U.S. DEPARTMENT OF COMMERCE
                          National Technical Information Service
                           PB-261 260
 Chemical Analyses  for
 Water Pollutants
 National Training &  Operational Technology Or., Cincinnati,  OH.
Nov 76

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        { b b 0 8 4
PB  261 260
EPA 430/1 IS 013
CHEMICAL  ANALYSES FOR WATER

POLLUTANTS
 TRAINING MANUAL




 U.S. ENVIRONMENTAL PROTECTION AGENCY


 OFFICE OF WATER PROGRAM OPERATIONS

               NAT|ON.V. "ECHNICAL
              INFC *'.' :>. SERVICE

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 BIBLIOGRAPHIC DATA
 SHEET
                     1. Report No.
                       eport No.
                       EPA-4UO/1-75-013
3. Recipient's Accession No.
 J!L
'4. Title and "Subtitle	

    Chemical Analyses for Water Pollutants
                                                                    5. Report Date
                                                                      November 1976
 '. Author(s)     ^
     Audrey E.  Kroner
                                                                    8- Performing Organization Kept.  !
                                                                      No.                  "   1
 '. Performing Organization Name and Address
    U.S.  Environmental Protection Agency, OWPO
    Municipal Operations and Training Division
   .National Training & Operational Technology Center
    Cincinnati, Ohio 45268
                                                                    10. Project/'TasK, Work Unit No.  ;
                                                                    11. Contract/Grant No.
12. Sponsoring Organization Name and Address
                                                                    13. Type of Report & Period
                                                                       Covered
                                                                     14.
 15. Supplementary Notes
 16. Abstracts

    This  training  manual contains outlined information used by Lecturers  when
    presenting topics in EPA-NTOTC Course 100. 3, "Chemical Analyses for Water
    Pollutants. "  The contents concern individual constituents found in water (e. g.
    dissolved oxygen) and information useful to analysts (e.g. statistics).  A section
    of laboratory  procedures adapted for class groups is included.
 17. Key Words and Document Analysis.  17a. Descriptors

    Acidity,  Alkalinity,  Ammonia, Biochemical  Oxygen Demand,  Carbon,
    Dissolved gases. Hardness,  Inorganic nitrates, Nitrites,  Nitrogen cycle,
    Oxygen,  Phosphorus, Quality Assurance,  Statistical Analysis, Data Collection
    and Evaluation.
 17b. Identifiers/Open-Ended Terms
 17e. COSATI Fie Id/Group
                                                          19. Security Class (This    |21. "No. of Pages
                                                            Report)                   '         j
                                                          	UNCLASSIFIED                    j
18. Availability Statement

   Release to public
                                                          20. Security Class (This
                                                            Page
                                                               UNCLASSIFIED
    1 NTIS-35 IRdV. 3-7ZI
                                                                               USCOMM-OC r

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                                                     EPA-430/ 1-75-01 3
                                                     December  1975
CHEMICAL ANALYSES  FOR  WATER POLLUTANTS
   This course is designed for chemists or technicians
   who will perform chemical analyses for water pollutants.

   The student will learn about analyses listed in the
   guidelines developed pursuant to Section 304(g) of the
   Federal Water Pollution Control Act Amendments of
   1972 for NPDES permit applications and reports and
   also for certifications issued by the States.

   The student will learn theoretical concepts and perform
   laboratory exercises pertaining to the following: Acidity
   and Alkalinity Determinations, pH Measurement,
   Biochemical Oxygen Demand, Dissolved Oxygen
   (Winkler and Probe), Calcium and Magnesium Hard-
   ness Determination, Nitrogen Analyses (Ammonia,
   Kjeldahl, Nitrate, Nitrite), Phosphorus Determinations,
   Total Organic Carbon Analysis, Total Solids Deter-
   mination.

   Discussion periods are scheduled after each laboratory
   assignment is completed.  Time is divided about equally
   between classroom and laboratory work.

   Persons attending should have fundamental knowledge
   of inorganic chemistry  and quantitative analysis.  They
   should also be able to use  an analytical balance,  volu-
   metric glassware and titration assemblies.
      U. S.  ENVIRONMENTAL PROTECTION AGENCY
            Office of Water Program Operations
                TRAINING  PROGRAM

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                            FOR KW OK l>
These  mainuals are prepai-ed for reference use of students enrolled in
scheduled training courses of the Office of Water Program Operations.
Environmental Protection Agency.

       Due to the limited availability of the manuals it is not
       appropriate to cite them as technical references  in
       bibliographies or other forms of publication.

       References to products and manufacturers are for
       illustration only; such references do not imply product
       endorsement by the Office of Water Program
       Operations, Environmental Protection Agency.

The reference outlines in this manual have been selected and  developed
with a goal of providing the student with a fund of the best available
current information pertinent to the  subject matter of the course.
Individual  instructors may provide additional material to cover special
aspects of their own  presentations.

This manual will  be useful to anyone who has need for information on
the subjects covered.  However,  it should be understood that  the manual
will have its greatest value as an adjunct to classroom presentations.
The inherent advantages of classroom presentation is in the give-and-
take discussions and exchange of information between and among
students and the instructional staff.

Constructive suggestions for improvement in the coverage, content,
and format of the manual are solicited and will be given full
consideration.

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                                         CONTENTS



 Title or Description                                                  Outline Number

 I  DATA COLLECTION AND EVALUATION

    Sample Handling - Field Through Laboratory                                 1

    Statistics for Chemists                                                      2

    Accuracy-Precision-Error                                                  ^

    Interlaboratory Quality Control Studies                                       4

 II  STANDARD SOLUTIONS, ACIDITY, ALKALINITY

    Volumetric Analysis of Water Quality                                        5

    Acidity.  Alkalinity, pH and Buffers                                          6

    Alkalinity and Relationships Among the Various Types of Alkalinities           7

III  OXYGEN TESTS

    Dissolved Oxygen - Factors Affecting DO Concentrations in Water             8

    Dissolved Oxygen Determination (Winkler lodometric Titration and             8
    Azide Modification)

    Dissolved Oxygen Determination - Electronic Measurements                   9

    Biochemical Oxygen Demand Test Procedures                               10

    BOD Determination - Reaerated Bottle Probe Technique                      10

    Effect of Some Variables on the BOD Test                                   10

    Mathematical  Basis of the Biochemical Oxygen Demand (BOD) Test           10

IV  SELECTED ANALYSES

    Sources and Analysis of Organic Nitrogen                                   11

    Ammonia,  Nitrites and Nitrates                                            11

    Total Carbon Analysis                                                     12

    Determination of Calcium and Magnesium Hardness                          13

    Solids Relations in Polluted Water                                          14

    Phosphorus in the Aqueous Environment                                     15
  100.3. 11.75

                                          II

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2	C o n_t e n t s	










Title or Description                                                   Outline Number





V  LABORATORY PROCEDURES




   Biochemical Oxygen Demand Test - Dilution Technique                      16




   Laboratory Procedure for Dissolved Oxygen (Winkler-Azide Procedure)      17




   Determination of Kjeldahl Nitrogen                                        18




   Determination of Nitrate-Nitrite Nitrogen                                  19




   Laboratory Procedure for Total Solids                                     20




   Laboratory Procedure for Total Hardness                                  21




   Laboratory Procedure for Phosphorus                                     22




   Acidity                                                                  23




   Laboratory Procedure for Total Alkalinity                                 24

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                    SAMPI.K HANDLING  - KII-: LP THROmi f I  \HOK\TOKY
I  PLANNING A SAMPLING PROGRAM

A  Factors to Consider:

   1  Locating sampling sites

   2  Sampling equipment

   3  Type of sample required

      a  grab
      b  composite

   4  Amount of sample  required

   5  Frequency of collection

   6  Preservation measures, if any

 B Decisive  Criteria

   1  Nature of the sample source

   2  Stability of constituent(s) to be measured

   3  Ultimate use of data


II  REPRESENTATIVE SAMPLES

   If a  sample is to provide meaningful and
   valid data about the parent population, it
   must be representative of the conditions
   existing in that parent source at the
   sampling location.

 A The container should be rinsed two or
   three times with the water to be collected.

 B Compositing Samples

   1  For some sources, a composite of
      samples is made which will represent
      the average situation for stable
      constituents.

   2  The nature of the constituent to be
      determined may require a series of
      separate samples.
 C  The equipment used to collect ;nc sample
    is an important factor to consider.
    ASTM* ' has a detailed section on various
    sampling devices and techniques.

 D  Great care must be exercised when
    collecting samples in sludge  or mud areas
    and near benthic deposits.  No definite
    procedure can be given,  but careful
    effort should be made to obtain a rep-
    resentative sample.
Ill  SAMP LE IDENTIFICATION

 A  Each sample must be unmistakably
    identified,  preferably with a tag or label.
    The required information should be planned
    in advance.

 B  An information form preprinted on the
    tags or labels provides uniformity of
    sample records,  assists the sampler, and
    helps  ensure that vital information will
    not be  omitted.

 C  Useful Identification Information includes:

    1  sample  identity code
    2  signature of sampler
    3  signature of witness
    4  description of sampling location de-
       tailed enough to accommodate repro-
       ducible  sampling.  (It may be more
       convenient to  record the details in the
       field record book).
    5  sampling equipment used
    6  date of  collection
    7  time of  collection
    8  type of  sample (grab or composite)
    9  water temperature
   10  sampling conditions such as weather,
       water level, flow rate of source, etc.
   11  any preservative additions or Techniques
   12  record  of any  determinations done in
       the field
   13  type of  analyses to be done in laboratory
 WP.SUR.sg. 6. 3.74
                                                                                       1-1

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 Sample Handling - Field Througji  Laboratory	
IV  SAMPLE CONTAINERS

 A  Available Materials

    1  glass
    2  plastic
    3  hard rubber

 B  Considerations

    1  Nature of the sample  -  Organics
       attack polyethylene.

    2  Nature of constituents) to be determined
       -  Cations can adsorb readily on soine
       plastics  and on certain  glassware.
       Metal or aluminum foil cap liners can
       interfere with metal analyses.

    3  Preservatives to be used - Mineral
       acids attack some plastics.

    4  Mailing Requirements - Containers
       should be large enough  to allow extra
       volume for effects of temperature
       changes  during transit.  All caps
       should be securely in place. Glass
       containers must be protected against
       breakage.  Styrofoam linings are
       useful for protecting glassware.

 C  Preliminary Check

    Any  question of possible interferences
    related to the sample container should
    be resolved before the study begins.  A
    preliminary check should be made using
    corresponding sample materials,  con-
    tainers, preservatives and analysis.

 D  Cleaning

    If new containers are to be used,  prelim-
    inary cleaning is usually not necessary.

    If the sample containers have been used
    previously,  they should be carefully
    cleaned before use.

    There are several cleaning methods
    available.   Choosing the best method in-
    volves careful consideration of the nature
    of the sample and of the constituent(s) to
    be determined.
   i   Phosphate detergents should r.oi bo
      used to clean containers for phosphorus
      samples.

   '2   Traces of dichromatic cleaning  solution
      will interfere v, ith metal analyses.

L  Storage

   Sample containers should be stored and
   transported in a manner to assure their
   readiness for use.
V  SAMPLE PRESERVATION

   Every effort should be made to achieve
   the  shortest possible  interval between
   sample collection and analyses.  If there
   must be a delay and it is long enough to
   produce significant changes in the sample,
   preservation measures are required.

   At best, however,  preservation efforts
   can only retard changes that inevitably
   continue after the sample is removed
   from the parent population.

A  Functions

   Methods of preservation  are relatively
   limited.  The  primary functions of those
   employed are:

   1  to retard biological action
   2  to retard precipitation or the hydrolysis
      of chemical compounds and  complexes
   3  to reduce volatility of constituents

B  General Methods

   1  pH control - This  affects precipitation
      of metals,  salt formation and can
      inhibit bacterial action.
   2  Chemical Addition - The choice of
      chemical depends on the change to be
      controlled.
      Mercuric chloride is commonly used
      as a bacterial inhibitor.  Disposal of
      the mercury-containing samples is a
      problem  and efforts to find a substitute
      for this toxicant are underway.
    1-2

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                                        Samp!< Handling -  I'lciii Tlinuigh I
      To dispose of solutions of inorganic
      mercury salts, a recommended
      procedure is to capture and retain the
      mercury salts as the sulfide at a high
      pH.  Several firms have tentatively
      agreed to accept the mercury sulfide for
      re-processing after preliminary con-
      ditions are
    3  Refrigeration and Freezing - This is
       the best preservation technique avail-
       able, but it is not applicable to all
       types of samples.  It is not always a
       practical technique for field operations.

 C  Specific Methods

    The EPA Methods Manual    includes a
    table summarizing the holding times and
    preservation techniques for several
    analytical procedures.  This information
    also can be found in the standard refer-
    ences (! 2 3) as part of the presentation
    of the individual procedures.
VI  METHODS OF ANALYSIS

    Standard reference books of analytical
    procedures to determine the physical
    and chemical characteristics of various
    types of water samples are available.

 A  EPA Methods Manual

    The Methods Development and Quality
    Assurance Research Laboratory of the
    Environmental Protection Agency, has
    published a manual of  analytical procedures
    to provide methodology for monitoring  the
    quality of our Nation's Waters and to deter-
    mine the impact of waste discharges.   The
    title of this manual is  "Methods for Chem-
    cal Analysis of Water  and Wastes. "<2>

    For some procedures, the analyst is
    referred to Standard Methods and/or to
    ASTM Standards.

 B  Standard Methods

    The American Public Health Association,
    the American Water Works Association
    and the Water Pollution Control Federation
    prepare and publish a volume describing
    methods of water analysis.   These include
    physical and chemical proceaures.  The
    title of this book is "Standard Methods
    for the Examination of Water and Waste -
    water. "<3>

C   ASTM Standards

    The American Society for Testing and
    Materials publishes an annual "book"
    of specifications and methods for testing
    materials.  The "book" currently con-
    sists of 33 parts.   The part applicable
    to water is a book titled,  "Annual Book of
    ASTM Standards,  Part 23. Water;
    Atmospheric Analysis".   '

D   Other References

    Current literature and other books of
    analytical procedures with related in-
    formation are available to the analyst.

 E  NPDES Methodology

    When gathering data for National Pollutant
    Discharge Elimination System report
    purposes,  the analyst must consult the
    Federal Register for a listing of approved
    analytical methodology.  There he will  be
    directed to pages in the above cited
    reference books  where acceptable pro-
    cedures can be found.  The Federal
    Register also provides information con-
    cerning the protocol for obtaining approval
    to use analytical procedures other than
    those listed.
VII  ORDER OF ANALYSES

     The ideal situation is to perform all
     analyses shortly after sample collection.
     In the practical order,  this is rarely
     possible.  The allowable holding time
     for preserved samples is the basis
     for scheduling analyses.

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  Sample Handling - Field Through Laboratory
 A   The allowable holding time for samples
     depends on the nature of the  sample, the
     stability of the constituent(s) to be de-
     termined and the conditions of storage.

     1  For some constituents and physical
        values, immediate determination is
        required, e.g. dissolved  oxygen, pH.

     2  Using preservation techniques, the
        holding times -for other determinations
        range from  6 hours (BOD) to 7 days
        (COD). Metals may be held up to 6
         months.^2'
                                 (2)
     3  The EPA Methods Manual   includes
        a table summarizing holding times and
        preservation techniques for several
        analytical procedures.  This information
        can also be  found in the standard
                  (123)
        references  ' '   as part of the
        presentation of the individual
        procedures.

     4  If dissolved concentrations are
        sought, filtration should be done in
        the field if at all possible.  Other-
        wise, the sample is filtered as soon
        as it is received in the laboratory.
        A 0.45 micron membrane filter is
        recommended for reproducible
        filtration.

 B   The time interval between collection
     and analysis is important and should be
     recorded in the laboratory record book.
VIII RECORD KEEPING

     The importance of maintaining a bound,
     legible record of pertinent information
     on samples cannot be over-emphasized.

 A   Field Operations

     A bound  notebook should be used. Informa-
     tion that should be recorded includes:
    1  Sample identification records (Sec
       Part UI)

    2  Any information requested by the
       analyst as significant

    3  Details of sample preservation

    4  A complete record of data on any
       determinations done in the field.
       (See  B, next)

    5  Shipping details and records

B   Laboratory Operations

    Samples should be logged in  as soon as
    received and the analyses performed
    as soon as possible.

    A bound notebook should be used.
    Preprinted data forms provide uniformity
    of records and help ensure that required
    information will be recorded.  Such sheets
    should be permanently bound.

    Items in the laboratory notebook would
    include:

    1  sample identifying code
    2  date and time  of collection
    3  date and time of analysis
    4  the analytical method used
    !i  any deviations from the analytical
       method used and why this was done
    6  data obtained during analysis
    7  results of quality control checks on the
       analysis
    8  any information useful to  those who
       interpret and use the data
    9  signature of the analyst
   1-4

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                                                Sample Handling - Field Through Laboratory
IX  SUMMARY

 Valid data can be obtained only from a repre-
 sentative sample,  unmistakably identified,
 carefully collected and stored.  A skilled
 analyst, using approved methods of analyses
 and performing the determinations within
 the prescribed time limits,  can produce data
 for the sample.  This data will be of value
 only if a written record exists to verify sample
 history from the field through the laboratory.

      REFERENCES

 1    ASTM Standards, Part 23,  Water; .
        Atmospheric Analysis.

 2    Methods for Chemical Analysis of Water
        and Wastes, EPA-MDQARL,
        Cincinnati, OH  45268.  1974.

 3    Standard Methods for the Examination of
        Water and Wastewater, 13th edition,
        APHA-AWWA-WPCF,  1971.
   Dean, R., Williams, R. and Wise, R.,
     Disposal of Mercury Wastes from
     Water Laboratories, Environmental
     Science and Technology, October,  1971.
This outline was prepared by A. Donahue,
Chemist, National Training Center, MPOD,
OWPO. EPA, Cincinnati, Ohio 45268.
Descriptors: On-Site Data Collections,
On-Site Investigations, Planning, Handling,
Sample, Sampling, Water Sampling,
Surface Waters, Preservation, Wastewater
                                                                                       1-5

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                                 STATISTICS FOR CHEMISTS
 I   INTRODUCTION

 A  Statistics may be defined, for our purpose,
    as a collection of methods which have been
    developed for handling numerical data
    pertaining to samples or portions of entire
    populations.

 B  The statistical methods with which we will
    concern ourselves deal with the presentation
    and analysis of numerical data from samples.
H   FREQUENCY

 A  Definitions

    1  Frequency - indicates how many times
      a particular score occurs in a collection
      of data
Frequency table -  a tabular arrange-
ment of data, ranked in ascending or
descending order of magnitude,
together with the corresponding
frequencies

Frequency histogram  - a set  of
rectangles having bases on a  horizontal
axis with centers at the given scores
and heights equal to the corresponding
frequencies (See Figure 1)

Frequency polygon - a line graph of
frequencies plotted against scores
(can be obtained by connecting mid-
points of tops of rectangles in the
frequency histogram)  (See Figure 1)
                                              Figure 1

                             Frequency Histogram & Frequency Polygon
Frequency
>-> CO GO 4^ O
1 1 1 1 1
X

X
X
X
j
X


^
V
s
X
\
s
\
\
\
1 1 1 1 1
98 99 100 101 102
Chloride ug/l
  ST.25b.11.75
                                                                                       2-1

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 Statistics For Chemists
B  Application

   Consider the application of the above
   definitions to the following set of data,
   obtained from twelve determinations for
   chloride in water.
               Results (tig/1)
       100        101          99

       101        100         100

        99        102         100
        98        101         102
                                                        numbcT of obscrvntions Hie iiiedian is
                                                            '-'!  *  I
                                                                     tin1 :i v t' ra^r < >1 LIi
                                                  middle two scores.

                                               4  Mean -  arithmetic average of all the
                                                  values in the sample distribution,  de-
                                                  noted by X.   The formula for calcula-
                                                  ting the sample mean is
                                                  X=  *!-
                                                                         x
                                                       n
                                                         X,
                 Table 1
              Frequency Table
Chloride (ng/1)
98
99
100
101
102
Frequency
1
2
4
3
2
II  MEASURES OF CENTRAL TENDENCY

 A  Definitions

    1  Central tendency - the tendency of
       values to cluster about a particular
       value in the distribution

    2  Mode - that value which occurs most
       frequently

    3  Median - midpoint of an array of
       scores.  If there is an odd number of
       observations, n,  the median is
        xn +1    where  Xn +
           2
          n+ 1
            2
      distribution.
                                    represents
the
                 value in the frequency

                     If there is an even
                                                  	      4
                                                  X = l   where there are n number
                                                            of values.
                                                   B  Aids in calculation of the mean

                                                      Application of the following two  statements
                                                      can reduce errors and amount of time
                                                      spent in calculating the mean  of a
                                                      distribution.

                                                      1   Adding or subtracting a constant to or
                                                         from each score in a distribution is
                                                         equivalent to adding or subtracting the
                                                         same constant to or from the mean of
                                                         the distribution.   Thus the  following
                                                         formula:
                                                         X  = X C
                                                          c
                                                                where the Xj's are the
values in the distribution with mean X,
and the Xj  C's are the_values in the
distribution with mean Xc.

Multiplying or dividing each score in
a distribution by a constant is equivalent
to multiplying or dividing the mean of
the distribution  by the same constant.
Thus the following formulas:

(1) Xc = CX

or

(2) Xc = -   where  the X^s are the

values in the distribution with mean X,
  2-2

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                                                                   Statistics for Chemists
      and the CXj's or the - "s are the

      v_alues in the distribution with  mean
      X
C  Application

   Consider the application of the above
   definitions to the previously mentioned
   set of data, obtained from twelve deter-
   minations for chloride in water, shown
   in Table 1.

   1  Mode =  100

100 + 100
2 2
2
= inn
_ X6 + X7
2
       Denote the mean of the distribution in
       Table  1 by X .   If we add 100 to each
       score in the distribution in Table 2, we
       obtain the scores in the distribution in
       Table 1; likewise if we add 100 to the
       mean,  X,  of the distribution  in Table 2,
       we obtain the mean, X , of the distri-
       bution in Table  1.
       Thus X  = X + 100
             c
X  = -=
 c    n
                         100
                                                  -  =  l(-2) + 2(-l) + 4(0) + 3(1) + 2(2)  +
                                                  X
                                                                     12
                                                                                         100
                                                  X   =  .25 + 100 = 100.25
                                                   c
   3  Mean
              n
    _  98 + 2 (99) + 4 (100) + 3 (101) + 2 (102)
                       12

    =  100.25

   4  Aid in Calculation

      Consulting Table 1  and observing that
      the values are in the neighborhood of
      100 we might subtract 100 from each
      score and obtain the following distribution:
                   Table 2

                Frequency Table

    Chloride (fig/1)           Frequency

         -2                       1

         -1                       2

          0                       4

          1                       3

          2                       2
IV  MEASURES OF DISPERSION

 A Definitions

    1  Dispersion - spread or variability of
       observations in a distribution

    2  Range - the difference between the
       highest value and the lowest value

       R =  max - min

    3  Average deviation - the sum of the
       deviations of the values from their
       mean, without  regard to sign, divided
       by the total number of data values (n)

       The formula for calculating the average
       deviation is:
                                                                                       2-3

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Statistics for Chemists
      Average deviation of the mean (D) -
      the average deviation of individual
      data items from the mean (d) divided
      by the square root of the number of
      data items (n)

      The. definition of the average deviation
      of the mean can be expressed by the
      formula:
               d
        D  =
      Variance - the sum of the squares of
      the deviations of the values from their
      mean divided by the total number of
      data values (n) minus 1

      The definition of the variance can be
      expressed by the following formula:

                 - X)2
              n  -  1
      Standard deviation - the square root
      of the variance

      The definition of the standard deviation
      can be expressed by the following
      formula:
               - X)2
     However, the formula commonly used
     because of its adaptability to the hand
     calculator is the following:
      a -
              n - 1
where there are
n number of values.
                              The dei'iuil.iim ui' the .stundur
                              deviation of the mean can be
                              expressed by the- formula:
                              S  =
       s

        n
                           8  Relative standard deviation - the
                              standard deviation (s) expressed as
                              a fraction of the mean,  s

                                                     X

                              The relative standard deviation is
                              often expressed as a percent. It is
                              then referred to as the coefficient
                              of variation (V):

                              V  = ; X 100  =  %
                                   X

                              The relative standard deviation is
                              particularly helpful when comparing
                              the precision of a number of deter-
                              minations on a given substance at
                              different levels of concentration.

                        B  Aids in Calculation

                           Application of the following statements
                           can reduce errors and amount of time
                           spent in calculating the variance or
                           standard deviation of a distribution.

                           1  Adding or  subtracting a constant to or
                              from each score in a distribution
                              doesn't affect the variance  or standard
                              deviation of the distribution.
                              Thus the following formulas:

                              (D
        2   2
      s   = s
       c
     Standard deviation of the mean (S) - the
     standard deviation of individual data
     items (s) divided by the square root of
     the number of data items (n)
(2)    s  = s
       c

      where the X^'s are the values in
      the distribution with variance s
      and standard deviation s, and the
      X.- +  C's are the values in the
       A                         o
      distribution with variance s
      and standard deviation s  .
  2-4

-------
                                                                    Statistics for Chemists
2 Multiplying or dividing each score in
a distribution by a constant is tMjuivnlent
to multiplying or dividing the variance
of that distribution by the square of the
same constant.
Thus the following formulas:

9 99
(1) B* * C S
C _
2 s2
(2) s 2 - 
c *

2

n
1
2
4
3
2



-*
98
99
100
101
102
X = 100.
deviation - d

IXi- X|

2.25
1.25
. 25
. 75
1.75
25


nlX

2
2
1
2
3
11

-------
Statistics for Chemists

f
5 Standard deviation - s = / 
si
n Xj nXj X ,
1 98 98 9604

2 99 198 9801
4 100 400 10000
3 101 303 10201
2 102 204 10404
1203

/ 2
/*> -3? /
a V n v
, (3)2
2 (XiH 2 . 12 _ 16.25 .
Xi - n  u n !

n - 1
nX^i
9604 /EX2- (EX )2 	
s = / i i /I 48 ^ 1  '"*
19602 / -5^ y
40000 * n - 1
30603
20808 7 Standard deviation of the mean -
120617 s
vTir

ci* ioncm Using calculations from number 6,
91 1 - llUOUl
11
    rTT     1.21
  6  Aid in calculation

     Recalling that adding or subtracting a
     constant to each score in the distri-
     bution doesn't affect the variance or
     the standard deviation of the distribu-
     tion we can simplify the computations
     by first subtracting 100 from each
     score in the distribution, thus obtain-
     ing the frequency distribution shown
     in Table 2.
  n

  1

  2

  4

  3

  2
               nCXj-C)  (XrC)2  n(XrC>2
            n - 1
8  Relative standard deviation expressed
   as a percent (coefficient of variation)

   v=-S-  xioo
       X
   Using calculations from number 6 for
   s = 1. 22 and from number 2 for
   X = 100.25,
                                                       V =-
                                                           X
              1.22
             100.25
X  100
               Figure 2
       Normal Distribution Curve
                                                              Quantity Measured
2-6

-------
                                                                    Statistics for Chemists
V  INTRODUCTION TO NORMAL
   DISTRIBUTION CURVE

 A Statistics deals with theoretical curves
   which are smoother than frequency
   polygons, obtained from experiments In
   real life.  However, frequency distribu-
   tions or frequency polygons of experimental
   data often approximate a mathematical
   function called the "normal" distribution
   curve.  (See Figure 2)
                             As shown in Figure 3,  the frequency polygon
                             for the  12 determinations for chloride in
                             water is a fairly good approximation of the
                             normal curve.  If, however,  in the chloride
                             determinations we had obtained 103  instead
                             of 98 and 104 instead of 99 this distribution
                             would not have- been a good approximation of
                             the normal curve, as is shown in Figure 4.
                                           Figure 3

                      Comparison of Normal Curve and Frequency Polygon
       o
       0)
       g.
       V
       (-,
       fc
97       98       99      100      101

                     Chloride ng/1
                                                             102
                                           Figure 4

                      Comparison of Normal Curve and Frequency Polygon
        o
        0)
        .
        0>
                        99
                                                                                       2-7

-------
Statistics for Chemists
  If a frequency distribution is a good
  approximation of the normal curve, we
  can use some facts about the normal
  curve to give us information about the
  frequency distribution.
Figure 5 shows the normal distribution
in terms of the population mean a,  and
the  standard deviation of the population
a ,  and gives the percent of area under
the  curve between certain points.
                                          Figure 5

                                 Normal Distribution Curve
                                                     + l
-------
                                                                 Statistics lot1 C
We may check the distribution of sample
data to see if it is a "normal" distribution
in the following manner.  Substitute the
value of the sample mean (X)  for the value
of the midline and substitute the value of
the sample standard deviation (s) for the
limits of the value  spans where we might
expect certain percentages of the data
items to occur.   Then we can check the
number of data items which actually do
occur within these  value spans.

Figure 6 demonstrates this application
using the chloride data values from Table 1.
The data values are marked on the hori-
zontal line and the frequency  of the
occurrence of each value is marked on the
vertical.  The midline of the  distribution
is marked at the value of the  sample mean
(X = 100, See m C 3). The value  of the
sample standard deviation (s  = 1.21, See
IV C 5) is used to mark value areas under
the curve where  different percentages of
data values will probably occur.   Thus,
for the area X + Is, X  - Is * 98. 79 and
X + 1 B - 101.21.  Therefore, according
to the normal distribution curve shown in
Figure 5, we might expect about 68% of the
data items to have values between 99 and
101. (The values are rounded to whole
numbers since the data values are thus
recorded).

Consulting Table 1. we find that 75% or 9
of the 12 data items have values in this
range.   This percentage is shown  in
Figure 6 by the frequency polygon for the
data shown earlier in Figure  3.

 Likewise assuming a normal distribution,
we would expect 95% of the observations
to lie within + 20 's from the  population
mean.  In fact,  100% of the observations
were within + 2  s's from the  sample mean.

In both cases the observed percentages are
 reasonably close to the expected percentages.
Other tests exist for determining whether
 or not a frequency distribution might
 reasonably be assumed to approximate
 the normal distribution.
   It would be good to become as familiar as
   possible with the' normal distribution siru
   an underlying normal distribution in
   assumed for many statistical tcwta  ol
   hypothesis.
REFERENCES

1  Bennett,  C.A. and Franklin, N. L.
      Statistical Analysis in Chemistry and
      the Chemical Industry.  John Wiley
      & Sons, Inc., New York.  1954.

2  Crow,  E. L., Davis, F.A., and Maxfielri,
      M.W.  Statistics Manual.   Dover
      Publications, Inc., New York.   1960.

3  Dixon, W.J.  and Massey, F. J.
      Introduction to Statictical Analysis.
      McGraw-Hill Book Co., Inc.,  New
      York.  1957.

4  Ostle,  B.  Statistics in Research.  The
      Iowa State University  Press, Iowa,
      1963.
   Youdon,  W.J.
      Chemists.
      New York.
 Statistical Methods for
John Wiley & Sons,  Inc
 1951.
 This outline was prepared by L. A. Lederer,
 Statistician,  formerly with Analytical
 Reference Service. Training Program,
 NCUIH,  SEC.  Revised by Audrey E.
 Donahue, Chemist, National Training  Center,
 MPOD, OWPO. USEPA. Cincinnati, OH 45268
 Descriptors:  Graphic Methods, Quality Control,
 Statistical Methods, Statistics
                                                                                    9-0

-------
                              ACCURACY-PRECISION-ERROR
I  INTRODUCTION

An analytical method is subject to errors.
These errors  may affect the accuracy of the
method because they introduce bias into the
results. There are other types of errors
which affect the precision of the method
because they produce random fluctuations in
the data.  The most desirable situation for
the analyst is  shown in the diagram in
Figure 1 where the results are both precise
and accurate.
                                  X

                                  X
        IMPRECISE  AND INACCURATE
          PRECISE BUT INACCURATE
          ACCURATE BUT IMPRECISE
           PRECISE AND  ACCURATE

   Figure 1.  PRECISION AND ACCURACY
   For results to be accurate,  the analysis
   used must give values close to the true
   value.(See Figure 1)

 B Precision

   Precision is the degree of agreement
   among results obtained by repeated
   measurements on a single sample under
   a given set of conditions.  It is a measure
   of the degree to which results "check. "
   (See Figure 1)

 C Note

   It is possible to have precision without
   accuracy. (See Figure 1)
II   DETERMINATE ERROR AND ACCURACY

 A determinate error is one which con-
 tributes a constant error or bias to results,
 causing them to be inaccurate.  This
 constant error makes it possible for results
 to agree with each other (be precise) and
 still be  inaccurate.( See Figure  1)

 Determinate errors have "assignable"
 causes which can usually be identified and
 either eliminated or controlled.  (The terms
 "determinate" error, "assignable" error,
 and "systematic" error are synonymous).

 A  Sources of Determinate Error

    1 Method error

      Method errors are those that are
      inherent in the procedure. These are
      very serious and  the hardest to detect
      and  correct. The most common
      method error is the presence of inter-
      ferences in the sample.  Other
      examples would be precipitation of
      substances other  than the  desired
      material, partial solubility of pre-
      cipitates, and entrainment as in a
      solvent extraction procedure.
 CH. MET. con. lc. 11.75
                                     v-1

-------
  Accuracy-Precision-Error
     2  Personal errors

        Personal errors are attributable to
        individual mistakes which are con-
        sistently made by an analyst.  These
        errors are the result of consistent
        carelessness,  lack of knowledge or
        personal bias.  Examples are errors
        in calculations, use of contaminated
        reagents, non-representative sampling,
        or poor calibration of standards and
        instruments.

     3  Instrumental errors

        Instrumental errors are those which
        are caused by an analytical instrument
        or by the effects of the  environment
        acting on the instrument.  Moisture in a
        G.C. column,  improper wavelength
        markings on a spectrophotometer or
        incorrect scoring on a buret would be
        examples.

  B  Effects of Determinate Error

     1  Additive

        An additive determinate error is one
        which has a constant value regardless of
        the amount of analytically sought con-
        stituent present in the sample.
        (See Figure 2)

     2  Proportional

       A proportional  determinate error
        changes value according to the amount
       of analytically sought constituent in the
       sample.   (See Figure 3)
IH  DETECTION OF DETERMINATE ERROR

 A Spiked Samples

    1  Samples which can be determined before
       and after the addition of a known con-
       stituent ( in the concentration range of
       interest) provide a way to detect
       determinate errors.  Spiked samples
       should be representative and resemble
       actual conditions as closely as possible.
       The quantitation of bias  can then be
       obtained with the following measures.
      a   Mean error - the difference between
         the mean of the data and the I nu-
         n-suit.

      b   Uelative error - the mean error ol
         a set of data expressed as a per-
         centage of the true  result.


         EXAMPLE:  An analyst determines
         the nitrate content of the effluent
         from his sewage treatment plant to be
         0.5 mg/1.  He then adds 1 mg/1 of
         standard nitrate solution to the
         sample.  Table  1 shows the replicate
         results obtained on the spiked
         sample,  and calculation of both
         mean and relative errors.
                 Table 1

      Sample: Effluent
Determination: Nitrate (Modified Brueine)

                  X.
                   i
                1.55 mg/1

                1.58

                1.60

                1.47

                1.35

  1  Calculation of mean error

           7.55
     X  =
I. 51 mg/1
     Mean error =  +0. 01 mg/1

  2  Calculation of relative error

                       0.01 X 100
                                                        Relative Error  =
                                                                            1.50
                                     0.7'
  2  Control charts

     Trends and shifts on control charts
     may also indicate determinant error.
     Using spiked samples, the standard
     deviation is calculated and control
     limits (usually + 3 standard deviations)
     for the analysis are set (see Figure 4.
     For further discussion of control limits,
     see reference 3, p.  62.
  3-2

-------
4r-
        1234
    THEORETICAL VALUE
  Fipure 2.  ADDITIVE ERROR
         1234
     THEORETICAL VALUE
Figure 3.  PROPORTIONAL ERROR
         300
               ,23456        89

         100
                     Figure 4. CONTROL CHART

-------
Accuracy-Precis ion-Error
   3  In some cases, it is impossible to
      spike a sample so that it resembles
      actual conditions (e.g., BOD and
      pesticide samples).  Youden '**'
      provides excellent techniques for
      detecting bias in this situation.

B  Unknown Samples

   1  Independent method

      Analysis of a sample for a desired
      constituent by two or more methods
      that are entirely different in principle
      (gravimetric  and volumetric) may aid
      in the estimation of determinate error.
      However,  another reliable method may
      not be available or may be laborious to
      perform.

   2  Control charts

      It is possible to plot a control chart
      (Figure 4) even when it is not possible
      to spike a sample.  One can use as a
      reference  value an average of a series
      of replicate determinations performed
      on a composite check sample.   Such a
      sample must  be preserved or stabilized
      in such a way that the concentration
      of the constituent being measured will
      not change from day to day (see
      reference  1).

   3  Aliquoting

     If the determinate error is additive,
     the magnitude may be estimated by
     plotting the measured quantity versus
     a range of  sample volumes or sample
     weights. If the error has a constant
     value regardless of the amount of
     analytically sought constituent,  then
     a straight line fitted to the points will
     not go through the origin.  (See Figure 5)

C   Youden's Graphical Technique(10, 11, 12)

     Dr. W. J. Youden has devised an
     approach to test for  determinant errors
     with a minimum of effort on the part
     of the analyst  and his laboratory.
     Samples used  may be of known (spiked)
     or unknown composition.
 1   Technique

    Two different test samples (X and V)
    are prepared and distributed for
    analysis to as many individuals or
    laboratories as possible.  lOach
    participant is asked to perform
    only one determination on each
    sample (NOTE:  It is important
    that the  samples be relatively
    similar  in concentration of the
    constituent being measured).
    Table 2  shows the results on two
    such samples analyzed for percent
    potassium by 14 different laboratories.
    The mean for each sample has been
    calculated.

2   Interpretation - Figure 6

    The vertical line drawn on the
    graph represents the mean (X)
    of all the results obtained on
    Sample X; the horizontal line was
    drawn through the mean (Y) of all
    the results obtained on sample Y.
    Each pair of laboratory results
    can then be plotted as a point on
    the graph (marked X. 1 etc.)

    If the ratio of the bias (error) to
    standard deviation is close to  zero
    for the determinations submitted
    by the participants, then one would
    expect the distribution of the paired
    values (or points) to be close to
    equal among the four quadrants.
    The fact that the majority  of the
   points fall in the (+, +) and (-, -)
    quadrants indicates that the results
   have been influenced by some source
   of bias or determinate error.

   Furthermore, one can even learn
   something about a participant's
   precision from the graph.  If all
   participants had perfect precision
   (no indeterminate error),  then all
   the paired points would fall on a
   450 line  passing through the  origin.
   Consequently the distance from such
   a 450 line of each participant's
   point provides an indication of  that
   participant's precision.
  3-4

-------
                                                            Accuracy-Precision-Error
Tin; Yoiirk-n method lf> ali-io  disi:ssi%tl
in thin manual in llu- Hec-tion on
Inter laboratory Quality Control
Studies.  A quantitative treatment
of this subject can be found in
references 11 and 12.
    10i-
 1  8
 Z 6

 O
     2
            1234
         SAMPLE VOLUME (ml)

            Figure 5
Laboratory
1













Sample X
9.74%
9.92
9.98
9.99
10.00
10.11
10.12
10.14
10.19
10.23
10.25
10.29
10.55
10.62
LI niplc N
8.50":,
8.28
8. 84
8.24
8.73
8.54
8.64
8.82
9.04
8.93
8.97
8.80
9.21
8.95
X = 10.15
Y = 8. 75
  9.40
  9.20
  9.00
  8 80 -
  8.60
  8.40
  8.20
  8.00




^
 QJ- -
a.
*
i/i



































Y




X
1






Tt

3




X
8
X.
S



)(.
X
X
6













2 X Y
SAf
4PLE X (


11
X
10









%K)

13















X
4























     9.00   9.20    9.40    9.60   9.80   10.00   10.20   10.40   10.60   10.80   11.00

                 Figure 6.  YOUDEN'S GRAPHICAL TECHNIQUE

-------
 Accuracy-Precision-Error
IV  ELIMINATION OF DETERMINATE
    ERROR

  Various approaches can be used to eliminate
  the source of determinate error. The
  approach used depends upon whether the
  source is personal, method, or  instrumental.

  A Personal

    Great care must be  used to avoid producing
    an unconfldent attitude in a technician.  It
    is undesirable for the analyst to feel he is
    being "policed. "

  B Method and Instrumental

    1  Blanks

       Blanks  can be used to correct for
       interferences from reagents,  sample
       color, etc.

    2  Correction factors

       Examples of correction factors used
       in environmental analyses to eliminate
       determinate errors are the following:

       a  Recovery factors in organic
          extractions

       b  Chemical yield values in gravimetric
          analyses

       c  Counting efficiencies for radiation
          counters

    3  Standard addition
       1 l|;un- 7.  Mil!','',
       AUDI ! IO\ (. li ' I''' 'C \ i  \ll I 111  * ' 
                  M- ni\i i \ ni\ i u .\
            t 90


            I 7

            g so
            \n
               ,.<<

    NOTK: The X value appearing along the
     abscissa (i.e., \ + 20, X > 40, etc.) re-
     fers to the unknown added to the strontium
     standard
    4.  Standard compensation

       Another approach is to prepare the
       standard so that its composition
       resembles that of the  sample as
       closely as possible.  The objective
       of the approach is exactly the same
       as that  of standard addition - to
       compensate for the presence of
       interfering substances in the unknown.
V  INDETERMINATE ERROR AND
   PRECISION
       Sample interferences producing deter-
       minate errors can be overcome by
       adding equal amounts of unknown
       sample to a series of standards. The
       concentration of the unknown can then
       be determined graphically from a plot
       of the measured quantity (absorption,
       emission, etc.) versus the standard
       concentration.  (See Figure 7)
 Even when all determinate errors are
 eliminated, every replicate analysis will
 not give the same value.   Such variation
 in results is due to indeterminate error,
 also known as random, chance or
 uncontrollable error.  Indeterminate
 error affects the precision or agreement
 among results.

-------
                                                                         v-Precision- Error
 A  Sources of Indeterminate Error

    Indeterminate errors are due to
    unassignable or "chance causes."
    Examples are Inadvertent contamination
    of sample or glassware, variation in
    reagent additions, or variations in
    instrument response (see Figure 8).

 B  Effects of Indeterminate Error

    Since the causes of these errors are
    random, the effects are also random.
    Fortunately, these random variations
    conform to the "Laws of Chance" so
    statistical measures of precision can be
    used to quantitate indeterminate errors.
VI  DETECTION OF INDETERMINATE
    ERROR

 A measure of the degree of agreement among
 results can be obtained by analyzing a single
 sample repeatedly under a given set of
 conditions.

 A Range

    The range of the replicate results
    (difference between the lowest and the
    highest value) provides a measure of
    indeterminate variations.

 B Standard Deviation

    An estimation of indeterminate error can
    be obtained through a calculation of the
    standard deviation.   The following formulas
    should be applied to  random data which
    follows a normal distribution.  Normality
    can be checked by ranking and plotting the
    data on normal probability paper; it should
    fall on a  straight line (see Figure 9). Any
    values which  do not fall close to the straight
line (those values entire-led) ohould be
rejected in the calculation oi standard
deviation.  Other statistical U:sts W
can be used to objectively evaluate the
rejection of outliers.  The value obtained
for standard deviation for a particular
method may vary with the analyst, the
concentration range of the constituent,
and the composition of the sample
analyzed.   The confidence of the
estimate is increased as the number of
results (n) used to compute the standard
deviation is increased.
1  Replicate results on the same sample:


                                   (1)
                                                         s  =
   Xi
   X  =
 f(X. - X)2
                                                                    n-1
value of single result

average (mean) of results on
same sample
   n  =  number of results
Example : Table 3 contains a set of 5- day
BOD results obtained on a synthetic
sample containing 150 mg/1 of glucose
and 150 mg/1 glutamic acid.  (Note:  A  1%
dilution was used in the actual test).  The
results are  those submitted by laboratories
participating in a colaborative study .
Calculate the standard deviation of these
results.
   X   =  192 mg/1
          36.0
              =  58,200

-------
Accuracy-Precision-Error
          2  3
                     BECKMAN B (650 m*)
                     METHYLENE  BLUE SOLUTION
                     1cm CELL
                     25 REPLICATE READINGS







                   .528        .530        .532         .534        .536

                                       ABSORBANCE
                        Figure 8. VARIATIONS IN SPECTROPIIOTOMETKR READINGS
FIGURES NORMAL PROBABILITY CURVE
, tMIM ft   fO > TO 0 90 40 )0 ZD


1*0
1M
340
uo
300
2*0
2ZO
ZOO
i.n
i.eo
140
I.ZO
1.00























































































































^
















^

















I 
















^*









































































































,-^x










4.M
0















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L  '




































































































oft aa


















a 0460J ojasil 9  toosoi?oio we M M*m* ntt
plclliiif mltnal - -^t * 2.0*3

-------
                                                       Accuracy-Freciaion-l^rror
SAMPLE;
Table 3
160 mg/1 gliu -use i 150ms /I ulutnmic ac'itl ( !" dilution)
DETERMINATION. 5-day Biocheinu al Oxygen IVmun<1
X
i
100 mg/i
117
125
132
142
147
153
160
165
165
167
173
(78
18'J
190
196
19C
1U7
'Data taken
Service, Tr

vx
-92 mg/1
-75
-67
-GO
-50
-45
-3J
-32
-27
-27
-25
-19
-14
- 3
- 2
4
4
5
(X| - X)" X.
8464 (mg/1)2 198 mg/1
5625 199
44H9 200
3600 200
2500 204
2025 210
1521 211
1024 212
729 215
729 223
625 224
361 227
196 229
9 238
4 247
16 250
16 ?59
U!j 274
Iron) ttnter. Oxygen Demand Report (Julj
unmg 1' run ram

H. A. Tuft Solitary l-'.np.n
Table 4 *
SAMPLE: Aqueous
DETERMINATION: Phosphate (Lucerna, Cet.du, and
Sample
A

B

C

D

E

F

G

H

I

J

K

Mg/1 P
51
53
33
39
53
54
47
47
50
51
48
47
50
50
47
47
42
42
50
51
59
60
2
d d Sample
2 4 L

0 0 M

1 1 N

0 0 O

1 1 P

1 1 Q

0 0 R

DOS

GOT

1 1 U

1 1 V

Vx
6 mg/1
7
3
8
12
18
19
20
23
31
32
35
37
46
55
58
67
82
, IPfiO), Analyi
leerinp CVuti'f.
(X. - X)"
36 (mg/1)2
49
64
64
144
326
361
400
529
961
1024
1225
1369
2116
3025
3::44
4489
67?.4
ii ,il Reference
Cuii'itmati, Ohio.
CONCENTRATION
RANGE:
Prat Method)
Mg/l P
54
56
38
37
52
52
58
58
54
54
54
54
43
48
52
51
53
53
46
47
42
42
30 - 70 mg/1
d d2
2 4

1 1

0 0

0 0

0 0

0 0

0 0

1 1

0 0

1 1

0 0

*Data obtained from Frank Scluckner. Proctor and Gamble Company.
                                                                            3

-------
A ccuracy-Precision- Error
   2  Duplicate results on different samples

      In a laboratory where duplicates are
      routinely run, it would be simple to
      use the following formula for evaluation
      of standard deviation.

             /  (d  )  (See reference  5,
      S  =  J  2k    page 654)         (2)

      d  =  difference between duplicates

      k  =  number of samples

   Example: Table 4 contains a set of
   phosphate results  obtained on aqueous
   samples in the range 100-200 mg/1.
   Calculate the  standard deviation of these
   results.
       :(d2)
     16
                 22
      8      =   .61 mg/1

    3 Duplicate and triplicate results on
      different samples
        i
2(X.  - X)2

    n - k
(See reference 7,
 page 73)      (3)
       Note:  X  =  average of results on the
             same sample

       Example:  Table 5 contains a set of %
       nitrogen results obtained on unknown
       organic compounds.

       Calculate the standard deviation.
                                         4  Use of range iO estimate standard
                                            deviation

                                            For a small number of replicates
                                            (n < 10), the range can be used to
                                            estimate the standard deviation (See
                                            Table 6 ).
                                                  R

                                                 dN
                                        = s
                                                              (4)
                              Example:  Table 7 contains a set of
                              replicate nitrate results.  Calculate
                              the standard deviation of these results.

                              a Use of formula (1)

                                X = 0.72 mg/1

                                n = 5

                        S(X. -X)2 = .0134

                                 S =t

                                 s =  . 058 mg/1


                              b  Use of formula (4)

                                 R  *   . 14 mg/1
                                                N
                                       2.33
                                       .14 mg/1
                                          2.33
                                 s   =   .060 mg/1

                        C  Coefficient of Variation

                           An estimation of indeterminate error
                           can also be made by calculating the
                           coefficient of variation.
                                      V = ;- X
                                           X
                                                               100
                                                     By comparing the standard deviation
                                                     (dispersion) to the average or mean
                                                     value (central tendency) in a set of data
                                                     and expressing this relative standard
                                                     deviation as a percentage,  the analyst
                                                     has a meaningful interpretation of the
                                                     degree of dispersion present.  Indeter-
   3-10

-------
Accuracy-Precision-Error
                        SAMPLE:
                        DETERMINATION:
       Table 5*

Unknown Organic Compounds
7. Nitrogc:. (Kjeldahl)
CONCENTRATION-
RANGE:  lOfc - 20%
Sample
A

B

C

D


E


F



G

H


I

%N
16.48
16.45
16. 50
16.49
16.72
16.57
17.52
17.60
17.63
16.31
16j30

16.40
16.31
16.35

17.56
17.54
14.96
14.66

19. 15
18.89
*
16.47

16.50

16.65

17.58


16. 3J


16.35



17.55

14.81


19.02

x - !t
i
.01
.01
0
.01
.07
.08
.06
.02
.05
0
.0;

.05
.04
0

.01
.01
.15
. 15

.13
. 13
(X - XT
i
.0001
.0001
0
.0001
.0049
.0064
.0036
.0004
.0025
0
.0001

.0025
.OOlfi
0

.0001
.0001
.0325
.0225

.0169
.0169
Sample
J

K

L

M


N


O


P

Q


R


ToN
13.93
13.56
10.34
10. 19
17. 16
17. 13
15.01
15.05

12.44
12.70
12.73
14.37
14.36

11.85
11.85
14.79
14.70
14.70
17. 19
17. 14

X
13.75

10.27

17. 15

15.03


12. G2


14.37


11.85

14.73


17. 17


X - X
i
. 18
, Ib
.07
.08
.01
.02
.02
.02

. 18
.08
. 11
0
.01

0
0
.06
.03
.63
.C2
.03

(X. - X)2
. 0324
. 0361
.0049
.0064
.0001
.0004
.0004
.0004

.0324
.0064
.0121
0
. 000 1

0
0
.0036
.0009
.0009
.0004
.0009

             *Dta obtained from Frank Schickner, Proctor  and Gamble Company.
FACTORS
Size
of Sample
2
3
4
5
6
7
8
9
10
*Natrella,
Table 6*
USED TO ESTIMATE THE STANDARD
DEVIATION FROM RANGE
(n) ^
1. 13
1.69
2.06
2.33
2. 53
2.70
2.85
2.97
3.08
(8)
Experimental Statistics, pp.
1
dN
.887
.591
.486
.430
.395
.370
.351
.337
.325
2-6.

Table 7

SAMPLE: Ohio River Water
DETERMINATION:
Xi
0.65 mg/lN
0.68
0.70
0.76
0.79
Nitrate (Modified
x.-x
-0.07
-0.04
-0.02
+0.04
+0.07
Brucine)
(X.-X)2
.0049
.0016
.0004
.0016
.0049
                                                                                                             11

-------
                                                               AOOUIMO% -Precision-Error
   minate error would be the cause of undue
   dispersion.

   Example:  Table 3

   s  =  41 mg/1

   X  =  192 mg/1

   V  =  ^  X 100

   V    21%
    An indication of an individual's analytical
    precision can be obtained by his partic-
    ipation in an interlaboratory study.  In
    this case,  the analyst performs only
    one determination on each of two samples.
    Using Youden's Graphical Technique as
    an indication of an individual's precision
    was discussed previously in this outline
    in m C.  Also see the outline on "Inter-
    laboratory Quality Control Studies."
REFERENCES

1  Accuracy in Clinical Chemistry.  Dade
      Reagents, Inc., Miami, Florida.

2  Allan, Douglas H.   Statistical Quality
      Control.  Reinhold Publishing Corp.,
      New York.   1959.

3  American Society for Testing Materials
      ASTM Manual on Quality Control of
      Materials.  Special Technical
      Publication 15-C.  1951.
   Bauer, E. L.
      Chemists.
      1960.
A Statistical Manual for
Academic Press, New York.
5  Bennett,  Carl A. and Franklin, Norman L.
      Statistical Analysis in Chemistry and
      the Chemical Industry.  John Wiley &
      Sons,  Inc., New York.  1954.

6  Chase and Rabinocvitz.   Principles of
      Radioisotope Methodology.  Minneapolis
      Burgess Publishing Company.   1964.
                               7  Mickley, Harold S.,  Sherwood, Thomas K.
                                    and Reed, Charles E.  Applied
                                    Mathematics in Chemical Engineering.
                                    McGraw-Hill Book Company, New York.
                                    1957.

                               8  Natrella, M.G.  Experimental Statistics,
                                    National Bureau of Standards Handbook
                                    91.  U.S. Dept. of Commerce.   1963.

                               9  Schickner,  Frank A.  Personal Com-
                                    munication.   The Procter & Gamble
                                    Company, Miami Valley Laboratories,
                                    Research & Development Department,
                                    P.O. Box39175, Cincinnati, OH 45239.

                               10  Youden, W.J.  The  Collaborative Test.
                                     JAOAC 46:55-62. January 1963.

                               11  Youden, W.J.   The Sample,  The
                                     Procedure, and the  Laboratory.
                                     Anal. Chem.  32:23-37A.   Dec.  1960.

                               12  Youden, W.V.   Statistical Techniques
                                     for Collaborative Tests.  Association
                                     of Official Analytical Chemists,  Box 540,
                                     Benjamin Franklin Station, Washington,
                                     DC .   1967.
                                 This outline was prepared  by Betty A.
                                 Punghorst,  Chemist, formerly with
                                 National Training Center, and revised by
                                 Audrey E.  Donahue,  Chemist,  National
                                 Training Center.  MPOD. OWPO, USEPA,
                                 Cincinnati,  Ohio  45268
Descriptors: Accuracy, Data Collections,
Data Processing, Error Analysis, Errors,
Graphic Methods, Measurement,  Monitoring,
Precision, Quality Control
   3-12

-------
                      INTERLABOHATOKY QUALITY CONTUOL STUOIKS
 I   DEFINITION OF INTERLABORATORY
    STUDY IN EPA

 A  It is a between-laboratory evaluation of
    exact physical, chemical or biological
    methods which yield a measurement of
    desired properties.

 B  It is not an interlaboratory evaluation of
    materials,  reagents, or different test
    conditions.  It is not an initial study of a
    method, nor a study to develop methods.
II  PURPOSES OF INTERLABORATORY
   STUDY

 A Selection of an analytical method.

 B Evaluation of an analytical method.

 C Evaluation of laboratory and analyst
   performance.
 I  PREPARATION FOR INTERLABORATORY
   STUDY

 A Laboratories are in control.  Between-
   replicate deviation will then be very
   small and uniform in all laboratories.

 B Ruggedness testing of a method has been
   completed by the method developer,  or
   by a single qualified laboratory.  Effects
   of small changes in time, temperature,
   pH,  reagent and sample volume measure-
   ments, etc.,  are known and corrected
   for"7

 C All laboratories and analysts are familiar
   with the method to be tested. Any special
   equipment or reagents required are
   known and available., If all analysts are
   not acquainted with the method,  a simple
   preliminary study is undertaken to
   accomplish this.   '
IV  INSTRUCTIONS FOR THE STUDV

A  Exact method write-ups are provided
    to each analyst.  The technical
    description of an analytical method
    is difficult.  The language and organiza-
    tion must be complete, yet simple and
    unambiguous.  These characteristics
    are often conflicting.

    Requirements in a method may seem
    trivial.  Yet if these are not recognized
    and controlled, the entire  study can be
    useless.

 B  Explicit directions for  sample preserva-
    tion, sample  make-up, time limitations,
    sequence of analyses,  etc. , are provided.

 C  Advance notice of tests is  given so that
    laboratories can integrate the test into
    their program and realistic deadlines
    can be established.  Once  established,
    deadlines for agreement to participate,
    for completion of analyses and for report-
    ing are followed closely.
 V  THE TEST SAMPLES

 A  The sample is carefully designed to refleo:
    the concentrations found in natural waters
    and yet be within the best portion of the
    concentration range for the method.

 B  Since precision of almost all methods
    varies with concentration, a compre-
    hensive study includes  several levels of
    concentration.

 C  Since accuracy of a  method is very im-
    portant, exactly known levels of con-
    stituents are added both to distilled and
    natural waters for testing.  Stabilized
    natural waters are not used as samples.
  CH.MET. con.7a. 11. 75
                                                                                       4-1

-------
 Interlaboratory Quality Control Stiali.-.s
 VI SAMPLE FORM AND CONTAINER

 A In the EPA Interlaboratory Program,
    samples are prepared as concentrates
    for final dilution at the testing laboratory.
    Use of concentrates has several ad-
    vantages.  .This greatly reduces space
    requirements and  thereby greatly re-
    duces cost of mailing.  With use of sealed
    glass ampules, preservation of con-
    centrates is easily obtained through
    steam sterilization.  Chemical preser-
    vatives can be used at fairly high levels
    in the concentrate and can be removed as
    interferences by the dilution of the con-
    centrate to final sample volume.  Theo-
    retically, a well designed sample proper-
    ly sealed in glass  is stable indefinitely.

 B Samples can be prepared as dilute sim-
    ulated or natural water samples to be
    analyzed, as is.   These samples  have an
    advantage in that they reach the analyst
    exactly as a routine sample.  There is
    no error in making a dilution and this
    source of variance in the method  is re-
    moved.

    Some disadvantages of such samples are:
    the serious logistics problem from
    storage if a large  number of samples is
    involved; a relatively high cost of mailing;
    and a limited choice of preservation
    methods because of the size of containers,
    the probable use of plastic containers,
    and only limited testing of water types.
VII INTERLABORATORY STUDY DESIGN

 A Sample concentrates are prepared in sim-
    ilar yet different concentrations at each
    of several levels of analysis.

 B True values are calculated from the
    amount of a constituent added, and are
    used to measure the accuracy of the study
    method.

 C Multiple constituents are prepared in a
    sample by related groups of  analyses.
     For example,  a mineral and physio..;]
     .sample is analy/.rd for pll, alkalinity/
     acidity,  specific- conductance,  total hard-
     ness,  sulfate,  chloride, fluoride,  solids,
     calcium, magnesium, sodium and
     potassium.   A nutrient sample is analyzed
     for ammonia nitrogen, nitrate nitrogen,
     Kjeldahl nitrogen,  orthophosphate and
     total phosphorus.

  D  The analyst dilutes separate aliquots to
     volume with distilled water and with
     natural water of his choice.

  E  Single analyses are made for each
     parameter.  Replicates are of limited
     value in evaluating a  method for  routine
     analyses because of data handling
     problems and failure to detect significant
     differences by replication.

  F  Recoveries are compared for distilled
     and for natural waters. Recovery from
     distilled water should indicate method
     bias.  Differences  in recoveries from
     distilled and natural waters should
     indicate interference by natural water
     samples.
VIII DATA EVALUATION AND REPORTING

 A  Rejection of Outliers-Accuracy.

   The T-Test is applied to all data using
   the standard deviation of all data and
   compared with a T value at the 99% con-
   fidence interval.  Accuracy is calculated
   after rejection of this data.

B Precision Measures

   A statistical summary is developed for
   computerized treatment of data as
   suggested by Larsen^3).  Method pre-
   cision is reported as standard deviation,
   95% confidence interval, and coefficient
   of variation.   Method precision is reported
   for  each sample since it varies with
   concentration.
4-2

-------
                                                  Intcrlaboratory Quality Control Studies
C Graphic Display

   Methods are evaluated usinj> two-sample
   charts described by Youden*^'.  The
   values reported by an analyst for a
   sample pair became the x and y coordi-
   nates for a single data point on the chart.
   Data form ellipses around the true value
   because of systematic error.  Random
   error is shown as the perpendicular
   distance from the 45  slope.  General
   scatter indicates poor method pre-
   cision.   Bias is shown by movement of
   the ellipse away from the true values.
   Outliers are visible at some distance
   from the major grouping of data.  Ex-
   amples shown on the following pages are:

   Figure  1 - Outliers,
   Figure  2 - Limited precision and
              limited accuracy,
   Figure  3 - Negative bias,
   Figure  4 - Positive bias,
   Figure  5 - Systematic error,
   Figure  6 - Good precision  and
              accuracy.
IX  INTER LABORATORY STUDY
    REPORT (5' 6)

 A  Reports are slanted toward audience.

 B  All data are coded by laboratory and
    analyst.

 C  Glossary of terms is necessary.

 D  Computer use increases statistical
    capabilities tremendously.  There is a
    danger of "over-evaluation".
li  Increased efficiency in reporting data is
   achieved by using the computer print-out
   directly in a report.  See Figures 7
   and 8.

            REFERENCES

1  Youden,  W.  J.  1961.  Experimental
   Design and ASTM Committees.  Materials
   Research & Standards, 1(11):862-867.

2  Preliminary Study,  Nutrient Analyses,
   September 1969, Analytical Quality
   Control Laboratory,  FWPCA,
   Cincinnati, Ohio.

3  Larson,  Kenneth E.,  The Summarization
   of Data,  J. of Quality Technology, Vol.
   1, No. 1, Jan.  1969.

4  Youden,  W.  J.  1967.  Statistical Tech-
   niques for Collaborative Tests.  The
   Association  of Official Analytical
   Chemists, Washington, D. C.

5  Winter,  J. A. and Midgett, M.  R. 1969.
   FWPCA  Method Study 1,  Mineral and
   Physical Analyses, Analytical Quality
   Control Laboratory, FWPCA,  Cincinnati,
   Ohio.

6  Winter,  J.  A. and Midgett, M.  R. 1970.
   Method Study 2, Nutrient Analyses, Manual
   Method,  EMSL.  USEPA,  Cincinnati, Ohio.
This outline was prepared by J. A. Winter,
Chief, Method & Performance Evaluation,
EMSL, USEPA, Cincinnati, Ohio 45268.
                                                   Descriptors:    Laboratory Tests,  Quality
                                                   Control,  Statistical Methods,  Testing
                                                   Testing Procedures, Error Analysis, Graphic
                                                   Methods
                                                                                       4-3

-------
2.70
2.46-
2.22-
 1.98
 1.74
.50
             FI6HE 1

 AMMONIA NITR06EN, t N/UTH, WUttBS

 REJECTED BY T-TEST AT fl% WWAIIUTY.

FROM: METHOD STUDY 2, NUTRIENT ANALYSES,
          MANUAL METHODS
                       SAMPLE 3
            1.74
       1.98
2.22   '  2.46
2.70
1.9
6.0
5.0
4.0

3.0
2.0
i i i i
, HMKS
" KJEIIAHL NITtifiEN, i
6081 PRECISION WfTI
' ERRtR. FROM: NETNO
NITRIENT ANALYSES.
METIODS
-
Lkl
-a.
_5
".
; 
2.0 3.
.
J


 

' .1
i i i ^ i
UN/LITER. .
SYSTEMATIC
I STMT 2,  
NANBAL .
* . 

-
-
-
SAMPLE 8
0 4.0 5.0 6.0 7.0
                                                                                                                             o
                                                                                                                             *!

                                                                                                                             f
                                                                                                                             CT



                                                                                                                             E.
                                                                                                                             O
                                                                                                                   O


                                                                                                                   V.
.Oil ' ' ' i
I F
- KJELDAHL NITROGEN,

.76


.52


.28

t04
0.?
- , - T 	 1 	 , 	 , 	 .
IGURE 2
HI N/LITER, POOR PRECISION. ~
FROM: METHOD STUDY 2, NUTRIENT ANALYSES,
MAN


CO
"UJ *
 J 
a.
. V*

^ * *
* 
1 1 1 1
UAL METHODS
t
t
_

** *
 *

*
SAMPLE 5


5




4

3



i
Fl(
i i
iURE 6
~ pH DATA, GOOD PRECISION AND GOOD ACCURACY. ~
FROM: METHOD STUDY 1, MINERAL AND PHYSICAL
AX



ALYSES


-
jrf
>fr

UJ
 SAMPLE 3
-., . 	 I
_

" i
    0.2
                  .28
                 .52
          .76      1.0,

-------
. M-
.56
.42
.28
.14

AMMONIA NITROGEI
~ AS NEGATIVE BIAS.
ANALYS

.
*
 
04 * *

0.
"^
i  i i
i i i i i i
FIGURE 3
1, mg N/LITER, LIMITED ACCURACY
FROM: METHOD STUDY 2, NUTRIENT ~
ES, MANUAL METHODS
.
-
-
SAMPLE 1
i i i i i i
0.0
.14
                               .28
.42
.56
.70
\
en
   28
   24
   20
                          FIGURE 4     .
         POTASSIUM ',  mg K/LITER,  POOR ACCURACY AS
           A POSITIVE  BIAS,  SODIUM INTERFERENCE.
           FROM: FWPCA METHOD STUDY I, MINERAL
                   AND PHYSICAL ANALYSES
        a.
        3E
        c
        CO
    16
                               SAMPLE 5
                             20
                    24
             28
                                                                                           I.IOQOO CON*. LLK.    L.171 I9S .!I
                                                                                                  FIGURE  7
                                                                                   BOD DATA,  STATISTICAL SUMMARY. PREPARED BY
                                                                                        COMPUTER FOR DIRECT USE IN REPORT.
                                                                                 FROM: METHOD RESEARCH STUDY 3,  DEMAND ANALYSES
                                                                                                                                                cr
                                                                                                                                                o
                                                                                                                                                           

-------
                              FIGURE  8
          AMMONIA NITROGEN DATA, YOUDEN'S TWO-SAMPLE CHARTS.
               PREPARED FOR REPORT BY COMPUTER PLOTTER
.6.
   1A
OBTUBIUTO
            SAMPLE 1
.6
.4.
.2
0
IB '
M
ill
*'' .
'. '.'

.
y|
^MPLE.l . .
J *2 4 o
                                      1
                                            10
                                         NA1UWLIUTR

                                                 SAMFJ.E 3

-------
                       VOLUMETRIC ANALYSIS OF WATER QUALITY
I   INTRODUCTION

A   A standard solution is one whose com-
    position and concentration are known
    to a high degree of accuracy.  In chem-
    ical analyses, it is used to determine
    the concentration of a particular com-
    ponent in a sample.

B   These chemical analyses very frequently
    involve an acid-base reaction or an
    oxidation-reduction reaction.

C   Three important terms  connected with
    these two types of chemical  reactions
    are:  mole,  equivalent weight,  and
    normality.  These  terms will be de-
    fined in  following sections.
II  ANALYTICAL CHEMICAL REACTIONS

A  As stated above, in a chemical analysis
    a volume of standard solution is brought
    into contact with a volume of sample
    in order to determine the concentration
    of some particular component of the
    sample.

B  For a given volume of sample, it is
    necessary to use a definite amount  of
    the standard solution--too much or too
    little would give erroneous results.

C  For example,  one cannot simply mix
    together random volumes of sodium
    hydroxide and hydrochloric acid solu-
    tions and expect the only two substances
    produced to be sodium chloride and
    water.

D  Unless the  concentrations of t h e two
    reagents are known and the  amounts
    measured accurately,  excess sodium
    hydroxide or hydrochloric acid will
    also  remain at the end of the reaction.

E  The reason for these limitations is
    that when molecules react with
    one another, they do so in definite ratio.
H
K
Unless the number of molecules of each
reactant is known,  there will always be
an excess of one of the reactants re-
maining at the end  of the chemical
reaction.  As mentioned before,  this
leads to erroneous analytical results.

Because of their size, it is not possible
to count out numbers of molecules.
However,  the number of molecules in
a quantity of a chemical may be found
by determining its  weight and consulting
a table which lists  the weights of the
atoms making up the chemical.

For  example, sodium hydroxide has the
formula NaOH.  It can also be stated
that  a molecule  of sodium hydroxide
consists of one sodium atom, one hy-
drogen atom and one oxygen atom.

One  sodium atom weighs 23 atomic
mass units (amu).  An oxygen atom
weighs 16 amu; and a hydrogen atom
weighs 1 amu.

A molecule of sodium hydroxide, there-
fore, weighs 40 amu; all amu values
have been rounded off.  Forty amu is
the molecular weight of sodium hydroxide.

A mole of any chemical is a number of
grams numerically equal to the molec-
ular weight of that chemical.  One mole
of sodium hydroxide,  therefore, con-
tains 40 grams (40 g).

Similarly, the atomic weight of a.
chlorine atom is 35 amu; that of a hy-
drogen atom is 1 amu; and the molec-
ular weight of the hydrogen chloride
molecule is 36 amu.  One mole of hy-
drogen chloride weighs 36 g.

Synonyms for mole are:  mol, gram
mol, gram mole, and gram molecular
weight.

Tables listing atomic weights of the
elements can be found in virtually all
 PC. 17 b. 11.75
                                                                                      5-1

-------
 Analytical Heaftions  - Standard Solutions
     of the texts used for high school and
     first year college chemistry courses.

     The remaining two terms mentioned in
     1C (equivalent weight and normality)
     willbe considered as they are related to
     acid-base and oxidation-reduction reac-
     tions .
Ill  ACID-BASE REACTIONS

A   Recall that an acid is a substance which
    donates a hydrogen ion,  or proton,  (H+)
    in a chemical reaction  and a ba-se is a
    substance which donates a hydroxide ion
    (OH~) in a chemical reaction. Reactions
    involving these two ions are termed
    acid-base reactions.

B   In the case of sodium hydroxide, the
    molecular weight  is 40 amu and one
    mole of sodium hydroxide  weighs 40 g.
    One hydroxide ion is contained in the
    sodium hydroxide molecule.

    1    For a base, the number of grams
         in a mole divided by the number of
         hydroxide ions equals a quantity
         called the equivalent weight.   Ex-
         amples are given below.  All amu
         values have been rounded off.
        a    Base - potassium hydroxide KOH

        Atoms  Number  Wt/atom
           K      .1      39 amu
           O       1      16
           H       1        1
                          Total
                           39 amu
                           16
                            1
                           56 amu
One mole of KOH = 56 g
Number of hydroxide, ions = 1
Equivalent weight of KOH = 56 g

b   Base - magnesium hydroxide
           Mg(OH)2
Atoms Number Wt/atom  Total
  Mg      1       24 amu   24 amu
  O       2       16       32
  H       2        1        2
                           58 amu
One mole of Mg(OH)2 = 58 g
Number of hydroxide  ions = 2
Equivalent weight of Mg(OH)2 = 29 g
                                                 For an acid, ih4_
         98 amu
        One mole of H2SO4 = 98 g
        Number of hydrogen ions = 2
        Equivalent weight of H2SO4  = 49 g

C   Normality is a method of expressing
    solution concentrations.  If one equiv-
    alent weight of a chemical is dissolved
    in a solvent and the volume brought to
    one liter (1),  the concentration of the
    solution is one normal (N).

    1   The equivalent weight  of KOH was
        calculated to be  56 g.  This amount
        of the solid dissolved in water and
        diluted to a liter would give a 1 N
        solution.

    2   The equivalent weight of HNO3 was
        found to be 63 g.  This quantity of
        acid diluted to a liter would give a
        1 N solution.
                                                 IV  OXIDATION-REDUCTION REACTIONS

                                                 A   The concepts of mole, equivalent weight
                                                     and normality, as described in previous
                                                     sections,  apply also to oxidation -
                                                     reduction reactions.
 5-2

-------
                                                  Analytical peai:iio.'i.s -_'^;a.'dar< 1 '-olu_: i_i
B
    One definition of an oxidation is that it
    involves an increase in the oxidation
    state (charge) of an atom.

    For example:  FeCl2   FeCls.  In this
    conversion the Fe has been oxidized
    from +2 to +3.

    A  reduction is a decrease in the oxida-
    tion state (charge) of an atom.

    For example:  KMnO4 -* MnO2-   In this
    conversion the Mn has been reduced
    from +7 to +4.

    The equivalent weight of an oxidizing
    or reducing agent is calculated by
    dividing the number of grams in a mole
    of the reagent by the change in charge
    involved.

    For example:  FeCl2 (used as a reducing
    agent).
Atoms
  Fe
  Cl
Number
   1
   2
                         Wt/atom  Total
                          56 anxu
                          70
                                    56 amu
                                    70
                                   126 amu
                         126 g
    One mole of
    Change in charge   1
    Equivalent weight of FeCl2
                                 126 g
    The concentration of a liter of solution
    which contains 126 g of FeCl2 is 1 N.


V   PRIMARY STANDARDS

A   A reagent of known  purity is called a
    primary standard.  Primary standard
    grade chemicals are available from
    chemical supply houses and the National
    Bureau of Standards.  An accurately
    measured quantity of a primary stand-
    ard is used for the preparation of
    standard solutions.

B   Other requirements of a  primary stand-
    are are:

    1    It must be stable at  105 C (the
         temperature used for drying).

    2    It should not be reactive with com-
         ponents of the air, such as C>2 and
         CO2-
                                                          It should have a high equivalent
                                                          weight so as to minimize any
                                                          errors in the analysis.

                                                          It should be  readily available at a
                                                          reasonable cost.
                                                 VI   STORAGE OF STANDARD SOLUTIONS

                                                 A   Standard solutions should be prepared
                                                      using high quality distilled water.

                                                 B   Care should be taken to insure the
                                                      cleanliness of the glass or plastic
                                                      bottle  used for storage.

                                                 C   Some solutions may decompose on ex-
                                                      posure to light and should be stored in
                                                      dark bottles.

                                                 D   The stopper or cap should fit tightly.
VII  CA LCULA TIONS

A   The basic formula used in volume! ri.-
     analysis is

        (1 XN)  of standard solution =

             (1 XN) of sample

     In a typical analysis, three of the four
     quantities are known, or  found, and
                                                  B
                                                          N  of sample
                                                             (1 XN) of standard
                                                              1 of sample
                                          This formula can be rearranged to give

                                              g =1 XN Xequivalent weight

                                          where g and equivalent weight (ew) re-
                                          fer to the component being analyzed
                                          and 1 and N to the standard solution.

                                          For example:  How many g of NaOH are
                                          present in a sample if 100.0ml of 0.2N
                                          HC1 are required for its titration?

                                              g = 1 XN Xequivalent weight =
                                               100.0 ml
                                              1000.0 ml/I
                                                                                   =  0.8

-------
Analytical Reactions - Standard Solutions
 REFERENCES                                   3    A.yros, li. II.    yiiatiiimi ivc
                                                           Analysis,   llnrpor atul Ufolhi-rs.
 1   Hamilton, S. B.  and Simpson. S. G.                     Now York.  1.
          Quantitative Chemical Analysis
          llth edition, The MacMillan Co.,         	
          New York.   1958.                        This ourline was prepared by C.  R. Feldniann,
                                                  Chemist,  National Training Center, MPOD,
 2   Blaedel, W.J. and  Meloche, V. W.             OWPO, USEPA.  Cincinnati, Ohio 45268
          Elementary Quantitative Analysis:
          Theory and Practice.    Row,            Descriptors: Analytical Techniques,  Chemical
          Peterson and Co., N. Y.  1957.          Analysis, Volumetric Analysis, Water Analysis
 5-4

-------
                         ACIDITY, ALKALINITY, pH AND BUFFERS
I   DEFINITIONS OF ACIDS AND BASES

A   Arrhenius Theory of Acids and Bases
    (Developed about 1887)

    1    Acid: A substance which produces,
         in aqueous  solution, a hydrogen ion
         (proton), IT*".

    2    Base: A substance which produces,
         in aqueous  solution,  a hydroxide
         ion,  OH".

    3    The Arrhenius  theory was confined
         to the use of water as a solvent.

B  Bronsted and Lowry Theory of Acids
    and Bases (Developed about 1923)

     1    Acid: A substance which donates,
         in chemical reaction, a hydrogen
    ion (proton).

     2    Base: A substance which accepts,
         in chemical reaction, a hydrogen
         ion (proton).

     3    Bronsted and Lowry had expanded
         the acid-base concept into non-
         aqueous media; i.e., the solvent
         could, but did not have to be .water.

 C  There are other acid-base theories. The
    two above are probably the most commonly
    used ones when discus sing  wastewater
    topics however.

 II   DEFINITIONS OF ACIDITY,
     ALKALINITY AND NEUTRALITY

 A   Acidity
     A  condition  in which there is a prepon-
     derance of acid materials present in
     the water.

 B   Alkalinity
     A  condition  in which there is a prepon-
     derance of alkaline (or basic) materials
     present in the water.
C   Neutrality
m
        It is possible to have  present  in
        the water chemically  equivalent
        amounts of acids and bases. The
        water would then be described as
        being neutral;  i.e.,  there is a
        preponderance of neither acid nor
        basic materials.  The occurrence
        of such a condition would be rare.
         The term "neutralization" refers to
         the combining of chemically equiv-
         alent amounts of acids and bases.
         The two products of neutralization
         are a salt and water.
         HC1
              NaOH
                              NaCl
       Hydro-    Sodium
       chloric   Hydroxide
       acid
                            Sodium
                            Chloride
                             (a salt)
     The key word in the above definitions
     is "preponderance."  It is possible to
     have a bas   ion of acidity while there
     are basic materials present in the
     water,  as well as conversely.
HOW ARE DEGREES OF ACIDITY AND
ALKALINITY EXPRESSED?
 The pH scale is used to express various
 degrees of acidity and alkalinity.  Values
 can range from Oto 14. These two ex-
 tremes are of theoretical interest and would
 never be encountered in a natural  water or
 in a wastewater.  pH readings from 0 to
 just under 7 indicate  an acidic condition;
 from just over  7 to 14, an alkaline condition.
 Neutrality exists if the pH value is exactly
 7.  pH paper, or a pH meter, provides the
 most convenient method of obtaining pH
 readings. It should be noted, that under NPDES
 Methodology, pH measurements are to be made
 using a pH meter. Some common liquids andpH
 values are listed in Table I.
 CH.ALK. 3.3. 75
                                   6-1

-------
Acidity. Alkalinity, pH and Buffers
TABLE 1. "pH Values of
Household lye
Bleach
Ammonia
Milk of magnesia
Borax
Baking soda
Sea water
Blood
Distilled water
Milk
Corn
Boric acid
Orange juice
Vinegar
Lemon juice
Battery acid
5.9 - 8.4 is the common
natural waters.
Common Liquids
13.7
12.7
11.3
10.2
9.2
8.3
8.0
7.3
7.0
6.8
6.2
5.0
4.2
2.8
2.2
0.2
pH range for most

IV   HARD AND SOFT WATERS

In addition to being acidic or basic,  water
can also be described as being hard or soft.

A   Hard water contains large amounts of
     calcium, magnesium, strontium, man-
     ganese and iron ions, relative to the
     amount of sodium and potassium ions
     present.  Hard water is objectionable
     because it forms insoluble compounds
     with ordinary  soap.

B   Soft water contains small amounts of
     calcium, magnesium, strontium, man-
     ganese and iron ions, relative to the
     amount of sodium and potassium ions
     present.   Soft water does not form in-
     soluble compounds with ordinary soap.
 V   TITRATIONS

 A   The conversion of pH readings into such
     quantities as milligrams (mg) of acidity,
     alkalinity,  or hardness, is not easily
     carried out.   These values are more
     easily obtained by means of a titration.

 B   In a titration, an accurately measured
     volume of sample (of unknown strength)
     is combined with an accurately measured
  6-2
    volume of standard solution (of known
    strength) in the presence of a suitable
    indicator.

C   The strength (called normality) of the
    sample is then found using the following
    expression:

    milliliters (ml) of sample X normality
    (N)  of sample *  ml of standard solu-
    tion X N of standard solution.

    Three of the four quantities are known,
    and

    N of sample = ml of standard solution
    X N of standard solution/ml of sample.

D  In modified form,  and a more specific
    application of the above equation, alka -
    linity  is calculated in the following
    manner (13th ed. Standard Methods).

    mg of alkalinity  as mg CaCO3/liter (1)
    * ml of standard H~SO4 X  N of standard
    H,SOd X 50 X 1000/ml sample.
VI   INDICATORS

The term  "suitable indicator" was used
above.  At the end of A titration, the pH of
the solution will not necessarily be 7.  It
may be above or below 7.  A suitable indi-
cator, therefore, is one which undergoes
its characteristic color change at the appro-
priate pH.  Below are a few examples of
indicators and the pH range in which they
undergo their characteristic color changes.
In some cases, mixed indicators may be
used in order to obtain a  sharper and more
definite color change. Again it should be noted
that under NPDES,  pH meters are to be used
for the measurement of pH.

Indicator
Methyl Yellow
Methyl Orange
Methyl Red
Cresol Purple
Phenolphthalein
Alizarine Yellow
TABLE 2. pH
Operational
pH Range
2.8 - 4.0
3.1 - 4.4
4.4 - 6.2
7.4 - 9.0
8.0 - 9.6
10.0 - 12.0
Range of Indicators

-------
                                                          Acidity,  Alkalinity ,  pH and Buffers
VII  BUFFERS

 A   A buffer is a combination of substances
     which, when dissolved in water, resists
     a pH change in the water,  as might be
     caused by the addition of acid or alkali.
     Listed below are a few chemicals
     which, when combined in the proper
     proportions, will tend to maintain the
     pH in the indicated range.
      Chemicals                 pH Range
  A cetic A cid + Sodium Acetate   3.7-  5.6
 Sodium Dihydrogen Phosphate +
 Disodium Hydrogen Phosphate   5.8-  8.0
 Boric Acid + Borax             6.8-  9.2

 Borax  + Sodium Hydroxide      9.2 -  11.0
  TABLE 3.   pH Range of Buffers
   A buffer functions by supplying  ions
   which will react with hydrogen ions
   (acid "spill"),  or with hydroxide ions
   (alkali "spill").

   In many instances, the buffer is composed
   of a weak acid and a salt of the weak acid;
   e.g., acetic acid and sodium acetate.
    1   In water,  acetic acid  ionizes or
        "breaks down" into hydrogen ions
        and acetate ions.
      HC2H3O2  -      H+   + C2H3O2"
    (acetic acid) (hydrogen ion) (acetate ion)
                    (proton)

        This ionization occurs to  only a
        slight  extent, however, most of the
        acetic acid remains in the form of
        HC2H3O2;  only a small amount of
        hydrogen and acetate ions is formed.

    2   Thus,  acetic acid is said to be a
        weak acid.
    3   In the  case of other acids,  ionization
        into the component ions occurs to a
        large degree, and the term strong acid
        is applied;  e.g., hydrochloric acid.
     HC1
(hydrochloric
    acid)
(hydrogen ion)
  (proton)
                                   cr
                                 (chloride
                                    ion)
                                                   D
                                     4    The terms "strong" and "weak" are
                                         also applied to bases.  In water
                                         solutions,  those which break down
                                         into their component ions to a large
                                         extent are termed "strong", and
                                         those which do notare "weak".
                                         Sodium hydroxide is a relatively
                                         strong base, while ammonium
                                         hydroxide is realtively weak.
                                     5   Sodium acetate (a salt of acetic acid)
                                         dissociates or "breaks down" into
                                         sodium ions and acetate ions  when
                                         placed in water.

                                     NaC2H302   -   Na+     +    C2H302-
                                    (sodium acetate) (sodium ion) (acetate ion)
                                         This dissociation occurs to a large
                                         extent, and practically all of  the
                                         sodium acetate is in the form of
                                         sodium ions and acetate ions.

                                     It would be difficult and expensive to
                                     prepare large quantities  of buffers for
                                     use in a treatment plant.   However,
                                     certain naturally occurring buffers may
                                     be available (carbon dioxide is an ex-
                                     ample).  It dissolves in water to form
                                     the species indicated below.
                                        C02  +      H20
                                   (carbon dioxide) (Water)
                                                                                 H2C03
                                                                              (carbonic acid)
                                                       H2C03
                                                    H
                    +    HC03-
          (hydrogenion)(hydrogen car-
            (proton)    bonate ion)
                       (bicarbonate)
The hydrogen ions react with hydroxide
ions which might appear in the water
as the result of an alkali "spill".
 H+     +    OH"     =     H20
(in the    (hydroxide ion
 buffer)     "spilled")
The hydrogen carbonate ions react with
hydrogen ions which might appear in
the water as the result of an acid "spill",

 H+     +     HCO3-   =    H2CO3
(hydrogen ion) (in the
(proton)        buffer)
 "spilled"

This buffering action will be in effect as
long as there is carbonic acid present.
                                                                                       6-*

-------
Acidity.  Alkalinity,  pH and Buffers
    Buffering action is not identical with	
    a process in which acid wastes are             This outline was prepared by cTR
    "neutralized" with alkali wastes, or          Chemist.  National Training Center, MPOD,
    conversely.  The  desired effect is             OWPO, EPA, Cincinnati, OH  45268
    achieved in both cases, however (i.e.,
    the pH is maintained within a desired
    range.)                                      Descriptors; Acids, Acidity, Alkalis,
                                                 Alkalinity,  Analytical Techniques, Buffers,
                                                 Buffering Capacity, Chemical Analysis,
                                                 Hydrogen Ion Concentration,  Indicators,
                                                 Neutralization, Water Analysis.
  6-4

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                       ALKALINITY AND RELATIONSHIPS AMONG THE
                              VARIOUS TYPES OF ALKALINITIES
I  PRELIMINARY

The property of water referred to as alkalinity
is usually caused by the presence of hydroxyl,
carbonate and bicarbonate ions.  To a lesser
extent,  borates,  phosphates and silicates
contribute but are generally present in
negligible amounts.
The concentration and ratio of the OH
C3
and HCO,  ions may be measured by titrating
a sample to certain specified pH's or end
points which are detected either by use of a
pH meter or by color indicators.  Phenol-
phthalein is used for visual detection of the
first end point,  (approximately pH 8) which
indicates the neutralization of NaOH and con-
version of CO  ~  to HCO3 . A number of
indicators  (methyl orange, methyl purple,
brom cresol green,etc.) are used for detection
of the  second end point (pH 3-5) which indicates
the complete conversion of HCO   to HO and
CO .   The final end point is determined by
the amount of CO-  " and HCO   originally
present in  the sample.  If the end points are
determined electrometrically they are taken
as the mid-point of the greatest rate of pH
change per unit volume of titrant.
II   RELATIONSHIPS BETWEEN HYDROXIDE,
    CARBONATE, AND BICARBONATE
    ALKALINITIES

 The results obtained from phenolphthalein
 and total alkalinity measurements offer a
 means of classification of the principal
 forms of alkalinity, if certain assumptions
 are made.  It must first be assumed that
 interferences are absent and that bicar-
 bonate and  hydroxide do not exist in the
 same solution.  According to the system
 presented in Standard Methods, 13th Edition:

 A  Hydroxide alkalinity is present if the
    phenolphthalein alkalinity is more than
    one-half the total alkalinity.

 B  Carbonate alkalinity is present if the
    phenolphthalein alkalinity is not zero
    but is less than the total alkalinity.

 C  Bicarbonate alkalinity is present if the
    phenolphthalein alkalinity is less than
    one-half the total alkalinity.
         Table 1.  Relationships Between Phenolphthalein Alkalinity, Total Alkalinity,
            Carbonate Alkalinity, Bicarbonate Alkalinity and Hydroxide Alkalinity
Lecture
Notes
Case 1
Case 2
Case 3
Case 4
Case 5
Result of
Titration
P * T
P  IT
P= O
P > \1
p < IT
OH~ Alkalinity
as CaCO3
T
0
O
2P-T
O
CO. " "Alkalinity
as CaCO_
O
O
2P
O
2(T-P)
2P
HCO " Alkalinity
as CaCO
O
O
0
T
O
T-2P
                P  =  Phenolphthalein Alkalinity
                  T  =  Total Alkalinity
 CH.ALK.2d. 11.75
                                                                                       7-1

-------
 Alkalinity and Relationships Among the Various Types oi AIkaUnities
             Table 2.  Stoichiometric Volumes of Solutions of Different Normalities
Standard Solution

Normality
Equivalent Volumes, ml



H0SO.
2 4
0.0200
10.0
9.4
10.0
6.3
NaOH

0.0189
10.6
10.0
10.5
6.6
Na,,CO,.
2 .1
O.Ol'.M)
9.9
9.5
10.0
6.3
Na H CO..

0.0125
16.0
15. 1
15.9
10.0
HI  CASE EXAMPLES

 The relationships involved in Table 1 may
 best be explained by  reference to the following
 graphs.  These were prepared by titrating
 volumes of standard  solutions of sodium
 hydroxide,  sodium carbonate, and sodium
 bicarbonate with standard sulfuric acid.
 The Stoichiometric volumes of the various
 solutions are summarized in Table 2 for
 convenience in the interpretation of the charts.
 A CASE 1 - Where phenolphthalein alkalinity
     total alkalinity
B  CASE 2 - Where phenolphthalein alkalinity
   = one-half the total alkalinity
                                                                      I      1	I	I	
                                                                 Ml, fl.n^o N II(S(>  ADOKO
   6
  pll
    3
          25 Mt ii.'(M-i N N.UHI

                     II' 
    The sharp break occurs at the point where
    all of the NaOH has been exactly neutral-
    ized by the acid.  The pH and concentration
    of the end products (Na SO and HO)
    determine the pH at the equivalence point
    between NaOH and H?SO.; in this case,
    approximately 7.0.
  The titration proceeds in 2 stages wherein
  all of the CO-  " is converted, first to
  HCO3"and finally to H2CO .  The first
  end point occurs at approximately pH 8,
  and at exactly half the volume of acid
  used for the total titration.  The end point
  which occurs at approximately pH 4
  represents the total alkalinity and requires
  exactly twice the volume of acid used for
  the first end point.

  If either HCO ~ or OH' ions had been
  present the titration volumes for the curves
  would not have been of equal magnitude.
  7-2

-------
                         Alkalinity and Relationships Among the Various Types of Alkalinities
C  CASE 3 - Where phenolphthalein alkalinity
   = 0
25 Ml. 0.0125 N Nnllc'O.j v  11,020 N
            (P  (1)
                                    j
T  The reaction proceeds in one stage with
   the initial pH at approximately 8. 5 and
   final pH at 4. 0.  In this case the phenol-
   phthalein alkalinity is  zero and since no
   conversion of CO- " to HCOg is noted the
   total alkalinity can only be due to the
   HCO3"  ion.

D CASE 4 - Where phenolphthalein alkalinity
   is greater than one-half the total alkalinity
    The volume of acid required for the first
    end point (phenolphthalein alkalinity) is
    due to the OH" neutralization and con-
    version of the CO "" to HCO, .   The
    second end point represents the complete
    conversion of HC03  to H2CO .   Referring
    to Case 2 where the volume of acid was
                                          similar for each end point,  it is apparent
                                          that a base responding to phenolphthalein
                                          but which is not CO,,   must be present.
                                          Since it was originally assumed that OH
                                          and HCO," do not exist in the same
                                          solution we must conclude that the total
                                          alkalinity is due to OH  and COg   .

                                        E CASE 5 - Where phenolphthalein alkalinity
                                          is less than one half of the total alkalinity
                                                                 MI.lMlliu N H.,SO} AOOK1I

                                                 pO Ml. 0.020 N N.-..,r<>.( I Hi Ml. <>. IW N N..WO.Q vs  "."20 N ",!><'.,

                                                                      11> < .'. T)
                                           If, in the reaction NaOH +
                                           Na  CO  + HO, the NaOH exists in
                                           excess quanuty, the final sample con-
                                           tains NaOH and Na CO . (Case 4) in
                                           which the volume of acid required for
                                           the phenolphthalein end point is greater
                                           than one half the total.  In this case,
                                           however,  the situation is reversed,
                                           wherein the volume of acid required
                                           for the HCO  " end point is greater than
                                           one half the total. Referring again to
                                           the reaction NaOH + NaHCO -* Na2C3
                                           HO, if NaHCOg is in excess the end
                                           products must consist of Na^COg and
                                           NaHCO  and OH~  must be absent.  The
                                           end points consist, therefore,  of Na^CO^
                                            -*  NaHCO (phenolphthalein end point)
                                           and NaHCOg  H CO
                                                                                        7-3

-------
Alkalinity and Relationships Among the Various Types of Alkaiinitics
F CASE 6 - Where phenolphthalein alkalinity
   is greater than one-half total alkalinity.
and
                                                          (10 +  12.6) _
               = volume of acid required

                                    The
second end point occurs at
l1P,.t
                                                      for conversion of CO    to HCO
         5     Id     II    -ii    -i'.
                 MI.O.IUU N llj""1, ADIIlil)
       PlOMl. 0.01HO N NiiOM t  HMI. (I.OIiiri N
                       (r
                                                  IV
                                     K'oH 
   Following the original assumption that
   OH" and HCO ~ are not compatible, with
                                                                 ml, the volume of
                                                          e,                             o
                                                      which is converted to H  CO  .  This then
                                                      becomes the same as Case \.
COMPARISON OF ANALYTICAL
METHODS FOR ALKALINITY (According
to the Analytical Reference Service
Report JAWWA Vol.  55,  No. 5,  1963)

            DETERMINATION METHODS

         TABLE 2SlaliaiaU Summary (cnU.)
a
G C
i
12
3
6
PH
3
0
t
condition similar to Case 4 (P > IT).
1ASE 7 - Where phenolphthalein alkalinitj
s greater than one -half total alkalinity.
ill ii i
 ^^^"*-*^^ A 	 (P) KN1> i'OIN 1 ~
	 -r ^^ p( r> I-:NJ> POINT __
III II 1
I in is :MI 2:1 :in r,
Ml.fl. H2U N ll..s<>, Al'li:i>
UttM
r
Vnr
No.o(
Vtton
R*fMnt
-------
                       Alkalinity and Relationships Among the Various Types of Alkalinities
               SUMMARY
Amount Added

Avg.  Deviation from
amount added

Standard Deviation
50% Range
17 mg/1 (as CaCO3)

3.1 mg/1

3.4 mg/1
2.0 mg/1
Method Most Commonly  Electrometric
Used
Method Preferred

   Total Number of
   Observations
Electrometric
     41
V  PROCEDURE

 The actual measurement of alkalinity is a
 very simple procedure requiring only titration
 of sample with a standardized acid and the
 proper indicator.  For phenolphthalein
 alkalinity the end point and indicator are well
 established.  For the bicarbonate titration
 the final end point is a function of the HCO3
 concentration.  With low amounts (< 50 mg/1
 as CaCOJ the pH at end point may be
 approximately 5. 0.  With high concentrations
 (> 250 mg/1 as CaCOJ the pH at end point may
 be 4.5 to 3. 8.  For all-purpose work, in
 which the highest degree of accuracy is not
 required, an end point at pH 4.5 using methyl
 purple as the indicator is recommended.

 The traditional methyl orange frequently
 proves to be unsatisfactory because of the
 indefinite color change at the end point and
 also because of the low pH (3.8  - 3.9)
 required to establish the change.
VI   EPA ANALYTICAL METHODS.

 A  The Environmental Protection Agency,
     Office of Water Program?. Analytical Quality
    Control Laboratory has compiled a manual
    of Analytical Methods which is to be used
    in Federal laboratories for the chemical
    analysis of water and waste samples.
    The title of this manual is "'Methods for
     Chemical Analysis of Water and Wastes,
     1971."

 B This manual lists two parameters which
    are related to the subject matter in this
    outline.  They are total alkalinity and
    total acidity.

 C  For  the measurement of both of these
    parameters, the recommended method
    is volumetric,  with the equivalence point
    being determined electrometrically.
     The  use of a color indicator (methyl
     orange) is recommended only for the
     automated method.

  D  The  procedure references cited by the
     EPA Methods manual are the 13th
     Edition of Standards Methods and ASTM
     Standards,  Part 23,  1970.

  REFERENCES

  1  Standards Methods for the Examination of
       Water and Wastewater,  13th Ed.
       APHA, Inc., New York.   1971.

  2  ASTM Standards,  Part 23.   1973.

  This outline was prepared by R. C.  Kroner, for-.r.
  Chief,  Physical and Chemical Methods,
  Analytical Quality Control Laboratory,
   National Environmental Research Center,
  Cincinnati, OH 45268.

  Descriptors: Alkalis, Alkalinity, Analytical
  Techniques, Buffers,  Buffering Capacity,
  Chemical Analysis, Water Analysis
                                                                                       7-5

-------
                                                OXVCKN
                       Factors Affecting  DO Concentration In Water
I  The Dissolved Oxygen determination is
a very important water quality criteria for
many reasons:                       \

A  Oxygen is an essential nutrient for all
   living organisms.  Dissolved oxygen is
   essential for survival of aerobic
   organisms and permits  facultative
   organisms to metabolize more effectively.
   Many desirable varieties of macro or
   micro organisms cannot survive at
   dissolved oxygen concentrations below
   certain minimum values.   These values
   vary with the type of organisms, stage
   in their life history,  activity,  and other
   factors.

 B Dissolved oxygen levels may be used as
   an indicator of pollution by oxygen
   demanding wastes.  Low DO concen-
   trations are likely to be associated with
   low quality waters.

 C The presence of dissolved oxygen
   prevents or minimizes  the onset of
   putrefactive decomposition and the
   production of objectionable amounts of
   malodorous sulfides,  mercaptans,
   amines, etc.

 D Dissolved oxygen is essential for
   terminal stabilization wastewaters.
   High  DO concentrations are normally
   associated with good quality water.

 E Dissolved oxygen changes with respect
   to time, depth or section of a water
   mass are useful to indicate the degree
   of stability or mixing characteristics
   of that situation.

 F The BOD or other respirometric test
   methods for water quality are commonly
   based upon the difference between an
   initial and final DO determination for a
   given sample time interval and con-
   dition.  These measurements are
   useful to indicate:
II
    1  The rate of biochemical activity in
      terms of oxygen demand for a
      given sample and conditions

    2  The degree of acceptability
      (a bioassay technique) for bio-
      chemical stabilization of a given
      microbiota in response to food,
      inhibitory agents or test conditions

    3  The degree of instability of a
      water mass on the basis of test
      sample DO changes  over an
      extended interval of time.

    4  Permissible load variations in
      surface water or treatment units
      in terms of DO depletion versus
      time,  concentration, or ratio of
      food to organism mass,  solids, en-
      volume ratios.

    5 Oxygenation requirements
      necessary to meet the oxygen
       demand  in treatment units or
       surface  water situations.

The DO  test is the only chemical test
included in all Water Quality Criteria.
Federal. State.  Regional or local

FACTORS AFFECTING THE DO
CONCENTRATION IN WATER
 A  Physical Factors:
           DO solubility in water for an
           air/water system is limited to
           about 9 mg DO/liter of water at
           20C.  This amounts to about
           0. 0009% as compared to 21% by
           weight of oxygen in air.

           Transfer of oxygen from air to
           water is limited by the interface
           area, the  oxygen deficit, partial
           pressure, the conditions at the
WP.NAP.25. 11.75
                                                                                       8-1

-------
Dissolved Oxygen Determination
       interface area, mixing phenomena
       and other items.

       Certain factors tend to confuse
       reoxygenation mechanisms of
       water aeration:

       a  The transfer of oxygen in air
          to dissolved molecular oxygen
          in water has two principal
          variables:

          1)  Area of the air-water
              interface.

          2)  Dispersion of the oxygen-
              saturated water at the
              interface into the body liquid.

          The first depends upon the surface
          area  of the air bubbles in the water
          or water drops in the air; the
          second depends upon the mixing
          energy in the liquid.   If diffusors
          are placed in a line along the wall,
          dead spots may develop in the core.
          Different diffusor placement or
          mixing energy may improve oxygen
          transfer to the liquid two or threefold.

       b Other variables in oxygen transfer
          include:

          3)   Oxygen deficit in the liquid.

          4)   Oxygen content of the gas phase.

          5)   Time.

          If the first four variables are
          favorable, the process of water
          oxygenation is rapid until the liquid
          approaches saturation.   Much more
          energy and time are required to
          increase oxygen saturation from
          about 95 to 100% then to increase
          oxygen saturation from 0 to about
    95%.  For example: t\n oxygen-
    depleted sample often will pick up
    significant DO during DO testing;
    changes are unlikely with a sample
    containing equilibrium amounts
    of DO.
c   The limited solubility of oxygen
    in water compared to the oxygen
    content of air does not require
    the interchange of a  large mass of
    oxygen per unit volume of water
    to change DO saturation. DO
    increases from zero to 50%
    saturation are common in passage
    over a weir.

 d   Aeration of dirty water is practiced
    for cleanup.  Aeration of clean
    water results in washing the air and
    transferring fine particulates and
    gaseous contaminants to the liquid.
     One liter of air at room temperature
     contains about 230 mg of oxygen.
     A 5 gal carboy of water with 2 liters
     of gas space above the  liquid has
     ample oxygen supply for equilibration
     of DO after storage for 2 or 3  days
     or  shaking for 30 sec.

     Aeration tends toward evaporative
     cooling. Oxygen content becomes
     higher than saturation values  at
     the test temperature, thus
     contributing to high blanks.
  Oxygen solubility varies with the
  temperature of the water.
  Solubility at lOOC is about two
  times that at 30 C.  Temperature
  often contributes to DO variations
  much greater than anticipated by
  8-2

-------
                                               Dissolved Oxygen Determination
solubility. A cold water often has
much more DO than the solubility
limits at laboratory temperature.
Standing during warmup commonly
results in a lose  of DO due to
oxygen diffusion from the super-
saturated sample.  Samples
warmer than laboratory tempera-
ture may decrease in volume due
to the contraction of liquid as
temperature is lowered.  The full
bottle at higher temperature will
be partially full after shrinkage
with air entrance around the stopper
to replace the void.  Oxygen in the
air may be transferred to raise the
sample DO.  For example, a
volumetric flask filled to the 1000 ml
mark at 30 C will show a water
level about 1/2 inch below the mark
when the water temperature is
reduced to 2QOC.  BOD dilutions
should be adjusted to 2QOC + or  -
1  1/2 before filling and testing.

Water density varies with tem-
perature  with maximum water
density at 4QC.  Colder or warmer
waters tend to promote stratification
of water that interferes with
distribution of DO because the
higher density waters tend to seek
the lower levels.

Oxygen diffusion in a water mass is
relatively slow,  hence vertical and
lateral mixing are essential to
maintain relatively uniform oxygen
concentrations in a water  mass.

Increasing salt concentration
decreases oxygen solubility
slightly but has a larger effect
upon density stratification in a
water mass.

The partial pressure of the oxygen
in the gas above the water interface
controls the oxygen solubility
limits in the water.   For example,
the equilibrium concentration of
oxygen in water  is about 9 mg DO/I
under one atmospheric pressure of
        air, about 42  mg DO/liter in
        contact with pure oxygen and  0 mj.;
        1X">/ liter  in contact with pure
        nitrogen ((a 20" C').

B   Biological or  Bio-Chemical Factors

    1   Aquatic life requires oxygen  for
        respiration to meet energ>
        requirements for growth, repro-
        duction, and motion.  The net
        effect is to deplete oxygen resources
        in the water at a rate controlled
        by the type, activity,  and mass of
        living materials present,  the
        availability of food and favor -
        ability of conditions.

    2   Algae, autotrophic bacteria,  plants
        or other organisms capable of
        photosynthesis may use light
        energy to synthesize cell materials
        from mineralized nutrients with
        oxygen released in process.

        a   Photosynthesis occurs only
             under the influence of aacq-a?.:,
             light  intensity.

        b   Respiration of alga is
             continuous.

         c   The  dominant effect in terms
             of oxygen assets or
             liabilities of alga depends upon
             algal activity, numbers  and
             light intensity.  Gross algal
             productivity contributes to
             significant diurnal DO
             variations.

     3    High rate deoxygenation commonly
         accompanies assimilation of
         readily available  nutrients and
         conversion into cell mass or
         storage products.   Deoxygenation
         due to cell mass respiration
         commonly occurs at some lower
         rate dependent upon the nature of
         the organisms present, the stage
         of decomposition and the degree
         of predation, lysis,  mixing and
         regrowth. Relatively high
                                                                           8-3

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Dissolved Oxygen Determination
        deoxygenation rates commonly are
        associated with significant growth
        or regrowth of organisms.

        Micro-organisms tend to flocculate
        or agglomerate to form settleable
        masses particularly at limiting
        nutrient levels (after available
        nutrients have been assimilated or
        the number of organisms are large
        in proportion to available food).

        a   Resulting benthic deposits
            continue to respire as bed
            loads.

        b   Oxygen availability is limited
            because the deposit is physically
            removed from the source of
            surface oxygenation and algal
            activity usually is more
            favorable near the surface.
            Stratification is likely to limit
            oxygen transfer to the bed load
            vicinity.

        c   The bed load commonly is
            oxygen deficient and decomposes
            by anaerobic action.

        d   Anaerobic action  commonly is
            characterized by  a  dominant
            hydrolytic or solubilizing action
            with relatively low  rate growth
            of organisms.

        e   The net effect is to produce low
            molecular weight products
            from cell mass with a. corre-
            spondingly large fraction of
            feedback of nutrients to the
            overlaying waters.   These
            lysis products have the  effect
            of a high rate or immediate
            oxygen demand upon mixture
            with oxygen containing waters.

        f   Turbulence favoring mixing of
            surface waters and benthic
            sediments commonly are
            associated with extremely
            rapid depletion of DO.
             Recurrent resuspension of
             thin benthic deposits may
             contribute to highly erratic
             DO patterns.

        g    Long term deposition areas
             commonly act like point
             sources of new pollution as
             a result of the feedback of
             nutrients from the deposit.
             Rate of reaction may be low
             for old materials  but a low
             percentage of a large mass of
             unstable material may produce
             excessive oxygen  demands.

C   Tremendous DO variations are likely
    in a polluted water in reference to
    depth,  cross section or time of day.
    More stabilized waters tend to show
    decreased DO variations although it  is
    likely that natural deposits such as leaf
    mold will produce differences related
    to depth in stratified deep waters,

A CKNOWLEDGMENTS

This  outline contains significant materials
from previous outlines by J. W. Mandia
Review and comments by C.  R. Hirth and
R.  L. Booth are greatly appreciated

REFERENCE

1  Methods for Chemical Analysis of
   Water and Wastes. EPA-MDQARL.
   1974.
 This outline was prepared by F. J.  Ludzack.
 Chemist, National Training Center, MPOD,
 OWPO, EPA. Cincinnati, OH  45268 and
 revised by Charles R.  Feldmann, Chemist,
 National Training Center
 Descriptors :    Aeration,  Aerobic Conditions,
 Air-Water  Interfaces, Anaerobic  Conditions,
 Benthos, Biolcg ical Oxygen Demand,  Dissolved
 Oxygen, Water Pollution,  Water Quality
 8-4

-------
                                  DISSOLVED OXYGEN
          Determination By The Winkler lodometric Titration and Azide Modification
The basic Winkler procedure (1888) has been
modified many times to improve its work-
ability in polluted waters. None of these
modifications have been completely
successful.  The most useful modification
was proposed by Alsterberg and consists of
the addition of sodium azide to control
nitrite interference during the iodometric
titration.   The A zide modification of the
iodometric titration is recommended as
official by the  EPA- WPO Quality Control
Committee for relatively clean waters.

A   Reactions

     1    The determination of DO involves
         a complex  series of interactions
         that must be quantitative to provide
         a valid DO result.  The number of
         sequential reactions also compli-
         cates interference control.  The
         reactions will be presented first
         followed by discussion of the
         functional aspects.
  MnSOx + 2 KOH -*Mn(OH)9 + K SO
2 Mn(OH)
  MnCKOH)2 + 2
                      MnO(OH>
             2 KI - MnS0
(a)

(b)

(c)

(d)

(e)
         Reaction sequence

         The series of reactions involves
         five different operational steps in
         converting dissolved oxygen in the
         water into a  form in which it can
         be measured.
       a   02-MnO(OH)2
                               Mn(SO4)2
             I -> Thiosulfate (thio) or

             phenylarsine oxide (PAO)
             titration.
b   All added reagents are in excess
    to improve contact possibilities
    and to force the reaction toward
    completion.

The first conversion, O_ ~*
MnCKOH)  (reactions a, 6) is an
oxygen transfer operation where
the dissolved oxygen in the water
combines with manganous
hydroxide to form an oxygenated
manganic hydroxide.

a   The manganous salt can react
    with oxygen only in a highly
    alkaline media.

b   The manganous salt and alkali
    must be added separately with
    addition below the surface of
    the sample to minimize reaction
    with atmospheric oxygen via
    air bubbles or surface contact.
    Reaction with sample dissolved
    oxygen is intended to occur
    upon mixing of the reagents arid
    sample after stoppering the
    full bottle (care should be used
    to allow entrained air bubbles
    to rise to the  surface before
    adding reagents to prevent
    high results due to including
    entrained oxygen).

c   Transfer of oxygen from the
    dissolved state to the pre-
    cipitate form  involves a two
    phase system of solution and
    precipitate requiring effective
    mixing for quantitative
    transfer.  Normally a gross
    excess of reagents is  used
    to limit mixing requirements.
    Mixing by rapid inversion 25
    to 35 times will accomplish
    the purpose.   Less energy is
    required by inversion 5 or 6
 CH,O.-do.31c. 11.75
                                                                                     8-5

-------
Dissolved Oxygen Determination
             times, allowing the solids to
             settle half way  and repeat the
             process.  Reaction is rapid;
             contact is the principal
             problem in the  two phase
             system.

         d   If the alkaline floe is white,
             no oxygen is present.

         Acidification {reactions c and d)
         changes the oxygenated manganic
         hydroxide to manganic sulfate
         which in turn reacts with
         potassium iodide to form elemental
         iodine.  Under acid conditions,
         oxygen cannot react directly with
         the excess manganous sulfate
         remaining in solution.

         Iodine (reaction e)  may be titrated
         with sodium thiosulfate standard
         solution to indicate the amount of
         dissolved oxygen originally
         present in the  sample.

         a    The blue color of the starch-
              iodine complex commonly is
              used as an indicator.  This
              blue  color disappears when
              elemental iodine has been
              reacted with an equivalent
              amount of thiosulfate.

         b    Phenylarsine  oxide solutions are
              more expensive to obtain but
              have better keeping qualities
              than thiosulfate solutions.
              Occasional use, field operations
              and situations where it is not
              feasible to calibrate thio
              solutions  regularly,  usually
              encourage use of purchased
              PAO reagents.

          For practical  purposes the DO
          determination scheme involves the
          following operations.
Fill a 300 ml bottle* under
conditions minimizing DO
changes.  This means that the
sample bottle must be flushed
with test solution to displace
the air in the bottle with water
characteristic of the tested
sample*.

*DO test bottle volumes should
be checked  - discard those
outside of the limits of 300 ml
+ or - 3 ml.

To the filled bottle.

 1)   Add MnSO4 reagent (2 ml)

 2)   Add KOH, KI, NaN  reagent
     (2 ml)
     Stopper, mix by inversion,
     allow to settle half way  and
     repeat the operation.
     Highly saline test waters
     commonly settle very
     slowly at this stage and
     may not settle to the half
     way point in the time
     allotted.

 To the alkaline mix (settled
 about half way) add 2 ml of
 sulfuric acid, stopper and mix
 until the precipitate  dissolves.
 Transfer the contents of the
 bottle to a  500 ml Erlenmeyer
 flask and titrate with 0. 0375
 Normal Thiosulfate*.  Each
 ml of reagent used represents
 1 mg of DO/liter of sample.
   S-6

-------
                                                    Dissolved Oxygen  I ^.'termination
             The same thing applies for
             other sample volumes when
             using an appropriate titrant
             normality such as:

             1)  For a 200 ml sample, use
                 0. 025 N Thio

             2)  For a 100 ml sample, use
                 0.0125 N Thio

             *EPA-WPO Method

         The addition of the first two DO
         reagents, (MnSO4 and the  KOH,  KI
         and NaN~ solutions) displaces an
         equal quantity of the sample. This
         is not the case when acid is added
         because the clear liquid above the
         floe does not contain dissolved
         oxygen as all of it  should be con-
         verted  to the particulate MnCXOH^.
         Some error is introduced  by this
         displacement of sample during
         dosage of the first two reagents.
         The error upon addition of 2 ml  of
         each reagent to a 300 ml sample
         is  -i-  X 100 or 1. 33% loss in DO.
            300
         This may be corrected by an
         appropriate factor or by adjust-
         ment of reagent normality.  It is
         generally considered small in
         relation to other errors in sampling,
         manipulation and interference,
         hence this error may be recognized
         but not corrected.

         Reagent preparation and pro-
          cedural details can be found in
          reference 1.
IV    The sequential reactions for the
 Chemical DO determination provides
 several situations  where significant inter-
 ference may occur in application on
 polluted water, such as:

 A   Sampling errors may not be strictly
      designated as  interference but have the
      same effect of changing sample DO.
      Inadequate flushing of the bottle con-
      tents or exposure to air may raise the
      DO of low oxygen samples or lower the
      DO of supersaturated samples.
B  Entrained air may be trapped in a DO
   bottle by:
1
       Rapid filling of vigorously mixed
       samples without allowing the
       entrained air to escape before
       closing the bottle and adding DO
       reagents.

       Filling a bottle with low temperature
       water holding more DO than that  in
       equilibrium after the samples warm
       to working temperature.

       Aeration is likely to cool the sampi-
       permitting more DO to be introduced
       than can be held at the  room or
       incubator temperatures.

       Samples warmer than working
       incubator temperatures will be
       only partially full at equilibriurr
       temperatures.

       Addition of DO reagents results in
       reaction with dissolved or entrained
       oxygen.  Results for DO are invalu
       if there is any evidence of gas
       bubbles in the sample bottle.
   The DO reagents respond to any
   or reductant in the sample capable 01
   reacting within the time allotted.  HOC1
   orH2O2 may raise the DO titration
   while H2S, & SH  may react with sample
   oxygen to lower the sample titration.
   The items mentioned react rapidly ana
   raise or lower the DO result promptly.
   Other items such as Fe   or SO,  may
   or may not react completely within the
   time allotted for reaction.  Many
   organic materials or complexes from
   benthic deposits  may have an effect upon
   DO results that are difficult to predict.
   They may have one effect during the
   alkaline stage to release iodine from
   Kl while favoring irreversible
   absorption of iodine during the acid
   stage.  Degree of effect may increase
   with reaction time. It  is generally
   inadvisable to use the iodometric
   titration on samples containing large
   amounts of organic contaminants  or
                                                                                     8-7

-------
Dissolved Oxygen Determination
      benthic residues.  It would be expected
      that benthic residues would tend toward
      low results because of the reduced iron
      and sulfur content - they commonly
      favor high results due to other factors
      that react more rapidly, often giving
      the same effect as in uncontrolled
      nitrite interference during titration.

      Nitrite is present to some extent in
      natural waters or partially oxidized
      treatment plant samples.  Nitrite is
      associated with a cyclic reaction during
      the acid stage of the DO determination
      that may lead to erroneous high results.

      1   These reactions may be repre-
          sented as follows:
2HN00 + 2 HI
     2
2HN02*-
                          4H O + NO   (a)
                            2^2

                          H0 + N0    (b)
           These reactions are time,  mixing
           and concentration dependent and
           can be minimized by rapid
           processing.

       2    Sodium azide (NaN,) reacts with
           nitrite under acid conditions to
           form a combination of N, + NgO
           which effectively blocks the
           cyclic reaction by converting the
           HNO- to noninterfering compounds
           of nitrogen.

       3    Sodium azide added to fresh
           alkaline KI reagent  is adequate to
           control interference up to about
           20mgof  NO."   N/liter of sample.
           The azide is unstable and grad-
           ually decomposes.  If resuspended
           benthic sediments are not detectable
           in a sample showing a returning
           blue color, it is likely that the
           azide has decomposed in the
           alkaline KI azide reagent.

       Surfactants, color and Fe+++ may
       confuse  endpoint detection if present
       in significant quantities.
                                             F Polluted water commonly contains
                                                significant interferences such as C.
                                                It is advisable to use a membrane
                                                protected sensor of the electronic type
                                                for DO determinations in the presence
                                                of these types of interference.

                                             G The order of reagent addition and prompt
                                                completion of the DO determination is
                                                critical.  Stable waters may give valid
                                                DO results after extended delay of
                                                titration during the acidified stage.  For
                                                unstable water, undue  delay at any stage
                                                of processing accentuates interference
                                                problems.
REFERENCE

1  Methods for Chemical Analysis of Water
      and Wastes. EPA-MDQARL.   1974.
                                            ACKNOWLEDGMENTS

                                            This outline contains significant materials
                                            from previous outlines by J. W. Mandia.
                                            Review and comments by C. R. Hirth and
                                            R. L. Booth are greatly appreciated.
                                            This outline was prepared by F. J. Ludzack,
                                            Chemist, National Training Center.  MPOD,
                                            OWPO,  USEPA, Cincinnati, Ohio 45268. and
                                            revised by C.  R.  Feldmann, Chemist, National
                                            Training Center.
                                            Descriptors: Chemical Analysis, Dissolved
                                            Oxygen, Oxygen, Water Analysis
8-8

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                                     DISSOLVED OXYGEN
                        DETERMINATION BY ELECTRONIC MEASUREMENT
I  INTRODUCTION

A  Electronic measurement of DO is attractive
   for several reasons:

   1  Electronic methods are more readily
      adaptable for automated analysis,  con-
      tinuous recording, remote sensing or
      portability.

   2  Application of electronic methods with
      membrane protection of sensors affords
      a high degree of interference control.

   3  Versatility of the electronic system
      permits design for a particular measure-
      ment,  situation or use.

   4  Many more determinations per man-
      hour are possible with a minor expend-
      iture of time for  calibration.

B  Electronic methods of analysis impose
   certain restrictions upon the analyst to
   insure that the response does,  in fact,
   indicate the item sought.

   1  The ease of reading the indicator tends
      to produce a false sense of security.
      Frequent and careful  calibrations are
      essential to establish workability of the
      apparatus and validity of its response.

   2  The use of electronic devices requires
      a greater  degree  of competence on the
      part of the analyst. Understanding of
      the behavior of oxygen must be supple-
      mented by an understanding of the
      particular instrument and its behavior
      during use.

C  Definitions

   1   Electrochemistry - a  branch of chemistry
      dealing with relationships  between
      electrical and chemical changes.
 Electronic measurements or electro-
 metric procedures - procedures using
 the measurement of potential difference^
 as an indicator of reactions taking
 place at an electrode or plate.

 Reduction - any process in which one
 or more electrons are added to an atom
 or an ijn, such as O + 2e -*  2O~
 The oxygen has been reduced.

 Oxidation - any process in which one
 or more electrons are removed from
 an atom or an ion,  such as ZnO - 2e
    Zn
       +2
The zinc has been oxidized.
Oxidation - reduction reactions - in a
strictly chemical reaction,  reduction
cannot occur unless an equivalent
amount of some oxidizable substance
has been oxidized.  For  example:
     - 4e  5
2+4e
   2H20

   4H    hydrogen oxidize.
     -2
   2O    oxygen reduced
Chemical reduction of oxygen may also
be accomplished by electrons supplied
to a noble metal electrode by a battery
or other energizer.

Anode - an electrode at which oxidation
of some reactable substance occurs.

Cathode - an electrode at which
reduction of some reactable substance
occurs.  For example in I. C. 3, the
reduction of oxygen occurs at the
cathode.

Electrochemical reaction - a reaction
involving simultaneous conversion of
chemical energy into electrical energy
or the reverse.  These conversions are
      Note:  Mention of Commercial Products and Manufacturers Oo-.s
             Not Imply Endorsement by the Environmental Protection
             Agency.
  CH.O.do.32a. 11.75
                                9-1

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Dissolved Oxygen Determination
     equivalent in terms of chemical and
     electrical energy and generally are
     reversible.

  9  Electrolyte a solution, gel, or mixture
     capable of conducting electrical energy
     and serving as a reacting media for
     chemical changes.  The  electrolyte
     commonly contains an appropriate
     concentration of selected mobile ions
     to promote the desired reactions.

 10  Electrochemical cell - a device con-
     sisting of an electrolyte  in which 2
     electrodes are immersed and connected
     via an external metallic  conductor.
     Tifc electrodes may be in separate
     compartments connected by tube con-
     taining electrolyte to complete the
     internal circuit.

     a  Galvanic (or voltaic)  cell - an
        electrochemical cell  operated in
        such a way as to produce electrical
        energy from a chemical change,
        such as a battery (See Figure 1).
                   Folarographic (electrolytic) cell -
                   an electrochemical coll operated in
                   such a way as to produce a  chemical
                   change from electrical energy
                   (See Figure 2).
            Cithitt
                         POLAROGRAPHIC CELL

                               Filiri 2
Auto
Citkide
              GALVANIC CELL

                  Flf HI 1
              As indicated in I. C. 10 the sign of an
              electrode may change as a result of the
              operating mode.  The conversion by the
              reactant of primary interest at a given
              electrode therefore designates terminology
              for that electrode and operating mode.
              In electronic oxygen analyzers, the
              electrode at which oxygen reduction occurs
              is designated the cathode.

              Each cell type has characteristic advantages
              and limitations. Both may be used
              effectively.

              1 The galvanic cell depends  upon
                measurement of electrical energy
                produced as a result of oxygen
t

-------
                                                       Dissolved Oxygen Determination
      reduction.  If the oxygen content of the
      sample is negligible, the measured
      current is very low and indicator driving
      force is negligible, therefore response
      time is longer.

   2  The polarographic  cell uses a standing
      current to provide  energy for oxygen
      reduction.  The indicator response
      depends upon a  change  in the standing
      current as a result of electrons
      released  during oxygen reduction.
      Indicator response time therefore is
      not dependent upon oxygen concentration.

   3  Choice may depend upon availability,
      habit,  accessories, or the situation.
      In each case it is necessary to use
      care and  judgment  both in  selection
      and use for the  objectives  desired.
H   ELECTRONIC MEASUREMENT OF DO

 A  Reduction of oxygen takes place in two
    steps as shown in the following equations:
       H22
+ 2e
                   2e
2OH
                                20H
    Both equations require electron input to
    activate reduction of oxygen.  The first
    reaction is more important for electronic
    DO measurement because it occurs at a
    potential (voltage) which is below that
    required to activate reduction of most
    interfering components (0.3 to 0.8 volts
    relative to the saturated calomel electrode -
    SCE).  Interferences that may be reduced
    at or below that  required for oxygen
    usually are present at lower  concentrations
    in water or may be minimized by the use
    of a selective membrane or other means.
    When reduction occurs, a definite quantity
    of electrical energy is produced that is
    proportional to the quantity of reductant
    entering the reaction.  Resulting current
    measurements thus are more specific for
    oxygen reduction.

 B  Most electronic  measurements of oxygen
    are based upon one of two techniques for
    evaluating oxygen reduction in line with
                                        equation II. A. 1.  Both require activating
                                        energy,  both produce a current propor-
                                        tional to the quantity of reacting reductant.
                                        The techniques differ in the means of
                                        supplying the activating potential; one
                                        employs a source of outside energy, the
                                        other uses spontaneous energy produced
                                        by the electrode pair.

                                         1  The polarographic oxygen  sensor
                                            relies upon an outside source of
                                            potential to activate oxygen reduction.
                                            Electron gain by oxygen changes the
                                            reference voltage.
  a  Traditionally, the dropping mercury
     electrode (DME) has been used for
     polarographic measurements.  Good
     results have been obtained for DO
     using the DME but the difficulty of
     maintaining a constant mercury drop
     rate, temperature control, and
     freedom from turbulence makes it
     impractical for field use.

  b  Solid electrodes are attractive
     because greater surface area
     improves sensitivity.  Poisoning
     of the solid surface electrodes is
     a recurrent problem.  The use of
     selective membranes over noble
     metal electrodes has minimized
     but not eliminated electrode con-
     tamination.  Feasibility has been
     improved sufficiently to make this
     type popular for regular use.

2  Galvanic oxygen electrodes consist of
   a  decomposable anode and a noble
   metal cathode in a suitable electrolyte
   to produce activating energy for oxygen
   reduction (an air cell or battery).  Lee. .-.
   is commonly used as the anode because
   its decomposition potential favors
   spontaneous reduction of oxygen.  The
   process is continuous as long as lead
   and oxygen are in contact in the electrolyte
   and the electrical energy released at
   the cathode may be dissipated by an
   outside circuit.  The anode may be
   conserved by limiting oxygen availability.
   Interrupting the outside  circuit may
   produce erratic behavior for a time
   after reconnection.  The resulting
                                                                                       9-3

-------
 Dissolved Oxygen Determination
nr
       current produced by oxygen reduction
       may be converted to oxygen concen-
       tration by use of a sensitivity coefficient
       obtained during calibration.  Provision
       of a pulsed or interrupted signal makes
       it possible to amplify or control the
       signal and adjust it for direct reading
       in terms of oxygen concentration or to
       compensate for temperature effects.
ELECTRONIC DO ANALYZER
APPLICATION FACTORS
 A Polarographic or galvanic DO instruments
    operate as a result of oxygen partial
    pressure at the sensor  surface to produce
    a signal characteristic  of oxygen reduced
    at the cathode of some  electrode pair.
    This signal is conveyed to an indicating
    device with or without modification for
    sensitivity and temperature or other
    influences depending upon the instrument
    capabilities and intended use.

    1  Many approaches and refinements have
       been used to improve workability,
       applicability, validity,  stability and
       control of variables. Developments
       are continuing.  It is possible to produce
       a device capable of meeting any reasonable
       situation, but situations differ.

    2  Most commercial DO instruments are
       designed for use under  specified con-
       ditions.  Some are more versatile than
       others.  Benefits are commonly reflected
       in the price.  It is essential to  deter-
       mine the requirements of the measure-
       ment situation and objectives for use.
       Evaluation of a given instrument in
       term* of sensitivity, response  time,
       portability, stability, service
       characteristics,  degree of automation,
       and consistency are  used for judgment
       oa a cost/benefit basis to select the
       most acceptable unit.

 B Variables Affecting Electronic DO
    Measurement

    1  Temperature affects the solubility of
       oxygen, the magnitude of the resulting
       signal and the permeability of the
   protective membrane.  A curve of
   oxygen solubility in water versus
   increasing temperature may be concave
   downward while a similar curve of
   sensor response versus temperature
   is concave upward.  Increasing
   temperature decreases oxygen solubility
   and increases probe sensitivity and
   membrane permeability.  Thermistor
   actuated compensation of probe
   response based upon a linear relation-
   ship or average of oxygen solubility
   and electrode sensitivity  is not precisely
   correct as the maximum  spread in
   curvature occurs at about 17 C with
   lower deviations from linearity above
   or below that temperature.   If the
   instrument is calibrated at  a temperature
   within + or - 5 C of working temperature,
   the compensated readout  is likely to be
   within 2% of the real value.  Depending
   upon probe geometry,  the laboratory
   sensor may require 4 to 6% correction
   of signal per  C change in liquid
   temperature.

2  Increasing pressure tends to increase
   electrode response by compression
   and contact effects upon the electrolyte,
   dissolved gases and electrode  surfaces.
   As long as entrained gases  are not
   contained in the electrolyte or under
   the membrane, these effects are
   negligible.

   Inclusion of entrained gases results in
   erratic response that increases with
   depth of immersion.

3  Electrode sensitivity changes occur as
   a result of the nature and concentration
   of contaminants at the electrode sur-
   faces and possible physical chemical or
   electronic side reactions produced.
   These may take the form of a physical
   barrier,  internal short, high residual
   current,  or chemical changes  in the
   metal surface.  The membrane is
   intended to allow dissolved  gas pene-
   tration but to exclude passage  of ions
   or particulates.  Apparently some ions
   or materials producing extraneous ions
   within the electrode vicinity are able
   to pass in limited amounts which
     9-4

-------
                                                    Dissolved Oxygen Determination
   become significant in time.  Dissolved
   gases include 1) oxygen, 2) nitrogen,
   3) carbon dioxide, 4) hydrogen sulfide,
   and certain others.  Item 4 is likely to
   be a major problem. Item 3 may pro-
   duce deposits in alkaline media; most
   electrolytes are alkaline or tend to
   become so in line with reaction n.A. 1.
   The usable life of the sensor varies
   with the type of electrode system,
   surface area, amount of electrolyte
   and type,  membrane characteristics,
   nature of the samples to which the
   system is exposed and the length of
   exposure.  For example,  galvanic
   electrodes used in activated sludge
   units showed that the time between
   cleanup was 4 to 6 months for electrodes
   used for intermittent daily checks of
   effluent DO; continuous use in the mixed
   liquor  required electrode cleanup in 2
   to 4 weeks.  Each electrometric cell
   configuration and operating mode has
   its own response characteristics.
   Some are more stable than others.
   It is necessary to check calibration
   frequency required under conditions
   of use  as none of them will maintain
   uniform response indefinitely.   Cali-
   bration before and after daily use is
   advisable.

4  Electrolytes  may consist of solutions
   or gels of ionizable  materials such as
   acids,  alkalies or salts.  Bicarbonates,
   KC1 and KI are frequently used.  The
   electrolyte is the transfer and  reaction
   media, hence,  it necessarily becomes
   contaminated before damage to the
   electrode surface may occur.  Electro-
   lyte concentration, nature,  amount and
   quality affect response time, sensitivity,
   stability,  and specificity of the sensor
   system.  Generally a small quantity of
   electrolyte gives a shorter response
   time and higher sensitivity but  also may
   be affected to a greater extent by a
   given quantity of contaminating sub-
   stances.

5  Membranes may  consist of teflon,
   polyethylene, rubber, and certain
   other polymeric films.  Thickness
   may vary from 0.5 to 3 mils (inches X
   1/1000).   A thinner membrane  will
   decrease response time and increase
   sensitivity but is less selective and
   may be ruptured more easily.  The
   choice of material and its uniformity
   affects response time, selectivity and
   durability.  The area of the membrane
   and its permeability are directly
   related to the quantity of transported
   materials that may produce a signal.
   The permeability of the membrane
   material is related to temperature and
   to residues accummulated on the
   membrane surface or interior. A
   cloudy membrane usually indicates
   deposition and more or less loss of
   signal.

6  Test media characteristics  control the
   interval of usable life between cleaning
   and rejuvenation for any type of
   electrode.  More frequent cleanup is
   essential in low quality waters than for
   high quality waters.  Reduced sulfur
   compounds are among the more
   troublesome contaminants.  Salinity
   affects the partial pressure of oxygen
   at any given temperature.  This effect
   is small compared to most other
   variables but is significant if salinity
   changes by more than 500 mg/1.

7  Agitation of the sample in the vicinity
   of the electrode is important because
   DO is reduced at the cathode.  Under
   quiescent conditions a gradient in
   dissolved oxygen  content would be
   established on the sample side  of the
   membrane as well as on the electrode
   side, resulting in atypical response.
   The sample should be agitated
   sufficiently to deliver a representative
   portion of the main body of the  liquid
   to the outer face of the membrane.
   It is commonly observed that no
   agitation will result in a very low or
   negigible response after a short period
   of time.  Increasing agitation will cause
   the response to rise  gradually until
   some minimum liquid velocity is reached
   that will not cause a further increase
   in response with increased mixing
   energy.  It is important to check
   mixing velocity to reach a stable high
   signal that is independent of a reasonable
   change in sample mixing.   Excessive
                                                                                   9-5

-------
Dissolved Oxygen Determination
             may create a vortex and expose
      the sensing surface to air rather than
      sample liquid.. This should be avoided.
      A linear liquid velocity of about 1 ft/sec
      at the sensing surface is usually
      adequate.

   8  DO sensor response represents a
      potential or current signal in the
      mill!-volt or milli-amp range in a
      high resistance system.  A high quality
      electronic instrument is essential to
      maintain a usable signal-to-noise ratio.
      Some of the more common difficulties
      include:

      a  Variable line voltage or low batteries
         in amplifier power circuits.

      b  Substandard or unsteady amplifier
         or resistor components.

      c  Undependsble contacts or junctions
         in the sensor, connecting cables, or
         Instrument control circuits.

      d  Inadequately shielded electronic
         components.

      e  Excessive exposure to moisture,
         fumes or chemicals in the wrong
         places  lead to stray currents,
         internal shorts or other malfunction.

C Desirable features in a Portable DO
   Analyzer

   1  The unit should include steady state
      performance electronic and indicating
      components In a convenient but sturdy
      package that is small enough to carry.

   2  There should be provisions for addition
      of special accessories such as bottle
      or field sensors,  agitators, recorders,
      line extensions,  if needed for  specific
      requirements.  Such additions should
      be readily attachable and detachable
      and my!1***1" good working characteristics.

   3  The instrument should include a
      sensitivity adjustment which upon
      calibration will provide for direct
      reading in terms of mg of DO/liter.
   4  Temperature compensation and temp-
      erature readout should be incorporated.

   5  Plug in contacts should be positive,
      sturdy, readily cleanable and situated
      to minimize contamination.  Water
      seals should be provided where
      necessary.

   6  The sensor should be suitably designed
      for the purpose intended in terms of
      sensitivity, response, stability,  and
      protection during use.  It should be
      easy to clean,  and reassemble for use
      with a minimum loss of service time.

   7  Switches, connecting plugs,  and con-
      tacts preferably should be located on
      or in the instrument box rather than
      at the  "wet" end of the line near the
      sensor.  Connecting cables should be
      multiple strand to minimize  separate
      lines.  Calibration controls should be
      convenient but designed so that it is
      not likely that they will be inadvertently
      shifted during use.

   8  Agitator accessories for bottle use
      impose special problems because they
      should be small,  self contained,  and
      readily detachable but sturdy enough
      to give positive agitation and electrical
      continuity in a wet zone.

   9  Major  load batteries should be
      rechargeable or readily replaceable.
      Line operation should be feasible
      wherever possible.

  10  Service and replacement parts avail-
      ability are a primary consideration.
      Drawings, parts identification and
      trouble shooting memos should be
      incorporated with applicable operating
      instructions in the instrument manual
      in an informative organized form.

D  Sensor and Instrument Calibration

   The instrument box is likely to have some
   form of check to verify electronics,
   battery or other power supply conditions
   for use.  The sensor commonly is not
   included in this check.  A known reference
    9-6

-------
                                                     Dissolved Oxygen Determination
sample used with the instrument in an
operating mode is the best available
method to compensate for sensor variables
under use conditions.  It is advisable to
calibrate before and after daily use under
test conditions.  Severe conditions.changes
in conditions, or possible damage call for
calibrations during the use period.  The
readout scale is likely to be labeled -
calibration  is the basis for this label.

The following procedure is recommended:

1  Turn the instrument on and allow  it to
   reach a stable condition.  Perform the
   recommended instrument  check as
   outlined  in the operating manual.

2  The instrument check usually includes
   an electronic zero  correction.  Check
   each instrument against the readout
   scale with the sensor immersed in an
   agitated  solution of sodium sulfite
   containing sufficient cobalt chloride to
   catalyze the reaction of sulfite and
   oxygen.  The indicator should stabilize
   on the zero reading.  If it does not,  it
   may be the result of residual or stray
   currents, internal  shorting in the
   electrode, or membrane rupture.
   Minor adjustments may be made using
   the indicator rather than the electronic
   controls.  Serious  imbalance requires
   electrode reconditioning if the electronic
   check is  O.K.  Sulfite must be carefully
   rinsed from  the sensor until the readout
   stabilizes to prevent carry over to the
   next sample.

3  Fill two  DO bottles with replicate
   samples  of clarified water similar to
   that to be tested.  This water should
   not contain significant test interferences.

4  Determine the DO in one by the azide
   modification of the  iodometric titration.

5  Insert a magnetic stirrer in the other
   bottle or use a probe agitator.  Start
   agitation after insertion of the sensor
   assembly and note the point of
   stabilization.
          Adjust the instrument calibration
          control if necessary to compare
          with the titrated DO.

          If sensitivity adjustment is not
          possible,  note the instrument
          stabilization point and designate
          it as ua.  A sensitivity coefficient,
is equal to
                            where DO is the
          titrated value for the sample on
          which ua was obtained.  An unknown
                                IIS
          DO then becomes DO    .  This
                                 
          factor is applicable as long as the
          sensitivity does not change.

       Objectives of the test program and the
       type of instrument influence calibration
       requirements.  Precise work may
       require calibration at 3 points in the
       DO range of  interest instead of at zero
       and high range DO.  One calibration
       point frequently may be adequate.

       Calibration of a DO sensor in air is a
       quick test for possible changes in
       sensor response.  The  difference in
       oxygen content of air and of water is
       too large for air calibration to be
       satisfactory  for precise calibration
       for use in water.
IV  This section reviews characteristics of
 several sample laboratory instruments.
 Mention of a soecific instrument does not
 imply   EPA   endorsement or recommendati.
 No attempt has been made to include all the
 available  instruments, those described are
 used to indicate the approach used at one
 stage of development which may or may not
 represent the current available model.

 A  The electrode described by  Carrit and
    Kanwisher (1) is illustrated in  Figure 3.
    This electrode was an early example of
    those using a membrane.  The anode was
    a silver  -  silver oxide reference cell with
    a platinum disc cathode (1-3 cm diameter).
    The salt bridge consisted of N/2 KC1 and
                                                                                   9-7

-------
Dissolved Oxygen Determination
   KOH.  The polyethylene membrane was
   held in place by a retaining ring.  An
   applied current was used in a polarographic
   mode.  Temperature effects were relatively
   large.  Thermistor correction was studied
   but not integrated with early models.
                Figure 3
   The Beckman oxygen electrode is another
   illustration of a polarographic DO sensor
   (Figure 4).  It consists of a gold cathode,
   a silver anode,  an electrolytic gel con-
   taining KC1,  covered by a teflon membrane.
   The instrument has a temperature readout
   and compensating thermistor, a source
   polarizing current,  amplifier with signal
   adjustment and  a readout DO scale with
   recorder contacts.
SENSOR ELECTRONICS
IX.-""1"

E

, 2
1


\N^


y


M
s

i3

t t 


k'
! M *f I..... r,.k.

                                                          A|ltt*r Art.. I.
                                                                           Afil.l.r ling

-------
                                                       Dissolved Oxygen Determination
  The Galvanic Cell Oxygen Analyzer (7,8)
  employs an indicator for proportional DO
  signal but does not include thermistor
  compensation or signal adjustment.
  Temperature readout is provided.  The
  sensor includes a  lead anode ring,  and
  a silver cathode with KOH electrolyte
  (4 molar)  covered by a  membrane film
  (Figure 7).
       rociiion Galvanic Call Oxyfon robe
                             Connecter Leodi
                              Thermlttor Cable
                                 Retainer
                             . Tapered Section
                              to Pit iOD ottlei
                              Plaitlc Membrane
                               Retainer Ring

                              lead Anode Ring
Silver Cathode 
Polyethylene Membrane
  The Weston and Stack Model 300 DO
  Analyzer (8) has a galvanic type sensor
  with a pulsed current amplifier adjustment
  to provide for signal and temperature
  compensation.   DO and temperature
  readout is provided.  The main power
  supply is a rechargeable battery.  The
  sensor (Figure 8) consists of a  lead anode
  coil recessed in the electrolyte cavity
  (50% KI) with a platinum cathode in the tip.
  The sensor is covered with a teflon mem-
  brane.  Membrane retention by rubber
  band or by a plastic retention ring may be
  used for the bottle agitator or depth
  sampler respectively.  The thermistor
  and agitator are mounted in a sleeve that
  also provides protection for the membrane.
                                               G The EIL Model 15 A sensor is illustrated
                                                  in Figure 9.  This is a galvanic cell with
                                                  thermistor  activated temperature com-
                                                  psnsation and readout.  Signal adjustment
                                                  is provided.  The illustration shows an
                                                  expanded scheme of the electrode which
                                                  when assembled  compresses into a  sensor
                                                  approximating 5/8 inch diameter and 4 inci
                                                  length exclusive  of the enlargement at the
                                                  upper end.  The anode consists of com-
                                                  pressed lead shot in a replaceable capsule
                                                  (later models used fine lead wire coils),
                                                  a perforated silver cathode sleeve around
                                                  the lead is covered by a membrane  film.
                                                  The electrolyte is saturated potassium
                                                  bicarbonate.  The large area  of lead
                                                  surface, silver and membrane provides
                                                  a current response of 200 to 300 micro-
                                                  amperes in oxygen saturated water  at
                                                  2DOC for periods of up to 100 days use (8).
                                                  The larger  electrode displacement favors
                                                  a scheme described by Eden (9) for
                                                  successive  DO readings for BOD purposes.
V   Table 1  summarizes major characteristic
 of the sample DO analyzers described in
 Section IV.  It must be noted that an ir.._-.;  .
 analyst may adapt any one of these for .-;.  .<
 purposes on a do-it-yourself program.  The
 sample instruments are mainly designer L>-
 laboratory  or portable field use.   Those
 designed for field monitoring purposes may
 include similar designs or alternate designs
 generally employing larger anode,  cathoce,
 and electrolyte capacity to approach better
 response stability with some sacrifice in
 response time and sensitivity.  The electrciv
 controls, recording, telemetering, and
 accessory apparatus generally are semi-
 permanent  installations of a complex nature.
                                               A CKNOW LEDGMENTS:

                                               This outline contains certain materials from
                                               previous outlines by D. G. Ballinger,
                                               N. C.  Malof,  andJ.W. Mandia.  Additional
                                               information was provided by C.R. Hirth,
                                               C.N.  Shadix,  D.F. Krawezyk, J.  V/oo.h-i.
                                               and other t;.

-------
Dissolved Oxygen Determination
      WESTON  &  STACK
          DO  PROBE
                 CORD

                 CORD RESTRAINER


                 SERVICE CAP

                 PROBE  SERVICE  CAP

                 ELECTROLYTE  FILL SCREW

                 PROBE  BODY

                 PLATINUM CATHODE

                 CONNECTOR PINS

                 PIN  HOUSING

                 LEAD ANODE

                 REMOVABLE PROBE SHIELD
                 AND THERMISTOR HOUSING
                               Figure 8
   9-10

-------
                                              Made! A1SA ELECTRODE COMPONENT PARTS
       Cable Sealing
            Nut
          A 15017
                       Cable
                     Connection
                       Cover
                      A15016A
'O' Ring
  R524
        'O' Ring
          R389
            R385
                    Membrane Securing
                          'O' Ring
                           R317
           Anode
          Contact
          A 4 5 0140
       (With Sleeve S24)

 Anode
Contact
 Holder
A15015A
Lead Anode
Co*f
(A150

ilete
24A)
 
1

S



'O' Ring
R612
                Membrane Securing
                      'O' Ring
                       R317
Silver Cathode
   A15013A
Filler Screw
   Z47I

.  ^ [III!

1 111 II ILL. 
 ' lllllll Illlllll'1 
                                                             O' Ring
                                                              R622
                         Note: Red wire of cable connects to Anode Contact Holder

                              Black wire of cable connects  to Anode Contact

                              Membrane  not shown E. I. L.  part number T22
                              'O' Ring
                               R622
                                                                                                  End Cap
                                                                                                   A15011A
                                                a
                                                en
                                                co
                                                O
                                                
                                                a
                                                                                         m
                                                                                         n>

                                                                                         O
                                                                                         a>
CD
I
                                                                                         O
                                                                                         a
                                                         FHI.K

-------
TAB1.K \
CHARACTERISTICS OF VAHIOHS LABORATUHY UO INSTRUMKNTS
DO Temp.


Carrit &
Kanwisher
Beckman

Yellow Springs
51

Yellow Springs
54

Precision
Sci
Weston tk
Stack
300 	


Delta
T5
Delta
85 	 _

Anode
silver -
silver ox.
ring
Aq*
ring
Ag
coil

"

Pb
ring 	
Pb
coil
Pb

Lead
Lead

Cathode
Pt
disc
Au
disc
Au
ring



silver
disc
Pt
disc
Ag

Silver
disc
Silver
	 disc

Elec
KC1
KOH
N/2
KC1
gel
KC1
so In
sat.


KOH
4N
ja
40%
KHCO,

KOH
IN
KOH
IN

Type
pol*
pol

pol



galv*

galv
galv

galv
galv

Membr
polyeth
teflon

teflon



*polyeth

teflon
teflon

teflon
teflon
Sig.
Adj.
no
yes

yes

yes

no

yes
yes

yes
yes
Comp.
Temp. Rdg.
no
ves
yes
no*
yes

yes
yes

no
yes
yes
ves_
yes
yes
no
yes
Accessories for
which designed
Recording temp.
& signal adj. self
assembled
recording

field and bottle
probe

recording field
bottle & agitator
probes


agit. probe
depth sampler
recording

field bottle &
agitator probe
field bottle &
agitator probe
 *Pdl - Polarographic (or amperometric)
**G*lv - Galvanic (or voltametric)
 REFERENCES

 1  Carrit, D.E. and Kanwisher, J.W.
       Anal. Chem.  31:5.   1959.

 2  Beckman Instrument Company.   Bulletin
       7015, A Dissolved Oxygen Primer,
       Fuller-ton. CA.   1962.

 3  Instructions for the YSI Model 51 Oxygen
       Meter,  Yellow Springs Instrument
       Company. Yellow Springs. OH 45387.

 4  Instructions for the YSI Model 54 Oxygen
       Meter,  Yellow Springs Instrument
       Company. Yellow Springs. OH 45387.
5  Technical Bulletin TS-68850 Precision
     Scientific Company, Chicago, IL  60647.

6  Mancy, K.H..  Okun,  D.A. and Reilley,
     C.N.  J. Electroanal. Chem.  4:65.
     1962.

7  Instruction Bulletin, Weston and Stack
     Model 300 Oxygen Analyzer.  Roy F.
     Weston.  West Chester, PA  19380.

8  Briggs. R.  and Viney, M.   Design and
     Performance of Temperature Com-
     pensated Electrodes for Oxygen
     Measurements.  Jour, of Sci.
     Instruments 41:78-83.   1964.
     9-12

-------
                                                       Dissolved Oxygen Determination
 9  Eden, R. E.  BOD Determination Using        11   Methods for Chemical Analysis of Water
     a Dissolved Oxygen Meter.  Water                and Wastes,  EPA-MDQARL. 1974.
     Pollution Control, pp. 537-539. 1967.

10.  Skoog,  D.A.  and West, D. M.  Fundamentals
     of Analytical Chemistry.  Holt,
     Rlnehart & Winston,  Inc.  1966.
                                                 This outline was prepared by F.  J. Ludzack
                                                 Chemist, National Training Center,  MPOD, OWPO,
                                                 USEPA,  Cincinnati,  OH  45268 and Nate
                                                 Malof, Chemist, EPA.OWPO, National
                                                 Field Investigations Center, Cincinnati, OH


                                                 Descriptors :   Chemical Analysis, Dissolved
                                                 Oxygen,  Dissolved Oxygen Analyzers,
                                                 Instrumentation, On-Site  Tests,  Water Analysis,
                                                 Analysis, Wastewater, Oxygen
                                                                                    9-13

-------
                    BIOCHEMICAL OXYGEN DEMAND TEST PROCEDURES
I  OXYGEN DEMAND OF POLLUTED
   WATERS

Established practice includes common use
of the BOD test as a tool for estimation of
the bio-oxidizable fraction of surface waters
or wastewaters discharged to them.  Any
index including a quantity per unit time such
as th BOD  is a rate  expression.  The
ultimate demand  is more important than any
one point on the progression.  The results
of a bottle test with minimum seeding and
quiescent storage are  not likely to be as
high as those on the same influent in a mix-
ing situation and  abundant seed of secondary
treatment or receiving waters. The BOD
is "a" fraction of total oxygen requirements.

A The particular technique used for BOD
   commonly is specified by State agencies
   and/or supervisors.  They are required
   to interpret the results as obtained by
   laboratory testing.   It is essential that
   the tester and the interpreters have a
   common understanding of what was done
   and how.  It is highly advisable to main-
   tain a given routine until all concerned
   agree upon a change.

   1  Each particular  routine has many un-
      deflnable factors. The particular
      routine is not as important as the con-
      sistency and capability with which the
      result was  obtained.

   2  This outline and Standard Methods
      discusses several valid approaches for
      obtaining BOD results.  Selection of
      "method" is not  intended in this outline
      or in the EPA Methods Manual*2'.

B The common 5-day incubation period for
   BOD  testing is a result of tradition and
   cost. Initial lags are likely to be over
   and some unknown fraction of the total
   oxidizable mass has been satisfied after
   5 days.

C A series of observations over a period of
   time makes it possible to estimate the
   total  oxidizable mass and the fraction
   oxidized or remaining to be oxidized at
   any given time.  The problem is to define
   the shape of the deoxygenation pattern and
   its limits. A fair estimate of the shape of
   the deoxygenation pattern is available by
   observations at 1, 2, or 3 days,  7 days
   and 14 days..   Increased observations are
   desirable for more valid estimates of
   curve shape, rate of oxidation and total
   oxidizable mass or ultimate BOD.

 D Increasing impoundment of surface waters
   and concurrent increases in complexity
   and stability of wastewater components
   emphasize the importance of long-term
   observation of BOD. The 5-day observation
   includes most of the readily oxidizable
   materials but a very small fraction of the
   stable components that are the main factors
   in impoundment behavior.
II   DIRECT METHOD

 A  With relatively clean surface waters, the
    BOD may be determined by incubation of
    the undiluted sample for the prescribed
    time interval.  This method is applicable
    only to those waters whose  BOD is less
    than 8 mg/1 and assumes the sample
    contains suitable organisms and accessory
    nutrients for optimum biological
    stabilization.

 B  Treated effluents, polluted  surface waters,
    household and industrial wastewaters
    commonly require dilution to provide the
    excess oxygen required for the oxygen
    demand determination.  General guidelines
    for dilution requirements for a given BOD
    range in terms of the percent of sample in
    BOD dilution water are:

    For a 5-day BOD of
      5-20 mg/1, use 25 to 100% sample

    For a BOD of
      20-100 mg/1, use 5 to 25% sample

    For a BOD of
      100-500 mg/ 1, use 1  to 5% sample

    For a BOD of
      500-5000 mg/1, use 0. 1  to 1. Q/c sample
III  PROCEDURES

 A Cylinder Dilution Technique
  CH.O.bod.57g. 11. 75
                                                                                        10-1

-------
 Biochemical Oxygen Demand Test Procedures
   1  Using an assumed or estimated BOU
      value as a guide, calculate the factors
      for a range of dilutions to cover the
      desired depletions.  Those dilutions
      ranging from a depletion of 2 mg/l
      and a residual of 1  mg/1 are most
      reliable.  At least three dilutions in
      duplicate should be used for an
      unknown sample.

   2  Into a one-liter graduate cylinder (or
      larger container if necessary)  measure
      accurately the required amount of mixed
      sample to give one liter of diluted waste.
      Fill to the one liter mark with dilution
      water. Carefully mix.   The initial DO by
      calculation includes IDOD (VIII) a
      determined initial does not.  Both are
      essential to estimate significance of
      IDOD.  Entrapment of air bubbles during
      manipulation must be avoided.

   3  Siphon the mixture  from the cylinder into
      three 300 ml glass  stoppered bottles,
      filling the bottles to overflowing.

   4  Determine the DO concentration on  one
      of the bottles by the appropriate
      Winkler modification and record as
      "Initial DO".

   5  Incubate the two remaining bottles at
      20C in complete darkness.  The
      incubated bottles should be water-sealed
      by immersion in a tray or by using  a
      special water-seal bottle.

   6  After 5 days of incubation, or other
      desired interval, determine the DO  on
      the bottles.  Average the DO concentration
      of the duplicates and report as  "Final DO".

B  Direct Dilution Technique

   1  It may be more convenient to make the
      dilution directly in  sample bottles of
      known capacity.  A  measured volume of
      sample may be added (as indicated in
      A-l) above',  and the bottle filled with dilution
      water to make the desired sample
      concentration for incubation.   In this
      case, the sample must be precisely
      measured, the bottle carefully  filled,
      but  not overfilled,  and the bottle volumes
      comparable and known.  Precision is
      likely to be poorer than for cylinder
      dilution.

   2  Continue the procedure as in A-4, 5,
      and 6 above.
C  Seeded Cylinder Dilution. Technique

   1   Many wastewaters may be panially or
      completely sterile as a result of
      chlorination, effects of other toxic
      chemicals, he&t,  unfavorable pH or
      other factors detrimental to biological
      activity.  Validity of the BOD result
      depends upon the presence of organisms
      capable of prompt and effective bio-
      degradation and favorable conditions
      during  the particular test.  Correction
      of the cause resulting in sterilization
      must be corrected by adjustment,
      dilution, etc. , prior to reinoculation to
      achieve meaningful BOD data.   Receiving
      water,  biologically treated effluents,  and
      soil suspensions are a good  source of
      organisms  likely to be adapted for
      stabilization of wastewaters.  Untreated
      wastewaters provide numerous organisms
      but are likely to contain nutrients
      contributing to excessive seed corrections
      and may require appreciable time for
      adaptation before  test waste oxidation
      becomes significant.

   2   The amount of added inoculant must be
      determined by trial.  The concentration
      added should initiate biochemical
      activity promptly but should  not exert
      enough  oxygen demand to unduly reduce
      the oxygen available for sample
      requirements.

   3   Estimate the sample concentration
      desired in accordance with A-l and
      C-2 above and add the sample aliquot to the
      dilution cylinder.

   4   Add approximately half of the required
      amount of dilution water to the sample
      and mix.  This is  necessary to assure
      that the concentrated waste does not
      exert a toxic effect on the seed organisms.

   5   Measure a suitable aliquot of seed into
      the bottle or cylinder and fill with
      dilution water.  Mix the combined sample,
      seed and dilution water without excessive
      air entrainment.

   6   Continue as in III-A steps 4, 5, and 6 above.
IV  INTERPRETATION OF RESULTS

 Standard Methods   includes a calculation
 section that is valid and concise.  Preceding
 it  are details of reagent preparation and
   10-2

-------
                                          Biochemical Oxygen Demand Test Procedures
procedures for the test.  These will not be
reprinted here.   This section considers
certain items that may cause concern about
the validity of results unless they are care-
fully considered and controlled.

A  The initial DO of the BOD test obviously
   should be high.  The method of attaining
   a high DO can trap the analyst.

   1  Aeration of dilution water is the most
      commonly  considered treatment.
      This technique does produce a high DO
      but it is a treacherous ally.

      a  Dirty air passing through clean
         dilution water can produce clean
         air and  dirty water.  This is a
         simple air-washing operation.
         Filtering the entering air stream
         may remove brickbats and 2 x  4's,
         but filters tend to  pass organic
         gases, fine aerosols, and partic-
         ulates.

      b  A stream of air passing through
         water tends to cool the water by
         evaporation 1 to 3  C below ambient
         temperature.  The cooled liquid
         picks up more DO than it can hold
         at ambient temperature.  The
         physical loss of oxygen may produce
         an erroneously high depletion value
         for a determined initial DO, or a
         low depletion on a calculated
         initial DO.  Erroneous blanks are
         a particular concern.  The dilution
         water temperature /DO shift is
         critical.

   2  Raising DO by allowing the sample to
      equilibrate in a cotton-plugged bottle
      for 2 or 3 days permits oxygenation
      with minimum air volume contact.

   3  Shaking a partially filled bottle for a
      few seconds also oxygenates with  min-
      imum opportunity of gas washing con-
      tamination, supersaturation,  or
      temperature  changes.

B  Seeding always is a precarious procedure
   but a very necessary one at times.  Often
   the application of seed corrections is a
            if you do,
if you don't"
   situation.  Hopefully,  seed corrections
   are small because each individual
   biological situation is a "universe" of
   its own.

   1   Unstable seeding materials such as
      fresh wastewater have "seed11 organisms
      characteristic of their origin and
      history.  Saprophytes resulting in
      surface water stabilization may be a
      small fraction of the population.  Re-
      actable oxygen-demanding components
      produce excessive  demands upon test
      oxygen resources.

   2   A seed containing viable organisms at
      a lower energy state because of limited
      nutritional availability theoretically is
      the best available seed source.  An
      organism population grown under
      similar conditions  should be most
      effective for initiating biochemical
      activity as soon as the nutrient situation
      favors more activity.  The population
      should not be stored too long because
      organism redistribution and die-out
      become limiting.  This type of seed
      would most likely be found in a surface
      water or a treatment plant effluent
      with a history of receiving the particular
      material under consideration.

   3   Seed sources and amounts can only be
      evaluated by trial.   Different seed
      sources and locations  require checkout
      to determine the best available material
      from a standpoint of rapid initiation of
      activity, low correction, and predictable
      high oxygen  depletion under test.

C  Chlorination and BOD results fundamentally
   are incompatible.  Chlorination objectives
   include disinfection as the number one
   goal.  Chlorine is notoriously non-specific
   in organism effects.  Chlorine acts like
   an oxidant in the DO determination.  Test
   organisms are  less suitable for activity
   than they were before chlorination.
   Nutrients may be less available after
   chlorination.   Certainly the conditions are
   less suitable for biological response after
   chlorination.   Dechlorination is feasible
   with respect to the oxidizing power of
                                                                                        10-3

-------
 Biochemical Oxygen Demand Test Procedures
    chlorine,  but many organic chlorine com-
    pounds that do not show strong oxidizing
    action still have toxic effects on biologic
    response.

    Numbers are obtainable after dechlorination
    and reseeding.  The meaning of these
    numbers is obscure.  At least two states
    (New York and New Jerssy) specify BOD's
    before chlorination only.
 V  PRECISION  OF THE BOD TEST
 A  The DO test precision often has been used
    to suggest precision of the BOD result.

    DO precision is a relatively minor and
    controllable factor contributing to BOD
    results.  Other factors  such as organism
    suitability, members,  adaptation and
    conditional variables are much more
    difficult to control or to evaluate.

 B  The Analytical Reference Service  report
    on Water Oxygen Demand,  July, 1960
    (Sample type VII) included the results of
    seeded samples of glucose-glutamic acid
    BOD results from 34 agencies on 2,  3,  5
    and 7 day incubations.

    The relative geometric  standard deviation
    (average) was 19% on 2% sample and 24%
    on 1% sample concentrations.   Rate
    coefficients ranged from 0. 10 to 0. 27 with
    a median of 0. 16 from 21 different
    laboratories that participated in rate studies.
VI  ALTERNATE BOD TECHNIQUES

 Reaeration methods are becoming increasingly
 popular in order to approach more nearly the
 actual waste concentration in the receiving
 water.   It is common to obtain "sliding" BOD
 results  related to the concentration of waste
 in a series of dilutions of the same sample.
 This may result from greater possibilities
 for toxic effects at higher concentrations, or
 to a different selection of organisms and change
 in oxidation characteristics at low concentrations
 of sample.  The most reliable estimate of stream
 behavior is likely to be from that dilution closest
 to the wastewater dilution in the receiving water.
    Reaeration can be accomplished by the
    usual series techniques by dumping all of
    the remaining sealed bottles into a common
   container when the residual DO reacfies about
   1. 0 mg/1.  After reoxygenation, the remaining
   bottles are refilled and a new initial DO
   determined.  Subsequent dissolved oxygen
   depletions are added incrementally as a
   summation of the total oxygen depletion
   from the start of the test.  If necessary,
   the reaeration technique may be performed
   several times but at  a sacrifice of double
   DO determinations for each day on which
   reaeration occurs.

B  Special methods of reaeration have evolved
   to  minimize the extra manipulation for
   reaeration of individual sample dilutions.

   1  Elm ore Method

      A relatively large volume  of the sample
      is stored in an unsealed bottle.   Small
      bottles are withdrawn in sets of 5 or
      more,  sealed, incubated,  and the DO
      determined at appropriate intervals.
      When the DO concentration in the smaller
      bottles reaches 1. 0 mg'l, a new set is
      withdrawn from the large  unsealed
      bottle, after reoxygenation if necessary.

   2  Orford Method

      The deoxygenation is carried out in a
      large sealed jug from which samples
      for  DO are withdrawn at appropriate
      intervals.  To maintain the waste level
      and a sufficient DO in the  jug, additional
      waste is added from  a second open
      container.   See diagram.

C  Excess oxygen may be provided by
   oxygenation with commercial oxygen
   instead of with air to increase the initial
   oxygen content for incubation while limiting
   the number of dilutions  or  reaeration steps
   When oxygen is used in  place of air the
   oxygen saturation in water at 20C is about
   40 mg/1 instead of 9 mg/1.  Limited results
   are available hence the  analyst must verify
   his technique.  The DO  tends to decrease
   as soon as the bottle is  opened hence,  about
   35 mg/1 of oxygen content  is the top of the
   practical working concentration.  There has
   been no evidence that the biota is inhibited
   by the  higher oxygen content  with respect
   to BOD progression.

D  Reaeration or Oxygenation Advantages and
   Limitations.

   1  Reaeration expands the  range of BOD
      results obtainable directly at field
      concentrations, but is not advisable for
      applications when the sample BOD
      exceeds 50 mg' 1.
   10-4

-------
                                                Biochemical O.xygen Demand Test ['rot edures
     2  Dilution water problems are eliminated,
       to the extent that the stream sample may
       be tested without dilution.

     3  Incubator storage space becomes a real
       problem i'or multiple sample routine.


VII  Dissolved oxygen electrodes, polarographic
  and others, are feasible for use in BOD
  determinations, often making it possible to
  make an estimate of DO or BOD when sample
  interference prevents a valid Winkler DO
  determination.

  Electronic probe DO makes it possible to
  determine many successive DO's at different
   time intervals on the same bottle with
   negligible sample loss.  Reaeration or
   extended time series, therefore, are more
   feasible.

   Another outline in this series describes
   response of reaerated BOD,, with electronic
   DO probes.
                       It is the rebponsibility of the analyst to
                       evaluate:

                       1  Applicability of the specified technique
                         and sample.

                       2  To determine requirements for mixing
                         and possible thermal  effects while
                         mixing in terms of instrument response
                         and biochemical reaction.

                       3  To evaluate long-term calibration or
                         standardization and their effects upon
                         precision and accuracy of the BOD
                         result.
                  VIH  IMMEDIATE DISSOLVED OXYGEN
                       DEMAND (IDOD)

                     Immediate dissolved oxygen demand include;
                     dissolved oxygen utilization requirements of
                     substances such as ferrous iron, sulfite
                     and sulfide which are susceptible to high
                     rate chemical oxidation.
          REAERATION METHODS FOR i.O.D. DETERMINATION
          reservoir
AA
                     AAAA
                    sealed bottles
               ELMORE METHOD
                                      d.o.  samples

                                OtFOiD METHOD
                                                                                   10-5

-------
Biochemical Oxygen Demand Test Procedures
A  The IDOD is an apparent response as
   indicated by a specified technique.  Since
   DO titration is based upon iodine titration,
   any factor that causes Io  response different
   from that produced by the reaction of KI
   and molecular oxygen confuses the IDOD
   determination,

B  IDOD Determination

   1  The IDOD determination includes the
      determination of DO on a sample and
      dilution water separately.  A waste
      likely to have a significant IDOD is
      unlikely to show a DO.

   2  According to mixing theory, it should
      be possible to calculate the DO of any
      definite mixture of the sample and
      dilution water from the DO of component
      parts and their proportion.
      The same relative proportions of sample
      and dilution water should be mixed
      without air entrainment and the DO
                       *6 arbitrarilv elected
   4  Any difference between the calculated
      initial DO as obtained in  2 above, and
      the DO determined in 3 above, may be
      designated as IDOD.

   5  Sample aeration.  DO interference,  and
      other factors affect   results for IDOD.

C Sample  Calculation of IDOD

   1  Sample DO checked and shown to
      be 0. 0 mg/ 1
Dilution water DO found to be 8. 2 mg! \

Assume a mixture of 9 parts of dilution
water and 1 part (V/V) of sample.
Calculated DO -

1X0=0

9 X 8.2 = 73.8

  10 parts of the mixture contain 73. 8/10
  or 7. 4 mg DO/1.  Note that mixing has
  reduced the DO concentration because
  the original amount is present in a
  larger package.

  The mixture described above  was held
  for 15 minutes and the DO determined
  was 4. 3 mg/1.

                               100
                           7c sample"
                             used
     IDOD = DO   . - DO . .  ->
                calc    detm

           = 7.4 - 4. 3 x 10

          = 31 mgIDOD/1

REFERENCES

1  Standard Methods, APHA-AWWA-WPCF
     13th fid.  1971.

2  Methods for Chemical Analysis of Water
     and Wastes,  EPA-MDQARL.  1974.

This outline was prepared by F. J.  Ludzack,
Chemist,  National Training Center, MPOD,
OWPO,  USEPA, Cincinnati, Ohio 45268.
                                                   Descriptors:  Biochemical Oxygen Demand,
                                                   Chemical Analysis,  Dissolved Oxygen,
                                                   Water Analysis,  Analysis, Wastewater
    10-6

-------
            BOD DETERMINATION - REAERATED BOTTLE PROBE TECHNIQUE
I  INTRODUCTION

A  Customary dilution technique has certain
   limitations with respect to the BOD
   determination such as a prior commit-
   ment on sample dilutions to be used,
   number of bottles to be included to permit
   Wlnkler DO determinations to be made
   throughout a predetermined test interval,
   (with sample destruction for each DO test),
   possibility for anomalous effects due to
   dilution water, dilution, or inconsistent
   response between test bottles.

 B DO probe technique offers the advantages
   of nondestructive DO testing, possibilities
   of adjusting routine according to sample
   behavior during test, and retaining the
   sample with minor losses for long term
   observation (if desired) and for supplemen-
   tary tests at the  end of the BOD test
   interval.

   Note:  Use of the probe results in a loss
   of about 1/2  ml of each 300 ml sample.
   Use dilution water  with a glass rod or
   bead to bring the sample volume back to
   300 ml for further  testing.

 C  This test was performed in triplicate on
    the same sample at 100% concentration
    with reaeration  as needed to illustrate
    results obtainable.
H  PROCEDURE

 A  Sample

    Final effluent (catch sample at 1100 hours)
    of the Advanced Waste Treatment Research
    Activated Sludge (Unit B) Sanitary Engineering
    Center Experimental Wing (September 3,  1970).
    One hundred percent concentration.

 B  DO Probe Calibration

    The probe was  calibrated daily in air
    saturated tap water versus the azide
  modification of the iodometric titration.
  A replicate test bottle of the titrated
  sample was retained for initial probe
  adjustment,  if necessary,  and to recheck
  sensor response at the end of the use
  period.  Zero sensor response in cobalt
  treated sulfite solution was checked at
  three day intervals.  Electronic response
  and battery condition were checked daily.

C Reaeration

   1  When the DO test indicated 1. 0 mg
      DO/liter or appeared likely to reach
      that point prior to the next test reading
      the test bottles were reaerated and a
      new initial DO was obtained.

   2  An adapter was inserted into the neck
      of the sample bottle indicating low DO
      and an  empty bottle inserted on the
      opposite end. The combination was
      shaken vigorously, while in an inverted
      position to allow the transfer of sample
      to the empty bottle; immediately after
      transfer the  combination was reinverted,
      shaken as before and sample returned
      to the original bottle.

   3  The sample was allowed to stand for
      at least five  minutes to allow entrained
      air to rise (generally formed a froth
      ring).   The froth ring was raised into
      the neck of the bottle by displacement
      with about 1/2 ml of dilution water.
      It usually was necessary to tilt the
      bottle  slightly and roll it to sweep
      fine air bubbles off the shoulder of the
      bottle and into the neck.  After careful
      reinsertion of the DO sensor these
      bubbles were displaced by tipping the
      assembly and discarding contents of
      the BOD bottle lip containing the frothy
      residue.

   4  It is recognized that surface active
      materials and possibly other components
      would concentrate in the  discarded froth.
      It would be possible to  limit this effect
  CH.O.bod.lab.2a. .11.75
                                                                                        10-7

-------
BOD Determination - Reaerated Bottle Probe Technique   _ __
      by allowing a longer time for the froth
      to break--say 15 to 30 minutes.  This
      was not attempted for this demonstration.

D No attempt was made to determine DO at
   any regular time interval.  Observation
   time ie recorded for each test on a
   24-hour clock basis with zero time at
   midnight.

E Results are recorded in tabular form
   including the date (Column 1), time in
   hours (2),  sample temperature (3), hours
   of incubation (4), and the triplicate sample
   data.   For each of the samples the
   observed DO, the change  since the last
   observation (ADO) and the summation of
   all the DO depletions observed during
   the incubated interval are recorded as
   L ADO.  A bold face line in the tabulated
   data in the DO column indicates DO before
   reaeration; the number below the line
   indicates DO after reaeration to be used
   as a new initial for the next observation.

F Near the end of the fifth day of incubation
   (116 hours) one of the triplicates (no.  3)
   was not reaerated to check the effect of
   complete depletion of oxygen.  It was
   reaerated on the following day and thereafter.

G The results plotted  in graphic form are
   presented following the BOD tabular data.

H The results of a similar respiration test
   on mixed liquor from the same activated
   sludge units are tabulated to show the
   effects of different types of feed on DO
   depletion (respiratory activity). In this
   case,  time is in minutes,  DO  is in mg/1
   and ADO/minute also is in mg/1.  The
   first test in Table 2 represented 100 ml
   of return sludge plus plant effluent to fill
   the bottle.  Approximately one percent
   nutrient agar solution was added by
   syringe (1.0 ml) to produce  the response
   noted in the second series; one ml of five
   percent mercuric chloride produced the
   effects in series 3.  A new replicate
   sample (100 ml sludge) and plant influent
   to fill the bottle produced the results in
   series 4.
Ill  SUMMARY

 A Table 1 shows that it is possible to obtain
    consistent results using a DO probe plus
    reaeration for BOD technique.  The
    major requirement is to carefully calibrate
    the DO instrument and sensor on a daily
    basis. It is not recommended to reaerate
    as many times as found necessary here.
    It could have been diluted at any time
    since the sample was available.    Had
    the BOD response at five days been
    substantially complete only two
    reaerations would have been necessary.
    However, since the sample was working
    into second stage BOD it proceeded to
    react accordingly.  The oxygen demand
    of 15 mg/liter at five days increased  to
    30 mg/liter in seven days and reached
    45 mg/liter in ten days.  The sample that
    was allowed to deplete on the fifth day
    slowly recovered but by the  twelfth day
    or thereafter equaled or exceeded  the
    BOD of the samples with residual DO at
    all times.  This technique requires much
    less incubator space, time and manip-
    ulation with an added advantage of
    retaining the sample for adjustment or
    extended observation.  The results do
    not  depend upon a preconceived guestimate
    which may or may not fit the situation.

 B  Table 2 indicates the possibility for
    estimating feed acceptability in an
    activated sludge  treatment plant.  On
    many occasions an inquiry from an
    industrial plant,  a different type of
    inflow or perhaps a new plant requires
    a decision regarding effects at the  treat-
    ment plant.  A respiration test by probe
    technique offers  an opportunity to rate
    the situation within ten minutes after
    probe  calibration.  A test of sludge and
    effluent oxygen demand gives an estimate
    sludge respiratory activity now.  When the
    same sludge is subjected to a new feed of
    an acceptable nature respiratory activity
    rises as in series 2.   If the feed happened
    to be highly toxic as in series 3 (mercuric
    chloride) respiratory activity stops--
 10-8

-------
                                  BOD Determination - Reaerated Bottle Probe Technique
obviously a dead or severely shocked
condition.  A replicate sludge (Series 4)
with the regular plant influent shows a
prompt increase in activity.  Oxygen
requirements,  permissible load ratio,
acceptable feed to sludge ratios (return
sludge adjustment) all may be estimated
on the basis of DO tests  related to overall
plant performance.

The graphic of BOD versus time includes
the COD--an estimate of first stage
oxygen demand and the TKN for the
composite effluent sample. If the total
Kjeldahl nitrogen  (TKN) is multiplied by
its oxygen equivalent (4. 57)  then the COD
plus TKN times 4,57 gives an estimate
of ultimate oxygen demand (138 mg/1).
  The twenty day BOD was about f>f> \
  or forty percent of this  value.  The effluent
  sample obtained at 1100 hours is unlikely
  to contain a significant portion of the high
  load period considering the primary,
  aerator and secondary clarifier detention.
  Also the COD and TKN  may have included
  materials that were  chemically oxidized
  but gave a poor response to biological
  oxidation.  The BOD gives the rapid
  respiratory oxygen demand.  COD plus
  the oxygen equivalent of N (on the same
  sample)  gives a quick estimate of potential
  ultimate demand.
This outline was prepared by F. J.  Ludzack,
Chemist, National Training Center, MPOD,
OWPO,  USEPA, Cincinnati, Ohio 45268.

Descriptors: Analysis,  Biochemical Oxygen
Demand, Chemical Analysis, Dissolved Oxvs
Wastewater, Water Analysis
                                                                                    10-9

-------
 BOD Determination - Reaerated Bottle Probe Technique
                                           TABLE 1
                     BOD Tabulated Results - Reaerated Sample; Probe DO

                                    9/3/70 100% Sample

Date
9/3/70
9/3/70
9/3/70
9/4/70

9/4/70
9/8/70

9/8/70

9/9/70

9/10/70

9/11/70

8/11/70


9/12/70

9/13/70

9/14/70

9/15/70

9/16/70
* 9/20/70

Time
1340
1355
1620
1010

1600
1340

0940

1235

1200

0800

1400


2035

1130

1200

1340

1200
1240

Toc
24.0
24.0
22. C
20.2

20.6
19.5

19.8

20.0

20.0

20.0

20.0


20.0

20.0

20.0

19.6

19.3
21.6
Hrs.
Incub
0
0.25
2.6
20.5

26.3
72

116

143

166

186

192


222

238

262

288

310
480
Sample 1 ,
DO
6.7
6.7
6.5
4.9
8.2
7.4
1.0
8. 1
0.6
8.6
1.1
8.2
1.0
8.2
1.2
8.4
5.0
8.1

0.5
8.2
6.3
8.3
7.0

5.7

5. 1
3.8
ADO

-
0.2
1.6

0.8
6.4

7.5

7.5

7.2

7.0

3.4


7.6

1.9

1.3

1.3

0.6
1.3
EADO


0.2
1.8

2.6
9.0

16.5

24.0

31.2

38.2

41.6


49.2

51. 1

52.4

53.7

54.3
55.6
Sample 2
DO
6.7
6.7
6.5
4.7
8.3
7.5
1.5
8.1
0.5
7.6
1. 1
8.2
1.2
8.6
1.1
8.3
5.3
8.4

0.6
8.2
3.8
8.4
6.9

6.2

5.8
4.6
ADO

-
0.2
1.8

0.8
6.0

7.6

6.5

7.0

7.5

3.0
EADO


0.2
2.0

2.8
8.8

16.4

22.9

29.9

37.4

40.4
I

7.8

4.4

1.5


48.2

52.6

53. 1

0.7 | 53.8

0.4 j 54.2
1.2 j 55.4
Sample 3
DO
6.8
6.8
6.6
4.8
7.9
6.9
1. 1
7.4
0.4

0.0
7.6
2.0
8.0
2.0
8.5
5.7
8.6

0.4
8.2
4.6
8.3
0.9
8.3
4.5
8.2
7.2
4.9
ADO

-
0.2
1.8

1.0
5.8

7.0

0.4

5.6

6.0

2.8

EADO


0.2
2.0

3.0
8. 8

15. 8

16.2

21.8

27. 8

30.6

I
8.2 ' 38.8

3.6

42.4

7.4 49 . 8

3.8

1.0
2.3

53.6

54.6
56.9
* Incubator power off
   10-10

-------
                               BOD Determination - Reaerated Bottle Probe Technique
       ACTIVATED SLUDGK HKSPIKATION DATA 1>O fliOHK
Time
Min
 0
 1
 2
 3
 4
 0
 1
 2
 3
 0
 1
 2
 3
 0
 1
 2
 3
Temp

 240



 24.5




 24.8



 24.2
DO


7.2
6.6
6.2
5.6
5. 1
4.7
3.8
2.7
1.7
4.7
4.8
4.7
4.7
5.9
4.6
3.3
2.0
  ADO

Series 1

  0.6
  0.4
  0.6
  0.5
Series 2
  0.9
  1.1
  1.0
Series 3

Nil

Series 4
  1.3
  1.3
  1.3
                                                          Sample
100 ml Return Sludge & Effluent
+ 1 ml Nutrient agar
+ 1 ml HgCl2 Soln
100 ml Sludge + Influent
                                                                              10-11

-------
o
I
                                   GRAPHIC OF REAERATED PROBE BOD RESULTS
                                    STARTING 9/3/70 SAMPLED 1100 HOURS

               COMPOSITE SAMPLE RESULTS FOR 9/3/70

                   COD-69 m/LITER

                   TKN-15 Hf/LITER

               ULTIMATE DEMAND 138 mg/LITER
                                                                   23 DO DETERMINATIONS

                                                                    1/2 ml DISPLACED PER

                                                                        DETERMINATION
LOSS
                                                                            ml *  100
       300
                                                                                    = 3.8%
                                    SAMPLE ALLOWED TO  DEPLETE
                                          INCUBATION DAYS jhrs)
                                         1        1       1
                                         8(216)   10(268)  12(312)
I
14
          I
          16
I
18
I
20

-------
                      EFFECT OF SOME VARIABLES ON THE BOD TEST
I  TIME
                                    ,-kt
A  The common equation y^=L(l-10 "") for
   BOD relationship indicates time as a
   variable.  The rate coefficient (k^lndicates
   that a specific  percentage of material
   initially present (oxygen) will be used
   during a given  time unit.  Each successive
   unit of time has less reactant present
   initially than the preceding interval,  hence
   a definite precentage decrease results in
   successively smaller amounts of reactant
   use per unit of lapsed time. Increasing
   kj results in a larger percentage oxygen
   use per unit of time and also increases the
   change in reactant mass among successive
   time intervals.

B  Adney's work for the British Royal Com-
   mission cited 5 days passage time from
   source to the ocean as maximum for
   English streams.   The 8th Report  (1909)
   largely established BOD philosophy in-
   cluding the 5-day interval. At 5 days,
   initial lags generally have terminated and
   a substantial fraction of the long-term
   oxygen demand has been exerted.   If only
   one time interval can be used, 7 days
   permits better scheduling. Any one time
   Interval is "a" fraction of the total oxygen
   requirement; this  is a poor reference
   point if we do not  know how it arrived.
   For example, the percentage of
   oxidizable material stabilized in terms
   of oxygen use at various rate  factors
   are:
                  % oxidized
           (logio>
    k.,   (loe,,,) in 5 days      KI (log )
   0.1)5
   0. 10
   0. 15
   0. 25
   0.50

This range (K   =2.3 k^ is commonly en-
countered in wasteWater stabilization with
the higher rates characteristic of fresh oxi-
dizable material that is  readily converted.
in 5 days
42%
67%
84%
94%
99+%
K! (L
0. 11
0.23
0.34
0.57
1. 15
   The lower coefficients are characteristic
   of cell mass at later stages of oxidation
   and of low-rate reactants in general.

   The oxygen utilization at specified inter-
   vals of time are required to estimate k.,..
   and L, the estimate of oxygen use at
   infinite time.  It is common to observe
   results at equal intervals of time but
   this is not  essential as long as
   the time intervals are accurately known.
   The initial time periods are critical as
   an error of a few hours in time represent-
   a relatively large change in  reactant mpss
   in a system at maximum instability.  Un-
   equal time periods can be plotted to define
   the curve from which any given interval
   can be selected as desired.
D  Increasing impoundment of surface wa-r:
   provides more time for stabilization or
   relatively inert soluble or suspended
   pollutants and for organism adaptation
   to the situation or pollutants.  Long te- ;.
   BOD's are essential to indicate changes
   in the pattern of oxygen demand vs. time.
   It may be expected that one or more
   plateaus will be evident in the BOD cur ,
   followed by a temporary rise in rate
   during second stage oxidation or thereaftr-
   Anaerobiosls may cause a rise in rate
   coefficient after aerobic conditions are
   re-established.  Eventually k  stabilize L.
   at very low values.

   1 Rate coefficients tend to be difficu.".:
     interpret  during long term BOD's
     because of progressive changes and
     other factors.

     a  The relative error of the DO test may
        be a large fraction of the incremental
        DO change during low rate periods.

     b  Cell mass may agglomerate under
        quiescent test conditions  and decrease
        nutrient availability.
 CH.O.bod.56d. 11.75
                                   10-13

-------
 Effect of Some Variables on the BOD Test
      c  U it not likely t)iat recycled nutrients
         under aerobic test conditions will
         Have as much effect as recycle from
         anaerobic benthic deposits in a
         stream.

    2  The BOD result tends to underestimate
      doxygenation relative to surface water
      behavior because of interchanges,
      turbvdence,  biota, and boundary  effects.
      Reeceokig doeo not occur in a sealed
      bottle but rcseeding IB inevitable in a
      atrwuu or Treatment unit.

U  TEMPERATURE

 A  Effect on Oxidation Hate

    Temperature is one of the important con-
    trolling labors in any biological system.
    In the BOD reaction,  changes in tempera-
    ture prodjce acceleration or depression
    of the rate  of oxidation.  Figure 1 shows
    the  changes in the value of k at tempera-
    tures from 0 - 25C on a common
    wastewater.
B  Teat Temperature

   In th BOD test procedure an arbitrary
   tempera U: re ie usually selected for
   convenience even though a wide temperature
   range exists under natural conditions.

   Incubctioa of the teat containers at 20C
   for tha  whole period is now accepted
   practice in the U.S.; 18. 5C is preferred
   in England.  Camp (ASCE, SA591.1,  Oct.
   65) recommended light  and dark bottle
   immersion  in the stream.

C  Temperature Cor rection

   When it is necessary to calculate the  rate
   of oxidation at a temperature other than
   20,  the following relationship may be
   used:


        ^ .  * 
    0, 3

    0, 2
    0. 02


                                          J
        LJi.
where:
IJJ
        j =  rate coefficient at temperature T

      kr> -rait? at coefficient al te rape ran; re T,,

        6 -  tempera lure t oa/f-;-lu;,;,  :'or which
           Streeter zA Phe'.p,-- obtained  w.e value
           i, 047,   e changes *ith temperature;  it
           appears to be higher in the  range of
           5-15C thar. ir. iht range  of 30 to 40C.
           The value given refers tc 15-30.


     The cited temperature coefficient appears
     reasonable for household wastes.  It may
     not apply lor other wastes where  developing
     or  seed organisms may not tolerate tem-
     perature changes  as readily. A given
     temperature coefficient should be checked
     for applicability under specified conditions.
  A The organisms involved in biochemical
    conversions apparently have an optimum
    response near a pH of 7. 0 providing other
    environmental factors are favorable; a pH
    range of about 5. 5 to 8. 3 apparently is
    acceptable (Figure 2).  Reactivity is likely
    to be significantly lower on both sides of the
    acceptable pH range but microbial adapta-
    tion may extend the limits appreciably.
    For example, trickling filters have operated
    with better than 50% treatment efficiency
    at pH 3 and 10 after adaptation.
   10-14

-------
                                                   Effect of Some Variables on the BOD Test

     2 C
     & >.


               Fvgur* 2



           4         6    pH   8
 B  Adjustment of Concentrated Samples

    When wastes are more acid than pH 6. 5 or
    more alkaline than pH 8. 3, adjustment to
    pH 7. 2 is advisable before reliable BOD
    values can be obtained.

 C  Dilution Samples
    Standard dilution water is buffered at pH
    7.2.  Sample-dilution water mixtures should
    be checked to make sure that the sample
    buffer capacity does not exceed the capacity
    of the dilution water for pH adjustment.


IV  ESSENTIA L MINERA L NUTRIENTS

 A  Importance
    In 1932 Butterfield reported on the role of
    certain minerals in the biochemical oxidation
    of sewage and concluded that deficient
    minerals often upset metabolic response.
    In addition, he found that inadequate nitrogen
    and/or phosphorus was a common cause of
    low BOD  results in industrial wastewaters.
    (Figure 3)
               J   S   4   5      T

                       Time In Dyi
                Effect of Minerml Nutrients on BOD
H  Standard Methods Dilution Water
   The dilution water specified for the BOD
   test approximates USGS estimates for an
   average U.S. mineral content of surface
   water except for added phosphate buffer.
   It is assumed to provide essential mineral
   nutrients for most wastewaters but cannot
   be expected to meet requirements for
   grossly deficient wastewater nutrients both
   mineral and organic.  Ruchhoft (S.W. J.
   13:669, 1941) summarized committee action
   leading to the present dilution water.

C Other Dilution Considerations
   There is a trend toward the use of receiving
   water, storage-stabilized if necessary,  to
   evaluate waste behavior.  It is advisable
   to minimize dilution and consider the
   nutrient level likely in the receiving  water
   as most valid.  Any change in the environ-
   ment,  such as dilution, upsets the
   microbial balance and requires adaptive
   changes.

V  MICROBIOLOGICA L POPU LA TION

A Need for Complex Flora and Fauna
   Butterfield, Purdy, and Theriault (Pub.
   Health Rep. 393,  1931) demonstrated that
   an isolated species of organisms was not
   as effective in biological stabilization as
   a variety of species.  Figure 4 summarires
   some of their data.   Bhatta and Gaudy
   (ASCE, SA3, 91:63, June 1965) reinvestigated
   this factor.  Many studies have emphasized
   the need for a mixed biota in the BOD test.
   It appears that bacteria are capable  of
   varied activities, but all species are not
   capable of synthesizing all required nutrients.
   Certain bacterial species may be capable
   of producing enzymes, amino acids, or
   growth factors needed for their use and by
   other species for optimum performance.
   It has been shown that oxygen demand
   becomes minimal when some limit of
   bacterial population has been reached.
   Predation prevents such  an approach to
   maximum numbers and maintains a con-
   tinuing bacterial growth and recycle of
   nutrients among a mixed population.  The
   net effect is a symbiotic  relation among
   mixed organisms tending to enhance the
   rate of stabilization or utilization of
   oxygen as in the BOD test.
                                                                                    10-15

-------
Effect of Some Variables on the BOD Test
B  Organism Adaptation

   1 Early invest!gallons in "elation to the
     BOD test considered domestic H a stew:-re i-;:-:
     primarily.  The saprophytic organisms
     involved in  stabilization eithf-r were
     prevent in adequate numbers or quickly
     multiplied to attain effective populations.
         <
   2 ThJperiod  of adjustment required to
     shift enzyme production needed to utilise
     an'energy source  differ ant from that
     previously utilized or to shift population
     variety from that  favored by one food tt>
     that favored by another food i,-- con-
     sidered an adaptation  period.  Dilution.
     temperature, oxygen tension,  pH,
     nutrient type,  inhibitory substances,
     light and other changes all are common
     inducements for microbial adaptation.
     Mutation of organisms nisy be encountered
     during adaptation but usual1,;- i? not a
     factor.

   3 The developments in industry and
     technology have resulted in discharge
     of new  and more varied waatewatc-r
     constituents.   Microorganisms may
     adapt themselves  to the use of a new
     substance as an energy source providing
     the energy and environment are favor-
     able.  The receiving stream usually shows
     development of an adapted microbiota
     for a new or different  discharge con-
     stituent within hours,  days or weeks
     after fairly regular discharge.  The
     time for adaptation depends on the na ure
     of the constituent, available energy..
     tolerance of the organisms, and environ-
     mental conditions.

C  Seedtnl

   The amount of seed and its selection must
   be determined experimentally.  The most
   effective inoculant would be that which
   WOttW produce the maximum BOD response
   with minimum lag period and negligible
   eed demand.  This  would mean some
   nfMExfrnum population adapted to feed and
   conditions at a minimum equilibrium energy
   nutrient supply.
  pro.'irrssiixi on s. synthetic feed with
   'vet s--a*?r am'- :-ttU^ sewage inoculants
  ai several cor?ce^trations.  The river
  \v?t*?r resulted  in higher BOD with
  >;eg?ieible I?.?? and set-d  correction.  The
  deed correction at 20%  concentration
  of inoculant was less than 0.3 mg.
  DO/lat 5 day a. i would be possible to
  use this river v/atsr as a diluent without
  excessive oxygen loss to  produce more
  valid BOD progression  for that receiving
  water.  The lower waetev/ater  inoculant
  concentration resulted in a  definite BOD
  lag.   Higher wastewater concentrations
  produced comparable BOD progression
  earlier but resulted in high seed
  corrections and lowered availability of
  rissolved oxygen for the sample.

2 A good secondary treated effluent
   ,j.roducd results similar tc river wai^r
  inoculation with higher  seoo corrections
  per increment  of applied tncculant.
  Soil suspensions also are v^ry effective
   sou; ces  of seed organisms  with minor
  s^ed corrections if chey are reasonablj
  stabilized surface soils.,

3 It appears that the BOD progression
  meat nearly  indicating  receiving water
  oxidation would be one based upon
  receiving water dilution or  inoculated
  with organisms from it.

1 A new or unusual wastewater may
  require adapted organisms  not present
  in sufficient  numbers in the receiving
  watei-.  Development of an adapted seed
  from soil suspensions,  plant effluents
  or receiving water  may be necessary to
  evaluate oxidation potential in  a plant
  or receiving water  at some future tims.
  Enrichment culture technique is  bene-
  ficial whore  small concentrations of the
  test  wastewater are applied regularly
  v.'ith increases in w&stewater concen-
  trations as BOD or respiration activity
  indicates increasing tolerance and
  oxidation of the test waste.  Both time
  and concentration limits are useful to
  characterize the waste-water and its
  acceptability for biological stabilization.
   10-16

-------
                                                   Effect  of Some Variables on the BOD Test
                   All tormi In river wtr
                   Mixed occur!* t> plankton
                   Pure culture B. Aerogene* at plankton
                   Mlied culturt ixrlerU
                   Pure culturt 8. A*rogtntm
                4  3  6   7  
                   Time in Daya
           l of Biological Form* on Oxygen Depletion
                    Figure 4
           DO DEPLETION VS SEED
6  CONCENTRATION & TYPE (ax oiucost-otuiAMtc AC.)
                                    RIVER WATER
                                     STALE W W
                       0.5%  STALE SEW  -
                       0.2%
                       0.1%
                     1-20%  RIVER WATER
                                       DAYS
                  Figui

     It must be recognized that BOD
     progressions are most likely to err
     on the low side.  A  meaningful BOD
     test should seek the highest consistent
     oxygen demand feasible for sample and
     conditions.
 D  Algae

    When large numbers of algae are present
    in surface waters,  they produce significant
    changes in the oxygen  content.  Under the
    influence of sunlight excess oxygen is
    produced while a net deficit occurs in the
    dark.  The result is a wide variation in
    surface water DO depending on sample
    time.

    When stream samples containing algae are
    incubated in the laboratory the algae
    survive for a time, then die because  of the
    lack of light.  Short-term BOD determina-
    tions may show  the influence of oxygen
    production by the algae.  When the algae
    die,  they release the stored organic load
    for  recycle and  increase the  BOD.  There-
    fore, samples incubated in the dark may
    not  be representative  of the  deoxygenation
    process in the stream, since the benefits
    of photosynthesis are lacking.  Conversely,
    samples incubated in the light, under
    conditions of continual photosynthesis,
    will yield low BOD values.

    The influence of algae on  BOD is one of
    the most difficult variables to evaluate.
    More research  is needed  to develop
    satisfactory  methods  for the accurate
    determination of BOD in the  presence o;
    large numbers  of algae.   Light and dark
    bottle incubations suggest the magnitude
    of effects.
IV  TOXICITY

  A  Effect

    Since satisfaction of the  DOD is accom-
    plished through the action of microorgan-
    isms,  the presence of toxic substances
    will result in depression of the oxidation
    rate.  In many cases,  toxicity will
    produce a lag period, until tolerant
    organism activity becomes significant.
    Figure 6 shows the effect of cyanide on the
    BOD curve.  A prominent lag period is
    exhibited in the 2 ppm curve, while at
    10 ppm the  lag extends beyonu the fifth day.
    An activated sludge may be adapted to work
    effectively in degradation of 60 mg C'N/1.

-------
Effect of Some Variables on the BOD Test
                                                                   Table I
                    i       i
                  TUB* ill D*jm
        Ctttct al Cyuld* on BOO at Domestic
          (M Scwaf* IB Formula C OUatiaa Wiur)

                Figure 6

   Heavy  metals have similar effects depending
   on history and environment. The effects of
   copper and chromium  are  illustrated in
   Figure 7.
Waste
cone.
10%
5%
2%
1%
0.5%
Depletion
3.51
4.53
2.80
1.52
0.74
5 day BOD
35
91
140
152
148
              EFFECT OF HEAW \IET\LS O\ BOD
                 Figure 7
 B Detection

   In laboratory determinations of BOD the
   absence of toxic substances including
   chlorine must be established before the
   results can be accepted as valid.
   Comparison of BOD values for several
   dilutions of the waste will indicate the
   presence or absence of toxicity.  In Table 1
   the calculated BOD for the dilutions show
   higher values in the more dilute concen-
   trations. It is apparent that toxicity was
   present  and that the toxic effect was diluted
   out at a  waste concentration of 2% or less.
VII NITRIFICATION

 A Mechanism

    The oxidation process, as exemplified by
    the equation:
                                                            y = L (l-10~kt)
    presumably involves the oxidation of
    carbonaceous matter or 1st stage oxygen
    demand.

          C  H O  -2   C02 + H20


    The rate coefficient is normally  high, giving
    nearly complete oxidation  in a few days.
    When nitrogenous material is present its
    oxidation can be shown as:
          NH3
                                                                         _ O     _

                                                                      N2     N3
     Nitrogen oxidation may be delayed for
     several days during BOD tests unless
     suitable micro-biota are initially available.
     Under some circumstances these two
     oxidations can proceed simultaneously and
     the resultant BOD curve will be a com-
     posite of the two reactions.

                                   k t
  v  =    (1-10  c)  + L  (1-10"  n
  't  L  c                n

  where yt =  the simultaneous BOD of the car-
  bonaceous and nitrogenous phases or 1st and
  2nd stage demands.
       kc and kn = the rate coefficients appli-
     cable to the carbonaceous and nitrogenous
     materials respectively.
   16-18

-------
                                                  Effect of Some Variables on the BOD Test
B
L  and L   the ultimate oxygen demands
characteristic of the two phases respectively.
This is the general formula for any system
characterized by two simultaneous reactions.

Principal conditions  governing simultaneous
carbon and nitrogen oxidation:

1  Presence  of an effective nitrifying
   culture at the beginning of the test
   interval (nitrifiers grow relatively
   slowly).

2  Maintenance of adequate DO, believed
   to be a minimum of 0. 5 to 1.0 mg/1,
   for nitrifier activity.

3  Available  nitrogen - in excess of that
   required for synthesis.  This is believed
   to require a minimum of about 7 mg/1
   to support active nitrification on a
   continuous basis.

4  Nitrifiers appear to be more sensitive
   to toxicity than most saprophytic
   organisms, hence are likely to be
   inhibited more readily.  This is
   particularly evident during nitrite to
   nitrate'conversion.

It  may require 5 to 10 days to establish
nitrification if the population was not
nitrifying initially.  This is the basis for
the sequential carbonaceous and nitrogenous
oxidation of  sewage oxidation.

1  Effects on the BOD curve indicate a
   typical pattern such as in Figure  8.
   The influence of nitrification in the
   production of a secondary rise in the
   BOD curve is so well known that  any
   secondary rise may be erroneously
   attributed to nitrification whether or
   not nitrification was involved.  Actually,
   a secondary rise in the curve may be
   due to any oxidation system assuming
   dominance after the initial oxidation
   system has been completed.

2  The nitrification phenomena occurs
   simultaneously in many  streams,
   treated effluents or partially stabilized
   samples.   The designation of a secondary
     BOD rise to nitrification should be
     based on analysis,  not curve shape.

C  The extent of nitrification is conclusively
   shown  only by periodic analysis of
   ammonia, organic, nitrite and nitrate
   nitrogen.  The conversion of ammonia
   and organic nitrogen to oxidized nitrogen
   is a definite indication of nitrification.

D  Nitrification Inhibition

   Plant efficiencies from a BOD standpoint
   can be  erroneous because nitrification
   generally is not established during the
   usual incubation of influent samples but
   may be a major factor in  effluent
   incubations.  It requires about 2 times
   the oxygen to convert NEL -N  to NO_ -N
   as to convert C to CO hence this is a
   major  fraction of stream oxygen use.
   Most secondary treated effluents are
   characterized by a larger fraction of
   carbon than nitrogen removal  which
   accentuates the problem.

   Pasteurization of samples, methylene
   blue, chromium, and acid treatment
   followed by neutralization have been used
   to inhibit nitrification for estimation of
   carbonaceous BOD only.  Any inhibition
   of nitrification also produces a change in
   the sample  or its behavior and may
   partially inhibit carbonaceous oxidation.
   Nitrification is a factor in stream self-
   purification and treatment.  It does not
   appear realistic to alter it for convenience.
   The most realistic approach to carbon-
   aceous oxidation is the measurement of
   CO2 or COD.
                                                                  Figure 8
                                                                                   10-19

-------
 Effect of Some Variables on the BOD Test
VIII  EFFECT OF DILUTION

When a series of dilutions are made on a
BOO sample usually the result a vary to the
extent that only an approximate BOD value
is obtained.

                 Table 2

      INTERPRETATION OF BOD DATA
Sample cone. DO
i
, Depletion

Initial ; 8. 2
Final:
1* , 5-5 2-7
2* 3. 3 ; 4.9
4% 0.'
) Complete
BOD

"
2TO
245

A For example, in Table 2,  1%,  2% and 4%
   concentrations of sample were used.  The
   4% concentration became anaerobic before
   the end of 5 days.  The 5-day BOD of the
   1% concentration was 270 and that of the
   2% concentration was 245.

B Statistically one value is more reliable
   than the other.
      Dilation

         1%

         2%
      Difference
DO depletion
  5.5 mg/1

  3. 3 mg/1
  2. 2 mg/1
   The difference in depletion between 1 and
   2% dilations is 2.2 mg/1.  This difference
   may be attributed to an additional 1% of
   sample added to the original 1%.  If the
   difference is multiplied by the dilution
   factor of 100 to obtain the BOD,  the result
   is 220 mg/L.

      1  We now have three estimates of the
         BOD on a one percent concentration
         basis from the two dilutions:

         a the actual 1% depletion gives 270
         b 2%/2 depletion gives 245
         c (2%- 1%) depletion gives 220
Statistically the probabilities of being
nearer the actual value goes with the
nearest two of three.  The 4% value
of 8.2 dupletion/4 as a minimum
possible BOD 1% concentration gives
a BOD of at least 200.

There is the possibility that higher
concentrations may reflect  significant
toxicity while lower  concentrations
tend to reflect a greater proportion of
dilution water.  The toxicity problem
does not appear to be significant since
the 4% sample concentration indicated
a BOD of at least 200.  The higher
BOD at 1% sample concentration may
be due to a contaminated dilution water
or to the fact that a  similar number of
seed organisms had less food and
utilized certain fractions that they had
passed by  when they had more choice
with the 2% sample concentration.
Data is insufficient to resolve this one.

Incubations having a depletion of at
least 2 mg DO/liter and a residual of
at least 1 mg DO/liter are  indicated
to be most valid**).  Both the 1 and
2% concentrations fit this requirement
in Table 2.  An average error of
+ or -0.1 ml on the  DO titration would
have a smaller relative error upon
the 2% depletion.

We have a reasonable presumption
that the sample BOD of  about 230 was
a good estimate.  We  do not have an
unequivocal basis for  so stating.
Possible variations in results with
different dilutions of a given sample
are subject to many uncertainties in
the test routine.

If some cause is known - such as a
titration eror, the inclusion of ex-
traneous substances producing high
or low response, or a definite procedural
error that rules out a valid estimate of
the sample BOD- that result should be
 labeled as a lost cause  or unreported.
 Otherwise,  report what was obtained
to the best of your ability with the
 provision of uncertainty for uncon-
trollables.
   10-20

-------
                                                   Effect of Some Variables on the BOD Test
A CKNOW LEDGM ENT:

Certain portions of this outline contain
training material from prior outlines by
D. G.  Ballinger and J. W.  Mandia.
REFERENCE:

Standard Methods, APHA-AWWA-WPCF,
   13th edition,  1971.
This outline was prepared by F. J. Ludzack,
Chemist,  National Training Center,  MPOD,
OWPO, USEPA, Cincinnati, Ohio 45268.

Descriptors:  Algae,  Ammonia, Bacteria,
Biochemical Oxygen Demand,  Essential
Nutrients, Microorganisms, Nitrates, Nitri-
fication, Nitrites, Nutrients,  Oxygen Demand,
Rates,  Time,  Toxicity, Waste Dilution,
Water Analysis, Chemical Analysis
                                                                                       10-21

-------
       MATHEMATICAL BASIS OF THE BIOCHEMICAL OXYGEN DEMAND (BOD) TEST
                                           Part 1
 I   FUNDAMENTAL CONCEPTS

 For a number of years, the oxidation of
 organic waste substances in surface waters
 haa been under investigation.  The first re-
 ported observation of oxygen depletion was
 on the Seine River below Paris (1877).  Other
 studies followed in Germany, England and
 the U.S.  Certain fundamentals are now
 universally accepted.  These are:

 A  Dissolved oxygen in the water is reduced
    during stabilization of the organic material.

 B  As long as dissolved oxygen is present,
    the  rate of oxidation is independent of the
    actual amount of oxygen available.

 C  The type and numbers of biological forms
    present is an important factor.

 D  Measurement of changes in oxygen content
    can be related to quantity and character
    of oxidizable organic matter.
                    II   FIRST ORDER RATE CONCEPT

                     In order to illustrate the mathematical
                     relationships in the BOO reaction, assume
                     the following laboratory observations:

                     A  A set of replicate bottles is filled with
                        river water and sealed so that outside
                        air is excluded.

                     B  Each day,  one of the bottles is analyzed
                        for DO  content, and the results tabulated.
                        (See Table 1.)

                        It will be  noted that on each successive
                        day, the DO concentration is less than
                        the day before.  That is, oxygen is being
                        consumed by biological or chemical
                        action in the water.  The oxygen residual
                        when plotted against time forms the
                        deoxygenation curve (Figure 1).

                        The oxygen demand or BOD curve is re-
                        presented by the summation of observed
                        oxygen  depletions ve time (Figure 2).  This
                                          Table 1.
        Days
DO
       Oxygen depletion
Per day                Cumulative
0
1
2
3
' 4
5
6
7
8
9
10
9.2
7.4
5.8
4.6
3. 7
2.9
2.3
1.8
1.5
1.2
0.9
0
1.8
1.6
1.2
0. 9
0. 8
0.6
0.5
0.3
0.3
0.3
-
1.8
3.4
4.6
5.5
6.3
6.9
7.4
7. 7
8.0
8.3
CH. O.bod. 58c.ll.75
                                                   Preceding page Hank
                                                                                 10-23

-------
Mathematical BatuB of the BOD Teat

                Figure 1
  is an inverted representation of the de-
  oxygenation curve.  Inclusion of the daily
  equilavents of oxygen demand in Figure 2
  shows that increasingly smaller amounts
  of oxygen normally are used during each
  successive day.

  If the reaction follows a Ist-order
  rate system, a constant percentage of the
  oxygen present at the beginning of each
  day will be used during that day.   There-
  fore,  the rate coefficient (K in nat. log.
  or k  in log,0) is constant from day to day.
  Kj =^.3 k
            :
   Similarly, the organic matter present in
   the sample is being oxidized,  so there is
   progressively less oxidizable  material
   present each successive day.  The rela-
   tionship between oxygen demand and the
   amount of oxidizable material present
   can be stated as follows:  The oxygen
   demand per unit time is proportional to
   the amount of unoxidized material present.
   Streeter and Phelps^ * ^stated the concept
   as - the rate of biochemical oxidation of
   organic matter is proportional to the con-
   centration of unoxidized substance,
   measured in terms of oxidizability.   Such
   a rate ia  termed a First Order Reaction
  Rate.  The same authors also  stated that
  there is no logical reason to expect one
   raie coefficient ["but iTlnay"appear so
I   of crude measurement and  the effects of
   many individual oxidation systems.
                                                                 TIMI IN MfTS
 Figure 2.  INTEGRAL BOD CURVE

 The curve as shown in Figure 2 is a
 typical BOD curve.  When interpreted in
 light of the fundamentals previously dis-
 cussed,  it is apparent that:

 1   With organic materials which are
    easily oxidized,  the reaction proceeds
    rapidly,  nearing stabilization in a
    few days.  The curve is therefore
    steep,  rapidly approaching a maximum.

2  As oxidation nears completion the BOD
   curve will approach a limit or maximuir
   value that can be  represented by a line
   above the time axis and parallel to it.
   This line represents the ultimate oxyger
   demand (L) for the sample and condi-
   tions of the test.  The curvature of  the
   BOD line as it approaches the maximum
   is a function of the rate of oxidation.

i   The BOD test commonly is applied for
   samples containing mixed components
   each of which  may differ in availability
   as a nutrient.  The population of organ-
   isms generally contains  many varieties.
  A progression of varieties occurs from
  those favored by an  excess of initial
  nutrients to those  favored by cell mass
  and eventually to those tolerating a low
  available energy level.

-------
                                                       Mathematical Basis of the BOD Test
   4  This type of system may have apparent
      characteristics of 1st order reaction
      mechanisms for limited periods of
      time.  It is likely that two or more 1st
      order mechanisms may be apparent if
      observation time, nutrients and condi-
      tions are appropriate.  The data also
      may be interpreted as a zero order, 2nd
      order or multiple order reaction
      mechanism.  It is not always possible
      to prove reaction order nor is  it essen-
      tial to  do so.  The main objectives are
      to be able to estimate the ultimate BOD,
      the amount of BOD remaining after a
      given time interval and to obtain rea-
      sonable agreement among observed and
      calculated data.  The first order
      reaction concept provides "a" means
      to meet the need without implying  that
      the reactions are necessarily 1st order
      or that it is the only means of approach.
      t
      L

      L1
      y

   then:
time in days

ultimate demand
demand remaining at time t

demand satisfied at time t
            fraction of L remaining at time t
          =  fraction of L oxidized at time t
 The relationships may be expressed as a
 differential equation with respect to "t"
             dL
             dt
                                                                     -KL
                           (1)
UI  BOD EQUATION

 Using the typical BOD reaction curve,  it is
 possible to develop equations expressing the
 various relationships.  Labeling the coordi-
 nates as in Figure 3.
 where K is a rate coefficient for deoxygena-
 tion as a loge.
                                                      changing to
                         -Kt
                                       (2)
                  Figure 3
                                                      Log
           (L-y) _
        10  L
                                                                      kt
                                                      and:
                                                                 Table 2
                                                    Equivalents in the BOD Equation
BOD remaining at
time t
Fraction of BOD
remaining

Conversion of loge
to log 10

Fraction of BOD
satisfied
                                                                        L-y/L   L
                                                                        K
                    t/.
                      L
                   2.3k
                           (3)
                          10
                                                                                          -kt
                                                                                        10-25

-------
 Mathematical Basis of the BOD Teat
   from Table 2
               1 - 10
                     -kt
                                    (4)
    multiplying both sides of equation (4) by
    L:
                  =  Ui  - icfkt)
rearranging and cancelling gives:

y s L(l - 10
                ~kt)
    This is the usual form of the BOI? equation.
    To avoid confusion with other "k" values,
    the deoxygenation rate is usually written
    as ki and the equation becomes.
        y =  L (1  -
                                         (6)
    The components in Figure 5 are as in HI
    where L is the ultimate oxygen demand at
    infinite  time and y refers to the demand
    satisfied at time t.  The term kj refers to
    a "velocity constant" in physical chemistry
    terminology.  Section n B3 indicates
    factors  that result in system changes
    during progression of the BOD test that
    may affect the value of kj.  For this
    reason the term "rate coefficient" is used
    for ki to recognize that kj may not be a
    "constant" except  for limited time periods.
    As given, kj is an expression of the slope
    dL /   as a log    and is related to the
    rate  of oxidation.

    The L value is a laboratory number re-
    garded as a theoretical rather than an
    actual limit.  The limit is useful for an
    estimation of  the effects of a waste upon
    the receiving  water where the total demand
    is more valid than a part of it as for
    example a 5 day BOD.


IV  SIGNIFICANCE OF BOD CONSTANTS

 A The effect of kj upon y is shown in
    Figure 4.

    The upper curve represents the BOD of a
    domestic sewage.   The kj is 0. 15.
At this rate,  50% of the demand will be
exerted in the first two days,  indicating
that the organic matter is readily avail-
able as food for the organisms.  In five
days, 83% of the demand has been satis-
fied and in 15 days, the oxidation is
essentially complete.

 The lower curve represents the BOD of
 a relatively  stable sample.  The kj
 in this case  is 0. 05.   In five days
 only 44%  of the demand is satisfied and
 at 15 days,  there  is still 17% of the
 material  unoxidized.   Note that at the
 lower  rate,  it takes 15 days to accom-
 plish 83% oxidation, while at  the higher
 rate, the same percentage can be accom 
 plished in 5  days.
                                                                    MVS
                                                          EFFECT OF R  RATE ON SHORT TERM MID.

                                                                Figure 4
                                                   Laboratory tests on the BOD of waste
                                                   materials are generally 5-day tests.
                                                   In the case of these two samples,  the
                                                   standard 5-day interval would  represent
                                                   vastly different degrees of oxidation.
                                               V  LOG OF % BOD REMAINING

                                               Using the mathematical relationships estab-
                                               lished above, a second BOD equation can be
                                               developed.
   10-26

-------
                                                           Mathematical Basis of the BOD Test
 From Table 2:
           L
                     -k t
                =  10  1  =  fraction remaining
       -k t
     10  1  X 100  " %  remaining

              -k  t
     log   (10   1 X 100)  = login% remaining
                               = 10'
     further:
               ,-k.t
        10g1Q(10   1 X 100)    2 - kjt

     then;                         ^
        log of % BOD remaining =  2  -  k^  (7)


VI  SUMMARY

 The relationships presented herein form the
 basis for development of engineering esti-
 mates from laboratory data.   Validity  of the
 estimate  depends upon sampling, prompt and
 correct BOD  technique, judicious data
 analysis, and realization that the laboratory
 BOD test does not include many factors
 controlling stream behavior.
Two examples of the estimation of k and L
values are presented in Part 2, which begins
on the next page.
ACKNOWLEDGEMENT:

Certain portions of this outline contain
materials made available by D. G.
Ballinger,  F.  P.  Nixon and D.  E.
Baumgartner.
REFERENCES

1  Streeter, H. W., and Phelps, E. B.
      Public Health Bulletin 46,  USPHS.
      1925.

2  Phelps, E. B.  Stream Sanitation.
      John Wiley & Sons, New York.  1944.
                                                                                      10-27

-------
         MATHEMATICAL BASIS OF THE BIOCHEMICAL OXYGEN DEMAND (BOD) TEST
                                 ESTIMATION OF k and L

                                          Part 2
I  Several methods are available that may
give a fair estimate of k  and L providing
that the observed data plots in a form that
suggests a first order reaction rate fit. The
fact that stream samples frequently consist
of partially stabilized river water mixed
with varying amounts of more  recent waste -
water additions leads to numerous situations
where observed ciata calculated as "a" first
order reaction does not give a good fit of
observed and calculated data.  The low rate
oxidation of the aged material  and high rate
oxidation of the new material simply cannot
be explained in terms of one k^ and L.

The BOD test is not precise enough to dis-
tinguish the infinite number of individual
rate systems included in deoxygenation of
a mixed wastewater by the mixed organisms
involved.  The persistence of the one first
order reaction rate concept (incorrectly
called a monomolecular rate) partially is due
to the practical limitations of the test.  High
assimilative oxidation rates generally have
been partially completed in the sewer, during
 compositing or sample storage before BOD
analysis, or in the receiving water.  Remain-
 ing deoxygenation may lake the form charac-
 teristic of cell mass and storage products,
 (endogenous oxidation) which for a few days
 shows a ^ of about 0. 1.   If the observation
period was extended beyond the usual 7 to
 10 days for rate estimation a. progressive
change   of k-, would become apparent as the
 more oxidizable components disappear and
 relatively inert biological or other residues
 became a larger and larger fraction of the
 total oxidizable mass.  The increased im-
poundment of surface water makes long-term
 deoxygenation more and more significant.

 The computed k-, and L are a result of the
 selected measurement routine, past history
 of the sample, and the manner of processing
 the result.  The validity  of the estimate
 depends upon engineering judgment and a
realization of the  variables and effects
involved.   No two individuals will derive
 precisely the same values from given data
 but common sense approaches using mathe-
 matics as a tool rather than as a deity, will
 produce useful estimates for resolution of
 the problem.
II   Useful methods of deriving k..
 include the least squares method of Reed and
 Theriault (Public Health Bulletin 127, 1927);
 the Thomas Slope Method (Sew. Works J.  9,
 No. 3,  425.  1937), the Moments Method of
 Moore, Thomas, and Snow (Sew. and Ind.
 Wastes 22, No. 10,  1343, 1950) and the
 Rapid Ratio Method of Sheehy (JWPCF  32,
 No. 6,  646,  1960).  The first three of
 these presume a singe rate coefficient
 and do not give a good fit of data when de -
 oxygenation  fails to  follow the pattern.   Cer
 tain other factors enter the picture even
 when the curve apparently follows first
 order characteristics (Ludzack et al, Sew.
 and Ind. Wastes 25, No.  8, 875, 1953).
 Sheehy's procedure elucidates the dis-
 appearance of high rate components during
 early stages and can be very useful for
 rapid calculations.

 The daily difference  method outlined by
 Tsivoglou (Oxygen Relations in Streams,
 SEC Tech. Report W-58-2. p.  151,  1958)
 is an adaptation of Fair and of Velz methods
 and gives  a graphic picture of observed data
 and predominant rate changes with time.
 The method is rapid, versatile, and gives a
 great deal of information in a simple package
 that is  readily assembled.  This technique
 was used to illustrate its applicability  as
 "one" means of estimation.

 A  Using the data given in Table 1, Part 1.
       Plot y vs.  t on cartisian coordinate
       paper.  Draw a curve of best fit through
       the observed points,  including lags,
       plateaus, if any, to get a picture of
       the deoxygenation curve.  If a majority
       of observed values describe a smooth
                                                       Preceding page blank
                                                                                      10-29

-------
Estimation of k and L
      progression, those that fail to fit may
      be considered erratic and curve values
      -may be used for subsequent operations.
      Lags should be eliminated by curve
      fitting and taking the observed points
      after fhe lag termination.  (Figure 2)

      Plot the daily differences,  corrected
      if necessary, on semilog paper with
      time on the linear scale and the daily
      difference on the  log scale.  The
      differences are conventionally plotted
      as 1/2, 1-1/2,  2-1/2 days.  etc. to
      illustrate intervals rather than points.
      (Figure 5)

      Examination of Figure 2 shows identi-
      cal daily  differences for the last 3 days
      or k^ - 0 for that interval in Figure  5.
      That la unlikely considering the short
      period of observation and the results
      of the first 6 days.

      It may be assumed that deoxygenation
      follows a smooth curve after consider-
      ing possible lag periods or multicom-
      ponent rates operating in combination
      or sequentially.  Daily differences,
      therefore, should be calculated from a
      line of best fit such as Figure 2 rather
      than from the individual points included
      in Table  1 which may be affected by
      experimental error.

      Figure  5  is a plot of the daily differences
      from Table  1 on the Iog10 scale vs
      time on the arithmetic scale.  The
      corrected curve of best fit was not used
      here to show the effects of the last 3
      points on the logarithmic pattern.  The
      slope of the  line was well defined on
      the basis of the first 7 points.  Note
      that the daily differences are plotted
      at the middle of the time interval,  i. e.,
      the deoxygenation occurring from  0 to
      1 day appears at the 1/2 day position
      and that occurring during the second
      day appears at the 1-1/2 day position.
   It is advisable to check arithmetic on
   the work sheet by comparing the sum
   of the daily differences in Table 1 with
   that resulting from the line of best fit
   in Figure 5.  In  Table 1:  1.8+1.6 +
   1. 2 + .  . . .  + 0. 3  = 8. 3; in Figure 5;
   1. 88 + 1. 50 + 1. 20  + . .  .  . + 0. 26 =
   8. 4.  Since the difference in sums is
   1 part in 84 it is apparent that serious
   errors did not occur in arithmetic or
   plotting.

   From Figure  5,  the number at the zero
   intercept of the daily difference slope
   is a function of L.  Other intercepts
   are a function of L^  using equivalents
   in TabLe 2. The  number representing
   L at 0 time =  2. 1; the number repre-
   senting Lt when t =  10 is 0. 23.  The
   percent BOD  remaining when t = 10 is
   L+/L X  100 or
   0. 23 X 100
     2. 1
             =  10.9 or !
8  From equation 7:

   LglO ^ *^e ft BOD remaining = 2 ~kjt

   The Log 10 of 11%   1. 04 and t - 10
   then 1.04  2 - k1  X  10

   or ki   2 - 1. 04/10 = - = 0. 096 or
                          10    0.10

9  From equation 6:

   yt = L (1 - 10-klt) = L  (1 -*- )

   From Table 1, y  = 8. 3, and

   (1 - ^  )  =0.89 (Step  7)

   The estimate of L becomes

   y1Q = L  (1 - i*)  or 8. 3 = L (0. 89)


       8.3
L =
                                                           0.89
             =  9. 34 or 9. 3 mg BOD/1.
   10-30

-------
                                                                 Estimation of k and  L
  10  The laboratory test gives estimates
      of the rate coefficient or k) of 0. 10 and
      of L or the Ultimate Oxygen Demand
      of 9. 3  mg of BOD per liter of sample.
      The data approximates a system con-
      sisting primarily of the oxidation of
      partially stabilized cell mass and
      storage products and plots as a one
      rate system during the observation
      period.

      Equations 6 and 7 provide  the funda-
      mental relationships and the equivalents
      in Table 2 may be read from Figure  5.

   The  data in. Table 3 present a different
   situation involving more recent
   contamination.
days

  1

  2

  3

  4

  5
  6
  7

  8
  9

 10
Table 3

 BOD

  2. 24

  3. iS

  3. 81

  4. 22

  4.56

  4. 88

  5. 12

  5. 32

  5. 49

  5. 61
per day

   2. 24

   0.94

   0. 63

   0. 41

   0. 34

   0. 32

   0. 24

   0. 20

   0. 17

   0. 12
                                       The differences among the first 3 ob-
                                       served daily differences and the line
                                       ab are 1. 55,  0. 36 and 0. 14 from
                                       Figure 7.  If the differences are plott-
                                       ed on Figure 7, (coded as x)  a new
                                       rate cd can be estimated that gives a
                                       reasonable fit with 2 of the 3 intervals.
                                       The first daily difference is above the
                                       slope cd suggesting a 3rd rate  group
                                       higher than ab or cd.   Since this would
                                       be based upon a single point in 1 series,
                                       in which some discrepancy is apparent
                                       in Figure  7 with respect to line fitting,
                                       it is advisable to regard it as a
                                       possibility as shown by the graphic
                                       but withhold judgement on the 3rd rate
                                       group for further evidence. The curve
                                       in Figure  6 may be approximated by a
                                       two rate system operating simultan-
                                       eously with one of the  two  nearly
                                       completed in 3 days.
3  Mathematically this situation may be
   expressed as:
Yt - L  .(1 -
      cd
+ L . (1 -
   ab
                                      or
= L  , (1 - )  + L . { 1 - -
   cd      L ,       ab     L ,
            cd               ab
where -~   may be obtained from
       L
Figure 7 at the designated time intervals
for each slope.

4  From Figure 7 the zero and 10 day
   intercepts of line ab  are 0. 75  and 0. 13.
      The line of best  fit of the observed
      BODs in Table 3 are shown as a
      smooth curve in Figure 6.

      Figure 7 shows the daily differences
      on the  serai log scale vs the time
      interval on the arithmetic  scale.  The
      line ab is a reasonable fit  for the slope
      described by 5 of the 7 daily differences
      after the 3rd day.  It is apparent that
      the curve in Figure 6 cannot be
      described as one first order reaction.
                                                 _ 0.13 X 100
                                              L      0.75
                                        remaining when t = 10.
                                                                               = 17.4%  BOD
                                        The log1Q of 17. 4 is 1.24.

                                        If the log   of the % BOD remaining =
                                        2 - kt
                                        then,  1. 24 = 2 - kab X 10 and kab  =

                                        2 - 1.24    0.76
                                          10
                                     10
                                                         =  0.076
                                                                                      10-31

-------
 5  From Figure 7 the zero and 2 day
    intercepts of line cd are 1.40 and 0. 22.
       'cd
              0.22 X 100
                1.40
                   15. 7% BOD
    remaining when t * 2.  The logio of

    15. 7 is 1. 20 as in Step 3:
           2 -  1. 20     0.80
    kcd  *   2       "2
                      = 0.40
  6  From the relation in B2,  Lcd may be
    approximated by substituting the ob-
    served value for the 2 day BOD in
    Table 2 or 3.18.  The  fraction ofLj
                                   L
    from *ero to 2 days is
                          0.53
                                ab
                           or 0. 71

    (i .  3. ) . o. 29.  therefore the
         Lab
use of the 2 day observed value for
line cd is incorrect because 0. 29 Lfib
is included in the observed value.
Since L . is unknown,  the first estimate
of L   wul be incorrect by the value
of 0^28 L h but may be approximated
later.
and
-it  =
Lcd
                      then a high approxi-
     mation of L-cd becomes.

        3. 18 = Lcd X 0. 84

        Lcd-05  "3.8mgBODA.

  7  A similar approximation of LaD may be
     set up for t  10 where the observed
     Y 10 from Table 2 of 5. 6 is corrected
     by substracting Led which should be
     almost complete by 10 days.
     becomes 5. 6 -  3. 8 =  1. 8 and
 Substituting the appropriate numbers
 as in step 5:
 T      1'8
 Lab r 0. 826
                  - 2.18 or 2.2 mg/1.
  8  A better approximation of Lcd is now
     possible assuming that the 2 day ob-
     served value included the following:
                                         = 3. 18 - 0. 29 X 2. 2 or 3. 18 - 0. 84 = 2. 34
                                              and
                                                   =  2. 9 mg BOD/1 instead of 3. 8
                                               A second approximation of Lab would
                                               raise the estimate of Lab because
                                               Y10&b = 5. 6 - 2.9 -  2. 7 and

                                               t    -  2'7
                                               ^ab "  0. 826
                                               and an estimate of the L values of the
                                               sample plotted in Figure 6 would be
                                               2. 9 + 3. 3 or 6.2 mg BOD/ liter.
                                                                 =  3- 28 or 3- 3
                                                                         BOD/1
                                       C The substitution of the derived values for
                                         the appropriate and ultimate oxygen
                                         demand into the expression given in B2
                                         should give values comparable to the ob-
                                         served BODs in Table 3 or Figure 6.
                                         Further,  it should be possible to approxi-
                                         mate curves for the individual rate systems
                                         to obtain daily vectors, the sum of which
                                         would compare with the observed points.
                                         For example:
                                                 Where L  , = 2. 9
                                                         cd
 L
.-k!
                                                              3.3
                                                                 and k  , * 0. 40
                                                                     cd
                                                                 and k   = 0. 076
                                                                     ab
                                             and 10"    may be approximated by -=*

                                             from Figure 7. (See Table 2)



                                                    2.9X  0.60+3.3 X 0. 16

                                                   = 1. 74 + 0. 53 = 2. 27. observed = 2. 24



                                                    2. 4 -t-  0. 9   3. 3 observed  3.18
                                                 I.. I. -
                                                                         * 1.3
                                                        = 2. 9 + 1. 3 = 4. 2    observed = 3. 81
                                                 2.9+ S.S 
-------
                   Estimation of k and L
    = 2. 9 + 2. 7 = 5. 6   observed 5. 61

These data show a reasonable approxi-
mation of the  observed BODs by a
reverse calculation from the derived
rate and ultimate BOD obtained from
them.  The agreement indicates that
approximations made were reasonably
correct and that the composite BOD
curve in Figure 6 may be described as
a 2 rate system as indicated by the
BODs in Figure 6. The largest error
occurred in calculation of the com-
ponents for 3 at the point in time where
the higher rate system  was being phased
out.  Curve fitting, approximations of
correction factors for estimation of L,
     plotting, slide rule and other errors
     contribute to estimates of kj and L in
     addition to the errors related to the
     BOD test.

     An approximation of kj is possible
     from 2 BOD values obtained at different
     intervals of time by using the data
     presented in the Theriault Tables  or
     that given nomographically by the  chart
     on the Rates of BOD Satisfaction.

This outline was prepared by F. J. Ludzack,
Chemist,  National Training Center,  MPOD,
OWPO. .USEPA,    Cincinnati, OH  45268.

Descriptors: Analysis, Analytical Techniques,
Biochemical Oxygen Demand.  Data Handling,
Graphical Analysis, Graphical Methods,
Mathematical Models,  Mathematical Studies,
Mathematics
                                    10-33

-------
Estimation of k and L
Figure 5. l.og.~ Slope of the Dailj Differences from Figurr 2
l o ...... __ -- 	 
iw.w
90
.w
a o
7 0
60
5 A
4M
V
w 3 O
W
u
2

w
 2.0
H-l
D
>>
d
s
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1








X


















X









1








X









2








v
N








3










^-JB
*N





MMMWW^M
D.












5
'X



B^^BiaB
5
fcYS













N,
S


i*MB**














N
^s
 o 

^^M
7















 ^N

















	 


8 9 K

-------
                                        Figure 6.   BOD of the Data from Table 2.
\








 CA
       6


                                                                    COMPOSITE BODs FROM TABLE 2
                                                 BODs AS DESCRIBED BY LINE cd FIGURE 7
                                                       BODs AS DESCRIBED  BY LINE ab FIGURE 7


                                                    DAYS

-------
                                                                  Estimation of k and L
10.0
      Figure 7.  Log10 Slopes of the Daily Differences  From Figure 6 BODs
 0.1

-------
Estimation of k and
Thi-riauli Table
NUMERICAL VALUES OF THE FUNCTION (1 - 10" S
FOR THE RANGE k = 0.040 to k = 0. 250
Period of
incubation
(days)






7 	 _ 	

8- _
i n -  - - 	 - -

1 9_____ 	



j.0--------
1 R-.---- 	
1 7___ 	 .


Ofl-- 	 	

99_ 	
00 _.  	



Period of
incubation
(days)



A 	

0.04
0.088
.168
.241
. 308
.369
.425
.475
. 563
. 602
.637
.669
.698
.725
.749
.771
.791
.809
.826
.842
.855
.868
.880
.890
.900

0.11
0.224
.397
.532
fi37

0.05
0. 109
0.206
0.292
.369
.438
.499
.553
.602
.645
.(384
.718
.749
. 776
.800
.822
.842
.859
.874
.888
.900
.911
.921
.929
.937
.944

0. 12
0.241
.425
.563
.669

0.06
0. 129
.241
.339
.425
.499
.563
.620
.669
.712
.749
.781
.809
.834
.855
.874
.890
.905
.917
.928
.937
.945
.952
.958
.964
.968

0. 13
0.259
.450
.593
.699
Value of k
0.07
0.149
.276
. 383
.475
.553
.620
.676
.725
.766
.800
.830
.855
.877
.895
.911
.924
.935
.945
.953
.960
.966
.971
.975
.979
.982
Value 01 k
0.14
0.276
.475
.620
.725

0.08
0.168
.308
.425
.521
.602
.669
.725
.771
.809
.842
.868
.890
.909
.924
.937
.943
.956
.964
.970
.975
.979
.983
.986
.988
.990

0.15
0. 292
.499
.645
.749

0.09
0.187
.339
.463
.563
.645
.712
.766
.809
.845
.874
.898
.917
.932
.945
.955
.964
.970
. 976
.981
.984
.987
.990
.991
.993
.994

0. 16
0.308
.521
.669
.771

0. 10
0. 206
.369
.499
.602
.684
.749
. 800
.842
.874
.900
.921
. 937
.950
.960
.968
.975
.980
.984
.987
.990
.992
.994
.996
.997

0. 17
0.324
.543
.691
.791
/C -57

-------
                                                                    Estimation of k and L
                                    Theriault Table
  Period of                                Value of k
 incubation
   ,.   .       0.11       0.12       0.13      0.14     0.15     0.16
   (days)	
 5	     .718       .749       .776      .800     .822
                67 01        fiflQ        QQ__L       fl R (^      P^ A
  ........     ^ IO1        OUo        OOrk       ODD     * OI *
 7	     .830       .855       .877      .895     .911     .924       .935
 8-	     .868       .890       .909      .924     .937     .948       .956
 9	     .898       .917       .932      .945     .955     .964       .970
10--------     .921       .937       .950      .980     .968     .975       .980
U	     .938       .952       .963      .971     .978     .983       .987
12	     .952       .964       .972      .979     .984     .988       .991
13	     .963       .972       .980      .985     .989     .992       .994
14	     .971       .979       .985      .989     .992     .994       .996
15-	     .978       .984       .989      .992     .994     .996       .997
16	     .983       .988       .992      .994     		       	
17	     .987       .991       .994      .996     		       	
18	     .990       .993       .995      .997     	     	       	
19	     .992       .995       .997      .998     	       	
2Q		.994	.996	.997	.998     -     		
 Period of"  ~~~~~                    Value of k
ln"bat*on      67T8oTTi6726     oT2i     Ol     57235724    oTlT
  (days)	
 1	     0.339    0.354    0.369    0.383    0.397    0.411    0.425   0.438
 2	      .563     .583     .602     .620     .637     .653     .669    .684
 3	      .712     .731     .749     .766     .781     .796     .809    .822
 4	      .809     .826     .842     .855     .868     .880     .890    .900
 5	      .874     .888     .900     .911     .921     .929     .937    .944
 6	      .917     .928     .937     .945     .952     .958     .934    .968
 7	      .945     .953     .960     .966     .971     .975     .979    .982
 8-	      .964     .970     .975     .979     .983     .986     .988    .990
 9	      .976     .981     .984     .987     .990     .991     .993    .994
10	      .984     .987     .990     .992     .994     .995     .996    .997
U	      .990     .992     .994     .995     	
12	      .993     .995     .996     .997     	     	     	
13	      .995     .997     .997     .998     	     	
14__	      .997     .998     .998     .999     	
15	      .998     .999     .999     .999     	     	     	    	

-------
Estimation of k arid L
                  RATES   OF  B.O.D.  SATISFACTION
                    FOR VALUES OF  K.  FROM  .05  TO.60



            100
            80
            60
            40



             8  =
                           '\JL \ V-\ T~T\"~V \~ I "\"" ""'"Xl ~' - ^-L ' J t ^i j - {
                                8   10   12   I4   '6   i8   20


-------
                       SOURCES AND ANALYSIS OK ORGANIC' NITROGEN
 I   INTRODUCTION

 A  Organic nitrogen refers to the nitrogen in
    combination with any organic radical.
    For sanitary engineering the main interest
    is the nitrogen contained in proteins,
    peptides,  amines, amino acids, amides
    and related compounds of animal or
    vegetable origin.
II   SOURCES OF ORGANIC NITROGEN

 A  Natural Origin

    1  Dead animal and plant residues

    2  Animal wastes - urea,  feces

    3  Autotrophic organisms - algae, s.bact.

    4  Heterotrophic organisms

 B Industrial Origin

     1  Food processing wastewater - meat,
       milk, vegetables

     2  Pharmaceutical wastes, antibiologicals

     3  Plastics - polyamides, nitriles

     4  Chemical intermediates or products

     5  Dye industry - azo, nitro
 III  TOTAL KJELDAHL NITROGEN
    PROCEDURE

  A Organic nitrogen is determined using the
    Total Kjeldahl Nitrogen (TKN) method.
    This determination includes both organic
    nitrogen and free ammonia.  By dis-
    tilling the free ammonia off the sample
    before the determination, organic nitrogen
    can be determined directly.

  B Scope

    The procedure converts nitrogen compon-
    ents of biological origin such as amino

   CH.N.Sa. 11.75
   acids, proteins and peptides to ammonia
   but it may not convert the nitrogenous
   compounds of some industrial wastes
   such as amines, nitro compounds,
   hydrazones,  oximes, semi-carbazones
   and some refractory tertiary amines.

C  Method Summary

   The sample is heated in the presence of
   a concentrated sulfuric acid-potassium
   sulfate-mercuric sulfate mixture and
   evaporated until sulfur trioxide fumes are
   obtained and the solution becomes color-
   less or pale yellow.  The residue is
   cooled and diluted, then treated and made
   alkaline with a hydroxide-thiosulfate
   solution.  The ammonia is distilled off
   and then determined either by nessler-
   ization or by titration.

   1  Nesslerization (colorimetric) is the
      method used when the ammonia-
      nitrogen concentration is less than 1. 0
      mg N/liter.

   2  For ammonia-nitrogen concentration?
      above 1. 0 mg N /liter, the ammonia i=?
      determined by titration with 0. 02 N
      H  SO  in the presence of a mixed
      indicator.

      For a detailed description of the
      procedure and reagent preparation,
      consult the EPA methods manual.

 D Precision and Accuracy

    1  Thirty-one analysts in 20 laboratories
      used the  Total Kjeldahl Nitrogen
      procedure to analyze natural water
      samples  containing the following increments
      of organic nitrogen:  0. 20,  0. 31,  4. 10
      and 4.61 mg N/liter.

      a  Precision results for a standard
         deviation were  0.197, 0.247,  1.056
         and 1. 191 mg N/liter, respectively.

      b  Accuracy as bias was +0. 03,  +0. 02,
         +0. 04 and -0. 08 mg N/liter,  respect-
         ively.
                                       11-1

-------
Sources and Analysis uf Organic Nitrogen
   2  The nature and composition of ex-
      traneous materials affect analytical
      recovery.  High salt concentrations
      may raise digestion temperature.
      High concentrations of organic sample
      components may require excessive acid
      during digestion tending to low nitrogen
      yield.

   3  The digestion temperature is critical.
      380 to 390C usually gives high
      analytical recovery on the more re-
      fractory nitrogen compounds of natural
      origin.  Nitrogen losses occur above
      42
-------
                             AMMONIA,  NITRITES AND NITRATES
 I    SOURCES AND SIGNIFICANCE OF
     AMMONIA,  NITRITES AND NITRATES
     IN WATER

 The natural  occurrence of nitrogen com-
 pounds is best demonstrated by the nitrogen
 cycle (Figure 1).

 A   Ammonia

      1    Occurrence

          Ammonia is a product of the micro-
          biological decay of animal and plant
          protein.   In turn, it can be used
          directly  to produce plant  protein.
          Many fertilizers contain ammonia.

      2    Significance
          The presence  of ammonia nitrogen
      in raw sura surface waters might indicate
          domestic pollution.  Its presence in
          waters used for drinking purposes
          may require the addition of large
          amounts  of chlorine in order  to
          produce  a free chlorine residual.
          The chlorine will first react with
          ammonia to form chloramines be-
          fore it exerts  its full bactericidal
          effects (free chlorine residual).

 B    Nitrites

      1    Occurrence
          Nitrite nitrogen occurs in water
          as an  intermediate  stage in the
          biological decomposition of organic
          nitrogen.  Nitrite formers (nitro-
          somonas) convert ammonia under
          aerobic conditions to nitrites. The
          bacterial reduction of nitrates can
          also produce nitrites under anaero-
          bic conditions.  Nitrite is used  as
          a corrosion inhibitor in industrial
          process  water.

      2    Significance
          Nitrites  are usually not  found  in
          surface  water to a  great  extent.
          Tiie presence of large quantities
          indicates a source of wastewater
          pollution.
     Nitrates
          Occurrence
          Nitrate formers convert nitrites
          under aerobic conditions to nitrates
          (nitrobacter).   During electrical
          storms, large amounts of nitrogen
          (No) are oxidized to form nitrates.
          Finally, nitrates can be found  in
          fertilizers.

          Significance

          Nitrates in water usually indicate
          the final stages of biological sta-
          bilization.   Nitrate rich  effluents
          discharging into  receiving waters
          can,  under proper environmental
          conditions,  cause stress to stream
          quality by producing algal bloomi-.
          Drinking water containing  exces-
          sive  amounts of nitrates can cause
          infant methemoglobinemia.
II   PRESERVATION OF AMMONIA, NITRATE
    AND NITRITE SAMPLES<8)

 A  If the sample is to be analyzed for Ammom;.
    or Nitrate,  it may be preserved with 2rr,I
    concentrated sulfuric acid per liter  and
    cooled to 4C.

 B  For nitrite,  cool to 4CC and analyze as soon
    as possible.

 C  Mercuric chloride is effective as a  preserv-
    ative but its use is discouraged because:

    1 The Hg ion interferes with some of the
      nitrogen tests.
    2 The Hg presents a disposal problem.

 D  Even when "preserved", conversion from one
    nitrogen form to another may occur.  Samples
    should be analyzed as soon as possible.
CH.N.6e. 11.75
                                                                                          11-3

-------
Ammonia, Nitrites and .Nitrates
                                    PLANT
                                   PROTEIN
                                        ANICN
 FOOOSTUFF

THE NITROGEN CYCLE
                                Figure 1
11-4

-------
                                                            Ammonia, Nitrites  and Nitrates
111   l)ET?:i

                                        1   Reaction
                                                The sample is distilled in
                                                the presence of a borate
                                                buffer at pH 9. 5
                                                              NH
                                                                   H+
                                            H+ + Na2 B4O7
                                                 Buffer
                                                         pH 9. 5 maintained
                                                The ammonia in the dis-
                                                tillate is then measured by
                                                either of two techniques.
                                                1)
                                                2)
                                                             Nesslerization is used
                                                             for samples containing
                                                             less than 1 mg/1 of
                                                             ammonia nitrogen.

                                                             Absorption of NH3 by
                                                             boric acid and back ti-
                                                             trationwith a standard
                                                             strong acid is  more
                                                             suitable for samples
                                                             containing more than
                                                             1 mg NH3 N/l.
                                        NH3 +HBO2-
                                                                            BO2"
                                                         H
                                                                 HB0
                                                Methylene Blue
                                        pH 7.8 - 8.0
                                          Green
                                                                  pH 6. 8 -  7.0
                                                                    Purple
                                                                                        11-5

-------
Ammonia,  Nitrites and Xitrates
    2  Interferences

       a  Cynate may hydrolyze, even at
          pH 9. 5.

       b  Volatile organics may come over in
          the distillate, causing an off-color
          for Nesslerization.  Aliphatic and
          aromatic amines cause positive
          interference by reacting in the acid
          titration.  Some of these can be
          boiled off at pH 2 to 3 prior to
          distillation.

       c  Residual chlorine must be  removed
          by pretreatment with sodium
          thiosulfate.

       d  If a mercury salt was used for
          preservation, the mercury ion
          must be complexed with sodium
          thiosulfate (0. 2 g) prior to distil-
          lation.

    3  Precision and Accuracy*")

       Twenty-four analysts in sixteen
       laboratories analyzed natural water
       samples  containing the following
       amounts  of ammonia nitrogen:
       0.21, 0.26, 1.71,  andl.92mg
       NH3-N/liter.

       a  Precision

          The standard deviation was:  0. 122,
          0.070, 0.244, and 0. 279 mg
          NH3-N/liter, respectively.

       b  Accuracy

          The bias was: -0.01, -0,05.
          + 0.01, and -0.04 mg NH3-N/liter,
          respectively.

 C Selective Ion Electrode(8)

    1  Principle

       A hydrophobic, gas-permeable mem-
       brane is  used to separate the  sample
       solution from an ammonium chloride
       internal solution.  The ammonia in
       the sample diffuses through the
       membrane and alters the pll of tin-
       internal solution, which is sensed by
       a pH electrode.  The constant level ot'
       chloride in the internal solution is
       sensed by a chloride selective ion
       electrode which acts as the reference
       electrode.

    2  Interferences

       a  Volatile amines are a positive
         interference.

       b  Mercury forms a complex with
         ammonia so it should not be used
         as a preservative.

    3  Precision and Accuracy

       In a single laboratory (EPA) four surface
       water samples were analyzed  containing
       the following amounts of ammonia
       nitrogen:  1.00,  0.77, 0.19,  and0.13mg
       NH3-N/liter.

       a  Precision

         The standard  deviations were 0.038,
         0.017,  0.007,  and 0.003  nig
         NHg-N/liter,  respectively.

       b  Accuracy

         The % recovery on the 0. 19 and 0. 13
         concentrations was 96% and 91%
         respectively.
IV  DETERMINATION OF NITRITE

    A  Diazotization' '

       1   Reaction

          a Under acid conditions,  nitrite
            ions react with sulfanilic acid to
            form a diazo compound.

          b The diazo compound then couples
            with  o-naphthylamine to form an
            intense red azo dye which exhibits
            maximum absorption at 540 nm.
 11-6

-------
                                                          Ammonia, Nitrites and! Nitrates
    NH,,
                               N^= N
                                                  OH
+ NO    -i- 2H	
             pH1.4
                                                    HSO
                                     * 2H^
    '3                        ""3*
  SULFANILICACID    DIAZO COMPOUND
                              N  N
                                                           OH
                                                        H+ + NOl
                                                                                          H,,0
                                                                                            2
                                                                                 SO H
                                                      b  In alkaline solution the nitro
                                                         derivative rearranges  to
                                                         form & yellow -colored com -
                                                         pound which exhibits maxi-
                                                         mum absorption at 410
                                                                + 3KOH
                                                                                             OK
       2  Interferences
                                             COLORLESS
YELLOW
          a  There are very few known
             interferences at concentrations
             less than 1000 times that of
             nitrite.

          b  High alkalinity (greater than
             600 mg/liter) will give low results
             due to a shift in pH.

       3  Precision

          On a synthetic  sample containing
          0. 25 mg nitrite nitrogen/1,  the ARS
          Water Minerals Study (1961) re-
          ported 125 results with a standard
          deviation of 0. 029 mg/1.
V   DETERMINATION OF NITRATE
                              /C \
    A   Phenoldisulfonic AcidVD'

         1   Reaction

             a    Phenoldisulfonic a c i d re-
                  acts with nitrate to produce
                  a nitro derivative.
                                                  2    Interferences
                                                          Chloride ion under the acid
                                                          conditions of the test intro-
                                                          duces negative interference.
                                                      6C1' + 2NO3~ + 8H+
                                                          3C12 f  + 2NO f + 4H20
                                                          Silver  sulfate can be used
                                                          to precipitate Cl", but due
                                                          to incomplete precipitation
                                                          of Ag+, an  offcolor or
                                                          turbidity is produced when
                                                          the final color is developed.
                                                          (Note:   This difficulty can
                                                          be overcome by  using
                                                          NH4OHas the alkali.)

                                                          Nitrites in concentrations
                                                          greater than 0.2 mg N/l
                                                          introduce positive interfer-
                                                          ence.  However, in most
                                                          waters, the concentration
                                                                               H-7

-------
Ammonia, Nitrites and Nitrates
                  of nitrite is insignificant
                  compared to nitrate.

              c    Color and turbidity may be
                  removed by using A1(OH>3
                  suspension or by floccula-
                  tion with ZnSO4 and alkali.

         3   Precision and accuracy
             On a synthetic sample con-
             taining 1. 0 mg nitrate N/l,
             and 200 mg Cl'/l.  the ARS
             Water Nutrients Study (1966)
             reported 46 results with a
             standard deviation of 10.399
             mg/1 and a mean error of
             -0.31 mg/1.

     B   Brucine Sulfate(6> 7'8)

         1   Reaction
             Brucine, a strychnine compound
             reacts with nitrate to form a
             yellow compound which exhibits
             maximum absorption at 410 nm.
             The reaction according to t h P
             procedure as outlined  in
             Standard Methods does not
             follow  Beer's Law.  However,
             a recent modification by Jenkins
             andMedsker (2) has been devel-
             oped.  Conditions are controlled
             in  the reaction so that Beer's Law
             is  followed and concentrations
             below 1 mg nitrate N/l can be
             determined.

         2   Interferences

             a    Nitrite may react the same
                  as nitrate but can be  elim-
                  inated by the addition  of sul-
                  fanilic acid to the brucine
                  reagent.

             b    Organic  nitrogen compounds
                  may hydrolyze and give
                  positive  interference at low
                  (less than 1 mg/1) nitrate
                  nitrogen concentrations.

              c   Residual chlorine may be
                  eliminated by the addition of
                  sodium arsenite.
   3  Preci.sii'ii umt A' rut-Be
                                                                                 (8)
     a  Twenty seven analysts in 15 lab-
        oratories analyzed in natural
        water samples containing the
        following increments of inorganic
        nitrate:  0.16,  0.19,  1.08 and
        1.24 mgN/liter.

     b  Precision result s as standard
        deviation were 0. 092,  0. 083,
        0.245, and 0.214 mg N/liter
        respectively.

     c  Accuracy expressed as bias was
        -0.01, +0.02, +0.04 and+0.04
        mg N/liter, respectively.

C  Cadmium Reduction*6- 8>

   1 Reaction

     A filtered sample is  passed through
     a  column containing granulated
     copper-cadmium to reduce nitrate to
     nitrite.  The nitrite (that originally
     present plus reduced nitrate) is deter-
     mined by diazotizing with sulfanilamide
     and coupling with N-(l-naphthyl)-
     ethylenediamine dihydrochloride to
     form an intensely red azo dye which
     is measured spectrophotometrically.

     To obtain the value for only nitrate,
     more of the filtered sample is tested
     using the same colorimetric reaction
     but without passing it through the
     reduction column.  The resulting value
     represents the nitrite originally present
     in the sample. Subtracting this nitrite
     value for the non-reduced sample from
     the nitrate + nitrite value for the re-
     duced sample gives the value for nitrate
     originally present in the sample.

   2 Interferences

     a  Build-up of suspended matter in the
        reduction column will restrict
        sample flow.  Filtration or floc-
        culation with zinc  sulfate should
        remove turbidity.
11-8

-------
                                                         Ammonia, Niu lies and Nitrates
     b  High concentrations of iron,
        copper or other metals may
        interfere.  EDTA may be used
        to complex  these.

     c  Large concentrations  of oil and
        grease in a sample can coat the
        surface of the cadmium.  Pre-
        extracting the sample with an
        organic solvent removes oil and
        grease.

   3 Precision and accuracy^  

     In 11 laboratories,  three samples
     were analyzed containing the follow-
     ing amounts of nitrate nitrogen-
     0.05,  0. 5,  and 5 mg
     NO3-N/liter.

     a  Precision

        The relative standard deviation
        was 96.4%, 25.6%,  and 9.2%,
        respectively.

     b  Accuracy

        The relative error was 47. 3%,
        6.4%, and 1.0%, respectively.

   4 Automated cadmium reduction

     The EPA Methods  Manual^8) contains
     details for the automated procedure.

D  Hydrazine Reduction

   A method using hydrazine to reduce
   nitrate to nitrite  followed by subsequent
   measurement of nitrite by diazotizatioii
   was reported by Fishman, et al. W

   The means to determine nitrate is the
   same as  above  in the  Cadmium Reduction
   Method.  Subtraction  of nitrite (deter-
   mined from non-reduced sample)  from
   the total  nitrite (reduced nitrate +
   original nitrite) will give the original
   nitrate nitrogen concentration.

   The procedure  was adapted  to the Auto
   Analyzer by Kamphake, et al. (3) and
   can be  found in detail in the 1971 edition
   of the EPA Methods Manual.(8)
REFERENCES

1  Fishman,  Marvin,].,  Skougstad, Marvin
     W , and Scarbio, George, .1 r
     Diazotization Method for  Nitrate and
     Nitrite.  JAWWA 56:633-638  May,  I960

2  Jenkins,  David and Medsker, Lloyd L
     Brucine Method for Determination of
     Nitrate in Ocean, Estuarine and Fresh
     Waters. Anal  Chem  36:610-612
     March, 1964.

3  Kamphake, L. J., Hannah,  S. and Cohen, J.
     Automated Analysis for Nitrate by
     Hydrazine Reduction.  Water Research,
      1,  205.  1967.

4  Lishka, R. J. , Lederer,  L. A., and
     McFarren,  E.  F.   Water Nutrients
     No.  1,  Analytical Reference Service
      1966.

5  Sawyer, Clair N,   Chemistry for Sanitary
     Engineers.  McGraw-Hill Book Co. ,
     New York.  1960.

6  Standard Methods for  the Examination of
     Water and Waste Water.  APHA. AWWA.
     WPCF.  13th Ed.  1971.

7    ASTM Book of Standards,  Part 23, 1970.

 8    Methods for Chemical Analysis of Water &
       Wastes,  EPA-MDQARL, Cincinnati,
       Ohio 45268.  1974.
 This outline was prepared by B.  A.  Punghorst,
 former Chemist, and C. R.  Feldmann,
 Chemist, and revised by A.  E. Donahue,
 Chemist. National  Training Center, MPOD,
 OWPO, USEPA, Cincinnati,  Ohio 45268.
 Descriptors:  Ammonia, Chemical Analysis,
 Nitrates, Nitrites, Nitrogen, Nitrogen
 Compounds, Nitrogen Cycle,  Nutrients, Water
 Analysis, Water Pollution Sources
                                                                                   11-9

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                                TOTA L CARBON ANALYSIS
I  INTRODUCTION

A  History of Carbon Analyses

   In the wake  of a rapid population, growth,
   and the increasing heavy use of our
   natural waterways, the nation, and indeed
   the world, is presented with the acute
   problem of  increased pollutional loads on
   streams, rivers and  other receiving
   bodies.  This has resulted in a growing
   awareness of the need to prevent the
   pollution of streams, rivers, lakes and
   even the oceans.  Along with this aware-
   ness has developed a desire for a more
   rapid and precise method of detecting and
   measuring pollution due to organic
   materials.

 B The Methods

   In the past,  two general approaches have
   been used in evaluating the degree of
   organic water pollution.

   1  The determination of the amount of
      oxygen or other oxidants required to
      react with organic impurities.

   2  The determination of the amount of
      total carbon present in these impurities.

 C Oxygen Demand Analyses

   The first approach is represented by
   conventional laboratory tests for deter-
   mining Chemical Oxygen  Demand (COD)
   and Biochemical Oxygen Demand (BOD).
   One of the principal disadvantages of these
   tests  is that they are limited primarily
   to historical significance, that is,  they
   tell what a treatment plant had been doing,
   since they require anywhere from two
   hours to five days to  complete.  Since up
   to now no faster method has been
   available, traditional BOD and COD
   determinations have become accepted
   standards of measure in water pollution
   control work even though they are
   essentially ineffective for process
   control purposes.

   Until the introduction of the Carbonaceous
   Analyzer, all methods taking the second
   approach, the total carbon method of
   evaluating water quality, also proved
   too slow.
II  THE ANALYSIS OF CARBON

A Pollution Indicator

   Now the carbonaceous analyzer provides
   a means to determine the total carbon
   content of a dilute water sample in
   approximately two minutes.  With proper
   sample preparation to remove inorganic
   carbonates, the instrument determines
   the total organic carbon content  in the
   sample.

 B Relationship of Carbon Analysis to BOD
   and COD

   This quantity varies with the structure
   from 27 percent for  oxalic acid through
   40 percent for glucose to 75 percent for
   methane.  The ratio of  COD to mg carbon
   also varies  widely from 0.67 for oxalic
   acid through 2.67 for glucose to 5. 33 for
   methane.  Representative secondary
   sewage effluents have given a ratio of
   COD to carbon content of between 2. 5 and
   3. 5 with the general average being 3.0.

   The BOD, COD and  carbon contents  of
   these and some other representative
   compounds  are summarized in the follow-
   ing table.
 OH. MET. 24a. 11.75
                                                                                   12-1

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  Total Carbon Analysis
Sample
Sioaric Acid - C -H^O
Glucose - C-H. 9Or
G 12 6
Oxalic Acid - C2H2O4
Benzoic Acid - C,HCO0
I D i
Phenol - CgHgO


Potassium Acid Phthalate
KHC.H O4
Salicylic Acid - C7HgO3
Secondary Effluent, Clarified
IT (1
It ft ft
5 -Day
BOD-mg/mg
.786
.73
. 14
1.38
. 05 to 2. 1 de-
pending upon
concentration

.95
1. 25
13*
23*
4*
COD-
mg/mg
2.91
1.07
.18
1.97
2.36



1. 15
1.60
75*
67*
36*
ro Carbon
76
40
27
69
77



47
61
21*
12*
7*
     * In units of mg/1
 III  THE CARBON ANALYZER

  A Principle of Operation

     Basically the  carbonaceous analyzer con-
     sists of three sections - a sampling and
     oxidizing system, a Beckman Model  315
     Infrared Analyzer, and a strip-chart
     recorder.
    C*rbon*ceou> Anal7*er Schematic
               a carrier from cylinder
A micro sample (20-40 fjtl) of the water to be
analyzed is injected into a catalytic com-
bustion tube which is enclosed by an electric
furnace thermostated at  950C. The water
is vaporized and the carbonaceous  material
is oxidized to carbon dioxide (CO2)  and
steam in a carrier  stream of pure  oxygen.
The oxygen flow carries the  steam and
CO2 out of the furnace where the steam is
condensed and the condensate removed.
The CO2, oxygen and remaining water vapor
enter an infrared analyzer sensitized to
provide a measure  of CO2-  The output of
the  infrared analyzer is  recorded on a strip
chart, after which, the curve produced can
be evaluated by comparing peak height with
a calibration curve based upon standard
solutions.  Results are obtained directly in
milligrams of carbon per liter.

Application
       (D
Results    show that the method is  applicable
for  most, if not all, water-soluble organic
compounds -- including those that contain
sulfur, nitrogen,  and volatiles.

Nonvolatile organic substances can be
differentiated from volatiles, such as
carbon dioxide or light hydrocarbons by
12-2

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                                                                    Total Carbon  Analysis
   determination of carbon both before and
   after the sample solution  has been blown
   with  an inert gas.

C  Sample  Preparation

   The  Carbonaceous  Analyzer is often
   referred to as a total  carbon analyzer
   because it  provides a measure of all the
   carbonaceous material in  a sample,  both
   organic and  inorganic.  However,  if a
   measure of organic carbon alone  is  de-
   sired,  the  inorganic carbon  content of
   the  sample  can be removed  during sample
   preparation.

    1   Removal of  inorganic  carbon

        The simplest procedure for removing
        inorganic carbon  from the sample is
        one of acidifying  and  blowing.  A  few
        drops  of HC1 per  100 ml of sample
        will normally  reduce  pH to 2  or less,
         releasing all the  inorganic  carbon as
         CC>2.     Five minutes  of blowing with
        a  gas  free  of  CC>2  sweeps out the CC>2
         formed  by  the inorganic carbon.   Only
        the organic carbon remains  in the
         sample  and may  be analyzed without
         the inorganic interference.

    2    Volatile carbonaceous material

         Volatile carbonaceous material that
         may be  lost by blowing is accounted
         for by  using a dual channel carbon
         analyzer.   Beckman's new  analyzer
         has the  previously detailed high
         temperature (950C)  furnace  plus  a
         low temperature  (150C) one.  Using
         quartz  chips wetted with phosphoric
         acid,  the low  temperature  channel
         senses  only the CO2  (freed by the
         acid) in the original  sample.   The
         remaining  organics and water are
         retained in  the condenser connected
         to  this  low  temperature furnace.
         None of the organics  are oxidized by
         the 150C  furnace.

         By injecting a  sample into  the low
         temperature furnace,  a peak repre-
         senting  the inorganic  carbon  is  ob-
         tained on the  strip chart.  Injecting
         a nonacidified sample into the high
      temperature furnace yields a peak
      representing the  total carbon.   The
      difference between the values deter-
      mined for the two  peaks  is the total
      organic carbon.

   3  Dilute samples

      If the  sample is  dilute (less than 100
      mg/liter carbon) and is a true solution
      (no suspended particles)  no further
      preparation is  required.

   4  Samples containing solids

      If the sample contains solids and/or
      fibers which are to be included in the
      determination,  these must be  reduced
      in size so that they will be able to pass
      through the needle which  has an opening
      of 170 microns (needles  having larger
      openings may be obtained if necessary).
      In most cases,  mixing the sample in a
      Waring Blender will reduce the particle
      size  sufficiently  for  sampling.

IV PROCEDURE FOR ANALYSIS

 A Interferences

   Water vapor resulting from  vaporization of
   the  sample,  causes a slight interference  in
   the  method.   Most  of the water is trapped
    out  by the air condenser positioned immed-
    iately after the combustion  furnace.  How-
    ever,  a portion of the water vapor passes
   through  the system into the  infrared de-
   tector and appears  on the  strip chart as
    carbon.   The  water blank  also appears on
    the  standard calibration  curve,  and is
    therefore removed  frorn the final calcu-
    lation.   In tests of solutions containing the
    following anions:   NO3, Cl", SO'2,  PO^,
    no interference was encountered with  con-
    centrations up to one percent.

 B  Precision and Accuracy

    The recovery  of carbon from standard
    solutions is 98.5 -  100.0 percent.  The
    minimum detectable concentration using
    the  prescribed operating instructions is 1
    mg/1 carbon.   Generally,  the data are
    reproducible to +  1 mg/1 with a  standard
    deviation of 0.7 mg/1 at the 100 mg/1 level.
                                                                                       12-3

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Total Carbon Analysis
 V  APPLICATIONS

 Several of the many research and industrial
 applications of .ho Carbonaceous Analyzer
 are listed below:

 A Determine the efficiency of various waste -
    water  renovation processes,  both in the
    laboratory and in the field.

  B Compare a plant's waste outlet with its
    water inlet to determine the  degree of
    contamination contributed.

  C  Monitoring a waste  stream to che.ck for
    product lose.

  D  Follow the rate of utilization of organic
     nutrients by micro-organisms,

  E  To detect organic impurities in inorganic
     compounds.
  VI ADVANTAGES OF CARBON ANALYZER

   A Speed

     The Carbonaceous Analyzer's most
     important advantage is its speed of
     analysis.  One analysis can be performed
     in 2-3 minutes.  This speed of analysis
     brings about another advantage,  economy
     of operation.  Working with dilute  samples,
     one man can run ten to twenty carbon
      determinations per  hour.  This is  probably
      more than the number of COD or BOD
      tests that can even be started, much less
      completed, in the same period of time.

    B Total Carbon

      Another advantage is that the measure of
      carbon  is a total one.  The oxidizing
      system of the analyzer brings about com-
      plete oxidation of any form of carbon.  No
      compound has been found to which the
      method is inapplicable.
VII CONCLUSIONS

  The Carbonaceous Analyzer provides a
  rapid and precise measurement of organic
  carbon in both liquid and air samples.  It
  should be found useful for many research
  and industrial applications, a few of which
  have been mentioned.

  Because of its rapidity it may be found more
  useful than the more time-consuming BOD
  and COD measurements for monitoring
  industrial waste streams or waste treatment
  processes.
  REFERENCES

  1  Van Hall,  C. E. ,  Safranko. John and
        Stenger,  V.  A.  Anal. Chem. 35,
        315-9.  1963.

  2  Van Hall,  C. E.,  and Stenger, V.  A.
        Draft of Final Report - Phase I - Con-
        tract PH  86-63-94, Analytical Research
        Toward Application of the Dow Total
        Carbon Determination Apparatus to the
        Measurement of Water Pollution.

  3  Van Hall,  C. E. .  Stenger, V.  A.
        Beckman Reprint  - R6215.  Taken from
        Paper Presented at the Symposium on
        Water Renovation, Sponsored by the
        Division  of Water and Waste Chemistry,
        ACS in Cincinnati. Jan. 14-16, 1963.
   This outline was prepared by Robert T.
   Williams, Chief,  and revised by Charles J.
   Moench,  Jr.,  Waste Identification and
   Analysis Section, MERL,  USE PA, Cincinnati,
   Ohio 45268.
                                                    Descriptors:   Biochemical Oxygen Demand,
                                                    Carbon, Chemical Analysis,  Chemical Oxygen
                                                    Demand, Organic Matter,  Organic Wastes,
                                                    Water Analysis, Instrumentation,  Nutrients
12-4

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               DETERMINATION OF CALCIUM AND MAGNESIUM HARDNESS
I   INTRODUCTION

A   Definition of Hardness

    USPHS - "in natural waters, hardness
    is a characteristic of water which re-
    presents the total concentration of just
    the calcium and magnesium ions .ex-
    pressed as calcium carbonate.  If
    present in significant amounts,  other
    hardness-producing metallic ions should
    be included. "

 B   Other Definitions in Use

    1 Some confusion exists in understanding
      the concept of hardness as a result of
      several definitions presently used.

    2 Soap hardness definition includes hy-
      drogen ion because it has the capacity
      to precipitate  soap.  Present definition
      excludes hydrogen ion because it is not
      considered metallic.

    3 Other agencies define  hardness as
       "the  property  attributable to presence
      of alkaline-earths".

    4 USPHS definition is beat in  relation
      to objections of hardness in water.
II   CAUSES OF HARDNESS IN WATERS OF
    VARIOUS REGIONS OF THE U. S.

A   Hardness will vary throughout the country
    depending on:

    1  Leaching action of water traversing
       over and through various types of
       geological formations.

    2  Discharge of industrial and domestic
       wastes to water courses.

    3  Uses  of water which result in change
       in hardness, such as irrigation and
       water softening process.
 B  Objections to Hardness

    1  Soap-destroying properties

    2  Scale formation

 C  Removal and Control

    Hardness may be removed and controlled
    through the use of various softening oper-
    ations such as zeolite, lime-soda, and
    hot phosphate processes.  It can also be
    removed by simple distillation or com-
    plex formation with surface active agents
    (detergents).

Ill  DETERMINATION OF HARDNESS
                      /c \
 A  Two Methods in Use

    1  Compleximetric method (EDTA)

    2  Calculation - by the use of appro-
       priate factors, the hardness due
       to ions other than calcium and
       magnesium may be converted to
       an equivalent calcium carbonate
       hardness.
 B   Compleximetric Method

     1   Principle of determination

        Ethylenediaminetetra acetic acid
        (EDTA) is a sparingly soluble ammo
        polycarboxylic acid which forms
        slightly ionized and very stable color-
        less complexes with the alkaline-
        earth metals.

     2   Interferences:  iron, manganese,
        nickel, and zinc.

     3   Procedure:  Time and pH considera-
        tions.

     4   Calculations of total hardness
        assuming a known volume of titrant
        of EDTA.

     5   Precision and accuracy.
 CH.HAR.3c. 11.75
                                                                                         13-1

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 Determination of Calcium ind Magnesium Hardness	
C   Determination of Calcium Hardness

    1  Principle of determinations

      Murexide indicator forms a salmon-
      colored complex with calcium whose
      ionization constant is of a higher
      value than that of the Ca EDTA complex.

    2  Interferences:  Heavy metals and Sr.

    3  Procedure: Time of tritation and
      proper lighting conditions are critical
      factors.

    4  Calculation of Ca  hardness.

    5  Precision and accuracy.

 D  Determination of Magnesium Hardness

    1  Calculation by difference method
       most commonly used.

    2  Equivalent of Ca hardness is sub-
       tracted from total hardness equiva-
       lent!, the difference attributable to
       magnesium equivalents.

    3  Other methods such as pyrophosphate
       method,  where calculation by differ-
       ence method cannot be used.


 IV ANALYTICAL QUALITY CONTROL
    METHODS

 A The Environmental Protection Agency,
    Analytical Quality Control Laboratory
    has published a manual titled Methods
    for Chemical Analysis of Water and
    Wastes.  1974.
B
  The procedure for the determination
  of hardness, as discussed in this
  manual,  was excerpted from the
  13th ed.  of Standard Methods*4'
  and part 23  of ASTM. <5>
REFERENCES

1  Barnard, A. J.  Jr.,  Broad, W. C. and
      Flaschka, H.  The EDTA Titration.
      J. T. Baker Company.  1957.

2  U. S. Public Health Service.   Drinking
      Water Standards. USPHS Report,
      Volume 61.  No.  11, 1946.

 3 Rainwater, R. H. and Thatcher, L. L.
      Methods for the Collection and Anal-
      ysis of Water Samples.  U. S. Geo-
      logical Survey Water Supply, Paper
      1454.  1960.
    Standard Methods for the Examination
       of Water and Wastewater,  13th ed.
       APHA, AWWA, WPCF,  Method
       122B, 1971.

    ASTM Standards,  Part 23,  Method
      D1126-67,  1973.

    Methods for Chemical Analysis of
      Water and Wastes, EPA-MDQARL.
      Cincinnati, OH, 1974.
 This outline was prepared by B.  V. Salotto,
 Research Chemist, Waste Identification and
 Analysis Section, MERL. EPA,  and revised
 by C. R.  Feldmann, Chemist,  National
 Training Center, MPOD,  OWPO, USEPA,
 Cincinnati, OH  45268.

 Descriptors:  Calcium, Chemical Analysis,
 Hardness, Magnesium, Water  Analysis
  13-2

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                         SOLIDS RELATIONS IN POLLUTED WATER
I  MPN, oxygen demand, and solids have
been major water pollution  control criteria
for many years.   This discussion ie con-
cerned primarily with solids  and their inter-
relations with oxygen demand.

A  Engineered treatment or surface water
   self-purification depends upon:

   1 The conversion of  soluble or colloidal
      contaminants into  agglomerated masses
      that may be separated from the-water.

   2 Oxidation of putrescible components
      into stable degradation  products.

   3 Item  1 is the major concern in most
      treatment systems because item 2
      requires a greater investment In time,
      manpower and capital costs.

B  Stress on  oxygen demand removal fre-
   quently  results in an  unduly small amount
   of attention to the contribution of solids
   in the oxygen demand picture.

   1 Oxygen demand formulations generally
      are specified to be applicable in the
      absence of significant deposition.

   2 Increasing impoundment, tidal estuaries,
      and Incomplete solids removal, gener-
      ally ensure that solids deposition will be
      significant.

   3 The BOD test stresses the fraction of
      oxygen demand that is exerted relatively
      rapidly.  It includes only the fraction of
      unstable material that is exerted under
     test conditions - usually short term un-
      der aerobic conditions.

   4 Contributions of a bed load of solids to
     oxygen  demand frequently are incom-
     pletely  recognized because they have a
     more local effect,  are difficult to measure,
     tend to  move, and  are incompletely un-
      derstood.  The onset  of an aerobic action
     in deposited solids increases hydrolysis
      rate. High molecular weight cell mass
     splits into small molecules that are
      soluble and more available for high
      rate oxidation upon feedback into oxy-
      gen containing water.

II  A given wastewater may have several
forms of solids in changeable proportions
with time and conditions.

A  Solids may be  classified among the charted
   forms according to biological chemical
   or physical properties.  It is not possible
   to precisely classify a given material into
   any one form because they usually are
   mixtures that may include or be converted
   into other forms.

B  Interrelationships are indicated by diagram
   in Figure  1. Some changes occur more
   readily than others.

   1 Settleable solids generally consist of
      a mixture of organic, inorganic,  en-
      trained dissolved or colloidal solids
      with living and dead organisms.

      a  They may be hydrolyzed into smaller
        sizes to aquire colloidal or
        dissolved characteristics.

      b  They may be  converted into a larger
        fraction of living material or vice
        versa.

   2 Colloidal solids also are likely to be a
      mixture of organic and inorganic
      materials, living or dead containing
      associated dissolved materials.

      a  They are likely to agglomerate to
        form settleable masses with time
        or changing conditions.

     b  Chemical reactions may solubilize
        the colloidal masses for recombina-
        tion into  other forms.

   3 Dissolved solids are most readily avail-
     able of all forms for biological, chemi-
     cal or physical conversion.
  PC.6b. 11.75
                                                                                         14-1

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Solids Relations in Polluted \Vaier
                    Figure 1.  SOLIDS INTERRELATIONSHIPS IN WATER
      a  They may be assimilated into cell
         mass to become colloidal during a
         state of rapid growth or form settle -
         able masses during slow growth.
         Surface phenomena are likely to en-
         courage inclusion of other forms of
         solids with cell mass.

    Stabilization occurs with each  conversion
    because energy is expended with each
    change.

    1  Biological changes involve oxidation to
      obtain enough energy to synthesize cells.
Products consist of cell mass and
degradation products.

Oxidation tends toward production
of CO2, H2O,  NOg, SO^^etc.  CO2
has limited water solubility and
partially leaves the aqueous environ-
ment.  More soluble constituents
tend to remain with it for recycle.

Biological  residues tend to recycle.
Eventually  the mass consists of
relatively inert and largely insolu-
ble residues.  These make up the
 14-2

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                                                             Solids Relations in Polluted Water
          bed load that may decompose at a
          rate of less than 1% per day, but
          accumulates in mass until it be-
          comes dominant  in water pollution
          control activities.
Ill  Treatment is an engineered operation
  designed to utilize events in surface water
  self-purification in a smaller package in
  terms of space and time.  Effective treat-
  ment presupposes oxidation either in the
  plant or in facilities to minimize feedback
  of solids components to the aqueous
  environment.

  A Primary treatment consists of a  separation
    of floatable or settleable solids and re-
    moval from  the used water.

    1  Prompt removal is essential to minimize
       return of solubilized or leached materi-
        als  from the sludge mass.

    2  Sludge and scum are highly putrescible
       and difficult to drain.

    3  Subsequent treatment prior to disposal
       serves to enhance drainability,  reduce
       solids or volume for burial or burning.

  B Secondary treatment generally involves
    some form of aerobic biological activity
    to oxidize part  of the colloidal and  dis-
    solved contaminants and convert most of
    the remainder into a settleable sludge.

    1  Aerobic systems favor assimilation of
       nutrients into cell mass at the expense
       of part of the available energy repre-
       sented by oxidation to products such as
       CC>2 and  water.

    2  Cell mass and intermediate degradation
       products  are only partially stabilized
       and tend  to recycle with death and decay.

    3  Rapid growth of cells tends to produce
       sludges that are highly hydrated and
        difficult to concentrate.

    4  A compromise must be reached to pro-
       duce a favorable balance among separa-
       tion of solids and feedback of lysed
       materials.
   a  Cell growth continues as long as
      energy and nutrients permit.

   b  Under limiting nutrient conditions
      the population may show a relatively
      low rate overall dieoff but variety
      is changing.

   c  Species most favored by the new con-
      ditions tend to grow while others
      die, lyse and release part of their
      stored nutrients for subsequent use.

Anaerobic digestion of solids separated
during treatment is one process used to
increase solids stability and drainability
while reducing total volume or mass for
disposal.

1  Growth of cell mass is relatively slow
   under anaerobic conditions while hy-
   drolytic cleavage is relatively large
   in comparison to that during aerobic
   metabolism,

   a  Feedback to the aqueous environment
      represents a significant fraction of
      the input in the form of ammonia,
      colloidal solids, low molecular
      weight acids, and other products.

   b  Mass  of the  sludge is reduced by the
      fraction of methane, carbon dioxide,
      and other gases produced in process.

   c  Remaining solids tend to be more
      concentrated,  are lower  in putresci-
      bility  and give up their water more
      readily.

2  The liquid fraction of the products re-
   maining after anaerobic digestion con-
   tain nutrients,  oxygen demand, solids,
   and malodorous constituents that are
   objectionable in surface waters if re-
   leased without further aerobic
   stabilization.

3  Digester liquids are much more con-
   centrated than raw sewage and nutrition-
   ally unfavorable, hence they are difficult
   to treat and tend to shock aerobic
   treatment processes.
                                                                                  14-3

-------
Solids Relations in Polluted Water
 D  Suitable disposal of solids resulting from
    treatment operations takes various forms
    of which the most desirable is to oxidize
    it completely to gaseous products of oxi-
    dation or inert ash.  The objective is to
    limit feedback into the aqueous environ-
    ment and delay the charted interchanges.

    1  Deposition of sludge as a cover on crop
      land is effective providing surface
      drainage is properly designed to limit
      washoff.

    2  Burial in areas above the flood plain
      provides a suitable disposal,

    3  Incineration under controlled con-
      ditions to produce complete burning
      of organics is effective but requires
      close control.

      a  Incineration of digested sludge is
         feasible but the digestion process
         results in removal of part of the
         heat content of the sludge as met-
         hane  carbon dioxide and water. Aux-
         Dary heat may be required for wa-
         ter evaporation if solids concentra-
         tion is below 25%.

      b  Raw sludge incineration relieves
         the auxilary heat problem but stor-
         age may lead to odor problems.  The
         raw sludge may require chemical
         treatment to enhance dew ate ring,
         secondary sludges are more diffi-
         cult to dewater and may require
         centrifugation, floatation, or other
         treatment to concentrate sludge for
         heat balance purposes.

      c  Incineration requires close control
         of complex equipment to maintain
         acceptable environmental control.
         Usually, after burners and off gas
         scrubbers are necessary to avoid
         air pollution.
IV ANALYTICAL PROCEDURES

A The Analytical Quality Control
   Laboratory, Office of Water Programs,
   Environmental Protection Agency has
   published a manual titled. Methods for
   Chemical Analysis of Water and Wastes,
   1974.

B In this manual solids are classified into
   four groups:

   1   Residue,  Total Filterable
   2   Residue,  Total Non-Filterable
   3   Residue.  Total
   4   Residue,  Volatile

C The procedure for the determination of
   1,2, and 3 above is given in the  EPA
   Manual.

   For the Volatile Residue procedure.
   Standard Methods for the Examination
   of Water and Wastewater, 13th Ed.,
   page 536, Method 2243(1971) is the
   reference cited by the EPA Manual.
This outline was prepared by F. J. Ludzack,
Chemist,  National Training Center,  MPOD,
OWPO, USEPA, Cincinnati, Ohio 45268.

Descriptors: Anaerobic Digestion, Oxygen,
Oxygen Demand, Sludge, Sludge Disposal,
Sludge Treatment, Solids,  Waste Water
Treatment,  Water Pollution Sources
   14-4

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                       PHOSPHORUS IN THE AQUEOUS ENVIRONMENT
I  Phosphorus is closely associated with
water quality because of (a) its role in
aquatic productivity such as algai blooms,
(b) its  sequestering action, which causes
interference in coagulation, (c) the difficulty
of removing phosphorus from water to some
desirable low concentration,  and (d) its
characteristic of converting from one to
another of many possible forms.

A  Phosphorus is one of the primary nutrients
   such as hydrogen (H), carbon (C)f
   nitrogen (N),  sulfur (S)  and phosphorus (P).

   1  Phosphorus is unique among  nutrients
      in that its oxidation does not  contribute
      significant energy because it commonly
      exists in oxidized form.

   2  Phosphorus is intimately involved in
      oxidative energy release from and
      synthesis of other  nutrients into cell
      mass via:

      a  Transport of nutrients across
         membranes into cell protoplasm is
         likely to include phosphoryiation.

      b  The release of  energy for meta-
         bolic purposes  is likely to
         include a triphosphate exchange
         mechanism.

B  Most natural waters  contain relatively low
   levels of P (0.01 to 0. 05 mg/1) in the
   soluble state during periods of significant
   productivity.

   1  Metabolic activity  tends to convert
      soluble P into cell mass (organic P) as
      a part of the protoplasm, intermediate
      products, or sorbed  material.

   2  Degradation of cell mass and incidental
      P compounds results in a feedback of
      lysed P to the water  at rates  governed
      by the type of P and the environment.
      Aquatic metabolic kinetics show  marked
      influences of this feedback.
        The concentrations of P in hydrosoils,
        sludges,  treatment plant samples and
        soils may range from 102 to 106 times
        that in stabilized surface water.  Both
        concentration and interfering compo-
        nents affect applicability of analytical
        techniques.
 II  The primary source of phosphorus in the
  aqueous system is of geological origin.
  Indirect sources are the processed mineral
  products for use in agriculture, household,
  industry or other activities,

  A  Agricultural fertilizer run-off is related
     to chemicals applied, farming practice
     and soil exchange capacity.

  B  Wastewaters primarily of domestic
     origin contain major amounts of P from:

     1  Human, animal and plant residues

     2  Surfactants (cleaning agent) discharge

     3  Microbial and other cell masses

  C  Waste*waters primarily of industrial
     origin contain P related to:

     1  Corrosion control

     2  Scale control additives

     3  Surfactants or dispersants

     4  Chemical processing of materials
        including P

     5  Liquors from clean-up operations of
        dusts, fumes,  stack gases, or other
        discharges
III   Phosphorus terminology is commonly
 confused because of the interrelations among
 biological, chemical, engineering,  physical,
 and analytical factors.
  CH.PHOS.4c. 11.75
                                                                                 15-1

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Phosphorua	injthc Aqueous Environnu nt
   Biologically,  phosphorus may be available
   as a nutrient, synthesized into living mass,
   stored in living or dead cells, agglomerates,
   or mineral complexes, or converted to
   degraded materials.

   Chemically,  P exists in several mineral
   and organic forms that may be converted
   from one to another under favorable
   conditions.  Analytical estimates  are
   based upon physical or chemical techniques
   necessary to convert various forms of P
   into  ortho phosphates which alone can be
   quantitftted In terms of the molybdenum
   blue colorimetric teat.

   Engineering interest in phosphorus is
   related to the prediction, treatment, or
   control of aqueous systems to favor
   acceptable water quality objectives.
   Phosphorus removal is associated with
   solids  removal.
   Solubility and temperature are major
   physical factors in phosphorus behavior.
   Soluble P is much more available than
   insoluble P for chemical or biological
   transformations and the rate of conversion
   from one to another is strongly influenced
   by temperature.

   Table 1 includes a classification of the
   four main types of chemical P and some
   of the relationships controlling solubility
   of each group.  It is apparent that no
   clear-cut separation can be made on a
   solubility basis as molecular weight,
   substituent and other factors affect
   solubility.
   Table 2 includes a scheme of analytical
   differentiation of various forms of P
   based upon:

   1  The technique required to convert an
      unknown variety of phosphorus into
      ortho P which is the only one quanti-
      tated by the colorimetric test.
   Solubility characteristics of the sample
   P or more precisely the means required
   to clarify the sample.


   a  Any clarification method is subject
      to incomplete separation.  Therefore,
      it is essential to specify the method
      used to interpret the yield factor of
      the separation technique.  The
      degree of separation of solubles
      and insolubles will he significantly
      different for:
      1  Membrane filter separation
         (0.5 micron pore size)

      2  Centrifugation (at some specified
         rpm and time)

      3  Paper filtration (specify paper
         identification)

      4  Subsidence (specify time and
         conditions)
Analytical separations (Table 2) like those
in Table 1,  do not give a precise separa-
tion of the various forms of P which may
be included  quantitatively with ortho or
poly P.  Conversely some  of the poly and
organic P will be included  with ortho P if
they have been partially hydrolyzed
during storage or analysis.  Insolubles
may likewise be included as a result of
poor separation and analytical conditions.
   The separation methods provide an
   operational type of definition adequate
   in most situations if the "operation"
   is known.  Table 2 indicates the nature
   of incidental P that may appear along
   with the type sought.
15-2

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                                                     Phosphorus in the Aqueous Environment
                                           Table  1


                         PHOSPHORUS  COMPOUNDS  CLASSIFIED  BY
                          CHEMICAL AND  SOLUBILITY RELATIONS
               Form
       Water Soluble
                                                         (1)
        Insoluble
                                                (1)
1.   Ortho phosphates
       (PO/3
Combined with monovalent
cations such as H,+ Na
Combined with multi
valent cations such
     +2 .,
as Ca  Al
                                                                                 ^
                                                                                 Fe
                                               +3
2.   Poly phosphates

    ' "4"5(P39
    and others  depending upon
    the degree  of dehydration.
as in 1 above
Increasing dehydration
decreases solubility
(a)as in 1 above
(b) multi P polyphosphates
   (high mol.  wt.) in-
   cluding the "glassy"
   phosphates
3.  Organic phosphorus
    R-P, R-P-R       (2)
    (unusually varied nature)
(a) certain chemicals
(b) degradation products
(c) enzyme P
(d) phosphorylated nutrients
(a) certain chemicals
(b) cell mass, may be
   colloidal or agglom-
   erated
(c) soluble P sorbed by
   insoluble residues
4.  Mineral phosphorus
(a) as in 1  above
(a) as in 1 above
(b) as in 2 above
(c) geological P such as
   phosphosilicates
(d) certain mineral com-
   plexes.
(1)  Used in reference to predominance under common conditions.
                                                       -3
(2)  R represents an organic radical, P represents P, PC>4> or its derivatives.
         Total P in Table 2 includes liquid and
         separated residue P that may exist in
         the whole sample including silt,  organic
         sludge, or hydrosoils.  This recognizes
                     that the feedback of soluble P from
                     deposited or suspended material has a
                     real effect upon the kinetics of the
                     aqueous environment.
                                                                                      15-3

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Phosphorus  in the Aqueous Environment
                                          Table  2
                       PHOSPHORUS COMPOUNDS CLASSIFIED  BY
                              ANALYTICAL METHODOLOGY
        Desired P Components
    Technique
                                                (1)
                               Incidental P Included
                                                  (2)
     1.  Ortho phosphates
No treatment on clear
samples
Easily hydrolyzed
(a) poly phosphates -
(b) organic -P,  -
(c) Mineral -P, + or
     2.  Polyphosphates
        (2)-
-------
                                                     Phosphorus  in the Aqueous Environment
IV   I'olyphosphates are oi major interest in
  cleaning agent formulation,  an dispfrmuit::,
  and for corrosion control.

  A  They are prepared by dehydration of ortho
     phosphates to form products having two or
     more phosphate derivatives per molecule.

     1   The simplest polyphosphate may be
        prepared as follows:
        NaO                 NaO

          ;/  =0

                    O
    O
                             HO

                             NaO'
                               \p
  mono sodium ortho
     phosphate (2)
disodium dihydrogen
   polyphosphate
     2  The general form for producing
        polyphosphates from mono substituted
        orthophosphates is:
     n  (NaH2PO4)

     3  Di-substituted ortho phosphates or
        mixtures of substituted ortho phosphates
        lead to other polyphosphates:
      insoluble polyphosphate than the
      cation in the form of insoluble ortho
      phowphute.  InsolubiJiiy incf-anc;-: with
      the number of I1 atoinw in thi-
      polyphosphate.  The  "glassy" poly
      phosphates are a special group with
      limited solubility that are used to aid
      corrosion resistance in pipe distribu-
      tion systems  and similar uses.

B  Polyphosphates tend to hydrolyze or
   "revert" to  the ortho P  form by addition
   of water.  This occurs whenever
   polyphosphates are found in the aqueous
   environment.

   1  The major factors affecting the rate  of
      reversion of poly to orthophosphates
      include:

      a) Temperature,  increased T increases
         rate

      b) pH, lower pH increases rate

      c) Enzymes,  hydrolase enzymes
         increase rate

      d) Colloidal gels,  increase rate

      e) Complexing cations and ionic
         concentration increase rate

      f) Concentration  of the polyp'nosphau
         increases rate
  disodium hydrogen
   ortho phosphate
                 Nail |PO<

                  mono SO!
         The polyphosphate series usually
         consist of the polyphosphate anion
         with a negative charge of 2 to 5.
         Hydrogen or metals commonly occupy
         these sites.  The polyphosphate can be
         further dehydrated by heat as long as
         hydrogen remains.  Di or trivalent
         cations generally produce a more
                              2  Items a, b and c have a large eiiect
                                upon reversion rate compared with
                                other factors listed.  The actual
                                reversion rate is a combination of
                                listed items and other conditions or
                                characteristics.

                              3  The differences among ortho and ortho
                                + polyphosphates commonly are close to
                                experimental  error of the colorimetric
                                test in stabilized surface water samples.
                                A  significant difference generally
                                indicates that the sample was obtained
                                relatively close to  a source of poly-
                                phosphates and was promptly analyzed.
                                This implies that the reversion rate of
                                polyphosphates is much higher than
                                generally believed.

-------
Phosphorus  in the Aqueous Environment
V SAMPLING AND PRESERVATION
   TECHNIQUES

A Sampling

   1  Great care should be exercised to
      exclude any benthic deposits from
      water samples.

   2  Glass containers should be acid rinsed
      before use.

   3  Certain plastic containers may be
      used.  Possible adsorption of low con-
      centrations of phosphorus should be
      checked.

   4  If a differentiation of phosphorus forms
      is to be made, filtration should be
      carried out immediately upon sample
      collection.  A membrane filter of
      0.450 pore size is recommended for
      reproducible separations.

B Preservation

   1  If at all possible, samples should be
      analyzed on the day of collection.  At
      beet, preservation measures only
      retard possible changes in the  sample.

      a  Possible physical changes include
        solubilization, precipitation,
        absorption on or desorption from
        suspended matter.

      b  Possible chemical changes include
        reversion of poly to ortho P and
        decomposition of organic or min- .
        eralP.

      c    Possible biological changes
          include microbial decomposition
          of  organic P and algal or
          bacterial growth forming organic
          P.

   2  Refrigeration at 4 C is recommended
      if samples are to be stored up  to
      24 hours.  This decreases hydrolysis
      and reaction rates and also losses due
      to volatility.
     3    Addition of 2 ml concentrated
         H2SO4 or 40 mg HgCl2/liter is
         recommended for longer storage
         periods.  This chemical limits
         biological  changes.

         a   HgC ! is an interference in the
             analyucal procedure if the
             chloride level is low (less than
             50 mg. Cl/1).  See Part VI
             B 3 below.


VI   THE EPA ANALYTICAL PROCEDURE*6}

 A   This is a colorimetric determination,
     specific for orthophosphate. Depending
     on the nature of the sample and on the
     type of data sought, the procedure in-
     volves two general operations:

    1    Conversion of phosphorus forms to
         soluble orthophosphate (See Fig.  1);

        a   sulfuric acid-hydrolysis for
            polyphosphates,  and some
            organic P compounds,

         b   persulfate digestion for organic
            P compounds.
     2  The color determination involves
       reacting dilute solutions of phosphorus
       with ammonium molybdate and
       potassium antimonyl tartrate in an
       acid medium to form an antimony-
       phosphomolybdate complex.  This
       complex is reduced to an intensely
       blue-colored complex by ascorbic
       acid.  The  color is proportional to
       the orthophosphate concentration.

       Color absorbance is measured at
       880 nm or 650 nm and a concentration
       value obtained us ing a standard curve.

       Reagent preparation and the detailed
       procedure can be found in the EPA
       manual.

       The methods described there are
       usable in the 0.01 to 0.5 mg/liter
       phosphorus range.
   15-6

-------
                                                 Phosphorus  in the Aqueous Environment
Totl KydtolyiabU
A Orthophoiphttta

total
?hoiphorui
                                 Filter  0.45 Micron Membrane Filter
Coloriwtry
V \
p
I Soluble Soluble Uydrol7bU
[ Ortbcphosphatt 6 Orthophophc
ltiorir.ic polyphosphat*
phosphorus + some orpuaic
c ocpound* phosphor u

I ColorlMtry
!To:l Soluble
inorganic +
organic
phCMpburu*
                                                     coapouo4
                                               FIGURE 1
                              ANALYTICAL SCHEME FOR DIFFEREKHATION OF PHOSPHORUS FORMS
B   Interferences

    1  Erroneous results from, contaminated
       glassware is avoided by cleaning it
       with hot 1:1 HC1, treating it with
       procedure reagents and rinsings
       with distilled water. Preferably
       this glassware should be used only
       for the determination of phosphorus
       and protected from dust during
       storage.  Commercial detergents
       should never be used.

    2  High iron concentrations in samples
       can precipitate phosphorus.

    3  If HgCl- is used as a preservative,
       it interferes if the chloride level of
       the sample is less than 50 mg
       Cl/liter.  Spiking with NaCl is then
       recommended.

    4  Others  have reported interference
       from chlorine, chromium, sulfides,
       nitrite, tannins,  lignin and other
       minerals and organics at high  con-
       centrations.
                           / e \
C   Precision and Accuracy

    1  Organic phosphate -  33 analysts in
       19 laboratories analyzed natural
       water samples containing exact in-
       crements of organic phosphate of
       0.110,  0.132,  0.772,  and 0.882 mg
       PI liter.

       Standard deviations obtained were
       0.033,  0.051,  0.130 and 0.128
       respectively.

       Accuracy results as bias, mg P/liter
       were: +0.003, +0.016, +0.023  and
       -  0.008, respectively.

    2  Orthophosphate was determined by
       26 analysts in  16 laboratories using
       samples containing orthophosphate
       in amounts of 0. 029, 0.038, 0.335
       and 0.383  mg P/liter.

       Standard deviations obtained were
       0.010,  0.008,  0.018 and 0.023
       respectively.
                                                                                        '7

-------
  Phosphorus  in the Aqueous Environment
         Accuracy results as bias, mg P/liter
         were -0.001,  -0.002.  -0.009 and
         -0. 007 respectively.

  O Automated Methods

     The EPA Manual also contains a
     procedure for an automated colorimetry
     method using the ascorbic acid reduction
     method.
VII  VARIABLES IN THE COLORIMETRIC
     PROCEDURE

     Several important variables affect
     formation of the yellow heteropoly
     acid and its reduced form,  molybdenum
     blue, in the colorimetric test for P.

 A   Acid Concentration during color develop-
     ment is critical.  Figure 2  shows that
     color will appear in a sample containing
     no phosphate if the acid concentration
     is low.  Interfering color is negligible
     when the normality with respect to
            approaches 0.4.
     1  Acid normality during color develop-
        ment of 0. 3 to slightly more than
        0.4 is feasible for use.  It is prefer-
        able to control acidity carefully and
        to seek a normality closer to the
        higher limits of the acceptable range.

     2  It is essential to add the acid and
        molybdate as one solution.

     3   The aliquot of sample must be
        neutralized prior to adding the
        color reagent.
U

iu
                 0.05 mg P
         /\
         \ /  STD-BIK
     BLANK
        O.I   0.2  0.3   0.4  OJ   0.6

                   NO*MAl(TY
             Figure 2

       0-PHOSPHATE COLOR
            VS ACIDITY

 Choice of Reductant - Reagent stability,
 effective reduction and freedom from
 deleterious side effects are the bases
 for reductant selection.  Several re-
 ductants have been used effectively.
 Ascorbic acid reduction is highly
 effective in both marine and fresh water.
 It is the reductant specified in the
 EPA method.

 Temperature affects the rate of color
 formation. Blank, standards, and
 samples must be adjusted to the same
 temperature (*  1C). (preferably room
 temperature), before addition of the
 acid molybdate  reagent.

 Time for Color Development  must be
 specified and consistent.   After addition
 of reductant,  the blue color develops
 rapidly for 10 minutes then fades grad-
 ually after 12 minutes.
   15-8

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                                                    Phosphorus  in the Aqueous Environment
VIH DETERMINATION OF TOTAL
     PHOSPHORUS

 A   Determination of total phosphorus
     content involves omission of any
     filtration procedure and using the acid-
     hydrolysis and persulfate treatments
     to convert all phosphorus forms to the
     test-sensitive orthophosphate form.

 B   Determining total phosphorus content
     yields the most meaningful data since
     the various forms of phosphorus may
     change from one form to another in a
     short period of time.  (See part V, Bl)
 K   DEVELOPMENT OF A STANDARD
      PROCEDURE

      Phosphorus analysis received intensive
      investigation; coordination and validation of
      methods is more difficult than changing
      technique.

  A  Part of the problem in  methods arose
     because of changes in analytical objectives
     such as:

     1  Methods suitable to  gather "survey"
        information may not be adequate for
        "standards".

     2  Methods acceptable  for water are not
        necessarily effective in the presence
        of significant mineral and organic
        interference characteristic of hydro-
        soils, marine samples, organic
        sludges and benthic deposits.


     3  Interest has been centered on "fresh"
        water. It was essential to extend them
        for marine waters.
   4  Instrumentation and automation have
     required adaptation of methodology.

B  Analysts have tended to work on their own
   special problems.  If the method
   apparently served their situations, it was
   used.  ,Each has a "favorite" scheme that
   may be quite effective but progress
   toward widespread application of "one
   method has been slow.  Consequently,
   many methods are  available.  Reagent
   'acidity, Mo content,  reductant and
   separation techniques are the major
   variables.

 C At the present time there is not sufficient
   data to warrant EPA endorsement of the
   P procedure for sediment-type samples,
   sludges, algal blooms,  etc.  Following
   is a procedure (not included in the EPA
   manual) which is useful when  solids
   are present in  samples:

   1  If sample contains large particles,
      grind and emulsify solids in a blender.

   2  Transfer 50 ml sample, or aliquot
      diluted to 50 ml, into a 250 ml
      Erie run ever flask.

   3  Add 6.  0 ml of 18N H2SO4,  5 ml
      concentrated HNO"o,  2 berl saddles
      and digest on hot plate.

   4  Digest until the disappearance of  nitric
      acid fumes and the appearance  of white
      503 fumes. Continue digestion for
      approximately 5 minutes.  Cool before
      proceeding with Step 5.

   5  Add 2 ml of HNO3-HCK>4 mixture
      and 5 ml concentrated HNO3.  Continue
      digestion until all of the nitric acid is
      driven off and dense fumes of perchloric
      evolve.  Perchloric acid requires
      dilution with sulfuric acid and prior
      destruction  of most organics for safety.
                                                                                       15-9

-------
Phosphorus  in the Aqueous Environment
     Cool.  Add approximately 40 ml
     distilled water and transfer to 100 ml
     volumetric flask.
      Add 2-3 drops of phenolphthalein and
      concentrated ammonium hydroxide
      until a pink color is seen.  Then
      discharge the pink color with the
      strong sulfuric acid. It is advisable
      to add an equivalent amount of salt
      formed during neutralization of digested
      samples to the calibration standards to
      equalize salt content during color
      development.

      Determine  orthophosphate according
      to the usual color procedure.
ACKNOWLEDGMENT:

Materials in this outline include significant
portions of previous outlines by J.  M. Cohen,
L.  J. Kamphake,  andR.  J. Lishka.  Important
contributions and assistance were made by
B.  C.  Kroner, E. F. Barth, W. Allen Moore,
Lloyd Kahn,  Clifford Risley, Lee Scarce.
John Winter, and Charles Feldmann.
 REFERENCES
2 Gales,  Morris E. .  Jr.,  Julian, Elmo C. ,
     and Kroner, Robert C. , Method for
     Quantitative Determination of Total
     Phosphorus in Water.  JAWWA 58:
     (10)  1363.  October 1966.

3 Lee, G. Fred, Clesceri, Nicholas L. and
     Fitzgerald,  George P., Studies on the
     Analysis of Phosphates in Algal Cultures.
     Int.  J. Air & Water Poll.   9:715.  1965.


 4   Barth, E. F. and Salotto,  V. V.,
       Procedure for  Total .Phosphorus
       in  Sewage and Sludge, Unpublished
       Memo, Cincinnati Water Research
       Laboratory, FWQA. April 1966.

 5  Moss, H. V., (Chairman, AASGP
       Committee) Determination of Ortho
       Phosphate, Hydrolyzable Phosphate
       and Total Phosphate in Surface Water,
       JAWWA 56:1563.  December  1958.


  6  Methods for Chemical Analysis of Water
     & Wastes, EPA,  MDQARL, Cincinnati.Ohio
     45268, 1974.

  This outline was prepared by Ferdinand J.
  Ludzack, Chemist,  Audrey E. Donahue,
  Chemist, both with National Training Center,
  MPOD,  OWPO, USEPA,  Cincinnati, Ohio
  45268.
   Jenkins, David, A Study of Methods
      Suitable for the Analysis and
      Preservation of Phosphorus Forms
      in the Estuarine Environment.  DHEW,
      Central Pacific River Basins Project.
      SERL Report No. 65-18. University
      of California.  Berkeley, Calif.
      November 1965.
  Descriptors :
  Chemical Analysis, Nutrients, Phosphates,
  Phosphorus, Phosphorus Compounds,
  Pollutant Identification, Sampling,  Water
  Analysis, Water Pollution Sources.
  15-10

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                            BIOCHEMICAL OXYGEN DEMAND TEST
                                     DILUTION TECHNIQUE
I  OBJECTIVES:  to prepare a sample dilution for series observation of oxygen depletion versus
   time for the estimation of BOD , rate anc
   Winkler iodometric titration of dissolved
   will be used to check the titrimetric DO.
time for the estimation of BOD ,  rate and ultimate demand.  The azide modification of the
Winkler iodometric titration of dissolved oxygen will be used.  An electronic DO analyzer
II   PROCEDURE:

 A  Each group will prepare 1 dilution of either effluent or influent at an assigned concentration
    to enable filling 7 standard BOD bottles.  One of these will be used for electronic DO mon-
    itoring by the lab instructor,  the other  Bix  are for daily student determinations. Identify
    each bottle with group number, I for influent or E for effluent and the  assigned concentration
    in %.

 B  Calculate the volume of sample to be used to make up a total diluted volume of 3500 ml at
    the assigned concentration.  Then dilute the sample as follows:

    1 Add dilution water to the dilution bottle to make up about 1/2 of the total amount of
      distilled water to be used.  Add 1 ml each of the four mineral supplements.  (These
      solutions  are prepared at 4 times the concentration of Standard Methods mineral
      supplement  reagents so the added volume is lower).   Mix the supplemented dilution
      water thoroughly.

    2 Carefully mix the bottle of sample to be used to a uniform suspension.  If large chunks
      are evident, homogenize.   Rapidly pour out,  with mixing, into a graduate, the
      amount of sample required and return  to lab  station.


      (Mixing, time, and precision  are important.  Your sample may be non-representative to
      the extent of carelessness in mixing, allowing settling while adjusting measurement or
      measurement errors.)

    3 Add the measured quantity of sample to the dilution bottle.

    4  Carefully add dilution  water to make up 3500 ml of diluted sample. Use some of the dilu-
       tion water to rinse the graduate into the bottle.
    5  Stopper and shake the dilution bottle to mix the  sample and to ensure that you have an
       acceptable initial DO.  This step will add DO or reduce supersaturation.  About 15
       seconds of mixing is usually  adequate.

 C  Filling bottles is simplified if you place the dilution bottle on the center pedestal,  insert
    the  siphon and line up your identified bottles with stoppers removed.  Have a container
    larger than the bottles available to accommodate the overflow while filling them.  Otherwise,
    move the apparatus near a sink.  Fill the siphon tube and waste some of the diluted sample
    to rinse  this tube.  Be sure that the siphon tube is used  in a manner so that it is always
    full of the diluted sample.  Agitate the  diluted sample regularly to avoid undue collection of
    solids on the bottom.
                                                                                         16-1
 CH, O. bod. lab. 3. 11.75

-------
Biochemical Oxygen Demand Test Dilution Technique
   1  Place first bottle to be filled in the overflow container or hold over sink.  Insert the
      siphon delivery tip all of the way down into the bottom of the test bottle.  Tilt the
      bottle to cover the delivery tip with water promptly.  Start the siphon flow slowly to
      decrease turbulence until the delivery tip is well-covered.  Fill to overflowing.  Slowly
      raise the delivery tip while overflowing the sample.  Stop the flow with the bottle com-
      pletely full.  Insert the bottle stopper and turn it slightly to seat it.  Fill the remaining
      bottles in the same manner.

   2  Each bottle will have the same DO  and sample mix as the next if you have followed a
      consistent procedure, mixed carefully, and have not changed technique enroute.

   3  One  of the seven bottles la to be retained for initial tit ration of DO by your group.  Another
      bottle is to be turned in to the lab instructor for use  in performance checks  on all class
      dilution*.  The remainder are to be placed into trays for 20  incubation.  Be certain
      that  all bottles are properly identified.

D Dissolved Oxygen Determination

   1  You  are encouraged to check the DO of your sample  on the  electronic analyzer.  A
      calibrated unit will be available with assistance.  The DO analyzer will not  result in
      more than  1/2 ml loss of sample on probe insertion.  You will be able to add DO reagents
      and  continue the Winkler titrimetric DO on the same bottle.

   2   An Initial DO value (DO ) using the Winkler method  must be determined today.  On succeed
       ing  days, the DO. will b% determined on the incubated samples for estimation of BOD
      progression.  (If reaeration is required,  the technique will be demonstrated).   All data
      is to be recorded on the sheet provided in this manual.

      The Winkler-Azide procedure as presented in outline 17 is to be used for these DO
      determinations.

   3  A blank value for each stock bottle of distilled water will be determined by the staff.
                                                  REFERENCE

                                                  Standard Methods for the Examination of
                                                     Water and Wastewater,  13th edition.
                                                     APHA, AWWA, WPCF, New York.
                                                     p.  489.   1971.
                                                   This outline was prepared by F.  J.  Ludzack.
                                                   Chemist, National Training Center, MPOD,
                                                   OWPO,  USEPA, Cincinnati. Ohio 45268.

                                                   Descriptors: Analytical Techniques,
                                                   Biochemical Oxygen Demand, Chemical
                                                   Analysis. Laboratory Tests,  Water Analysis
  16-2

-------
                                 Biochemical Oxygen Demand Test Dilution Technique
                                     HOD DATA SHEKT
Group No.
                                         Initial Date
Buret
Readings
DO
 Buret
Readings
DO,
ADO
Sf/ID
                                                   BOD
Buret readings - initial and final
DO - initial DO in mg/liter
   o
DO  =  DO at indicated incubation time t
DO  DO change (DO  - DOt); a depletion
Sf   =  the sample content as a decimal fraction in the dilution
ID   =  sample identification (influent or effluent)
BOD=   DO   -  DO  (according to Standard Methods,  13th edition, 1971)
             Sf
                                                                                       16-3

-------
                     LABORATORY PROCEDURE FOR DISSOLVED OXYGEN

                                     Winkler-Azide Procedure
I  INTRODUCTION

The azide modification is used for most
sewage, effluents, and streams which con-
tain nitrate nitrogen and not more than 1 mg
of ferrous iron/1.  If 1 ml 40% KF solution
is added before acidifying the  sample and
there is no delay in titration,  the method is
also applicable in the presence of 100-200
mg/1 ferric iron.

The "Methods for Chemical Analysis of
Water & Wastes, 1974, " published by the
Environmental Protection Agency,
recommends the Winkler-Azide
jmethod using the full bottle technique.
   REAGENTS
A  Manganous Sulfate Solution

   Dissolve 480 g MnSO
   water and dilute to
 - - 4H  O in distilled
1 liter.2
B Alkali-Iodide-Azide Reagent

   Dissolve 500  g  sodium hydroxide and 150 g
   potassium iodide in distilled water and
   dilute to 1 liter. To this solution add 10 g
   sodium azide.  NaNg dissolved in 40 ml
   distilled water.

C Sulfuric Acid, Cone.

  . The strength of this acid is 36 N.

D Starch Solution

   Prepare an emulsion of 10 g of soluble
   starch in a mortar or beaker with a small
   quantity of distilled water.  Pour this
   emulsion into 1 liter of boiling water,
   allow to boil a few  minutes,  and let settle
   overnight.  Use the clear supernate.
   This solution may be preserved by the
   addition of 5 ml per liter of chloroform
   and storage in a 10 C refrigerator.
                            E  Sodium Thiosulfate Stock Solution 0.75 N

                               Dissolve 186. 15 g Na^Og- 5 HO in boiled
                               and cooled distilled water and dilute to
                               1 liter.  Preserve by adding 5 ml chloroform.
                            F  Sodium Thiosulfate Standard Titrant 0. 0375N

                               Dilute 50. 0 ml of stock solution to 1 liter.
                               Preserve  by adding 5 ml of chloroform.
                               Standardize with potassium biiodate.
HI  PROCEDURE

 A Addition of Reagents

    1  Manganous sulfate and alkali- iodide-
       azide

       To a full sample bottle (300 ml + 3 ml
       BOD incubation),  add 2 ml manganous
       sulfate solution and 2 ml alkaline-
       iodide reagent with the tip of  each
       pipette below the surface of the sample.

    2  Replace the stopper.
    3  Rinse under running water.
    4  Mix well by inverting 4-5 times.
    5  Allow the precipitate to settle until at
       least  100 ml of clear supernate have
       been produced.
    6  Repeat steps 4 and 5.

    7  Add 2 ml cone, sulfuric acid  with the
       tip of the pipette above the surface of
       the sample.

    8  Stopper the bottle.
    9  Rinse under running water.
   ] 0  Mix by inverting several times to dissolve
       the precipitate.
    1 1  Pour contents of bottle into a  wide  mouth
       500 ml Erlenmeyer flask.
 B Titration

    1  Titrate with 0. 0375 N thiosulfate to a
       pale straw color.
 CH.O.do.lab. 3b. 11.75
                                                                 17-1

-------
 Laboratory Procedure for Dissolved Oxygen
    2 Add 2 ml starch solution indicator.

    3 Continue titrating to the disappearance
      of the blue color.

C   Calculation
mla tttrant X 0.0375 X8000
            300
                               fl
                              *
 mla titrantX 0.0125 X80  mg/1 DO

 mis tltrant X 1  mg/1 DO

 mis titrant  mg/1 DO
REFERENCE
Methods for Chemical Analysis of Water and
   Wastes, EFA-MDQARL,. Cincinnati, Ohio.
   1974.

This outline was prepared by J.  W. Mandia,
Chemist,  formerly with National Training
Center, and revised by C.  R.  Feldmann,
Chemist,  National Training Center, MPOD.
OWPO,  EPA,    Cincinnati, OH 45268.

Descriptors:  Analytical Techniques,  Chemical
Analysis, Dissolved Oxygen, Laboratory
Tests,  Oxygen. Water Analysis

-------
                       DETERMINATION OF KJELDAHL NITROGEN
                         (MICRO APPARATUS-NESSLERIZATION)
I  REAGENTS

A  Distilled Water

   This should be ammonia-free.  Pass
   distilled water through an ion exchange
   column with strongly acidic cation resin
   mixed with a strongly basic anion -resin.

B  Sulfuric Acid (20%)

   20 ml  acid/ 100 ml distilled water

C  Mercuric Sulfate Solution

   Dissolve 8g mercuric oxide in 50 ml of
   20% sulfuric acid.   Dilute to 100 ml with
   distilled water.

D  Digestion Reagent

   Dissolve  134g potassium sulfate in about
   650 ml distilled water.  Add 200 ml
   concentrated sulfuric acid.   Add 25 ml of
   mercuric sulfate solution (C above) and
   dilute  to 1 liter.

E  Sodium Hydroxide  - Sodium Thiosulfate
   Solution

   Dissolve  500g sodium hydroxide and 25g
   sodium thiosulfate pentahydrate in distilled
   water  and dilute to 1 liter.

F  Mixed Indicator

   Mix 100 ml of methyl red indicator
   (200 mg in 100 ml  95% ethyl alcohol) with
   50 ml  of methylene blue indicator solution
   (200 mg in 100 ml  95% ethyl alcohol).

G  Boric  Acid Solution, 2%

   Dissolve  20g boric acid in distilled water
   and dilute to 1 liter.

H  Ammonium Chloride Stock Solution

   Dissolve  3. 819g NH4C1 in distilled water
   and dilute to 1 liter.  1.0 ml =  1.0 mg
   NH3-N.

CH.N.lab.8. 11.75
I  Ammonium Chloride Standard Solution

   Dilute 10. 0 ml of the  stock solution with
   distilled water to 1 liter.
   1.0 ml = 0.01  mg NH3-N

J  Sodium Hydroxide Solution

   Dissolve 160 g sodium hydroxide in 500 ml
   distilled water.

K Nessler Reagent

   Dissolve lOOg mercuric iodide and 70g
   potassium iodide in a small volume of
   water.  Add this mixture slowly to the
   sodium hydroxide solution (J above),  then
   dilute to 1 liter.
II  EQUIPMENT PREPARATION

 A This procedure should be used if the
   apparatus has been out of service for 4
   hours or more.

   1  Add  about 50 ml of a 1:1 mixture of
      ammonia-free distilled water and
      sodium hydroxide-sodium thiosulfate
      solution to each of the micro Kjeldahl
      flasks to be used.

   2  Add  glass beads to each flask.

   3  Attach a flask to the steam distillation
      apparatus and distill about half the
      mixture.

   4  Add  1 ml of the Nessler reagent to the
      distillate to check for ammonia.

      a  If the distillate is colorless, the
         equipment is ammonia-free and the
         procedure can be repeated with the 
         next flask to be used.

      b  If the distillate is yellow, discard  it,
         distill another half of the mixture  and
         check  this distillate with 1 ml  Nessler
         reagent.  Repeat the process until the
         distillate is colorless.

                                       18-1

-------
 Determination of Kjeldahl Nitrogen
HI  DIGESTION OF SAMPLE

 A Preparation of Digestion Mixture

    1  Shake the sample.

    2  Measure 50.0 ml sample into a 100 ml
       Kjeldahl flask.

    3  Add 10 glass beads.

    4  Add 10 ml of the digestion reagent.

  B Digestion

    1  Place the flask to a properly ventilated
       Kjeldahl digestion apparatus.

    2  Turn on the heat source.

    3  Evaporate the mixture until sulfur
       trioxide (SO3) fumes are given off.
            fumes are white.  Also, the solution
       will be pale yellow. )

     4 Continue heating for an additional 30
       minutes.

     5 Turn off the heat source.

     6 Cool the residue in the flask.


 IV  STEAM DISTILLATION

  A  Preparation of the Digestion Residue

     1 Add 30 ml of ammonia-free distilled
       water to the digested residue in the
       Kjeldahl flask.

     2 Connect the flask to the ground glass
       joint of the micro steam distillation
       apparatus.

     3 Add 5 ml of 2% boric acid to a 50 ml
       Erlenmeyer receiving flask.

     4  I'osition the Erlenmeyer flask so that
       the tip of the condenser is below the
       level of the boric acid solution in the
        receiving flask.
   5  Carefully add 10 ml oJ  the flodlum hyHroxidr-
     sodium thiosull'ate solution from the
     dropping funncL.

B  Distillation

   1  Turn on the heat source.

   2  Distill at a rate of 6-10 ml/minute up to
     the 30 ml mark on the  Erlenmeyer
     receiving flask.

   3  Remove the receiving flask.

   4  Put a small beaker under the condenser
     tip to receive any additional distillate.

   5  Turn off the heat source if there are no
      more digestion residues to distill.
V COLORIMETRY FOR AMMONIA-
   NESSLERIZATION

If the ammonia content is found to be greater
than 1 mg/liter,  a titration procedure should
be used^ rather than Nesslerization.

A Preparation of Standards and Sample

   1  Label nine 50. 0 ml Nessler tubes with
      the following:  0, 0.5, 1. 2,  4, 5, 8,
      10, and S.

   2  Pipet the following volumes of ammonium
      chloride standard solution into the corre-
      spondingly labeled tubes:  0. 5 ml, 1. 0 ml,
      2.0ml, 4.0ml,  5.0ml. 8.0ml,  and
      10.0 ml.

   3  Pour the 30 ml of distillate collected
      from the sample into the Nessler tube
      marked "S".

   4  Pour ammonia-free distilled water into
      the tube labeled "0", bringing the volume
      to the 50.0 ml line.

   5  Add ammonia-free distilled water to each
      of the remaining 8 tubes, bringing the
      volume of  each to the 50. 0 ml line.
  18-2

-------
                                                         Determination of Kjeldahl Nitrogen
   6  Add 1.0 ml of Nessler Reagent to each
      of the 9 tubes.

   7  Mix the solution in the "0" tube using a
      cap or a rubber stopper on the top, then
      inverting the tube three times.

   8  Remove, rinse, and dry the cap  or
      stopper.

   9  Repeat steps 7 and 8 to mix the contents
      of the other 8 tubes.

  10  After mixing, let the tubes stand in a
      rack for 20 minutes before getting
      absorbance readings.

B Spectrophotometric Readings

   1  Turn the instrument on.

   2  Set the wavelength  at 425 nm.

   3  After the 20 minute time span, use the
      contents of the "0" tube to adjust to zero
      absorbance on the spectrophotometer.

   4  Using the contents  of the tube labeled
      0. 5, rinse, then fill an instrument cell.

   5  Place the cell  in the holder and record
      the absorbance value from the instrument.

   6  Discard the contents from the cell.

   7  Use ammonia-free distilled water to
      rinse the cell three times.

   8  Repeat steps 4 through 7 to obtain
      absorbance values  for the rest of the
      standards  and for the sample.

   9  Turn off the instrument.
VI  CALIBRATION CURVE

 A  Constructing the Curve

    1  Calculate the concentration of each
      standard by multiplying the ml of
      working standard used times 0.01 mg/ml,
      which is  the concentration  of the
      standard solution.  This was diluted to
       50. 0 ml in the Nessler tube, so the
       result is mg NH3-N/50.0 ml.  For
       example, if 0. 5 ml of standard was used,
       the  concentration is
       (0.5)(0.01) =  0.005  mg NH3-N/50.0 ml.

    2  Plot the absorbance values for the
       standards against these calculated
       concentrations,

    3  Draw the best straight line from zero
       through all the points.

  B Using the Curve

    1  To find the NHj-N concentration in the
       sample, locate its absorbance value on
       the  curve.

    2  Find the corresponding mg NH3-N/50.0 ml
       by dropping a vertical line to the
       concentration axis.

    3  Record this result.
VII FINAL CALCULATIONS

  A Use this formula to calculate Total Kjeldahl
    Nitrogen:
TKN. mg/1 =
     Where:
                           X
                             B
                 ml sample  C
     A =  mg NH,,-N/50. 0 ml from curve
     B =  ml total distillate including boric acid
     C =  ml distillate taken for Nesslerization
     ml sample = ml  of original sample taken

  B  An example calculation using the value from
     the calibration curve would be:

     TKN,  mg/1 = AX 1000    B_
                  ml sample  C
     If:
     A = 0.045
     B = 30 ml (25 ml distillate +  5 ml boric acid)
     C = 30 ml
     ml sample - 50 ml
                                                                                       18-3

-------
Determination of Kjeldahl Nitrogen
   Then:
   TKN, mg/1 =
                        20
                0.045X  J000
I?
 1
              =  0.045X20X 1
              =  0.045 X 20
              =  0.90
   TKN   0. 90 mg/1
                 This outline was prepared by A. E. Donahue,
                 Chemist, National Training Center, MPOD,
                 OWPO.  USEPA. Cincinnati. Ohio 45268.
REFERENCE

1  Methods for Chemical Analysis of Water
      and Wastes,  EPA-MDQARL.   1974.
                 Descriptors: Ammonia, Analytical Techniques,
                 Chemical Analysis, Laboratory Tests. Nitrogen
                 Nitrogen Compounds, Nutrients. Water Analysis
18-4

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                        LABORATORY PROCEDURE FOR TOTAL SOLIDS
  I    INTRODUCTION

  A   This procedure  was excerpted from methods
      for Chemical Analysis of Water and Wastes,
      1971, Environmental Protection Agency,
      Office of Water  Programs,  Analytical Quality
      Control Laboratory.

  B   The procedure is applicable to surface and
      saline waters, domestic and industrial wastes.

  C   The practical range of the determination is
      10-30000 mg/1.

  D   Nonhomogenous  materials (large floating
      particles or submerged agglomerates) should
      be excluded from the sample.  Floating grease
      and oil should be included in the sample and
      dispersed in a blender before measuring the
      aliquot.

  E   Samples should  be analyzed as soon as
      possible.
 H    EQUIPMENT

 A   Porcelain, Vycor, or platinum evaporating
      dishes,  100 ml capacity; smaller sizes may
      be used as required.

 B   Muffle furnace,  550- 50C

 C   Drying oven, 103 - 105 C

 D   Desiccator

 E   Analytical balance

 F   Steam bath or Oven at 98C
 C Store the dish in the desiccator and weigh
   just before use.

 D Weigh the dish on an analytical balance.

 E Shake the sample container vigorously.

 F Measure 100 ml of the well mixed sample
   in a graduated cylinder.  (At least 25 mg
   of residue should be obtained; less volume
   of sample may be used if the sample appears
   to be high in solids content.   If it is low in
   solids content, more sample may be added
   to the dish after drying).

 G Rapidly, but without spilling, pour the
   sample into the evaporating dish.

 H Dry the sample on a steam bath,  or at 98 C
   (to prevent boiling and splattering) in the
   oven.
 I  Dry the evaporated sample in the oven at
   103 - 105 C for at least one hour.
 J Cool the dish in the desiccator and then
   weigh it.

 K Repeat the heating at 103 - 105C, cooling
   and weighing until the weight loss is less
   than 4% of the previous weight,  or 0. 5 mg,
   whichever is less.
IV
CALCULATIONS
 mg total solids/1 = (wt dish + residue)* -
                   (wt dish)*x 1000 x 1000
                      ml of sample

 * in grams
III    PROCEDURE

 A    Heat the clean evaporating dish in a muffle
      furnace at 550- 50C for 1 hour.

 H    Cool the dish in a desiccator.
 This outline was prepared by C. R. Feldmann,
 Chemist, National Training Center,  MPOD,
 OWPO, USEPA,  Cincinnati, Ohio 45268.

 Descriptors: Analytical Techniques,  Chemical
 Analysis, Laboratory Tests, Solids,  Water
 Analysis
 PC. lab. 16a. 11.75
                                                                                        20-1

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                     I,AROHATOHY PHOCKIMiKi:  !'()]( TOTAL
                 JNICSS
I  REAGENTS

A  Buffer Solution:

   1  Dissolve 16. 9 g of NH Cl in 143 ml of
      cone. NH4OH.        4

   2  Dissolve 1. 179 g of analytical reagent
      grade disodium  ethylenediamine tetra-
      acetic acid dihydrate(Na9EDTA-2H9O)
      and 0. 644 g of MgCl  6K9O in 50 ml of
      distilled water.     z    i

   3  Add the solution from (2) to the solution
      from (1) with mixing,  and dilute to 250
      ml with distilled water.  Addition of
      small amounts of Na_EDTA-2H2O or
      MgCl-- 6H2O may be  necessary to attain
      exact equivalence.

   4  The buffer should be  stored in a plastic
      or resistant glass container tightly
      stoppered  to prevent  CO  absorption
      and NH, loss.   Discard  me buffer when
      1 or 2 ml added to the sample fails to
      produce  a pH of 10. 0 + 0.1 at the end
      point of the titration.

 B Inhibitor:

   1  Most water samples  do  not require the
      use of an inhibitor.  In the presence of
      certain interfering ions,  however,  an
      inhibitor may be needed to sharpen the
      endpolnt color change.  Several types
      of Inhibitors may be  prepared or pur-
      chased.  Sodium cyanide,  NaCN, is one
      of the  simpler inhibitors to use.

   2  Add 0. 25 g of powdered NaCN to the
      sample and adjust the pH to 9. 9-10. 1.
      Caution:  NaCN is poisonous.  Use
      Targe amounts of water  when flushing
      solutions containing NaCN down the
      drain.  Do not acidify solutions contain-
      ing NaCN; volatile, poisonous hydrogen
      cyanide, HCN,  would be liberated.

 C Indicator:

    1  Eriochrome Black-T dye  (EBT) is use-
      ful for the determination.   Other commer-
      cial grades or laboratory formulations
      of the dye  are also satisfactory.
      Prepare the indicator in dry powder
      form by grinding together 0.5 g of the
      dye and  100 g of NaCl.
D  Standard Calcium Carbonate,
   Weigh 1. 000 g of anhydrous, primary
   standard grade CaCO,, and transfer it to
   a 500 ml Erlenmeyer'rlask.  Add 1:1 HCI
   (equal volumes of cone.  HCI and water)
   dropwise and with swirling of the flask
   until the CaCO  has dissolved.  Bring
   the volume of liquid to about 200 ml with
   water,  boil a few seconds to dispel CO.,
   cool, and add a few drops of methyl rea
   indicator.  Adjust the color of the solution
   to an intermediate orange by the dropwise
   addition of  1:1 HCI or 1:4 NH OH (1 volume
   of cone.  NH OH + 4 volumes of water).
   Transfer the solution quantitatively to a
   one liter volumetric flask and  dilute to the
   mark with water. (1.0 ml = 1.0 mgCaCOJ
                                          O

E Na EDTA-2H9O (0. 01 M):
      i        
   Dissolve 3. 723 g of the dry reagent grade
   Na EDTA-2HO in distilled water and
   dilate to 1 liter.  1. 0 ml of the 0. 01 M
   solution = 1. 0 mg of CaCO,.   Check the
   concentration of this solution by titration
   against the standard calcium carbonate
   solution as described in n below.
n   STANDARDIZATION OF THE i\a_EDTA'
    2H20:

 A  Dilute 25. 0 ml of CaCO  standard to about
    50 ml with distilled water in a 125 ml
    Erlenmeyer flask against a white background.

 B  Add 1-2 ml of the buffer solution and check
    the pH to ensure that it is 9. 9 to 10. 1.

 C  Add approximately 0. 2 g of the indicator.

 D  Add the  Na EDTA^HoO slowly and with
    stirring until the color changes from a
    rose to a blue color.   If the color change
    is not sharp, repeat the determination
    using the inhibitor.  If the endpoint is  still
    not sharp, prepare a fresh supply of
    indicator.

 E  The titration should take less than 5 minutes,
    measured from the time of buffer addition.
 CH. HAR.lab. 3b. 11.75
                                                                                          21-1

-------
 Laboratory Procedure for Total hardness
    In an analysis of this type it is advant-
    ageous to carry out a preliminary, rapid
    titratton in order to determine approxi-
    mately how much titrant will be required.
    This is accomplished by adding the
    NftalpDTA-ZHoO at a fast dropwise rate
    unurehe color change is observed.
    PROCEDURE

 Repeat steps n A through n F using sample
 in place of CaCOg standard.  The amount of
 sample taken should require less than 15 ml
 of NaBtfA'aO titrant.
IV  CALCULATION

 A Standardisation of the Na2EDTA-2H2O:
REFERENCES

1  Standard Methods for the Examination
      of Water and Wastewater. 13th Edition,
      A PHA,-AWWA-WPCF (1971)
      p. 179, Method 122B.

2  ASTM Standards. Part 23,  Water;
      Atmospheric Analysis 1972
      p. 170,  Method Dl 128-67.
3  Methods for Chemical Analysis of
     Water and Wastes.  1974,
     Environmental Protection Agency,
     MDQARL, Cincinnati. OH.
    ml of CaCO3 equal to 1. 0 ml of the Na2EDTA-2H2O
    (symbol B>
                ml of CaCO,
                ml oma-EDTA'ZHgO required tor tttration

  B Total Hardness
                   ^ r*n  /1 - A x B x 1000
             as mg CaC03/1 - ml of sample
This outline was prepared by C. R. Feldmann,
Chemist. National Training Center, MPOD,
OWPO, USEPA. Cincinnati. Ohio 45268.
                                                 Descriptors: Analytical Techniques,  Calcium,
    A  ml of Na2BDTA-2H2O for tttration of sample Chemical Analysis, Hardness. Laboratory
                                                 Tests,  Magnesium, Water Analysis
  21-2

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                                          ACIDITY
1. Scope and Application
   1. 1   This method is applicable to surface waters,  sewages and industrial wastes,
         particularly mine drainage  and receiving streams, and other waters containing
         ferrous iron or other polyvalent cations in a reduced state.
   1. 2   The method covers the range from approximately 10 mg/1 acidity to approxi-
         mately 1000 mg/1 as CaCOs, usmS a *> nil sample.
2. Summary of Method
   2. 1   The pH of the sample is determined and a measured amount of standard acid is
         added, as needed, to lower the pH to 4 or less.  Hydrogen peroxide is added, the
         solution boiled for several minutes, cooled, and titrated electrometrically with
         standard alkali to pH 8.2.
3. Definitions
   3.1   This method measures the mineral acidity of a sample plus the acidity resulting
         from  oxidation and hydrolysis of polyvalent cations, including salts of iron and
         aluminum.
4. Interferences
   4.1   Suspended matter present in the sample, or precipitates formed during the
         titration may cause a sluggish electrode response.  This may be offset by allowing a
         15-20 second pause between additions of titrant or by slow dropwise addition of
         titrant as  the endpoint pH is approached.
5. Apparatus
   5. 1   pH meter,  suitable for electrometric titrations.
6. Reagents
   6. 1   Hydrogen  peroxide  (H2O2-  30% solution).
   6. 2   Standard sodium  hydroxide,  0.02 N.
   6. 3   Standard sulfuric acid, 0. 02 N.
7. Procedure
   7.1   Pipet 50 ml of the sample into a 250 ml beaker.
   7. 2   Measure the pH of the  sample.  If the pH is above 4. 0 add standard sulfuric acid
         in 5. 0 ml  increments to lower the pH to 4.0 or less.  If the initial  pH of the sample
         is less than 4. 0,  the incremental addition of sulfuric acid is not required.
   7. 3   Add 5 drops of hydrogen peroxide.
CH.ALK.lab.5. 11.75                                                                 23"1

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    7. 4  Heat the sample to boiling and continue boiling for 2 to 4 minutes.  In some
         instances, the concentration of ferrous iron in a sample is such that an additional
         amount of hydrogen peroxide and a slightly longer boiling time may be required.
    7. 5  Cool the sample to room temperature and titrate electrometrically with standard
         alkali to pH 8. 2.
 8. Calculations
    8.,
         where:
         A =  vol. of standard alkali used in titration
         B *  normality of standard alkali
         C =  volume of standard acid used to reduce pH to 4 or less
         D =  normality of standard acid
    8. 2  If it is desired to report acidity in millequivalents per liter, the reported values
         as CaCC*3 are divided by 50. as follows:
                        Acidity as meq/1 = "^ CaC3
                                              50

 9. Precision
    9. 1  On a round robin conducted by ASTM on 4 acid mine waters, including
         concentrations up to 2000 mg/1. the precision was found to be  1 10 mg/1.
10. References
    10.1 The procedure to be used for this determination can be found in:
         ASTM Standards,  Part 23. Water; Atmospheric Analysis, p 124,  D-1067, Method
         E (1973).
         Standard Methods for the Examination of Water and Wastewater,  13th Edition.
         p 370. Method 201 (Acidity and Alkalinity) (1971).
                                                  (The preceding is a copy of pp 1 and 2 from
                                                   Methods for Chemical Analysis of Water
                                                   and Wastes. EPA-MDQARL.  1974.)
                                                  Descriptors: Acids, Acidity,  Analytical
                                                  Techniques, Chemical Analysis,  Laboratory
                                                  Tests, Neutralization, Water Analysis
 23-2

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                    LABORATORY PROCEDURE FOR TOTAL ALKALINITY
I  REAGENTS

For detailed discussion of reagent preparation,
consult method reference 3.

A Carbon Dioxide - Free Distilled Water

B Standard Sodium Carbonate Solution

C Hydrochloric Acid Titrant (0. 02 N)
H   STANDARDIZATION OF THE HYDRO-
    CHLORIC ACID TITRANT

 A  Set the temperature reading on the pH
    meter dial to match the temperature of
    the buffer and sample solutions.

 B  Standardize the pH meter against a
    reference buffer solution. Check against
    a second buffer solution.
 C  Weigh accurately 0. 088 + 0. 001 g of the
    dried sodium carbonate and transfer it to
    a 500 ml conical flask.

 D  Add 50 ml of water and swirl to dissolve
    the carbonate.

 E  While stirring the solution (magnetic bar
    and sttrrer),  add the hydrochloric acid
    titrant from a 100 ml buret until a pH of
    4. 5 is attained.

 F  Calculate the normality of the hydrochloric
    acid solution as follows:
                                         III  PROCEDURE

                                           A Pipette 50 ml of the sample into a 150 ml
                                             beaker.

                                           B Titrate with the hydrochloric acid to
                                             pH4.5.

                                           C Calculation

                                             Total alkalinity as mg of CaCO3/l  =

                                                      A X N X 50000
            A =
                  0.053  X C
                                                            B


                                              A =  ml of standard HC1 titrant

                                              N =  N of standard HC1 titrant

                                              B =  ml of sample

                                              50 * equivalent weight of CaCOg

                                              1000 -  converts ml to liters


                                           REFERENCES

                                           1  Methods for Chemical Analysis of Water
                                                and Wastes,  EPA-MDQARL, Cincinnati,
                                                Ohio 45268, 1974. p.  3.

                                           2  Standard Methods for the Examination of
                                                Water and Wastewater, 13th ed.,  1971,
                                                p.370.

                                           3  Book of ASTM Standards Part 23, 1972,
                                                p. 143.
       A  =  normality of the hydrochloric acid

       B  =  g of sodium carbonate used

       C  =  ml of hydrochloric acid consumed
                                           This outline was prepared by C. R. Feldmann,
                                           Chemist,  National Training Center,  MPOD,
                                           OWPO, USEPA. Cincinnati, Ohio 45268.
                                           Descriptors: Alkalis, Alkalinity, Analytical
                                           Techniques,  Chemical Analysis,  Laboratory
0.053  = milliequivalent weight of Na2C03   Tests, Neutralization, Water Analysis
   CH.ALK.]ab.2a.ll.75
                                                                                        24-1

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