9.4
10.0
9.5
15.1
10.0
10.5
10.0
15.9
6.3
6.6
6.3
10. 0
IE CASE EXAMPLES
The relationships involved in Table 1 may
best be explained by reference to the following
graphs. These were prepared by titrating
volumes of standard solutions of sodium
hydroxide, sodium carbonate, and sodium
bicarbonate with standard sulfuric acid.
The stoichiometric volumes of the various
solutions are summarized in Table 2 for
convenience in the interpretation of the charts.
CASE 1 - Where phenolphthalein alkalinity
= total alkalinity
B CASE 2 - Where phenolphthalein alkalinity
= one-half the total alkalinity
IP) i:no point
(T) PND POINT
(p ¦ i t>
12
0
-
6
H^P) FND POINT
pll
V.
3
-
—. -
0
1 1
1 1 1 1
Zr> ML 0 nut1) N NnOII
(P e T)
The sharp break occurs at the point where
all of the NaOH has been exactly neutral-
ized by the acid. The pH and concentration
of the end products (NagSO^and HgO)
determine the pH at the equivalence point
between NaOH and HgSO^, in this case,
approximately 7. 0.
The titration proceeds in 2 stages wherein
all of the CO is converted, first to
HCOg'and fimlly to H2C03> The first
end point occurs at approximately pH 8,
and at exactly half the volume of acid
used for the total titration. The end point
which occurs at approximately pH 4
represents the total alkalinity and requires
exactly twice the volume of acid used for
the first end point.
If either H<^0„ or OH ions had been
present the titration volumes for the curves
would not have been of equal magnitude.
25-2
-------
Alkalinity and Relationships Among the Various Types of Alkalinities
C CASE 3 - Where phenolphthalein alkalinity
= 0
12
0
(P) FND POINT
G
pH
3
0
30
35
0
in
Ml. 0 020 N Jf2SO< ADDED
IS
25 ML 0 0liS N NnllCO. vm O 020 N II „bO
J 2 4
(V • O)
T The reaction proceeds m one stage with
the initial pH at approximately 8. 5 and
final pH at 4. 0. In this case the phenol-
phthalein alkalinityis zero and since no
conversion of CO,j to HCO^ is noted the
total alkalinity can only be due to the
HC03" ion.
D CASE 4 - Where phenolphthalein alkalinity
is greater than one-half the total alkalinity
IP) END POINT
(T) END POINT _
30
15 20
ML 0 020 N HjSO^ ADDFD
5
10
0
0 020 N H^>04
iT)
The volume of acid required for the first
end point (phenolphthalein alkalinity) is
due to the OH neutralization and con-
version of the COg to HCO„ . The
second end point represents tne complete
conversion of HCOg to HgCO . Referring
to Case 2 where the volume of acid was
similar for each end point, it is apparent
that a base responding to phenolphthalein
but which is not CO,, must be present.
Since it was originally assumed that OH
and HCOg do not exist in the same
solution we must conclude that the total
alkalinity is due to OH and CO^
E CASE 5 - Where phenolphthalein alkalinity
is less than one half of the total alkalinity
12
0
-------
Alkalinity and Relationships Among the Various Types of Alkalinities
F CASE 6 - Where phenolphthalein alkalinity
is greater than one-half total alkalinity.
and
(10 + 12.6) _
(p> i:nd point
(T) END POINT
= volume of acid required
The
second end point occurs at
(10 + 12.6)
for conversion of CO^ to HCO^
ml, the volume of HCO_
It O
which is converted to H^CO
becomes the same as Case
2- i:
This then
QlOMLO 0180 N NoOrt ~ 10 ML 0 012«> N NnllCOgH vs
0 020 N ll^SO^
(p> in
Following the original assumption that
OH" and HCO„" are not compatible, with
T T/""I/"\ ¦ Ik m a ^ f*~\ 1>> A Kn « m
IV COMPARISON OF ANALYTICAL
METHODS FOR ALKALINITY (According
to the Analytical Reference Service
Report JAWWA Vol. 55, No. 5, 1963)
Mnmt
DETERMINATION METHODS
TABLE 2—Statistical Summary (contd )
a condition similar to Case 4 (P > 2 T).
Method
Year
No 01
Vaieet
Cones
Added
Concn Determined
mt/l
50%
«,T
Standard
D» vial too
G CASE 7 - Where phenolphthalein alkalinity
Reported
•l/J
Mean
Low
Hl«h
¦l/l
(P) END POINT
T) PND POINT
[lOMLO 0180 N NiOH + 10 ML O 020 N NaaC03 * 10 ML
0 0125 N NnllCO_TvR 0 020 N ILSO,
2 4
(P> 1 T)
Alkalinity
Methyl orange
1956
38
19
20 0
11 23
30 0
± 2
5 049
1958
19
17
195
16
24
± 1
2 483
1961
27
42 5
43 7
38
50
db 1.5
3 250
Clectrometre
1956
14
19
19 7
190
210
± 1
0.911
1958
53
17
194
15
25
db 2
2 740
1961
88
42 5
44 2
38 9
57
±25
3 605
Methyl purple
1956
8
19
14 7
140
15 2
± 4
0475
1958
10
17
193
16
21
± 2
0 823
1961
18
42 5
44 8
40
50
± 1 5
3 081
Muted indicator
1958
4
17
195
18
20
± 3
1 000
1961
15
42 5
42 8
39
49
ds 3
3 550
Brom cresol green
1958
1
17
19
19
19
± 2
All method*
1956
60
19
19 2
II 23
300
± 2
4 460
1958
92
17
196
15
25
± 2
2 320
1961
178
42 5
419
25
57
db 19
S.335
The first end point occurs at the stoichio-
metric sum of the equivalent volumes as
follows:
(9.4
-6.3)-"°t12-6'
where (9. 4 - 6.3) = volume of N acid
required for excess OH after OH +
HCO reaction,
0
Alkalinity - The methods for alkalinity
measurement varied only in the choice
of indicator or pH for determining the
end point of the titration. The indicators
used included methyl orange, methyl
purple, and mixed indicator. The data
shows that as the use of electrometric
end point increased, the use of methyl
orange decreased.
25-4
-------
Alkalinity and Relationships Among the Various Types of Alkalinities
SUMMARY
VI EPA ANALYTICAL METHODS
Amount Added 17 mg/1 (as CaCO^)
Avg. Deviation from 3.1 mg/1
amount added
Standard Deviation 3.4 mg/1
50% Range 2.0 mg/1
Method Most Commonly Electrometric
Used
Method Preferred
Total Number of
Observations
Electrometric
41
V PROCEDURE
The actual measurement of alkalinity is a
very simple procedure requiring only titration
of sample with a standardized acid and the
proper indicator. For phenolphthalein
alkalinity the end point and indicator are well
established. For the bicarbonate titration
the final end point is a function of the HCOg
concentration. With low amounts (< 50 mg/1
as CaCOg) the pH at end point may be
approximately 5. 0. With high concentrations
(> 250 mg/1 as CaCO„) the pH at end point may
be 4.5 to 3.8. For all-purpose work, in
which the highest degree of accuracy is not
required, an end point at pH 4. 5 using methyl
purple as the indicator is recommended.
The traditional methyl orange frequently
proves to be unsatisfactory because of the
indefinite color change at the end point and
also because of the low pH (3.8 - 3.9)
required to establish the change.
A The Environmental Protection Agency,
Office of Water Programs, Analytical Quality
Control Laboratory has compiled a manual
of Analytical Methods which is to be used
in Federal laboratories for the chemical
analysis of water and waste samples.
The title of this manual is '"Methods for
Chemical Analysis of Water and Wastes,.
1971."
B This manual lists two parameters which
are related to the subject matter in this
outline. They are total alkalinity and
total acidity.
C For the measurement of both of these
parameters, the recommended method
is volumetric, with the equivalence point
being determined electrometrically.
The use of a color indicator (methyl
orange) is recommended only for the
automated method.
D The procedure references cited by the
i EPA Methods manual are the 13th
Edition of Standards Methods and ASTM
Standards, Part 23, 1970.
REFERENCES
1 Standards Methods for the Examination of
Water and Wastewater, 12th Ed.
APHA, Inc., New York. 1965.
2 ASTM Standards, Part 23. 1968,
This outline was prepared by R. C. Kroner,
Chief, Physical and Chemical Methods,
Analytical Quality Control Laboratory,
Water Programs Operations, EPA,
Cincinnati, OH 45268.
25-5
-------
LABORATORY PROCEDURE FOR TOTAL ALKALINITY
I METHOD SUMMARY
A In the current methods manual^ of the
Environmental Protection Agency, the
following method is specified for use in
Office of Water Programs laboratories
The sample is titrated with 0. 02N
hydrochloric or sulfuric acid to a final
pH of 4 5, the end point being determined
electrometrically.
1 The sample must not be filtered,
diluted, concentrated or altered in
any way
2 Results are reported as mgCaCOg/liter
3 The procedure can be found in Standard
Methods(2) and ASTM Book of Standards.*3*
B The procedure as given in the above
references has been adapted for this
laboratory session to accommodate group
participation and to accomplish in-
structional objectives.
II DISCUSSION
In this manual, outline 8 considers the
relationships among the 3 forms of alkalinity
commonly found in water Refer to the
graphs in outline 8 for the following-
For Case 1, (OH) alkalinity only, pH
values change very gradually until the
approach of pH 9 Then an abrupt change
occurs and the addition of a few ml of
titrant causes the value to change rapidly
to about pH 4
For Case 2, (COg) alkalinity only, there
is a gradual change of pH values as the
acid titrant is added. Around pH 9 there
is an abrupt change of the scale reading
Around pH 6, the change is again gradual.
Around pH 5, another rapid change occurs
until pH 3 is reached
A pH of 8 3 represents the average pH when
(OH) has been totally neutralized and
(CO_) has been half neutralized to
(HCOg) . A pH of 4 5 represents the
average pH occurring when (HCO^) has
been neutralized to CO_ and H O.
(See Case 3)
In order to obtain standardized data, the
EPA method specifies stopping alkalinity
titrations at qH 4 5. At this pH, any
(OH) , (COg) and (HCOg) in the sample
will be neutralized. Since all three of
these alkalinity forms may be present,
the results are expressed as total
alkalinity
It is useful to express total alkalinity as
mg CaCOg/liter.
Ill INSTRUCTIONAL OBJECTIVES
A You will use the adapted EPA method to
determine total alkalinity by titrating 3
solutions of known alkalinities and one
unknown to an end point of pH 4 5 You
will then use the data to calculate the
total alkalinity for each as mg CaCO„/liter
0
1 By slowly adding the HC1 titrant to
solutions of (OH)", of (COg) and of
(HCOg) and observing the pH scale,
the characterizing response of each
alkalinity at different pH values can
be observed.
2 Recording the volume of HC1 used to
reach pH 8 3 and then pH 4 5 will
also illustrate the pH characteristics
of the three forms of alkalinity
3 Calculating the total alkalinity of these
three solutions will familiarize you
with the calculations for the determination.
B You will also do titrations on the same
solutions using color indicators —
phenolphthalein (colorless at pH 8) and
methyl orange (amber-orange at pH 4 6)
— in order to contrast values obtained
by the pH determination.
Cn AI
-------
Laboratory Procedure for Total Alkalinity
C Using experience gained in the lab:
session, you will estimate the types of
alkalinity present in the unknown sample.
IV REAGENTS
A Carbon Dioxide-Free Distilled Water
B Anhydrous Sodium Carbonate (primary
standard)
C Hydrochloric Acid Titrant (0. 02N)
D Phenolphthalein
E Methyl Orange
V STANDARDIZATION of the HYDROCHLORIC
ACID TITRANT
A Set the temperature reading on the pH
meter dial to match the temperature of >
the buffer and sample solutions.
B Standardize the pH meter against a
reference buffer solution.
C Weigh accurately 0. 088 + 0. 001 g of the
dried sodium carbonate and transfer it,
to a 500 ml conical flask.
D Add 50 ml of water and swirl to dissolve
the carbonate.
E While stirring the solution, add the
hydrochloric acid titrant from a 100 ml
buret until a pH of 4.5 is attained.
F Calculate the normality of the hydrochloric
acid solution as follows-
A B
0.053 X C
A = normality of the hydrochloric acid
B = g of sodium carbonate used
C = ml of hydrochloric acid consumed
0.053 = millequivalent weight of Na^O^
VI TITRATIONS to pH END POINTS
For each of the solutions marked Case 1
. (OH)", Case 2 (COg)* and Case 3 (HCOg)~
and for the assigned unknown, A or B,
the following procedure is to be followed
All data and the calculation results should
be recorded on the Data Table provided
A Calibrating the pH meter
1 Pour about 50 ml of the buffer solution
into a small beaker.
2 Set the temperature reading on the pH
meter dial to match the temperature
of the reference buffer.
3 Immerse the electrode(s) into the
buffer solution.
4 Turn the meter to "ONM(or "Read"
or "pH").
5 Using the "Balance" (or "Asymm")
knob, set the scale reading to 6. 84.
6 Turn the meter to "Off" (or "Hold"
or "Standby").
7 Remove electrode(s) from buffer and
save this solution for possible later
use.
8 Rinse electrode(s) with distilled
water, then immerse them in a small
beaker of distilled water until time
to use
B Prepare the buret by rinsing 3 times using
about 15 ml HC1 each time. Then fill the
buret with HC1 and adjust the volume so
that the top of the liquid column is positioned
for a reading.
C Titration Procedure-
1 Each titration will require up to 25 ml
0. 02N HC1 titrant. Refill the buret as
needed before beginning each titration.
2 Pipet 50. 0 ml of the solution to be used
into a beaker
26-2
-------
Laboratory Procedure for Total Alkalinity
3 Remove the pH electrode(s) from the
beaker of distilled water and immerse
electrode(s) m the solution to be
titrated. The sample solution should
cover the glass bulb(s).
4 Turn pH meter to "ON" (or "Read"
or "pH").
5 On the Data Table, record the ml of
HC1 in the buret at the start of the
titration.
6 Record the initial pH of the sample
on the Data Table.
7 While gently swirling either the
electrode or the sample in the beaker,
slowly add the HC1 titrant, observing
the scale on the pH meter Stop
adding titrant when pH is about 8. 3
and record the buret reading (This
step is not part of the EPA procedure.
It is included here for instructional
purposes).
8 Again gently swirling either the
electrode or the beaker, continue
slow addition of the HC1 titrant, ob-
serving the meter scale Be very
cautious when the scale approaches
ph 5 0. Stop titrating at pH 4. 5 If
a lower pH is inadvertently reached,
note this on the Data Table.
9 On the Data Table, record the ml HC1
remaining in the buret.
10 Set pH meter to "OFF" (or "Hold"
or "Standby").
11 Rinse the pH electrode(s) with dis-
tilled water and immerse in the beaker
of distilled water
12 Empty the sample solution and rinse
the beaker with distilled water.
13 Using the next solution, repeat this
procedure (C, steps 1 through 12)
until data has been recorded for the
3 knowns and the 1 unknown sample.
Then proceed to part VII.
DATA TABLE
TITRATIONS TO pH ENE
POINTS
CASE 1
(OH)"
CASE 2
(c°3)_
CASE 3
(HC03)"
SAMPLE
Initial pH
ml@pH 4. 5
ml@pH 8. 3
ml@Start
TOTAL
ml.HCl
TOTAL
ALKALINITY
(mg CaCO.
/ liter)
VII TITRATIONS to COLOR END POINTS
If time permits, the following procedure
should be used to titrate the 3 solutions
of known constituents and the assigned
unknown, A or B. At least, do one of
the known solutions and the unknown
used for the pH titration All data and
the calculation results should be recorded
on the Data Table provided.
A Indicators Used
1 Phenolphthalein indicator is a deep
pink around pH 10 and becomes
colorless at pH 8 In the Data Table,
this colorless end point is designated
as P. End Pt.
2 Methyl orange indicator gives a yellow
color to a colorless solution At
pH 4. 6 (referred to as M. O. End Pt
on Data Table) it becomes amber-
orange. This end point color requires
practice to detect If the solution
acquires a pinkish cast, you are past
the desired end point.
B Titration Procedure
1 Each titration will require up to 25 ml
0. 02N HC1 titrant. (If pH determinations
have been done, refer to these results
for approximations of HCL titrant to
be used). Refill the buret as needed
before beginning each titration.
26-3
-------
Laboratory Procedure for Total Alkalinity
2 Pipet 50.0 ml of the solution to be used
into a beaker
3 Place a piece of white paper under
the beaker.
4 Add 3 drops phenolphthalem indicator
to the sample and swirl to mix
5 On the Data Table record the ml of
HC1 m the buret at the start of the
titration.
6 Using the known relationship of the
color of phenolphthalem to different
pH values, record your estimate of
the initial pH of the sample on the Data
Table.
7 If the solution remains colorless,
record 0 0 for P End Pt and continue
with step 9.
If there is pink color in the solution,
slowly add HC1 titrant while stirring
the sample
As the end point is approached, a
lingering colorless region where the
titrant first contacts the sample will
be observed Add the titrant very
cautiously at this time, stirring the
sample solution and allowing time for
total mixing and contact before adding
more HC1 titrant.
The end point occurs when the entire
solution is colorless. Stop adding
the HC1 titrant.
8 Record the ml of HC1 in the buret for
the P. End Pt. on the Data Table
9 Add 3 drops of methyl orange indicator
to the same sample solution.
10 If the sample solution becomes amber-
orange, record 0 0 for M.O. End Pt.
and continue at step 12
If the solution becomes yellow, slowly
add HC1 titrant while stirring the
sample.
As the end point is approached, a
a lingering amber-orange region where
the titrant first contacts the sample
will be observed Add the titrant very
cautiously at this time, stirring the
sample solution and allowing time for
total mixing and contact before adding
more HC1 titrant.
The end point occurs when the entire
solution is amber orange Stop adding
the HC1 titrant. (If the solution develops
a pinkish cast, you are past the desired
end point and should note this on the
Data Table).
11 Record the ml of HC1 in the buret for
the M. O. End Pt on the Data Table
12 Empty the sample solution and rinse
the beaker with distilled water
13 Choose the next solution according to
time available and repeat this procedure
(B, steps 1 thru 12).
DATA TABLE
TITRATIONS TO COLOR END POINTS
ZlASE 1
(OH)"
CASE 2
-------
Laboratory Procedure for Total Alkalinity
VIII CALCULATIONS
A Calculate Total Alkalinity for each
solution titrated, using the following-
mg CaCOg/ 1 = A x N x 50, 000
ml. of sample
A = total ml of standard acid required
for titration to pH 4 5 end point
N = normality of standard acid
50 = equivalent weight of CaC03
1000 converts ml to liters
B Record calculation results on the
Data Tables.
REFERENCES
1 Methods for Chemical Analysis of
Water and Wastes, EPA-AQCL,
Cincinnati, OH 45268, (1971)
2 Standard Methods for the Examination
of Water and Wastewater, 13th edition,
APHA-AWWA-WPCF, Method 102 (1971)
3 ASTM Book of Standards, Part 23,
p 154 (1970)
This outline was prepared by A. Donahue,
Chemist, National Training Center, DTTB,
MDS, OWP, EPA, Cincinnati, OH 45268.
26-5
-------
LABORATORY PROCEDURE FOR TOTAL ACIDITY
I REAGENTS
IH PROCEDURE
For detailed discussion of reagent preparation,
consult reference 2.
I
A Carbon Dioxide - Free Distilled Water
B Potassium Hydrogen Phthalate
(primary standard)
C Sodium Hydroxide Titrant (0.02 N)
A Pipet 100 ml of the sample into a 400 ml
beaker.
B Titrate with the sodium hydroxide to
pH 8.3
C Calculation
Total acidity as mg of CaCO^/l =
H STANDARDIZATION OF THE SODIUM
HYDROXIDE TITRANT
A Set the temperature reading on the pH
meter dial to match the temperature of
the buffer and sample solutions.
B Standardize the pH meter against a
reference buffer solution.
C Weigh accurately 0.19 + 0. 005 g of the
dried potassium hydrogen phthalate and
transfer it to a 500 ml conical flask.
D Add 100 ml of reagent water and swirl
gently to dissolve the phthalate.
E While stirring the solution(magnetic bar
and stirrer), add the sodium hydroxide
from a 100 ml buret until a pH of 8.3 is
attained.
F Calculate the normality of the sodium
hydroxide solution as follows-
A = §
0. 20423XC
A = normality of the sodium hydroxide
B = g of potassium hydrogen phthalate
C = ml of sodium hydroxide solution
0.20423 = milliequivalent weight of
potassium hydrogen phthalate
(khc8h4o4)
A X N X 50000
ml of sample
A = ml of standard NaOH titrant
N = N of standard NaOH titrant
50 = equivalent weight of CaCO^
1000 - converts ml to liters
REFERENCES
1 Methods for Chemical Analysis of
Water & Wastes, EPA-AQCL, 1971,
Cincinnati, OH 45268.
2 A.S. T. M. Book of Standards, Part 23,
D 1067-70, pp. 155-158.
This outline was prepared by Charles R.
Feldmann, Chemist, National Training
Center, OWP, MDS, DTTB, EPA,
Cincinnati, OH 45268.
CH.ALK.lab. 3b. 12.71
27-1
-------
DETERMINATION OF CHLORIDE IN WATER SUPPLIES
I OCCURRENCE
In fresh waters, high chloride values may
indicate the presence of animal pollution.
However, the chloride test as an indication
of animal pollution should be confirmed by
bacteriological and sanitary analyses. A
high chloride value can be due to other
sources such as oil field brines and other
industrial wastes, also from the passage
of water through a natural salt formation,
or from agricultural return wastes
n SIGNIFICANCE
Chloride compounds may break down,
specially under boiler pressures, to form
HC1, thus causing corrosion problems
Chlorides are undesirable in ice making
as they spoil the appearance of the ice. Ap-
proximately 500 mg of salt/1 imparts an
undesirable taste to drinking water For
brewing or soft drinks, the salt content
should not exceed 275 mg/1, as the concen-
tration may be increased in the procefes.
The U. S. Public Health Service Drinking
Water Standards for potable waters recom-
mend a maximum chloride content of 250 mg
per liter, because of taste effects
III ANALYTICAL METHODS
A Volhard
B Mohr
The neutral or weakly alkaline sample
is treated with chromate indicator and
titrated with silver nitrate Silver
chloride precipitates and at the end
point, red silver chromate is formed
Iodide and bromide register as equiva-
lent chloride. Phosphate, sulfide and
cyanide interfere Sulfite interferes
but can be removed with hydrogen per-
oxide. Color if present, can be
removed with aluminum hydroxide
suspension.
C Mercuric Nitrate
The sample, adjusted to pH 3.1*, is
titrated with mercuric nitrate solution.
Since slightly dissociated mercuric
chloride is formed, no precipitation
occurs At the end point, the excess
mercuric ions produce a violet color
with diphenylcarbazone indicator.
Bromphenol blue is added to the indi-
cator solution for pH adjustment It
improves the sharpness of the endpoint
by masking the pale color produced by
diphenylcarbazone during the titration
Iodide and bromide register as equiva-
lent chloride Sulfite in concentrations
greater than 10 mg/1 interferes, but
can be removed with hydorgen peroxide.
Chromate and feric ions interfere
when in excess of 10 mg/1
An excess of silver nitrate is added to
the acidified sample to precipitate
chlorides The excess silver is titrated
with thiocyanate in the presence of
ferric ion and nitrobenzene. At the end
point, red ferric thiocyanate is formed.
Iodide and bromide register as equiva-
lent chloride. Phosphate and sulfite do
not interfere, but sulfide does.
IV ENVIRONMENTAL PROTECTION AGENCY
METHOD
A The Analytical Quality Control Laboratory,
Office of Water Programs, Environmental
Protection Agency, has published a manual
of analytical chemical procedures.
B This manual cites references (2) or (3)
for the determination of chloride; the two
procedures both involve titration with
mercuric nitrate.
CH.HAL. cl Gf 9.72
28-1
-------
Determination of Chloride in Water Supplies
An automated ferricyanide method is
described in detail m reference (1).
V PRECISION
Using the mercuric nitrate titration,
forty-two analysts in eighteen laboratories
analyzed synthetic water samples containing
exact increments of chloride with the
following results:
Increment as
Chloride, mg/1
17
18
91
97
382
398
Precision as Standard
Deviation, mg/1
1. 54
1. 32
2. 92
3. 16
11. 7
11. 8
REFERENCES
1 Methods for Chemical Analysis of
Water and Wastes, 1971, Environmental
Protection Agency, Water Quality Office,
Analytical Quality Control Laboratory
2 Standard Methods for the Examination
of Water and Wastewater, 13th Ed.,
page 97, Method 112B (1971).
3 ASTM Methods, Part 23, Water;
Atmospheric Analysis, page 24,
Method 512-67 (1970).
This outline was prepared by D. G.
Ballinger, Director, Analytical Quality
Control Laboratory, NERC, EPA, and
revised by C. R. Feldmann, Chemist,
National Training Center, WPO, EPA,
Cincinnati, OH 45268.
28-2
-------
DETERMINATION OF SULFATE IN WATER SUPPLIES
I DEFINITION AND OCCURRENCE
Sulfate is found in all fresh waters as a
result of solvent action of water on gypsum
(CaSO^ • 2H2O) and other common minerals
such as epsom salt (MgS04 • THgO).
Sulfates also occur as the final oxidized
stage of sulfides, sulfites, and thiosulfates.
They may also occur as the oxidized state
of organic matter in the sulfur cycle, and,
in turn serve as sources of energy for
sulfate-reducing bacteria, at lower pH these
will form hydrogen sulfide, which is quite
undesirable.
Industrial wastes, such as those from
sulfate-pulp mills, tanneries, pickling
operations, and other plants that use sul-
fates or sulfuric acid, contribute to the
natural sulfate content of raw waters.
Sulfuric acid is the heaviest tonnage
chemical manufactured.
II SIGNIFICANCE
PHS Drinking Water Standards call for not
more than 250 mg S04°/l. Public water
supplies with high sulfate content are
commonly used with no adverse effects,
this limit does not appear to be based on
tests or physiological effects other than a
laxative action for new users. The taste
threshold of magnesium sulfate is 400 - 600
mg/1, calcium sulfate is reported to be
250 -900 mg/1. Excessive concentrations
(1000 - 2000 mg/1) of magnesium sulfate
may have purgative effects.
Sulfates may be either beneficial or detri-
mental in w a t e r used for manufacturing.
In the brewing industry, the presence of
sulfate is advantageous, as it aids in pro-
ducing desirable flavor. On the other hand,
sulfates are not desirable in the ice industry
because of the formation of white butts.
In domestic water systems, sulfates do not
appear to cause any increased corrosion
on brass fittings, but concentrations above
200 mg/1 do increase the amount of lead
dissolved from lead pipes.
Normally, calcium sulfate scale is not en-
countered in once-through cooling water
systems since it is qu 11 e soluble at the
temperatures usually existing. In re-
circulating systems, where concentration
takes place, the sulfate content of the
circulating water may become high enough
to precipitate calcium sulfate in the form
of gypsum (CaS04* 2H2O).
Treatment measures for preventing scale
formation are directed primarily at calcium
carbonate precipitation, since it is less
soluble than the sulfate. The usual treat-
ment uses sulfuric acid which increases
the sulfate content. This, under certain
conditions, may create the additional
problem of calcium sulfate scale formation.
The precipitation of calcium sulfate can be
hindered by surface-active agents such as
the polyphosphates and organics. If nec-
essary, sulfates can be removed by
evaporation, demineralization, or preci-
pitation with barium salts.
The publication, "Water Quality Criteria"
of the California State Water Pollution
Control Board, lists the recommended
limits on sulfate in mg/1, as shown in
Table 1.
Ill ANALYTICAL METHODS
The gravimetric method is recognized as
the standard procedure and is the most
accurate and time-consuming. It should
be used for sulfate in greater concentrations
than 60 mg/1. The turbidimetric procedure
is rapid and more accurate for concen-
trations less than 50 mg/1, but can be used
up to 60 mg/1. The most rapid method is
the titrimetric, which is applicable to
solutions containing 100 mg S04n/1, or more
where an accuracy of ^10% is acceptable
as in boiler water analysis.
This procedure is not applicable to Basic
Data Network samples. Obviously, dilution
CH. SUL. Id. 2. 71
29-1
-------
Determination of Sulfate in Water Supplies
Table 1. RECOMMENDED LIMITS ON SULFATE CONTENT OF INDUSTRIAL WATER
INDUSTRIAL PROCESS
Milligrams per liter
S04
CaS04
MgS04
Na2S04
Brewing
-
100 - 500
100 - 200
100
Carbonated Beverages
250
-
-
-
Concrete Corrosion
25
-
-
-
Ice Making
-
300
130 - 300
300
Milk Industry
60
-
-
-
Photographic Process
a
100
-
-
Sugar Making
20
-
-
-
Textiles
100
-
-
-
or concentration of the sample will bring
most waters into the desired range for any
of the methods.
A Gravimetric Procedure
The gravimetric procedure involves the
addition of a dilute solution o f barium
chloride to the sample to precipitate
barium sulfate. The precipitation is
made in a solution slightly acidified with
HC1 and near the boiling temperature.
The precipitate is filtered off, washed in
water until free of chloride ions, ignited
at 800° C and weighed as barium sulfate.
mg S04=/1
mg BaSQ4X411.5
ml of sample
B Turbidimetric Method
In the more rapid turbidimetric method,
sulfate ion is precipitated with barium
ion m acid solution in such a manner as
to form barium sulfate crystals of uni-
form size. No other ions are found in
normal waters that will precipitate
with barium in acid solution. Light
transmitted by the turbid solution is
measured with a photometer and the
sulfate ion concentration is read from
a standard curve. Color and turbidity
must be removed first. The procedure
described in Standard Methods involves
very careful control of stirring and the
time interval before reading. A modifi-
cation used by the ARS Laboratory at
SEC has shown more consistent results.
IV PRECISION AND ACCURACY
The ARS Water Mineral Study of 1961 re-
ported on a reference sample containing
259 mg S04"/l that the gravimetric pro-
cedure remains the most commonly used,
with the turbidimetric method next.
The standard deviation for the gravimetric
methodwas 111.9 mg/1 and for the turbid-
imetric method it was ^23.9 mg/1.
V FWQA METHODS
A The Federal Water Quality Administration,
Division of Research, Analytical Quality
Control Branch, has compiled a manual
of analytical methods which is to be used
in Federal laboratories when-analyzing
surface waters. The title of this manual
is "FWQA Methods for Chemitai Analysis
of Water & Wastes, 1970".
B In this manual the turbidimetric method
is recommended (see HI B); the gravi-
metric procedure is not recommended,
C The 12th Edison of Standard Methods
and A. S. T. M. Book of Standards, 1968
are cited in the FWQA manual as the
references for the turbidimetric method.
29-2
-------
Determination of Sulfate in Water Supplies
REFERENCES
1 Standard Methods for the Examination
of Water and Wastewater. 12 Ed.
APHA, AWWA, WPCF. 1 965.
2 Water Mineral Study of 1961.
Analytical Reference Service, SEC.
3 Water Quality Criteria. State Water
Pollution Control Board, Sacra-
mento, California. 1963.
4 Public Health Service Interstate
Quarantine Drinking Water Stand-
ards (Revised) 1961. Federal
Register pp 6737-6740. July27, 1961.
5 FWQA Methods for Chemical
Analysis of Water & Wastes, 1970.
This outline was prepared by
D. G. Ballinger, Director, Analytical
Quality Control Laboratory, NERC,
EPA, Cincinnati, OH 45268.
29-3
-------
SOURCES AND ANALYSIS OF ORGANIC NITROGEN
I INTRODUCTION
A Organic nitrogen refers to the nitrogen in
combination with any organic radical.
For sanitary engineering the main interest
is the nitrogen contained in proteins,
peptides, amines, amino acids, amides
and related compounds of animal or
vegetable origin
II SOURCES OF ORGANIC NITROGEN
A Natural Origin
1 Dead animal and plant residues
2 Animal wastes - urea, feces
3 Autotrophic organisms - algae, s.bact
4 Heterotrophic organisms
B Industrial Origin
1 Food processing wastewater - meat,
milk, vegetables
2 Pharmaceutical wastes, antibiologicals
3 Plastics - polyamides, nitriles
4 Chemical intermediates or products
5 Dye industry - azo, nitro
acids, proteins and peptides to ammonia
but it may not convert the nitrogenous
compounds of some industrial wastes
such as amines, nitro compounds,
hydrazones, oximes, semi-carbazones
and some refractory tertiary amines.
C Method Summary^'
The sample is heated in the presence of
a concentrated sulfuric acid-potassium
sulfate-mercuric sulfate mixture and
evaporated until sulfur tnoxide fumes are
obtained and the solution becomes color-
less or pale yellow. The residue is
cooled and diluted, then treated and made
alkaline with a hydroxide-thiosulfate
solution. The ammonia is distilled off
and then determined either by nessler-
ization or by titration.
1 Nesslerization (colorimetric) is the
method used when the ammonia-
nitrogen concentration is less than 1.0
mg N/liter.
2 For ammonia-nitrogen concentrations
above 1. 0 mg N/liter, the ammonia is
determined by titration with 0. 02 N
H SO in the presence of a mixed
indicsrtor.
For a detailed description of the
procedure and reagent preparation,
consult the EPA methods manual.
Ill TOTAL KJELDAHL NITROGEN
PROCEDURE
A Organic nitrogen is determined using the
Total Kjeldahl Nitrogen (TKN) method.
This determination includes both organic
nitrogen and free ammonia By dis-
tilling the free ammonia off the sample
before the determination, organic nitrogen
can be determined directly
B Scope
The procedure converts nitrogen compon-
ents of biological origin such as amino
CH.N. 8. 12 71
D Precision and Accuracy
1 Thirty-one analysts in 20 laboratories^
used the Total Kjeldahl Nitrogen
procedure to analyze natural water
samples containing the following increments
of organic nitrogen: 0.20, 0.31, 4 10
and 4. 61 mg N/liter.
a Precision results for a standard
deviation were 0. 197, 0.247, 1.056
and 1. 191 mg N/liter, respectively.
b Accuracy as bias was +0. 03, +0. 02,
+0. 04 and -0. 08 mg N/liter, respect-
ively.
30-1
-------
Sources and Analysis of Organic Nitrogen
2 The nature and composition of ex-
traneous materials affect analytical
recovery. High salt concentrations
may raise digestion temperature.
High concentrations of organic sample
components may require excessive acid
during digestion tending to low nitrogen
yield.
3 The digestion temperature is critical.
380 to 390°C usually gives high
analytical recovery on the more re-
fractory nitrogen compounds of natural
origin. Nitrogen losses occur above
420 C.
E Optimum temperature is associated with
a digestion mix containing 1 gram of
potassium sulfate for each ml of sulfuric
acid. However, the digestion mixture
usually contains a fraction of HgSO^
greater than this.
During heating, the water boils off first,
followed by fuming as the HgSO^ becomes
concentrated. Fuming and oxidation of
the sample components lead to loss of
H2SO4 and a progressive decrease m acid
content during heating. As the acid de-
creases, the temperature of the heated
mixture rises.
Sodium nitroprusside, which increases
the intensity of the color, is added to
obtain necessary sensitivity for measure-
ment of low level nitrogen.
V PRESERVATION OF SAMPLES
A Most nitrogen compounds are characterized
by rapid conversion from one form to
another by biological and chemical action.
Hydrolysis, deamination, peptide formation,
and other reactions may appreciably alter
the original form of sample nitrogen
within a short time Organic nitrogen
tends to hydrolyze to free NH^ during
storage Therefore, valid results require
prompt analysis.
B Addition of 40 mg HgCL^/liter of sample
and storage at 4 C are preservation
measures for samples. Even when so
preserved, samples are unstable and
should be analyzed as soon as possible.
REFERENCE
1 Methods for Chemical Analysis of Water
and Wastes, EPA-AQCL. Cincinnati,
OH 45268 (1971).
Valid nitrogen determinations require a
slight excess of acid to retain NH as
NH.HSO. rather than the more volatile
(NH ) SO. Concurrently, excess acid
will tend foward incomplete oxidation of
sample components as a result of lowering
digestion temperature
IV AUTOMATED PHENOLATE METHOD
The EPA manual' ^ also presents an
automated (phenolate) method for organic
nitrogen The sample is automatically
digested with a sulfuric acid solution con-
taining potassium sulfate and mercuric
sulfate as a catalyst, then neutralized with
sodium hydroxide solution and treated with
alkaline phenol reagent and sodium hypoch-
lorite reagent This treatment forms a
blue color designated as indophenol.
This outline was prepared by Audrey E
Donahue, Chemist, National Training Center,
DTTB, MDS, OWP, EPA, Cincinnati, OH 45268.
30-2
-------
AMMONIA, NITRITES AND NITRATES
I SOURCES AND SIGNIFICANCE OF
AMMONIA, NITRITES AND NITRATES
IN WATER
The natural occurrence of nitrogencom-
pounds is best demonstrated by the nitrogen
cycle (Figure 1)
A Ammonia
1 Occurrence
Ammonia is a product of the micro-
biological decay of animal and plant
protein In turn, it can b e used
directly t o produce plant protein
Many fertilizers contain ammonia
2 Significance
The presence of ammonia nitrogen,
in raw surface waters might indicate
domestic pollution. Its presence in
waters used for drinking purposes
may require the addition of large
amounts of chlorine in order to
produce a free chlorine residual.
The chlorine will first react with
ammonia to form chloramines be-
fore it exerts its full bactericidal
effects (free chlorine residual).
B Nitrites
The presence of large quantities
indicates a source of wastewater
pollution.
C Nitrates
1 Occurrence
Nitrate formers convert n i t r l tes
under aerobic conditions to nitrates
(nitrobacter). During electrical
storms, large amounts of nitrogen
(N2) are oxidized to form nitrates.
Finally, nitrates can be found in
fertilizers.
2 Significance
Nitrates in water usually indicate
the final stages of biological sta-
bilization. Nitrate rich effluents
discharging into receiving waters
can, under proper environmental
conditions, cause stress to stream
quality by producing algal blooms.
Drinking water containing exces-
sive amounts of nitrates can cause
infant methemoglobinemia.
II ANALYSIS OF AMMONIA, NITRITES
AND NITRATES IN WATER
1 Occurrence
Nitrite nitrogen occurs in water
as a n intermediate stage in the
biological decomposition of organic
nitrogen. Nitrite formers (nitro-
somonas) convert ammonia under
aerobic conditions to nitrites. The
bacterial reduction of nitrates can
also produce nitrites under anaero-
bic conditions. Nitrite is used as
a corrosion inhibitor in industrial
process water.
2 Significance
Nitrites are usually not found in
surface water to a great extent.
A Sample Preservation
1 If only ammonia is to be determined on
the sample, it may be preserved with
1. 0 ml of concentrated sulfuric acid
per liter and stored at 4°C.
2 To preserve samples for any of the
nitrogen procedures, add 40 mg HgClg
and store at 4° C. Even when so
preserved, conversion of organic
nitrogen to ammonia may occur.
Preserved samples should be analyzed
as soon possible.
CH.N.6d. 12.71
31-1
-------
Ammonia, Nitrites and Nitrates
<2
aCterial_Oxidation)
NH3
lAmmonia
NH,"*"
i
O ,
O ,
o
>
X
Urine
Urea
Animal
Protein
rial Reduction)
Nitrite
2
Atmosp
Nitrate
NO
.Am
mal Food
Plant
Protein
THE NITROGEN CYCLE
Figure 1
31-2
-------
Ammonia, Nitrites and Nitrates
B Determination of Ammonia
1 Nesslerization
a Reaction
Nessler's reagent, a strong
alkaline solution of potassium
mercuric iodide, combines
with NH3 in alkaline solution
to form a yellowish brown
colloidal dispersion.
2K2HgI4 + NH3 + 3KOH *-
I
Hg/
+ 7KI + 2HpO
<
\nh2
Yellow-Brown
The intensity of the color
follows the Beer-Lambert Law
and exhibits maximum absorp-
tion at 425 nm.
b Interferences
1) Nessler's reagent forms
a precipitate with some
10ns (e.g., Oa"1"1", Mg"1"*",
Fe1 1 1, and S=). These
10ns can be eliminated in a
pretreatment flocculation
step with zinc sulfate and
alkali. Also, EDTA or
Rochelle salt solution pre-
vents precipitation with
Ca++ or Mg++.
2) Residual chlorine indicates
ammonia maybe present
in the form of chloramines.
The addition of sodium thio-
sulfate will convert these
chloramines to ammonia.
3) Certain o r ganic s may
produce an off color with
Nessler's reagent. If
these compounds are not
steam distillable, the inter-
ference may be eliminated
in the distillation method.
4) If the turbidity and natural
color of the sample cannot
be eliminated with floccula-
tion, it is then necessary
to use the following distil-
lation method.
2 Distillation
a Reaction
1)
The sample is distilled in
the presence of a phosphate
buffer at pH 7.2 - 7.4.
nh4 +•
¦NH3 + H+
H+ + Na2HP04 *-NaH2P04 + Na+
Buffer
2)
pH maintained be-
tween 7.2 - 7.4
The ammonia in the dis-
tillate is then measured by
either of two techniques.
a)
b)
Nesslerization is used
for samples containing
less than 1 mg/1 o f
ammonia nitrogen.
Absorption of NH3 by
boric acid and back ti-
tration with a standard
strong acid is more
suitable for samples
containing more than
1 mg NH3 N/1.
nh3 +hbo2-
nh4+ + bo2"
HQ - + H+ Methyl Red)
Methylene Blue
pH 7.8 - 8.0
Green
pH 6. 8 - 7. 0
Purple
31-3
-------
Ammonia, Nitrites and Nitrates
b Interferences
1) Calcium reacts with the
phosphate buffer releasing
H+ and lowering the pH in
the distillation step. When
the sample contains more
than 250mgCa++/l, it may
be necessary to adjust the
pH to 7.4 after adding the
buffer.
2) Residual chlorine.
3) Aliphatic and aromatic
amines may distill over
and cause positive inter-
ference by reacting in the
acid titration.
Precision and Accuracy
On a synthetic sample containing
0. 8 mg ammonia nitrogen/1, the
ARS Water Nutrients Study (1966)
reported results as shown in
Table 1.
Standard
Mean
Method
Deviation
Error
Direct
Nesslerization
i 0. 077 mg/1
0.00 mg/1
Distillation with
Nesslerization
lo. 100 mg/1
+ 0.02 mg/1
Distillation with
Titration
io. 167 mg/1
+ 0.04 mg/1
Table 1. ARS WATER NUTRIENTS, 1966
C Determination of Nitrite - Diazotization
Reaction
Under acid conditions, nitrite
ions react with sulfanilic acid
to form a diazo compound.
NH„
SO_H
N= N
+ NC>2 + 2H
pH 1.4
+ 2H2°
s^h'
SULFANILIC ACID DIAZO COMPOUND
S°3H
pH
2.0-
2. 5
Interferences
a Certain ions (e. g. , Hg++, Ag+,
Bi+++, Sb+++, Fe+++ Pb++)
may interfere by precipitating
during the test. A 0.5% EDTA
solution may be used to com-
plex iron.
b Cupric ion introduces negative
interference by catalyzing de-
composition of the diazo com-
pound.
c Colored ions and turbidity may
be removed by using Al(OH)g
suspension or by flocculation
with zinc sulfate and alkali.
Precision
The diazo compound then
couples with a -naphthylamine
to form an intense red azo dye
which exhibits maximum ab-
sorption at 520 nm.
On a synthetic sample containing
0.25 mg nitrite nitrogen/1, the ARS
Water Minerals Study (1961) re-
ported 125 results with a standard
deviation of +0. 029 mg/1.
31-4
-------
Ammonia, Nitrites and Nitrates
Determination of Nitrate
1 Phenoldisulfonic Acid
a Reaction
1) Phenoldisulfonic a c i d re-
acts with nitrate to produce
a mtro derivative.
HSCX
OH
OH
HSO,
+ H + NO„
SO3H
S°3h
+ H2°
2) In alkaline solution the nitro
derivative rearranges to
form a yellow-colored com-
pound which exhibits maxi-
mum absorption at 410 nm.,
OH
O
HSO,
N02 KS03
+ 3KOH »-
so3h
COLORLESS
-N—OK
+ 3H2°
of Ag , an offcolor or
turbidity is produced when
the final color is developed.
(Note- This difficulty can
be overcome by using
NH^OH as the alkali .)
2} Nitrites in concentrations
greater than 0.2 mg N/1
introduce positive interfer-
ence. However, in most
waters, the concentration
of nitrite is insignificant
compared to nitrate.
3) Color and turbidity may be
removed by using Al(OH)g
suspension or by floccula-
tionwith ZnS04 and alkali.
Precision and accuracy
On a synthetic sample con-
taining 1. 0 mg nitrate N/1,
and 200 mg Cl"/1, the ARS
Water Nutrients Study (1966)
reported 46 results with a
standard deviation of + 0.399
mg/1 and a mean error of
-0.31 mg/1.
Brucine
so3k
YELLOW
Interferences
1) Chlorine ion under the acid
conditions of the test intro-
duces negative interference.
6C1" + 2N03" + 8H+
3C12 I + 2NO f + 4H20
Silver sulfate can be used
to precipitate CI", but due
to incomplete precipitation
Reaction
Brucine, a strychnine compound
reacts with nitrate to form a
yellow compound which exhibits
maximum absorption at 410 nm.
The reaction according to the
procedure as outlined in Stand-
ard Methods (page 178) does not
follow Beer's Law. However,
a recent modification by Jenkins
and Medsker (2) has been devel-
oped. Conditions are controlled
in the reaction so that Beer's Law
is followed and concentrations
below 1 mg nitrate N/1 can be
determined.
Interferences
1) Nitrite may react the same
as nitrate but can be elim-
31-5
-------
Ammonia, Nitrites and Nitrate?
mated by the addition of sul-
fanilic acid to the brucine reagent
2) Organic nitrogen compounds may
hydrolyze and give positive
interference at low (less than lmg'l)
nitrate nitrogen concentrations
3) Residual chlorine may be
eliminated by the addition of sodium
arsenite
„ ¦ . - <8)
c Precision and Accuracy
1) Twenty-seven analysts in 15 lab-
oratories analyzed in natural
water samples containing the
following increments of inorganic
nitrate' 0.16, 0.19, 1.08 and
1. 24 mg N/liter
2) Precision results as standard
deviation were 0. 092, 0. 083,
0.245, and 0. 214 mg N/liter
respectively
3) Accuracy expressed as bias was
-0.01, +0.02, +0. 04 and+0. 04
mg N/liter, respectively.
3 Hydrazine Reduction
A method using hydrazine to reduce
nitrate to nitrite followed by sub-
sequent measurement of nitrite by
diazotization was recently reported
by Fishman, et al.1 The procedure
has been successfully adapted to the
Auto Analyzer where a high degree
of control of reaction conditions
can be achieved ^)
HI EPA METHODS
A The Environmental Protection Agency,
Office of Water Programs, Analytical
Quality Control Laboratory, has com-
piled a manual of analytical methods
for use by Federal laboratories. The
title of this manual is "Methods for
Chemical Analysis of Water & Wastes",
1971.
B The manual lists four nitrogen con-
stituents These are Ammonia Nitrogen,
Total Kjeldahl Nitrogen, Nitrate Nitrogen
and Nitrite Nitrogen.
1 Ammonia Nitrogen
The recommended procedures are
either an automated method or
distillation followed by titration (for
high concentration samples) or
Nesslerization (for low concentration
samples)
2 Total Kjeldahl Nitrogen
The total Kieldahl nitrogen is determined
by an automated phenolate procedure,
or by mercuric sulfate-sulfuric acid-
potassium sulfate digestion followed
by titration or colorimetric analysis
3 Nitrate Nitrogen
The automated method of determination
involves reduction of the nitrate to
nitrite with hydrazine, followed by
colorimetric analysis The manual
method is a spectrophotometry
procedure in which a yellow color is
produced by the reaction the nitrate
ions with brucine sulfate and
sulfanilic acid
4 Nitrite Nitrogen
Nitrite is determined by diazotizing
sulfanilamide with nitrite ions under
acid conditions, coupling the product
with N '1-Naphthyl) ethylenediamine,
and reading the resultant red-purple
color at 540 nm.
REFERENCES
1 Fishman, Marvin J , Skougstad, Marvin
W , and Scarbio, George, Jr
Diazotization Method for Nitrate and
Nitrite JAWWA 56-633-638 May, 1960
2 Jenkins, David and Medsker, Lloyd L
Brucine Method for Determination of
Nitrate in Ocean, Estuanne and Fresh
Waters Anal Chem 36-610-612
March, 1964
3 Kamphake, L J , Chemist, Engineering
Section, Basic and Applied Sciences
Branch, DWS & PC, Robert A Taft
Sanitary Engineering Center, Personal
Communication
31-6
-------
Ammonia, Nitrites and Nitrates
4 Lishka, R J., Lederer, L A , and
McFarren, E F Water Nutrients
No. 1, Analytical Reference Service
1966
5 Sawyer, Clair N Chemistry for Sanitary
Engineers. McGraw-Hill Book Co ,
New York 1960
6 Standard Methods for the Examination of
Water and Waste Water APHA. AWWA.
WPCF. 13th Ed. 1971.
7 ASTM Book of Standards, Part 23, 1970.
8 Methods for Chemical Analysis of Water &
Wastes, EPA-AQCL, Cincinnati,
Ohio 45268
This outline was prepared by Betty Ann
Punghorst, former Chemist, National
Training Center and Charles Feldmann,
Chemist, NTC, DTTB, MDS, OWP, EPA,
Cincinnati, OH 45268.
31-7
-------
LABORATORY PROCEDURE FOR NITRATE NITROGEN
MODIFIED BRUCINE METHOD
(Range 0. 1 to 2 mg nitrate nitrogen/1)
I REAGENTS
A Water
Distilled water free of nitrite and nitrate
should be used in the preparation of all
reagents and standards.
B Stock Potassium Nitrate
(1. 0 ml = 0.1 mg NOg" N):
Dissolve 0. 7218 g of anhydrous KNO,
in distilled water and dilate to 1 liter.
C Standard Potassium Nitrate
(1. 0 ml = 0. 001 mg N03~ N):
Dilute 10. 0 ml of the stock solution to
1 liter with distilled water. This solution
should be prepared fresh weekly.
D Sulfuric Acid:
Carefully add 500 ml of concentrated
^2^4 Sr• to *25 ml of distilled
water. Coor and keep tightly stoppered.
E Sodium Chloride <30%):
Dissolve 30 g of NaCl in distilled water
and dilute to 100 ml.
F Brucine - sulfanilic acid reagent:
Dissolve 1 g of brucine sulfate,
^C23H26N2°4*2 ' H2S°4 • 7 HgO, and
0. 1 g of sulfanilic acid (NH„CgH^S03H •
in 70 ml of hot distilled wafer.
Add 3 ml of concentrated HC1, cool, and
dilute to 100 ml. This solution is stable
for several months if stored in a dark bottle
at 5°c. The pink color which develops
slowly does not effect the usefulness of the
solution. The bottle should be marked "toxic".
G Acetic Acid:
Dilute 1 vol of glacial HCgHgOg with 3 vols
of distilled water.
II SAMPLE PREPARATION
A In the case of highly alkaline waters, it
is necessary to adjust the pH to
approximately 7 with the acetic acid.
B Filter through a 0. 45 micron pore
size filter if necessary.
Ill PROCEDURE
A PipetO.O, 2.0, 5.0, 7.0, and 10 0 ml
of the standard KNO, solution into
50 ml test tubes held in a suitable rack.
Add sufficient distilled water to bring
the volume to 10.0 ml.
B Pipet2.0, 3.0, and 5.0 ml. of the
sample into 50 ml test tubes. The
purpose of using three different volumes
of the sample is to ensure that when the
colors are developed, at least one of the
three samples will give an absorbance
which lies within the range of the
calibration curve Add sufficient dis-
tilled water to bring the volume to
10 ml.
C If the samples are saline, pipet 2. 0 ml
of 30% NaCl into the standard and
sample tubes. Mix well This addition
is unnecessary for fresh water samples.
D Chill all of the tubes to 0 - 10<>C in a
cold water bath.
E Pipet 10. 0 ml of the H^SO solution into
each standard and sample tube, mix
well by swirling, and again chill to
0 - 10OC.
CH. N. lab.2e. 12. 71
32-1
-------
Laboratory Procedure for Nitrate Nitrogen
F Pipet 0. 5 ml of the brucine - sulfanilic
acid reagent into the standard and
sample tubes, mix well by swirling, and
place the tubes in a boiling water bath
for 25 minutes.
G Remove the tubes from the boiling water
bath and cool to 20-25° C in a water bath.
H If color or turbidity develops in the
sample while the tubes are in the boiling
water bath, a blank must be prepared
using all reagents except the brucine -
sulfanilic acid.
K Determine the mg of NO^ N present
in the samples using the calibration
curve and then calculate the mg of
NOg N/1 using the formula:
mg of NO. N/1 ¦
tJ
mg of NOg N from curve X 1000 ml/1
ml of sample
I Measure the absorbance values of the
standards and samples at 410 nm.,"
on a suitable spectrophotometer.
Before each solution is read, rinse the
spectrophotometer cell at least twice
with the solution.
J Prepare a calibration curve of absorbance
values for the standards vs. mg of
N03 N. For example- if 2. 0 ml of the
standard KNO^ solution are used, and
its concentration isO. OOl.mg of NO„ N/ml,
then0.002mg of NOg~ is the value
plotted on the calibration curve vs. the
corresponding absorbance value.
REFERENCES
Methods for Chemical Analysis of
Water & Wastes, EPA-AQCL, 1971. /
This outline was prepared by Charles R.
Feldmann, Chemist, National Training
Center, MDS, WPO EPA,
Cincinnati, OH 45268.
32-2
-------
DETERMINATION OF PHOSPHORUS IN THE AQUEOUS ENVIRONMENT
I Phosphorus is closely associated with
water quality because of (a) its role in
aquatic productivity such as algal blooms,
(b) its sequestering action, which causes
interference in coagulation, (c) the difficulty
of removing phosphorus from water to some
desirable low concentration, and (d) its
characteristic of converting from one to
another of many possible forms.
A Phosphorus is one of the primary nutrients
such as hydrogen (H), carbon (C),
nitrogen (N), sulfur (S) and phosphorus (P).
1 Phosphorus is unique among nutrients
in that its oxidation does not contribute
significant energy because it commonly
exists in oxidized form.
2 Phosphorus is intimately involved in
oxidative energy release from and
synthesis of other nutrients into cell
mass via:
a Transport of nutrients across
membranes into cell protoplasm is
likely to include phosphorylation.
3 The concentrations of P in hydro soils,
sludges, treatment plant samples and
soils may range from 10^ to 10® times
that in stabilized surface water. Both
concentration and interfering compo-
nents affect applicability of analytical
techniques.
II The primary source of phosphorus in the
aqueous system is of geological origin.
Indirect sources are the processed mineral
products for use in agriculture, household,
industry or other activities.
A Agricultural fertilizer run-off is related
to chemicals applied, farming practice
and soil exchange capacity.
B Wastewaters primarily of domestic
origin contain major amounts of P from-
1 Human, animal and plant residues
2 Surfactants (cleaning agent) discharge
3 Microbial and other cell masses
b The release of energy for meta-
bolic purposes is likely to
include a triphosphate exchange
mechanism.
C Wastewaters primarily of industrial
origin contain P related to:
1 Corrosion control
B Most natural waters contain relatively low
levels of P (0.01 to 0.05 mg/1) in the
soluble state during periods of significant
productivity.
1 Metabolic activity tends to convert
soluble P into cell mass (organic P) as
a part of the protoplasm, intermediate
products, or sorbed material.
2 Degradation of cell mass and incidental
P compounds results in a feedback of
lysed P to the water at rates governed
by the type of P and the environment.
Aquatic metabolic kinetics show marked
influences of this feedback.
2 Scale control additives
3 Surfactants or dispersants
4 Chemical processing of materials
including P
5 Liquors from clean-up operations of
dusts, fumes, stack gases, or other
discharges
III Phosphorus terminology is commonly
confused because of the interrelations among
biological, chemical, engineering, physical,
and analytical factors.
CH.PHOS.4c.12.71 33-1
-------
Determination of Phosphorus in the Aqueous Environment
A Biologically, phosphorus may be available
as a nutrientj synthesized into living mass,
stored in living or dead cells, agglomerates,
or mineral complexes, or converted to
degraded materials.
B Chemically, P exists in several mineral
and organic forms that may be converted
from one to another under favorable
conditions. Analytical estimates are
based upon physical or chemical techniques
necessary to convert various forms of P
into ortho phosphates which alone can be
quantitated in terms of the molybdenum
blue colorimetric test.
C Engineering interest in phosphorus is
related to the prediction, treatment, or
control of aqueous systems to favor
acceptable water quality objectives.
Phosphorus removal is associated with
solids removal.
D Solubility and temperature are major
physical factors in phosphorus behavior.
Soluble P is much more available than
insoluble P for chemical or biological
transformations and the rate of conversion
from one to another is strongly influenced
by temperature.
E Table 1 includes a classification of the
four main types of chemical P and some
of the relationships controlling solubility
of each group. It is apparent that no
clear-cut separation can be made on a
solubility basis as molecular weight,
substituent and other factors affect
solubility.
F Table 2 includes a scheme of analytical
differentiation of various forms of P
based upon:
1 The technique required to convert an
unknown variety of phosphorus into
ortho P which is the only one quanti-
tated by the colorimetric test.
33-2
2 Solubility characteristics of the sample
P or more precisely the means required
to clarify the sample.
a Any clarification method is subject
to incomplete separation. Therefore,
it is essential to specify the method
used to interpret the yield factor of
the separation technique. The
degree of separation of solubles
and insolubles will be significantly
different for-
1 Membrane filter separation
(0. 5 micron pore size)
2 Centrifugation (at some specified
rpm and time)
3 Paper filtration (specify paper
identification)
4 Subsidence (specify time and
conditions)
G Analytical separations (Table 2) like those
m Table 1, do not give a precise separa-
tion of the various forms of P which may
be included quantitatively with ortho or
poly P. Conversely some of the poly and
organic P will be included with ortho P if
they have been partially hydrolyzed
during storage or analysis. Insolubles
may likewise be included as a result of
poor separation and analytical conditions.
1 The separation methods provide an
operational type of definition adequate
in most situations if the "operation"
is known. Table 2 indicates the nature
of incidental P that may appear along
with the type sought.
-------
Determination of Phosphorus in the Aqueous Environment
Table 1
PHOSPHORUS COMPOUNDS CLASSIFIED BY
CHEMICAL AND SOLUBILITY RELATIONS
Form
Water Soluble
(1)
Insoluble
(1)
1. Ortho phosphates
(PO f3
4
2. Poly phosphates
' W' "4< W^W"'
and others depending upon
the degree of dehydration.
3. Organic phosphorus
R-P, R-P-R (2)
(unusually varied nature)
Combined with monovalent
cations such as H, Na, K, NH
as in 1
Increasing dehydration
decreases solubility
(a) certain chemicals
(b) degradation products
(c) enzyme P
(d) phosphorylated nutrients
Combined with multi
valent cations such
^ +2.,+3^ +3
as Ca A1 Fe
(a) as in 1
(b) multi P polyphosphates
(high mol. wt.) in-
cluding the "glassy"
phosphates
(a) certain chemicals
(b) cell mass, may be
colloidal or agglom-
erated
(c) soluble P sorbed by
insoluble residues
4. Mineral phosphorus
(a) as in 1
(a) as in 1
(b) as in 2
(c) geological P such as
phosphosilicates
(d) certain mineral com-
plexes.
(1) Used in reference to predominance under common conditions.
-3
(2) R represents an organic radical, Prepresents P, P04» °r derivatives.
Total P in Table 2 includes liquid and
separated residue P that may exist in
the whole sample including silt, organic
sludge, or hydrosoils. This recognizes
that the feedback of soluble P from
deposited or suspended material has a
real effect upon the kinetics of the
aqueous environment.
33-3
-------
Determination of Phosphorus in the Aqueous Environment
Table 2
PHOSPHORUS COMPOUNDS CLASSIFIED BY
ANALYTICAL METHODOLOGY
Desired P Components
Technique
(2)
Incidental P Included
1. Ortho phosphates
No treatment on clear
samples
Easily hydrolyzed
(a) poly phosphates -
(b) organic -P, -
(c) Mineral -P, + or -
2. Polyphosphates
(2)-( 1) = poly P
(hydrolyzable)
acid hydrolysis on clear
samples, dilute
(a) H2S04
(b) HC1
heated
(a) ortho-P +
(b) organic -P + or -
(c) mineral -P + or -
3. Organic phosphorus
(3) - (2) + org P
(hydrolyzable)
acid + oxidizing hydrolysis
on whole sample, dilute
(a) H2SO + HNOg
ft>) H2so, ~ (NH4)2s2oa
heated
(a) ortho P +
(b) poly P +
(c) mineral P + or -
4. Soluble phosphorus
(preferably classified
by clarification method)
clarified liquid following
filtration, centrifugation
or subsidence
generally includes
(a) 1, 2, or 3
(b) particulates not
completely separated
5. Insoluble phosphorus
(residue from clari-
fication)
Retained residues separated
during clarification
See (6)
(a) generally includes
sorbed or complexed
solubles.
6. Total phosphorus Strong acid + oxidant all components in
digestion 1, 2, 3, 4, 5 in the
(a) H SO + HNO whole sample
2 4 3
(b) H SO + HNO + HCIO .
.4 4 j 4
(c) H202 + Mg
-------
Determination of Phosphorus in the Aqueous Environment
IV Polyphosphates are of major interest in
cleaning agent formulation, as dispersants,
and for corrosion control.
A They are prepared by dehydration of ortho
phosphates to form products having two or
more phosphate derivatives per molecule.
1 The simplest polyphosphate may be
prepared as follows
NaO NaO
HO"^P * ° heaU "°^P - O
/ + H2°
= o o
\
1
NaO
H O'J \P = O
HO'
mono sodium ortho disodium dihydrogen
phosphate (2) polyphosphate
2 The general form for producing
polyphosphates from mono substituted
orthophosphates is
° „+ " H2°
3 Di-substituted ortho phosphates or
mixtures of substituted ortho phosphates
lead to other polyphosphates
insoluble polyphosphate than the same
cation in the form of insoluble ortho
phosphate. Insolubility increases with
the number of P atoms in the
polyphosphate. The "glassy" poly-
phosphates are a special group with
limited solubility that are used to aid
corrosion resistance in pipe distribu-
tion systems and similar uses.
B Polyphosphates tend to hydrolyze or
"revert" to the ortho P form by addition
of water. This occurs whenever
polyphosphates are found m the aqueous
environment.
1 The major factors affecting the rate of
reversion of poly to orthophosphates
include-
a) Temperature, increased T increases
rate
b) pH, lower pH increases rate
c) Enzymes, hydrolase enzymes
increase rate
d) Colloidal gels, increase rate
e) Complexing cations and ionic
concentration increase rate
f) Concentration of the polyphosphate
increases rate
heat.
disodium hydrogen
orlho phosphate
NaH PO.
2 4 7
mono sodium
di hydrogen
ortho phosphate
%P3°10
penta sodium
tn-phosphate
2 Items a, b and c have a large effect
upon reversion rate compared with
6ther factors listed. The actual
reversion rate is a combination of
listed items and other conditions or
characteristics.
4 The polyphosphate series usually
consist of the polyphosphate anion
with a negative charge of 2 to 5.
Hydrogen or metals commonly occupy
these sites. The polyphosphate can be
further dehydrated by heat as long as
hydrogen remains. Di or trivalent
cations generally produce a more
3 The differences among ortho and ortho
+ polyphosphates commonly are close to
experimental error of the colorimetric
test in stabilized surface water samples.
A significant difference generally
indicates that the sample was obtained
relatively close to a source of poly-
phosphates and was promptly analyzed.
This implies that the reversion rate of
polyphosphates is much higher than
generally believed.
33-5
-------
Determination of Phosphorus in the Aqueous Environment
V SAMPLING AND PRESERVATION
TECHNIQUES
A Sampling
1 Great care should be exercised to
exclude any benthic deposits from
water samples.
2 Glass containers should be acid rinsed
before use.
3 Certain plastic containers may be
used. Possible adsorption of low con-
centrations of phosphorus should be
checked.
4 If a differentiation of phosphorus forms
is to be made, filtration should be
carried out immediately upon sample
collection. A membrane filter of
0.45m pore size is recommended for
reproducible separations.
B Preservation
1 If at all possible, samples should be
analyzed on the day of collection. At
best, preservation measures only
retard possible changes in the sample.
3 Addition of 40 mg HgClg/liter is
recommended for longer storage
periods. This chemical limits
biological changes.
a HgCl„ is an interference in the
analytical procedure if the
chloride level is low (See Part
VI, B3).
VI THE EPA ANALYTICAL PROCEDURE^6'
A This is a colorimetnc determination,
specific for orthophosphate. Depending
on the nature of the sample and on the
type of data sought, the procedure in-
volves two general operations:
1 Conversion of phosphorus forms to
soluble orthophosphate (See Fig. 1)'
a sulfuric-acid-hydrolysis for
polyphosphates, and some
organic P compounds,-
b persulfate digestion for organic
P compounds.
a Possible physical changes include
solubilization, precipitation,
absorption on or desorption from
suspended matter.
b Possible chemical changes include
reversion of poly to ortho P and
decomposition of organic or min-
eral P,
c Possible biological changes
include microbial decomposition
of organic P and algal or
bacterial growth forming organic
P.
2 Refrigeration at 4°C is recommended
if samples are to be stored. This
decreases hydrolysis and reaction
rates and also losses due to volatility.
2 The color determination involves
reacting dilute solutions of phosphorus
with ammonium molybdate and
potassium antimonyl tartrate in an
acid medium to form an antimony-
phosphomolybdate complex. This
complex is reduced to an intensely
blue-colored complex by ascorbic
acid. The color is proportional to
the orthophosphate concentration.
Color absorbance is measured at
880 nm and a concentration value
obtained using a standard curve.
Reagent preparation and the detailed
procedure can be found in the EPA
manual.
The methods described there are
usable in the 0.01 to 0.5 mg/liter
phosphorus range.
33-6
-------
Determination of Phosphorus in the Aqueous Environment
fotwl Un
0 45 Micron Membrane Filter
FllCAT
SolubU
Ortho?ho«phat«
Total Hydroljinbla
t Orthophe*pb*c«
Total Soluble
PSwp'iorus
Total
OiChophoaphiC*
Soluble Uydrolysibla
6 Orchophosphac*
Jnoi tnic
phosphorus
cocpounds
polyphosphate
+ soma organic
phosphorus
coDpouads
inorganic «¦
organic
phosphorus
compounds
ANALYTICAL SCHEME FOR DIFFER EN HATION OF PHOSPHORUS FORMS
B Interferences
Erroneous results from contaminated
glassware is avoided by cleaning it
with hot 1:1 HC1, treating it with
procedure reagents and rinsings
with distilled water. Preferably
this glassware should be used only
for the determination of phosphorus
and protected from dust during
storage. Commercial detergents
should never be used.
High iron concentrations in samples
can precipitate phosphorus.
If HgCl„ is used as a preservative,
it interferes if the chloride level of
the sample is less than 50 mg
Cl/liter. Spiking with NaCl is then
recommended.
Others have reported interference
from chlorine, chromium, sulfides,
nitrite, tannins, lignin and other
minerals and organics at high con-
centrations.
/ g \
C Precision and Accuracy
1 Organic phosphate - 33 analysts in
19 laboratories analyzed natural
water samples containing exact in-
crements of organic phosphate of
0. 110, 0. 132, 0.772, and 0.882 mg
P/liter.
Standard deviations obtained were
0.033, 0.051, 0.130 and 0.128
respectively.
Accuracy results as bias, mg P/liter
were: +0.003, +0.016, +0.023 and
- 0.008, respectively.
2 Orthophosphate was determined by
26 analysts in 16 laboratories using
samples containing orthophosphate
in amounts of 0. 029, 0. 038, 0.335
and 0.383 mg P/liter.
Standard deviations obtained were
0.010, 0.008, 0.018 and 0.023
respectively.
33-7
-------
Determination of Phosphorus in the Aqueous Environment
Accuracy results as bias, mg P/liter
were -0.001, -0.002, -0.009 and
-0.007 respectively.
VII VARIABLES IN THE COLORIMETRIC
PROCEDURE
Several important variables affect
formation of the yellow heteropoly
acid and its reduced form, molybdenum
blue, in the colorimetnc test for P.
A Acid Concentration during color develop-
ment is critical. Figure 2 shows that
color will appear in a sample containing
no phosphate if the acid concentration
is low. Interfering color is negligible
when the normality with respect to
HgSO^ approaches 0.4.
1 Acid normality during color develop-
ment of 0.3 to slightly more than
0.4 is feasible for use. It is prefer-
able to control acidity carefully and
to seek a normality closer to the
higher limits of the acceptable range.
2 It is essential to add the acid and
molybdate as one solution.
3 The aliquot of sample must be
neutralized prior to adding the
color reagent.
0.05 mg P
STD-BLK
BLANK
0.2 03 04 0.5 06
H.SO. NORMALITY
Figure 2
0-PHOSPHATE COLOR
VS ACIDITY
B Choice of Reductant - Reagent stability,
effective reduction and freedom from
deleterious side effects are the bases
for reductant selection. Several re-
ductants have been used effectively.
Ascorbic acid reduction is highly
effective in both marine and fresh water.
It is the reductant specified in the
EPA method.
C Temperature affects the rate of color
formation. Blank, standards, and
o
samples must be adjusted to + 1 C,
preferably room temperature, before
addition of the acid molybdate reagent.
D Time for Color Development must be
specified and consistent. After addition
of reductant, the blue color develops
rapidly for 10 minutes then fades grad-
ually after 12 minutes.
VIII DETERMINATION OF TOTAL
PHOSPHORUS
A Determination of total phosphorus
content involves omission of any
filtration procedure and using the acid-
hydrolysis and persulfate treatments
to convert all phosphorus forms to the
test-sensitive orthophosphate form.
B Determining total phosphorus content
yields the most meaningful data since
the various forms of phosphorus may
change from one form to another in a
short period of time. (See part V, Bl)
IX DEVELOPMENT OF A STANDARD
PROCEDURE
Phosphorus analysis received intensive
investigation, coordination and validation of
methods is more difficult than changing
technique.
33-8
-------
Determination of Phosphorus 111 the Aqueous Environment
A Part of the problem in methods arose
because of changes in analytical objectives
such as:
1 Methods suitable to gather "survey"
information may not be adequate for
"standards".
2 Methods acceptable for water are not
necessarily effective in the presence
of significant mineral and organic
interference characteristic of hydro-
soils, marine samples, organic
sludges and benthic deposits.
3 Interest has been centered on "fresh"
water, it was essential to extend them
for marine waters.
4 Instrumentation and automation have
required adaptation of methodology.
B Analysts have tended to work on their own
special problems. If the method
apparently served their situations, it was
used. Each has a "favorite" scheme that
may be quite effective but progress
toward widespread application of "one
method has been slow. Consequently,
many methods are available. Reagent
acidity. Mo content, reductant and
separation techniques are the major
variables.
C At the present time there is not sufficient
data to warrant EPA endorsement of the
P procedure for sediment-type samples,
sludges, algal blooms, etc. Following
is a procedure (not included in the EPA
manual) which is useful when solids
are present in samples.
1 If sample contains large particles,
grind and emulsify solids in a blender.
2 Transfer 50 ml sample, or aliquot
diluted to 50 ml, into a 250 ml
Erlenmeyer flask
3 Add 6. 0 ml of 18N H2SO4, 5 ml
concentrated HN(5q, 2 berl saddles
and digest on hot plate
4 Digest until the disappearance of nitric
acid fumes and the appearance of white
SO3 fumes. Continue digestion for
approximately 5 minutes. Cool before
proceeding with Step 5.
5 Add 2 ml of HNO3-HCIO4 mixture
and 5 ml concentrated HNO3. Continue
digestion until all of the nitric acid is
driven off and dense fumes of perchloric
evolve. Perchloric acid requires
dilution with sulfuric acid and prior
destruction of most organics for safety.
6 Cool. Add approximately 40 ml
distilled water and transfer to 100 ml
volumetric flask.
7 Add 2-3 drops of phenolphthalein and
concentrated ammonium hydroxide
until a pink color is seen. Then
discharge the pink color with the
strong sulfuric acid. It is advisable
to add an equivalent amount of salt
formed during neutralization of digested
samples to the calibration standards to
equalize salt content during color
development.
8 Determine orthophosphate according
to the usual color procedure.
ACKNOWLEDGMENT:
Materials in this outline include significant
portions of previous outlines by J. M. Cohen,
L. J. Kamphake, and R. J. Lishka. Important
contributions and assistance were made by
R. C. Kroner, E. F. Barth, W. Allen Moore,
Lloyd Kahn, Clifford Riejey, Lee Scarce,
John Winter, Ferd Ludzack and Charles
Feldmann*.
REFERENCES
1 Jenkins, David, A Study of Methods
Suitable for the Analysis and
Preservation of Phosphorus Forms
in the Estuarine Environment. DHEW,
Central Pacific River Basins Project.
SERL Report No. 65-18, University
of California, Berkeley, Calif.
November 1965.
33-9
-------
Determination of Phosphorus in the Aqueous Environment
2 Gales, Morris E. , Jr. , Julian, Elmo C. ,
and Kroner, Robert C., Method for
Quantitative Determination of Total
Phosphorus in Water. JAWWA 58:
(10) 1363. October 1966.
3 Lee, G. Fred, Clesceri, Nicholas L. and
Fitzgerald, George P., Studies on the
Analysis of Phosphates in Algal Cultures.
Int. J. Air & Water Poll. 9:715. 1965.
4 Barth, E. F. and Salotto, V. V.,
Procedure for Total .Phosphorus
in Sewage and Sludge, Unpublished
Memo, Cincinnati Water Research
Laboratory, FWQA. April 1966
5 Moss, H. V. , (Chairman, AASGP
Committee) Determination of Ortho
Phosphate, Hydrolyzable Phosphate
and Total Phosphate in Surface Water,
JAWWA 56:1563. December 1958.
6 Methods for Chemical Analysis of Water
& Wastes, EPA-AQCL, 1971.
This outline was prepared by Audrey E.
Donahue, Chemist, National Training Center,
DTTB, MDS, WPQ EPA, Cincinnati,
CH 45268.
33-10
-------
LABORATORY PROCEDURE FOR PHOSPHORUS
I OBJECTIVE
The concentration of phosphorus (including available mineral, organic, poly and ortho
phosphate) in the whole sample from a wastewater treatment plant is sought.
II METHOD SUMMARY:
A The EPA Official Method is used. Certain non-official procedures are included to adapt
this exercise for group participation and according to sample characteristics. Class use
of 1 cm cells necessitates a stronger reference solution. Omitting the digestion of reference
standards also deviates from the official method.
B Mixed phosphorus forms in the sample will be converted into ortho phosphate by digestion at
boiling temperature for 30 minutes in the presence of sulfuric acid and the oxidant, ammonium
persulfate.
Excess acid will be neutralized, the sample diluted to its original volume and aliquots diluted
to 50 ml for comparison with a series of reference sample dilutions and a reagent blank.
Both sample and reference dilutions are adjusted to the same volume and mixed reagent
added at the same time for comparative color development.
All solutions used in the procedure should be at room temperature or within + 1°C of the
same temperature.
C You may encounter 0. 5 to 15 mg P/l in a sample of the type provided. Several small sample
aliquots diluted to volume should be prepared along with your reference series to obtain at
least one sample aliquot withm the readable absorbance range of the references. Suggested
sample aliquots in the procedure will give at least one readable result for a sample range
of 0.1 to 20 mg P/liter
IE ANALYTICAL PROCEDURE
A The conversion of phosphorus forms to orthophosphate
1 Measure 50 ml of the well mixed sample provided into one of the 125 ml erlenmeyer flasks.
2 Add one of the berl saddles to the sample aliquot.
3 Add one ml or 20 drops of strong acid to the sample aliquot.
4 Add 1 scoopful or approximately 0.4 g of dry powder ammonium persulfate,
(NH )„So0_, to the sample aliquot.
4 2 6 o
5 Mix and place the sample flask on a preheated hot plate. Adjust heat to obtain a gentle
boiling rate for 30 minutes. Add distilled water if necessary to prevent the sample volume
from evaporating to a volume of less than 10 ml.
6 After 30 minutes boiling remove the digestion flask from the hot plate and cool it. If you
cannot complete the analysis at this time, hold it at this stage after covering the flask
with aluminum foil or an inverted beaker to protect it from contamination
CH.PHOS.lab.3.12.71
34-1
-------
Laboratory Procedure for Phosphorus
B Colorimetric Determination of Orthophosphate
1 Add 1 drop of phenolphthalein solution. Add NaOH solution dropwise to neutralize excess
acid until the mixed digestate develops a red color. Add one drop of strong acid to
eliminate the indicator color.
2 Adjust the volume of the digestate to its original volume of 50 ml by addition of distilled
water. Mix.
3 Identify each of the seven remaining 125 ml erlenmeyer flasks as follows: ' Blank, 10-R,
20-R, 40-R, 0.5-S, 2.0-Sand 10.0-S.
4 Add 50 ml of distilled water to the blank. Add 10 ml of reference solution + 40 ml of
distilled water to the 10-R flask. Add appropriate amounts of reference solution or
sample as indicated to the remaining flasks, plus enough distilled water to make a final
volume of 50 ml in each flask. (It will not be necessary to add the indicator and adjust
pH with NaOH or acid because all mixtures are approximately neutral at this stage. If
you are not sure - check it).
5 Add 8 ml of mixed reagent to each of the reference and sample aliquots and to the 50 ml
blank. Mix each one after addition.
6 After a 10 minute color development time (and not longer than 30 minutes), measure
absorbancy of the color produced versus the reagent blank. (Adjust the instrument to
zero absorbance (A) with the reagent blank in the light path).
Record results on the following data table:
Item
ml R or S
Mg P/50 ml
mg P/l
A
Blank
0
0
0
0
10-R
10-R
12.5
0.25
20-R
20-R
25.0
0.50
40-R
40-R
50.0
1.00
0.5-S
0.5-S
2.0-S
2.0-S
10 0-S
' 10.0-S
These data items require measurement of the absorbancy produced versus the reagent
blank (adjust instrument to zero A for the blank) to estimate the mg P/l m the sample.
Note that both the |Ltg P/50 ml and the equivalent mg P/l are listed in separate columns
in the data record. The ng column is for check purposes to avoid calculation errors.
Data is reported as mg P/liter
34-2
-------
Laboratory Procedure for Phosphorus
IV EVALUATING PHOSPHORUS CONCENTRATION
A Procedure
1 Consulting the data table for a range of values, mark absorbance vs mg P/liter scales
on the coordinate paper provided. Also, mark the ng P/50 ml values below the mg
P/liter scale values.
2 Plot the data for the Reference samples and draw a line of best fit
3 Plot the data for the Sample aliquots and choose the valid result{s).
4 Evaluate the valid Sample points on the curve as mg P/liter.
5 Check calculations using the ng P/50 ml scale.
6 An example of this whole procedure follows.
B The Standard Curve
Reference and sample values have been plotted on this graph:
Absorbance values for References
Absorbance values for Samples
.0
©10 ml S
.8
.6
.4
2.0 ml S
0.2
u. 05
0.25 0.50 0.75 1.00
mg P/liter mg P/liter mg P/liter
(12.5 Mg P/50 ml) (25. 0 ng P/50 ml) (50. 0 Mg P/50 ml)
In this example, there is only one sample volume that falls on the most readable range
of the instrument and within the calibrated range of the Absorbance plot - the 2. 0 ml
sample. The other two sample volumes gave invalid results.
34-3
-------
Laboratory Procedure for Phosphorus
C Using the Standard Curve as shown in B above, the next step is to find mg P/liter
of the sample when absorbance on the 2 ml sample diluted to 50 ml was 0. 35: Draw a
dashed horizontal line from the absorbance scale reading of 0. 35 to the calibration line.
Draw a vertical line from that intersection to the concentration scale. This reads approx-
imately 0.45 mg P/liter for the diluted aliquot color. Since this sample was diluted from
2 to 50 ml, the original sample concentration was 0. 45 x 50 = 0. 45 x 25 or 11.25 mg P/1.
2
D Checking Calculations:
With the same observed values and manipulation but using the /ug P/50 ml scale, the vertical
intercept falls at about 22 ng P/50 ml in the sample. For a sample containing 22 ng P in
the diluted 2 ml aliquot, the volume correction becomes 22 x 1000 = 22 x 500 = 11000 Mg P/l
or 11. 00 mg P/1. 2
It is evident that the estimation of intercept positions on the small scales with infrequently
marked positions yields a lack of precise agreement for the two independent calculations.
The general agreement, however, does eliminate the question of a possible serious
dilution error which frequently plagues the results.
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, DTTB,
MDS, WPQ EPA, Cincinnati, OH 45268.
34-4
-------
VII SOLIDS RELATIONSHIPS IN POLLUTED WATER -
GREASE AND OIL
Solids control is a prime necessity and the major problem
in wastewater treatment. Solids are intimately related to
oxygen demand and feed back or recycle of polluting
materials depending upon the degree of contact with the
aqueous system. Solids separation from water is difficult
and comprises a major part of treatment dynamics.
Stabilization and disposal poses a two pronged difficulty
of what to do with it and where to put it to minimize feedback.
Contents of Section VII
Outline Number
Solids Relations in Polluted Water
35
Determination of Suspended Solids
36
Laboratory Procedure for the
Determination of Total Solids
37
Laboratory Procedure for Non-
filterable (Suspended) Solids
38
Laboratory Procedure for
Filterable (Dissolved) Solids
39
Laboratory Procedure for Volatile
Solids
40
The Determination of Oil and
Grease
41
-------
SOLIDS RELATIONS IN POLLUTED WATER
I MPN, oxygen demand, and solids have
been major water pollution control criteria
for many years. This discussion is con-
cerned primarily with solids and their inter-
relations with oxygen demand.
A Engineered treatment or surface water
self-purification depends upon:
1 The conversion of soluble or colloidal
contaminants into agglomerated masses
that may be separated from the water.
2 Oxidation of putrescible components
into stable degradation products.
3 Item 1 is the major concern in most
treatment systems because item 2
requires a greater investment in time,
manpower and capital costs.
II A given wastewater may have several
forms of solids in changeable proportions
with time and conditions.
A Solids may be classified among the charted
forms according to biological, chemical
or physical properties. It is not possible
to precisely classify a given material into
any one form because they usually are
mixtures that may include or be converted
mto other forms.
B Interrelationships are indicated by diagram
in Figure 1. Some changes occur more
readily than others.
1 Settleable solids generally consist of
a mixture of organic, inorganic, en-
trained dissolved or colloidal solids
with living and dead organisms.
B Stress on oxygen demand removal frequently
results in an unduely small amount of
attention to the contribution of solids in
the oxygen demand picture.
1 Oxygen demand formulations generally
are specified to be applicable in the
absence of significant deposition.
2 Increasing impoundment, tidal estu-
aries, and incomplete solids removal,
generally ensure that solids deposition
will be significant.
3 The BOD test stresses the fraction of
oxygen demand that is exerted relatively
rapidly. It includes only the fraction
of unstable material that is exerted
under test conditions - usually short
term.
4 Contributions of a bed load of solids to
oxygen demand frequently are incom-
pletely recognized because they have a
more local effect, are difficult to meas-
ure, tend to move, and are incompletely
understood.
a They may be hydrolyzed into smaller
molecules to acquire colloidal or
dissolved characteristics.
b They may be converted into a larger
fraction of living material or vice
versa.
2 Colloidal solids also are likely to be a
mixture of organic and inorganic
materials, living or dead containing
associated dissolved materials.
a They are likely to agglomerate to
form settleable masses with time
or changing conditions.
b Chemical reactions may solubilize
the colloidal masses for recombina-
tion into other forms.
3 Dissolved solids are most readily avail-
able of all forms for biological, chemi-
cal or physical conversion.
PC. 6a. 9. 72
35-1
-------
Solids Relations in Polluted Water
'agglom-
eratlon
slow
growth
orption
disper
sion i
Colloidal
Settleable
Dead Organic
Inorganic
Dissolved
Figure 1. SOLIDS INTERRELATIONSHIPS IN WATER
a They may be assimilated into cell
mass to become colloidal during a
state of rapid growth or form settle -
able masses during slow growth.
Surface phenomena are likely to en-
courage inclusion of other forms of
solids with cell mass.
Stabilization occurs with each conversion
because energy is expended with each
change.
1 Biological changes involve oxidation to
obtain enough energy to synthesize cells.
a Products consist of cell mass and
degradation products.
b Oxidation tends toward production
of C02, H20, NO^, S04"2etc. C02
has limited water solubility and
partially leaves the aqueous environ-
ment. More soluble constituents
tend to remain with it for recycle.
c Biological residues tend to recycle.
Eventually the mass consists of
relatively inert and largely insolu-
ble residues. These make up the
35-2
-------
I
Solids Relations in Polluted Water
bed load that may decompose at a
rate of less than 1% per day, but
accumulates in mass until it be-
comes dominant in water pollution
control activities.
Ill Treatment is an engineered operation
designed to utilize events in surface water
self-purification in a smaller package in
terms of space and time. Effective treat-
ment presupposes oxidation either in the
plant or in facilities to minimize feedback
of solids components to the aqueous C
environment.
A Primary treatment consists of a separation
of floatable or settleable solids and re-
moval from the used water.
1 Prompt removal is essential to minimize
return of solubilized or leached materi-
als from the sludge mass.
2 Sludge and scum are highly putrescible
and difficult to dram.
3 Subsequent treatment prior to disposal
serves to enhance drainability, reduce
solids or volume for burial or burning.
B Secondary treatment generally involves
some form of aerobic biological activity
to oxidize part of the colloidal and dis-
solved contaminants and convert most of
the remainder into a settleable sludge.
1 Aerobic systems favor assimilation of
nutrients into cell mass at the expense
of part of the available energy repre-
sented by oxidation to products such as
CC>2 and water.
2 Cell mass and intermediate degradation
products are only partially stabilized
and tend to recycle with death and decay.
3 Rapid growth of cells tends to produce
sludges that are highly hydrated and
thin.
4 A compromise must be reached to pro-
duce a favorable balance among separa-
tion of solids and feedback of lysed
materials.
a Cell growth continues as long as
essential nutrients are available.
b Under limiting nutrient conditions
the population may show a relatively
low rate overall dieoff but variety
is changing.
c Species most favored by the new con-
ditions tend to grow while others
die, lyse and release part of their
stored nutrients for subsequent use.
Anaerobic digestion of solids separated
during treatment is one process used to
increase solids stability and drainability
while reducing total volume or mass for
disposal.
1 Growth of cell mass is relatively slow
under anaerobic conditions while hy-
drolytic cleavage is relatively large
in comparison to that during aerobic
metabolism.
a Feedback to the aqueous environment
represents a significant fraction of
the input in the form of ammonia,
colloidal solids, low molecular
weight acids, and other products.
b Mass of the sludge is reduced by the
fraction of methane, carbon dioxide,
and other gases produced in process.
c Remaining solids tend to be more
concentrated, are lower in putresci-
bility and give up their water more
readily.
2 The liquid fraction of the products re-
maining after anaerobic digestion con-
tain nutrients, oxygen demand, solids,
and malodorous constituents that are
objectionable in surface waters if re-
leased without further aerobic
stabilization.
/
3 Digester liquids are much more con-
centrated than raw sewage and nutrition-
ally unfavorable, hence they are difficult
to treat and tend to shock aerobic
treatment processes.
35-3
-------
Solids Relations in Polluted Water
D Suitable disposal of solids resulting from
treatment operations takes various forms
of which the most desirable is to oxidize
it completely to gaseous products of oxida-
tion or inert ash. The objective is to limit
feedback, into the aqueous environment and
delay the charted interchanges.
1 Deposition of sludge as a cover on crop
land is effective providing surface
drainage is properly designed to limit
washoff.
2 Burial in areas above the flood plain
provides a suitable disposal.
3 Incineration under controlled conditions
to produce complete burning of orgamcs
is preferable.
a Incineration of digested sludge im-
plies substantial feedback in process.
b Incineration of freshly formed sludge
is difficult because of drainability
and concentration problems. It is
preferable from the standpoint of
maximum destruction of organic
pollutants and minimum feedback in
process. Current investigations are
directed toward improving feasibility
of this means of disposal of solids.
IV ANALYTICAL PROCEDURES
A The Analytical Quality Control
Laboratory, Office of Water Programs,
Environmental Protection Agency has
published a manual titled, Methods for
Chemical Analysis of Water and Wastes,
1971.
B In this manual solids are classified into
four groups:
1 Total solids
2 Non-filterable (suspended) solids
3 Filterable (dissolved) solids
4 Volatile solids
C The procedure for the determination of
1, 2, and 3 above is given in the
Analytical Quality Control Laboratory
'Manual.
Standard Methods for the Examination
of Water and Wastewater, 13th Ed.,
page 538, Method 224D (1971) is the
reference cited by the Analytical Quality
Control Laboratory Manual
This outline was prepared by F. J. Ludzack,
Chemist, National Training Center, Water
Programs Operations, EPA, Cincinnati, OH
45268.
35-4
-------
DETERMINATION OF SUSPENDED SOLIDS
I Wastewater stabilization includes the
conversion of solid, liquid, or gaseous
pollutants to (a) materials at a higher oxida-
tion state or(b) to a more removable form.
Both are interrelated, but (b) is favored
during conventional treatment.
A Economics of treatment are more favorable
when pollutants can be removed and treated
in more concentrated form.
B Pollutant characteristics guide selection
of operations applicable for contaminant
conversion to suspended solids.
C Control of suspended solids removal is a
major factor in treatment efficiency.
Incomplete removal leads to feedback of
nutrients from the unstable solids during
later stages of stabilization.
II Characterization of the various forms of
solids that may exist or be formed during
stabilization of polluting materials necessarily
depends upon operational factors. These are
usually conditional, but their appreciation is
essential.
A Dissolved solids include solid, liquid, or
gaseous materials that are dispersed in
the aqueous system in molecular or ionic
form. The "solution" may be colored but
it is likely to be clear unless the con-
centration of some items in the mixture
approaches their solubility limits.
B Colloidal solids include a stage or condition
of suspension where the solubility limit
has been reached and the ionic or molecular
dispersion tends to agglomerate to form
"clumps" of insoluble materials or partic-
ulates. These particulates are large
enough to scatter light and form cloudy or
turbid suspensions but are not large
enough to subside without further agglom-
eration.
C Suspended solids include the colloidally
dispersed solids and the suspended solids
that have agglomerated to form particulates
PC. 15c. 3 71
that will subside or settle under quiescent
conditions to form a solids concentrate
or sludge. Size is a major criterion but
density, shape, and interface character-
istics among the liquid and solid are
important factors.
D Total solids include A, B and C that may
be present in the whole sample.
E Solids interrelationships are discussed in
a prior outline. Determination of sus-
pended solids is the object of this one.
Ill Sources of suspended solids include both
Natural and man-made operations.
A Rainfall washes particulate material from
the air and from the surfaces of forest,
farmland, undeveloped land and urban or
industrial centers.
B Municipal water supplies are controlled
to maintain negligible suspended solids.
Some may be formed in distribution
systems.
C Groundwaters may contain dissolved solids
that form suspended solids upon exposure
to air or oxidation.
D Wastewaters are likely to contain varied
suspended solids or components that may
be converted to suspended solids by
biological, chemical or physical trans-
formations. Their nature varies with
the origin such as household, commercial,
industrial process, mining, washing, or
other operations.
E Breaks below the groundwater level in
partially filled sewers allow infiltration
of suspended materials characteristic of
the surrounding soils and water movement
in them.
F Combined sewer systems, either-
lntentional or otherwise, permit surface
drainage from roofs, streets, parking lots,
lawns, etc. to enter sanitary sewers
36-1
-------
Determination of Suspended Solids
IV The determination of suspended solids
relate to operational factors determining the
particular result obtained.
A A fence of wire or bars of appropriate
sized openings may serve as a barrier
to passage of horses and cattle but fail
to stop cats or dogs.
B Smaller sized particles are commonly
separated by filtration, centnfugation,
subsidence, or other hydraulic gradation.
1 Roots, branches, rags, etc., usually
are separated by bar racks or screens
with relatively large openings.
2 Grit, sand and small, but heavy,
particles generally are separated
hydraulically in chambers allowing 1
to 3 minutes detention time.
3 Filtration is the most commonly applied
technique for determination of the
relatively low density particulates that
tend to remain in aqueous suspension
largely as a result of turbulence. Since
pore sizes of filters differ, it is
essential to specify the media used to
interpret results.
V Standard Methods refers to residues from
whole sample evaporation as total residue,
suspended solids as the non-filterable residue
retained on or in the filter and dissolved
residue as that appearing in the filtrate.
A Many factors influence the results obtained
during suspended solids determinations
such as
1 Chemical and physical nature of the
solids
2 Sampling techniques
3 Pore size of the filter
A membrane filter of 0. 5m pore size
may retain particulates that are not
retained on paper.
4 Area, condition, and consistency of
filter media.
5 Technique and manipulative care in
drying, weighing, storage, transfer,
and temperature.
B Standard Methods includes three methods
for non-filterable residue or suspended
solids.
1 Gooch crucible
2 3
2 Membrane filter '
3 Buechner funnel-aluminum dish (aerator
or sludge suspended solids).
C A spectrophotometric method was 4
described by Krawczyk and Gonglewski .
The importance of homogenization to
improve sampling uniformity was stressed.
5
D Chanin, et. al. described the use of
glass fiber media for the determination
of suspended solids.
VI Precision, sensitivity, and applicability
of various methods for determination of
suspended solids are indicated in the cited
references.
A Table I indicates data from several
references c.i precision of he Gooch
crucible, asbestos fiber mat determination.
B Table 2 presents data published on the
precision of the Gooch crucible, glass
fiber determination.
7
C Smith and Greenberg compared results
of 5 replicates each of eight samples
(influents, primary effluents, final
effluents and industrial waste samples)
by five different methods of determination
including-
1 Gooch crucible, asbestos mat
2 Gooch crucible, glass fiber mat
3 Buechner funnel, wire cloth, glass
fiber mat
36-2
-------
Determination of Suspended Solids
TABLE 1
GOOCH CRUCIBLE
ASBESTOS MAT
Range
of Values
mfi/1
Rcf
Number
of
Replicates
M can
mir/1
Standard
Deviation
mg/1
Sample
Size
F iltered
ml
17U-308
6
10
224
+ or ¦
47
25
218-288
6
10
242
24
25
-
7
5
300
13
7
100
-
7
5
207
27
50
-
7
96
2
200
14-^6
n
10
17
4
100
1 DO-2 13
(d) 4
L0
201
7
100
100-132
-------
Determination of Suspended Solids
4 Membrane filter 0.4 to 0. 5 M pore size
5 Gooch crucible, glass fiber mat, celite
filter aid
a Their conclusions based upon care-
fully controlled technique indicated
that yield and precision were not
statistically different among the five
tested methods.
b Determining factors other than in
(a) included- manipulation time,
required skills, speed of filtration,
consistence of mat quality, equipment
cost and availability.
c Methods 2 and 3 more frequently gave
a lower coefficient of variation
among those tested and were more
favorable with respect to (b).
VII Section VI indicates that good results are
possible among any of the tested methods but
it was easier to obtain precise results with
certain methods. The Gooch Crucible -
asbestos mat requires a high degree of skill
and manipulative time to provide consistent
filtration. The membrane filter tends toward
slow filtration, sampling or manipulative
difficulties, and is more expensive.
A Certain precautions essential to consistent
results with Gooch crucible-asbestos mats
include
1 A thin suspension of asbestos in
distilled water is essential. A walnut-
sized wad of fiber/1 is desirable.
2 Shake the suspension vigorously, allow
it to settle 10-20 seconds. Pour the
suspension into another container and
discard asbestos slivers.
3 A more uniform suspension of asbestos
is achieved after 10-30 seconds treat-
ment in a Waring blendor or equivalent.
4 Allow to stand overnight or for a
sufficient time to form an asbestos
fiber concentrate on the bottom.
Discard any milky fines in suspension
and rewash, if necessary until a
reasonably clear supernatant is
obtained.
5 Insert the clean gooch crucible (30 ml
or other selected size and diameter)
into a Walters crucible adapter fitted
to an 0.5 or 1 1 suction flask.
6 Shake the asbestos suspension
vigorously, add the mixed suspension
to the crucible until it is 1/2 to 2/3
full.
7 Allow the fiber to "mat" for 1/2 to
2 minutes, then apply vacuum slowly.
If a hole forms in the mat the vacuum
was applied too soon and too rapidly.
8 Add additional suspension (vacuum off)
and apply vacuum to withdraw water and
form a mat of 2-3 mm. Waiting period
before vacuum application is not
essential once a mat has formed.
Water penetration rate is an effective
means to judge mat quality. Good
filtering is obtained if the exit stream
from the Walters adapter is continuous
but slow enough to be slightly off axis.
9 Wash the mat with successive portions
of distilled water (200-250 ml) to clear
the mat of fines.
10 Remove tne crucible, wipe the outside
with clean tissue or cloth and dry and
weigh.
B Precautions and aids for glass fiber mat
usage
1 Gooch crucibles employing glass fiber
mats of 2. 4 cm diameter may encourage
slow filtration, different shaped units
such as the asphalt filtration crucible
are designed to use larger diameter
mats which may be desirable for
aerator or sludge solids tests.
2 It is advisable to insert glass fiber mats
with the smooth side up. A second mat
may be required if the prewetting rinse
36-4
-------
Determination of Suspended Solids
suggests unusually rapid water passage.
Careful placement of the mat is essential.
C Membrane filter precautions include
1 The filters are fragile and must be
carefully handled to prevent damage
during use.
2 Prewashing in distilled water for 24
hours helps to remove glycerol or
other softening agent that is likely to
be eluted by the sample.
3 Filters tend to curl on drying. An
individual desiccator is described"^
that helps to control drying at ambient
temperature and curling in process.
D Beuchner funnel suspended solids deter-
minations include the (a) aluminum
dish-paper, (b) wire cloth-glass fiber and
(c) paper filtration. The aluminum dish
is designed to support the paper and
prevent losses during drying. The wire
cloth promotes more rapid filtration and
support. When neither supports are used,
the paper or glass fiber may be folded once
with the solids inside during drying.
1 Buechner funnel filtrations always
present the problem of solids adherence
to funnel sidewalls instead of the filter
media. Solids losses due to incomplete
transfer to the filter may be minimized
but not eliminated. A square ended
spatula aids transfer. It is advisable
to adjust sample size to yield > 100 mg
of non-filterable residue to reduce
percentage error for Buechner funnel
tests.
E General precautions applicable for precise
results on suspended solids include:
1 Filters or crucibles must be clearly
identified, dried, generally at 103OC,
and stored in a desiccator until weighed
and used.
2 The balance should contain a desiccant
to control atmospheric moisture during
weighing. This should be changed
regularly and the doors opened only as
necessary.
3 Filters may be dried in a desiccator
(preferably 24 hours).
4 For rapid, control work with Buechner
funnel-paper filtering, the papers may
be oven dried and equilibrated with
room moisture for 10 minutes to
reduce changes due to rapid moisture
pickup after oven drying.
5 Manufactured filters must be handled
carefully with forceps, polished if
necessary to prevent puncture or
damage to the filter surface.
6 Place the filter media carefully in the
crucible covering all openings in the
support.
a Special care must be used for
membrane filter holders to clamp
the filter in place firmly without
rupture or displacement.
7 Wet the filter media with distilled
water preferably directed at the center
of the mat first. Allow the mat to
become thoroughly wet before applying
vacuum then prewash the filter with
about 100 ml of distilled water.
8 With the vacuum on, add the selected
aliquot of sample.
a The sample volume should be large
enough to minimize percentage
errors during weighing. Less
precise technique requires more
weight difference between the pre
and post filtration weights. Three
to five mg weight difference is
adequate for Gooch crucible technique,
> 100 mg required for Buechner
funnel tests. Homogenize the sample
if sampling uniformity is questionable.
9 Rinse the non-filterable residue with
distilled water to wash sidewall solids
down onto the filter mat and to
minimize occluded solubles in the
solids and filter mat.
10 Dry and weigh under the same conditions
as used for preweighing.
36-5
-------
Determination of Suspended Solids
11 Final weight - Initial weight (mg)
x looo =
ml sample aliquot
suspended solids inmg/1
Results are usually expressed in mg/1
(gXlOOO).
12 It is not feasible to dry suspended
solids to a constant weight because of
the interchange of moisture, volatiles
or changes due to oxidation in process.
Weight must be defined in terms of a
routine that minimizes weight change
errors.
13 It is not advisable to pre-igmte glass
fiber mats for the determination of
sample volatile solids as recommended
for asbestos fiber mats. Glass fiber
generally will soften at about 400° C
but is likely to tolerate ignition at
550 to 600O C without fusion. It is
advisable to check a given supply of
fiber glass circles by igniting 5 or 6
under carefully controlled ignition
temperatures and time sequence. If
the glass fiber mats are uniform in
weight and composition their weight
loss on ignition will be comparable.
This amount of ignition loss may be
subtracted from the ignition loss for
samples to obtain a corrected value for
sample volatile suspended solids (VSS)
if the loss is acceptably consistent.
14 The aluminum dish may be fabricated
from an aluminum milk dish (listed in
apparatus supply catalogues) by punching
or drilling 1/16 inch holes in the
bottom comparable to those of the
Buechner funnel into which it is inserted.
The aluminum dish and filter may be
weighed before and after sample
filtration and drying. An O ring of
silicone M rubber slightly smaller than
the dish may be used to provide a
vacuum seal with the Buechner funnel.
Sample contents must not overfill the
dish during manipulation to avoid loss
of solids by adherence to the funnel
surfaces.
VIII The FWPCA "Methods for Chemical
Analysis of Water and Wastes", method
for non-filterable solids (Storet number
00530) prescribes filtration through glass
fiber media with drying at 105°C.
(1969, in press)
REFERENCES
1 Standard Methods, Water and Wastewater,
APHA, 12th Ed (1965).
2 Engelbrecht, R.S. and McKinney, Ross E.
Membrane Filter Method Applied to
Activated Sludge Suspended Solids
Determinations, Sewage and Ind.
Wastes, 28.(11) 1321, November 1956.
3 Winneberger, J.H., Austin, J.H., and
Klett, Carol A., Membrane Filter
Weight Determinations. 35: (6), 807
June 1963.
4 Krawczyk, D. L. and Gonglewski, N.,
Determination of Suspended Solids
Using a Spectrophotometer, JWPCF
31: (10) 1159, October 1959.
5 Chanin, G., Chow, E.H., Alexander,
R.B., and Powers, J. Use of Glass
Fiber Media in the Suspended Solids
Determination. Sew. and Ind. Wastes
30: (8) 1062. August 1958.
6 Laboratoi 0 Tnvestigation Report # 1,
Technical Advisory and Investigations,
DHEW, PHS, Div. of WS & PC (1963).
7 Smith, A.L., and Greenberg, A.E.,
Evaluation of Methods for Determining
Suspended Solids, JWPCF 35: (7) 940,
July 1963.
I'his outline was prepared by D. F. Krawczyk,
Acting Chief Consultant, Laboratory Services,
Pacific Northwest Water Laboratory, Corvallis,
Oregon and F. J. Ludzack, Chemist, National
Training Center, Environmental Protection
Agency, WPO, Cincinnati, OH 45268
36-6
-------
LABORATORY PROCEDURE FOR TOTAL SOLIDS
I INTRODUCTION
A This procedure was excerpted from methods
for Chemical Analysis of Water and Wastes,
1971, Environmental Protection Agency,
Office of Water Programs, Analytical Quality
Control Laboratory
B The procedure is applicable to surface and
saline waters, domestic and industrial wastes.
C The practical range of the determination is
10-30000 mg/1.
D Nonhomogenous materials (large floating
particles or submerged agglomerates) should
be excluded from the sample. Floating grease
and oil should be included in the sample and
dispersed in a blender before measuring the
aliquot.
E Samples should be analyzed as soon as
possible.
II EQUIPMENT
A Porcelain, Vycor, or platinum evaporating
dishes, 100 ml capacity; smaller sizes may
be used as required.
B Muffle furnace, 550— 50°C
C Drying oven, 103 - 105°C
D Desiccator
E Analytical balance
F Steam bath
C Weigh the dish on an analytical balance.
D Store the dish in the desiccator and weigh
just before use.
E Shake the sample container vigorously.
F Measure 100 ml of the well mixed sample
in a graduated cylinder. (At least 25 mg
of residue should be obtained, less volume
of sample may be used if the sample appears
to be high in solids content. If it is low in
solids content, more sample may be added
to the dish after drying).
G Rapidly, but without spilling, pour the
sample into the evaporating dish.
H Dry the sample on a steam bath, or at 98°C
(to prevent boiling and splattering) in the
oven.
I Dry the evaporated sample in the oven at
103 - 105 C for at least one hour,
J Cool the dish in the desiccator and then
weigh it.
K Repeat the heating at 103 - 105°C, cooling
and weighing until the weight loss is less
than 4% of the previous weight, or 0. 5 mg,
whichever is less.
IV CALCULATIONS
mg total solids/1 = (wt dish + residue)* -
(wt dish)* x 1000 x 1000
ml of sample
* in grams
IH PROCEDURE
A Heat the clean ^vaporating dish in a muffle
furnace at 550—50 C for 1 hour
B Cool the dish in a desiccator.
This outline was prepared by C. R. Feldmann,
Chemist, National Training Center, WPO, EPA,
Cincinnati, OH 45268.
PC. lab. 16.9.72
37-1
-------
LABORATORY PROCEDURE FOR NONFILTERABLE (SUSPENDED) SOLIDS
I INTRODUCTION
A This procedure was excerpted from Methods
for Chemical Analysis of Water and Wastes,
1971, Environmental Protection Agency,
Office of Water Programs, Analytical Quality
Control Laboratory.
B The procedure is applicable to surface and
saline waters, domestic and industrial wastes.
C The practical range of the determination is
20 - 20000 mg/1.
D All nonhomogeneous particulates (such as
leaves, sticks, fish and lumps of fecal matter)
should be excluded from the sample.
E Sample preservation is not practical; the
analysis should be done as soon as possible.
H EQUIPMENT
A Glass fiber filter discs, 4. 7 cm or 2. 2cm,
without organic binder. Reeve Angel type
984H, 934H, Gelman type A, or equivalent.
B Membrane filter funnel (for use with the 4. 7
cm disc), or
C 25 ml Gooch crucible (for use with the 2.2 cm
disc).
D Gooch crucible adapter.
E 500 ml suction flask
F Drying oven, 103 - 105°C
G Desiccator
H Analytical balance, 200 g capacity, capability
of weighing to 0.1 mg.
B Apply suction to the flask and wash the
disc with three successive 20 ml portions
of distilled water.
C Continue the suction until all water has
passed through the disc.
D Remove the Gooch crucible plus 2,2 cm disc
and dry in the oven at 103 - 105 C for one
hour, or,
E Remove the 4. 7 cm disc from the membrane
filter funnel and dry in the oven at 103 - 105°C
for one hour. In D or E, if the disc Js not
to be used immediately, store it in the
desiccator.
F Weigh the 4.7 cm disc or 2.2 cm disc plus
Gooch crucible just before use.
G Assemble the filtering apparatus.
H Apply suction to the flask.
I Shake the sample container vigorously.
J Measure 100 ml of the well mixed sample
in a 100 ml graduated cylinder. (If the
sample appears to be low in solids, a
larger volume may be used).
K Rapidly, but without spilling, pour the
sample into the funnel or crucible.
L Continue the suction until all of the water
has passed through the disc.
M Remove the 4. 7 cm disc from the funnel %
and dry in the oven at 103 - 105°C to
constant weight, or
N Remove the Gooch crucible plus 2. 2 cm
disc and dry in the oven at 103 - 105 C
to constant weight.
m PROCEDURE
A Assemble the filtering apparatus and suction
flask (either the 2.2 cm disc, Gooch crucible
and adapter, or the 4.7 cm disc and membrane
filter funnel).
Note: Drying to constant weight refers to the
process of:
1) drying the disc (or disc plus crucible)
at 103 - 105°C, 2) cooling in the desiccator,
3) weighing, 4) repeating steps 1), 2), and
3). If there is no difference in the two
PC. lab. 17.9.72
38-1
-------
Laboratory Procedure for Nonfilterable (Suspended) Solids
final weights, the disc (or disc plus
crucible) has been dried to constant
weight. In practice, the initial drying
period is sometimes extended to several
hours. A second heating step is then
generally not carried out.
IV CALCULATIONS
mg nonfilterable (suspended) solids /1 =
(wt of 4. 7 cm disc + residue)* - (wt of 4. 7 cm disc)* x 1000 x 1000
ml of sample filtered
or
(wt of 2. 2 cm disc + Gooch crucible + residue)* - (wt of 2. 2 cm disc + Gooch cruclble)*x 1000 x 1000
ml of sample filtered
*in grams
This outline was prepared by C. R. Feldmann,
Chemist, National Training Center, WPO, EPA,
Cincinnati, OH 45268. 1
38-2
-------
LABORATORY PROCEDURE FOR FILTERABLE (DISSOLVED) SOLIDS
I INTRODUCTION
A This procedure was excerpted from Methods
for Chemical Analysis of Water and Wastes,
1971, Environmental Protection Agency,
Office of Water Programs, Analytical Quality
Control Laboratory.
B The procedure is applicable to surface and
saline waters domestic and industrial wastes.
C The practical range of the determination is
10-20000 mg/1.
D Samples high in calcium, magnesium,
chloride, and/or sulfate may be hygroscopic
and require prolonged drying and desiccation,
and quick weighing.
E To insure complete conversion of bicarbonate
to carbonate, samples high in bicarbonate
content will require careful and possibly
prolonged drying at 180 C.
F Excessive residue in the evaporating dish
will crust over and entrap water that will not
be driven off during drying.
G Samples should be analyzed as soon as
possible.
H EQUIPMENT
A Glass fiber filter discs, 4.7 cm or 2.2 cm,
without organic binder. Reeve Angel type
984H Gelman type A, or equivalent.
B Membrane filter funnel (for use with the
4. 7 cm disc), or
C 25 ml Gooch crucible (for use with the
2.2 cm disc).
D Gooch crucible adapter
E 500 ml suction flask
F Drying oven, 180 + 2°C
G Muffle furnace, 550°C
H Drying oven, 180 + 2°C
I Desiccator
J Porcelain, Vycor, or platinum evaporating
dish, 100 ml capacity.
K Steam bath
L Analytical balance, 200 g capacity,
capability of weighing to 0. 1 mg.
IE PROCEDURE
A Assemble the filtering apparatus and
suction flask (either the 2. 2 cm disc, Gooch
crucible and adapter, or the 4. 7 cm disc
and membrane filter funnel).
B Apply suction to the flask and wash the
disc with three successive 20 ml portions
of distilled water.
C Continue the suction until all water has
passed through the disc.
D Remove the Gooch crucible plus 2. 2 cm
disc and dry in the oven at 103 - 105°C
for 1 hour, or,
E Remove the 4. 7 cm disc from the membrane
filter funnel and dry in the oven at 103 - 105°C
for 1 hour. In D or E, if the disc is not to
be used immediately, store it in the desiccator
F Weigh the 4. 7 cm disc or 2. 2 cm disc plus
, Gooch crucible just before use.
G Heat the clean evaporating dish in the muffle
furnace at 500 C for 1 hour.
H Store the dish in the desiccator and weigh
just before use.
I Assemble the filtering apparatus.
PC. lab. 18.9.72
39-1
-------
Laboratory Procedure for Filterable (Dissolved) Solids
J Apply suction to the flask.
K Shake the sample container vigorously.
L Measure 100 ml of the well mixed sample
in a 100 ml graduated cylinder. (A larger
or smaller volume may be used if the
dissolved solids content is thought to be low
or high).
M Rapidly, but without spilling, pour the
sample into the funnel or crucible.
N Continue the suction for at least three
minutes to assure water removal from
the disc.
O Using a 100 ml graduated cylinder, transfer
100 ml fo the filtrate to the evaporating dish.
P Evaporate the filtrate to dryness on the
steam bath.
Note: The filtrate from the Non-Filterable
(Suspended) Solids determination may
be used m this procedure.
IV CALCULATIONS
mg filterable (dissolved) solids/1 =
(wt dish + residue)* - (wt dish)* x 1000 x 1000
ml of sample
*in grams
Q Dry the evaporated sample for at least 1
hour at 180 + 2°C.
R Cool the dish in the desiccator.
S Weigh the dish. This outline was prepared by Charles R.
Feldmann, Chemist, National Training
T Repeat the drying, cooling, and weighing Center, WPO, EPA, Cincinnati, OH 45268.
steps until two successive weighings are the
same, or the weight loss is less than
0. 5 mg.
39-2
-------
LABORATORY PROCEDURE FOR VOLATILE SOLIDS
I INTRODUCTION
A In the manual, Methods for Chemical
Analysis of Water and Wastes, 1971,
Environmental Protection Agency, Office
of Water Programs, Analytical Quality
Control Laboratory, the reference given
for the determination of volatile solids is
Standard Methods for the Examination of
Water and Wastewater, 13th ed., p 538,
Method 224D, 1971.
B The Analytical Quality Control Laboratory
manual states that the test is subject to
many errors due to:
1 Loss of water of crystallization.
2 Loss of volatile organic material prior
to combustion.
3 Incomplete oxidation of certain complex
organics.
4 Decomposition of mineral salts during
combustion.
The principal source of error is said to be
failure to obtain a representative sample.
C The procedure in the 13th ed of Standard
Methods involves ignition of the filter disc
from the determination o^non-filterable
(suspended) solids at 550 C for 15 minutes,
cooling the disc in a desiccator and weighing.
D The equipment and procedure paragraphs
below are therefore those from the Analytical
Quality Control Laboratory manual.
H EQUIPMENT
A Glass fiber filter discs, 4. 7 cm or 2.2 cm,
without organic binder Reeve Angel type
984H, Gelman type A, or equivalent.
B Membrane filter funnel (for use with the
4. 7 cm disc), or
C 25 ml Gooch crucible (for use with the
2. 2 cm disc).
D Gooch crucible adapter.
E 500 ml suction flask.
F Desiccator.
G Analytical balance, 200 g capacity,
capability of weighing to 0.1 mg.
H Muffle furnace, 550°C.
IE PROCEDURE
A Assemble the filtering apparatus and
suction flask (either the 2.2 cm disc,
Gooch crucible and adapter, or the
4. 7 cm disc and membrane filter
funnel).
B Apply suction to the flask and wash the
disc with three successive 20 ml portions
of distilled water.
C Continue the suction until all water has
passed through the disc.
D Remove the Gooch crucible plus 2.2 cm
disc and dry in the oven at 103 - 105 C
for 1 hour, or,
E Remove the 4. 7 cm disc from the membrane
filter funnel and dry in the oven at 103 - 105°C
for 1 hour. In D or E, if the disc is not to
be used Immediately, store it in the
desiccator.
F Weigh the 4. 7 cm disc or 2. 2 cm disc
plus Gooch crucible just before use.
G Assemble the filtering apparatus.
H Apply suction to the flask.
I Shake the sample container vigorously.
PC. lab. 19.9.72
40-1
-------
Laboratory Procedure for Volatile Solids
J Measure 100 ml of the well mixed sample
in a 100 ml graduated cylinder. (If the
sample appears to be low in solids, a
larger volume may be used).
K Rapidly, but without spilling, pour the
sample into the funnel or crucible.
L Continue the suction until all of the water
has passed through the disc.
M Remove the 4. 7 cm disc from the funnel
and ignite in the muffle furnace at 550 C
for 1 hour, or,
N Remove the Gooch crucible and 2.2 cm
disc and ignite in the muffle furnace at
550°C for 1 hour.
O Cool the 4.7 cm disc or 2. 2 cm disc plus
Gooch crucible in the desiccator.
P Weigh the 4. 7 cm disc or 2.2 cm disc
plus Gooch crucible.
IV CALCULATIONS
mg volatile solids/1 =
(wt of 4. 7 cm disc + residue)* - (wt of 4. 7 cm disc)* x 1000 x 1000
ml of sample filtered
(wt of 2. 2 cm disc + Gooch crucible + residue)* - {wt of 2. 2 cm disc + Gooch crucible)* x 1000 x 1000
ml of sample filtered
*in grams
This outline was prepared by Charles R.
Feldmann, Chemist, National Training Center,
WPO, EPA, Cincinnati, OH 45268.
40-2
-------
THE DETERMINATION OF OIL AND GREASE
I INTRODUCTION
The terms grease and oil are not clearly defined.
Hydrocarbons of mineral oil sources and oxygen-
ated hydrocarbons of plant or vegetable origin
are included. Low molecular weight volatile
oils and high molecular weight solid fats, esters,
or waxes may be included. All have a greasy or
oily feeling and tend to adhere to extranenous
surfaces. Solubility is a major characteristic.
If the materials are preferentially more soluble
in relatively nonpolar solvents such as petroleum
ether or hexane than in water, they probably will
be considered as grease and oil. This type of
classification is part of the problem in grease
and oil analysis in wastewater treatment technology.
Many materials such as coloring agents having the
same solubility characteristics are included as
grease and oil.
H OCCURRENCE AND SIGNIFICANCE
A Grease and oil may be present as an oil-in-
water or water-in-oil emulsion, as a sorbate,
on suspended or deposited particulates as a
metallic soap in true solution, surface film,
or mixtures of these. Usually the solubility
in water is small but measureable, particu-
larly, for low molecular weight or combination
products.
B Greases and oils are of interest in wastewater
treatment operations because of their wide-
spread distribution resulting from natural
and industrial activities. The produce an
unusual variety of undesired effects that
are readily observed, and difficult to correct
during wastewater treatment.
1 Most greases and oils have a low specific
gravity and tend to float on the surface to
produce slicks, haze or scum.
2 Oil and grease tend to coat particulate
material in water to produce a sticky
mess that is unsightly, odorous, and
difficult to clean.
a Floating grease balls of fiber, sticks,
grease and miscellaneous solids tend
to build up into unsightly smelly
messes that are difficult to correct.
b Floating scum tends to collect grease,
fiber, and digestible material into an
impervious mass that limits surface
contact and biodegradability. Anaerobic
digester scum is a common operating
problem and is difficult to break up.
c Grease tends to adhere to equipment
surface such as pipelines, pumps,
screens, etc. Reduced capacity and
plugging results.
d Grease and oils in solids deposits
reduces efficiency of coagulation,
filter ability, and drying action.
3 Grease and oil are a definite safety
hazard on walkways, equipment, or
other surfaces.
H ANALYTICAL METHODS
A Standard Methods (1) lists three determina-
tions for oil and grease, based on the sample
coming from water, wastewater, or sludge.
1 In the case of a water sample, an
extraction is made in a separatory
funnel using a suitable solvent such
as trichlorofluoromethane or petroleum
ether. The solvent, containing the
grease and oil (and any other materials
extracted by the solvent), is transferred
to a previously weighed distilling flask.
All but about 10 ml of the solvent is
removed by distillation. The final 10 ml
is removed by use of a steam or hot
water bath. The distilling flask is again
weighed, and the increase of weight is due
to materials extracted from the sample
by the solvent.
WP.POL.ha. 3. 9.72
41-1
-------
The Determination of Oil and Grease
2 For sewage samples, 1 1 of sample
is acidified to pH 1.0 and then vacuum
filtered through muslin cloth overlaid
with filter paper. The filter paper is
carefully transferred to a Soxhlet
extraction thimble. Any grease or oil
materials adhering to the inside of the
funnel or the sample container are wiped
with pieces of filter paper soaked in
solvent (n-hexane or trichlorotrifluoroe-
thane). These pieces are also added to
the thimble. The extraction flask is
weighed, the thimble dried at 103°C
for 30 minutes, placed in the Soxhlet
extractor, and extracted at the rate of
20 cycles per hour for 4 hours.
The solvent is distilled slowly from the
flask at 85 C using a water bath or
heating mantle. Final drying is accom-
plished by means of a steam bath and
vacuum. The flask is weighed and the
weight gain recorded.
IV REFERENCES
1 Standard Methods for the Examination
of Water and Wastewater, 13th ed,
APHA, AWWA, WPCF, 1971.
2 Annual Book of ASTM Standards, Part
23 Water, Atmospheric Analysis, 1971.
3 Methods for Chemical Analysis of Water
and Wastes, 1971. Environmental
Protection Agency, Water Quality Office,
Analytical Quality Control Laboratory.
3 For sludge samples, the dry solids
content must be known. Magnesium
sulfate monohydrate is added to the
weighed, acidified sample. The mixture
is stirred to form a fine paste which
solidifies in 15-30 minutes. The mass
is ground in a porcelain mortar and the
powder transferred to a Soxhlet extraction
thimble. The remainder of the procedure
is essentially the same as in paragraph 2
above.
B ASTM (2) lists no specific procedure for oil
and grease. A method is given for the
extraction of organic matter from water.
It involves extracting the neutral sample
with a suitable solvent, acidifying to pH 3,
reextracting, making the solution alkaline
(pH 11), and carrying out a third extraction.
The three portions of solvent are combined,
evaporated, and the previously weighed
flask reweighed.
This outline was prepared by F. J. Ludzack,
Chemist, and Charles R. Feldmann, Chemist,
National Training Center, WPO, EPA,
Cincinnati, OH 45268.
C The Analytical Quality Control Laboratory
Office of Water Programs, Environmental
Protection Agency (3) method for the
determination of oil and grease is essentially
the same as given in paragraph A 2 above.
The major difference is that n-hexane is
specified as the extracting solvent.
41-2
-------
VIII CHLORINATION AND CHLORINE DETERMINATION
Increasing emphasis on disinfection of discharged
effluents requires consideration of special require-
ments of chlorination and chlorine determinations of
wastewater treatment plant samples. This section
presents background information and techniques
related to the situation.
Contents of Section VIII
Outline Numbers
The Basis for Chlorination of
Wastewaters 42
Chlorine Determinations and
Their Interpretation 43
-------
THE BASIS FOR CHLORINATION OF WASTEWATERS
I INTRODUCTION
A In a recent 1968 survey of all municipal
sewage treatment plants, it was reported
that an overall 30% of treatment plants
were provided with facilities for introduction
of chlorine in connection with treatment
plant operations.
Briefly, the survey of treatment plants
using chlorine may be summarized as
follows.
CHLORINE USED IN TREATMENT PLANTS
Population Served Total Number Number Plants % of Total Plants
of Plants With Chlorination Producing Chlorination
<500
1727
323
18. 7
500- 1000
2060
550
26.7
1000- 5000
4995
1903
38. 1
5000- 10, 000
1438
821
57. 1
10, 000- 25, 000
1164
695
59. 7
25,000- 50, 000
473
318
67.2
50, 000-100, 000
269
187
69. 5
100,000-250, 000
230
170
73.9
250, 000-500, 000
149
97
65. 1
> 500, 000
73
43
58.9
B In reviewing publications relating to the
use of chlorine with relation to wastewaters,
the uninitiated worker quickly can gain an
impression that chlorine is used in waste-
water treatment operations as an almost-
universal panacea, as a means of solving
whatever problems seem to be plaguing
the operations of the particular plant at
the time.
C The purpose of this discussion is to
review the common applications of
chlorine to wastewaters and in the treat-
ment of wastewaters, with special
reference to the forms of chlorine that
may be applied, places where chlorine
may be applied, and the purposes or
consequences of chlorination of wastewaters.
PC. 10 a. 7.71
42-1
-------
The Basis for Chlorination of Wastewaters
II USEFUL PROPERTIES OF CHLORINE
In terms of its significance in wastewater
treatment processes, the important
properties of chlorine include the following:
A Chlorine is a powerful oxidizing agent.
B Chlorine is poisonous to living organisms.
1 The poisonous properties of chlorine
probably are exerted through its
oxidizing ability, through which enzyme
systems essential to life are irreversibly
oxidized, or at least are inactivated.
2 According to the category of organisms
for which chlorine is being used as a
poison, chlorine could be called a
germicide, a bactericide, a disinfecting
agent, an algicide, an ovocide, a
cysticide, or by any of several other
"-cide" terms.
3 Different categories of organisms differ
widely in their susceptibility to chlorine.
Figure 1 illustrates the relative sus-
ceptibility of Escherichia coll and three
different kinds of viruses, to hypochlorous
acid.
4 The rate at which the disinfecting
(killing) process takes place is variable,
and subject to control through manage-
ment of such interrelated factors as
temperature, concentration, pH, and
the amount, kind, and physical state of
other suspended and dissolved substances
present in the water.
C Chlorine is highly soluble in water, and
can be introduced economically into water
and wastewater with accuracy and with
adequate provision to protect the health
and safety of operational personnel and
the population which will be exposed to
contact with the chlorine-treated waters.
IV USEFUL FORMS OF CHLORINE
A Most commonly, chlorine is added to water
in solution, being kept in tanks in liquid
form under pressure, the liquid chlorine
is converted to gas which in turn is dissolved
into the water or wastewater being treated.
a
E
u
O
x
(A
<
Ul
Z
5
o
X
w
COXSACKIE\
VIRUS A2 \
POUOVIRUS 1
E COLt
ADENOVIRUS 3
-------
The Basis for Chlorination of Wastewaters
B Molecular chlorine, Clg, in the concen-
trations applied, is quickly converted into
other forms, characterized by the following
examples:
1 Hydrolysis occurs:
C12 + H2° * HOC1 + H+ + C1~
2 Ionization of the hypochlorous acid
occurs with increasing pH:
HOC1 +=+ H+ + OCl"
At pH 5 and lower, practically all the
chlorine is in the form of hypochlorous
acid; at higher pH levels, increasing
ionization occurs until practically all
the chlorine is in the form of the hypo-
chlorite ion at pH 9. Hypochlorous
acid is far more active chemically
than the hypochlorite ion.
C Chlorine also may be added in the form of
sodium or calcium hypochlorite, as
chloramine, or as chlorine dioxide.
1 Hypochlorite ions enter into the same
sort of equilibrium reaction in water
as indicated above-
Ca(OCl)2 Ca++ + 20C1~
H+ + OCl" « » HOC1
2 Chlorine dioxide (CI O,,) is a gas, and
is extremely active chemically and as
a germicide.
3 Chloramines are formed in water con-
taining ammonia; when chlorine is
added, a mixture of monochloramine
(NHgCl), and dichloramine (NHC^),
are formed.
If enough chlorine is added to react
with all the available NHg and still
leave an excess of chlorme, then
nitrogen trichloride (NCI3) may be
formed. Chloramines are less reactive
chemically and less effective as germi-
cides, than are the free forms of
chlorine.
V REASONS FOR APPLYING CHLORINE
TO WASTEWATERS
A Odor Control
1 If sewage becomes anaerobic, whether
in the collecting system of sewers, in
any unit of a wastewater treatment plant,
or in a long outfall line, then unpleasant
odors may be developed. These odors
are due to the formation of such foul-
smelling substances as hydrogen
sulfide, indole, skatole, mercaptans,
and cadaverin.
Hydrogen sulfide usually is regarded as
the forerunner of the other foul-odor
substances. Accordingly, odor-control
measures are quite likely to result in
control of unpleasant odors from all
the substances listed.
2 Role of chlorine. Chlorine, when
applied, reacts with H2S, thus-
Cl2 + H2S > 2 HC1 + S
In addition, the addition of chlorine
results in a slowing of biological
activity, thereby reducing the rate
at which oxygen is removed from the
sewage.
3 Mode of application
a Any of the forms of chlorine indicated
above may be added to sewage for
odor control. However, since
chlorine also reacts indiscriminately
with other materials present, some
workers prefer to add ferrous
chloride for a more selective re-
moval of H2S, thus:
H S + FeCl ¥ FeS + 2 HC1
Chlorinated benzols also have been
applied in sewer systems for odor
control.
42-3
-------
The Basis for Chlorination of Wastewaters
b To control odors in sewer lines,
chlorine is added as far up-sewer
from the treatment plant as possible.
4 Chlorine treatment is highly regarded
for odor control. In the concentrations
used (usually 4-5 mg/liter, and some-
times over as wide a range as 3 - 30
mg/liter), a residual chlorine should
not be produced, and treatment pro-
cesses should not be impaired.
B Corrosion Control
1 Besides being an important contributor
to unpleasant odors, HgS formation can
result in damage to concrete, mortar,
and iron pipes. H2S, when oxidized,
results in production of sulfuric acid.
Therefore, any measures taken to pre-
vent formation of H2S also contribute to.
corrosion control.
2 Dosages sufficient to prevent formation
of HgS, as above, are indicated.
C Control of Operational Problems
1 Types of operational problems
a Ponding, on trickling filters, some-
times results from formation of
surface growths of organisms. These
organisms partially or completely
occlude the passageways for sewage
through the tricklingfilters.
b Bulking, or failure of satisfactory
settling of activated sludge, some-
times results when filamentous
organisms, such as Sphaerotilus
natans, and possibly other kinds of
organisms, become quite prominant
in the biota of activated sludge
systems.
c Foaming sometimes occurs in
Imhoff tanks.
d Septic, or anaerobic, conditions in
various parts of a treatment plant
may occur when the rate of flow or
holding time in tanks or conduits is
excessive and where reaeration is
not provided.
2 The application of chlorine often is a
useful and practical means of solving
the above types of problems. The
principle of chlorine application is to
add a limited amount of chlorine,
sufficient to inactivate or kill (select-
ively) the troublesome organisms, but
not to sterilize the unit. In this respect,
it is pointed out that there is a protec-
tion to the organisms most important
in sewage treatment due to (a) the
tremendous numbers of organisms
present, (b) inherently greater resist-
ance of the treatment organisms than
the nuisance organisms to chlorination
(debatable), and (c) treatment organisms
usually are deeply embedded in a slime
matrix or activated floe, and thereby
are somewhat less accessible to
chlorine than other forms.
D Disinfection of Sewage Effluents
1 Sewage is known to contain tremendous
numbers of microorganisms of intestinal
origin. When individuals harboring
pathogenic organisms, whether bacteria,
viruses, protozoa, and other forms of
disease-causing organisms, will be
present. Such organisms, when present,
constitute a hazard to the health and
safety of individuals coming into contact
with such sewage or treatment plant
effluents unless measures are taken
to destroy such organisms.
a In recent years, an epidemic of
infectious hepatitis was traced to
shellfish harvested from polluted
waters in Raritan Bay. A pollution
study of some magnitude was initi-
ated, and considerable numbers of
bacteria attributable to waste treat-
ment plants adjoining Raritan Bay
were demonstrated in the study.
During the period when wastewater
treatment plants chlorinated the
treatment plant effluents, enteric
42-4
-------
The Basis for Chlorination of Wastewaters
pathogenic bacteria could not be
recovered. However, when chlori-
nation of treatment plant effluents
was discontinued, Salmonella were
discovered with some regularity at
sampling points related to the above
waste treatment plant discharges.
b In studies of the Red River of the
North, Salmonella has been dis-
covered with great regularity in
waters polluted through discharge
of inadequately treated wastewaters.
In some cases, Salmonella has been
discovered where the fecal coliform
count was only a few hundreds per
100 ml.
2 Chlorine functions as a disinfectant in
the sense that it is applied in dosages
considered sufficient to destroy the
pathogenic (disease-causing) organisms.
Disinfection is not construed to mean
total destruction of all living organisms
present in the sewage effluents
(sterilization).
3 For disinfection, chlorine must be
added in sufficient concentration, and
with a sufficient contact time, to en-
sure destruction of the pathogenic
bacteria.
a One basis of chlorine application for
disinfection, widely noted in text-
books of sanitary engineering prac-
tices, provides recommendations for
adequate disinfection by adding
chlorine in an amount sufficient to
kill 99. 9% of all coliform bacteria
in the sewage, or sewage effluent,
after a contact time of 15 minutes,
and to have a chlorine residual of
0. 5 mg/liter (according to one re-
commendation, the residual chlorine
should be 2 mg/liter). A widely-
quoted table of chlorine application
follows
Table. Amounts of chlorine required for disinfection of sewage and sewage effluents,
with chlorine residual 0. 5 mg/liter after contact time of 15 minutes.
Type of Sewage or Effluent
Probable Chlorine
Requirements
mg/liter lb/day/1000
persons
Chlorinator
Capacities*
mg/liter lb/day/1000
persons
Raw sewage, depending on strength
and staleness
6-25
5-21
30
25
Settled sewage
5-20
4-17
25
20
Chemically precipitated sewage
3-20
3-17
25
20
Trickling filter effluent
CO
1
to
o
3-17
25
20
Activated sludge effluent
2-20
2-17
25
20
Intermittent sand filter effluent
1 - 10
1 - 3
15
12
*For sewage flow of 100 gallons per capita per day.
42* 5
-------
The Basis for Chlorination of Wastewaters
b It is known that the time-concentration -
temperature relationship of disinfec-
tion can be formulated in a manner
analogous to demonstration of the pro-
gressive exertion of the biochemical
oxygen demand. Accordingly, there
has been some interest in deter-
mining amounts of chlorine to be
added, based on the bacterial re-
sults to be achieved in the receiving
water at some point downstream
from the discharge of the treatment
plant effluent.
c The attached figure, based on
chlorine requirements for potability
treatment of clear water, with a 30
minute contact time, are quite at
variance with the requirements for
producing a relatively safe effluent
from a wastewater treatment plant.
E Reduction of BOD
1 It is emphasized that the action of
chlorine is indiscriminate when intro-
duced into wastewaters and treatment
plant effluents. Chlorine simultaneously
is killing or inactivating living organ-
isms with which it comes into contact
in sufficient concentration, for a suffi-
cient time, and it reacts with inanimate
inorganic and organic matter which
also is present.
In reacting with organic material,
chlorine has been stated tc^ reduce
from 10% to 35% of BOD when suffi-
cient chlorine has been added to pro-
vide a residual after 15 minutes of
contact time.
2 In reducing BOD through chlorination,
the action has been attributed to
a Outright chemical oxidation of the
organic material,
b Combination of chlorine with organic
matter to form inherently toxic com-
pounds (such as organic chloramines),
and
c Reaction of chlorine with organic
matter to form chlorinated compounds
which are biologically oxidized more
slowly than their predecessors.
3 Chlorination of sewage for the purpose
of reducing BOD is not a recommended
practice, except possibly in emergencies,
pending installation of adequate treat-
ment. The principal need for chlori-
nation for BOD reduction is to delay
(and hopefully, to diffuse) the oxygen-
depleting material in the receiving
water to an extent such that anaerobic
conditions will not develop in the aquatic
environment.
42-6
-------
The Basis for Chlonnation of Wastewaters
"C nvwrx^l ?j« of m ~ttrt c< mfamity hi#
c tlarint tudugi: is mi liktly io 6*
A Ttri.53 ¦ fhtv /se tv< c dt
-------
The Basis for Chlormation of Wastewaters
REFERENCES
1 The Federation of Sewage and Industrial
Wastes Association. Sewage Treat-
ment Plant Design. 1959.
2 Great-Lakes-Upper Mississippi River
Board of Sanitary Engineers. Re-
commended Standards for Sewage
Works. 1960.
3 Cosens, Kenneth W. The Operation of
Sewage Treatment Plants. A reprint
from Public Works Magazine.
4 Imhoff and Fair. Sewage Treatment.
1956, Book.
This outline was prepared by H. L. Jeter,
Director, National Training Center, Office
of Water Programs, EPA, Cincinnati, OH 45226.
42-8
-------
CHLORINE DETERMINATIONS AND THEIR INTERPRETATION
I INTRODUCTION
Chlorine normally is applied to water as a
bactericidal agent, it reacts with water con-
taminants to form a variety of products con-
taining chlorine. The difference among
applied and residual chlorine represents the
chlorine demand of the water under conditions
specified. Wastewater chlorination is parti-
cularly difficult because the concentration of
organisms and components susceptible to
interaction with chlorine are high and variable.
Interferences with the chlorine determination
in wastewater confuse interpretation with
respect to the chlorine residual at a given
time and condition, its bactericidal potency,
or the future behavior.
H CHEMISTRY OF CHLORINATION
A Chlorine compounds (CI2) dissolve in water,
and hydrolyze immediately according to the
reaction.
Cl2 + HgO ~ HOC1 + H+ + Cl"
The products of this reaction are hypo-
chlorous and hydrochloric acid. The re-
action is reversible, but at pH values above
3. 0 and concentrations of chlorine below
1000 mg/1 the shift is predominantly to the
right leading to hypochlorous acid (HOC1).
Hypochlorous acid is a weak acid and con-
sequently ionizes in water according to the
equation-
HOC1 -r H+ + OCl"
This reaction is reversible. At a pH value
of 5. 0 or below almost all of the chlorine
is present as hypochlorous acid (HOC1)
whereas above pH 10.0 nearly all of it
exists as hypochlorite ion (OCl ). The pH
value that will control is the pH value
reached after the addition of chlorine.
Chlorine addition tends to lower the pH
and the addition of alkali hypochlorites
tends to raise the pH,
B The initial reactions on adding chlorine to
wastewaters may be assumed to be funda-
mentally the same as when chlorine is
added to water except for the additional
complications due to contaminants and
their concentration.
Hypochlorous acid (HOC1) reacts with
ammonia and with many other complex
derivatives of ammonia to produce com-
pounds known as chloramines. Formation
of the simple ammonia chloramines includes:
1 nh3 + HOC1 — nh2ci + h2o
monochloramme
2 NH2C1 + HOC1 — NHC12 + HgO
dichloramine
3 NH2C1 + NHC12 — N2 + 3 HC1
The distribution of the ammonia chloramines
is dependent on pH, as illustrated below
Percentage of Chlorine Present as
pH Monochloramme Dichloramine
5 16 84
6 38 62
7 65 35
t
8 85 15
9 94 6
PC. 11a. 12.71
43-1
-------
Chlorine Determination and Their Interpretation
The formation of the ammonia chloramines
are dependent on pH, temperature, and
chlorine-ammonia ratio. Chlorine re-
actions with amino acids are likely, pro-
duct disinfecting powers are lower than
those of chlorine or of ammonia chloramines.
IE TERMINOLOGY
A Terms used with Respect to Application
Site
1 Pre-chlonnation - chlorine added
prior to any other treatment.
2 Post-chlorination - chlorine added
after other treatment.
3 Split chlorination - chlorine added at
different points in the plant - may in-
clude pre- and post-chlorination.
B Terms used in Designating Chlorine
Fractions
1 Free available residual chlorine - the
residual chlorine present as hypo-
chlorous acid and hypochlorite ion.
2 Combined available residual chlorine -
the residual chlorine present as chlor-
amines and organic chlorine containing
compounds.
3 Total available residual chlorine - the
free available residual chlorine + the
combined available residual chlorine -
may represent total amount of chlorine
residual present without regard to type.
In ordinary usage these terms are
shortened to free residual chlorine, com-
bined residual chlorine and total
residual chlorine. In the chlorination
of wastewaters only combined residual
chlorine is ordinarily present and is
often improperly termed chlorine
residual.
C Breakpoint chlorination specifically refers
to the ammonia-chlorine reaction where
applied chlorine hydrolyzes and reacts to
form chloramines and HC1 with the
chloramines eventually forming + HC1
as in I. B. 3. Assuming no other chlorine
demand, the total chlorine residual will
rise, decrease to zero and rise again with
increasing increments of applied chlorine.
Other substances may produce humps in
the applied chlorine vs residual chlorine
plot due to oxidation of materials other
than ammonia. Sometimes these are
erroneously considered as a breakpoint.
IV ANALYTICAL METHODS
The o-Tolidme color test and Iodometric
titration methods are the basis for numerous
modifications for determining chlorine
residuals in water. The relative advantages
of a specific determination depends upon the
form in which the reactable chlorine exists
and the amount and nature of interferences
in the water.
Iodometric titration using the amperometric
endpoint appears to be the most accurate
residual chlorine method available (See current
editions of Standard Methods APHAU) and ASTM
Standards (2)). The O-Tolidine and o-
Tolidine Arsenite methods require" little
apparatus, and are readily adapted as a field
or control test. The Starch Iodine color
titration endpoint for iodometric titration
is suitable for use on clean water or stock
solutions and may be useful on certain types
of wastewatc™ residuals. Selection of a suit-
able method of determining chlorine residuals
depends upon the correlation of the determined
residual and the bacterial kill in the presence
of existing interferences under applied
conditions.
A Iodometric Method
1 Scope and application
This method is applicable to the deter-
mination of total chlorine residual in
wastewaters, polluted waters and some
industrial wastewaters.
2 Summary of method
When a sample is treated with a measured
excess of standard phenylarsine oxide
43-2
-------
Chlorine Determination and Their Interpretation
solution, or a standard thiosulfate
solution, followed by the addition of
iodide, the iodine liberated at the
proper pH is stoichiometrically pro-
portional to the total chlorine present.
The liberated iodine reacts with the
phenylarsine oxide or thiosulfate
before any is lost to other extraneous
reactions. The excess phenylarsine
oxide or thiosulfate is titrated with
standard iodine solution in the presence
of starch until the phenylarsine oxide
or thiosulfate is completely oxidized.
The end-point of the titration is the next
addition of standard iodine solution that
causes a faint blue color to persist in
the sample.
3 Interferences
a Organic matter - reacts with liber-
ated iodine.
b Manganic manganese - liberates
iodine from iodide at pH 4. 0.
c Ferric iron, femcyanide and nitrites
up to 100 mg/1 do not interfere at a
pH of 4.0.
d Chromates - reduce phenylarsine oxide
or thiosulfate to an appreciable extent
before the excess can be titrated with
standard iodine.
e Excessive color and turbidity
B Iodometric Method with Amperometric
End-Point
1 Scope and application
This method is applicable to the deter-
mination of total chlorine residual in
wastewaters, polluted waters and some
industrial wastewaters. The back-
titration method is essential for waste-
waters in contrast to the direct titration
with phenylarsine oxide in clean waters.
2 Summary of method
When a sample is treated with a meas-
ured excess of standard phenylarsine
oxide solution followed by the addition
of iodide, the iodine liberated at the
proper pH is stoic hiometncally propor-
tional to the total chlorine present.
The iodine liberated reacts with the
phenylarsine oxide before any is lost to
other extraneous reactions. When the
cell is immersed in a sample so treated,
no current is generated due to halogens
nor is any further current generated,
as the excess phenylarseneoxide is
titrated with standard iodine solution
until the phenylarsine oxide is completely
oxidized. The end-point of the titration
is the next addition of standard iodine
solution that causes further current
to be generated and a microammeter
response or pointer deflection.
NOTE: As the end-point is approached
each increment of standard iodine solu-
tion causes a temporary deflection of the
microammeter, but the pointer drops
back to about its original position. The
true end-point is reached when a small
addition of standard iodine solution gives
a definite and permanent pointer deflection.
3 Interferences
a Organic matter - reacts with the
liberated iodine.
b Manganic manganese - liberates
iodine from iodide at a pH of 4. 0.
c Ferric iron, ferricyanide and
nitrites up to 100 mg/1 do not inter-
fere at a pH of 4. 0.
d Chromates - reduces phenylarsine '
oxide to an appreciable extent before
the excess can be titrated with standard
iodine solution.
e Cupric ions may cause erratic be-
havior of the apparatus.
43-3
-------
Chlorine Determination and Their Interpretation
f Cuprous and silver ions tend to
poison the electrode.
C Orthotolidine Method
1 Scope and application
This method is applicable to the deter-
mination of total chlorine residual in
wastewaters, polluted waters and some
industrial wastewaters.
2 Summary of method
When a sample is treated with a meas-
ured amount of orthotolidine reagent, the
orthotolidine is oxidized in the resulting
acid solution by chlorine and chloramines
and other oxidizing agents to produce a
yellow-colored compound (Holoquinone).
The color produced at pH values of less
than 1. 8 is proportional to the amount
of chlorine present and is suitable for
quantitative measurement. The chlorine
residual in mg/1 is read directly from
the colored glass disks or sealed
colored liquid standards or is calculated
from a previously prepared standard
curve. NOTE: Particular attention
is called to the importance of warming
the sample to 20°C after the addition
of orthotolidine reagent in order to
complete the reaction.
3 Interferences
a Organic matter - oxidizes orthotolidine
to produce a yellow color (Holo-
quinone) .
b Manganic manganese - in concentra-
tions above 0.01 mg/1 oxidizes
orthotolidine to produce a yellow
color (Holoquinone).
c Ferric iron - in concentrations above
0. 3 mg/1 oxidizes orthotolidine to
produce a yellow color (Holoquinone).
d Nitrites - in concentrations above
0. 10 mg/1 of nitrite nitrogen oxidizes
orthotolidine to produce a yellow
color (Holoquinone).
e Excessive color and turbidity.
D Orthotolidine- Arsenite Method
1 Scope and application
This method is applicable to the deter-
mination of free residue chlorine and
combined residual chlorine in waste-
waters, polluted waters and industrial
wastewaters, but as normally earned
out for wastewaters, etc. total residual
chlorine is measured.
2 Summary of method
A sample is split into two fractions (a)
and (b). Sample (a) is treated with a
measured amount of arsenite reagent,
followed by the addition of orthotolidine
reagent. The arsenite reacts with
chlorine while orthotolidine reacts with
ferric iron, manganic manganese and
nitrite nitrogen to produce additional
color (represents interfering color).
Sample (b) is treated with a measured
amount of orthotolidine reagent. The
orthotolidine is oxidized in the resulting
acid solution by chlorine, chloramines
and other oxidizing agents to produce
a yellow-colored compound (Holoquinone)
as described in the orthotolidine method
(represents total amount of residual
chlorire present and interfering color).
Mg/1 total residual chlorine = b - a if
color compensation is not made directly.
NOTE: more accurate readings may be
obtained if cells containing sample
fractions (a) and (b) are placed in the
comparator in such relative positions
that color compensation is made
directly.
3 Interferences
a High color and turbidity
V INTERPRETATION
In general residual chlorine concentrations
obtained by the lodometnc titration method
(starch iodide end-point and amperometric
43-4
-------
Chlorine Determination and Their Interpretation
end-point) will be higher than the concentrations
obtained from the orthotolidme and the orthoto-
lidine-arsenite methods. Some of the reasons
for the difference have been listed under the
individual methods. Extensive studies have
indicated that consistently better correlation
between bacterial kill and chlorine residual
found is possible when the titration method
is used.
REFERENCES
1 Standard Methods for the Examination of
Water and Wastewaters, 13th Ed.,
APHA, AWWA, WPCF. 1971.
2 Book of ASTM Standards, Part 23-
Industrial Water; Atmospheric
Analysis. American Society for
Testing and Materials.
Philadelphia, Pa., 1970.
3 Sawyer, C.N. Chemistry for Sanitary
Engineers. McGraw-Hill Book Com-
pany, New York. 1960.
4 Moore, E. W. Fundamentals of Chlori-
nation of Sewage and Wastes. Water
and Sewage Works. Vol. 98, No. 3,
March 1951.
5 Day, R. V., Horchler, D. H., and Marks,
H. C. Residual Chlorine Methods and
Disinfection of Sewage. Industrial and
Engineering Chemistry, May 1953.
6 Marks, H. C., Joiner, R. R., and
Strandskov, F. B. Amperometnc
Titration of Residual Chlorine in
Sewage. Water and Sewage Works,
May 1948.
This outline was prepared by J. L. Holdaway,
Chemist, Technical Program, EPA, Region HI,
Charlottesville, VA 22901.
43-5
-------
IX COMPUTATION AIDS AND GLOSSARY
The chart presented as a computation aid is intended for
use to provide quick and rough estimates for conversion
purposes and to check decimal point control for other
calculations.
The Glossary is an attempt to bridge the terminology
gap for recent recruits in wastewater treatment or
those interested in enhancing under standing or expression
of a larger fraction of the field.
Contents of Section IX
Powers Data Sheet No. 397
Glossary - Wastewater Treatment
Technology
A US. GOVERNMENT PAINTING OFFICE. 1972 — 759-548/1028
-------
POWERS DATA SHEET No. 397
Hlllion rowda
0.6 Million Gallon*
kr« FMt w
Aer« FMt Par 30 Days<
Gtllonsfrr Hiirata
-a. 5
'"Coble Fact I^r Second"
Flow Of Fluids Conversion Chart
With this chart you can conveniently deter-
mine equivalent units of discharge for fluids.
Merely line up a straight edge with the middle
of the chart and a known discharge; read the
equivalent on the other scales.
Example: Discharge from a given pipe is
1100 gallons per minute. How many gallons
per day are discharged? Lining up 1100 on
the gallons per minute scale with the chart
center, we read 1, 580, 000 gallons per day on
the appropriate scale. On other scales we
can determine that the flow is equivalent to
2. 45 cu ft per sec.
The scales cover sufficient range to allow you
to find values of any magnitude by multiplying
or dividing the scales by factors of ten. With
a little ingenuity, you can determine several
units not shown directly.
x
"Reprinted with permission from POWER, October 1965"
\i<2opyright McGraw-Hill, Inc., 1965. "
S. R. Ross, Denver, Colorado
f- US GOYHNMWT WIKIWCOfflCE. 197J— 759-396/87
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GLOSSARY - WASTEWATER TREATMENT TECHNOLOGY
This is a selected list containing the key
ideas of terms likely to be encountered
in treatment technology.
ABSORPTION - The taking up of one sub-
stance into the body of another.
ACRE FOOT - A volume term referring
to that amount of liquid 1 acre in area
and a depth of 1 foot. 43, 560 cu. ft.
ACID - Most commonly refers to a large
class of chemicals having a sour taste
in water, ability to dissolve certain
metals, bases or alkalies to form salts
and to turn certain acid-base indicators
to their acid form. Characterized by
the hydrated H+ ion.
ACTIVATED SLUDGE - A process used for
purification and stabilization of waste*
waters by mixing of the solids concen-
trate from previous contact with raw
or settled wastewaters under turbulent
oxygenating conditions for sufficient
time to permit transfer of nutrients
to the solids phase, partial biodegra-
dation and clarification of the water
before discharge.
ADSORPTION - The taking up of one sub-
stance upon the surface of or interface
zone of another substance.
ADVANCED WASTE TREATMENT -
Renovation of used water by biological,
chemical or physical methods that are
applied to upgrade water quality for
specific reuse requirements. May
include more efficient cleanup of a
general nature or the removal of com-
ponents that are inefficiently removed
by conventional treatment processes.
AERATION - The operation of adding oxygen
to, removing volatile constituents from,
or mixing a liquid by intimate contact
with air.
AERATION PERIOD - A theoretical time
usually expressed in hours equal to
the volume of the tank divided by the
volumetric rate of flow.
AEROBIC - A condition characterized by
an excess of dissolved oxygen in the
aquatic environment.
For more scientific definitions or unlisted
terms, consult the list of references at the
end of the glossary.
AEROBIC BACTERIA - Organisms that
require dissolved oxygen in the
aquatic environment to enable them
to metabolize or to grow.
AGGLOMERATION - An action by which
small particles gather into larger
particles that are more readily
separated from the liquid by sedi-
mentation or other means. May be
the result of biological, chemical
or physical factors.
AIR LIFT - A pump consisting of a vertical
pipe immersed in a liquid into which
air is mixed to reduce specific gravity
of the air-liquid mixture. The net
effect is to raise the liquid level in
the discharge pipe.
ALGAE - Primitive plants, one or many
celled, usually aquatic and capable
of growth on mineral materials via
energy from the sun and the green
coloring material, chlorophyll.
Generally considered as the primary
source of food for all other organisms.
ALKALINJTY - A term used to represent
the sum of the effects opposite in
reaction to acids in water. Usually
due to carbonates, bicarbonates and
hydroxides; also including borates,
silicates and phosphates.
AMPEROMETRIC CHLORINE RESIDUAL -
A means of determining residual
available chlorine with phenyl arsene
oxide (PAO) titration using current
response as an indicator of equiva-
lence. For wastewater, the PAO
preferably is used in excess with
iodine backtitration.
ANAEROBIC - A condition in which dis-
solved oxygen is not detectable in
the aquatic environment. Commonly
characterized by the formation of
reduced sulfur compounds from the
use of bound oxygen from sulfates
as an hydrogen acceptor.
AT. 2. 8. 69
1
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Glossary - Wastewater Treatment Technology
ANAEROBIC BACTERIA - Organisms that
can metabolize and grow in the absence
of dissolved oxygen. Their oxygen
supply is obtained from the bound oxy-
gen such as in sulfates, carbonates,
or other oxygen-containing compounds.
ANION - A negatively charged ion in water
solution. May be a single element or
a combination of elements, such as
the CI" ion in a water solution of NaCl
(common table salt) or S04= ion in a
sulfuric acid solution.
ASSESSMENT - A legal financial obligation
of the property owner in an irrigation,
water, drainage or sanitary district,
created for the purpose of financing
the construction and operation of
facilities required to protect and en-
hance public benefit within the district.
ATTACHED GROWTH - Plant or animal
growth that tends to seek a solid sur-
face for a point of attachment from
which to grow, in contrast with free-
swimming or suspended organisms.
ATOM - An extremely small unit or particle
of an element consisting of a positively
charged nucleus and one or more
negatively charged electrons. Atoms
of different elements are different in
mass and the number of electrons.
Electrons may be located in the nucleus
or externally. The external electrons
determine chemical combining power.
ATOMIC WEIGHT - A relative mass of an
atom of an element compared to
carbon-12. May be expressed in
grams (g), pounds or other consistent
weight units when used for process
control.
AVAILABLE RESIDUAL CHLORINE -
Generally refers to that part of the
chlorine that will react with ortho -
tolidine or amperometric tests and
exhibits significant germicidal activity.
A WW A (AMERICAN WATER WORKS
ASSOCIATION) - An organization' composed
of individuals engaged in research,
design, operation and control in the
advancement of knowledge related
to potable water supply
BACTERIA - Primitive organisms having
some of the features of plants and
animals. Generally included amofcg
the fungi. Usually do not contain
chlorophyll, hence commonly require
preformed organic nutrients among
their foods. May exist as single cfells,
groups, filaments, or colonies.
BACTERIACIDE - Any component that
will kill or destroy bacteria.
BACTERIOSTATIC - A condition during
which the normal metabolic functions
of bacteria are arrested until favor-
able conditions are restored.
BACTERIOLOGY - See Microbiology.
BAFFLE - A deflector or check such as
a vane, guide boards, plates, grids,
grating, or similar devices used
to control the flow distribution or
velocity of liquid in a channel or
basin.
BAR RACKS (SCREENS) - A coarse
screen usually consisting of bars
spaced with 1 to 5 inch openings
to trap roots, branches, rocks,
rags, and other large materials
that may be encountered in the flow
of a channel or conduit.
BASE - A foundation, plate, natural or
engineered support upon which, a
structure, channel, machine, or
other device is mounted.
Chemical: A base includes a large
variety of chemicals opposite in re-
action to acids (alkali).
BED LOAD - Generally refers to the
oxygen demand requirements of
benthic deposits, sludge, muck,
attached growths, moving materials,
living or dead that are exerted upon
waters as a result of bottom or
boundary dynamics.
BENTHIC DEPOSIT (BENTHOS) - Refers
to the accumulated deposition of cell
mass living or dead that collects at
the bottom of a stream impoundment
where velocity or catchment permits.
2
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Glossary - Wastewater Treatment Technology
BIO-CHEMICAL - Resulting from the
combined activities of biological
and chemical transformations.
Usually measured in terms of the
ensuing chemical changes.
BIODEGRADATION - The stabilization
of wastewater contaminants by bio-
logical conversion of pollutants into
separatable materials at a higher
oxidation state.
BIO FILTER - See Trickling Filter.
BIOLOGICAL PROCESSES - Activities of
living organisms to sustain life,
growth, and reproduction. Commonly
the processes by which organisms
degrade complex organic material
into simpler substances at a higher
oxidation state to obtain energy for
life processes and growth of new
cell mass.
BIOLOGY - The science and study of living
organisms, characteristics and be-
havior.
BOD - Biological or biochemical oxygen
demand. A test for estimation of
wastewater polluting effects in terms
of the oxygen requirements for bio-
chemical stabilization under specified
conditions and time.
BRIDGING - A condition in which particu-
lates or solids concentrates that would
normally seek the lowest level of a
restricted channel or basin, tend to
hang up on sidewalls. The bridged
material commonly may settle again
with vibration, agitation, a change
in flow direction, or increased flow
velocity.
BTU (BRITISH THERMAL UNIT) - That
amount of heat that will raise the
temperature of one pound of water
one degree Fahrenheit.
BUFFER ACTION - An action exhibited
by certain chemicals that limits the
change in pH upon addition of acid
or alkaline materials to the system.
In surface water, the primary buffer
action is related to carbon dioxide,
bicarbonate and carbonate equilibria.
BULKING - A condition, usually related
to activated sludge processing, in
which the sludge solids separation
from the liquid is inhibited.
Rapid growth, filamentous organisms,
and certain other factors that are but
vaguely understood, tend to produce
a low density thin sludge that settles
very slowly and has limited compact-
ability.
BURNER, WASTE GAS - A device for
burning the excess gas from sludge
digestion.
CALORIE - That amount of heat required
to raise one gram of water one
degree Centigrade, or Celsius.
CARBOHYDRATES - Naturally occurring
compounds consisting of carbon,
hydrogen and oxygen, that are con-
sidered as energy foods and precursors
of proteins and Fats in the natural
food chain.
CATALYST - A substance that influences
the rate of chemical change but either
remains unchanged during the reaction
or is regenerated thereafter.
Generally applies to acceleration of
reaction rates.
CATCH BASIN. - A chamber, well or other
enlargement of a channel, designed
to retain grit and detritus below the
point of liquid overflow.
CATION - A positively charged ion in
water solution. May be a single
element or a combination of elements,
such as Na+ in a water solution of
NaCl (common table salt).
CENTI - An expression used to indicate
1/100 of a given standard unit
centimeter (cm). 1/100 meter.
CENTIGRADE - A temperature measure-
ment scale in which the freezing point
of pure water at sea level is desig-
nated as 0°C and the temperature of
boiling water is designated as 100°C.
This is more properly termed the
Celsius scale.
CENTRIFUGAL PUMP - A pump consisting
of a rotating impeller within a casing
having an inlet near the center and
an outlet or discharge at the tip of
the impeller where centrifugal force
is greatest.
3
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Glossary - Wastewater Treatment Technology
CENTRIFUGE - A device for separation of
solids or liquids of different densities
by rotational energy; heavy materials
move outward, less dense materials
move toward a central take-off port.
CHANNEL - A natural or artificial waterway
which continuously or periodically con-
tains flowing water. A connecting link
between two bodies of water with a
definite bed and sidewalls to confine
the flow.
CHANNELING. - A condition in which certain
portions of the flow within a channel or
basin tend to seek a more limited dis-
tribution than that resulting from the
confining bed or sidewalls, i. e., the
flow may channel along the top, bottom
or mid channel depth due to density,
temperature, or some form of obstruc-
tion to uniform cross sectional flow.
CHECK VALVE (FLAP GATE) - A device
to control flow in a pipe or channel
limiting it to one direction. Commonly
a gate hinged at the top that is limited
in movement by a seat in a near
vertical position so that it can open
for flow in one direction but closed
by reverse flow.
CHEMISTRY - A science that deals with the
composition and characteristics of
substances and their behavior, i. e.,
the transformations that they undergo.
CHLORAMINES - Products of the combination
of chlorine and ammonia. Commonly
classified as combined available chlorine.
CHLORINE - A greenish yellow gaseous
element having strong disinfecting
and oxidizing properties in water
solution. It is commercially available
as compressed gas, liquid, or in
combined form as a powder. It is
highly toxic and irritating to skin, eyes,
and lungs in significant concentrations.
CHLORINATION - The application of chlorine
to water or wastewater for the purposes
of disinfection, oxidation, odor control,
or other effects. Pre-chlorination -
before treatment; post-chlorination -
after treatment; in-process chlorination
- during treatment.
CHLORINATION CHAMBER - A basin or
tank where chlorine is applied to the
liquid.
CHLORINE TEST - Commonly refers
to one of two methods separately
listed: see Ortho tolidine test;
see Amperometric test.
CHLORINE DEMAND - The difference
between applied chlorine and residual
available chlorine in aqueous media
under specified conditions and
contact time. Chlorine demand
varies with dosage, time, temperature,
nature and amount of the water im-
purities.
CHLORO ORGANIC COMPOUNDS -
A broad group of compounds containing
chlorine.carbon, hydrogen__and some- (v
times otner elements. Generally
originating from or associated with
living or dead organic materials. This
group shows a wide range of toxicities
but usually have relatively little oxi-
dizing energy compared to chlorine.
CHLOROPHYLL - The green coloring
material or pigments in plants that
promotes the photosynthetic reactions
forming organic materials from in-
organic nutrients and light energy
within the living cells.
CLAREFEER - A basin or chamber servinjg
as an enlargement of a channel to re-
duce flow velocity sufficiently to per-
mit separation of settleable or
floatable materials from the carrier
water (a sedimentation basin).
COAGULANT - A chemical, or chemicals,
which when added to water suspensibns
will cause finely dispersed materials
to gather into larger masses of im-
proved filterability, settleability, or
drainability.
COAGULATION - The process of modifying
chemical, physical, or biological
conditions to cause flocculation or
agglomeration of particulates.
COD - A test for the estimation of the
contamination of a wastewater in
terms of oxygen requirements from
a strong chemical oxidant under
specified conditions, i. e., Dichromate,
50% sulfuric acid and 145°C for 2 hours.
COLIFORM GROUP - A group of bacteria
that inhabits the intestinal tract of
man, warm-blooded animals, and
may be found in plants, soil, air and
4
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Glossary - Wastewater Treatment Technology
the aquatic environment. Includes
aerobic and facultative gram negative
non-spore forming bacilli that ferment
lactose with gas formation.
COLLECTION SYSTEM - The sewerage
collection system is comprised of the
conduits controlled by public agencies
to intercept house, commercisJ or
industrial discharges and transport
them to a treatment facility or dis-
charge point.
COLLOID, COLLOIDAL STATE - A state
of suspension in which the particulate
or insoluble material is in a finely
divided form that reamins dispersed
in the liquid for extended time periods.
Usually cloudy or turbid suspensions
requiring flocculation before clarifi-
cation.
COMBINED AVAILABLE CHLORINE -
Generally refers to chlorine-ammonia
compounds exhibiting a slower reaction
with ortho tolidine, determinable with
phenyl arsene oxide after addition of
potassium iodide under acid conditions
and usually requires higher concentra-
tion and longer time to kill in com-
parison with free available chlorine.
COMBINED SEWER - A sewer designed to
carry wastewaters and storm waters
in the same channel.
COMBINED SEWAGE - Consists of house-
hold, commercial or industrial wastes
in combination with roof and surface
storm drainage.
COMMINUTION -
a) The act of cutting and screening
materials contained in wastewaters.
b) To reduce the size of fibrous or
amorphous materials.
COMMINUTOR - A device for cutting sew-
age solids until they pass through an
acceptable screen opening to improve
pumping and wastewater processing.
COMPOUND -
a) A combination of two or more atoms
having definite physical and chemical
characteristics and mutually attracted
to each other.
b) Atoms in the elemental state are
electrically neutral but the number
of external electrons may be increased
or decreased in response to conditions
and nature of the atom. An atom that
becomes electrically charged may
combine with another atom of opposite
charge to form an electrically neutral
compound.
CONCENTRATION -
a) The act of increasing the mass
per unit volume of one substance
with respect to another, such as
concentrating the solids in a sludge
from 3% to 67c.
b) A means of designating the ratio
of one substance with respect to
another, such as 15 mg of suspended
solids per liter of water.
CONDITIONING - An action of improving
possibilities for subsequent process-
ing such as chemical treatment to ini-
prove sludge dewaterlng or filtering.
CONING - A condition inaclorifier sludge
hopper where the solids concentrate
or sludge is partially withdrawn to
form a cone or channel through which
clarified liquid is pumped out while
most of the solids remain behind
around the cone. Infrequent sludge
pumping tends to encourage this
condition where the sludge ten(js to
solidify and is resistant to fluid flow.
CONTAMINATION - A general term re-
ferring to the introduction of materials
into water that make the water less
desirable for its intended use. Also
introduction of undesired substances
into air, solutions, or other defined
media (chemical or biological)
CRITERION (pi. CRITERIA) - Something
which can be measured. Commonly
used as a basis for standards.
CROSS CONNECTION - In plumbing, a
physical connectionbetween two dif-
ferent water systems, such as be-
tween potable and polluted water lines.
CUBIC FOOT PER SECOND (c. f. s.) -
A unit of discharge rate such as
one cubic foot of gas per second
past a given point.
5
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Glossary - Wastewater Treatment Technology
CURVE -
a) A graphic plotted to represent
changes in value of one quantity in
reference to another.
b) A deviation from a straight line
without a sharp break or angularity.
DATA - Records of observations or
measurements of facts, occurrences
and conditions in written, graphical
or tabular form.
DEBRIS -
a) The remains of something broken
down or destroyed.
b) An accumulation of fragments of
rocks.
DECAY -
a) To undergo decomposition.
b) Implies a slow change from a state
of soundness or perfection.
c) To decay.
DEGRADE - To reduce the complexity of
a chemical compound.
DEIONIZED WATER - Water that has been
treated by ion exchange resins or com-
pounds to remove cations and anions
present in the form of dissolved salts.
DENITRIFICATION -
a) The conversion of oxidized nitrogen
(nitrate and nitrite-N) to nitrogen gas
by contact with septic wastewater solids
or other reducing chemicals.
b) A reduction process with respect
to oxidized nitrogen.
DETENTION PERIOD - The theoretical
time required to displace the entire
volume of a tank or basin at a given
rate of discharge. Tank volume -
rate of discharge.
DETENTION PERIOD, ACTUAL - The
actual time required for a given unit
of liquid to flow through the tank or
process unit. Usually determined
by tracer method and depends upon
inlet and outlet geometry, temperature,
specific gravity, stratification, and
other factors.
DETERGENT - Something used for clean-
ing. Commonly consists of soap or,
surfactant plus various additives or
associated materials.
DETRITUS - The heavier material moved
by natural flow, usually along the
channel bed. Sand, grit or other
coarse material.
DIAPHRAGM PUMP - A pump consisting
of a rubber diaphragm (generally)
fastened to a cylindrical casing
having inlet and outlet valves. When
the diaphragm is raised, liquid |
enters to be forced out the discharge
valve on the reverse stroke.
DIFFUSED AERATION - Aeration pro-
duced by introducing air through a
dispersing mechanism into a liquid.
Sufficient air pressure must be
applied to overcome hydrostatic head
and diffusor or pipe back pressure.
DIFFUSOR - A porous plate, tube, bag,
or other device, through which air
is forced into a liquid In the form
of small bubbles.
DIGESTED SLUDGE - Solids concentrated
stabilized under aerobic or anaerobic
conditions to preferentially decompose
the more unstable fractions and pro-
duce a residue of satisfactory dispoisal
characteristics. To reduce the
volatile fraction of the sludge.
DILUTION -
a) To make thinner or more liquid.
b) A ratio, volume or weight of a
more concentrated sample or effluent'
flow compared to that into which it
is discharged.
DISINFECTION -
a) To make free of infectious
organisms.
b) To kill disease organisms.
DISPOSAL -
a) The discarding or throwing away..
b) For wastewaters, this may repre-
sent any method of disposing, but
usually involves some degree of
degradation and discard in a non-
pollutional manner.
6
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Glossary - Wastewater Treatment Technology
DISSOLVED -
a) Those materials dispersed in water
in ionic, atomic, or molecular form;
an homogenous mixture or solution.
b) Generally clear but may be colored.
c) Present in true solution form.
DISSOLVED OXYGEN (D. O.) - Dissolved
molecular oxygen usually expressed
in mg DO/1 or percent of saturation.
DISTILLED WATER - A purified water
resultirfg from heat vaporization
followed later by vapor condensation
to separate the water from non-
volatile impurities.
DISTRIBUTOR -
a) A device to control flow into some
desired direction or place.
b) A device used to spread the flow
evenly across a trickling filter surface
or other process unit.
DIVERSION CHAMBER - A basin or tank
that may be used to divert part of the
flow from a channel. May or may not
contain treatment capabilities or a
means of returning the diverted flow
to the treatment plant when a shock
load has passed.
DOSING SIPHON - A device to permit inter-
mittent dosing, such as for a trickling
filter. Consists of a chamber that will
fill gradually to a fixed level before
starting a siphon that permits rapid
drainage to the filter or other treat-
ment unit.
DRAINAGE TILE (Filter, or bottom tile) -
A vitrified tile underdrainage system
laid on the bottom to support trickling
filter stone, sand, or other filter
media, including sludge drying beds.
These are specially prepared blocks
or half-tiles containing slots for
passage of water or air but restricting
bed media penetration.
DRYING - The removal of water by natural
or engineered means.
DYNAMIC HEAD, TOTAL - The difference
in pressure at the elevation of the
pump discharge and the elevation at
the pump suction flange, plus the
velocity head at the discharge minus
the velocity head at the suction flange,
all corrected to the same units and
datum points.
ECOLOGY - The relation of an organism
to its environment, i. e., how is an
organism affected by his surround-
ings such as air, water, heat, noise,
contamination, etc.
EFFICIENCY - The ratio of materials
out of a process to those into that
process usually expressed as a
percentage.
EFFLUENT - A liquid or product water
discharged from a chamber, basin
or other treatment operation.
ELEMENT -
a) Elementary substance.
b) A substance or kind of matter
in which all atoms are alike in that
they will have the same average
relative weight and the same number
of external electrons.
ELUTRIATION - A washing operation.
Sludge elutriation is an action wheri
digested or process sludge is washed
with sewage or effluent to remove
fine particulates or certain soluble
components. The elutriate is re-
cycled to process waters, the elu-
triated solids are more readily
filtered.
ENDOGENOUS METABOLISM - A dimin-
ished level of metabolism in which
various materials previously stored
by the cells are oxidized.
ENTERIC ORGANISMS - Those organisms
commonly associated with the in-
testinal tract.
ENTRAINMENT - A condition or action
that will cause an immiscible sub-
stance to be mixed with another.
Usually the result of turbulence or
entrapment, i.e., air bubbles in
aqueous media.
ENZYME - A soluble or colloidal organic
catalyst produced by a living organ-
ism. Usually they are simple or
conjugated proteins that catalyze
specific reactions.
EQUIVALENT -
a) Equal in force, amount, or value.
b) Chemical The atomic or molec-
ular weight of one substance that will
react with one unit of weight of another
substance, i. e., that weight of an
7
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Glossary - Wastewater Treatment Technology
alkali necessary to precisely equalize
1 gram atomic wt. of H+ion.
EUTROPHIC - Well nourished, rich in dis-
solved nutrients.
EUTROPHICATION - An action involving
the aging of lakes characterized by
nutrient enrichment and increasing
growth of plant and animal organisms.
The net effect is to decrease depth
until the lake becomes a bog and
eventually dry land. Man-made pol-
lution tends to hasten the process.
FACULTATIVE BACTERIA - Bacteria
that can adapt themselves to growth
and metabolism under aerobic or
anaerobic conditions. Many organ-
isms of interest in wastewater
stabilization are among this group.
FAHRENHEIT - A temperature scale in
which pure water at sea level has
a freezing point at 32 0 and the
boiling point is 212 °.
FATS - Naturally occurring compounds
functioning as storage products in
the living organisms. Consist of
carbon, hydrogen and oxygen in the
form of fatty acid esters. Generally
semi-solid or oily at normal temper-
atures.
FECAL COLIFORM - A group of organisms
belonging to the coliform group and
whose presence denotes recent fecal
pollution from warm-blooded animals.
Standard tests are available to differ-
entiate the fecal coliform group from
the other members of the group which
have a lesser sanitary significance.
FERMENTATION - A form of respiration
by organisms which requires little or
no free oxygen, yielding alcohol and
carbon dioxide as end products and
releasing only part of the food energy
available; i.e., the conversion of
sugars into alcohol by enzymes from
yeasts.
FILTER - A porous media through which
a liquid may be passed to effect re-
moval of suspended materials. Filter
media may include paper, cloth, sand,
prepared membranes, gravel, as-
bestos fiber, or other granular or
fibrous material.
FILTER CLOTH - Fabric, wire or other
material stretched over the drum
of a vacuum filter and accessories
to support the solids during cake
formation and discharge the solids
when and where desired.
FILTER FLOODING - The filling of a
trickling filter with liquid to a level
above the media by closing all out-
let parts. Generally to control
nuisance organisms such as flies.
FILTER FLY - Small black flies commohly
found in or near the trickling filter.
Commonly the Psychoda group.
FILTER LOADING - The mass (or volume)
of applied oxygen demand or solids
per unit of filter area or volume.
See load ratio.
FILTER MEDIUM - Any material over
which water sewage or other liquid
is passed for purification purposes
by chemical, biologioal or physical
processes.
FILTER PONDING or CLOGGING - The
effect of fine particles on sand
filters or organic growth on trlck-
ling filters that restricts normal
passage of liquid through the filter
as a result of filling void spaces.
FILTER RESIDUE - That material which
is retained on or in a filter.
FILTER UNLOADING - A phenomenon
in which normally attached growth
or slime on trickling filter media
becomes detached and either par-
tially or completely sloughs off.
FILTRATE - That liquid which has
passed through a filter.
FILTRATION RATE - A rate of applica-
tion of water or wastewater to a
filter. Commonly expressed in
million gallons per acre per day
or gallons per square foot per min.
FINAL SETTLER, CLAREFIER - A
settling basin or chamber for the
mixed liquor following secondary
treatment.
FLAME ARRESTOR - A safety device
in the handling of flammable gases.
Usually consists of an enlargement
in a pipe line containing a metallic
8
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Glossary-Wastewater Treatment Technology
grid that allows passage of gas but
acts as a barrier to the passage of
flame.
FLIGHTS - A cross member of a conveyor
system used for collection and trans-
port of the collected material, i. e.,
the boards fastened to a chain loop
on either side of a primary clarifier
that pushes scum along the surface
to a collector trough and sludge along
the bottom to the sludge collector.
FLOAT CONTROL - Commonly a device
to control a pump or pumps according
to the water level in a chamber or
well as indicated by the float. Usually
operates a relay to control pump power,
number, or speed of pumps in operation.
FLOATATION - A process for separation
of solids from clarified liquid that
causes particulates to be floated to
the surface by means of attached air
globules.
FLOATING COVER - A gas tight cover
with a water seal supported by digester
gas pressure and capable of moving
upward or downward with liquid and
gas content of the digester.
FLOC - Gelatinous or amorphous solids
formed by chemical, biological or
physical agglomeration of fine ma-
terials into larger masses that are
more readily separated from the liquid.
FLOCCULATION - The gathering together
of fine particulate materials in a sus-
pension to form loosely associated
larger masses of solids agglomerates.
FLUME - A long narrow channel for gravity
flow of liquid from one point to another.
FLY AWAY BOD - Wastewater stabilization
operations such as trickling filters en-
courage the development of insect larvae
that serve as scavengers during their
development. If the adult form of the
larvae have functionable wings, the
equivalent of oxygen demand consumed
during development becomes fly-away
BOD.
FREE AVAILABLE CHLORINE - Generally
includes that chlorine existing in water
as the hypochlorous acid. Character-
ized by rapid color formation with
ortho tolidine. Can be titrated in
neutral solution with phenyl arsene
oxide and produces a rapid organism
kill in low concentrations.
FREE BOARD - The vertical distance
from the normal water level in a
flume, conduit, channel, basin, or
other water enclosure, to the top
of the confining structure.
FRESH SLUDGE - Recently deposited
sludge from sedimentation tanks
that has not been conditioned,
processed, or progressed mater-
ially into the anaerobic action stage.
FUNGI - Simple or complex organisms
without chlorophyll. The simpler
forms are one-celled, higher forma
have branched filaments and com-
plicated life cycles. Examples are
molds, yeasts and mushrooms.
FWPCA - Federal Water Pollution Control
Administration, U. S. Department of
the Interior.
GAS DOME - A chamber usually mounted
on top of the digester cover for
separation of gas from scum, foam
or liquid.
GAS HOLDER - A tank used for storage
of gas from sludge digestion units
for the purpose of meeting the gas
demand for burners, engines, or
other use during non-steady pro-
duction or use periods.
GATE CHAMBER or GATE HOUSE -
A chamber installed for housing
devices for controlling flow to
various part3 of a collection, treat-
ment or distribution system,
including valves, gates, and auto-
matic or manual controls.
GAUGE -
a) The action of measuring some
item such as flow, level, size,
rate, etc.
b) A device for gauging.
c) A size designation or indicator
for some definite item such as 20-
gauge wire, 16-gauge sheet steel.
GERMICIDE - An agent that kills micro-
organisms.
9
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Glossary - Wastewater Treatment Technology
GRAVITY SYSTEM - A system of open or
closed conduits in which the liquid
flows by gravity (without pumping).
GRIT - The heavy material in water or
sewage such as sand, gravel, cinders,
etc.
GRIT CHAMBER - An enlargement of a
channel designed to reduce flow ve-
~ locity adequately to permit differential
separation of sand or grit from organic
suspended material. Usually approaches
a linear flow velocity of 1 to 3 ft[sec.
GRIT COLLECTOR - A device placed in a
grit chamber to collect and to convey
tiie more coarse and dense grit parti-
cles out of the chamber and permit
return of most of the organic or liquid
materials.
HARDNESS - Commonly refers to the
chemicals interfering with soap action
or producing scale in boilers or
heating units. Specifically refers to
Calcium and Magnesium salts, some-
times including iron, aluminum, and
silica.
HEAD LOSS - The difference in pressure
between the inlet and outlet pressure
of a given process unit arising as a
result of flow resistance within the
process unit, such as the head loss
due to friction of a filter media and
filter residue.
HEAT EXCHANGER COILS - A piping lay-
out designed to circulate a liquid media
within the contents of the process unit
but without mixing with the process
media for the purpose of adding or re-
moving heat, i. e., hot liquids may be
circulated within a digester to raise
digester temperature.
HUMUS - A brown or black complex and
variable material resulting from de-
composition of plant or animal mattef.
HYDROLYSIS - The addition of water to
any chemical compound. Commonly
involved in splitting complex sub-
stances by addition of water to form
more simple compounds.
HYDROSTATIC HEAD - The pressure
exerted by a given height of liquid
above a given datum point. May be
listed in feet of head, pounds per
square inch, or other criteria.
IMPELLER - A rotating set of vanes to
impart motion to a fluid, commonly
within a casing where dynamic energy
of fluid increases from the center
to the tip of the vanes. May be closed
or open depending on a tube or paddle
configuration.
IMHOFF CONE - A conical glass container
commonly one liter capacity, having
the upper larger diameter end open
and the closed apex downward with
graduations to assist estimation of
the volume of settleable solids after
an arbitrary time interval for set-
tling (usually one hour).
IMHOFF TANK - A deep two-story tank
originally patented by Karl Imhoff.
The floor of the upper chamber is
s lotted for transfer of settleable
solids from the settling chamber.
The lower chamber serves for an-
aerobic digestion and storage of
solids.
INDICATOR - May include the color change
of a dye, electronic sensor response,
or other means of estimating the
equivalence point of a reaction be-
tween two different materials.
INFILTRATION -
a) The entrance of ground water into
a sewer through breakB, defective
joints, or porous walls.
b) The penetration of water through
the soil from surface precipitation,
stream or impoundment boundaries.
INFLUENT - That material entering a
process unit or operation.
INORGANIC - Being composed of material
other than plant or animal materials.
Forming or belonging to the inanimate
world.
INTERCEPTOR - An intercepting sewer
designed to carry the dry weather
flow from a community to a treatment
plant, but not large enough to carry
storm water above some preset ratio
to dry weather flow. May be used
to collect lateral sewer flows.
10
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Glossary - Wastewater Treatment Technology
LAGOON - A natural or artificial basin used
for storage and/or stabilization of
wastewater or sludge. Sometimes
used for indefinite storage for dis-
posal purposes. Commonly the lagoon
depth is greater than a wadable depth
but not greater than twenty feet.
LATERAL SEWER - A sewer that dis-
charges into a branch or main sewer
and has no other tributaries other
than individual house connections.
LIQUID SLUDGE - An organic solids con-
centrate usually formed by deposition
from wastewaters. The water content
varies with the origin and nature of
the sludge, usually has enough water
to permit pumping but does not contain
significant separatable free water.
LOAD - The load to a process is that which
is contained in the inflow to that process.
It may be expressed as hydraulic,
oxygen demand, solids, or other
criteria.
LOAD RATIO - An index of loading, in-
cluding mass input per unit of capacity
per unit of time. Mass may be ex-
pressed in lbs., BOD, COD, Susp.
or volatile solids, capacity in volume,
weight of solids or volatile solids in
process.and time,usually in days.
LYSIS - To decompose, loosen, or separate
into component parts.
MANHOLE - An opening by which access
may be achieved for inspection,
maintenance, or repair of a sewer,
conduit, or other buried structure
or appurtenance.
MANOMETER - An instrument for measuring
pressure. Usually consists of a U-
shaped tube containing a liquid, the
surface of which moves proportionally
in one open end with changes in
pressure exerted upon the other end.
MECHANICAL AERATION - Aeration
produced by mechanical energy of
the turbine, pump, paddle, or other
device that imparts an intimate mix-
ture of liquid and air.
MEMBRANE FILTER - A flat, highlyporous
flexible plastic disc, commonly about
0.15 mm in thickness and 47-50 mm in
diameter. Membrane filters having
a pore size of 0. 45 n are used in
water microbiology to trap organisms,
and,by use of standard media and
conditions, direct enumeration by
colony count of selected organisms.
MENISCUS - The curved upper surface
of a liquid in a tube that is concave
upward when the containing walls
are wetted by the confined liquid,
and convex upward when they are not.
MESOPHILLIC - Medium temperature
loving. Organisms capable of opti-
mum metabolic activities at temper-
atures from about SO0-!!!)0^, 26 -
42°C.
METER - The length of a reference
platinum bar used as a standard
unit of measurement of length in
the metric system. 1 meter ¦
39. 37 inches.
MICRO - 1/1,000, 000 of a unit of measure-
ment, such as microgram, microliter.
MICROBIOLOGY - The science and study
of microbiological organisms and
t heir behavior. Commonly related
to the study of pathogenic organisms.
MICROORGANISM - Commonly an organ-
ism too small to be observed indi-
vidually by the human eye without
optical aid.
MILLI- - An expression used to indicate
1/1000 of a standard unit of weight,
length or capacity (metric system).
Milliliter (ml) 1/1000 liter (1)
Milligram (mg) 1/ 1000 gram (g)
Millimeter (mm) Tl 1000 meter (m)
MILLI EQUIVALENT WEIGHT - 1/1000
of the equivalent weight, usually
expressed in milligrams (mg).
MG/L - A unit of concentration on a
weight/volume basis: Milligrams
per liter. Equivalent to ppm when
the specific gravity of the liquid is
1. 0.
MIXED LIQUOR - A mixture of return
sludge and wastewater in the aerator
of an activated sludge plant. Also
may be used in reference to mixed
aerobic or anaerobic digesters.
11
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Glossary - Wastewater Treatment Technology
MIXING ZONE - An area where two or more
substances of different characteristics
blend to form a uniform mixture; i. e.,
chlorine application, heated water, or
other discharged materials entering a
water mass will show significant dif-
ferences of chlorine residual, tem-
perature or other criteria, depending
upon the sampling location throughout
the mixing zone and approach uniform
results with respect to lateral,longi-
tudinal^and vertical sampling positions
when mixing has been completed.
MOISTURE CONTENT - The content of
water in some other material. Com-
monly expressed in percentage of
moisture in soil, sludge or screenings.
MOST PROBABLE NUMBER (MPN) - A
statistical method of determining
microbial populations. A multiple
dilution tube technique is utilized
with a standard medium and observa-
tions are made for specific individual
tube effects. Resultant coding is
translated by mathematical probability
tables into population numbers.
NITRIFICATION - The biochemical con-
version of unoxidized nitrogen
(ammonia and organic N) to oxidized
nitrogen (usually nitrate).
NORMALITY -
a) A means of expressing the concen-
tration of a standard solution in terms
of the gram equivalents of reacting
substances per liter.
b) Generally expressed as a decimal
fraction, such as 0.1 or 0. 02 N.
NUTRIENTS -
a) Anything essential to support life.
b) Includes many common elements
and combinations of them. The major
nutrients include carbon, hydrogen,
oxygen, nitrogen, sulfur, and phosphorus.
c) Nitrogen and phosphorus are of
major concern because they tend to
recycle and are hard to control.
ODOR CONTROL - In wastewater treatment
this generally refers to good house-
keeping in the plant and aeration,
chlorination or other operations to
prevent onset of malodorous septicity
in the wastewater flow.
OILS -
a) Liquid fats of animal or vegetable
origin.
b) Oily or waxy mineral oils.
ORGANIC - Substances formed as a result
of living plant or animal organisms.
Generally contain carbon as a major
constituent.
ORGANIC CHLORINE - Compounds con-
taining chlorine in combination with
carbon, hydrogen and certain other
elements.
ORIFICE METER - A device consisting
of a flange set in a pipe section
containing an opening smaller than
the pipe. Pressure readings above
and below the orifice may be related
to flow.
ORTHO TOLIDINE CHLORINE TEST -
The dye, ortho toll dine, under highly
acid conditions, produces a yellow
color proportional In Intensity to the
concentration of available residual
chlorine and certain other oxidants
or Interfering materials.
OUTFALL SEWER - The outlet or channel
through which sewage effluent is dis-
charged.
OXIDATION - Chemically: The addition
of oxygen, removal of hydrogen, or
the removal of electrons from an
element or compound.
OXIDATION POND - A shallow basin
employed for the stabilization of
wastewaters.
OXYGEN AVAILABLE - That part of the
oxygen available for aerobic stabil-
ization of organic matter. Includes
dissolved oxygen and that available
in nitrite or nitrates, peroxides,
ozone, and certain other forms of
oxygen.
OXYGEN BALANCE - Refers to the dy-
namic relationship among the avail-
able oxygen assets and the oxygen
requirements for stabilization of
oxygen demanding materials in a
treatment plant or receiving water.
12
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Glossary - Wastewater Treatment Technology
OXYGEN DEPLETION - The loss of oxygen
from water or sewage due to biological,
chemical or physical action.
PARASITE - A living organism deriving its
nutrients at the expense of another
living organism, giving nothing in re-
turn.
PARSHALL FLUME - A device for estima-
tion of the flow in an open conduit.
Consist? of a constricting section,
a throat, and an expanding section.
The throat contains a sill over which
the liquid passes. The pressure
change over the sill can be related
to quantity of flow.
PARTICULATE MATERIAL - Refers to
detectable solid material dispersed
in a gas or liquid. Small sized par-
ticulates may produce a smoky or hazy
appearance in a gas; milky or turbid
appearance in a liquid. Larger partic-
ulates are more readily detected and
separated by sedimentation or filtration.
PARTICULATES - Pertaining to small sus-
pended solids in a gaseous or liquid media.
PARTS PER MILLION (PPM) - A unit of
concentration signifying parts of some
substance per million parts of dis-
persing medium. Equivalent numer-
ically to mg11 only when the specific
gravity of the solution is 1.0.
PATHOGENIC ORGANISMS - Bacterial,
fungal, viral, or other organisms
directly involved with diseases of
plants, animals, or man, are in-
cluded among this group.
PERCENTAGE TREATMENT - The ratio
expressed as a percentage of the
material removed from process water
in terms of the material entering.
Sometimes referred to as reduction.
pH - An index of hydrogen ion activity.
Defined as the negative logarithm
(base 10) of H+ ion concentration at
a given instant. On a scale of 0 to 14
pH 7. 0 is neutral, pH less than 7. 0
indicates a predominance of H or
acid ions, pH greater than 7. 0 indi-
cates a predominance of OH" or
alkaline ions.
PNEUMATIC EJECTOR - A device for
pumping sludge, sewage, or other
liquid by admitting the fluid into a
chamber through one check valve
and forcing it out of another by air
pressure in the chamber above the
liquid.
POLLUTION - Anything appearing in water
that renders it unacceptable in terms
of established water quality standards.
Commonly conditions or contaminants
that interfere with subsequent bene-
ficial uses of the water.
POND - A basin or catchment used for
retention of water for equalization,
stabilization, or other purposes.
Commonly less than five feet in depth.
PONDING - With reference to trickling
filtration, ponding refers to a plugging
of the filter media by slimes or solids
to restrict downward movement of
wastewater sufficiently to cause sur-
face accumulation of liquid either
partially or completely.
PRECIPITATE - The formation of solid
particles in a solution, or the solids
that settle as a result of chemical
or physical action that caused solids
suspension from solution.
PRESSURE - The total load or force acting
upon a surface. In hydraulics, the
term commonly means pounds per
square inch of surface, or kilograms
per square cm above atmospheric
pressure on site. (Atmospheric
pressure at sea level is about 14. 7
pounds per square inch.)
PRIMARY SLUDGE - Sludge obtained
from a primary sedimentation tank.
PRIMARY TREATMENT - Commonly the
separation of settleable or floatable
materials from carrier water.
Usually preceded by pretreatment
such as coarse screens, grit sep-
aration, comminution.
PROCESS - A series of operations or
actions that lead to a particular
result. A combination of unit oper-
ations that may be assembled and
used for a given treatment objective.
13
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Glossary - Wastewater Treatment Technology
PROTEINS - Naturally occurring compounds
containing carbon, hydrogen, nitrogen,
and oxygen, with smaller amounts of
sulfur and phosphorus, and trace com-
ponents essential to the living cells.
An essential food associated with
meat and eggs.
PROTOZOA - Single cell or multiple cell
organisms, such as amoeba, celiates,
and flagellates. Commonly aquatic
and generally derive most of their
nutrition from preformed organic food.
PSYCHROPHfLLIC ORGANISMS - Low
temperature loving organisms, or
having a competitive advantage
over other organisms at lower tem-
peratures; i. e., from about 10°C
downward to the freezing point.
PUTREFACTION - Biological decomposition
of organic matter with the formation
of ill-smelling products, such as H2S,
amines, mercaptans. Associated with
anaerobic conditions.
QUIESCENT - Characterized by a lack of
or negligible movement of the sus-
pending media, such as liquid or gas.
Still or absence of turbulence.
b) An individual who tabulates or
maintains records of events, actions
or measurements.
REDUCTION -
a) To make smaller or to remove
from a given amount of material
b) Chemistry: The removal of
oxygen, addition of hydrogen, or
the addition of electrons to an ele-
ment or compound.
RELIEF SEWER - A sewer built to carry
the flow in excess of the capacity of
an existing sewer.
RESIDUAL CHLORINE - See Available
Residual Chlorine.
RETURN SLUDGE - Sludge returned from
process to the influent flow. Com-
monly return activated sludge from
a secondary clarifier. Also may
include sludge from a clarifier after
trickling filtration.
ROTARY DISTRIBUTOR - A device usually
mounted on a center post with hori-
zontal arms extending to the edge of
a circular trickling filter for distri-
bution of flow over the entire bed
surface.
RAW WASTEWATER (SEWAGE) - Used
wastewater prior to treatment.
RECIPROCATING PUMP - A pump device
using a piston within a casing fitted
with suction and discharge valves.
Movement of the piston in one direction
fills the casing, the reverse movement
forces liquid into the discharge line.
May be vertical or horizontal.
RECIRCULATION - The return of effluent
to the influent of a process unit to
reduce influent concentration, sta-
bilize the system, maintain hydraulic
flow, to reprocess^ or for other bene-
ficial reasons.
RECORDER -
a) A device to keep a continuous or
Intermittent record of some measured
item such as flow, velocity, applied
power, etc.
SALT - A chemical compound formed as
a result of the interaction of an acid
and an alkali (base). The common-
est salt is sodium chloride formed
from hydrochloric acid and sodium
hydroxide. This ionizes in water
solution to form Na+ and CI".
SANITARY SEWER - A sewer designed
to receive and to convey household,
commercial or industrial waste-
water mixtures.
SAPROBIC, SAPROPHYTIC - Organisms
living upon dead or decaying organic
matter. Organisms that utilize non-
living organic matter as a food.
SATURATION - Commonly refers to the
maximum amount of any material
that can be dissolved in water or
other liquid at a given temperature
and pressure. For oxygen, this
commonly refers to a percentage
saturation in terms of the saturation
value, such as about 9 mg Oo/l at
20° c.
14
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Glossary - Wastewater Treatment Technology
SCAVENGERS - Organisms that feed
habitually upon refuse or carrion.
In water pollution, this commonly
refers to worms, insect larvae,
bloodworms, sow bugs, and crusta-
ceans. Or, more properly, oligo-
chaetes, chironomids and isopods.
SCREEN - A device with openings, gener-
ally having a relatively uniform size,
that permit liquid to pass but retain
larger particles. The screen may
consist of bars, coarse to fine wire,
rods, gratings, paper, membranes,
etc., depending upon particle size
to'be retained.
SCREENINGS - Material removed by the
screens.
SCUM BOARD - A vertical baffle, above
and below the liquid surface of a basin
or tank, designed to prevent the
passage of or to contain floating
material within designated limits.
SCUM BREAKER - A device installed in a
sludge digestion tank to disperse sur-
face accumulations. Generally
accomplished by means of mechanical
agitation, gas or liquid recirculation,
to promote mixing and destratification.
SCUM COLLECTOR - A mechanical device
for skimming and removing scum or
floatable material from the surface
of a tank.
SECOND FOOT - An abbreviation for
cubic foot per second. A rate term.
SECONDARY TREATMENT - Processes
used to convert dissolved and colloidal
materials in wastewater to a form that
may be separated from the water.
Commonly consists of biodegradation
and conversion to cell mass in a
separatable form with partial oxida-
tion, such as in activated sludge,
trickling filtration, or oxidation ponds.
SEDIMENTATION - The process of subsi-
dence and deposition of suspended
matter from wastewater by gravity.
Also called clarification, settling.
SEPTIC SLUDGE - That sludge which has
reached a stage of anaerobic putre-
faction (sulfate reduction). Includes
that from Imhoff, septic, or sludge
digestion tanks.
SEPTIC WASTEWATER (SEWAGE) -
Wastewater in which available oxy-
gen has been depleted and the
reduction of sulfates has begun. A
result of anaerobic putrefaction.
SETTLEABLE SOLIDS -
a) Includes materials that will settle
by gravity under low flow velocities.
b) Commonly expressed in terms
of the volume of solids accumulating
in an Imhoff cone after one hour on
a volume basis.
SETTLING BASIN - A natural or engineered
enlargement of a channel that reduces
velocity sufficiently to permit sedi-
mentation of settleable particulates.
SEWAGE - See Wastewater.
SEWAGE GAS, DIGESTER GAS - The gas
produced from anaerobic (septic)
sewage solids. Generally contains
marsn gas (methane) and carbon
dioxide with hydrogen sulfide and
other components in minor propor-
tions.
SEWER - A pipe or conduit,generally
coveredjfor the purposes of con-
veying wastewaters from the point
of origin to a point of treatment or
discharge.
SEWERAGE SYSTEM - A system of
sewers and appurtenances for the
collection, transportation and
pumping of used waters for a given
area or basin. Any treatment de-
vice or facility and its outfall con-
duit are a part of the system.
SHORT CIRCUITING - Hydraulic: A con-
dition in which one part or unit of
flow into the basin reaches the outlet
in much less time than that required
for a uniformly mixed flow.
Electrical: A situation in which an
electric current is out of place in
relation to its controlled pathway.
SLIME - SEWAGE SLIMES - Consisting
of organisms growing on wastewater
nutrients with the formation of mu-
cilaginous covering, streamers or
clumps. May consist of bacteria,
molds, protozoa or algae.
15
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Glossary - Wastewater Treatment Technology
SLOUGHING - A phenomenon associated
with trickling filters and contact
aeration units where slimes build up
to a varying degree, then slip off
into the effluent.
SLUDGE - Accumulated or concentrated
solids from sedimentation or clari-
fication of wastewater. Contains
varying proportions of solids in
wastewater depending upon source,
process, and nature.
SLUDGE BANKS - An accumulation of solids
including silt, mineral, organic, and
cell mass particulate material, that
is produced in the aquatic system
characterized by low current velocity.
Generally refers to gross deposits of
appreciable depth.
SLUDGE CAKE - The solids remaining
after dewatering sludge by vacuum,
filtration, or sludge drying beds.
Usually forkable or spadable, ,with
a water content of 30 to 80%. Also
may occur on the boundaries of
surface water.
SLUDGE COLLECTOR - A mechanical
device, including rake, drag, or
suction, for collecting settled sludge
from the bottom of a clarifier into a
sump or other withdrawal system.
SLUDGE DIGESTION - A process by which
organic matter in sludge is converted
into more stable or separatable form
through the action of living organisms.
May be the result of aerobic or an-
aerobic digestion.
SLUDGE DRYING BED - An area used to
discharge wet sludge for drainage
and drying. Generally prepared of
porous bed material surrounded by
sidewalls to contain the sludge while
the liquid percolates into an under-
drain system. May be covered or
uncovered.
SLUDGE FILTER - A device to effect
partial water removal from wet
sludge, usually with the aid of vacuum
or pressure of preconditioned sludge.
SLUDGE SYNTHESIS - The net gain in
sludge mass in a process over a
period of time as a result of simul-
taneous growth of cell mass and
endogenous oxidation within it.
SLUICE GATE - A gate constructed for
adjustment to control the flow In a
channel by gate position.
SOLUTION -
a) An homogenous mixture, commonly
gas, liquid, or solid in a liquid that
remains clear Indefinitely.
b) Generally an atomic, ionic, or
molecular dispersion in a liquid (may
be colored).
c) A water solution of dissolved
material.
SPECIFIC GRAVITY (Sp. Gr.) -
a) The weight of a material per unit
volume in reference to the weight
of water at maximum density.
b) Water at 4°C has a weight of lg
per ml. The weight ratio of any
substance divided by the weight of
water is the specific gravity.
SQUEEGEE -
a) A device, generally rubber, used
for dislodging and removing solids
scum or other materials from a
surface.
b) Metal or wood blades to move
sludge solids along the bottom of
a clarifier.
STABILITY -
a) The ability of any substance to
resist putrefaction.
b) Ability of an engineered structure
to resist distortion or overturn when
loaded.
STABILIZATION -
a) The activity proceeding along the
pathway to stability.
b) In organic wastes, generally
refers to oxidation via biochemical
pathways and conversion to gaseous
or insoluble materials relatively
inert to further change.
STANDARD - Something set by authority.
Having qualities or attributes re-
quired by law and defined by mini-
mum or maximum limits of accept-
ability in terms of established
criteria or measurable.indices.
16
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Glossary - Wastewater Treatment Technology
STANDARD METHODS - Methods of analysis
prescribed by joint action of APHA,
ASCE, AWWA, and FWPCA. Methods
accepted by authority.
STEP AERATION - A procedure for adding
increments of wastewater at various
points along the line of flow in an
activated sludge aerator.
STERILIZATION - The process of making
a medium free of living organisms
such as by killing them, filtering
through a porous medium fine enough
to be a barrier to the passage of
organisms, etc.
STORM OVERFLOW - A device such as a
weir, dam, or orifice, in a combined
sewer that will intercept design flow
but permit excess storm flow to dis-
charge directly. The overflow con-
tains a mixed discharge of storm and
other sewer components.
STORM SEWER - A sewer which carries
storm water from roofs, surface wash
and street drainage.
STUCK DIGESTER - Any of a series of
events that results in serious mal-
function of the digester. Commonly
refers to anaerobic digestion where
overloading, temperature control,
toxicity, or other factors, result in
an excessive acid production with
serious limitations of gasification,
stabilization, and solids concentration.
SUBSTRATE -
a) The base or media in which an
organisms lives.
b) The liquid in an activated sludge
aeration tank.
SUPERNATANT LIQUOR - The liquid over-
lying deposited sludge. Commonly
tnat fraction of liquid in an anaerobic
digester located over the deposited
material and beneath possible surface
floating material.
SURFACTANT -
a) A chemical that, when added to
water, will greatly reduce the surface
tension of the solution.
b) The surface active component in
a detergent mixture.
SUSPENDED SOLIDS - The concentration
of Insoluble materials suspended or
dispersed in waste or used water.
Generally expressed in mg/liter on
a dry weight basis. Usually deter-
mined by filtration methods.
SYNERGISM - Refers to the action pro-
duced when two or more substances
in combination have a greater effect
than that produced by the additive
effects of each one separately.
TAPERED AERATION - A procedure fori
adjusting air input along the line of
flow of an activated sludge aerator
according to need. Usually requires
addition of more air per unit of
volume at the inlet end of the aeratotr.
TERTIARY TREATMENT - See Advanced
Waste Treatment.
THERMAL POLLUTION - Refers to
heated discharges to surface water*
ways. The largest contributor of
heated discharges is associated
with power production.
THERMOPHILIC - High temperature
loving organisms. Generally con-
sidered to include organisms having
a favorable competitive advantage
at temperatures above llO^F or42°C.
THIEF - A term applied to a sampling
tube used to remove a core of
sample from a bag or bulk material.
TITRATION - The careful addition of a
standard solution of known concen-
tration of reacting substance to an
equivalence point to estimate the
concentration of a desired materiall
in a sample.
TOC - Total Organic Carbon. A test
expressing wastewater contaminant
concentration in terms of the car-
bon content.
TOTAL SOLIDS - Refers to the solids
contained in dissolved and suspended
form in waters Commonly deter-
mined on a weight basis by evapora-
tion to dryness.
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Glossary - Wastewater Treatment Technology
TRICKLING FILTER - A treatment process
employing downward flow of wastewater
over tne surfaces of a rock or grid
system with a large void space for
upward movement of air. Slime
organisms accumulate to effect bio-
logical stabilization.
UNIT OPERATION - A particular kind of
a physical change that is repeatedly
and frequently encountered as a step
in a process such as filtration,
aeration, evaporation, mixing, or
pumping.
UNITS OF MEASUREMENT -
English - foot, pound, second.
Metric - centimeter, grafn, second.
Abbreviations: ft., lb., sec.
cm., g.., sec.
USPHS - United States Public Health
Service, Department of Health,
Education and Welfare.
USPHS DRINKING WATER STANDARDS -
A list of standards prescribed for
potable water acceptable for use on
interstate carriers. Deal with
sources, protection, and bacterio-
logical, biological, chemical and
physical criteria—some mandatory,
seme desired. Official for municipal
use only upon acceptance by State and
local authorities.
VELOCITY (FLOW) - A rate term expressed
in terms of linear movement per unit
of time. Commonlv expressed in
ft per sec (English) or cm/ sec (Metric).
VENTURI METER - A device for estimating
flow of fluid in closed conduits or pipes.
Generally based upon constricting and
enlarging pipe sections with pressure
at the full size and the point of maximum
constriction. Differences in pressure
can be related to flow.
VIRUS - A term generally used to designate
organisms that pass filtration media
capable of removing bacteria. Tech-
nically described as a collective term
covering disease stimuli held by some
to be living organisms and by others
to be nucleic acids capable of repro-
duction and growth.
VOLATILE ACIDS - A group of low molec-
ular weight acids such as acetic and
propionic, that are distillable from
acidified solution.
VOLATILE MATERIAL -
a) Refers to those chemicals having
a vapor pressure low enough to
evaporate from water readily at
normal temperatures.
b) With reference to dry solids, the
term includes loss in weight upon
ignition at 600°C.
VOLATILE SOLIDS - The quantity of
solids in water that represents a
loss in weight upon ignition at 600°C.
WASTEWATER - Refers to the used water
of a community. Generally contam-
inated by the waste products from
household, commercial or lnduetrl41
activities. Often contains surface
wash, storm water and infiltrations
water.
WASTE SLUDGE -
a) Commonly refers to activated
sludge produced in excess of that
required for return process.
b) A ny solids concentrate to be
routed for disposal.
WATER QUALITY CRITERIA - Includes
selected analytical measurements
with limits designated to be accept-
able or unacceptable in reference
to water quality standards.
WATER QUALITY STANDARDS - Limits
set by authority on the basis of water
quality criteria required for bene-
ficial uses.
WEIR - A device used for surface over-
flow from a tank, basin or chamber.
Generally designed to smooth out
discharge flow to minimiz e turbu-
lence within the detention basin.
May be used to measure discharged
flow.
WEER BOX - An enlargement of the
channel upstream of a weir to re-
duce the velocity and turbulence
before reaching the weir.
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Glossary - Wastewater Treatment Technology
WELL -
a) An artificial excavation or shaft
that collects water from interstices
of the soil or rock.
b) Also an engineered structure for
the housing of pumps or other equip-
ment below ground level.
WET OXIDATION - Oxidation of substances
such as organic contaminants in water
media. Includes biological oxidation
and physical chemical oxidation, such
as that obtained at elevated tempera-
ture, pressure .catalyst or other pro-
moters.
WPCF - Water Pollution Control Federation.
An organization composed of individuals
engaged in the advancement of knowledge
in research, design, operation, and
control of water pollution in relation
to man and his environment.
YIELD -
a) To give up or relinquish.
b) To bear or bring forth as a result
of cell division.
c) To produce as a result of invest-
ment of energy, materials, or time.
d) The amount or quantity produced
per unit of raw material.
YIELD FACTOR - A decimal fraction or
percentage of product per unit of input.
REFERENCES:
1 Any of the standard dictionaries.
2 Glossary, Water and Sewage Control
Engineering. APHA, ASCE,
AWWA, FSWA. (1969)
3 Glossary, Ohio Operator Training
Committee.
4 Jacobs, MorrisB., Gerstein, Maurice J.,
and Walter, William G. Dictionary
of Microbiology. D. Van Nostrand,
Inc. (1957)
5 Rose, Arthur and Elizabeth. Condensed
Chemical Dictionary, 7th Ed.,
Reinhold Publishing Co. (1961)
6 U. S. Dept. of Interior, Office of Water
Resources Research. Water Re-
sources Thesaurus. (Nov. 1966)
7 Geckler, JackR., Mackenthun, K. M.,
and Ingram, W. M. Glossary of
Commonly Used Biological and
Related Terms In Water and Water
Control. Env. Health Series, UJ S.
Dept. HEW (July 1863)
8 Mathews, John E. Glossary of Eco-
logical Terms. Roberts. Kerr
Water Research Center, Ada, OK
In press.
ZOOGLOEA - A jelly-like matrix developed
by certain microorganisms at some
stage In their life cycle. Commonly
associated with sludge flocculation in
biochemical treatment operations.
ACKNOWLEDGMENTS:
Many individuals, unpublished memoranda
and literature sources contributed to the
selection of terms and key ideas Included — T ,
in this glossary. Contributors are too This outline was prepared by F. J. Ludzack,
numerous to list individually, but their Chemist, National Training Center, Office
assistance is gratefully acknowledged. of Water Programs, EPA, Cincinnati, OH 45268.
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