WASTEWATER
LABORATORY PROCEDURES
        & CHEMISTRY
 U.S. ENVIRONMENTAL PROTECTION AGENCY
             REGION VII

           1735 BALTIMORE
      KANSAS CITY, MISSOURI - 64108
         OFFICE OF INTERMEDIA PROGRAMS
         MANPOWER & TRAINING PROGRAM
         SURVEILLANCE & ANALYSIS DIVISION
          TECHNICAL SERVICES BRANCH


              JUNE 1975

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                                                 EPA 907/9-75-002
                            WASTEWATER

                LABORATORY PROCEDURES AND CHEMISTRY



     This manual has been adapted from Chapter 14 (by James Patterson)

of "Operation of Wastewater Treatment Plants - A Field Study Course."

     The complete Field Study Course has been prepared for EPA by

California State University - Sacramento and is available at a nominal

charge.  For information on ordering the complete course, write to:
                   Dr. Kenneth D. Kerri
                   Department of Civil Engineering
                   California State University
                   6000 Jay Street
                   Sacramento, California  95819
                   Environmental  Protection Agency
                   Region VII
                   1735 Baltimore
                   Kansas City, Missouri  64108
Office of Intermedia Programs               Surveillance & Analysis Division
Manpower & Training Program                 Technical  Services Branch
                             JUNE 1975

                                                Znwwmn^-i r- -  -
                                                     v;-'  .  ; -Lo0bl°n Agency

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The Superintendent of Documents
  classification number is:
        EP.1.8:   111

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                EPA REVIEW NOTICE
This manual has been reviewed by the Office of Water
Programs, EPA, and approved for publication.  Approval
does not signify that the contents necessarily reflect
the views and policies of the Environmental Protection
Agency.  Mention of trade names or commercial products
does not constitute endorsement or recommendation for
use by the Environmental Protection Agency or California
State University, Sacramento.
                          n

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                          TABLE OF CONTENTS

           CHAPTER 14  LABORATORY PROCEDURES AND CHEMISTRY

                                                                Page

14.0  Introduction	14-1

      14.00  Should You Start This Lesson Now?	14-1
      14.01  Material in This Lesson	14-2
      14.02  References	14-3
      14.03  Acknowledgements	14-4

14.1  Glossary of Terms and Equipment	14-4

      14.10  Terminology	14-4
      14.11  Equipment	14-6

14.2  Safety and Hygiene	14-12

      14.20  Laboratory Safety 	   14-12
      14.21  Personal Hygiene for Wastewater
             Treatment Plant Personnel  	   14-17

14.3  Sampling	14-21

      14.30  Importance	14-21
      14.31  Accuracy of Laboratory Equipment	14-21
      14.32  Selection of a Good Sampling Point
             to Obtain a Representative Sample .	14-22
      14.33  Time of Sampling	14-23
      14.34  Composition and Preservation of Samples  	   14-23
      14.35  Sludge Sampling	'	14-25
      14.36  Sampling Devices	14-26
      14.37  Summary	14-27

14.4  Laboratory Work Sheet	14-30

14.5  Plant Control Tests	14-35

      Test No.    Title

          1      Total  Alkalinity	14-37
          2      Biochemical  Oxygen Demand or BOD	14-37
          3      Carbon Dioxide (C02) in  Digester Gas	14-38
          4      Chemical  Oxygen Demand or COD 	   14-43
          5      Chlorine Residual  	   14-50

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      Test  No.    Title                                        Page
         6        Clarity	14-59
         7        Coliform Group Bacteria  	  14-62
         8        Dissolved Oxygen  or  DO	14-82
                 I.    In  Water	14-82
                 II.   In  Aerator	14-90
         9        Hydrogen Sulfide  (H2S).-	14-103
                 I.    In  Atmosphere.  . .	14-103
                 II.   In  Wastewater	14-104
        10        pH	14-107
        11        Settleability  of  Activated  Sludge  Solids.  .  14-113
                 I.    Settleability	14-113
                 II.   Sludge  Volume  Index (SVI)	14-115
                 III.  Sludge  Density  Index (SDI)  	  14-117
        12        Settleable Solids 	  14-119
        13        Sludge Age	14-123
        14        Sludge  (Digested) Dewatering
                 Characteristics  ....  	  14-126
        1^        Supernatant  Graduate Evaluation  	  14-129
        16        Suspended Solids	14-133
                 I.    Gooch Crucible  	  14-133
                 II.   Centrifuge  	  14-146
        17        Temperature  	  14-150
                 I.    Wastewater  	  14-150
                 II.   Digester  Sludge	14-154
        18        Total and Volatile  Solids (Sludge)	14-156
        19        Turbidity (See Clarity)
        20        Volatile Acids	14-164
        21        Volatile Solids  (See Total  Solids)
14.6  Recommended General Laboratory  Supplies	14-181
14.7  Additional  Reading  	  14-185
                                 IV

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                         OBJECTIVES


Chapter 14.  Laboratory Procedures and Chemistry

Following completion of Chapter 14 you should be able to:

1.  Work safely in a laboratory.

2.  Know how to operate laboratory equipment.

3,  Collect representative samples of influents to and effluents
    from a treatment process as well as sample the process.

4.  Prepare samples for analysis.

5.  Perform plant control tests.

6.  Recognize shortcomings or precautions for the plant control
    tests.

7,  Record laboratory results.

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       CHAPTER 14.  LABORATORY PROCEDURES AND CHEMISTRY
14.0  INTRODUCTION
14.00  Should You Start This Lesson Now?

Laboratory procedures and results are the means by which we control
the efficiency of our treatment processes and measure the effective-
ness of the processes.  To operate your plant as efficiently as
possible, you must understand the laboratory procedures and relate
them to the actual operation of your plant.

This lesson has been given to you at this time mainly for reference
purposes.  When you read the lessons on the treatment processes you
should begin to wonder how certain tests are performed that are
essential for proper plant operation.  At this time you should refer
to this lesson for a general discussion and a description of the
laboratory procedure.
It might seem logical to you to complete this lesson first in order
to better understand the operational aspects of the treatment process
lessons.  Many operators and potential operators who were interested
in this profession have taken this course.  Most of them have said
that they wanted to learn about the treatment processes first and
then learn how to apply the lab procedures to plant operation.  Many
potential operators experienced difficulty with the terminology when
they tried to work this chapter before completing the lessons on the
treatment processes.  If you are an experienced operator arid are
anxious to learn more chemistry and to obtain a better understanding
Of lab procedures, you may decide to try this lesson first.
                            14-1

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 Past  experience  has  indicated that most operators  prefer to use
 tjiis  section  as  a reference while  studying  the lessons  on treat-
 ment  processes.   You are  the  operator who wants to learn more
 abou"t treatment  plant operation, and you are encouraged to use
 this  material in any manner that you feel best fits ycur par-
 ticular  situation and professional goals.   Now is  the time for
 you to decide whether you are going to:

   1.   Thumb through  this  lesson, proceed through the chapters .
       on treatment processes, and  then complete this lesson;

   2.   Complete the lessons on treatment processes, referring
       to this lesson when interested,  and then complete this
       lesson;

   3.   Complete this  lesson and then the lessons on treatment
       processes;  or

   4.   Follow  your own plan.
 14..01  Material  in This  Lesson

 A  few of the' lab procedures  outlined, in  this  chapter are,.not
."Standard Methods" (4),1 but are  used by many operators  because
.they, are simple  and  easy to  perform.  Some  of these  procedures
 are  not accurate enough for  scientific investigations, but are
 satisfactory for successful  plant control and operation.   When
 lab  data must be submitted to regulatory agencies  for;monitoring
 and  enforcement purposes, you should  request  the agency,to ',   ,
 provide you  with a list  of approved test procedures.

 Each test section contains the  following information:

   1.  Discussion of  test.
   2.  What is tested?
   3.  Apparatus.
   4.  Reagents.
   5.  Procedures.
   6.  Precautions.
   7.  Examples.
   8.  Calculations.
  Numbers  in parentheses  refer  to  references  in  Section  14.02,
                              14-2

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If you would like to read an introductory discussion on laboratory
equipment and analysis, the Water Pollution Control Federation has
a good publication entitled "Simplified Laboratory Procedures" (3).
Good discussions on the use of the analytical balance may be found
in "Laboratory Procedures" (1) or "Simplified Procedures" (3),

14.02 References

1,  "Laboratory Procedures for Operators of Water Pollution Control
    Plants" by Joe Nagano.  Obtain from Secretary-Treasurer,
    California Water Pollution Control Association, P.O. Box 61,
    Lemon Grove, California  92045.  Price $3.25 to members of CWPCA;
    $4.25 to others.

2.  "EPA Methods for Chemical Analysis of Water and Wastes", Ana-
    lytical Quality Control Laboratory, 1014 Broadway, Cincinnati,
    Ohio  45202 (October 1974)

3.  "Simplified Laboratory Procedures for Wastewater Examination,"
    WPCF Publication No. 18,  1968, 60 pages.  $2 to WPCF members;
    $3 to others.

4-  "Standard Methods for Examination of Water and Wastewater,"
    13th Edition, 1971, 874 pages.  $16.50 to WPCF members;
    $22.50 plus postage to others.

Both References 3 and 4 may be obtained by writing:

    Water Pollution Control Federation
    3900 Wisconsin Avenue
    Washington, D.C.  20016

Order forms may be found in the Journal of the Water Pollution Control
Federation.
                            14-3

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14.03  Acknow1edgments

Many of the illustrated laboratory procedures were provided by
Mr. Joe Nagano, Laboratory Director, Hyperion Treatment Plant,
City of Los Angeles, California.  These procedures originally
appeared in Laboratory Procedures for Operators of Water
Pollution Control Plants, prepared by Mr. Nagano and published
by the California Water Pollution Control Association.  The
lists of equipment, reagents, and procedures outlined in this
chapter are similar to those listed in the references in
Section 14.02.  Use of information from these references is
gratefully acknowledged.
14.1  GLOSSARY OF TERMS AND EQUIPMENT
                                        14.10  Terminology
                                        > Greater than.

                                        DO > 5 mg/1, would-be
                                        read as DO greater than
                                        5 mg/1,              ~~
                                        < Less than.

                                        DO < 5 mg/1, would be
                                        read as DO less than
                                        5 mg/1.
                                        Aliquot (AL-li-kwot).
                                        Portion of a sample.
Ambient Temperature (AM-bee-ent).  Temperature of the surroundings.

Amperpmetric (am-PURR-o-MET-rick).  A method of measurement that
records electric current flowing or generated, rather than record-
ing voltage.  Amperoraetric titration is an electrometric means of
measuring concentrations of substances in water.

Anaerobic Environment (AN-air-G-bick).  A condition in which "free"
or dissolved oxygen is not present.
                             14-4

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Blank.  A bottle containing dilution water or distilled water,
but the sample being tested is not added.  Identical tests are
frequently run on a sample and a blank and the differences
compared.

Buffer.  A measure of the ability or capacity of a solution or
liquid to neutralize acids or bases.  This is a measure of the
capacity of water or wastewater for offering a resistance to
changes in the pH.

Composite (proportional) Samples (coir,-POZ-j.t).  Samples collected
at regular intervals in proportion to the existing flow and then
combined to form a sample representative, of the entire period of
flow over a given period of time.

Pistillate.  In the distillation of a sample, a portion is
evaporated; the part that is condensed afterwards is the distillate.

End Point.  Samples are titrated to the end point.  This means
that a chemical is added, drop by drop, to a sample until a
certain color change (blue to clear, for example) occurs which
is called the end point of the titration.  In addition tq a color
change, an end point may be reached by the formation of a precipi-
tate or the reaching of a specified pH,  An end point iray be
detected by the use of an electronic device such as a pH meter.

Flame Polished.  Sharp or broken edges of glass (such as the end
of a glass tube)  are flame polished by placing the edge in a flane
and rotating it.   By allowing the edge to melt slightly, it will
become smooth.

M pr^ Molar.  A molar solution consists of one gram molecular
weight of a compound dissolved in enough water to make one liter
of solution.  A gram molecular weight is the molecular weight of
a compound in grams.  For example, the molecular weight of sulfuric
acid (P^SOiJ is 98.  A 1M solution of sulfuric acid would consist
of 98 grams of H^SCV dissolved in enough distilled water to make
one liter of solution.

Molecular Weight.   The molecular weight of a compound in grains is
the sum of the atomic weights of the elements in the compound.  The"
molecular weight  of sulfuric acid (H2SQLf) in grams is 98.

                     Atomic  •      Number        Molecular
       Element       Weight       of Atoms        Weight

          H             1            2               2
          S            32  .          1              32

          0            16            4              64_
                                                    98
                             14-5

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N or Normal.  A normal solution contains one gram equivalent
weight of a reactant (compound) per liter of solution.  The
equivalent weight of an acid is that weight of a compound which
contains one gram atom of ionizable hydrogen or its chemical
equivalent.  For example, the equivalent weight of sulfuric
acid (^SOtJ is 49 (98 divided by 2 because there are two re-
placeable hydrogen.ions),  A IN solution of sulfuric acid
would consist of 49 grams of F^SO^ dissolved in enough water
to make one liter.

Oxidation (ox-i-DAY-shun).   Oxidation is the addition of oxygen,
removal of hydrogen, or 'the removal of electrons from an element
or compound.  In wastewater treatment, organic ma'tter is oxidized
to more stable substances.

Percent Saturation.   Liquids can contain in solution limited
amounts of compounds and elements.  100% saturation is the
maximum theoretical amount  that can be dissolved in the solution.
If more than the maximum theoretical amount is present, the
solution is supersaturated.


     o  f, „.   J_.       Amount in Solution    ,„„„
     % Saturation  =  rr—:	-r	—•	1 * 100%
                      Maximum Theoretical
                      Amount in Solution

Reagent (re-A-gent).  A substance which takes part in a chemical
reaction that is used to measure, detect, or examine other sub-
stances.

Representative Sample.  A portion of material or water identical
in content to that in the larger body of material or water being
sampled.

Titrate.  To titrate a sample, a chemical solution of known
strength is added on a drop-by-drop basis until a color change,
precipitate, or pH in the sample is observed (end point).
Titration is the process of adding the chemical solution to
completion of the reaction  as signaled by the end point.
14.11  Equipment

Equipment can be better described by a photo or a sketch than
a written description; consequently, this portion of the
glossary will describe equipment in this manner.  Photos of
equipment shown were provided by Van Waters § Rogers.
                            14-6

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             ILLUSTRATIONS OF  LABORATORY
60809-021 Series

 Test Tube
 60824-116 Series

Culture Tube
Without Lip
    13912-207

    Beaker
   30209-025

   Funnel
     29140-023
     Flask,
   Erlenraeyer
 (ER-len-MY-er)
   Wide Mouth
   29619-642
   Flask,
Volumetric
          29110-102

           Flask,
          Boiling
        Flat Bottom
     23130-049
     23131-020
   Condenser
          29126-022

          Flask,
         Boiling
       Round Bottom
        Short Neck
  29209-083
  Flask,
Distilling
           29415-100

           Flask,
         Filtering
      30294-024

      Funnel,
      Buchner
       With
Perforated Plate
                                14-7

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Bottle,
Reagent
 23835-000
Crucible
 Gooch
(GOO-ch)
Porcelain
 g
                   y *•/
     17685-005
Support,  Buret
§ Buret  Clamp
Bottle,
 BOD
 24707-2S5

Cylinder,
Graduated
 23810-021

Crucible
Porcelain
      25310-019
       Dish,,  '
    Evaporating
                                   -v-
                17454-443
                Buret
              (bur-RET)
               25313-017
                Dish,
             Evaporating
            Shallow  Form
                        17590-044
                        Buret
                      Automatic
                                14-8

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                Clamp, Beaker,
                Safety Tongs
                                                          217SO-009
                             ,  Dish
                       Safety  Tongs
                  21792.904
              Clamp, Flask,
              Safety Tpngs
               21611-046

           Clamp, Utility
    62765-029 Series

Tripod, Concentric
      Ring
                                                            21770-028

                                                      Clamp,  Test  Tube
                         21877-000
                     Clamp  Holder
    17951-029

Burner,  Bunsen
62730-024 Series

 Triangle
  Fused
                                14-9

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 66187-004
  Cone,
 Irahoff
(IM-hoff)
    66190-009
Cone Support
                        25353-248

                     Dish,  Petri
     68176-325
Color  Comparison
 Tubes,  Nessler
         25026-026
       Desiccator
  (DES-ick-kay-tor)
       52368-022 Series
Oven,  Mechanical Convection
                                                    53047-024 Series
                                                     r.Pipette   ;
                                                    (P IE-pet)
                                                    Volumetric  ,
     53224-028 Series
Pipet,  Serological
                                                 61048-033 Series
                                             Thermometer,  Dial
         33976-009
        Hot Plate
                               30632-003
                       Muffle Furnace, Electric
                               14-10

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       35960-000
     BOD Cabinet
     34114-055

  pH Meter
           57980-000
Spectrophotometer
   Weight = 95.5580 gm.
11274-008 Reading Scale
              11274-008
Balance, Analytical
                  54906-001
       Pump,  Air Pressure  $ Vacuum
                  60776-002
           Test  Paper,  pH 1-11
                             14-11

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        CHAPTER 14  LABORATORY PROCEDURES AND CHEMISTRY

                   (Lesson 1 of 8 Lessons)
14.2  SAFETY AND HYGIENE, by A.E. Greenberg from California Water
      Pollution Control Association Operators Laboratory Manual
14.20  Laboratory Safety

Safety is important in the laboratory as well as in the rest
of the treatment plant.  Therefore, each employee working in
a laboratory should be thoroughly familiar with this section.

On questions of safety, consult your state's General Industrial
Safety Orders or similar document and Sax's "Dangerous Chemicals".2

Personnel working in a wastewater treatment plant laboratory
must realize that a number of hazardous materials and conditions
exist.  _PREVENT_ ACCIDENTS..  Be alert and careful..  Be aware of
potential Dangers at all times.  The major threats to you are
listed for your safety,

1.  Infectious Materials
    Wastewater and sludge contain millions of bacteria, some
    of which are infectious and dangerous, and can cause
    diseases such as tetanus, typhoid, dysentery, poliomeiytds",
    and hepatitis.  Personnel handling these materials should
    thoroughly wash their hands with soap and water, particularly
    before handling food.  Do not pipette wastewater or polluted
    samples by mouth.  Use a rubber bulb.  Though not mandatory,
    inoculations by your County Health Department are recommended
    for each employee.
2 See Sax, N.I,, Dangerous Properties of Industrial Materials
  Third Edition, ReinholdY New York',' 1968", price $35.
                        14-12

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2.  Corrosive Chemicals
    A.   Acids
        (1)   Examples:   Sulfuric,  hydrochloric,  nitric,  glacial
             acetic,  Pomerpy solutions  Nos.  1 and 2,  and chromic
             acid cleaning solutions,

        (2)   Acids are  extremely corrosive  to human tissue,  metals,
             clothing,  wood, cement,  stone,  and  concrete.  Use  glass-
             ware or  polyethylene  containers.
        (3)   In  case  of accidental  spills,  immediately  dilute
             large  portion?  of water  and neutralize  the acid
             sodium carbonate  or bicarbonate
             unto,!  bubbling  and foaming stops.
             Clean  up neutralized material.
             If  spills  occur on bench  tops,
             dilute,  neutralize, and  squeegee
             into sink.   If  spills  occur on
             person,  immediately wash  pff
             with water,   If spills occur
             on  face  (spills pf concentrated
             acid), immediately flood  with
             large  quantities  of cold  water.
             Notify supervisor.  Remember to
             add acid to water, but not" reverse.
             Pour and pipette  carefu1ly to
             prevent  spilling  and dropping.
             Prevent  contact with metals,
             particularly equipment.
 with
with
   B.  Bases
       (1)  Examples:  Sodium hydroxide, potassium hydroxide,
            ammonium hydroxide, alkaline iodide	sodium azide
            solution.

       (2)  Handle with extra care and respect,  They are extremely
            corrosive to skin, clothing, and leather.  Use glass-"
            ware and polyethylene containers,.

            Ammonium hydroxide is extremely irritating to the
            eyes and respiratory system.  Ppur ammonium hydrpxide
            under a laboratory hood with fan in operation.
                          14-13

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        (3)   In case of accident, wash with large quantities
             of water and use saturated boric acid solution,
             to neutralize.
        Miscellaneous

        (1)   Chlorine gas solution	avoid inhalation.  Handle
             in hood.  Secure cover to prevent escape of vapors.

        (2)   Ferric salts, Ferric chloridf	very corrosive to
             metals.  Avoid body contact and wash off imme-
             diately,

        (3)   Strong oxddants^---avoid body contact.  1,'ash off
             immediately.  Use of perchloric acid by untrained
             personnel must be prohibited.
3.  TQXJC Materials

    Avoid ingesting qr inhaling,

    A.  Solids:   Cyanides, chromium, cadmium, and other heavy
        metal compounds.

    B.  Liquids:   Use in  vented hood.  Carbon tetrachlori'Je,
        ammqnium hydroxide, nitric acid, bromine, chlorine
        water, aniline dyes, formaldehyde, chloroform, and
        carbon disulfide.   Carbon tetraehloride is absorbed
        into skin on contact; its vapors will damage the lungs;
        and it will build up in your body to a dangerous level.

    C,  Gases:  Use in vented hood.  Hydrogen sulfide, chlorine,
        ammonia,  nitric,  hydrochloric acid.

    D.  Most laboratory chemicals have toxicity warnings and
        antidotes on their labels.  Learn about the materials
        you use,   Don't breathe, eat, or drink them; and if
        they come in contact with your body, quietly apply
        large quantities  of water to wash the substance away.
                           14-14

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 4.   Explosive or Inflammable Materials                ;

     A.   Gases:-  Acetylene,  hydrogen,

     B,   Li quids:   Carbon disulfide,  benzene, ethyl ether,
         petroleum ether, acetone,  gasoline.  ,   >:

     Store these  materials according  to fire  regulations^to
     prevent  fire  hazards.  If large  quantities must T?e "stxired,
     they should be located  in a separate storage building.
     Do  not  uss  near open flame or exposed heating elements-.'
     Use under a vended laboratory hood.   Do not,distill t'$ dry-
     ness  or explosive  mixtures may result.   Use .face mask.  Do
     not throw flammable liquids into sinks.  Cigarette-.discard
     may cttuse fire.  Do not  let gas cylinders fa IT.  ^"~"
5.  Broken Equipment

    A.   Inexpensive^Iterns--Beakers  and  flasks  should be dis-
         carded/ except  for minor  chips  which .can be flame
        polished3 easily,

    Bt   cxpensiye Iterns--Shou1d be  set  aside  for salvage if
        possible.   Discard if damaged beyond  repair.  ~  ?-%
" Flame Polished.   Sharp or broken edges of glass (such as the
  end of a glass tube)  are flame polished by placing the edge
  }.n^a flame and rotating it.   By allowing the edge to melt
  slightly,  it will become smooth.
                              14-15

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6.  Mi see 11 aneous
                         A.  Use safety goggles or face mask
                             in any experiment in which there
                             is danger to the eyes.  Never look
                             into the end of the test i ube during
                             reaction or heating.

                             Use care in making rubber- to- glass
                             connections.  Lengths of glass
                             tilting should be supported uhile
                             they are being inserted into rubber.
                             The ends of the glass should be
                             flame polished, and either wetted
                             or coverec.' with a lubricating jelly
                             for ease in joining connections.
                             Never use grease or oil.  Gloves
                             or grippers should be worn when
                             making such connections, and the
                             tubing should be held as close to
                             the e-.d being inserted as possible
                             to prevent bending or breaking.

                             Never try to fo::ce rubber tubing
                             or stoppers from glassware.  Cut
                             the rubber or material off,

                         B.  Always check labels on bottles to
                             make sure that the chemical selected
                             is correct.  All chemicals and bottles
                             should be cleaVly 'labeled'.  Never "
                             handle chemicals with bare hands .
                             Use spatula, spoon, or tongs.

                         C,  Never work in a poorly ventiJated
                             area.  Toxic fumes even in mild
                             concentrations can knock you out.
                             Be sure you have adequate venti-
                             lation before you start work in tm
                             laboratory.

    .0.   Stioking and eating should be avoifei when working with
        infectious materials such as \«/astewater ano sludge.  Never
        use laboratory glassware for serving the food.

    E.   Always use the proper type of equipment for handling hot
        containers, such as ^rc^ective gloves, tongs, clothing,
        glasses, etc,
                              14-16

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    F.  Where cylinders of oxygen or other compressed gases
        are used in the laboratory, they should be stored in
        separated and ventilated sections.  They should be
        chained or clamped in an upright position while being
        used.  The protective caps should never be removed until
        the cylinder is set and clamped in place, ready for
        attachment of valve gage  and connections.  Always use
        fittings approved for the cylinder being used and care-
        fully follow instructions.

    G.  Iji working in the plant, be carefijl around:

        (1)  Digesters--Do not smoke.

        (2)  Chlprinators—B® aware of chlorine leaks.  Chlorine
             may be detected by its odor, or a white mist will
             form near a rag soaked in ammonia.

        (3)  Power and BJ,ower--Wear ear plugs or ear covers if
             working over one hour in engine room.

        (4)  Open Wastewater Tanks—Be carefulj don't fall in.

        (5)  Closed Wastewater Tanks—Avoid running over tank
             covers by foot or vehicle.

        (6)  In Tanks or Ne,ar Construction—Wear hard hats.
14.21  Personal Hygiene for Wastewater Treatment Plant Personnel

Although it is highly unlikely that personnel can contract diseases
by working in wastewater treatment plants, such a possibility does
exist with certain diseases.

1,  Some diseases are contracted through breaks in the skin, cuts,
    or puncture wounds.  In such cases the bacteria causing the
    disease may be covered pver and trapped by flesh, creating a
    suitable anaerobic; environment1* in which the bacteria may
    thrive and spread throughput the body.
  Anaerobic Environment (AN-air-0-bick).  A condition in which
  "free" or dissolved oxygen is not present.
                            14-17

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     For projection against diseases contracted through breaks
     in the skin, cuts, or puncture wounds/ everyone working in
     or around wastewater must receive immunization from tetanus.
     Immunization must be received before the infection occurs.
     To prevent diseases from entering open wounds, care must be
     taken to keep wounds protected either with band aids or, if
     necessary, with rubber gloves or waterproof protective
     clothing.                 !       .

2.   Diseases that may be contracted through the gastrointestinal
     system or through the mouth are typhoid, cholera, dysentery,
     amebiases, worms, salmonella, infectious hepatitis, and
     polio virus.  These diseases are transmitted by the infected
     wastewater materials being ingested or swallowed by careless
     persons.  The best protection against these diseases is
     furnished by thorough c1eansing.  Hands, face, and body
     should be thoroughly washed wl'th soap and water, particularly
     the hands, in order to prevent the transfer of any unsanitary
     materials or germs to the mouth while eating.  A change of
     working clothes into street clothes before Leaving work is
     highly recommended to prevent carrying unsanitary materials
     to the employee's home.  Personal hygiene, thorough cleansing,
     and washing of the hands are effective means of protection.

     Immunization is provided for typhoid and polio.  Little is
     known about infectious hepatitis except that it can be trans-
     mitted by wastewater.  It is frequently associated with gross
     wastewater pollution.

3,   Diseases that may be contracted by breathing contaminated
     air include (1) tuberculosis, (2) infectious hepatitis, and
     (3) San Joaquin fever.  There has been no past evidence to
     indicate the transmission of tuberculosis through the air
     at wastewater treatment plants.  However, there was one
     case of tuberculosis being contracted by an employee who
     fell into wastewater and, while swimming, inhaled waste-
     water into his lungs.  San Joaquin fever is caused by a
     fungus which may be present in wastewater.  However, there
     is no record of operators contracting the disease while on
     the job.
                               14-18

-------
    bfst insurtnee against these diseases is proper personal
hygiene an4 imnnaniESfion.  Your plant should have an immunization
program aga;in?t (l); tetapus, (20 typhoid, (3) polio, and (4) small-
pox (although smallpox i^ not related to wastewater) .  The
imm«#ti Cation;* shwjtf be provided to protect you.  Check with your
IqcaJ, Or sWte health department for recommendations regarding
In the washing of hands, the kind of soap is less important than
th$ though BSe of the soap,  (Special disinfectant soaps are
not essential.)

The u«e of protective cjpthing is very important, particularly
gloves and boots.  The protection of wounds and cuts is also
important.  Report injuries and take care of them.

The responsibility rests upon you.

There is no absolute insurance against contraction of disease
in a wast^water treatfnent plant.  However, the likelihood of
transmission is practically negligible.  There appears to be no
special risk in working at treatment plants,  In fact, operators
may receive a natural immunization by working in this environment.
                            14-19

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                    QUESTIONS
14.2A  Why should you always use a rubb'er bulb to" -
       pipette wastewater or polluted wate'r?
                                                     ' f,  ;
14.2B  Why are inoculations against disease recommended
       for people working around wastewatei4?  "   '

14.2C  What would you do if you spilled a concentrated '
       acid on your hand?

14.2D  True or False:  You may add acid to water, but
       never water to acid.

14,2E  If you are working in a', wastewater treatment
       plant, why should you change your clothes b-ef-bre
       going home at night?
                       14-20

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 14.3   SAMPLING, by Joe Nagano,  from California  Water  Pollution
       Control  Association Operator? Laboratory  Manual   ,.',.,
 14.30   Importance                 .              ...-•''',

 Before  any  laboratory tests  are performed,  it  is highly  important
 to  obtain a proper, representative sample.  Without  a  representative
 sample,  a test shpuld not even be attempted because  the  test .result
 will be  incorrect  and meaningless.  A  laboratory test  without, a good
 sample will most likely  lead to erroneous conclusion?  and  confusion.
 The largest errors produced  in laboratory tests .are  usually,, caused
 by  imprp^er sampling, poor preservation, 'or lack of  enough mixing"
 diirlrif  corn  Ositlng^and  testing.    '   ,   T1     -^ •-•-•-      — •   -
14.31  Accuracy of Laboratory Equipment

Laboratory equipment, in itself, is generally quite accurate.
Analytical balances weigh to 0.1 milligram.  Graduated cylinders,
pipettes, and burettes usually measure to  1% accuracy, so that the
errors introduced by these items should total less than .5%,. and
under the worst possible conditions only 10%,  Under ideal  conditions
let us assume that a test of raw wastewater- for suspended solids
should run about 300 mg/1.  Because of the previously mentioned
equipment or apparatus variables, the value may actually range
fro|a £70 to 330 mg/1.  Results in this range are reasonable for
operation.  Other less obvious factors are usually present which
make it quite possible to obtain results which are 25, SO,  o.r'even
100% in error, unless certain precautions are taken.  Some examples
will illustrate hpw these errors are produced.

The City of Los Angeles Terminal Island Treatment Plant is a
primary treatment facility with a flow of 8 million gallons per
day.  It has an aerated grit chamber, two circular 85-foot clari-
fiers of 750,000 gallon capacity, and two digesters 100 and 75 feet
in diameter.   '      ,                     "
  Composite (Proportional) Samples (com-POZ-it.).  Samples collected
  at regular intervals in proportion,to the existing flow and;then
  cpmbined to form a sample representative of the entire-,period of
  flow over a given period of time,     "  *  Y*        '•.,;"? *\
                            14-21

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Monthly summary calculations based upon the suspended solids test
showed that about 8,000 pounds of suspended solids were being
captured per day during sedimentation assuming 200 mg/1 for the
influent and 100 mg/1 for the effluent.  However, it also appeared
that 12,000 pounds per day of raw sludge solids were being pumped
out of the clarifier and to the digester.  Obviously, if sampling
and analyses had been perfect, these weights would have balanced.
The capture should equal the removal of solids.  A study was made
to determine why the variance in these values was so great.  It
would seem logical to expect that the problem could be due to
(1) incorrect testing procedures, (2) poor sampling, (3) incorrect
metering of, the wastewater or sludge flow, or (4) any combination
of the three or all of them.           .          .   .

In the first case, the, equipment was in excellent condition.
The operator was a conscientious and able employee who was
found to have carried out the laboratory procedures carefully
and who had previously run successful tests on comparative
samples.  It was concluded that the equipment and test proce-
dures were Completely satisfactory.


14.32  Selection of a Good Sampling Point to Obtain  .     ,  ;>-
       a.Representative Sample              	  ,  .

A survey was the1* made.to determine if sampling stations were in
need of relocation.  By using Imhoff cones and running settleable
solids tests along the influent channel and the aerated,grit.,
chamber, one could quickly recognize that the best mixed, and
most representative samples were to be taken from the aerated
grit chamber rather than the influent channel.

The settleable solids ran 13 ml/1 ;i.n the aerated grit chamber
against 10 ml/I in the channel.  By the simple process of
determining the best sampling station, the suspended solids
value in the influent was corrected from 200 mg/1 to the more
representative 300 mg/1.  Calculations, using the correct
figures,, changed the solids capture from 8,000 pounds to 12,000
pounds per day and a balance was obtained.

This study clearly illustrates the importance of selecting a
good sampling point in securing a truly representative sample.
It emphasizes the point that even though a test is accurately
performed, the result may be enjb^rely" ^rr^ph^us' and meaningless
insofar as use' for process control isT concerned, unless a good
represgrit.aHve saniple is taken.  Furthermore, a good sample is
highly dependent: upon the sampling' station.  Whenever possible,
                            14-22

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select a place where mixing is thorough and the wastewater quality
is uniform.  As the solids concentration increases, above about
200 mg/1, mixing becomes even more significant because the waste-
water solids will tend to separate rapidly with the heavier solids
settling toward the bottom, the lighter solids in the middle, and
the floatables rising toward the surface.   If, as is usual, a
one-gallon portion is taken as representative of a million-gallon
flow, the job of sample location and sampling must be taken
seriously.
14.33  Time of Sampling

Let us consider next the time and frequency of sampling.   In
carrying out a testing program, particularly where personnel
and time are limited due to the press of operational responsi-
bilities, testing may necessarily be restricted to about  one
test day per week.  If the operator should decide to start his
tests early in the week, by taking samples early on Monday
morning he may wind up with some very odd results.

One such incident will be cited.  During a test for ABS (alkyl
benzene sulfonate), samples were taken early on Monday morning
and rushed into the laboratory for testing.  Due to the detention
time in the sewers, these wastewater samples actually represented
Sunday flow on the graveyard shift, the weakest wastewater obtain-
able.  The ABS content was only 1 mg/1, whereas it would  normally
run 8 to 10 mg/1.  So the time and day of sampling is quite important,
and the samples should be taken to represent typical weekdays or
even varied from day to day within the week for a good cross-section
of the characteristics of the wastewater.
14,34  Compositing and Preservation of Samples

Since the wastewater quality changes from moment to moment and
hour to hour, the best results would be obtained by using some
sort of continuous sampler-analyzer.  However, since operators
are usually the sampler-analyzer, continuous analysis would
leave little time for anything but sampling and testing.   Except for
tests which cannot wait due to rapid chemical or biological changa -
of the sample, such as tests for dissolved oxygen and sulfides,  a
fair compromise may be reached by taking samples throughout the
day at hourly or two-hour intervals.

When the samples are taken, they should be immediately refrigerated
to preserve them from continued bacterial decomposition.   When all
of the samples have been collected for a 24-hour period,  the samples
from a specific location should be combined or composited together
according to flow to form a single 24-hour composite sample.
                           14-23

-------
To prepare a composite sample, (1)  the rate of wastewater flow
must be metered and (2)  each- grab sample must then be taken
and measured out in direct proportion to the volume of flow
at that time.  For example, Table I illustrates the hourly flow
and sample volume to be measured out for a 12-hour proportional
composite sample.

                            TABLE I

   DATA COLLECTED TO PREPARE, PROPORTIONAL COMPOSITE SAMPLE

Time
6 AM
7 AM
8 Mi
9 AM
10 AM
11 AM

Flow
MGD
0.2
0.4
0.6
1.0
1.2
1.4


Factor
100
100
100
100
100
100

A sample composited

Sa£vple
20
40
60
100
120
140

in this

Vol Time
12 N
1 PM
2 PM
3 PM
4 PM
5 PM

manner would
Flow
MGD
1.5
1.2
1.0
1.0
1.0
0.9

total

Factor
100
100
100
100
100
100

1140 ml.

SampleVol
150
120
100
100
100
90
1140

Large wastewater solids should be excluded from a sample,, particu-
larly those greater than one-quarter inch in diameter,
Pur i n g compos i t in g
A very important point should be emphasized.
and at the exact moment of testing, the
remixed so that they w i 1 1 beof the s age cgnroosit . i on and _as wejLL_
T!dxeda£whenthCT_wereori^ra^ly sampled.  Sometimes sue!- re^ixii
may become lax., so that all the solids are not uniformly suspended.
Lack of mixing can cause low results in samples of solids that
settle out rapidly, such as those in activated sludge or raw vjasto
water.  Samples must therefore be mixed thoroughly and poured
quickly before any settling occurs.  If this is not done,, errors
of 25 to 50% may easily occur.  For example,, on the same mixed
liquor sample, one person may find 3,000 mg/1 suspended solids
while another person may determine that there are only 2,000 mg/1
due to poor mixing,,  When such, a composite sample is tested, a
reasonably accurate measurement of the quality of the day's flow
can be made.

If a 24-hour sampling program is not possible, perhaps due to
insufficient personnel or the absence of a night shift, single
representative samples should be taken at a time when typical
characteristic qualities are present in the wastewater.  The
samples should be taken in 'accordance with the detention time
                            14-24

-------
required for treatment.  For example, this period may exist
between 10 AM and 5 PM for the sampling of raw influent.   If
a sample is taken at 12 Noon, other samples should be taken
in accordance with the detention periods of the serial processes
of treatment in order to follow this slug of wastewater or plug
flow.  In primary settling, if the detention time in the pri-
maries is two hours, the primary effluent should be sampled at
2 PM.  If the detention time in the succeeding secondary treat-
ment process required three hours, this sample should be taken
at 5 PM.
14.35  Sludge Sampling

In sampling raw sludge and feeding a digester, a few important
points should be kept in mind as shown in the following illus-
trative table.

For raw sludge from a primary clarifier at Los Angeles' Terminal
Island Plant, the sludge solids varied considerably with pumping
time as shown by samples withdrawn every one-half minute,

                           TABLE II

        DECREASE IN PERCENT TOTAL SOLIDS DURING PUMPING
                                                 Cumulative
       Pumping Time         Total Solids           Solids
        In Minutes            Percent: 	          Average

           0.5                  7.0                 7.0
           1.0                  7.1                 7.1

           1.5                  7.4                 7.2
           2.0                  7.3                 7.2

           2.5                  6.7                 7.1
           3.0                  5.3                 6.8

           3.5                  4.0                 6.4
           4.0                  2.3                 5.9

           4.5                  2.0                 5.5
           5.0                  1.5                 5.1
                           14-25

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      Table II shows that the solids were heavy during the first
      2.5 minutes, and thereafter rapidly became thinner and
      watery.   Since sludge solids should be fed to a digester
      with solids as heavy as possible and a minimum of water.
      the pumping should probably have been stopped at about
      3 minutes.  After 3 minutes, the water content did become
      greater that desirable.

      In sampling this sludge, the sample should be taken as a
      composite by mixing small equal portions taken every 0.5
      minutes  during pumping.  If only a single portion of sludge
      is taken for the sample, there is a chance that the sludge
      sample may be too thick or too thin, depending upon the
      moment the sample is taken,  A composite sample will pre-
      vent this possibility.

      It should also be emphasized again that as a sludge sample
      stands,  the solids and liquid separate due to gasification
      and flotation or settling of the solids, and that it is
      absolutely necessary to thoroughly remix the sample back
      into its original form as a mixture before pouring it for
      a test.

      When individual samples are taken at regular intervals
      in this  manner, they shorld be carefully preserved to
      prevent sample del ?::-•:• ,t~'.-r If bacterial action.  Re-
      frigeration is an ox-ellent method of preservation an-
      is generally preft.-ii.ble to chemicals since chenucais aay
      interfere with tests such as BOD and COD.
14.36
Automatic sampling devices are wonderful timesavers and s'loul3 be
en,ployed where possible.  However,, like anything automatic,
problems of which the operator should be aware do arise in their
use.  Sample lines to auto-samplers may build up growths which
may periodically slough off and contaminate the sample with a
high solids content.  Very regular cleanout of the intake line
is required.  Another problem occurred at Los Angeles' Hyperion
Plant when the reservoir for the automatic sampler was attacked
by sulfides.  Metal sulfides flaked off and entered the sample
container producing misleading high solids results.  The
reservoir was cleaned and coa.ted with coal-tar epoxy and little
further difficulty has been experienced.
                          14-26

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Manual sampling equipment includes dippers, weighted bottles,
hand-operated pumps, and cross-section samplers.  Dippers con-
sist of wide-mouth corrosion resistant containers (such as
cans or jars) on long handles that collect a sample for testing.
A weighted bottle is a collection container which is lowered
to a desired depth.  At this location a cord or wire removes
the bottle stopper so the bottle can be filled.  Sampling pumps
allow the inlet to the suction hose to be lowered to the sampling
depth.  Cross-sectional samplers are used to sample where the
wastewater and sludge may be in layers, such as in a digester or
clarifier.  The sampler consists of a tube, open at both ends,
that is lowered at the sampling location.  When the tube is at
the proper depth, the ends of the tube are closed and a sample
is obtained from different layers.

Many operators build their own sampler (Fig. 14.1) using the
material described below:

1.  Sampling Bucket.  A coffee can attached to an eight-foot
    length of 1/2-inch electrical conduit or a wooden broom
    handle with a 1/4-inch diameter spring in a four-inch loop.

2.  Samp1ing Bottle,  Plastic bottle with rubber stopper equipped
    wi'tn two"3/8-1nch glass tubes, one ending near bottom of
    bottle to allow sample to enter and the other ending at the
    bottom of the stoppe~ to allow the air in the bottle to
    escape while the sample is £.'11 ing the bottle.

For sample containers, wide-mouth plastic bottles are recommended.
Plastic bottles, though somewhat expensive initially, not cnly
greatly reduce the problem of breakage and metal contamination,
but are much safer to use.  The wide- mouth bottles ease the
washing problem.  For regular samples, sets of plastic bottles
bearing identification labels should be used.
14,37  Summary

1.  Representative samples must be taken before any tests are
    made.

2.  Select a good sampling location.

3.  Collect samples and preserve them by refrigeration.

4.  If possible, prepare 24-hour composite samples.  Mix samples
    thoroughly before compositing and at the time of the test.
                           14-27

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1/2" Conduit
Length to Suit A.
                              1/4" Spring to Retain Sample Bottle
                                  Coffee Can

Quart
Plastic
Bottle
/



A


-<-
f!f
                                              Jl
                              Rubber Stopper V|_j  j !/
                                                            Glass Tube  Von
                                                         Glass  Tube  -  Cut  to
                                                    fit  1/2"  clearance from
                                                    bottom  of bottle
                  Fig.  14.1  Sampling bottle
                              14-28

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                    QUESTIONS
14.3A  What are the largest sources of errors found in
       laboratory results?

14.3B  Why must a representative sample be collected?

14.3C  How would you prepare a proportional composite
       sample?
                      14-29

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14.4  LABORATORY WORK SHEET
All laboratory results should be recorded immediately alter a
sample has been measured.  There is no standard laboratory form;
however, your plant or the agency that regulates your discharge
may have a preferred form.  Figure 14.2 is a typical laboratory
work sheet (sometimes called a bench sheet)  and will be referred
to throughout the chapter.
                            14-30

-------
                                            PLANT
                                            DATE
                         SUSPENDED SOLIDS $ DISSOLVED SOLIDS
SAMPLE
Crucib le
Ml Sample
Wt Dry § Dish
Wt Dish
Wt Dry
M Wt Dry,, gm x 1,000,000
mg/ ~ - Ml Sample
Wt. Dish § Dry
Wt Dish § Ash
Wt Volatile
o, „ i _ Wt Vol „
Wt Dry
























































                                         BOD
# Blank
SAMPLE
DO Sample
Bottle #
% Sample
Blank or adj blank
DO after incubation
Depletion, 5 days
Dep %











































	 I

i
i










i 	 i



Nitrate N03
Sample
Graph Reading
Sett. Solids
Sample
Direct Ml/1
COD
Sample
Blank Titration
Sample Titration
Depletion
  ,,   Dep x N FAS x 8000
mg/1 =  . f.	
           Ml Sample
                 Fig. 14.2  Typical laboratory work sheet
                                    14-31

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                                      TOTAL SOLIDS
SAMPLE
  Dish No.
  Wt Dish
  Wt Dish
  Wt Wet
  Wt Dish +
  Wt Dish
  Wt Dry
  % Solids »
          Wet
          Dry
           Wt
           Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
% Volatile - Wt Vo1
             Wt Dry
PH
Vol. Acid
Alkalinity as CaC03
                  x  100%
                      x 100%

  Grease (Soxlet)
    Sample
    Ml Sample
    Wt Flask + Grease
    Wt Flask
    Wt Grease
    me/1 = Wtl-GrJe_ase» m.g_ x
                 Ml Sample
H2S (Gas) (Starch-Iodine)
  Blank            	
  Sample           	
  Diff             	
  Diff x .68       	
  mg/1 x 43.6
                           * OOP
                               Ml
                               Ml
                               Ml
                               mg/1
                               grain/TOO  cu  ft
            Fig. 14.2  Typical  laboratory work sheet (continued)
                                     14-32

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                 END OF LESSON 1 OF 8 LESSONS

                              on

               Laboratory Procedures and Chemistry
        EXPLANATION OF DISCUSSION AND REVIEW QUESTIONS
Work this portion ,o£ the discussion and review questions after you
have completed answering the questions in Lesson 1.  At the end of
each lesson in this chapter you will find some discussion and review
questions that you should complete before continuing.

The purpose of these questions is to indicate to you how well you
understand the material in this chapter.
                           14-33

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                DISCUSSION AND REVIEW QUESTIONS

                    (Lesson 1 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry



Name                                              Date
Write the answers to these questions in your notebook before continuing.


1.  What precautions should an operator take to protect himself
    from diseases when working in a wastewater treatment plant?

2.  Why should work with certain chemicals be conducted under a
    ventilated laboratory hood?

3.  What is meant by a representative sample?

4.  How would you obtain a representative sample?
                          14-3:4

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       CHAPTER 14.  LABORATORY PROCEDURES AND CHEMISTRY

                   (Lesson 2 of 8 Lessons)
14.5  PLANT CONTROL TESTS

Tests in this section are listed in alphabetical order.  Many
of the tests are conducted at primary, secondary, and advanced
wastewater treatment plants.  Certain tests are commonly used
to control digester operation and activated sludge plants.
Typical plant and special plant control tests are summarized below,

A.  Typical PlantL Control Tests

    TEST NO.                   TITLE
        2
        4
        5
        6
        7
        8
        9
       10
       12
       16
       17
Biochemical Oxygen Demand or BOD, Procedure with DC
Chemical Oxygen Demand or COD
Chlorine Residual
Clarity
Coliform Group Bacteria,
Dissolved Oxygen or DO
Hydrogen Sulfide
PH
Settleable Solids
Suspended Solit>s (Gooch Crucible)
Temperature (Wastewater)
    Digester Control Tests

    TEST NO.
        1
        3
       14
       15
       17
       20
       21
             TITLE
Alkalinity, Procedure with Volatile Acids
Carbon Dioxide (C02) in Digester Gas
Sludge Dewatering Characteristics
Supernatant Graduate Evaluation
Temperature (Digester Sludge)
Volatile Acids
Total and Volatile Solids (Sludge)
                          14-35

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C.  Activated Sludge Control Tests

    TEST NO.                  TITLE

        8        Dissolved Oxygen (In Aerator)
       11        Settleability
       13        Sludge Age
       11        Sludge Density Index (SDI)
       11        Sludge Volume Index (SVI)
       16        Suspended Solids (Centrifuge)
                            14-36

-------
                                                    (Total Alkalinity)
                                                    (BOD)
    Total Alkalinity

    The alkalinity test is located with the volatile acid test be-
    cause the volatile acid/alkalinity relationship is critical in
    the successful operation of sludge digesters.
2.   Biochemical Oxygen Demand or BOD

    The BOD test is placed with the dissolved oxygen (DO)  test be-
    cause to measure the rate of oxygen uptake in the BOD test, the
    DO must be measured.
                           14-37

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3.  Carbon Dioxide (C02)  in Digester Gas
A.  Discussion

Changes in the anaerobic sludge digestion process will be observed
in the gas quality and are usually noted after the volatile acids
or volatile acid/alkalinity relationship starts to increase.  The
C02 content of a properly operating digester will range from 30%
to 40% by volume.  If the percent is above 44%, the gas will not
burn.  The easiest test procedure for determining this change is
with a C02 analyzer.
B.  What is Tested?

            Sample                    Preferred

      C02 in Digester Gas          30% - 35% by Volume


                         METHOD A

C,,  Apparatus

1.  One Bunsen burner

2.  Plastic tubing

3.  100 ml graduated cylinder

4.  250 ml beaker

                                                 \
D.  Reagents

C02 Absorbent (KOH).  Add 500 g potassium hydroxide (KOH) per liter
of water.
                           14-38

-------
                                                                        Cco2)
    E.  Outline of Procedure
    Clean out sampling line
    by allowing gas from
    sampling outlet to burn
    until line is full of
    gas from digester.
 Gas
Outlet
            Bunsen
            Burner
2.  Displace air in
    graduated
    cylinder.
3.   Place graduate upside
    down in beaker containinj
    C02 absor-
    bent.
   Insert hose in graduate
   and run gas for 60 seconds.
5.  Remove hose from
    graduate ar)d then
    turn off gas.
    Wait 10 minutes.
zbt
                 O
                  a
                 O
                             R .id volu:.'.c
                             gas rerr - •*?* .
                             nearest ml.,
                                                                            to
        PRECAUTIONS

        1.  Avoid any open flames near the digester.

        2.  Work in a well ventilated area to avoid the  formation  of  ex-
            plosive mixtures of methane gas.

        3,  If your gas sampling outlet is on top of your  digester, turn
            on outlet and vent the gas to the atmosphere for  several
            minutes to clear the line of old gas.  Start with step 2S
            displace air in graduated cylinder,  NEVER ALLOW  ANY SMOKI_NG_
            OR FLAMES NEAR THE DIGESTER AT ANY TIME.
                                       14-39

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                                                            (C02)


                            PROCEDURE
 1.   Measure  total  volume  of a  100  ml  graduate  by filling it  to
     the  top  with water  (approximately 125  ml) .   Record this
     volume.

 2.   Pour approximately  125  ml  of C02  absorbent in a 250 ml beaker.

     CAUTION:   Do not  get  any of this  chemical  on your skin
     or clothes.  Wash immediately  with running water until
     slippery feeling  is gone or severe burns  can occur.

 3.   Collect  a representative sample  of gas from the gas dome on
     the  digester,  a hot water  heater  using digester gas to heat
     the  sludge, or any  other gas outlet.   Before collecting  the
     sample  for the test,  attach one  end of a  gas hose to the gas
     outlet  and the other  end to a  Bunsen burner.  Turn on the
     gas, ignite the burner, and allow it to burn digester gas
     for  a sufficient  length of time  to insure  collecting a
     representative gas  sample.

 4.   With gas running  through hose  from gas sampling outlet,  place
     hose inside inverted  calibrated  graduated  cylinder and allow
     digester gas to displace air in  graduate.   Turn off gas,

     CAUTION:   The  proper  mixture of  digester  gas and air
     is explosive when exposed  to a flame,

 5.   Place graduate full of  digester  gas upside down in beaker
     containing C02 absorbent.

 6.   Insert  gas hose inside  upside  down graduate.

 7,   Turn on gas, but  donot blow out  liquid.   Run gas for at
     least 60 seconds.

 8.   Carefully remove  hose from graduate with  gas still running.

 9.   I mme di a t e T
10.   Wait for ten minutes and shake gently.   If liquid continues
     to rise, wait until it stops.

11.   Read gas remaining in graduate to nearest ml.   (Fig.  14,3)
                          14-40

-------
                                                              (C02)
           Fig. 14.3  C02 measurement using  in-
                      verted graduated  cylinder
F.  Example
           Total Volume of Graduate   =   126 ml

           Gas Remaining in Graduate  ~   80 ml
G.  Calculation
      CO   =
                                  Gas
                         Total Volume, ml
           =  (126 ml - 80 ml)
_46_
126


37%
                   126 ml
                  x 100%
                               x 100%
                                                     x 100%
       .365
126 /46.0
     37 8
      8 20
      7 56
        640
        630
                            14-41

-------
                                                             (C02)
                          METHOD B
                          (ORSAT)
The Orsat gas analyzer can measure the concentrations of carbon
dioxide, oxygen, and methane by volume in digester gas.  To
analyze digester gas by the Orsat method, follow equipment manu-
facturer's instructions.  This procedure is not recommended  for
the inexperienced operator.
                         QUESTIONS
     3. A  What are the dangers involved in running the
          C02 in digester gas test?
     3.B  What is the percent COa in a digester gas if
          the total volume of the graduated cylinder is
          128 ml and the gas remaining in the cylinder
          after the test is 73 ml?
                           14-42

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4.  Chemical Oxygen Demand or COD


A.  Discussion

COD is a good estimate of the first-stage oxygen demand for most
municipal wastewaters.  An advantage of the COD test over the BOD
test is that you do not have to wait for five days for the results.
The COD test also is used to measure the strength of wastes that
are too toxic for the BOD test.  COD is usually higher than the
BOD, but the amount will vaiy from waste to waste.  The method
related here is a quick, effective measure of the strength of a
waste.


B.  What is Tested?

          Sample                     Common Range, mg/1

         Influent                       200 - 400

         Effluent                        40 -  80

         Industrial Waste               200 - 4000


C.  Apparatus

Two 50 ml graduated cylinders

10 ml pipette

50 ml burette

Boiling flask

Reflux condenser

Hot plate
                          14-43

-------
                                                             (COD).
D.  Reagents
1.  Standard potassium dichromate  (I^C^Oy) 0.250 N,  Dissolve
    12.259 g dried K2Cr207 in distilled water and make up to
    1 liter.

2.  Surfuric acid-silver sulfate reagent.  Add 22 g of silver
    sulfate (Ag2SOzt) to a 9-lb bottle of concentrated sulfuric
    acid (H2S<\).   It takes one to two days to dissolve.

3.  Standard ferrous ammonium sulfate solution, 0.25 N.  Dissolve
    98 g FeCNHit)2(SOit)2'6H20 in distilled water, add 20 ml
    concentrated f-^SO^, cool and dilute to 1 liter.  This solution
    is unstable and must be standardized daily.

4.  Ferroin Indicator.  Dissolve 1.485 g of 1,10 phenanthroline
    (Ci2H8N2«H20), together with 0.695 g ferrous sulfate crystals
    (FeSOit«7H20) ,  in water and make up to 100 ml.

5.  Silver sulfate, reagent powder,

6.  Mercuric sulfate  (HgSO^) analytical grade crystals.
                           14-44

-------
                                                                   (COD)
       E.  Outline of F'rocedure
                5.   Add 30 ml
                    H2S(VAg2SOu
                    Solution
4.   Add 10 ml
    0.25 N K,Cr207

3.   Add 2 ml
    cone, H9
2.  Add
    20 ml
    Sample
  1.
                                               ,Cooling Water
                                   Vent  -
                               7.
                                 Reflux Two
                                 Hours, Cool
                                 $ Wash Down
                                                     Add Ferroin  9,
                                                     Indicator
                                                                    Titrate
                                                                    to red
                                                                    end point.
           Reflux condenser,  Friedrichs,  VWR - 23157-001
           Flask, boiling,  flat bottom,  VWR - 29113-068
                               PROCEDURE

       1.   Place 0.4 g mercuric sulfate into a 250 ml Erlenmeyer flask
           with a ground glas?  neck.

       2.   Measure 20.0 ml  sample  into the flask.

       3.   Add 2.0 ml concentrated sulf iric acid.   Swirl until  contents
           are welL mixed.

       4.   Pipette 10.0 ml  standard potassium dichromate solution into
           the flask.
                                  14-45

-------
                                                           (COD)
 5.   Carefully add 30  ml  sulfuric acid-silver sulfate  reagent
     into the  flask while swirling the  flask.   Use  caution.
     Make sure contents of the  flask are  thoroughly mixed before
     heat is  applied.

 6.   Add a few glass beads to reduce bumping and connect  to  con-
     denser.   The reflux  mixture must be  thoroughly mixed before
     heat is  applied.   If this  is not dene,  local hot  spots  on
     bottom of flask may  cause  mixture  to be blown  out of flask.

 7.   Prepare  a blank5  by  repeating above  steps and  by  substituting
     distilled water for  the  sample.

 8.   Reflux samples and blank for two hours.  (If sample  mixture
     turns completely  green,  the sample was  too strong.   Dilute
     sample with distilled water and repeat  above steps substi-
     tuting diluted sample.)

 9.   While the samples and blank are refluxing, standardize  the
     ferrous  ammonium  sulfate solution:

     a.   Pipette 10.0  ml  standard potassium  dichromate solution
         into  a 250 ml Erlenmeyer flask.   Add about 100 ml of
         water.

     b.   Add  30 ml concentrated HpSO^ with mixing.   Let cool,

     c.   Add  2-3 drops ferroin  indicator, titrate wiuh ferrous
         ammonium sulfate (FAS)  solution. Color change o^ solution
         is from orange to greenish to  red.

         ml FAS
                                  10 ml Ki,Cr207
         Concentration Ratio,  R =
                                     mi
10.   After refluxing mixture for two hours,  -;ash down, condenser.
     Let cool.   Add distilled water to about 140 ml,

11.   Titrate reflux mixtures with standard FAS.

         Blank   - ml FAS __	

         Sample - ml FAS ^	
   Blank.   A bottle containing dilution water or distilled water,
   but the sample being testec is not added.   Tests are frequently
   run on  a sample and a b1ank and the differences compared.
                            14-46

-------
                                                      (COD)


F.  Precautions

1.  Wastewater sample should be well mixed.  If large particles
    are present, sample should be homogenized.

2.  Flasks and condensers should be clean and free from grease
    or other oxidizable materials, otherwise erratic results
    would be obtained.

3.  The standard ferrous ammonium sulfate solution is unstable
    and should be standardized daily or each time the COD test
    is performed.

4.  Use extreme caution in handling concentrated H^SOt,..   Spillage
    on skin or clothing should be immediately washed off and
    neutralized.

5.  The solution must be well mixed before it is heated.  If the
    acid is not completely mixed in the solution when it is heated^
    the mixture could spatter and some of it will pass out the vent,
    thus ruining the test.

6.  Mercury sulfate is very toxic.  Avoid skin contact and
    breathing of this chemical.


G.  Example

1.  Standardization of ferrous amrucnium sulfate, FAS.

    ml 0.25 N K2Cr207 =  10.0

    ml FAS            =  11.0

    Concentration     _  ml KoCr207
    Ratio, R          =  —J-—


                         ICU^
                         11.0

2.  Sample test.

    Sample Taken                =  20.0 ml

    A = ml FAS used for blank   =  10.0 ml

    B = ml FAS used for sample  =   3.0 ml
                          14-47

-------
                                                              (COD)



H.  Calculation for COD
\   	'	


Method 1
COD, mg/1  =  (A - B) x R x 100


           =  (10.0 - 3.0) (10/11)  (100)


           =  635 mg/1



Method 2 (According to Standard Methods)


COD, mg/1  .  (A - B) x CjcJOOO
                  ml Sample


where


        C  =  Normality of FAS


        N  =  Normality of K2Cr207  Standard


              ml KCr20?
                         x N
                ml FAS
           =  0.227
COD, mg/1  =  M-i-_ I" °)i°-'-227J



           =  635 mg/1
                             14-48

-------
                           QUESTIONS
4.A  What does the COD test measure?

4.B  What are some of the advantages of the COD test
     over the BOD test?
                           14-49

-------
5.  Chlorine Residual
A.  Discussion

A chlorine residual should be maintained in a plant effluent
for disinfection purposes.  The amount of residual remaining
in the treated wastewater after passing through a contact
basin or chamber may be related to the numbers of bacteria
allowed in the effluent by regulatory agencies.

Method A (lodometric) is used for samples containing waste-
water, such as plant effluents or receiving waters.  Method B
(Deleted).  Method C (Amperometric? Titration) gives the best
results, but the titrator is expensive.

B.  What is Tested?
                                         Common Range, mg/1
            Sample                       (After 30 MinutesJ

           Effluent                        0.5 - 2.0 mg/1
C.  Apparatus

METHOD A (lodometric)

1.  One 250 ml graduated cylinder

2.  One 10 ml measuring pipette

3.  One 500 ml Erlenmeyer flask

4.  Two 5 ml measuring pipettes

5.  One 50 ml Buret
 Amperometric (am-PURR-o-MET-rick).  A method of measurement
 that records electric current flowing or generated, rather
 than recording voltage.  Amperometric titration is an electro-
 metric means of measuring concentrations of substances in water.
                              14-50

-------
                                                (Chlorine Residual)
METHOD B (Orthotolidine-Arsenite or OTA)

One permanent glass color comparator
Three comparator cells
METHOD C (Amperometric Titratioi j

See Standard Methods



D.  Reagents


                           METHOD A

1.  Standard phenylarsine oxide solution, 0.00564 N.  Dissolve
    approximately 0.8 g phenylarsine oxide powder in 150 ml
    0.3 N NaOH solution.  After settling, remove upper 110 ml
    of this solution into 800 ml distilled water and mix thoroughly,
    Adjust pH up to between 6 and 7 with 6 N HC1 and dilute to
    950 ml with distilled water.  To standardize this solution
    accurately measure 5 to 10 ml of freshly standardized
    0.0282 N iodine solution into a flask and add 1 ml KI
    solution.  Titrate with phenylarsine oxide solution, using
    starch solution as an indicator.  Adjust to exactly 0.00564 N
    and recheck against the standard iodine solution; 1.00 ml =
    200 yg available chlorine.  CAUTION:  Toxic - avoid ingestion.

2.  Potassium iodide, crystals.

3.  Acetate buffer solution, pH 4.0.  Dissolve 146 g anhydrous
    NaC2H302, or 243 g NaC2H302 • 3H20, in 400 ml distilled water,
    add 480 g concentrated acetic acid, and dilute to 1 liter
    with distilled water.

4.  Standard iodine titrant, O.Q282 N.  Dissolve 25 g KI in a
    little distilled water in a 1-liter volumetric flask, add
    the proper amount of 0.1 N iodine solution exactly standard-
    ized to yield a 0.0282 N solution, and dilute to 1 liter.
    Store in amber bottles or in the dark, protecting the solution
    from direct sunlight at all times and keeping it from all
    contact with rubber.

5.  Starch indicator.  Make a thin paste of 6 g of potato starch
    in a small quantity of distilled water.  Pour this paste into
    one liter of boiling, distilled water.  Allow to boil for a
    few minutes, then settle overnight.  Remove the clear super-
    natant and save; discard the rest.  For preservation, add two
    drops of toluene (C6H5CH3).
                            14-51

-------
                                          (Chlorine Residual)
                        METHOD B




Deleted









                        METHOD C




See Standard Methods
                                14-52

-------
                                                                         (Chlorine Residual)
        E.   Procedure
                                            METHOD A
1\   Place 5.00 ml
    phenylarsine
    oxide solution
    to Erlenmeyer
    flask
       2.  Add excess
              KI
           (approx 1 g)
        3.  Add 4 ml
            acetate buffer
            solution
    Add  200  ml
    sample
5.   Mix with
    stirring
    rod
6.
Add 1 ml
starch
solution
                                                             mi
Titrate until
blue color
first appears
and remains
after mixing
                                            14-53

-------
                                                    (Chlorine Residual)
                           METHOD A
1.  Place 5.00 ml 0.00564 N phenylarsine oxide solution in an
    Erlenmeyer flask.

2.  Add excess KI (approx. 1 g).

3.  Add 4 ml acetate buffer solution, or enough to lower the
    pH to between 3.5 and 4.2 .

4.  Pour in 200 ml of sample.

5.  Mix with a stirring rod.

6.  Add 1 ml starch solution just before titration.

7.  Titrate to the first appearance of blue color, which
    remains after complete mixing.
                           METHOD B

1.  Label the three comparator cells "A," "B," and "C."  Use
    0.5 ml of orthotolidine reagent in 10-ml cells, 0.75 ml in
    15-ml cells, and the same ratio for other volumes of sample.
    Use the same volume of arsenite solution as orthotolidine.

2.  Add orthotolidine reagent to Cell A.

3.  Add sample to mark on wall of Cell A.  Mix quickly, and
    immediately (within 5 seconds)  add arsenite solution.  Mix
    quickly again and compare with color standards as rapidly
    as possible.

          Free available chlorine and
           interfering colors, A      =  	   mg/1

4.  Add arsenite solution to Cell B.
                             14-54

-------
                                              (Chlorine Residual)


5.  Add sample to mark on wall of Cell B.  Mix quickly, ami
    immediately add orthotolidine reagent.  Mix quickly again
    and compare with color standards as rapidly as possible.

            Interfering colors present  _             ,,
            in immediate reading, B}    "  - - -

6.  Compare with color standards again in exactly 5 minutes.

            Interfering colors present  _             ...
            in 5 -minute reading, B2        -

7.  Add orthotolidine reagent to Cell C.

8.  Add sample to mark on wall of Cell C.  Mix quickly and compare
    with color standards in exactly 5 minutes.

            Total amount of residual chlorine  _                ,,
            and interfering colors present, C  ~  -  °
F.  Examples and Calculations

Method A

Titration of a 200 ml sample required 0.4 ml of 0.0282 N I.

    „ 1  .    „  .  ,  ,     ,,     (1 - ml I) 1000
    Chlorine Residual, me/1  =  £• •  \ ..... • •.,• < ........ ,
                     '  &       Sample Volume, ml

                                (1 - 0.4)  (1000}
                                      200

                             =  CO. 6) (5)

                             =  3.0 mg/1
NOTE:   The larger the ml of I used in the titration, the smaller
       the (1 - ml I) term and thus the lower the chlorine residual.
       This is why this test is sometimes called the back titration
       test for chlorine residual.  If 1 ml of I is used in the
       titration, you have titrated back to a zero chlorine residual,
                            14-55

-------
                                                    (Chlorine Residual)
Method B

Results from the OTA test on a plant effluent.

             A  =  0.5 mg/1

             B! =  0.2 mg/1

             B2 =  0.3 mg/1

             C  =  1.4 mg/1
Total Available Residual
Chlorine, mg/1
                          ~       2
                          =  1.4 mg/1 - 0.3 mg/1

                          =  1.1 mg/1
Free Available Residual
Chlorine, mg/1
                          ~       l
                          =  0.5 mg/1 - 0.2 mg/1

                          =  0.3 mg/1
Combined Available
Residual Chlorine, mg/1
                             Total Available   -
                             Residual Cl, mg/1

                             1.1 mg/1 - 0.3 mg/1

                             0.8 mg/1
                                                   Free Available
                                                   Residual Cl,  mg/1
Total available residual chlorine consists of free available chlorine
(HOC1 and OC1~) and combined available chlorine (chloramines--compounds
formed by the reaction of chlorine with ammonia).
                            14-56

-------
                                                  (Chlorine Residual)
                            QUESTIONS
 5.A  Why should plant effluents be chlorinated?

 5.B  Discuss the important differences between the lodometric
      titration, orthotolodine, and amperometric titration
      methods of measuring chlorine residual.
                 END OF LESSON 2 OF 8 LESSONS
                              on
              Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 3.
                            14-57

-------
                DISCUSSION AND REVIEW QUESTIONS

                    (Lesson 2 of 8 Lessons)

       Chapter 14.  Laboratqry Procedures and Chemistry




Name                                             Date
Write the answers to these questions in your notebook.  The problem
numbering continues from Lesson 1.
5.  How can you obtain a representative sample of digester gas?

6.  Why is the COD test run?

7.  Why should a chlorine residual be maintained in a plant effluent?
                             14-58

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         CHAPTER 14.   LABORATORY PROCEDURES  AND CHEMISTRY

                     (Lesson  3 of 8 Lessons)
 6.   Clarity


 A.   Discussion

 All  high  quality  effluents  should have  a clarity reading taken
 at high noon  or some  other  specific  time.   This  test  is  based
 on how far  you can  see  through  your  plant  effluent  under similar
 conditions  at the same  time every day.   The objective of the  test
 is to indicate the  clearness  or clarity of the plant  effluent.
 The  test  can  be performed either in  the lab by looking down
 through the effluent  in a graduated  cylinder, or in the  field
 by looking  down through the effluent  in a  clarifier or chlorine
 contact basin.  Sometimes this  test  is  referred  to  as a  tur-
 bidity measurement, but you are interested in the clarity of
 your effluent.


 B.   What  is Tested?

                                        Common Range
          Sample                         QField^Test)_

     Secondary Clarifiers:               Poor     Good

      Trickling Filter                  1 ft     3 ft
    • Activated Sludge                  3 ft     6 ft

     Activated Sludge  Blanket
     in Secondary  Clarifier              1 ft     4 ft

     Chlorine Contact  Basins             1 ft      5 ft
C.  Apparatus

1.  One clarity unit (Secchi (SECK-key) Disc) and attached
    cord marked in one-foot units.

2.  One 1000 ml graduated cylinder

3.  Hach Turbidimeter,  Model 2100 A


D.  Reagents

    None


                            14-59

-------
E.  Procedures
1.  Field Test.  Tie end of marked nylon rope to handrail where
    tests will be run, for example, in final sedimentation unit.
    Always take tests at the same time each day for comparable
    results.  Lower disc slowly until you just lose sight of it.
    Stop.  Bring up slowly until just visible.  Stop,  Look at
    the marks on the rope to see the depth of water that you can
    see the disc through.  Bring up disc and store.  Record results,
       -.   Use a clean 100° ml graduate.  Fill with a well-
    mixed sample up to the 1000 ml mark.  During every test the
    same lighting conditions in the lab should be maintained.
    Look down through the liquid in the cylinder and read the
    last visible number etched on the side of the graduate and
    record results.

    Hach Turbidimeter.  Follow manufacturer's instructions.
Whether you use one or each of these tests, you should run
either test at the same time every day and under similar
conditions for comparable results.
                            14-60

-------
                                                    (Clarity)


F.  Example and Calculation

1.  Each foot of depth is better clarity with Secchi disc.

2.  Each 100 ml seen in depth is better clarity.

3.  Turbidimeter reading indicates degree of clarity.
                           QUESTION
6.A  What does the clarity test tell you about
     the quality of effluent?

6,B  What happens when you attempt to measure
     clarity under different conditions, such
     as lighting and clarifier loadings?
                           14-61

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7.  Coliform Group Bacteria

A.  Discussion

Coliform bacteria are measured to indicate the presence of
bacteria originating in the intestines of warm-blooded animals.
High coliform counts indicate the usefulness of water may have
been impaired by fecal contamination.  Coliform bacteria are con-
sidered harmless, but their presence may be indicative of the
presence of disease-producing organisms that may be found with
them.

B.  What is Tested?
           Samole_       '                 Usual Range, MPN/100 ml

   Effluent:

     Primary                               5,000 to 1,000,000

     Nonchlorinated Secondary                   >240,000

     Chlorinated Secondary                    50 to 500

   Receiving Waters                        1,000 to 1,000,000

C.  Sampling Bottles

Polypropylene wide-mouthed bottles with 200 to 400 ml capacity are
used to collect samples.  Before sterilization by autoclave, add
sodium thiosulfate (0.1 ml of a 10% solution per 4 ounce bottle) to
the bottles to neutralize any chlorine residual in the samples.
When filling bottles in the field, do not flush out sodium thio-
sulfate or contaminate sample or bottle.  Fill bottles approximately
three-quarters full, maintain at 4° C with ice during transport and
start test in lab within eight hours after sampling,

D.  Media Preparation

1.  General Discussion

Careful media preparation is necessary to meaningful bacterio-
logical testing.  Attention must be given to the quality, mixing,
and sterilization of the ingredients.  The purpose of this care
is to assure that if the bacteria being tested for are indeed
present in a sample, every opportunity is presented for their
                               14-62

-------
                                                        (Coliform)


 development and ultimate identification.  Much bacteriological
 identification is done by noting changes in the medium; conse-
 quently, the composition of the medium must be standardized.
 Much of the tedium of media preparation can be avoided by purchase
 of dehydrated media (Difco, BBL, or equivalent).  The operator
 is advised to make use of these products; and, if only a limited
 amount of testing is to be done, consider using tubed, prepared
 media.

 2.  Glassware

 All glassware must be thoroughly cleansed using a suitable detergent
 and hot water (160°F), rinsed with hot water (180°F) to remove all
 traces of residual detergent, and finally rinsed with distilled or
 deionized water.

 3.  Water

 Only distilled water or demineralized water which has been tested
 and found free from traces of dissolved metals and bactericidal
 and inhibitory compounds may be used for preparation of culture
 media.

 4.  Buffered8 Dilution Water

 Prepare a stock solution by dissolving 34 grams of KH^POt,. in
 500 ml distilled water,  adjusting the pH to 7.2 with IN NaOH.
 Prepare dilution water by adding 1.25 ml of the stock solution
per liter of distilled water.   This  solution can be dispersed
 into various size dilution blanks or used as a sterile rinse water
 for the membrane filter test.
  Buffer.   A measure  of the  ability  or  capacity  of  a  solution
  or liquid to neutralize  acids  or bases.   This  is  a  measure
  of the  capacity of  water or wastewater  for  offering a
  resistance to changes in the pH.
                           14-63

-------
                                                   (Colifom?)


5.  Co liform Test—Fermentation Tube Method

a.  Lactose Broth or Lauryl Tryptose Broth

For the presumptive coliform test, dissolve the recommended
amount of the dehydrated medium in distilled water.  Dispense
solution into fermentation tubes containing an inverted glass
vial.  Autoclave the capped tubes at 121°C for 15 minutes.

b.  Brilliant Green Bile Lactose Broth

For the confirmed coliform test, dissolve 40 grams of the
dehydrated medium in one liter of distilled water.  Dispense
and sterilize as with Lactose Broth.

c.  Compensation for Diluting Effect of Samples

Large volumes of samples can dilute the medium in the fermen-
tation tube.  Use the concentrations listed below to compensate
for diluting effects when using lauryl tryptose broth.

  No. ml      Ml of sample     Nominal           No. grams
  medium      or dilution      concentration     dehydrated
  in tube                      before            medium per
                               inoculation       liter


    10        0.1 to 1.0          Ix               35.6

    10            10              2x               71.2
    20            10              1.5x             53.4

    35           100              4x              137.3

6.  Coliform Test-ElevatedTemperature for Fecal Coliforms

EC Broth

For the fecal coliform test, dissolve 37 grams of the dehydrated
medium in one liter of distilled water.  Dispense and sterilize
as with Lactose Broth.


7.  Co li form Tes t- -Memb rang Fi Iter Method

M-Endo Broth

Prepare this medium by dissolving 48 grams of the dehydrated
product in one liter of distilled water which contains 20 ml
 f ethyl alcohol per liter.  Heat solution to boiling only--
JO NOT AUTOCLAV^.  Prepared media should be stored in a
refrigerator and used within 96 hours.
                            14-64

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                                                             (Coliform)
 8.  Autoclaving
Steam autoclaves are used for the sterilization of the liquid media
and associated apparatus.  They sterilize  (killing of all organisms)
at a relatively low temperature of 121°C within 15 minutes by
utilizing moist heat.

Components of the media, particularly sugars such as lactose, may
decompose at higher temperatures or longer heating times.  For this
reason adherence to time and temperature schedules is vital.

Autoclaves operate in a manner similar to the familiar kitchen
pressure cooker:

1.  Water is heated in a boiler to produce steam.

2.  The steam is vented to drive out air.

3.  The steam vent is closed when the air is gone.

4.  Continued heat raises the pressure to 15 lbs/in2 (at this
    pressure, pure steam has a temperature of 121°C) .

5.  The pressure is maintained for the required time.
       V.
6.  The steam vent is opened and the steam is slowly vented
    until atmospheric pressure is reached.  (Fast venting will
    cause the liquids to boil.)

7.  Sterile material is removed to cool.
                                               \
In autoclaving fermentation tubes, a vacuum is formed in the inner
tubes.   As the tubes cool,  the inner tubes are filled with sterile
medium.   Capture of gas in  this  inner tube from the culture of
bacteria is the evidence of fermentation.
                           14-65

-------
                                                      CColiform)


E.  Test for Coliform Bacteria


1.  General Discussion

The test for coliform bacteria is used to measure the suitability
of a water for human use.  The test is not only useful in determin-
ing the bacterial quality of a finished water, but it can be used
by the operator in the treatment plant to guide him in achieving
a desired degree of treatment,

2.  Multitube Fermentation Technique

Coliform bacteria are detected in water by placing portions of
a sample of the water in lactose broth.  Lactose broth is a
standard bacteriological medium containing lactose (milk) sugar
in tryptose broth.  The coliform bacteria are those which will
grow in this medium at 35 °C temperature and ferment and produce
gas from the sugar within 48 hours.  Thus to detect these bac-
teria the operator need only inspect fermentation tubes for gas.
In practice, multiple fermentation tubes are used in a decimal
dilution for each sample.


3.  Materials Needed

1.  Fifteen sterile tubes of lactose broth are needed
    for each sample.

2.  Use five tubes for each dilution.

3.  Dilution tubes or blanks containing 9 ml or 99 ml
    of sterile buffered distilled water.

4.  Quantity of one and 10 ml sterile pipettes.
                             14-66

-------
                                          (Coliform)
4.  Technique for Inoculation and/or Dilution of Sample (Fig. 14.4)

All inoculations and dilutions of wastewater specimens must be
accurate and should be made so that no contaminants from the air,
equipment, clothes or fingers reach the specimen, either directly
or by way of the contaminated pipette.

1.  Shake the specimen bottle vigorously 20 times before removing
sample volumes.
2.  Into the first five lactose tubes pipette 1.0 ml of sample
directly into each tube.  (It is important to realize that the
sample volume applied to the first 5 tubes will depend upon the
type of water being tested.   The sample volume applied to each
tube can vary from 10 ml (or more) for high quality waters to
as low as 10  or 0.00001 ml (applied as 1ml of a diluted sample)
for raw wastewater specimens).

Note:  When delivering the sample into the culture medium, deliver
sample portions of 1ml or less  down into the culture tube near
the surface of the medium.  Do_ not deliver small sample volumes
at the top of the tube and allow them to run down inside the tube;
too much of the sample will fail to reach the culture medium.

Note:  Use 10 ml pipette for 10 ml sample portions, and 1 ml pipette
for portions of 1 ml or less.  Handle sterile pipettes only near the
mouthpiece, and protect the delivery end from external contamination.

3.  Pipette 1/10 ml or 0.1 ml of raw sample into each of the next
5 lactose broth tubes.  This makes a 0.1 dilution.

4.  To make the 0.01 dilution,  place 1 ml of well mixed raw sample
into 99 ml of sterile buffered  dilution water.  Mix thoroughly
by shaking.  This bottle will be labeled bottle A.

5.  Into each of the next 5 lactose broth tubes place directly 1 ml
of the 0.01 dilution, from bottle A.

At this point you have 15 tubes inoculated and can place these
three sets of tubes in the incubator; however, your sample specimen
may show gas production in all  15 fermentation tubes.

This means your sample was not  diluted enough and you have no
usable results.  To obtain usable results it is recommended that the
first time a sample is analysed that 30 tubes having a range of
six dilutions be setup.  In most cases this will give usable results.

6.  To make a 1/1000 or 0.001 dilution add 0.1 ml from the 1/100
dilution bottle (Bottle A) directly into each tube of five more
lactose broth tubes.
                                14-67

-------
                                                 (Coliforw^
7.  To make a 1/10000 or 0.0001 dilution take 1 ml from Bottle A
and place this 1 ml into 99 ml of sterile buffered dilution water.
Mix diluted sample thoroughly by shaking.  This bottle will be
called Bottle B.

8.  From the 0.0001 dilution (Bottle B)  pipette 1.0 ml of sample
directly into each tube.  Set 5 tubes up with this dilution.

9.  To make a 1/100000 or 0.00001 dilution pipette 0.1 ml of
sample directly into each tube.  Set 5 tubes up with this dilution.

The first time a sample is analysed 30 tubes of lactose broth
should be prepared.  Once the appropriate dilutions are established
that give usable results for determining the MPM Index only 15 tubes
need by prepared for subsequent samples  to be analysed.
                              14-67a

-------
                      COLIFORM BACTERIA  TEST   FIG. 14.4

                                       1  ml  TO EACH TUBE
 n
                                      O.t ml  TO EACH TUBE
      WATER SAMPLE
1  ml RAW SAMPLE
                                       1 ml TO EACH TUBE
                                              I       I
          f~\
0
0
                u
                                      0.1  ml TO EACH TUBE
     BOTTLE A
              99 ml  STERILE
              BUFFERED
              DILUTION
              WATER
p
                                       t ml TO BCti
                                     0.1 ml TO EACH TUBE
     BOTTLE
             99ml STERILE
             BUFFERED
             DILUTION
             WATER
                              ' &       S
                                                      9        W
                                                             u
                              DILUTION
                             NO DILUTION
                                                                          0.1
                             0.01
                                                                          0.001
                                                                          0.0001
                                                 1
                            IKCUBATE ALL TUBES AT 35°  C ±  0.5°C FOR  24 HOURS
                      GAS IN INNER VIAL
                      IS A + TEST RESULT
                                  14-68

-------
5.  24-Hour Lacotse Broth Presumptive Test

Place all inoculated lactose broth tubes in 35 C ^ 0.5°C incubator.
After 24+2 hours have elapsed, examine each tube for gas formation
in inverted vial (inner tube).   Mark + on report form for all tubes
that show presence of gas.  Mark - for all tubes showing no gas
formation.  Save all positive tubes for confirmation test.  The
negative tubes must be reincubated for an additional 24 hours.
6.  48-Hour Lactose Broth Presumptive Test

Record both positive and negative tubes at the end of 48 + 3 hours.
Save all positive tubes for confirmation test.
7.  24-Hour Brilliant Green Bile Confirmation. Test

Confirm all presumptive tubes that show gas at 24 or 48 hours.
Transfer, with the aid of a sterile 3 mm platinum wire loop,
one loop-full of the broth from the lactose tubes showing gas,
and inoculate a corresponding tube of BGB (Brilliant Green Bile)
broth by mixing the loop of broth in the BGB broth.  "Discard"
all positive lactose broth tubes after transferring is completed.

Always sterilize inoculation loops and needles in flame immediately
before transfer of culture; do not lay loop down or touch it to any
nonsterile object before making the transfer.  After sterilization
in a flame, allow suffic;e™.t time for cooling, in the air, to pre-
vent the heat of the loop from killing the bacterial cells being
transferred.  Sterila wooden applicator sticks also are used to
transfer cultures, expecially in the field where a flame is not
available for sterilization.

After 24 hours has elapsed, inspect each of the BGB tubes for gas
formation.  Those with sny amount of gas are considered positive
and are so recorded on the data sheet.  Negative BTB tubes are
reincubated for an additional 24 hours.
8.  _48^Hpur Brilliarit green Bile Confirmation Test

1.  Examine tubes for gas at the end of the 48+3 hour period.
    Record both positive and negative tubes.

2.  Complete reports by decoding MPN index and recording MPN
    on work sheets.
                               14-69

-------
9.  Methods of calculations of the most probable number

Select the highest dilution with all positive tubes, before a
negative tube occurs, plus the next two dilutions, (See
Example No. 1).
                             14-70

-------
                              EXAMPLE  NO. 1
m...*,...         6AS      6AS      6*S      6AS      GAS
DILUTION        ^    ^    ^    ^     __        RESULTS
   o.
                                                            5  out of  5
   0.1
                 GAS      GAS     GAS     GAS      GAS
5 out of 5
   0.01
                GAS     GAS     GAS     GAS      GAS
5 out  of 5    •—i
   0.001
                 GAS    MO GAS  NO GAS  NO GAS    GAS
                                                           2 out of 5
   0.0001
               NO GAS  NO GAS  SO GAS  NO GAS  MO SAS
0 ouf o! 5
   0.00001
               NO GAS  NO GAS  m GAS  NO GAS  NO GAS
0 out of 5
                                14-70a

-------
From the code 5-2-0 in the MPN Table (Table III) the MPN index
is 49.   Using the following formula the # of coliform bacteria
/100 ml is determined.

                         MPN                a constant
            MPN/100 ml = Index from table x 10

                            Dilution giving all positive,
                            before a negative tube occurs

                       = 49 x 10 = 49000
                           0.01
                            14-70b

-------
Example number 2

Dilutions
No. of tubes positive
out of 5 tubes set up
at each dilution
                          0   0.1   0.01   0.001   0.0001   0.00001
                                                   0
                                                            0
Our code in example 2 is 5-0-0.  Going to the MPN index (Table III)
we read the MPN Index of 23.  Using the formula as in example number
1
                                 23 x 10
                 MPN/100 ml   =   0.001
                 MPN/100 ml   =  230,000
                 MPN
                          or
                              =  230,000 per 100 ml
Example number 3

Dilutions
No. of tubes positive
out of 5 tubes set up
at each dilution
                          0   0.1   0.01   0.001   0.0001   0.00001
In example number 3 the code 5-1-0-1 is unreasonable because probability
would indicate that the 0.01 dilution should have 1 tube in 5 giving a
positive result.  In this case it is assumed that the 0.01 dilution
should have given 1 tube in 5 as positive and the code 5-1-1 is used to
determine the MPN/100 ml.
Using the formula:
                 MPN/100 ml
                                    46 x 10
                                       0
                 MPN
                                    460/100 ml
                             14-70C

-------
                                                  (Coliform)
                           TABLE III

MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
       IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
                FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
Five 1-ml
portions
0
0
0
1
1
1
2
2
3
0
0
0
0
1
1
1
2
2
2
3
3
4
0
0
0
0
1
1
1
2
2
2
Five 0. 1 ml
portions
0
1
2
0
1
2
0
1
0
0
' 1
2
3 '
0
1
2
0
1
2
0
1
0
1
1
2
3
0
1
2
0
1
2



































MPN Index
(organisms
per 100 ml)
<2
2
4
2
4
6 |
4
6
6
2
4
6 ]
8
4
6
8
6
8
10
8
10
11
5
7
9 :
12
7
9
12
9
12
14
                           14-71

-------
                                                     (Coliform)
                      TABLE III (cont'd.)

MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
       IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
                FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
2
2
2
3
3
3
3
3
3
3
3
3
3

3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
Five 1-ml
portions
3
3
4
0
0
0
1
1
1
1
2
2
Five 0.1 ml
portions
0
1
0
0
1
2
0
1
2
3
0
1
2 i 2
















!
5 0
3
4
4
5
0
0
0
0
1
1
1
2
2
2
3
3
3
1
0
1
0
0
1
2
3
0
1
2
0
1
2
0
1
2


















MPN Index
(organisms
per 100 ml)
12
14
15
8
11
13
11
14
17
20
14
17
20


















17 i
21
21
24
25
13
17
21
25
17
21
26
22
26
32
27
33
39

















                             14-72

-------
                                                        (Coliform)
                      TABLE III (cont'd.)

MPN INDEX FOR VARIOUS COMBINATIONS OF POSITIVE AND NEGATIVE RESULTS
       IN A PLANTING SERIES OF FIVE 10-ml, FIVE 1-ml AND
                FIVE 0.1-ml PORTIONS OF SAMPLE
Number of tubes giving positive
reaction out of
Five 10-ml
portions
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Five 1-ml
portions
4
4
5
5
0
0
0
0
0
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
4
5
5
5
5
5
5
Five 0.1 ml
portions
0
1
0
1
0
1
2
3
4
0
i
2
3
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
























MPN Index
(organisms
per 100 ml)
34
40
41
48
23
31
43
58
76
33
46
63
84
49
70
94
120
148
177
79
109
141
175
I 212













253
130
172
221
278
345
426
240
348
542
920
1600
>2400







j






























                            14-73

-------
                                                      (Coliform)


F.  Test for Fecal Coliform Bacteria


1.  General Discussion
Many regulatory agencies are measuring the bacteriological
quality of water using the fecal coliform test because this
test is a more reliable test for indicating the potential
presence of pathogenic organisms than is the coliform group
of organisms.  The procedure described is an elevated temper-
ature test for fecal coliform bacteria.
2.  Materials Needed

Equipment required for the tests are the same as those required
for the 24-Hour Lactose Broth Presumptive Test, a water bath,
and EC Broth.
3.  Procedure

    1.  Run lactose broth or lauryl tryptose broth presumptive
        test.

    2.  After 24 hours temporarily retain all gas-positive tubes,

    3.  Label a tube of EC broth to correspond with each
        gas-positive tube of broth from presumptive test.

    4,  Transfer one loop-full of culture from each ga?-
        positive culture in presumptive test to the
        correspondingly labeled tube of EC broth.

    5.  Incubate EC broth tubes 24 t 2 hours at 44.5°C i
        0.2°C in a waterbath with water depth sufficient
        to come up at least as high as the top of the culture
        medium in the tubes.  Place in waterbath as soon as
        possible after inoculation and always within 30 minutes
        after inoculation.

    6,  After 24 hours remove the rack of EC cultures from
        the waterbath, shake gently, and record gas pro-
        duction for each tube.  Gas in any quantity is a
        positive test.

    7.  As soon as results are recorded, discard all tubes.
        This is a 24-hour test for EC broth inoculations and
        not a 48-hour test.
                             14-74

-------
                                                       (Coliform)
        Transfer any additional 48-hour gas positive tubes
        from the presumptive test to correspondingly labeled
        tubes of EC broth.  Incubate for 24 ± 2 hours at
        44.5°C ± 0.2°C and record results on data sheet.

        Codify results and determine MPN of fecal coliforms
        per 100 ml of sample.
G.  Membrane Filter Method
 1.  General Discussion
In addition to the fermentation tube test for coliform bacteria,
another test is used for these same bacteria in water analysis.
This test uses a cellulose ester filter, called a membrane filter,
the pore size of which can be manufactured to close tolerances.
Not only can the pore size be made to selectively trap bacteria
from water filtered through the membrane, but nutrients can be
diffused up through the membrane to grow these bacteria into
colonies.  These colonies are recognizable as coliform because
the nutrients include fuchsin dye which peculiarly colors the
colony.  Knowing the number of colonies and the volume of water
filtered, the operator can then compare the water tested with
water quality standards.
2.  Materials Needed

1.  One sterile membrane filter having a 0.45y pore size.

2.  One sterile 47 mm Petri dish with lid.

3.  One sterile funnel and support stand.

4.  One sterile pad.

5.  One receiving flask (side-arm, 1000 ml).

6.  Vacuum pump, trap, suction or vacuum gage, connecting sections
    of plastic tubing, Glass "T" hose clamp to adjust pressure by-
    pass.

7.  Tweezers, alcohol, Bunsen Burner, grease pencil.
                           14-75

-------
                                              (Coliform)
 8.  Sterile buffered distilled water for rinsing made up in
     100-500 ml quantities.

 9.  M-Endo Media.

10.  Sterile pipettes—two 5 ml graduated, one 1 ml for aliquot
     or one 10 ml for larger aliquot.  Quantity of one ml pipettes
     if dilution of sample is necessary.   Also, quantity of dilution
     water blanks if dilution of sample is necessary.

11.  One moist incubator at  35° C temperature.  Auxiliary incubator
     dish with cover.
                               14-76

-------
                                                                   (Coliform)
                  3.  Illustration of Inoculation of Membrane Filter
                          Fig. I
             1.  Center membrane filter on
                 filter holder.  Handle mem-
                 brane only on outer 3/16
                 inch with tweezers sterilized
                 before use in ethyl or methyl
                 alcohol and passed lightly
                 through a flame.
                           Fig.  II

                     2.   Place funnel
                         onto filter
                         holder.
            Fig.  Ill
3,   Pour or pipette sample
    aliquot into funnel.
 s  Avoid spattering.   After
    suction is applied rinse
    two  times with sterile
    buffered distilled water.
Fig. IV
    Fig.  V
Remove membrane filter from
filter holder with sterile
tweezers.  Place membrane
on pad.  Cover with Petri
top.
Incubate in
inverted
position for
22+2 hours.

Count colonies
on membrane.
                                        14-77

-------
                                                 (Coliform
 4.   Procedure for Inoculation of Membrane Filter

 All filtrations and dilutions of water specimens  must be  accurate
 and should be made so that  no contaminants from the  air,  equipment,
 clothes or fingers reach the specimen either directly or  by way  of
 the contaminated pipette.

 1.   Secure tubing from pump and bypass to receiving  flask.  Place
     palm of hand on flask opening and start pump.  Adjust
     suction to % atmosphere with hose clamp on pressure bypass.
     Turn pump switch to OFF.

 2.   Set sterile filter-support-stand and funnel on receiving  flask.
     Loosen wrapper.  Rotate funnel counter-clockwise to disengage
     pin.  Recover with wrapper.

 3.   Place Petri Dish on bench with lid up.  Write identification
     on lid with grease pencil.

 4.   Open sterile filter pad package.  Light Bunsen burner.

 5.   Sterilize tweezers by dipping in alcohol and  passing  quickly
     through Bensen burner.

 6.   Center membrane filter  on filter stand with  tweezers after
     lifting funnel.  Membrane filter with printed grid should
     show grid uppermost (Fig. 1).

 7.   Replace funnel and lock against pin (Fig. 11).

 80   Add a small amount of the sterile dilution water to funnel.
     This will help check for leakage and also aid in dispersing
     small volumes (Fig. Ill).
                                                            o
 9,   Shake sample or diluted sample.  Measure proper  aliquot
     with sterile pipette and add to funnel.

10.   Now start vacuum pump.
 9
  Aliquot (AL-li-kwot).   Portion of sample.
                               14-78

-------
                                                (Coliform)
12.  After filtration of entire sample is finished,  rinse two
     times wit1-1 sterile buffered distilled water,  pouring just
     below inner lip of funnel.  Allow each rinse  to completely
     pass through funnel before proceeding to next rinse.

13.  When membrane filter appears barely moist,  switch pump to
     OFF.

14.  Sterilize tweezers as before.

15.  Remove membrane filter with tweezers after  first removing
     funnel as before (Fig. 1).

16.  Center membrane filter on pad containing M-Endo medium
     with a rolling motion to insure water seal.  Inspect
     membrane to insure no captured air bubbles  are present.
     (Fig. IV).

17.  Place inverted Petri Dish in incubator for  22+2 hours.

 5.  Procedure for Counting Membrane Filter Colonies

 1.  Remove Petri Dish from incubator.

 2.  Remove lid from Petri Dish.

 3.  Place Petri Dish with filter under illuminating light.   Tilt
     membrane filter in base of Petri Dish so that green and
     yellow-green colonies are most apparent. Direct sunlight
     has too much red to facilitate counting.

 4.  Count individual colonies utilizing an overhead fluorescent
     light.  The coliform colony is characterized  by a "metallic
     sheen" and only those colonies showing ANY  amount of this
     sheen are considered to be coliforas,

 5.  Report total number of "coliform colonies"  on work sheet.
     Use the membranes that show from 20 to 80 colonies and do
     not have more than 200 colonies of all types  (including  non-
     sheen or, in other words, non-coliforms).
                              14-79

-------
Example:

A total of 42 colonies grew after filtering 10 ml of the undiluted
sample.

                No. of colonies counted x 100 ml
Bacteria/100 ml=Volume of sample filtered

Example:       =(42 colonies) (100ml)     »(4.2  (100 ml)    = 420 per 100 ml.
                  (10 ml) (100 ml)               100 ml

A total of 30 colonies grew after filtering 20 ml of a 1/100 or 0.01
diluted sample.

Bacteria/100 ml=No. of colonies counted x 100 ml
                Volume of sample filtered x dilution factor
                =30 x 100 ml
                 20 ml x 0.01

                =15000 bacteria/100 ml

                      QUESTIONS

7.A  Why should sodium thiosulfate crystals be added to
     sample bottles for coliform tests before sterilization?

7.B  Steam autoclaves effect sterilization (killing of all
     organisms) at a relatively low temperature (p	   C)
     within _^	minutes by utilizing moist heat.

7.C  Calculate the Most Probable Number (MPN) of coliform
     group bacteria from the following test results:

     Dilutions   0      -1    -2      -3      -4   •   -5

     Readings    555120

7.D  How is the number of coliforms estimated by the membrane
     for filter method ?
                END OF LESSON 3 of 8 LESSONS

                             on

             Laboratory Procedures and Chemistry

Work the next portion of the.discussion and review questions
before continuing with Lesson 4.
                                14-80

-------
                 DISCUSSION AND REVIEW QUESTIONS

                     (Lesson 3 of 8 Lessons)

        Chapter 14.'  Laboratory Procedures and Chemistry



 Name                                        Date
 Write the answers to these questions in your notebook.  The
 problem numbering continues from Lesson 2.
 8.  Why must the clarity test always be run under the
     same conditions?

 9.  What is the purpose of the coliform group bacteria
     test?

10.  What does MPN mean?
                             14-81

-------
 The following graphical method may be  helpful  and  is  added here
 as an alternate:

                      PROCEDURE FOR USE OF  GRAPH

 Step 1:   Locate the point  on the  horizontal  axis which  corresponds
          to the coliform value obtained from the laboratory  analysis.

 Step 2:   Draw a vertical line from the point located  in Step 1 up
          to the diagonal line on  the graph.

 Step 3:   Draw a horizontal line from the point on  the diagonal line
          wh^ch was  located in Step 2 to the  vertical  axis on the
          left side  of the  graph.

 Step 4:   The point  on the  vertical axis which  was  located in Step 3
          corresponds to the logarithm  of the colifprm value.

 Step 5:   Repeat Steps 1 through 4 for  each coliform value which was
          obtained in the given time  period.

 Step 6:   Sum all of the logarithm values obtained  in  Step 4.

 Step 7:   Divide the sum of the logarithms  by the number of  logarithms
          summed in  Step 6.

 Step 8:   Locate the point  on the  vertical  axis which  corresponds  to
          the value  obtained in Step  7.

 Step 9:   Draw a horizontal line from the point located  in Step 8  to
          the diagonal line on the graph.

Step 10:   Draw a vertical line from the point on,  the diagonal line
          which was  located in Step 9,  down to  the  horizontal axis
          of the graph.

Step 11:   The point  on the  horizontal axis  which was located in Step
          10 corresponds to the geometric mean  coliform  value.
                                  14-81 a

-------
The following graphical method may be helpful and is added here
as an alternate:

                      EXAMPLE OF USE OF GRAPH
Given;  The following coliform levels (in numbers per 100 ml) were
        determined for eight effluent samples collected during a
        1-month period: 375, 425, 78, 17, 1098, 8, 9327, and 172.

jPindj   The monthly geometric mean value of th$ e,ight effluent
        coliform samples.

        A.  The logarithm of each coliform determination i§ selected
        from the graph using Steps 1 through 5 of the procedure.

Sample Coliform Determination        Logarithm of Coliform Determination

           1.    375                                2.57

           2.    425                                2.63

           3.     78                                1.89

           4.     17                                1.23

           5.  1,098                                3.04

           6.      8                                0.90

           7.  9,327                                3.97

           8.    172                                2/24

                                        Total =    18.47

        B.  The sum of the logarithm determinations is obtained (Step 6
        of the procedure).

        C.  The arithmetic average of the logarithm is obtained (Step 7
        of the procedure).  Arithmetic average of logarithms =

                            Total	 - 18,47 = 2.31
                            Number of samples     8

        D.  The geometric mean value is selected from the graph using
        Steps 8 through 11 of the procedure.  Geometric mean value =
        200/100 ml.                            ~~"      '
                                 14-81b

-------
             ooi aid  siuno)

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       CHAPTER 14.  LABORATORY PROCEDURES AND CHEMISTRY

                     (Lesson 4 of 8 Lessons)


3.  Dissolved Oxygen or DO and Biochemical Oxygen Demand or POD


                         I.  IN WATER
A.  Discussion

The dissolved oxygen (DO) test is, as the name inplies, the testing
procedure to determine the amount pf oxygen dissolved in samples of
water or wastewater.  There are various types of tests that can be
run to obtain the amount of dissolved oxygen.  This procedure is the
Sodium Azide Modification of the Winkler Method and is best suited for
relatively clean waters.  Interfering substances include color,
organics, suspended solids, sulfides, chlorine, and ferrous and
ferric iron.  Nitrites will not interefere with the test if fresh
azide is used.

The generalized principle is that iodine will be released in pro-
portion to the amount of dissolved oxygen present in the sample.
By using sodium thiosulfate with starch as the indicator, one can
titrate the sample and determine the amount of dissolved oxygen.
B.  What is Tested?

         Sample

    Influent

    Primary Clar.  Effluent


    Secondary Effluent


    Oxidation Ponds

    Activated Sludge--
     Aeration Tank Outlet
    Common Range, mg/1

Usually 0,>1 is very good.

Usually 0, Recirculated from
filters > 2 is good,

50% to 95% Saturation, 3 to
>8 is good.

1 to 25+*


>2 desirable
(> means greater than)
(* supersaturated with  oxygen)
                            14-82

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                                                (DO and BOD)
                                                              !

C.  Apparatus

METHOD A (Sodium,Azide Modification of Winkler Method)

1.  Buret, graduated to 0.1 ml.                               j
                                                              I
2.  Three 300 ml glass-stoppered BOD bottles

3.  Wide-mouth Erlenmeyer flask, 500 ml.

4.  One 10 ml measuring pipette.

5.  One 1-liter reagent bottle to collect activated sludge.


METHOD B (DO Probe)

Follow manufacturer's instructions.  See Section H for Discussion,
Calibration, and Precautions.


D.  Reagents
1.  Manganous sulfate solution.  Dissolve 480 g rcanganous sulfate
    crystals (MnSO^. 4H20)  in 400 to 600 nil distilled water.  Filter
    through filter paper.,  then add distilled water to the filtered
    liquid to make a 1-liter volume.

2.  Alkaline iodide-sodium azide solution.  Dissolve 500 g sodium
    hydroxide (NaOH) in 500 to 600 ml distilled water; dissolve
    150 g potassium iodide (KI) in 200 to 300 ml distilled water
    in a separate container.  Exercise caution.  Mix chemicals in
    pyrex glass bottles using a magnetic stirrer.  Add the chemicals
    to the distilled water slowly and cautiously.  Avoid breathing
    the fumes and body contact with the solution.  Heat is pro-
    duced when the water is added, and the solution is very
    caustic.  Place an inverted beaker over the top of the mixing
    container and allow the container to cool at room temperature.

  •  Mix both solutions when they ar,e cool.
    Dissolve 10 g sodium azide (NaNs) in 40 ml of distilled water.
    Exercise caution again.  This solution is poisonous.

    Add the sodium azide solution with constant stirring to the
    cooled .solution of alkaline iodide; ther, add distilled water
    to the mixture to make a 1-liter volume.  Sod:um azide will
    decompose in time and is no good after tnree months.
                          14-83

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                                                   (DO and BOD)
3.  Sulfuric acid.   Use concentrated reagent-grade acid .v
    Handle carefully, since this material will burn hands  and
    clothes.  Rinse affected parts with tap water to prevent
    injury.

    CAUTION:  When working with alkaline azide and sulfuric acid,
    keep a nearby water faucet running for frequent hand rinsing.

4.  0.0375 N sodium thiosulfate solution.  Dissolve exactly
    9.308 g sodium thiosulfate crystals (Na2S203'5H20)  in
    freshly boiled and cooled water and make up to 1 liter.
    For preservation, add 0.4 g or 1 pellet of sodium hydroxide
    (NaOH).  Solutions of "thio" should be used within two weeks
    to avoid loss of accuracy due to decomposition of solution.

5.  Starch solution.  Make a thin paste of 6 g of potato starch
    in a small quantity of distilled water.  Pour this paste
    into one liter of boiling, distilled water, allow to boil
    for a few minutes, then settle overnight.  Remove the clear
    supernatant and save; discard the rest.  For preservation,
    add two drops toluene (C6H5CH3).

6.  Copper sulfate solution.  Make a 10 percent solution by dis-
    solving 10 grams of copper sulfate in 100 ml of water.

Sodium Azide Modification of the Winkler Method

NOTE:  The sodium azide destroys nitrates which will
       interfere with this test.
E.  Outline of Procedure
                                  4.  Mix by
                                  Inverting
                                         White floe
                                          NO no
1.
Take
300 ml
Sample
2.
Add
2 ml
MnSO^
below
surface
Add
2 ml
KI +
NaOH
below
surface
                                             0 0
                                            #00
                                             0 o
                                         Brown floe
                                         DO present
Reddish-
Brown
Iodine
Solution
                            14-84

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                                                                       (DOand BOD)
         Titration of Iodine Solution:
I.  Pour Bottle
   Contents
   into Flask.
                         Reddish-
                          Brown
 Pale
Yellow    /*•
Blue,
               2. Titrate
     3. Add 5tarch
        Indicator
                                                                          "ft
   /    V Clear

End Point
                                    14-85

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                                                        (DO  and BOD)


                            PROCEDURE

 The reagents  are to be  added in  the quantities,  order,  and  methods
 as follows:

 1.  Collect  a sample to be  tested in  300  ml  (BOU) bottle  taking
     special  care to avoid aeration of the liquid being  collected.
     Fill bottle completely  and add cap.

 2.  Remove cap and add  2 ml of manganous  sulfate solution below
     surface  of the liquid.

 3.  Add /' ml  of alkaline-iodide-sodium azide  solution below the
     surface  of the liquid.

 4.  Replace  the stopper, avoid trapping air bubbles, and  shake
     well by  inverting the bottle several  times.  Repeat this
     shaking  after the floe  has settled halfway.  Allow  the  floe
     to settle halfway a second time.

 5.  Acidify  with 2 ml of concentrated sulfuric acid by  allowing
     the acid to run down the neck of  the  bottle  above the surface
     of the liquid.

 6.  Restopper and shake well until the precipitate  has  dissolved.
     The solution will then  be ready to titrate.  Handle the
     bottle carefully to avoid acid burns.

 7.  Pour contents of bottle into an Erlenmeyer flask.

 8.  If the solution is  brown in  color, titrate with 0.0375  N
     sodium thiosulfate  until the solution is  pale yellow  color.
     Add a small quantity of starch indicator  and proceed  to
     step 10.

 9.  If the solution has no  brown color, or is only  slightly
     colored,  add a small quantity of  starch  indicator.  If
     no blue  color develops,1 there is  zero Dissolved Oxygen.
     If, -a blue color does develop, proceed to  step 10.

10.  Titrate  to the first disappearance of the blue  color.   Record
     the number of ml of sodium thiosulfate used.

11.  The amount of oxygen dissolved in the original  solution will
     be equal  to the number  of ml of sodium thiosulfate  used in
     the titration provided  significant interfering  substances are
     not present.

     mg/1DO  = ml sodium thiosulfate
                             14-86

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                                                              JC and 10D)
     Example
The DO titration of a 300 ml  sample  requires  5.0  ml  of 0.0375 N
Sodium Thiosulfate.  Therefore, the  dissolved oxygen concentra-
tion in the sample is 5 mg/1.
G.  Calculation

You 'will want to find the percent  saturation  of DO in  the
i-ffiuent of your secondary plant,   ^lie  DO  3.5  5.0 mg/1  and  the
temperature is 20CC.  At 209C,  100%  DO  saturation is 9.2 mg/1.

The dissolved oxygen saturation values  are  given in Table  IV,
Note that as the temperature of water increases, the DO satura-
tion value (100% Saturation Column)  decreases.   Table  IV gives
hVi";, DO saturation  values for temperatures  in °C and C'F.


iX.) Saturation, %  =  -SP °f S^Ple>  "8/1. x, P0%
                     00 at  :'">ra; Saturation, mg/1
H.  DO Probe
    Discussion

Measurement of the dissolved oxygen  (DO) concentration  with  a
probe and electronic readout meter is a satisfactory  substitute
for the Sodium Azide Modification of the Winkler Method, under
many circumstances.  The probe is recommended when  samples con-
t. in substances -,.-hj.ch interfere with the modified Winkler procedure,
such as sulfite, tniosulfate, polythionate, mercaptans,  free
chlorine or hypochlof-te, organic substances -eadily  hydrplyzed
in alkaline solutions, free iodine,  intense color o^  turbidity,
»nd biological  "Iocs.  \ continuous  record of the dissolved
                            14-87

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                                    (DO  t!nd  BOD)


                 TABLE IV
LFFECT OP 1TMPHKATURE ON OXYGEN SATURATION
 FOR A CHLORIDE CONCENTRATION OF ZERO Mg/":


°c
0
1
•>
.i.
3
4
5
6
7
8
-1
' 10
11
12
13
'14 '
15
16
17
18
' '
2U_
21
22
23
24'
i. - -

°F
32.0
33.8
35.6
37.4
39.2
41.0
42.8
44.6
46.4
48.2
50.0
51.8
53.6
55.4
57.2
60.0
61.8
63.6
65.4
67. J
68.0
69.8
71.6
73.4
75.2
77.0
mg/1 DO at
saturation
14.6
14.2
13.8
13.5
13.1
12.8
12.5
12.2
11.9
11.6
11.3
11.1
10.8
10.6
ID. 4
10.2
10.0
9. ~
9.b
9.4
9^2
9.0
8.8
8.7
8.5
8.4

                 14-88

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                                                  [Dp and 30f>i
oxygen content of aeration tanks and receiving waters may be
obtained -using a probe.  In determining the BOD of sapples.,
a probe may be used to determine the DO initially and u/te,r
the five-day incubatior period of the blanks and samp.e «i. t;.:
2.  Procedure

Follow manufacturer's instructions.


3.  Calibration

To be assured that the DO prone reading provides the dissolved
oxygen content of the sample, the probe must be calibrated.  lake
a sample that does not contain substances that interfe/e with
either the probe reading or the ipodified Winkler procedure.
Split the sample.  Measure the DO in one portion of the sample
using the modified Winkler procedure and compare this result with
the DO probe reading on the other portion of the sample.  Adjust
the probe reading to agree with the results from the modified
Winkler procedure.

When calibrating the probe in an aeration tank of the activated
sludge process, do not attempt to measure the dissolvec oxygen
in the aerator and then adjust the probe.  The biological floes
in the aerator will interfere with the modified Winkler procedure,
and the copper sulfate-sulfamic acid procedure is not sufficiently
accurate to calibrate the probe.  An aeration tank probe may be
calibrated by splitting an effluent sample, measuring the DO by
the modified Winkler procedure, and comparing results with the
probe readings.  Always keep the membrane in the tip of the r>ro;>e
frpm drying because the probe can lose its accuracy until re-
conditioned.


4.  Precautions
1.  Periodically check the calibration of the probe.

2.  Keep the membrane in the tip of the probe from drying out.

3.  Dissolved inorganic salts, sucn as found in sea water, can
    influence the readings from a probe.

4.  Reactive compounds, such as reactive gases and sultur coru-
    poxands, can interfere with the output of a probe.

.-;,  Don't place the probe directly over a diffuser because you
    want to measure the dissolved oxygen in the water being
    treated, not the oxygen in the air supply to the aerator.
                            14-89

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                                                   (DO  and BOD)


 8.  Dissolved Oxygen


                         II.   IN AERATOR
Copper Sulfate-Sulfamic Acid Flocculation, page 413,  12th Edition,
1965, "Standard Methods".
A,  Discussion

This modification is used for biological floes  that have high
oxygen utilization rates in the activated sludge process,  and
when a DO probe is not available.  It is very important that
some oxygen be present in aeration tanks at all times to maintain
aerobic conditions.

This test is similar to the regular DO test except that copper
sulfate is added to kill oxygen-consuming organisms, and sulfamic
acid is added to combat nitrites before the regular Dp test is run.

NOTE;'  If the results indicate a DO of less than 1 mg/j, it is
       possible that''the DO in the aera'tion tank is ZERO1
       When the DO in" the aeration tank' is near zero, consider-
       able DO from the surrounding atmosphere can mix with the
       sample when it is collected, when the inhibitor is  addend,
       while the solids are settling, and when the sample  is
       transferred to a BOD bottle for the DO test.  If you use
       this test, use a deep container and avoid stirring.  See
       article by Hughes and Reynolds JWPCF, Vol. 41, pg.  184,
       January 1969, for a discussion of the shortcomings  of
       this test.
B.  What is Tested?

           Sample                        C ommon DO Range,

    Aerator Mixed Liquor                      0.1-3.0

C.  Apparatus

1.  One tall bottle, approximately WOO ml.

2.  Regular DO apparatus.
                            14-90

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  (DO
                                                          BOD)
!>.   Reagt p.ts

1.   Copper sulfate-sulfamic acid inhibitor solution.   .^ssolve
    32 g technical grade sulfamic acid O!H2S02OH) without heat
    in 475 ml distilled water.  Dissolve 50 g copper sulfate,
    CuSOi4-5H20, in 5f'r; ml water.  Mix the two solution5? together
    and add 23 ml concentrated acetic acid.

2.   Regular DO reagents.
    Outline of Procedure
 1.  Add  10  ml  of
    inhibitor.
Jip  into  mixed  3.  Settle
liquor             sairpjc,
:.to7r;;er  settle.
4.  Siphon over 300 ml
   of sample into
   SOD : j-;t:e.
1.   Add at least 10 ml of inhibitor (5 ml copper sulfate and
    5 ml sulfamic acid) to any TALL bottle  (1-cmart milk oottle)
    fcith an approximate volume oTTdOO ml.  °lace filling  tube
    near the bottom.  An emptying tube is placed approximately
    1/4 inch from the top of the bottle cork.  Attach bottle  to
    rod or alum: nura conduit and lower into  aeration tank.

2.   Allow bottle to fill and then withdraw.

3.   Let stand, un* •.J clear s.pernatant liquor can be siphoned  into
    a 300 ml " ? botx .e.  Do not aerate in  transfer,

4.   Then run regular DO.
                             ^4-91

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                                                   fOQ and HOP!


F. and G.  Example and Calculations


Same as regular DO test.
                           QUESTIONS
R,A  Calculate the percent dissolved oxygen saturation if
     the receiving water DO is 7.Q rag/1 and the temperature
     is 10 °C.

8.B  How would you calibrate the DO probe in an aeration tan*.1'

8.C  What are the limitations of the copper sulfate-suiramie  acid
     procedure for measuring DO in an aeration tank when the  DO
     in the tank is very low?
                            14-92

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                                                   (DO and BOD)


Biochemical Oxygen Demand or BOD


A.  Discussion                                                   i

The BOD test gives the amount of oxygen  used by microorganisms   j
to utilize the substrate (food) in wastewater when placed in a  j
Controlled temperature for five days.  The  DO (dissolved oxygen))
is measured at the beginning and recorded.   After the 5-day     '
jncubation period the DO is again determined.   The BOD is then
calculated on the oasis of the reduction of DO and the size of
sample.  This test is an estimate of the availability of food   !
in the sample ffoo^  .or organisms that take up oxygen)  expressed
ir. terms of oxv , .  use.  Results of a BOD test indicate the rate
of oxidation a.nu provide an indirect estimate of the Availability
to ov*eanistns o*1 concentration of the waste.

Samples are incubaxoa i«..>r a standard period of five days because
a vra.:tiou of the total BO'.  «•.•>. M be exerted curing this period.
fhe :':t;ir,ate or total. BOD i... normally never run for plant control,
•  Disadvantage of t;>e BU1 *• st is that 'che  res Jits are not *.v'ail-
:-'•• ie until f: ve. days , at't^r tne sample was collected,


.<•.  iVhat is Tested?

             Sample                    Common Range, mg/1

        Influent                          15C   >'^

        Primary Effinen*                    60 - 160

        Secondary Efflueir                  10 - 60

        j.tester Supernatant              1000 - 4000-r

        Industrial Wastes                 100 - 3000-


C.  Apparatus

    300 ml BOD bottles with ground glass stoppers

:,  Incubator, 20 °(

7.  Pipettes, 10 ml  gr;  .Uct'ced, 1/32 to 1/16-inch diameter tip

*,  Burette and stand

:,  Erlenmeyer flask, ?nO ml
                             14-93

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                                                   (DO and BOD)
D.  Reagents^
See Section U, page 14-°
-------
                                                          (DO and BOD)
1.   Fill 2 BOD bottles
    with BOD dilution
    water.
                           OUTLINE OF PROCEDURE
4.
20°C



A
1 Incubate
I 5 days

I
i



                                Test  for D.O.
                                         3. Fill with
                                         dilution water
                                            I
                                            2.  Add
                                             sample
              5.   Immediately  test  2  §  4  for initial  D.O.
   6.   Add
  2 ml
  below
  surface
                                    Test  for D.O.
                                                     0.375  N
                                                     Na2S203
7.  Add 2 ml
Alkaline KI
below
surface
8.  Add 2 ml  9. Transfer  10. Titrate
   H2SOtt     Bottle Con-
             tents to
    14-95    Flask

-------
                                                         (DO and BOD)


E.  Outline of Procedure

The test is made by measuring the oxygen used or depleted during
a 5-day period at 20°C by a measured quantity of wastewater sample
seeded into a reservoir of dilution water saturated with oxygen.
This is compared to an unseeded or blank reservoir of dilution
water by subtracting the difference and multiplying by a factor
for dilution.  See outline on Page 14-107.


                           PROCEDURE

1.  BOD bottles should be of 300 ml capacity with .ground glass
    stoppers and numbers.  To clean the bottles, carefully rinse
    with tap water followed by distilled water.

2.  Fill two bottles completely with dilution water and insert
    the stopper tightly so that no air is trapped beneath the
    stopper.  Siphon dilution water from its container when
    filling BOD bottles.

3.  Set up one or more dilutions of the sample to cover the
    estimated range of BOD values.  From the estimated BOD,
    calculate the volume of raw sample to be added to the BOD
    bottle based on the fact that:


    The most valid DO depletion is 4 mg/1.   Therefore,
    ml of sample added  _   (4 mg/1)  (300  ml)
    per 300 ml          ~  Estimated  BOD,  mg/1

                                 1200
                           Estimated BOD,  mg/1
    Examples:

    a.   Estimated BOD =  400  mg/1

        ml  of  sample  added  _   1200
        to  BOD bottle       ~   400

                            =   3 ml

-------
                                                   (DO and BOD)
    b.   Estimated BOD  =  200 rog/1:   use  6 ml
                          100 mg/1:   use 12 ml
                           20 mg/1:   use 60 ml

    When the BOD is unknown,  select  more than pne sample size.
    For example, place several samples--! ml, 3 ml, 6 ml, and
    12  mi—into four BOD bottles,

    For samples with very high BOD values, it may be, difficult
    to  accurately measure small volumes or to get a truly repre-
    sentative sample.  In such a case, initial dilution should
    first be made on the sample.  A dilution of 1:10 is convenient.

4.  To  perform the BOD test,  first fill two BOD bottles with
    BOD dilution water.  Nos. (1)  and (2) in illustration,
    Page 14-107

5.  Next, for each sample to be tested, carefully measure out the
    two portions of sample and place, them into two new BOD bottles,
    Nos. (3) and (4).  Add dilution water until the bottles are
    completely filled.  Insert the stoppers.  Avoid entrapping air
    bubbles.  Be sure that there are water seals on the stoppers.

6,  On  bottles (2) and (4) immediately determine the initial
    dissolved oxygen.

7.  Incubate the remaining dilution water blank and diluted sample
    at  20°C for five days.  These are bottle? (1) and (3).

8.  At  the end of exactly five days (± 3 hours), test bottles
    (1) and (3) for their dissolved oxygen by using the sodium
    azide modification of the WinkTer" method or a DO probe.
    At the end of five days, the oxygen content should be at
    least 1 mg/1.  Also, a depletion of 2 mg/1 or more is
    desirable.  Bottles  (1) and (2)  are only used to check the
    dilution water quality.  Their difference should be  less
    than 0.2 mg/1 if the quality is good and free of impurities.
                              14-97

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                                                  (DO and BOD)


F.  Precautions

Since this is a bioassay (BUY-o-ass-SAY),  that is, living organisms
are used for the test, environmental conditions must be quite exact.

1.  The temperature of the incubator must  be at 20°C.  Other
    temperatures will change the rate of oxygen used.

2.  The dilution water should be made according to Standard
    Methods for the most favorable growth rate of the bacteria.
    This water must be free of copper which is often present
    when copper stills are used by commercial dealers.  Use all
    glass or stainless steel stills.

3.  The wastewater must also be free of toxic wastes, such as
    hexavalent chromium.

4.  If you use a cleaning solution to wash BOD bottles, be sure
    to rinse the bottles several times.  Cleaning agents are
    toxic and if any residue remains in a BOD bottle, a BOD
    test could be ruined.

5.  Wastewater normally contains an ample supply of seed bacteria;
    therefore seeding is usually not necessary.


G.  Chlorinated Samples

It is very difficult to obtain reliable and reproducible results
from the BOD test, and a chlorinated sample is even more difficult.
For this reason, samples for BOD tests should be collected before
chlorination.
H.  Example

     BOD Bottle Volume          =  300 ml

     Sample Volume              =  15 ml

     Initial DO of                 0 _   /n
     Diluted Sample             =  8'° mg/1

     DO of Sample and Dilution     A n   ,,
     After 5-day Incubation     =  4'U mg/i
                           14-98

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                                                  (DO and BOD)
I.  Calculations
HOD,
mg/1
Initial DO of
Diluted Sam-
pie, ng/1
            DO of Diluted"')
            Sample After
            5-Day Incuba-
            tion, mg/1
                                             30D Bottle Vol., xil
                                              Sample Volume, ml
                                f      \
          (8.0 mg/1 - 4.0 mg/1) H^ "*!
          (4.0) (300)
              15

          SO mg/1

For acceptable results, the percent depletion of oxygen  in  the  BOD
test should range from 30% to 80% depletion.
„  n  ,  _.
%  Deplete on
DO of Diluted Sample, mg/f|
  -^_ DO After^ 5 Jays, mg/lj
DO of Diluted Sample, mg/1
                 (3.0 ing/]. - 4.0 mg/1) x 1Q{J%
                       8.0 mg/1


                 4 x 100%
              =  50%

When a sample reo^ires a large volume in the BOD test  and  a  small
amount of dilution water, or if a sample has a high  DO (plant  or
pond effluent), the initial JO of the mixture may  be determined
as fellows.
Example:'  BOD Bottle Volume
          Sample Volume
          Sample DO
          DO of Dilution Water

          DC of Sample and Dilution
          After 5-Day Incubation
                       300 ml

                       60 ml

                       2.0 ii'g/1

                       8.0 mg/1

                       4.0 mg/1
                             14-99

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                                                  (DO and BOD
DO of Initial
Mi xture of
Dilution Water
and Sample, mg/1
m] of Sample x DO of Sample + ml of
 Dilution H20 x DO of pilution HzO
         BOD Bottle Volume
BOD, mg/1
60 ml x 2.0 nig/1 + 24Q ml x 8.0 mg/1
               300 ml
                     120 + 1920
                        300


                     6.8 mg/1
                             6.8
                    300/2040.0
                          18CO
                           240.0
                           240.0
DO o£
Diluted
Sample,
mg/1
N
DO After
- 5 Days,
mg/1
J
                                                   ^ Bottle Vol. ,^ ml
                                                  Sample \/ol., ml
                  =  (6.8 mg/1 - 4.0 mg/1)
                        500 ml
                         '60 ml
NOTES
                  =  (2.8),
       200
        60
                  =  14.0 mg/1
1,  On effluent samples where the DO is run on the sample and the
    blue bounces back on the end point titration, this indicates
    nitrite interference and can cause the BOD to be higher than
    actual by as much as 10% to 15% of the answer.  This fact
    should be considered in interpreting your results.  The end
    point also may waver because of decomposition of azide in an
    old reagent or resuspension of sample solids.  To correct a
    wavering end point, try preparing a new alkaline-azide solution
    or more of the old solution should be used because it may be
    decomposing.

2.  Researchers and equipment manufacturers are continually striving
    to develop quicker and easier tests to measure BOD.   If you find
    a test procedure that provides you with an effective operational
    control test, use it.  Be sure to check with your regulatory
    agencies for the procedures they require you to use  in your
    effluent monitoring program.
                           14-100

-------
                           QUESTIONS
8.D  How would you determine the ampunt of organic
     material in wastewater?

8.E  How would you prepare dilutions to measure the
     BOD of cannery waste having an expected BOD of
     2000 mg/1?

8.F  What is the BOD of a sample of wastewater if
     a 2 ml sample in a 300 ml BOD bottle had an
     initial DO of 7.5 mg/1 and a final DP of 3.9 mg/1?

8.G  Why should samples for the BOD be collected
     before chlorination?

8.H  Why should opened bottles of "Thio" be used or
     restandard!zed within two weeks?
                 END OF LESSON 4 OF 8 LESSONS

                               on

              Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 5.
                             14-101

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                DISCUSSION AND REVIEW QUESTIONS

                    (Lesson 4 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry



Name                                            Date
Write the answers to these questions in your notebook.   The problem
numbering continues from Lesson 3.
11.  What is the fbrmula'for calculating the percent
     saturation of DO?

12.  What precautions should be exercised when using
     a DO probe?

13.  What is a blank, as referred to in laboratory
     procedures?  .

14.  What are some of the disadvantages of the BOD test?

15.  What precautions should be taken when running a
     BOD test?

16.  Calculate the BOD of a 5 ml sample if the initial
     DO of the diluted sample was 7.5 mg/1 and the DO
     of diluted sample after 5-day incubation was 3.0 mg/1?
                           14-102

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       CHAPTER 14.  LABORATORY PROCEDURES AND CHEMISTRY

                   (Lesson 5 of 8 Lessons)
9,.  Hydrogen Sulfide (H2S)
                       I.  IN ATMOSPHERE


A.  Discussion

The rate of concrete corrosion is often directly related to the rate
of H2S production or amount of H2S in the atmosphere.  This test deals
with the time it takes a paper tape or unglazed tile to turn black.
It is a qualitative measurement of the H2S present in the sewer atmos-
phere.  H2S is recognized by its characteristic odor of rotten eggs.


B.  What is Tested?

           Samle                              Common
    Atmosphere in sewers, out-         Not black in     ,,   ,  0.  ,
    •, ^  ,-    c       .       ,          -, , ,           =  Good. 24+ hr
    lets from force mains, wet         24 hours
    pits, pumping stations, and
    influent areas to treatment        Black in less    R  ,    , ,
    plants.                            than 1 hour          '
C,  Apparatus

    Lead acetate paper or unglazed tile soaked in lead acetate.


D,  Reagents

    Saturated lead acetate solution.


E.  Procedure

1.  Obtain pieces of unglazed tile or use lead acetate paper.  Cut
    tile with hacksaw into % inch strips.

2,  Soak strips in tile in lead acetate solution.
                            14-103

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                                                           LH2S)
3.  Dry tile in drying oven or air dry.
4.  An open manhole or any point where wastewater is exposed to the
    atmosphere is a good test site.  Drive a nail between metal crown
    ring of manhole, concrete, or other convenient place.  Tie paper
    or tile with cotton string to nail and then replace it and return
    in half an hour or less.  If tile is not black or substantially
    colored, return periodically until black,  If H2S is present as
    indicated by a color change, then measure flow, temperature, pH,
    and BOD for further evaluation of problem.
                    II.  IN WASTEWATER

A,  Piscussion

In sewers, when there is no longer any dissolved oxygen, H2S tests
are run to determine the rate of H2S increase as the wastewater
travels to a pumping station or treatment plant.  If the wastewater
is exposed to the atmosphere, H2S will be released and a typical
rotten egg odor will be detected.  Anaerobic bacteria found in
wastewater can liberate H2S from the solids.  When the gas leaves
the wastewater stream and comes in contact with moisture and
oxygen, sulfuric acid is formed which is very corrosive to concrete.
Not all odors in wastewater are from H2S, and there is no correlation
between H2S and other odors.  The total H2S procedure is good up to
18 mg/1, and higher concentrations must be diluted before testing.
H2S production can be controlled by up-sewer aeration which reduces
US formation and also stabilizes the wastewater in the collection
svstem.
                             14-104

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                                                 (H2S)


B.  What is Tested?

    Sample Wastewater From               Possible Results, mg/1
    the Following Locations              GocKl'   ~        "    Bad

    Sewers                                ,1                 1

    Outlets from force mains              .1                 1

    Wet pits, pumping stations            .1                .5

    Influents to treatment plants    Preferably 0           .5

    All of the above locations should be sampled, if pertinent, when
    using up-stream aeration to control H2S.


C.  Apparatus

1.  One LaMotte-Pomeroy Sulfide Testing Kit to test:

    a.  Total Sulfides

    b.  Dissolved Sulfides

    c.  Hydrogen Sulfide in solution

    Obtain from LaMotte Chemical Products Company.  Order by Code
    #4630, $27.50, FOB, Chestertown, Maryland  21620.

2.  One LaMotte-Pomeroy Accessory Hydrogen Sulfide Kit for testing
    H2S in air and gases (not essential).  Obtain from LaMotte
    Chemical Products Company.  Order by Code #4632, $22.00, FOB,
    Chestertown, Maryland  21620.


D.  Reagents

    The instructions are in the kit.


E,  Procedure
    The instructions are in the kit.

Note:  No EPA evaluation of test kit was available at time this
       manual was prepared.
                           14-105

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                                                           (H2S)
F.  Example



    The instructions are in the kit.






G.  Calculations
    The instructions are in the kit.
                          QUESTION






   9.A  Why would you measure the H2S concentration:




             1.  In wastewater?




             2.  In the atmosphere?
                           14-106

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10.  p_H


A.  Discussion

The intensity of the alkaline or acid strength of water is
expressed by its pH.

Mathematically, pH is the logarithm of the reciprocal of the
hydrogen ion concentration, or the negative logarithm of the
hydrogen ion concentration.

                                     1              .
                        pH  =  log TTTTV  =  -log (FT)
For Example

If a wastewater has a pH of 1, then the hydrogen ion concentration
(H+) = 10'1 = 0.1.

If pH = 7, then (H+) = 1Q-7 = 0.0000001.


                          pH Scale
0 increasing acidity -- 7 -- increasing alkalinity 1
1 +- 2 «-
• 3 +- 4 •*- 5 •*- 6 x\ 8 •> 9 -
Neutral
6 through 8
*• 10 ->• 11 -> 12 -> 13
In a solution, both hydrogen ions (H+) and the hydroxyl ions  (OH")
are always present.  At a pH of 7, the concentration of both byiU-oqon
and hydroxyl ions equals 10~7 moles per liter.  When the pH is  loss
than 7, the concentration of hydrogen ions is greater tli.T; the hyiroxyl
ions.  The hydroxyl ion concentration is greater than the hydrogen  ions
in solutions with a pH greater than 7.

The pH test indicates whether a treatment process may continue  to
function properly at the pH measured.  Each process in the plant has
its own favorable range of pH which must be checked routinely,
Generally a pH value from 6 to 8 is acceptable for best organism
activity.
                           14-107

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                                                             fpH)
The paper tape colorimetric comparison method is explained in
this section.  This is not considered a "Standard Method" but   j
will give a rough indication of the pH.  Most wastewater contains
many dissolved solids and buffers which tend to minimize pH changes,

There are many ranges of pH tapes available.  Normally a range of
5 to 8 will cover the inplant control testing.
B.  What is Tested?
              Wasewater                     Common
    Influent or Raw Wastewater  (domestic)     6.8 to 8.0

    Raw Sludge (domestic)                     5.6 to 7.0

    Digester Recirculated Sludge or
      Supernatant                             6.8 to 7.2

    Plant Effluent Depending on
      Type of Treatment                       6.0 to 8.0



C.  Minimum Apparatus List

    1.  pH Meter.

 or 2.  Three rolls of paper tapes (range 5 to 8) .

 or 3.  Colorimetric set (range 6.8 to 8.4) — permanent glass
        can be used with chlorine comparator or liquid color tubes
        that are less stable.


D.  Reagents

    (to be used with corresponding apparatus listed under Section C)

1.  Buffer tablets of various pH values.   Distilled water.

2 .  None .

3,  Brom thyml blue (for pH 6.2 to 7.6).
    Phenol red (for 6,4 to 8.0).
                           14-108

-------
                                                              (pi I)


E,  Procedures

    Use the same samples used for the other tests.


                    METHOD A (pH Meter)

Procedure         '
1.  Due to the differences between the various makes and models
    of pH meters commercially available, specific instructions
    cannot be provided for the correct operation of all instru-
    ments.  In each case, follow the manufacturer's instructions
    for preparing the electrodes and operating the instrument.

2.  Standardize the instrument against a buffer solution with a
    pH approaching that of the sample.

3.  Rinse electrodes thoroughly with distilled water after re-
    moval from buffer solution.

4.  Place electrodes in sample and measure pH.

5.  Remove electrodes from sample, rinse thoroughly with dis-
    tilled water.

6.  Immerse electrode ends in beaker of pH 7 buffer solution.

7.  Shut off meter.
Precautions

1,  To avoid faulty instrument calibration, prepare fresh buffer
    solutions as needed, once per week, from commercially avail-
    able buffer tablets.

2.  pH meter, buffer solution, and samples should all be at the
    same temperature (constant) because temperature variations
    will give erroneous results,

3.  Watch for erratic results arising from electrodes, faulty
    connections, or fouling of electrodes with oily or precipitated
    matter."
                            14-109

-------
                                                  (pH)


                     METHOD B (Paper Tape)
Procedure
1.  Measure pH directly in tank or immediately after collecting
    sample.

2.  Tear off tape lh to 2" long.  Dip half of tape in tank or
    sample and quickly read results.                             '

3.  Remove tape and compare color with colors on package, and
    record pH on Laboratory Work Sheet in proper column from
    which" the sample came.  For example, if the sample came
    from the plant influent and the color of the portion of the
    tape wetted by the sample matches a color on the package
    indicating a pH of 7.2, then record 7.2 on Laboratory Work
    Sheet in the influent column on the pH row.  (See Fig. 14.2
    second page of work sheet).

This procedure applies to liquids that have solids which separate
(settle or float) easily.
       METHOD B (Paper Tape-High Solids Cone, in Sample)
Procedure

The following procedure is for samples containing higher solid
concentrations such as found in the raw sludge, digester recircu-
lated sludge, digester supernatant, and digested sludge samples,

1.  Obtain representative samples and identify them.

2.  Allow samples to stand until some of the solids have settled
    and water is visible above the solids.  Sufficient water
    should be above the solids to allow the tape to be dipped in
    the sample and not discolored by the solids.

3.  Bend the tape by making a sharp crease ^ from end.  Very carp-
    fully allow tape to touch liquid surface.

    End of                   4.  Remove tape from liquid surface
    bent           I	         and compare the color with pH
                ~»~ I—
tape
color standard on the package.
Record on Laboratory Work Sheet,
                          14-110

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                                                   (pH)


              METHOD C (Colorimetric Comparitor)


Procedure                              •                         :
                                                                i
1.  Fill the three tubes or two rectangular bottles provided with
   -the comparitor unit to the indicator line with a portion of
    the sample being tested.

2.  Add the recommended amount of indicator solution.

3.  Place the tubes in the comparitor in such a way that the color
    standards are opposite the tubes not containing the indicator
    s olut i on.

4.  Compare the colors by rotating the comparitor disk or changing
    the standard color solution vials.  Read the pH of the indi-
    cator having the color closest to the color of the sample.
    Record results on Laboratory Work Sheet.

5.  Thoroughly wash and dry sample tubes when test is completed
    and before returning tubes to comparitor unit for storage.


F.  Precautions
                                                           a
1.  Collect fresh samples and test immediately.  The pH of „
    sample can change rapidly due to loss of C0~ .and biological
    activity.  A fresh effluent sample could have a pH of 6.5
    and after standing overnight the pH could be 8,0.

2.  Always measure aerator pH directly in the aerator.

3.  The pH of a composite sample will not accurately describe pii
    conditions in your plant.  A ten-minute slug of a highly acid
    waste can upset plant performance for a day or longer, but
    you may not notice it in a composite sample.  Measure pH in
    place, frequently and quickly, for best description of
    environment encountered' by'"brgahisms' in' treatment' processes .
                            14-111

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                           QUESTIONS
10.A  How would you measure the pH by the  paper tape
      colorimetric comparison method for:

      1.   Plant influent?

      2.   Raw sludge?
10.B  What precautions should be exercised when using
      a pH meter?
                          14-112

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11.  Settleability of Activated Sludge Solids
                       I.  SETTLEABILITY
A,  Discussion

This test is run on mixed liquor or return sludge and plotted on
attached graph (Fig. 14.5).   All pertinent information is filled
in for process control of aerators.
       -p
        o
       _Q
        a
        Q)
            0
               0
60
                          10    15    20

                           Time, minutes

           14.5  Settleability of activated sludge solids

Settleability is important in determining the ability of the solids
to separate from the liquid in the final clarifier.  The activated
sludge solids should be returned to the aeration tank, and the
quality of the effluent is dependent upon the absence of solids
flowing over the effluent weir.

The suspended solids should be run on the same sample of mixed
liquor that the Settleability test is run.  This will allow.you Lo
calculate the Sludge Volume Index (SVI)  or the Sludge Density Index
(SDI)  which are explained in other sections.
                           14-113

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                                                               (Settleability)
            The 2000 ml graduate that is filled with mixed  liquor  in  the
            settleability test is supposed to indicate what will happen to
            the mixed liquor in the final clarifier--the  rate  of sludge
            settling, turbidity, color, and volume of sludge at the end of
            60 minutes.
            B.  What is Tested?

                  Sample

                Mixed Liquor or
                Return Sludge
   Working Range

Depends on desirable mixed
liquor concentration
            C.  Apparatus

                2000 ml graduated cylinder.10
            D.  Reagents

                None.
            E.  Procedure
  --Mix sample and pour
   into 2000 ml graduate.
Sample
                               2.   Record settleable solids, %, at 5-minute intervals,
                                   950-
                                 -I   l
                                            730-
                                           -1   L.
                                                     550 -
                      Time,
                      min.     0
     J  C
                                                              510-

•'. •'
$%fc2
'W%#.
u'S ; 'A?': ;M
^'s&N;
/
4
                                                                                 470-
 10
15
20
30
60
            10 Mailory Direct Reading Settleometer (a 2 liter graduated cylinder
               approximately 5 inches in diameter and 7 inches high).   Obtain from
               Scientific Glass Apparatus Co., Inc., 735 Broad Street, Bloomfield,
               New Jersey.   Catalog No.  JS-1035.   Price $16.50 each.
                                        14-114

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                                                  (Settieabllj-ty)
1.  Collect a sample of mixed liquor or return sludge,

2.  Carefully mix sample and pour into 2000 ml graduate.  Vigorous
    shaking or mixing tends to break up floe and produces slower
    settling or poorer separation.                         '

3,  Record settleable solids, %, at regular intervals.     ;
F.  Example and Calculation                                J

The percent settling rate can be compared for the various days of
the week and with other measurements—suspended solids, SVI, per-
cent sludge solids returned, aeration rate, and plant inflow.  A
very slow settling mixed liquor usually requires air and solids
adjustment to encourage increased stabilization during aeration.
A very rapidly settling mixed liquor usually gives poor effluent
clarification.
                II.  SLUDGE VOLUME INDEX (SVI)


A.  Discussion

The Sludge Volume Index (SVI) is used to indicate the condition
of sludge (aeration solids or suspended solids) for settle-
ability in a secondary or final clarifier.   The SVI is the volume
in ml occupied by one gram of mixed liquor suspended solids""a*fTer"
30 minutes of settling.  It is a useful test to indicate changes
in sludge characteristics.  The proper SVI  range for your plant
is determined at the time your final effluent is in the best con-
dition regarding solids and BOD removals and clarity.


B.  What_is Tested?

       Sample                        Preferable Range, SVI

    Aerator Solids or                    ..      ~
    Suspended Solids
                           14-115

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C.  Apparatus



    See 11.  Settleability of Activated Sludge Solids, Part I,

    Settleability, and 16.  Suspended Solids,
D.  Reagents



    None.                   ,                                      I





E.  Procedure



    See Section 11, I, on Settleability, and 16, Suspended Solids^





F.  Example



    30-minute settleable solids test = 360 ml or 18%.



    Mixed liquor suspended solids = 1500 mg/1.             -       :





G.  Calculations
    Sludge Volume  _     % Settleable Solids x 10^.^

    Index, SVI        Mixed Liquor Suspended' Sol id's / ing/~i


                      18 x 10,000


                   "
                   -  18Q°                    •

                   "   15                      1
                   =  120
                           14-116

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                                                   (Settleability -  SDI)


                III.   SLUDGE  DENSITY INDEX (SDI)


 A.   Discussion

 The Sludge Density Index (SDI)  is  used in a way  similar  to  the  SVI
 to  indicate the  settleability  of a sludge in a secondary clarifie?  or
 effluent.   The calculation of  the  SDI  requires the  same  information
 as  the SVI test.


       SDI   =  mg/l °£ suspended solids in mixed  liquor
               ml/1 of settled"  mixed liqupr solids x ID

        or

       SDI   =  100/SVI


 B.   What is  Tested?

        Sample                     Preferable  Range, SDI

     Aerator Solids or
     Suspended  Solids                   0.4-1.0


 C.   through  G.

 These  items  are not included because of their  similarity  to the
 SVI  test.

                           QUESTIONS

     11.A   Why  should you run settleability tests on mixed liquor?

     11.B   What is  the Sludge Volume Index  (SVI)?

     11.C   Why  is the SVI test run?

     11.D   What is  the relationship between the Sludge Density
           Index (SDI)  and SVI?



                 END OF LESSON 5 OF 8  LESSONS

                              on

               Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 6.

                         14-117

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                DISCUSSION AND REVIEW QUESTIONS

                    (Lesson 5 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry



Name                                            Date
Write the answers to these questions in your notebook.  The problem
numbering continues from Lesson 4.
17.  Hydrogen sulfide is measured because it causes
18.  What factors promote H2S production in sewers?

19.  The pH scale runs from 	to 	, with 7 being neutral.

20.  Calculate the SVI if the mixed liquor suspended solids are
     2000 mg/1 and the 30-minute settleable solids test is 500 ml
     or 25%.

21.  Calculate the SDI if the SVI is 125.
                           14-118

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       CHAPTER 14.   LABORATORY PROCEDURES AND CHEMISTRY

                   (Lesson 6 of 8 Lessons)


12.  Settleable Solids


A.  Discussion

The settleable solids test is the volume of settleable solids  in
one liter of sample that will settle to the bottom of an Imhoff
cone during a specific time period.   The test is an indication of
the volume of solids removed by sedimentation in sedimentation
tanks, clarifiers,  or ponds.  The results are read directly in
milliliters from the Imhoff cone.


B.  What is Tested?

       Sample                         Common Ranges Found

                                   12 ml/1 medium wastewater
    Influent                       20 ml/1 strong wastewater
                                    8 ml/1 weak wastewater

    Primary Effluent              0,1 ml/1 - 3 ml/1

    Secondary Effluent            Trace —0.5 ml/1
                                  Over .5 ml/1 poor


C.  Apparatus

1.  Imhoff Cones.

2,  Rack for holding Imhoff Cones.

3.  Glass stirring rod, or wire.
                           14-119

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   D.  Outline of Procedure
                                                (Settleable Solids)
Mix well and
pour 1 liter
into Irnhoff
Cone.
                    Settle
                  45 Minutes
                                     Gently Stir
                                        Sides
                                                                   1 Liter
                                                                 Read-
                                                                 Sludge
                                                                 Volume
                                  Settle
                                15 Minuses
   1.


   2.

   3.

   4.


   5.
                     PROCEDURE

Thoroughly mix the wastewater sample by shaking and immediately
fill an Imhoff cone to the liter mark.
Record the time of day that the cone was filled.  T =

Allow the waste sample to settle for 45 minutes.
Gently spin the cone to facilitate settling of material adhering
to the side of the cone.

After one hour, record the number of milliliters of settleable
solids in the Imhoff cone.  Make allowance for voids among the
settled material.
                               14-120

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                                               (Settleable Solids)
6.  Record the settleable solids as ml/1 or milliliters per
    liter.
    Settleable Solids, Influent  =  ______ ml/1
    Settleable Solids, Effluent  =	 ml/1

    Settleable Solids, Removal   =          ml/1
E.  ExajnpJLe

Samples were collected from the influent and effluent of a primary
clarifier,  After one hour, the following results were recorded:

                              Sette able
            Influent    '               12.0
            Effluent                    0,2
F.  Calculations
1.  Calculate the efficiency or percent removal of the  above primary
    clarifier in removing settleable solids.

% Removal   _  (Inf 1. Set Sol , ml/1 - Ef f 1. Set^ Sol, ml/1)  x  ]OQ%
of Set Sol  ~            Influent Set Sol, ml/1


               12 ml/1 - 0.2 ml/1   Inn0             -12,0
            =   - » • • « ..—— -—- «-. .»,..»^..«.      '
                    12 ml /I
                                      ____
            =  liil x 100%         12 / 11,8
                12                     10^3
                                         1 00

            *  98%                      -_£
                                           40


2.  Estimate the gallons per day of sludge pumped  to  a  digester
    from the above primary clarifier  if the flow is 1 MGD (1  million
    gallons per day) .  In your plant, the Imhoff cone may not
    measure or indicate the exact performance of your clarifier
                            14-121

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                                                  (Settleable Solids)


or sedimentation tank, but with some experience you should
be able to relate or compare your lab tests with actual
performance.

Sludge Removed by Clarifier, ml/1

     =  Influent Set Sol, ml/1 - Effluent Set Sol, ml/1

     =  12 ml/1 - 0.2 ml/1

     =  11.8 ml/1

To estimate the gpd (gallons per day) of sludge pumped to a
digester, use the following formula:

Sludge to Digester, gpd

     =  Total Set Sol Removed, ml/1 x 1000 x Flow, MGD

     -  n °  mA  Y 1000 mg „ 1 M gal
     —  i, 1 . O . .    A   L -.,    A    ,
             M mg     ml        day

     =  11,800 gpd

This value may be reduced by 30 to 75% due to compaction of
the sludge in the clarifier.

If you figure sludge removed as a percentage (1.18%), the sludge pumped
to the digester would be calculated as follows:

                                 Sludge to Digester, gpd
                                    Flow of 1,0(30 ,'OoTTgpct

     „, ,      „.            ,     1.18% x 1,000,000 gpd
     Sludge to Digester, gpd  =         -   - *    w.
                              =  11,800 gpd
G.  Clinical Centrifuge
Settleable solids also may be measured by a small clinical centri-
fuge.  A mixed sample is placed in 15 ml graduate API tubes and
spun for 15 minutes.  The solid deposition in the tip of the tube
is related to plant performance for plant control.  A centrifuge
also is used in Section 16, Suspended Solids, II, Centrifuge.

                           QUESTION
                 «t
12. A  Estimate the volume of solids pumped to a digester
      in gallons per day (gpd)  if the flow is 1 MGD, the
      influent settleable solids is 10 ml/1, and the eff-
      luent settleable solids is 0.4 ml/1 for a primary
      clarifier.

                          14-122

-------
 13,  Sliulpe Are
A.  Discussion

Sludge age Is a control guide that is widely used and  is  a  rough
indicator of the length of time a pound of solids is rraintai ncd
nudcr aeration in the system.  The basis for calculating  the  sludge
age is weight of suspended solids in the mixed  liquor  in  the  aeration
tank divided by weight of suspended solids added per day  to  the
aerator.

                Suspended Solids in Mixed Liquor, mg/1
Sludge Age,  =   x._ Aerat or Vojume^ in_ MG^ x_ _8Jj4_ Ibs / g a 1
d:iys                SS in Primary Effluent, mg/1*
                   x Daily Flow, HGD x 8.34 Ibs/gal

Any significant additional loading placed on the aerator  by  the
digester supernatant liquor must be added to the above  loadings by
considering the additional flow (MGD)  and concentration (mg/1).
The selection of the method of determining sludge age  is  discussed
in Chapter 7, Activated Sludge.
                                       Common Range, ii'g/1

    Suspended solids in aerator        Depends on p
    and BOD or suspended solids
    in primary effluent

    Sludge age                         Conventional
                                       2.5-6 days,
* NOTE:  Sludge age is calculated by three different meUiods:

     1.  Suspended solids in primary effluent, mg/1

     2.  Suspended solids removed from primary effluent.s mg/1, cr
         primary effluent, suspended solids, ing/1 - final effluent
         suspended solids, mg/1

     3.  BOD or COD in primary effluent, mg/1
                            14-123

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                                                     (Sludge  Age)
C.  Apparatus

    See 16, Suspended Solids Test.


D.  Reagents

    None.


E.  Procedure
    See 16, Suspended Solids Test.


F.  Example

    Suspended Solids in Mixed Liquor  =  1500 mg/1

    Aeration Tank Volume              =  0.50 MG

    Suspended Solids in Primary Effl. =  100 mg/1

    Daily Flow                        =2.0 MGD


G.  Calculations
                    Susp. Solids in Mixed Liquor, mg/1
    Sludge Age,  _  x Aerator Vol., MGx_ 8.34 Ibs/gal
    days            Susp. Solids in Primary Effl., mg/1
                        x Flow, MGD x 8.34 Ibs/gal


                 -    Mixed Liquor Susp. Solids,  Ibs
                    Primary Effluent SS, fbT/day


                    1500 mg/1 x 0.50 MG x 8.54 Ibs/gal
                     100 mg/1 x 2.0 MGD x 8.34 Ibs/gal


                    1500 x 0.50
                     100 x 2.0


                    7.5
                    2.0


                 =   3.75 days
                           14-124

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                           QUESTION
13.A  Determine the sludge age in an activated sludge process
      if the volume of the aeration tank is 200,000 gallons
      and the suspended solids in the mixed liquor equals
      2000 mg/1.  The primary effluent SS is 115 mg/1, and
      the average daily flow is 1.8 MGD.
                            14-425

-------
 14.   Sludge  (Digested)  Dewatering  Characteristics
 A.   Discussion
     The  dewatering  characteristics  of  digested  sludge  are  very
     important.  The better  the  dewatering  characteristics  or
     drainability  of the  sludge,  the quicker  it  will  dry  and the
     less  area will  be  required  for  sludge  drying  beds.
 B,  What  is Tested?

                                       PREFERRED  RANGE
       Sample                   .   Method  A        Method  B

    Digested Sludge                Depends  on      100-200  ml
                                   appearance
C.

    METHOD A

    1000 ml graduated cylinder.

    METHOD B

1.  lir.hoff cone with tip removed,

2,  Sand from drying bed.

3.  500 ml beaker.


D.  Reagents

    None.


E.  Procedure

Two methods are presented in this section.  Method A relies on a
visual observation and is quick and simple.  The only problem is that
operators on different shifts might record the same sludge draining
characteristics differently.  Method B requires 24 hours, but the
results are recorded by measuring the vplume of liquid that passed
through the sand.   Method B would be indicative of what would happen
if you had sand drying beds.
                            14-126

-------
                                                         (Sludge Dewatering)
                                  METHOD A
1.   Add digested  sludge
    to 1000  ml  graduate.
   Sample
 Container
Pour sample from graduate
back into container.
3.   Watch solids
    adhere to
    cylinder wall;
                                                                     ^p
                                                                     S&KI
                                                                    aJ&^feAUv
        1.   Add  sample  of digested sludge to 1000 ml graduate.

        2,   Pour sample back into sample container.  Set graduated
            cylinder  down.

        3.   Watch graduate.  If solids adhere to cylinder wall and
            water leaves solids in form of rivulets, this is a
            good dewatering sludge on a sand drying bed  (Fig, 14.6).
                                                Fig. 14.6

                                                Sludge on graduated
                                                cylinder walls for
                                                sludge dewatering
                                                test

-------
                                                         ^Sludge Uewatering)
                                  METHOD B
       1.  Pour digested sludge  2.  Place beaker under  3.  Measure liquid
           on top of sand in         tip and wait 24         that has passed
           Imhoff cone.              hours.                  through the sand.
Broken Tip
       1.


       2.

       3.

       4,

       5.
Broken glass Imhoff cone that has tip removed and a glass  wool
plug in the end to hold the sand in the cone.

Fall halfway with sand from sand drying bed.

Fill remainder to 1 liter with digested sludge.

Place 500 ml beaker under cone tip and wait 24 hours.

Record liquid that has passed through sand in ml.  If
less than 100 ml has passed through sand,  you have poor
sludge drainability.
                                   QUESTION

       14.A  What are the differences in the use of (1)  a graduated
             cylinder and (2)  an Imhoff cone, filled with sand,  that
             has a broken tip, to neasure the dewaterJng characteristics
             of digested sludge?
                                  14-128

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15.  Supernatcnt. Graduate evaluation
A.  Discussion

The digester supernatant solids test measures the percent of
settleable solids being returned to the plant heaclworks.   The
settleable solids falling to the bottom of a graduate should
not exceed the bottom 5no of the graduate in most secondary
plants.  When this happens, you are imposing a load on the
primary settling tanks that they were not designed to handle.
If the solids exceed 5% you should run a suspended solids
Gooch crucible test (Section 16) on the sample and calculate
the recycle load on the plant that is originating from the
digester.
B.  What is Tested?

      Sample                         Common Values

    Supernatant                  % Solids should be <5°



C.  Apparatus

    100 ml graduated cylinder.



D.  Reagents

    None.
                           14-129

-------
  F;.  Procedure
    Fill 100 ml graduate
    with supernatant.
Supernatant
  Sample
          100 ml Graduate
                                                     (Supernatant)
                                     2.   After 60  minutes,
                                         read ml  of solids
                                         at  bottom.
                                                        JL
                                                           10 ml
  1.

  2.


  3.
Fill a 100 ml graduated cylinder with supernatant sample.

After 60 minutes, read the ml of solids that have settled
to the bottom.

Calculate supernatant solids, %.

Supernatant Solids, %  -  ml of Solids
  F.   Ivx a mp 1 e

      Solids on bottom of cylinder, 10 ml.



  G.   CaIculations

      Supernatant Solids, %  =  ml of Solids

                             =  10 ml

                             =  10% Solids (High)  by Volume
                              14-130

-------
                           QUESTION
15.A  Why should the results of the supernatant solids test
      be less than 5% solids?
                 END OF LESSON 6 OF 8 LESSONS

                              on

             Laboratory Procedures and Chemistry
Work the next portion of discussion and review questions before
continuing with Lesson 7.
                             14-131

-------
                DISCUSSION AND REVIEW QUESTIONS

                   (Lesson 6 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry



Name                                          Date
Write the answers to these questions in your notebook.   The
problem numbering continues from Lesson 5.
22.  Calculate the efficiency or percent removal of a
     primary clarifier when the influent settleable
     solids are 10 ml/1 and the effluent settleable
     solids are 0.3 ml/1.

23.  Why does the actual volume of sludge pumped from
     a clarifier not agree exactly with calculations
     based on the settleable solids test?                                  '

24.  What does sludge age measure?

25.  Why should the dewatering characteristics of
     digested sludge be measured?

26.  What happens to the plant when the supernatant
     from the digester is  high in solids?
                            14-132

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           Ji 14.  LABORATORY PROCEDURES AND CHEMISTRY

                 (Lesson 7 of 8 Lessons)



16'.  Suspended Solids


                     I.  GOOCH CRUCIBLE

A.  Discussion

One of the tests run on w%astewater is to determine the amount of
material suspended within the sample.  The result obtained from
the suspended solids test does not mean that all of the suspended
solids settle out in the primary clarifier or, for that matter, in
the final clarifier.  Some of the particles are of such size and
weight that they will not settle without additional treatment.
Therefore, suspended solids are a combination of settleable solids
and those solids that remain in suspension.


B.  What is Tested?

      SampJLe                      Common r Ran_ges,^ mg/JL

    Influent               .     Weak 150  -  400+ Strong

    Primary Effluent            Weak  60-  -  150+ Strong

    Secondary Effluent          10 Good   -  60+ Bad

    Activated Sludge Tests      Depending on Type of Process

    Mixed Liquor                1000  -   < 5,000

    Return or Waste Sludge      2000  -  < 12,000

    Digester Tests:

    Supernatant                 3000  -  < 10,000

    When supernatant suspended solids are greater than 10,000 mg/1,
    the total solids test is usually performed.
                            14-133

-------
                                       (Suspended Solids  - Gooch)
1.  2.4 cm glass fiber filter.




2.  No. 4 Gooch crucible.




3.  Distilled water.




4.  Filter flask.




5.  Graduated cylinder.




6.  Vacuum pump or aspirator.




7.  Oven.




8.  Analytical balance.
0.  Outline of Procedure




The procedure is outlined on Page 14-157.




(Method with Gooch Crucible and Glass Fiber Filter)
                           14-134

-------
                                              (Suspended Solids  - Gooch)
                   'iltering Flask

                  Seat filter, by add-
                  ing distilled water
                  and applying vacuum.
                                                n  n   n
                                                                 O
     5.   Dry crucibles in oven
         at 103CC.
           4.  Cool
                                       5,  Wei^h crucible.
Pour
measured
volume of
sample in
Gooch
crucible.
 7.  Filter out suspended
     solids with vacuum.

 8.  Wash graduate, crucible,
     and filter with distilled
     water to complete  solids
     transfer.


n n n


a
O
[y \ vj
                                                          Drv crucibles  plus
                                                          suspended solids
                                                          at  103CC,
                      11.   iVeigh  crucible
                           plus suspended
                           solids.
nrm
                                  10.   Cool.
                                    14-135

-------
                                             (Suspended Solids  - Gooch)


t.   Preparation of Gooch Crucible

1.   Put a No. 4 Gooch  crucible into  filtering apparatus.

2.   Insert 2.4 cm glass fiber filter and center it.

3.   Apply suction.

4.   Wash filter with 100 ml of distilled water to seat well.

5.   Dry at 103°C for one hour.

6,   If volatile suspended solids are to be determined, ignite
     crucible in muffle furnace for one hour  at 550°C.

7,   Cool in desiccator.

8.   Weigh and record tare weight.


1;.   How to Perform the Test

1.   Depending on the suspended solids content, measure out a
     25, 50, or 100 ml portion of a well mixed sample into a
     graduated cylinder.  Use 25 ml if sample filters slowly.
     Use larger volumes of sample if  samples  filter easily,
     such as secondary effluent.  Try to limit filtration time
     to about 15 minutes or less.

2.  Wet prepared Gooch crucible with distilled water and apply
     suction.

3.   Filter sample through the Gooch crucible.

4.  Wash out dissolved solids on the filter with about 20 ml
     of distilled water.  (Use two 10 ml porLions.)

5.  Dry crucible at 103°C for one hour or other specified time.
    Some samples may require up to three hours to dry if the
    residue is thick.

6.  Cool crucible in desiccator for 20-30 minutes.

7.  Weigh  and record weight.

8.  Total  Weight   = 	 g

    Tare Weight     = 	 g

    Solids  Weight   =          g
                         14-136

-------
                                      {.Suspended Solids - Gooch)
    Precautions
1.  Check and regulate the oven temperature at 103° - 105°C.

2.  Observe crucible and glass fiber for any possible leaks.  A
    leak will cause solids to pass through and give low results.
    The glass fiber filter may become unseated and leaky when the
    crucible is placed on the filter flask.  The filter should be
    reseated by adding distilled water to the filter in the crucible
    and applying vacuum before filtering the sample.

3. ' Mix the sample thoroughly so that it is completely uniform in
    suspended solids when measured into a graduated cylinder before
    sample can settle out.  This is especially true of samples heavy
    in suspended s'o'lTdsV'such as raw wastewater and mixed liquor in
    activated sludge which settle rapidly.  The test can be no better
    than the mix.

4.  It is a good practice to prepare a number of extra Cooch crucibles
    for additional tests if the need arises.  If a test result appears
    faulty or questionable, the test should be repeated. Check filtration
    rate and clarity of water passing through the filter.

II.  Example and Calculations

This section is provided to show you the detailed calculations.  After
some practice, most operators use the lab work sheet as shown at the
end of the calculation-s.

           CALCULATIONS FOR SUSPENDED SOLIDS TEST
       (or use lab work sheet at end of calculations)

F.xamplc:  Assume the following data.

    Volume of sample  =  50 ml.
                                      Recorded Heights
    Crucible weight                      21.6329 g
    Crucible plus dry solids             21.6531 g

    Crucible plus ash11                  21.6360 g
11 Obtained by placing the crucible plus dry solids in  a muffle
   furnace at 550°C for one hour.  The crucible plus remaining
   ash are cooled and weighed.
                              14-137

-------
                                        (Suspended Solids - Gooch)
1.  Compute total suspended solids.
      21.6531 g          Weight of Crucible plus Dry Solids, grams
    ~ 21.6529 g        - Weight of Crucible, grams _,....,..,	
    =  0.0202 g        = Weight of Dry Solids, grams

      or

    = 20.2 mg

    1000 milligrams (mg)  =  1 gram (g)

      or

    20.2 mg  =  0.0202 g
    Total
    Suspended  _  Weight of Dry Solids.max 1000
    Solids,                Sample Volume, ml
    mg/1

                  __ _      1000 ml/1              404.
               =  20.2 mg x 	-—           /	—
                              50 ml          507 20200.
                                                 200
                                                   200
               =  404 mg/1                         200
2.  Compute volatile or organic suspended solids.

      21.6531 g          Weight of Crucible plus Dry Solids, g
    - 21.6360 g_        - Weight of Crucible plus Ash, g _ 	

    =  0.0171 g        = Weight of Volatile Solids, g

      or

    = 17.1 me
    Volatile
    Suspended  =  Weight of Volatile Solids^ ^ mg^ _x 1000 ml/I
    Solids,                   Sample Volume, nil
    mg/1

                  17.1 mg x 1000 ml/1         	342_
                         50 ml             507 17100
                                               150
               =  342 mg/1                      21Q
                                                200
                                                 100
                                                 100

                           14-138

-------
                                        (Suspended Solids  -  Gooch)
3.  Compute-the percent volatile solids.

    Volatile   =  (height Volatile, mg) 100%
    Solids, °6     Weight Total Dry Solids, mj
                       mg
                            100%
               =  84.71
                              	.8465
                        20.2 •' 17.10
                              16  16
                                  940
                                  808
                                  1320
                                  1212
                                  1080
                                  1010
4.  Compute fixed or inorganic suspended solids.
      21.6360 g
    - 21.6529 g

    =  0.0031 g
      or
    = 3.1 m
          Weight of Crucible plus Ash,
        - Weight of Crucible, g
        = Weight of Fixed Solids, g
    Fixed
    Suspended
    Solids,
    nig/I
   Weight of Fixed^ Solids, mg x 1000 ml/1
             Sample Volume, ml

   5.1 mg x 1000 ml/1
         50 ml

=  62 mg/1
    To check your work:

    Fixed Susp.  Solids
            Total Susp. Solids, mg/1 - Volatile
                     Susp. Solids, mg/I
                        =  40-4 mg/1 - 342 mg/1

                        =  62 mg/1  (Check)
                                          404
                                         -342
                                           62
                             14-139

-------
                                       (Suspended Solids - C-ooch)


5.  Compute the percent  fixed  solids,

    .,.   , c i-j   o      (Weight  Fixed,  ing)  x 100%
    f'ixed Solids, %  =   — •• P-- — • — • - '— •••••• — — — •— -
                            Weight Total,  mg

                                   100%
                        20.2 mg

                    " =  15 . 3%

The above calculations are also performed  on  a Laboratory Work
Sheet (Fig. 14.7) to illustrate the  use  of the work sheet.
         CALCULATIONS- FOR OVERALL  PLANT  REMOVAL OF
                SUSPENDED SOLIDS IN  PERCENT

Example :  Assume the  following data.

    Influent suspended solids                     202 mg/1

    Primary Effluent  suspended solids             110 mg/1

    Secondary Effluent suspended solids            52 mg/1

    Final Effluent suspended solids                12 mg/1

To calculate the percent removal or  treatment  efficiency for a
particular process or the overall  plant,  use the following formula:
            Removal, %  =  -. ."...     x  100%
                               In
Compute percentage removed between  influent  and  primary effluent
    Removal, %  =  C,1?1. ,". Out) x  10Q%
                       In
                   (202 mg/1 -  110 mg/1)
                         202 mg/1
                =  -   x 100%
                   202                            -110
                                                   92
                =  45,5%
                            14-140

-------
                                         (Suspended'.Solids  -  Gooch)


Compute percentage  removed  between influent and secondary effluent:


    Removal,  %   -   (In  "  Out)  x 100%     	'
                        In

                 -   (202 mg/1  -  52  mg/1)                   202
                 —   	'	"' "" • *' "'• '  • rr' • '  X lUU-o
                          202  mg/1                ,         -52
                                                 '  .   •    150

                    150    100%             *74
                _  	„ x  JLUU-8        /	
                   202            202/  150.00
                                       141.4

                                         8 60
                =  74%                   8 08

                                           52
Compute percentage removed between  influent  and final effluent
(overall plant percentage removed):


    Removal, %  =  dp. .-. Out? x  100%                  '
                       In

                ,  .(202 mg/1 - 12. mg/1)  y lop%
                         202 mg/1


                »  122 x 100%
                   202

                =  94.1% removal  for the  plant  in  suspended solids
   CALCULATIONS FOR POUNDS SUSPENDED SOLIDS  REMOVED  PER DAY

Example:   Assume the following data.

    Influent suspended solids                200  mg/1

    Effluent suspended solids                10 mg/1

    Flow in million gallons/day              2 MGD

    1 gallon of water weighs                 8.34 Ibs
                            14-141

-------
                                       (Suspended Solids - Gooch)
Compute pounds suspended solids removed:
The general formula for computing pounds removed is

    R     d       (Concentration In, mg/1 - Concentration Out, mg/1)
    Ibs/day                  x Flow, MGD x 8.34 Ib/gal
                  (200 mg/1 - 10 mg/1) x 2 MGD x 8.34 Ib/gal
              =  190 x 2 x 8.34

              =  3169 Ibs/day of suspended
                 solids removed by plant
   8.34
    380
    000
  6672
 2502
3169.20
                         DERIVATION

This section is not essential to efficient plant operation, but is
provided to furnish you with a better understanding of the calcu-
lation if you are interested.  For practical purposes,

    1 mg/1  =  1 ppm or 1 part per million

      or    =1 mg/million mg, because 1 liter = 1,000,000 mg

Therefore:

    Ibs  _   mg    M gal   Ibs
    day     M mg    day    gal

         =  Ibs/day
                            14-142

-------
                                                  [Suspended Solids - Gooch)
                                                PLANT
                                                DATE
                                                      CLEAN WATER
                          SUSPENDED SOLIDS § DISSOLVED SOLIDS
  SAMPLE
    Crucible
    Ml Sample
    Wt Dry $ Dish
    Wt Dish
mc/l =
 S
  Wt Dry
     m
                          OOP
             Ml Sample
    Wt Dish § Dry
    Wt Dish § Ash
    Wt Volatile
    vol =
            Wt Dry
                   x 100
                            INFL.
                             #015
                               50
                            21.6531
                            21.6329
                               0.0202
                             404 mg/1
                            21.6531
                            21.6360
                             0.0171
84.73
                                          BOD
                                                      # Blank
  SAMPLE
    DO Sample
    Bottle #
    % Sample
    Blank or adj blank
    DO after incubation
    Depletion, 5 days
    Dep %
  Nitrate N03
  Sample         	
  Graph Reading
                                           Sett. Solids
                                           Sample
                                           Direct Ml/1
COD
Sample
Blank Titration
Sample Titration
Depletion
       DeP x N FAS
                       8000
            Ml Sample
                      Fig. 14.7  Calculation of solids content
                                 on Laboratory Work Sheet
                                        14-143

-------
                                                 (Suspended Solids - Gooch)
                                      TOTAL SOLIDS
SAMPLG
Dish No.
"Wt Dish $ Wet
Wt Dish
Wt Wet
Wt Dish + Dry
Wt Di sh
Wt Dry
, o-lid_ _ Wt Dry x 100%
Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
- V-latilc - Wt V01 X 10°%
Wt Dry
pH
Vol. Acid
alinity as CaC03














































































Grease (Soxlet)
  Sample
  Ml Sample
  Wt Flask + Grease
  Wt Flask
  Wt Grease
         Wt Grease, mg x 1000
              Ml Sample
ing/1
  H2S (Gas)  (Starch-Iodine)
    Blank
    Sample
    Diff
    Diff x .68
    mg/1 x 43.6
                           Ml
                          .Ml
                           Ml
                           mg/1
                           grain/100 cu ft
                   Fig.  14.7  Calculation of solids content on
                              Laboratory Work Sheet (continued)
                                      14-144

-------
                         QUESTIONS


16.A  Why does some of the suspended material in wastewater fail
      to be removed by settling or flotation within one hour?

16.B  Given the following data:

        100 ml of sample

        Crucible weight                 19.3241 g

        Crucible plus dry solids        19.3902 g

        Crucible plus ash               19.3469 g

      Compute:

        a.  Total suspended solids
        b.  Volatile suspended solids
        c.  Percent volatile
        d.  Fixed suspended solids
        e.  Percent fixed

16.C  Compute the percent removal of suspended solids by the
      primary clarifier,  secondary process (removal between
      primary effluent and secondary effluent), and overall
      plant:

        Influent suspended solids  =  221 mg/1

        Primary effluent  SS        =  159 mg/1

        Final effluent SS          =   33 mg/1

16.D  If the data in problem 16.C is from a 1,5 MGD plant,,,
      calculate the pounds of suspended solids removed:

        a.  By the primary unit
        b.  By the secondary unit
        c.  By the overall plant
                            14-145

-------
 16.  Suspended Solids
                        II.  CENTRIFUGE
A.  Discussion

This procedure is frequently used in plants as H. quid-; and easy
method to estimate the suspended solids concentration of the
mixed liquor in the aeration tank instead of the regular suspended
solids test.  Many operators control the solids in their aerator
on the basis of centrifuge readings.  Others prefer to control
solids using Fig. 14.8.  Irs either case, the operator should
periodically compare centrifuge readings with values obtained
from suspended solids tests.  If the solids are in a good settling
condition, a 1% centrifuge solids reading could have a suspended
solids concentration of 1000 mg/1.  However, if the sludge is
feathery, a 1% centrifuge solids reading could have a suspended
solids concentration of 600 ir.g/1.

The centrifuge reading versus mg/1 suspended solids chart (Fig 14.8)
Must be developed for each plant by comparing centrifuge readings
with suspended solids determined by the regular Gooch crucible
method.  The points are plotted and a line of best fit is drawn
as shown in Fig, 14.8.  This line must be periodically checked by
comparing centrifuge readings with regular suspended solids tests
because of the large number of variables influencing the relation-  .
ship, such as characteristics of influent waste, mixing in aerator,
and organisms in aerator.  If you don't have a centrifuge or if
your solids content is over 1500 mg/1, determine suspended solids
by the regular method.
B.  What is^ Tested?

        Sample                              Common Range

    Suspended Solids in Mechani-           800 - 1200 mg/1
    cal Aeration Tanks

    Suspended Solids in Diffused          1000 - 3000 mg/1
    Aeration Tanks
C.  Apparatus

1.  Centrifuge.

2.  Graduated centrifuge tubes, IS ml,




                           14-146

-------
                                 (Suspended Solids - Centrifuge)
I).  Reagents

    None.


B.  Procedure
1.  Collect sample in regular sampling can.

2,  Mix sample well and fill each centrifuge tube to the 15 ml
    line with sample.

3.  Place filled sample tubes in centrifuge holders.

4.  Crank centrifuge at fast speed as you count slowly to 60.
    Be s".re tc count and cruik at the same speed for all tests.
    It is extremely important to peiform each step exactly the
    sane every time.

5.  Remove one tube and read the amount of suspended solids con-
    centrated in the bottom of the tube.  This reading will be
    1/10 of ml.  Results in other tubes should be compared.

6.  Refer to the conversion graph to determine suspended solids
    in mg/1.

NOTF.:   The reason for filling tubes to the 15 ml mark is that  the
       graph (Fig. 14.8) is computed for samples of this size.
'•',  Example

Suspended solids concentration on bottom of centrifuge tube is 0.4 ml.


G.  Calculations

From Fig. 14.8, find 0.4 ml on centrifuge reading side and follow
line horizontally to line on chart.  Drop downward from  line  on  chart
to mg/1 suspended solids and read result of 900 mg/1.

If the suspended solids concentration is above or below  the desired
range, then you should make the proper changes in the pumping rate
of the waste and return sludge.  For details on controlling the  solids
concentration, refer to Chapter 7, Activated Sludge.
                           14-147

-------
CO
          200
400
500          600          700

   Suspended Solids 
                                                                                                                          a.

                                                                                                                          CO
                                                                                                                          o
                                                                                                                          i—'
                                                                                                                          H-
                                                                                                                          a-
                                                                                                                          m
n
(T>

r+
i-i
H-
H5

00
CD

-------
                                        (Suspended Solids - Centrifuge)
    Development of Fig. 14.8
To develop Fig. 14.8 take a sample from the aeration tank and
measure suspended solids and also centrifuge a portion of the
sample to obtain the centrifuge sludge reading in ml pf sludge
at the bottom of the tube.  Obtain other samples of different
solids concentrations to obtain the points on the graph.  Draw
a line of best fit through the points.  Periodically the points
should be checked because the influent characteristics and con-
ditions in the aeration tank change.
                           QUESTION
16.E  What is the advantage of the centrifuge test
      for determining suspended solids in an aeration
      tank in comparison with other methods of measuring
      suspended solids?
                          14-149

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17.  Temperature


                        I.   WASTEWATER

A.  Discussion

This is one of the most frequently taken tests.  One of the many
uses is to calculate the percent saturation of dissolved oxygen
in the DO test.  (Refer to DO Test for procedure.)

Changes of plus or minus 4°F from the average or expected value
should be investigated and the cause corrected if possible.

For example, an influent temperature drop may indicate large
volumes of cold water from infiltration.  An increase in temperature
may indicate hot water discharged by industry is reaching your plant.

A temperature measurement should be taken where samples are
collected for other tests.   This test is always immediately
performed on a grab sample because it changes so rapidly.
Always leave the thermometer in the liquid while reading
the temperature.  Record temperature on suitable work sheet,
including time, location, and sampler's name.


B.  What is Tested?

      Sample                            Common Range

    Influent12                          65°F to 85°F13

    Effluent12           '              60°F to 95°F or
                                        higher from ponds
    Receiving Water12                   60°F to ambient
                                        temperature1 **

    Digester (Recirculated               60°F to 100°F
    Sludge before Heat Ex-
    changer- -Supernatant)
12 If dissolved oxygen (DO)  measurements are performed on any
   samples,  the temperature  should be measured and recorded.
13 Depends  on season,  location,  and temperature of water supply.
14 Ambient  Temperature (AM-bee-ent).   Temperature of the
   surroundings.
                            14-150

-------
                                                   (Temperature)


C.  Apparatus
1.  One NBS (National Bureau of Standards) thermometer for
    calibration of the other thermometers.

2.  One Fahrenheit mercury-filled, 1° subdivided thermometer.

3.  One Celsius (formerly called Centigrade) mercury-filled,
    1° subdivided thermometer.

4.  One metal case to fit each thermometer.
There are three types of thermometers and two scales.

Scales

1.  Fahrenheit, marked °F.

2.  Celsius, marked °C (formerly Centigrade).

Tliermome_ters

1.  Total immersion.  This type of thermometer must be totally
    immersed when read.  This will change most rapidly when
    removed from the liquid to be recorded.

2.  Partial immersion.  This type thermometer will have a solid
    line around the stem below the point where the scale starts.

3.  Dial.  This type,has a dial that can be easily read while
    the thermometer is still immersed.  Dial thermometer readings
    should be checked (calibrated) against the NBS thermometer.
    Some dial thermometers can be recalibrated (adjusted) to read
    the correct temperature of the NBS thermometer.
13.  Reagents

    None.
                          14-151

-------
                                                   (Temperature)
E.  Procedures
Use a large volume of sample, preferably at least a 2-pound coffee
can or equivalent volume.  The temperature will have less chance
to change in a large volume than in a small container.  Collect
sample in container and immediately measure and record temperature.
Do not touch the bottom or sides of the sample container with the
thermometer.  To avoid breaking or damaging glass thermometer,
store it in a shielded metal case.  Check your thermometer accuracy
against the NBS certified thermometer by measuring the temperature
of a sample with both thermometers simultaneously.  Some of the
poorer quality thermometers are substantially inaccurate (off as
much as 6°F),
F.  Example

To measure influent temperature, obtain sample in large coffee can,
immediately immerse thermometer in can, and record temperature when
reading becomes constant.  For example, 72°F.

-------
                                               (Temperature)



  °C  =  5/9 (OF - 32°)
                                77
      =  5/9 (77° - 32°)        -32_
                                45
      =  5/9 (45°)                         , 5
                                        9 < 45
      =  25°                                        5
                                                   X:;
                                                   25"
                     QUESTIONS
17.A  What could a change in influent temperature indicate?

17.B  Why should the thermometer remain immersed in the
      liquid while being read?

17.C  Why should thermometers be calibrated against an
      accurate NBS certified thermometer?
                        14-153

-------
 17.  Temperature                       -.  , t-  <    -    '-  .-'


                      II.   DIGESTER SLUDGE

 A.  Discussion

 The rate of sludge digestion  in a digester  is  a  function of  the
 digester temperature.  The normal temperature  range  in  a digester
 is around 95 to 98°F.  The temperature of a digester should  not
 be changed by more than  1°F per day because then the helpful
 organisms in the digester  are unable  to  adjust to  rapid temperature
 changes.


 B.  Apparatus and Procedure

 Ref-r to I., WASTEWATER.
                 END OF LESSON 7 OF 8 LESSONS

                               on

              Laboratory Procedures and Chemistry
Work the next portion of the discussion and review questions
before continuing with Lesson 8.
                            14-154

-------
                DISCUSSION^ AND REVIEW QUESTIONS

                    (Lesson 7 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry
Name
                                            Date
Write the answers to these questions in your notebook.
numbering continues from Lesson 6.
                                                   The problem
27.  Given the following data:

         100 ml of sample
         Crucible weight
         Crucible plus dry solids
         Crucible plus ash

     Compute:

     1.  Total suspended solids
     2.  Volatile suspended solids
     3.  Percent volatile
28.
29.

30.

31.
                               19.9850 g
                               20,0503 g
                               20.0068 g
Estimate the pounds of solids removed per day by a primary
clarifier if the influent suspended solids is 220 mg/1 and
the effluent suspended solids is 120 mg/1 when the flow is
1.5 MGD.

What is the ambient temperature?

Convert a temperature reading of 50°F to °C.

Why should the temperature of a digester not be changed by
more than 1°F per day?
                           14-155

-------
     CHAPTER 14.  LABORATORY PROCEDURES AND CHEMISTRY

                 (Lesson 8 of 8 Lessons)
18.  Total and Volatile Solids (Sludge)
A.  Discussion

Total solids measure the combined amount of suspended and dissolved
materials in the sample.

This test is used for wastewater sludges or where the solids can be
expressed in percentages by weight and the weight can be measured
on an inexpensive beam balance to the nearest .01 of a gram.  The
total solids are composed of two components, volatile and fixed
solids.   Volatile solids are composed of organic compounds which
are of either plant or animal origin.  Fixed solids are inorganic
compounds such as sand, gravel, minerals, or salts.
B.   What is Tested?
                                  COMMON RANGE, % BY WEIGHT
        Sample

    Raw Sludge

    Raw Sludge plus Waste
    Activated Sludge

    Recirculated Sludge

    Supernatant:
      Good Quality, has
      Suspended Solids

      Poor Quality

    Digested Sludge
    to Air Dry


1


Total
6% to 9%
2% to 5%
.5% to 3%
< 1%
> 5%
3% too Thin
to < 8%
Volatile
75%
80%
75%
50%

50%
  Fixed

25% ± 6%


20% ± 5%

25% ±5%
50% ± 10%
50% ±
                           14-156

-------
          C.  Apparatus


          1.  Evaporating dish.

          2.  Analytical balance.

          3.  Drying oven, 103° - 105°C.

          4.  Measuring device—graduated cylinder.

          5.  Muffle furnace, 550°C.



          D.  Outline of Procedure
                     v
                                  2.   Cool
1.   Ignite empty dish
    in muffle furnace
          4.   Measure out
              sludge
3.  Weigh
   dish
                              5.   Evaporate water at
                                  103-105°C
\
7.  Weigh dish
   + residue
                                                               6.  Cool dish
                                                                   + residue
                                     14-157

-------
                                        (Total and Volatile Solids)
                           PROCEDURE
1.  Dry the dish by ignition in a muffle furnace at 550°C for
    one hour.  Cool dish in desiccator.

2.  Tare the evaporating dish to the nearest 10 milligrams, or
    0.01 g on the Mettler single pan balance.  Record the weight
    as Tare Weight =  _ _ J _ _ a ; __. _.,  .     Sms<

3.  Weigh dish plus 50 to 100 ml of well mixed sludge sample.
    Record total weight to nearest 0.01 gram as Gross Weight =
    	 gms.

4.  Evaporate the sludge sample to dryness in the 103°C drying
    oven.

5.  Weigh the dried residue in the evaporating dish to the
    nearest 10 milligrams, or 0.01 g.  Record the weight as
    Dry Sample and Dish = 	 gms.

6.  Compute the net weight of the residue by subtracting the
    tare weight of the dish from the dry sample and dish.
E.  Precautions
1.  Be sure that the sample is thoroughly mixed and is representative
    of the sludge being pumped.Generally, where sludge pumping is
    intermittent, sludge is much heavier at the beginning and is less
    dense toward the end of pumping.  Take several equal portions of
    sludge at regular intervals and mix for a good sample.

2.  Take a large enough sample.  Measuring a 50 or 100 ml sample
    which is closely equal to 50 or 100 grams is recommended.
    Since this material is so heterogeneous (non-uniform), it is
    difficult to obtain a good representative sample with less
    volume.  Smaller volumes will show greater variations in answers,
    due to the uneven and lumpy nature of the material.

3.  Control oven temperature closely at 103° - 105°C.  Some solids
    are lost at any drying temperature.  Close control of oven
    temperature is necessary because higher temperatures increase
    the losses of volatile solids in addition to the evaporated
    water.
                           14-158

-------
           Heat  dish  long enough  to  insure  evaporation  of water,
           usually about  3-4 hours.   If heat  drying  and weighing
           are repeated,  stop when the  weight change becomes  small
           per unit of drying time.   The  oxidation,  dehydration,
           and degradation of the volatile  fraction  won't completely
           stabilize  until it is  carbonized or becomes  ash.

           Since sludge is so non-uniform,  weighing  on  the  analytical
           balance should probably be made  only  to the  nearest  0.01
           grams or 10 milligrams.
           Outline  of Procedure  for  Volatile  Solids
                     (continue  from  total  solids  test)
1,   Ignite dried solids
    at 550°C
                                     2.   Cool
                                               3,  Weigh fixed solids
       1.

       2.


       3.

       4.
                       PROCEDURE

Determine the total solids as previously described in Section D.

Ignite the dish and residue from total solids test at 550°C for
one hour or until a white ash remains.
Cool in desiccator for about 30 minutes.

Weigh and record weight of Dish Plus Ash  =
gms,
                                 14-159

-------
                                     (Total and Volatile Solids)
G.  Example

          Weight of Dish (Tare)     =     20.31 g

          Weight of Dish plus
          Wet Solids (Gross)        =     70.31 g

          Weight of Dish plus
          Dry Solids                =     22.81 g

          Weight of Dish plus Ash   =     20.93 g
H.  Calculations
See Laboratory Work Sheet (Fig. 14.9) or calculations shown below.

1.  Find weight of sample.

    Weight of Dish plus Wet Solids (Gross)  =  70.31 g
    Weight of Dish (Tare)                   =  20.31^ g

    Weight of Sample                        =  50.00 g

2.  Find weight of total solids.

    Weight of Dish plus Dry Solids          =  22.81 g
    Weight of Dish (Tare)                   =  20.31 g

    Weight of Total Solids                  =   2.50 g

3,  Find % solids.

    % solids  =  (Weight of Solids, g) 100%
                    Weight of Sample, g

                 (2.50 g) 100%
                    50.00 g

              =  5%

4.  Find weight of volatile solids.

    Weight of Dish plus Dry Solids          =  22.81 g
    Weight of Dish plus Ash                 =  20.95 _g

    Weight of Volatile Solids               =   1.88 g
                            14-160

-------
5.  Find % volatile solids.

    % Volatile Solids  =  (Weight of Volatile Solids, gjj.00%
                               Weight of Total Solids, g

                          (1.88 g) 100%
                             2.50 g

                       =  76%
                          QUESTION
    18.A  What is the origin of the volatile solids found in a
          digester?

    18.B  What is the significance of volatile solids in a
          treatment plant?
19.   Turbidity

     See Clarity.
                           14-161

-------
                                              PLANT
                                              DATE
                       SUSPENDED SOLIDS § DISSOLVED SOLIDS
SAMPLE
Crucible
Ml Sample
Wt Dry § Dish
Wt Dish
" Wt Dry
-._/, _ Wt Dry, gm x 1,000,000
6 Ml Sample
Wt Dish $ Dry
Wt Dish $ Ash
Wt Volatile
% vol - -l^2i x 100%
Wt Dry

























































                                          BOD
                                                     # Blank
SAMPLE
  % Sample
  Dep %

Nitrate N03
Sample
Graph Reading
COD
Scimple
Blank Titration
Sample Titration
Depletion
rag/1 =
le
#
e
r adj blank
r incubation
on, 5 days




























































                                            Sett. Solids
                                            Sample
                                            Direct Ml/1
                   X80°
           Ml Sample
                       Fig.  14.9  Calculation of total solids
                                  on Laboratory Work Sheet
                                         14-162

-------
                                       TOTAL SOLIDS
SAMPLE
Dish No.
Wt Dish § Wet
Wt Dish
Wt Wet
Wt Dish + Dry
Wt Dish
Wt 'Dry
?- c -. • !„ _ wt Dry x i°°%
Wt Wet
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
* Volatile - Wt Vo1 x 100%
Wt Dry
pH
Vol. Acid
Alkalinity as CaC03
RAW
7
70.31
20.31
50.00
22.81
20.31
2.50
5.0%
22.81
20.93
1.88
76%




































































Grease (Soxlet)
  Sample
  Ml Sample
  Wt Flask + Grease
  Wt Flask
  Wt Grease
  mg/1 = Wt Grease, mg x 1000
              Ml Sample

H2S (Gas) (Starch-Iodine)
  Blank
  Sample
  Diff               	
  Diff x .68         	
  mg/1 x 43.6        	
Ml
Ml
Ml
mg/1
grain/100 cu ft
                       Fig. 14.9  Calculation of total solids on
                                  Laboratory Work Sheet (continued)
                                          14-163

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20.  VoJLatile Acids and Total Alkalinity


A.  Discussion

Volatile acids are determined on sludge samples from the digesters.
Most modern digesters have sampling pipes where you can draw a
sample from various levels of the tank.  Be sure to allow the
sludge in the line to run for a few minutes in order to obtain a
representative sample of the digester contents.  Samples also may
be collected from supernatant draw-off tubes, or thief holes.15

The concentrations of volatile acids and alkalinity are the first
measurable changes that take place when the process of digestion
is becoming upset.  The volatile acid/alkalinity relationship can
vary from 0.1 to about 0.5 without significant changes in digester
performance.  When the relationship starts to increase, this is a
warning that undesirable changes will occur unless the increase is
stopped.  If the relationship increases above 0.5, the composition
of the gas produced can change very rapidly, followed by changes
in the rate of gas production, and finally pH.

In a healthy and properly functioning digester, the processes or
biological action taking place inside the digester are in equilibrium.
When fresh sludge is pumped into a digester, some of the organisru
in the digester convert this material to volatile (organic)  acid.?,
In a properly operated digester, other organisms feed on the newly-
produced volatile acids and eventually convert the acids to methane
(CHiJ  gas, which is burnable and carbon dioxide (C02).  If too much
raw sludge is pumped to the digester or the digester is not function-
ing properly, an excess of volatile acids are produced.  If excessive
amounts of volatile acids are produced, an acid environment unsuitable
for some of the organisms in the digester will develop and the digeste.
may cease to function properly unless the alkalinity increases too.

Routine volatile acids and alkalinity determinations during trie
start-up process for a new digester are a must in bringing the
Oigester to a state of satisfactory digestion,

Routine volatile acids and alkalinity determinations during digestion
are important in providing the information which will  enable the
operator to determine the health of the digester.
15 Thief Hole.   A digester sampling well.
                             14-164

-------
For digester control purposes, the volatile acid/alkalinity relation-
ship should be determined.   When the volatile acid/alkalinity
relationship is from less than 0.1/1.0 to 0.5/1.0, the loading and
seed retention of the digester are under control.  When the relation-
ship starts increasing and becomes greater than 0.5/1.0,  the digester
is out of control and will become "stuck" unless effective corrective
action is taken.
B.  What is Tested?

        Sample                           Desirable Range

    Recirculated Sludge                  150 - 600 mg/1
                             (expect trouble if alkalinity less than
                              two times volatile acids]
                           METHOD A

                      (Silic Acid Method)



C.  Apparatus

1.  Centrifuge or filtering apparatus.

2.  Two 50 ml graduated cylinders.

3.  Two medicine droppers.

4.  Crucibles, Gooch or fritted glass

5.  Filter flask

6.  Vacuum source

7.  One 50 ml beaker

8.  Two 5 ml pipettes

9.  Buret
                           14-165

-------
D.  Reagents

1.  Silicic acid, solids, 100-mesh.  Remove fines from solid
    portion of acid by slurrying the acid in distilled water
    and removing the supernatant after allowing settling for
    15 minutes.   Repeat the process several times,   Dry the
    washed acid solids in an oven at 103°C aid then store in
    a desiccator.

2.  Chloroform-butanol reagent.  Mix 300 ml chloroform, 100 ml
    n-butanol, and 80 ml 0.5 N H  SO  in separatory funnel and
    allow the water and organic Iaye?s to separate.  Drain off
    the lower organic layer through filter paper into a dry
    bottle.

3.  Thymol blue indicator solution.  Dissolve 80 mg thymol
    blue in 100 ml absolute methanol.

4.  Phenolphthalein indicator solution.  Dissolve 80 mg
    phenolphthalein in 100 ml absolute methanol.

5.  Sulfuric acid, 10 N.

6.  Standard solium hydroxide reagent, 0.02 N.  Prepare in
    absolute methanol from cone. NaOH stock solution in water.
                             14-166

-------
     12.  Outline of Procedure
                                                3.
                             Add a
                             few drops
                             of thymol
                             blue.
Separate solids by
centrifuging or
filtering sample.
2. Measure 10-15 ml
   of sample into
   beaker.
4. Add
   10 N H2S04
   dropwise until
         thymol
      blue turns
            red
Add 5 ml
acidified
sample.
               7.  Add 50 ml
                  chloroform-butanol
Place 10 g silic
acid in crucible
and apply suction,
                                        Apply suction until
                                        all of reagent has
                                        entered solid acid
                                        column.
               9,  Remove filter flask.
                  10.  Add a few
                      drops of
                      phenoIphthale in
                                 11. Titrate with
                                     0.02N NaOH,
                               14-167

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                      PROCEDURE

 1.  Centrifuge or filter enough sludge to obtain a sample of
     10 to 15 ml.  This same sample and filtrate should be
     used for both the volatile acids test and the total
     alkalinity test.

 2.  Measure volume  (10 to 15 inl) of sample and place in a
     beaker.

 3.  Add a few drops of thymol blue indicator solution.

 4.  Add 10 N H^SO^, dropwise, until thymol blue color just turns
     to red.

 5.  Place 10 grams of silicic acid (solid acid) in crucible
     and apply suction.  This will pack the acid material
     and the packed material is sometimes called a column.

 6.  With a pipette, distribute 5.0 ml acidified sample
     (from step 4) as uniformly  as possible over the column.
     Apply suction briefly to draw the acidified sample into
     the silicic acid column.  Release the vacuum as soon as
     the sample enters the column.

 7.  Quickly add 50 ml chloroform-butanol reagent to the column.

 8.  Apply suction and stop just before the last of the reagent
     enters the column.

 9.  Remove the filter flask from the crucible.

10.  Add a few drops of phenolphthalein indicator solution to
     the liquid in the filter flask.

11.  Titrate with 0.02 N NaOH titrant in absolute methanol, taking
     care to avoid aerating the sample.  Nitrogen gas or C0£ - free
     air delivered through a small glass tube may be used both to
     mix the sample and to prevent contact with  atmospheric C0~
     during titration [ CC-2 - free air may be obtained by passing
     air through ascarite or equivalent].

          Volume of NaOH used in sample titration,  a =-	ml.

12.  Repeat the above procedure using a blank of distilled water.

          Volume of NaOH used in blank titration, b = 	ml.
                             14-168

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F.  Precautions

1.  The sludge sample must be representative of the digester.
    The sample line should be allowed to run for a few minutes
    before the sample is taken.   The sample temperature should
    be as warm as the digester itself.

2.  The sample for the volatile  acids test should not be taken
    immediately after charging the digester with raw sludge.
    Should this be done, the raw sludge may short-circuit to
    the withdrawal point and result in the withdrawal of raw
    sludge rather than digested  sludge.  Therefore, after the
    raw sludge has been fed into the tank, the tank should be
    well mixed by recirculation  or other means before a sample
    is taken.

3.  If a digester is performing  well with low volatile acids
    and then if one sample should unexpectedly and suddenly
    give a high value, say over  1000 mg/1 of volatile acids,
    do not become alarmed.  The  high result may be caused by
    a poor, nonrepresentative sample of raw sludge instead
    of digested sludge.  Resample and retest.  The second
    test may give a more typical value.  When increasing
    volatile acids and decreasing alkalinity are observed,
    this is a definite warning of approaching control problems.
    Corrective action should be  taken immediately, such  ^
    reducing the feed rate, reseeding from another digest--
    maintaining optimum temperatures, improving digester mixmg,,
    decreasing sludge withdrawal rate, or cleaning the tank
    of grit and scum.
                            14-169

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G.  Example


    bquivalent Weight  of Acetic  Acid,  A          =60  mg/ml


    Volume of Sample,  B                          =  10  ml


    Normality of NaOH  tit rant, N                =  0.02 N


    Volume of NaOH used  in  sample  titration,  a  =  2.3 ml


    Volume of NaOH used  in  blank titration, b   =  0.5 ml



H.  Calculation


    Volatile Acids, mg/1  _ A x 1000  ml/1^ x  N (a - b)
    (as acetic acid)                     B
                              60 mg/ml  x  100°. P1/.1. x, P.'P2  Q2'5 ml. ". P_\5
                                                  10  ml


                          =   216 mg/1
                           METHOD B
                 (Nonstandard Titration Method)
C.  Apparatus


1.  One pH meter.


2.  One adjustable hot plate.


3.  Two Burets and stand.


4.  One 100 mi beaker.



D.  Reagents


1.  pH 7.0 buffer solution


2.  pH 4.0 buffer solution


3.  Standard acid.


I.  Standard base.
                            14-170

-------
            E.  Outline of Procedure
                                            3. Titrate with
1. Separate solids by
   centrifuging or re-
   moving water above
   ye'.T led sample.
o ., SUlf
2. Measure
50 ml $ J°.a
, . 4.0.
place in
beaker.


	
Vv?j *~
uric ac
pH of
/T\
o oo| \\
V


id '

* r
A
i rnn\
If •/;••.-.
                                           4. Note acid used
                                              and continue
                                              titrating to
                                              pH 3.5 to 3.3.
                                                                                      *•*
                                                                                      .)
5,  lightly boil
   -  r  -' : for
       'nutes.
6. Cool in water bath.
7.  Titrate to ra of 4.0,
   with 0.05 ;\ NaOH, note
   buret reading, and com-
   plete titration to - pi!
   of 7.0.
                                         14-171

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                         PROCEDURE
1.  Buffer the pll meter at 7.0 and check pH before treatment
    of sample to remove the solids.  Filtration is not necessary.
    Decanting (removing water above settled material) or centri-
    fuging sample is satisfactory.  Do not add any coagulant aids.

2.  Titrate 50 ml of the sample in a 100 ml beaker to pH 4.0
    with the appropriate strength sulfuric acid (depends on
    alkalinity), note acid used, and continue to pH 3.5 to 3.3.
    A magnetic mixer is extremely useful for this titration.

3.  Carefully buffer pH meter at 4.00 while lightly boiling the
    sample a minimum of three minutes.  Cool in cold water bath
    to original temperature.

4.  Titrate sample with standard 0.050 N sodium hydroxide up to
    pH 4,00, and note buret reading.   Complete the titration at
    pH 7,0.  (If this titration consistently takes more than
    10 ml of the standard hydroxide,  use 0.100 N NaOH.)

5.  Calculate volatile acid alkalinity (alkalinity between pH
    4.0 and 7.0).

    Volatile Acid  _  ml 0.050 N NaOH x 2500
    Alkalinity              mi Sample

    For a 50 ml sample the volatile acid alkalinity equals
    50 x ml 0.050  N NaOH,  or 100 x ml 0.100 N NaOH.

6.  Calculate volatile acids.

    Case 1:  > 180 mg/1 volatile ncid alkalinity.

      Volatile Acids = Volatile Acid  Alkalinity x 1.50

    Case 2:  < 180 mg/1 volatile acid alkalinity,

      Volatile Acids = Volatile Acid  Alkalinity x l.on

    Steps 1 and 2  will give the analyst the pH and total alkalinity,
    two control  tests normally run on digesters.   The difference
    between the  total and  the  volatile acid alkalinity is bicarbonate
    alkalinity.  The time  required for Steps  3 and 4 is  about ten
    minutes.

    This  is an  acceptable  method for  digester  control to determine
    the  volatile acid/alkalinity relationship,  but not of sufficient
    accuracy  for research  work.
                           14-172

-------
For details regarding this test see DeLallo, R., and Albertson,
O.E., Volatile Acids by Direct TitTatian^ Water Pollution Control
Federation, Vol. 33, No. 4, pp 356-365, April  1961.  The procedure
is reproduced from the article.
F.  Example and Calculation

Titration of pH 4.0 to 7.0 of a 50 ml sample required  8 ml of
0.05 N NaOH.

Step 5 - Calculate volatile acid alkalinity  (alkalinity between
pH 470 and 7.0).

Volatile Acid     _  ml 0.05 N NaOH x 2500
Alkalinity, mg/1  "        ml Sample

                     8 ml x^ 2500
                        50 ml

                  =  400 mg/1


Step_j5_ - Calculate volatile acids.

Case 1:  400 mg/1 > ?. f':" mg/1.  '.herefore,

Volatile             ., , ..,  . .  , ...   ,.  ..     ,  _„
, . ,      ,.       =  Volatile Acid Alkalinity x  1.50
Acids, mg/1                                 J

                  -  400 mg/1 x 1.50

                  =  600 mg/1
                          QUESTION
    20.A  What is the volatile acid concentre" ' ir  -" /;  a
          digester if a 50 ml sample required  :  .:   oC 0.05
          NaOH for a titration from a  pH  of  4.','  to 7.0V
                             14-173

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Total A Ika Unity


A.  Discussion

Tests for total alkalinity of digesters are normally run on settled
supernatant samples.  The alkalinity of the recirculated sludge is
a measure of the buffer capacity in the digester.  When organic
matter in a digester is decomposed anaerobically, organic acids
are formed which could lower the pH, if buffering materials
(buffer capacity) were not present.  If the pH drops too low, the
organisms in the digester could become inactive or die and the
digester becomes upset (no longer capable of decomposing organic
matter).

For digester control purposes, the volatile acid/alkalinity relation-
ship should be determined.  When the volatile acid/alkalinity
relationship is from less than 0.1/1.0 to 0.5/1.0, the loading and
seed retention of the digester are under control.  When the relation-
ship starts increasing and becomes greater than 0.5/1.0, the digester
is out of control and will become stuck unless effective corrective
action is taken.  The pH will not be out of range as long as the
volatile acid/alkalinity relationship is low.  This relationship
gives a warning before trouble starts.

All samples must be settled so that a liquid free of solids is availir:
for the test.   Tests cannot be calculated correctly if solids are in
the sample.
B.  What is Tested?

              Sample                          Common Ra-.ge

        Recirculated Sludge             2-10 Times Vo'atMe
                           14-174

-------
                                   (Volatile Acids and Total Alkalinity)


C.  Apparatus


1.  Centrifuge and centrifuge tubes,  or settling cylinder.

2.  Graduated cylinders (25 ml and 100 ml)

3.  50 ml Buret

4.  400 ml Erlenmeyer Flask or 400 ml beaker

5.  pH Meter or a methyl orange chemical color
    indicator may be used (see Procedure)




D.  Reagents
1.  Sulfuric Acid, 0.2 N.  Prepare stock solution of approximately
    0.1 N by cautiously adding 2.8 ml of concentrated sulfuric
    acid (H^OiO to 1 liter of distilled water.  Dilute 200 ml of
    the 0.1 N stock solution to 1 liter with boiled distilled
    water.   Standardize against 0.02 N sodium carbonate (Step 2).

2.  Sodium Carbonate, 0.02 N.  Dry in oven before weighing.  Dis-
    solve 1.06 g of anhydrous sodium carbonate (Na2CC>3) in boiled
    distilled water and dilute to 1 liter with distilled water.

3.  Methyl Orange Chemical Color Indicator.  Dissolve 0.5 •:>
    methyl orange in 1 liter of distilled water.
                          14-175

-------
E.  Procedure
                                                           4.   Titrate
   Centrifuge
   or settle
3.   Place electrodes  of
    pH meter in beaker
              2.  Add 190 ml of
                 distilled water
                                            Add  2  drops of
                                            methyl  orange
                                                               TZJT7
This proceuure is followed to measure  the  alkalinity  of a sample
and also the alkalinity of a distilled water blank.

1.  Take a clean 400 ml beaker and add 10  ml or  less  of clear
    supernatant (in case of water or distilled water, use 200 ml
    sample).  Select a sample volume that  will give a useable
    titration volume.  If the liquid will  not separate from the
    sludge by standing and a centrifuge is not available, use
    the top portion of the sample.   This same sample  and filtrate
    should be used for both the total  alkalinity test and the
    volatile acids test.

2.  Add 190 ml distilled water (in case of water or distilled
    water determination skip this step).
                            14-176

-------
                                    (Volatile Acids and Total Alkalinity)
.">.  Place the electrodes of pH meter into the  400 ml beaker
    containing the sample.

4.  Titrate to a pH of 4.5 with 0.02 N sulfuric  acid.   (In
    case of a lack of pH meter, add 2 drops of methyl orange
    indicator.  In this case, titrate to the first permanent
    change of color to a, red-orange color.  Care must be
    exercised in determining the change of color and your
    ability to detect the change will improve  with experience.)

5.  The alkalinity of the distilled water should be checked
    and if significant, subtracted from the calculation,

6.  Calculate alkalinity.

    Alkalinity of
    Distilled      =  ml of 0.02 N H2S04 x 5*
    Water, mg/1

    Total Alka-    _ " ml of 0.02 N H2SOk x 100*  - mg/1
    linity, mg/1   ~  alkalinity of distilled  H20
F.  Example

Results from alkalinity titrations on

1.  Distilled Water            4 ml 0.02 N H2SOtt

2.  Recirculated Sludge     19.8 ml 0.0? N H:,S04


G,  C aIculations

Alkalinity of            .  ,, -  ,_ ., ., „...
r. f.  1 j T, n     /i  =  ml (rf" O.i)2 N HobU,, x 'a
Distilled H20, mg/1                   z   +

                     =  4 n'l x 5

                     =  20 >'!.-'!
Hlse 5 if measuring alkalinity of water or distilled wat-:>r
 (200 ml sample) and 100 if measuring alkalinity of sludge
 (10 ml sample).
                           14-177

-------
Total Alka-
linity, mg/1,
of recircu-
lated sludge
ml of 0.02 N H2SOi4 x 100 - mg/1 alka-
linity of distilled H20

19.8 ml x 100 - 20 mg/1

1980 mg/1 - 20 mg/1

1960 mg/1
                           QUESTIONS
20.B  Why would you run a total alkalinity test on
      recirculated sludge?

20.C  What is meant by the buffer capacity in a
      digester?

20.D  If the total alkalinity in a digester is
      2000 mg/1 and the volatile acids concen-
      tration is 300 mg/1 per liter, what is
      the volatile acid/alkalinity relationship?
                            14-178

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2 * •
       See Total Solids.
                 END OF  LESSON  8  OF  8  LESSONS




                              on




             Laboratory  Procedures  and Chemistry
                             14-179

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                DISCUSSION AND REVIEW QUESTIONS

                    (Lesson 8 of 8 Lessons)

       Chapter 14.  Laboratory Procedures and Chemistry



Name                                         Date
Write the answers to these questions in your notebook.  The
problem numbering continues from Lesson 7.
32.  Why are solids only weighed to the nearest 0.01 gram
     when determining the total and volatile solids content
     of digesters?

33.  What is a thief hole?

34.  What relationship is the critical control factor
     in digester operation?
                          14-180

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14.6  KECOMMENI'O GENERAL LABORATORY SUPPLIES
Supplies needed in addition to apparatus listed for tests.  Source:
WPCF Publication No. 18, Simplified Laboratory Procedures for Waste-
water Examination.
Quantity     Description

   12        Pinch clamps, medium
  200        Corks, assorted
    1        Cork borer set, sizes 1 through 6

    1        Cork borer sharpener
 2 Ib        Glass tubing, 8 mm
    4        Thermometers, -20° to 100°C

40 ft        Rubber tubing, 1/4-in. ID, 3/32-in. wall
 2 Ib        Rubber stoppers, assorted (sizes 6 through 12)
    1        Tripod, concentric ring, 6 in. OD

    1        Latest edition, Standard Methods for the Examination oj
               Water & Wastewater
    2        Funnels, 50 mm
    2        Funnels, 100 mm

2 pair       Balance watch glasses, 3 in.
    4        Beakers, Pyrex, 1000 ml
    4        Beakers, Pyrex, 600 ml

    6        Beakers, Pyrex, -in-; ;, i
    •t        Beakers, Pyrex, 250 nJ
             Beakers, Pyrex, 100 ml

    4        Beakers, Pyrexs SO ml
    2        Bunsen burner>
    2        Brushes, medium

    2        Brush, B
    2        Brush, A
    2        Brush, Flask

    2        Aprons, plastic, 42 in, length
    3        Wire gauzes, 4 x 4 in.
    3        Triangles, 2-1/2 in, per side
1 tube       Stopcock lubricant
                           14-181

-------
            SUPPLEMENTAL EQUIPMENT FOR THE BOD TEST
Quantity    Desc ripti on

   12       Flask, Erlenmeyer, 500 ml
   12       Flask, Erlenmeyer, 250 ml
    2       Pipettes, volumetric, 25 ml
    2       Pipettes, volumetric, 10 ml
    2       Pipettes, volumetric, 5 ml

    2       Flasks, volumetric, graduated to contain and
              deliver 1000 ml
    2       Flasks, volumetric, graduated to contain and
              deliver 500 ml
    2       Flasks, volumetric, graduated to contain and
              deliver 100 ml

    6       Bottles, 32 oz
    6       Bottles, 16 oz
    6       Bottles, 8 oz

   24       BOD bottles, with funnel opening
    2       Burets, 50 ml
    1       Buret clamp, double

    2       Bottles, dropping, 30 ml
    2       Spatulas, 75-mm blade
    3       Bottles, storage, 2-1/2 gal

    1       Buret support, medium
 9 Ib       Sulfuric acid, CP
 5 Ib       Sodium hydroxide pellets, CP

   j'2       Bulb, rubber, pipette, 2 ml
   24       Holder, rubber, stor.per
    4       Flask, volumetric, w'o stopper,  100 ml

 2 Ib       Potassium iodide, CP
 1 Ib       Starch, soluble potato
 1 Ib       Sodium thiosulfi; te, CP

 5 Ib       Manganous sulfate, CP
100 g       Sodium azide, CP
 1 Ib       Magnesium sulfate

1/4 Ib      Ferric chloride
 1 Ib       Potassium phosphate,  mono-basic
 1 Ib       Potassium phosphate,  dibasic
                          14-182

-------
Quantity
Description
 ] Ih        Sodium phosphate, dibasic heptahydrate
1/4 Ib       Ammonium chloride
 1 oz        Potassium bi-iodate, primary standard

 1 Ib        Potassium dichromate
 10 g        Sodium diethyldithio carbamate
    1        Incubator, BOD

    1        Refrigerator
 1 Ib        Calcium chloride, 20 mesh
    1
    1
 6 Ib
 25 g
             SUPPLEMENTAL EQUIPMENT FOR THE CHLORINE RESIDUAL TEST
Quantity     Description
Comparator, water analysis
Disc for comparator, chlorine
Hydrochloride acid, CP
Orthotolidine dihydrochloride
             SUPPLEMENTAL EQUIPMENT FOR SOLIDS ANALr  ..S

Quantity     Description

    1        Brush, camel hair, 1 - \ a, vide
    1        Balance wit,-\ cove °
    ]        Weights, balance set, 50 g

   12        Crucibles, Gooc1  , No. 4
    2        Holders, cruciMe
    2        Cylinder, graduated, 1000 ml

    2        Cylinder, gradua* •', Snn ml
    2        Cylinder, gradual  '  "50 ml
    4        Cylinder, graduated, 100 ml

    4        Cylinder, graduated, 50 ml
    2        Cylinder/ graduated, 25 ml
    1        Cylinder, graduated, 10 ml

    1        Desiccator, 250 mm
    1        Desiccator olate
   12        Dishes, r-vaporating, size 0
                           14-183

-------
Quantity
Description
    3        Flask, filtering, 500 ml
    2        Pipettes, 25 ml
    6        Pipettes, 10 ir.l

    2        Pipettes, 5 rcl
    1        Hot plate, 660 w
    2        Tongs, crucible

    1        Tongs, furnace, 18 in,
 8 ft        Tubing, rubber, (heavy) 1/4-in. ID
    2        Filter pumps

    1        Clock, interval timer, 2 hr
    1        Furnace, muffle
2 boxes      Paper, filter, glass fiber, 2.4 cm

    1        Water baths, four-hole
    1        Balance, platform, triple beam
    2        Bottles, washing, polyethylene, 500 ml

   ; 6        Pencils, wax, red
2 boxes      Filter paper, 12.5 cm, Whatman No. 41
1 bottle     Ink, marking, black

 1 Ib        Rod, glass, 6 mm
    1        File, triangular, 4 in.
   12  .      Bulb, rubber, pipet, 2 oz

    1  '      Balance desiccator
    1        Oven, drying
   24        2.4 cm glass fiber filter

    2        Buchner funnel, size 2A
    6        Tube "T", connecting, 1/4-in.
 5 Ib        Drierite
             SUPPLEMENTAL EQUIPMENT FOR COLIFORM GROUP
             BACTERIA ANALYSES

Quantity     Description

    1        Sterilizer or autoclave
   12        3 mm wire transfer loop
   24   ,     Pipets, measuring, 10 ml
   48        Pipets, measuring, 1 ml, or quantity of disposable
               sterile pipets
                            14-184

-------
 Many equipment suppliers  will  furnish suggested equipment  lists
 upon request and indication of size  of plant  and tests  being
 performed.   Lists may be  obtained from:

 Central Scientific Company       Van Waters § Rogers
 1700 Irving Park Road-            Post Office  Box 2062
 Chicago,  lllinios  '              Terminal  Annex
                        , .         Los Angeles,  California   90054
•14.7   ADDITIONAL  READING


 a.  MOP  11

b.  New  York Manual, pages  127-148

 c.  Texas Manual, pages 565-587

d.  Laboratory Procedures for Operators of Water Pollution Control
    Plants,'- Nagano, Joe.  Obtain  from Secretary-Treasurer, California
    Water Pollution Control Association, P.O. Box 61, Lemon Grove,
    California  °2045.  Price $3.25 to members of the CWPCA; $4.25
    to others.

e.  Simplified Laboratory Procedures for Wastewater Examination,
    WPCF Publication No. 18, Water Pollution Control Federation,
    3900 Wisconsin Avenue, Washington, D.C.  20016.  Price $2.00
    to members; $4.00 to others.  Indicate your member association
    when ordering.

f.  Standard Methods for Examination of Water and Wastewater, pro-
    duced by APHA, AWWA, and WPCF, Water Pollution Control Federation,
    3900 Wisconsin Avenue, Washington, D.C.  20016.  Price $16.50 to
    members prepaid only-; otherwise $22.50 plus postage.  Indicate
    your member association when ordering.

g.  Chemistry for Sanitary Engineers, Sawyer, Clair N. and
    McCarty, Perry L., McGraw-Hill Book Company, New York, 1967.
    Price $13.50.

h.  Methods  for Chemioal Analysis of Water and Wastes,  1971,
    Environmental  Protection Agency,  Water Quality Office, Analytical
    Quality Control  Laboratory,  1014 Broadway,  Cincinnati, Ohio  45202.
    For sale by Superintendent  of Documents,  Government  Printing Office,
    Washington,  D.C.   20402, Stock Number 5501-0667.   Price $3.00.
                           14-185

-------
14-186

-------
                       SUGGESTED ANSWERS
       Chapter 14.   Laboratory Procedures arid Chemistry
14.2A  A bulb should always be used to pipette wastewater
       or polluted water to prevent infectious materials
       from entering your mouth..

14.2B  Inoculations are recommended to reduce the possibility
       of contracting diseases.

14.2C  Immediately wash area where acid spilled with water and
       neutralize the acid with  sodium carbonate or bicarbonate,

14.2D  True.   You may add acid to water, but never reverse,

14.2E  Work clothes should be changed before going home at
       night  to prevent carrying unsanitary materials and
       diseases home which could infect you and your family.

14.3A  The largest sources of errors -.found in laboratory results
       are usually caused by improper sampling; poor preservation;
       and lack of sufficient mixing, compositing, and testing.
                                                    IB
14.3R  A representative sample must be collected or the test
       results will not have any significant meaning.  To
       efficiently operate a wastewater treatment plant, the
       operator must rely on test results to indicate to him
       what is happening.

14.3C  A proportional composite  sample may be prepared by collect-
       ing a  sample every hour.   The size of this sample is
       proportional to the flow  when the sample is .collected.
       All of these proportional samples are mixed together  to
       produce a proportional composite sample.  If an equal
       volume of sample was collected each hour and mixed, this
       would  be simply a composite sample.

  3.A  The dangers encountered in running t-he CC>2 on digester
       gas include:

       1.  Digester gas contains methane, which is
           explosive when mixed with air.
       2.  The C02 gas absorbent is hafmful to your skin.
                            14-187

-------
     »      -  (Total Volume, ml - Gas Remaining, ml) x 100%
3.B  a LU2  -                 Total Volume, ml

               (128 ml - 73 ml) x 100%               128
                       128 ml                       - 75
                                                      55

            =  !§ x 100%
4.A  The COD test is a measure of the strength of a waste
     in terms of its chemical oxygen demand.  It is a good
     estimate of the first-stage oxygen demand.  (Either
     answer is acceptable.)

4.B  The advantage of the COD test over the BOD test is
     that you don't have to wait five days for the results.

5. A  Plant effluents should be chlorinated for disinfection
     purposes to protect the bacteriological quality of the
     receiving waters.

5.B  The idometric method gives good results with samples
     containing wastewater, such as plant effluent or re-
     ceiving waters.  Orthotolidine will give satisfactory
     results if used within 20 minutes of the application
     of chlorine; however, the entire chlorine demand may
     not yet have been satisfied.  Amperometric titration
     gives satisfactory results, but the equipment is ex-
     pensive.

6.A  The clarity test indicates the relative change of depth
     you can see down in the final clarifier or contact basin
     This reflects a visual comparison of color, solids, and
     turbidity from one test to the next.  OR_  Indication of
     quality of effluent.

6.B  When clarity is measured under different conditions the
     results can not be compared.  You won't be able to tell
     whether your plant performance is improving, staying the
     same, or deteriorating.
                           14-188

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7,A  Sodium thiosulfate crystals should be added to sample
     bottles for coliform bacteria tests before sterilization
     to neutralize any chlorine that may be present when the
     sample is collected.  Care must be taken not to wash the
     bottles out when a sample is collected.

7.B  121°C within 15 minutes.

7.C  Dilutions     -2     -3     -,4     -5

     Readings       5	__1	2_      0

     MPN  =  63,000/100 ml

7.D  The number of coliforms is estimated by counting the
     number of colonies grown on the membrane filter.

8.A  DO Saturation, %  =  DO of Sample, mg/1 x 100%,
                           DO at Saturation, mg/1

                          (7.9 mg/1) 100%                     .699
                             11.3 mg/1

                       =  70%
8.B  To calibrate the DO probe in an aeration tank, a
     sample of effluent can be collected and split.  The
     DO of the effluent is measured by the modified Winkler
     procedure, and the probe DO reading is adjusted to agree
     with the Winkler results.

8.C  When the DO in the aeration tank is very low, the
     copper sulfate-sulfamic acid procedure can give high
     results.  The results are high because oxygen enters
     the sample from the air when the sample is collected,
     when the copper sulfate-sulfamic acid inhibitor is
     added, while the solids are settling, and when the
     sample is transferred to a BOD bottle for the DO test.

8.D  BOD test or volatile solids test.
                           14-189

-------
 8.F.  To prepare dilutions for a cannery waste with an expected
      BOD of 2000 mg/1, take 10 ml of sample and add 90 ml of
      dilution water to obtain a new sample with an estimated
      BOD of 200 mg/1 (10 to 1 dilution);
      BOD Dilution, ml  «*
                                  1200
                           EstimateTd BOD/ mg/l
                           1200
 8.F
     BOD,
     mg/1
                           6 ml
Initial DO of
Diluted
pie, mg/1
                                   pO of Diluted
                                   Sample After
BOD Bottle Vol., ml
 Sample Volume/ mJ
            *  (7.5 mg/1 - 3.9 mg/1)
                                      500ml
           *  (3.6 mg/1) (130)

           *  540 mg/1

8.G  Samples for the BOD test should be collected before chlori
     nation because chlorine interferes with the organisms in
     the test.  It is difficult to obtain accurate results with
     dechlorinated samples,

8.H  A solution of sodium thipsulfate at 0.0375 N is very weak
     and unstable and will not remain accurate over two weeks.

9. A  (1)
           You wpuld measure thp fyS in the wastewater to know
           the strength of H2S and an indication of the corrosion
           taking place on the concrete.
      (2)   HaS in the atmosphere produces a rotten egg odor.   It
           is indicative of anaerobic decomposition of organics
           in wastewater which occurs in the absence of oxygen.

10. A  (1)   To measure plant influent pH with a paper tape,
           collect representative sample, mix sample with a
           clean stirring rod, and dip tape in sample while
           it is still moving.  Compare tape color with pack-
           age color and record resu 1 ts ,

      (2)   To measure raw sludge pH with a paper tape first
           allow raw sludge sample to settle.   Dip tape in
           liquid at top, compare resulting color, and record
           results.

      pH of both samples should be measured in place or as  soon
      as possible.

                            14-190

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10. B  Precautions to be exercised when usiftg  a  pH wet^r include: >

      (1)  Prepare fresh .buffer solution weekly for
           calibration purposes.-.

      (2)  pH meter, samples, and buffer solutions should
           all be at the same" temperature.

      (3)  Watch for erratic results  arising  from faulty*
           operation of pH meter or fouling of  electrodes
           with interfering matter.

11. A  Settleability tests should be run on the  mixed  liquor to
      determine the settling characteristics  of the sludge  floe
      at regular intervals for 60 minutes.  The results -are used
      in the SVI and SDI determinations.

11. B  The SVI is the volume in ml occupied by one gram of mixed
      liquor suspended solids, after 30 minutes,  of settling.

ll.C  The SVI test is used to indicate changes  in sludge
      characteristics.

11. D  Sludge Density Index (SDI)  =   100/SVI  .  - -

      Sludge         ,         '••.",   ••-.'•••

12. A,  t;° 1)1" :...=  ;(Tot,al Vet ''So f. Removed, ml/1)  (1000)  (Flow,  MGD)
                         -
      gpd      =  (10 ml/1 - 0.4 ml/1)  (1000 nig/ml)  (1  M Gal/day)
                  9.6-,ml
                   M mg
               -=  9600 gpd,
1000 ms
  ml
  Cal
day
      This value may be reduced by 30 to  75%  due  to
      compaction of the sludge•in -the clarifier.

13.A  The sludge age of.a 200,000 gallon  aeration tank  that has
      2000 mg/1 mixed liquor suspended solids,  a  primary effluent
      of 115 mg/1 SS, and an Average.flow of  1.8  MGD:
                          '-,''•'    •     *"  -
      Sludge   ,  '  Vol'of Aeration' Tank   '  •"'"   ,. ,   onnn    /^
      Age,      -   ;:     >2MG  ..  -    ^-Sus Solids, 2000  mg/1
      days         Flow, MGD,, .,1..,8 :x. Primary Effl','T15 rag/1

                   0.2 MG x 2000 mg/1    '  •
                ~  1.8-MGD'X 115 mg/1

                =  1.93
                            14-191

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14.A  (1)  Results from the graduated cylinder are available
          immediately, but different operators may interpret
          the results differently.

      (2)  Results are not available until the next day,  but
          different operators will  record the same result.

15.A  If the supernatant solids test is greater than 5%,
      the  supernatant could be placing a heavy solids load  on
      the  plant and the appropriate operational adjustments
      should be made.

16.A  The  specific gravity is very  near that of H20 and is  not
      light enough to float nor heavy enough to settle.

16.B  Solids calculations will be shown in detail here to illus-
      trate the computational approach and the units involved.
      After you understand this approach, use of the laboratory
      work sheet on the following pages is more convenient.

      a;   Total Suspended Solids

      Volume of Sample, ml  =  100  ml

      Weight of Dried Sample 5 Dish, grams  =  19.3902 g
      Weight of Dish (Tare Weight), grams   =  19.3241^g

                                Dry Weight  =   0.0661 g
                                or           =    66.1 mg
      Total
      Suspended _  Weight of Solids^ m^jx J-OOO ml/1
      Solids,             Volume of Sample,  ml
      mg/1
                   66.1 mg x 1000 ml/1
                         100 ml

                =  661  mg/1
      b.   Volatile Suspended Solids

      Weight  of Dried Sample § Dish,  grams   =  19,3902 g

      Weight  of Ash PT Dish,  grams            =  19.3469 g

                    Weight Volatile,  grains   =   0.0433 g

                    or                           43.3 mg
                          14-192

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Volatile
Suspended  _  Weight of Volatile,  mg x 1000 ml
Solids,             Volume  of  Sample,  ml
mg/1
              (43.3 mg)  (10_0p|  ml/1)
                     100 ml

           =  433 mg/1
c.  Percent Volatile Solids

o  ,, ,.,..-,  c TJ      Weight  Volatile,  mg x 100%
% Volatile Solids  =  ~~-~e~————...-. ..>\,..A...	,—_
                            Weight  Dry, nig
                              x 100%
                      661 mg             661  433.0
                                                  6
                   =  65.5%                    36 40
                                               33 05
                                               " 3 350
                                                3 305
d.  Fixed Solids

Total Suspended Solids, mg/1      =   661 mg/1
Volatile Suspended Solids, rng/1   =   433 mg/1

Fixed Solids, mg/1                =   228 mg/1


e.  Percent Fixed Solids

Total Solids, %      =  100.00%
Volatile Solids, %   =  _65_._50%

Fixed Solids, %      =    34.5  %

or
o  ,-•  j     Fixed, mg      „
% Fixed  =  —>	*—~ x 100%
            Total, mg
         =  228jng x 10(J%
            661 mg

         =  34.5%  (Check)


                     14-193

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16. C  Calculate Percent Reduction through Primary:
      % Removal  =      -..Out)  x 100%       In = Infl»ent tp
                        jn                       plant or unit

                                           Out = What is leaving
                                                 plant or unit
                 -  C221 mg/ 1-159 wg/1)
                 --- __ - _ x 100,

                     ,7                                 ,28
                          100%                    221 '62.0 "
                                                      44 2
                 =  28% reduction through  primary     ^
                                                      17 63
      Calculate  Percent Removal by Secondary System:

      o  r,      !      (In - Out)    ,_,.0        In = 159
      %  Removal   =  -	- x 100%               .       __,
                        In                       primary effluent
                                           Out = 33 mg/1 SS in
                                                 final effluent
                 =  (159 mg/1 - 33 mg/1)       «,
                          159 mg/1
                                                         .79
                              ,  *      -          159/ 126.0
                 =   79%  removal  from primary
                    effluent  to  final effluent        >  - • •
                                                       14 70
                                                       J4 31

      Calculate Overall  Plant Efficiency:
     %  Removal   -   (In. T. PPtt. x  100%        In  =  2^ 1>g/l  SS in
                        In                        plant influent

                                           Out  =  33 mg/1  SS in
                                                 plant effluent

                              - 33 m/1)
                          221 mg/1

                       x  100%


                =  85.5%  overall plant removal
                          14-194

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16,D  Calculate the pounds of solids removed per day by each unit:

      Amount
      Removed,  =  Cone. Reduction, mg/1 x Flow, MGD x 8.34 Ib/gal
      Ib/day

          where MGD  =  million gallons per day
  A.   Influent, mg/1          =  221 mg/1

      Primary Effluent, mg/1  =  159 mg/1

      Primary Removal, mg/1   =   62 mg/1

      Amount Removed,      ,,„    ,.,. ,. _ .,„.,-.  fa -.
      Ib/day (Primary)  =  <62 mg/1) (1'5 MGD)  (8"34

                        =  775.6 Ibs/day
                           removed by primary


  B.   Primary Effluent, mg/1   =  159 mg/1

      Final Effluent, mg/1     =   53^ mg/1

      Secondary Removal, mg/1  =  126 mg/1

      Amount Removed,        /i-./-    /i-, ri r *tr-^ rn ~,»  TL /   i-.
      Ib/day (Secondly)  =  (126 ^/D (1'5 MGD) (8'34  lb/gal)

                         • =  1576 Ib/day
                             removed by secondary


  C.   Influent, mg/1         =  221 mg/1

      Final Effluent, mg/1   =   55 mg/1

      Overall Removal, mg/1  =  188 mg/1

      Amount
      Removed,   =  (188 mg/1) (1.5 MGD) (8.34 Ib/gal)
      mg/1
                =  2351 Ibs/day
                   removed by plant

      or        =  Primary Removal, Ib/day + Secondary,  Ib/day

                =  775 + 1576

                =  2351  (Check)
                           14-195

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16.E  The advantages of the centrifuge over the regular
      suspended solids test are:

      (1)  Speed of answer!  Not as accurate as
           other methods, but results are sufficiently
           close.

      (2)  Answers very acceptable if suspended solids
           concentration is below 1000 mg/1.

      Disadvantage:  Small plants cannot always afford
      the' $500 or "more cost of the centrifuge.

17.A  Changes in influent temperature could indicate a new
      influent source.  A drop in temperature could be caused
      by cold water from infiltration, and an increase in
      temperature could be caused by an industrial waste dis-
      charge.

17.B  The thermometer should remain immersed in the liquid
      while being read for accurate results.  When removed
      from the liquid, the reading will change.

17.C  All thermometers should be calibrated against an
      accurate National Bureau of Standards thermometer
      because some thermometers can be purchased that
      are substantially inaccurate (off as much as 6°).

18.A  Volatile solids found in a digester are organic compounds
      of either plant or animal origin,

18.B  Volatile solids in a treatment plant represent the waste
      material that may be treated by biological processes.

20.A  Volatile Acid        ml 0.05 N NaOH x 2500
      Alkalinity, mg/1           ml Sample

                           5 ml x 2500
                              50 ml

                        =  250 mg/1

      Since 250 mg/1 > 180 mg/1,

      Volatile Acids,      .. . . .,   .-,»,,,-  -^    ,  ™
        ,,               =  Volatile Acid Alkalinity x 1.50

                        =  250 mg/1 x 1.50

                        =  375 mg/1
                           14-196

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20.B  The alkalinity test is run to determine the buffer capacity
      and the volatile acids/alkalinity relationship in a digester.

20.C  The buffer capacity in a digester as measured by the total
      alkalinity tests indicates the capacity of the digester to
      resist changes in pH.

      VoJLatile Acid  =   500 mg/1
       Alkalinity       2000 mg/1

                     =  0.15
                           14-197

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                           OBJECTIVE TEST

         Chapter 14.  Laboratory Procedures and Chemistry
Name                                              Date
Please write your name and mark the correct answers on the IBM answer
sheet.  There may be more than one correct answer to each question.


TRUE OR FALSE (1-10):

1.  A rubber bulb should be used to pipette wastewater or polluted
    water.

    1.  True
    2.  False

2.  Acid may be added to water, but not the reverse.

    1.  True
    2.  False

3.  Always, wear safety goggles when conducting any experiment in which
    there may be danger to the eyes.

    1.  True
    2.  False

4.  Smoking and eating should be avoided when working with infectious
    material such as wastewater and sludge.

    1.  True
    2.  False

5.  In the washing of hands after working with wastewater, the kind
    of soap is less important than the thorough use of soap.

    1.  True
    2.  False

6.  The pH scale may range from 0 to 14, with 7 being a neutral  solution.

    1.   True
    2.   False
                            14-198

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 7.   If at all  possible,  samples  for the BOD test  should
     be collected before  chlorination.

     1.  True
     2.  False

 8,   The COD test is a measure of the chemical  oxygen
     demand of  wastewater.

     1.  True
     2.  False

 9.   The BOD test is a measure of the organic content  of
     wastewater.

     1.  True
     2.  False

10.   The answers  from the total  solids  and suspended solids
     test are always the  same.

     1.  True
     2.  False
 Possible definitions of the words  listed below are  given  on  the
 right.  For each word listed on the left, try to find  its defini-
 tion on the right.  Mark the number of the definition  in  the
 answer column for each word.  For  example, if the definition of
 a word is after the number 2, mark column 2 on your answer sheet
 after the word.

      Word                          Definition

                               1.   Surrounding

 11.  Aliquot                  2.   Capacity to resist pH change

 12.  Ambient                  3,   Portion of a sample

 13.  Blank                    4.   Inside

 14.  Buffer                   5.   Test run without  sample


 15.  Large errors in laboratory tests may be caused by:

      1.  Improper sampling
      2.  Large samples
      3.  Poor preservation
      4.  Poor quality effluent
      5.  Lack of mixing during compositing

                              14-199

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16.  The most critical factor in controlling digester
     operation is the:

     1.  C02
     2.  Gas production
     3.  Volatile solids
     4.  Volatile acids/alkalinity relationship
     5.  pH

17.  The COD test:

     1.  Measures the biochemical oxygen demand
     2.  Estimates the first-stage oxygen demand
     3.  Measures the carbon oxygen demand
     4.  Estimates the total oxygen demand
     5.  Provides results quicker than the BOD test

18.  A clarity test on plant effluent:

     1.  TellsMf the effluent is safe to drink
     2.  Is measured by an amperemeter
     3.  Should always be measured at the same time
     4.  Should always be measured under the same light conditions
     5.  Is measured by a Secchi Disc

19.  Coliform group bacteria are:

     1.  Measured by the membrane filter method
     2.  Measured by the multiple fermentation technique
     3.  Measured by the modified Winkler procedure
     4.  Harmful to humans
     5.  Indicative of the potential  presence of bacteria
         originating in ttie intestines of warm-blooded animals

20.  The saturation concentration of dissolved oxygen in
     water does not vary with temperature.

     1.  True
     2.  False

21.  DO probes are commonly used to measure dissolved oxygen
     in water in:

     1.  Aeration tanks
     2.  Sludge digesters
     3.  Manholes
     4.  Streams
     5.  BOD bottles
                             14-200

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22.  Hydrogen sulfide:

     1.  Reacts with moisture and oxygen to form a substance
         corrosive to concrete
     2.  Is sometimes written as I^S
     3.  Smells like rotten eggs
     4.  Is formed under aerobic conditions
     5.  Should not be controlled in the collection system.

23.  Results from the settleability test of activated sludge
     solids may be used to:

     1.  Calculate SVI
     2.  Calculate SDI
     3.  Calculate sludge age
     4.  Determine ability of solids to separate from liquid
         in final clarifier
     5.  Calculate mixed liquor suspended solids.

24.  Results of the settleable solids test run using Imhoff
     cones may be used to:

     1.  Calculate the Imhoff Settling Index
     2.  Calculate the efficiency of a treatment process.
     3.  Calculate the pounds of solids pumped to the digester
     4.  Indicate the quality of the influent
     5.  Indicate the quality of the effluent

25.  Precautions that must be observed in running the suspended
     solids-Gooch crucible test include:

     1.  Collecting and testing a representative sample
     2.  Proper temperature level in oven at all times
     3.  Lack of leaks around and through the glass fiber
     4.  Thoroughly mixing sample before testing
     5.  Discarding any large chunks of material in sample

26.  A chlorine residual should be maintained in a plant effluent:

     1.  To keep the chlorinator working
     2.  For disinfection purposes
     3.  For testing purposes
     4.  To protect the bacteriological quality of the receiving
         waters
     5.  None of these
                             14-201

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                                              PLANT
                                              DATE
                       SUSPENDED SOLIDS & DISSOLVED SOLIDS
, SAMPLE
Crucible
Ml Sample
Wt Dry & Dish
Wt Dish
Wt Dry
/I _ Wt Dry, gm x 1 ,000,000
; Ml Sample
: ' Wt Dish & Dry
Wt.Dish & Ash
-.- . yt-V.Qla.t11e
1 Vnl - Wt Vo1 x 100
-•. ^•"rM-.vWfr.Dry x IUU

























































                                         BOD
Nitrate
Sample
Graph Reading _

COD
Sample
Blank Titration
Sample Titration
Depletion
mnn   Pep x N FAS x 8000
mg/1 =     Ml Sample -
                                                   # Blank
'SAMPLE
DO Samole
Bottle #
% Sample
Blank or adj blank
DO after incubation
Depletion, 5 days
Dep %




























































Sett. Solids
Sample

Direct Ml/I
                       Typical Laboratory Work  Sheet

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                                            TOTAL SOLIDS
SAMPLE
  Dish No.
  Wt Dish & Wet
  Wt Dish
  Wt Wet
  Wt Dish +
  Wt Dish
  Wt Dry
  % Solids
          Dry
          Wt Dry
          Wt Wet
x 100%
                   o1
                      x 100%
Wt Dish + Dry
Wt Dish + Ash
Wt Volatile
% Volatile = *  n
             Wt Dry
pH
Vol.  Acid
Alkalinity as CaC03

Grease (Soxlet)
  Sample
  Ml  Sample
  Wt Flask + Grease
  Wt Flask
  Wt Grease
  ma/1  =  Wt Grease, mg x 1000
   y           Ml  Sample
  H2S (Gas)   (Starch-Iodine)
    Blank
    Sample
    Diff               ZZ
    Diff x .68
    mg/1  x 43.6
                                  Ml
                                  Ml
                                  Ml
                                  mg/1
                                  grain/100 cu  ft
                      Typical  Laboratory Work Sheet (Continued)

                                          14-203

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
1. REPORT NO.
                                                             3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE

 Wastewater Laboratory  Procedures and Chemistry
                                                             5. REPORT DATE
             6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
                                                             8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
                                                             1O. PROGRAM ELEMENT NO.
 U. S. Environmental Protection Agency
 Office  of Intermedia Programs
 Manpower and Training  Program
 Region  VII,  1735 Baltimore, Kansas City, Missouri  64108
             11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
                                                             13. TYPE OF REPORT AND PERIOD COVERED
                                                             14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
 This manual has been adapted from Chapter 14 of "Operation of Wastewater Treatment
 Plants  - A Field Study Course" for  limited distribution in Region VII.   This manual
 is intended to serve as a training  reference material for personnel of the Regional
^Surveillance and Analysis Program to  assist them  in providing assistance to treatment
 plant operators who have been identified as being in need of greater skills to perform
 necessary  laboratory analyses.
17.
                                 KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                               b.IDENTIFIERS/OPEN ENDED TERMS
                            c. COS AT I Field/Group
 Water Pollution Control  Laboratory
 Analysis
  Water Pollution Control
   DISTRIBUTION STATEMENT


 LIMITED
19. SECURITY CLASS (This Report)'
  UNCLASSIFIED
21. NO. OF PAGES
       240
20. SECURITY CLASS (This page)
  UNCLASSIFIED
                            22. PRICE
EPA Form 2220-1 (9-73)
                                            14-204

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