EPA-430/1-75-003
STREAM SURVEILLANCE AND MONITORING:
 FIELD AND LABORATORY PROCEDURES

  TRAINING MANUAL


  U.S. ENVIRONMENTAL PROTECTION AGENCY
  OFFICE OF WATER PROGRAMS

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                                                   EPA-430/1-75-003
                                                   March 1975
STREAM SURVEILLANCE  AND  MONITORING:
   FIELD AND  LABORATORY  PROCEDURES
      This course is designed for personnel engaged in programs
      concerned with monitoring the quality of surface waters.
      Interdisciplinary needs and responsibilities in the accum-
      ulation and interpretation of data from field and laboratory
      activities are emphasized.

      Upon  completion of the course the student will be able to
      apply suitable  methods,  techniques,  and instrumentation
      used in field sampling and measurements, and will be famil-
      iar with basic laboratory techniques and procedures in the
      chemical, biological, andbacteriological areas.  Advantages
      and limitations of equipment,  methods and techniques will
      be considered.
            ENVIRONMENTAL PROTECTION AGENCY
               Office of Water Program Operations
                   TRAINING  PROGRAM

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                                      CONTENTS



Title or Description                                                      Outline Number


   Flow Measurement                                                           1

   Water Level Recorders        '                                              2

   Tracing Natural Waters                                                      3

   Sampling in Water Quality Studies                                             4

   Sample Handling - Field through Laboratory                                   5

   Sample Preservation                                                         6

   Bacteriological Sampling in the Field                                         7

   Biological Field Methods                                                     8

   The Aquatic Environment (Part 1 through 5)                                   9

   The Identification of Aquatic Organisms                                       10

   Aquatic Organisms of Significance  in Pollution Surveys                         11

   Key to Selected Groups of Freshwater Animals                                 12

   Using Benthic Biota in Water Quality Evaluation                               13

   Automatic Instruments for Water Quality Measurements                        14

   DO - Determination by the Winkler lodometric Titratiori                        15
    and Azide Modification

   DO - Determination by Electronic Measurement                               16

   Dissolved Oxygen: Factors Affecting DO Concentration                         17
    in Water

   COD and COD/BOD Relationships                                             18

   Laboratory Procedures for Routine Level COD                                 19

   Acidity,  Alkalinity, pH and Buffers                                           20

   Operating Characteristics and Use of the pH Meter                             21
  161.3.75

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                                       Contents
Title or Description                                                      Outline Number


   Specific Conductance                                  •                      22

   Calibration and Use of a Conductivity Meter                                   23

   Chlorine Determinations and Their Interpretation                              24

   Phosphorus in the Aqueous Environment                                      25

   Chemical Tests, Observations,  and Measurements                            26
    in the Field

   Bacteriological Indicators of Water Pollution                                  27

   Examination of Water for Coliform and Fecal Streptococcus                    28
    Groups (Multiple Dilution Tube Methods)

   Examination of Water for Coliform and Fecal .Streptococcus                    29
    Groups (Membrane Filter Methods)

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                                  FLOW MEASUREMENT
 I  INTRODUCTION

 Flow measurements are among the more
 important data collected during a water
 quality survey.  Such measurements ar'e
 used to interpret data variations, calculate
 loadings, and expedite survey planning. If
 the analysis of survey data involves estima-
 tion of loads, the accurate measurement of
 discharge assumes a level of importance equal
 to that of laboratory and analytical results.

 In the following discussion, procedures for
 measurement of stream flow and waste dis-
 charge are described.  Some of these pro-
 cedures are used in long-term, very detailed
 water quality and supply studies; others are
 more suited to short-term pollution surveys.
II  PLANNING

 A Station Location

   Four factors influence location of gauging
   or flow measurement stations:

   1  Survey objectives

   2  Physical accessibility

   3  Characteristics of the stream bed

   4  Hydrologic effects

   Survey objectives represent the major
   -influence on station location; depending
   upon objectives,  gauging stations may be
   located above and/ or below confluences
   and  outfalls.

   Physical accessibility determines the
   ease and cost of installation and main-
   tenance of the station.  The characteristics
   of the stream bed may greatly influence
   the obtainable accuracy of measurement.
   For instance, rocky bottoms greatly
   reduce the accuracy of current meters.
   Sedimentation in pools behind control
   structures may influence stage-discharge
   relationships.  Hydrologic variations in
   stream flow may cause washout or bypass
   of the gauging station.  In the Southwest,
   flash floods have been known to wash out
   or bypass gauging stations by assuming
   different channels of flow.
 B  Methodology

    Choice of a specific measurement pro-
    cedure is dependent upon at least three
    considerations:

    1  The relation between obtainable and
       desired accuracy

    2  Overall cost of measurement

    3  The quantity of flow to be measured

    Ideally, discharge measurements  should
    be reported to a specific degree of accuracy;
    the gauging procedure greatly influences
    this accuracy.  The influence  of overall
    cost on the gauging program is readily
    apparent. Extensive, detailed studies are
    usually characterized by high  costs for
    automatic instrumentation and low personnel
    cost; the opposite is usually true for less
    detailed studies.  The range of flows to be
    measured (within acceptable accuracy) is,
    of course, not known prior to  the survey.
    However, experienced personnel usually
    can make reasonable estimates of expected
    flows from visual observations and other
    data, and may recommend appropriate
    gauging procedures.  In this regard,
    experienced personnel always should be
    consulted.

Ill  MEASUREMENT

 A  Streams, Rivers, and Open Channels

    1  Current Meter
       The current meter is a device for
       measuring the velocity of a flowing body .
       of water.  The stream cross section is
       divided into a number of smaller sections,
       and the average velocity in each section
       is determined.  The discharge  is then
       found by summing the products of area
       and velocity of each section.

    2  Stage-discharge relationships

       Large flows usually are measured by
       development of and reference to a stage-
       discharge curve; this procedure has long
       been used by the U. S.  Geological Survey.
       Such gauging stations are composed of a
       control structure located downstream of
       the location of measurement and some
       type of water level indicator which iden-
       tifies the height of the water surface
       above a previously determined  datum.
 IN. SG. 13a.3.74
                                       1-1

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Flow Measurement
      Location of the control structure so
      that reliable measurements of flow
      will be obtained at all river stages  is •
      particularly important.  The water level
      may be continuously recorded by an
      automatic recorder located in a wet
      well or may be indicated directly on a
      staff gauge located at the bank of the
      river.  Such stations must be calibrated
      by measurement of flow by velocity-
      area methods (current meter) at all
      expected stages of river flow.

   3  Weirs

      A weir may be  defined as  a dam or
      impediment to flow,  over  which the
      discharge conforms  to an  equation.
      The edge or top surface over which the
      liquid flows is called the weir crest.
      The sheet of liquid falling over the weir
      is called the nappe.  The difference in
      elevation between the crest and the
      liquid surface at a specified location,
      usually a point  upstream,  is  called the
      weir head. Head-discharge equations
      based on precise installation require-
      ments have been developed for each
      type of weir.   Weirs so installed are
      called standard weirs.  Equations for
      non-standard installations or unusual
      types may be derived empirically.

      Weirs are simple, reliable measure-
      ment devices and have been investigated
      extensively in controlled experiments.
      They are usually installed to obtain
      continuous or semi-continuous records
      of discharge.  Limitations of weirs
      include difficulty during installation,
      potential siltation in the weir pond,
      and a relatively high head requirement,
      0. 4 - 2.0 feet.  Frequent errors in
      weir installation include insufficient
      attention to standard installation re-
      quirements and failure to assure com-
      pletely free discharge of the  nappe.

      a  Standard suppressed rectangular
         weir

         This type of weir is essentially a dam
         placed across a channel.  The height
         of the crest is so controlled that con-
         struction of the nappe in the vertical
         direction is  fully  developed.   Since
         the ends of the weir are coincident
         with the sides of the channel lateral
         contraction is impossible.  This weir
         requires a channel of rectangular
         cross section, other special instal-
         lation conditions, and is rarely used
         in plant survey work.   It is more
   commonly used to measure the dis-
   charge of small streams.

   The standard equation for discharge
   of a suppressed rectangular weir
   (Francis equation) is:

     Q  =   3.33 LH3/2

   where

     Q  =   discharge, cfs
      L  =   length of the weir crest, feet
     H  =   weir head, feet

   The performance of this type of weir
   has been experimentally investigated
   more intensively than that  of other
   weirs.  At least six forms of the dis-
   charge equation are commonly
   employed.  The standard suppressed
   weir is  sometimes used when data
   must be unusually reliable.

b  Standard contracted rectangular weir

   The crest of this  type of weir is
   shaped like a rectangular notch.
   The sides and level edge of the crest
   are so removed from the sides and
   bottom of the channel that contraction
   of the nappe is fully developed in all
   directions.   This weir is commonly
   used in both plant surveys  and meas-
   urement of stream discharge.

   The standard equation for discharge
   of a contracted rectangular weir
   (corrected Francis equation) is
      Q  =   3.33 (L - 0. 2H)H
                            3/2
   where

      Q =
      L =

      H =
   0. 2H =
discharge, cfs
length of the level crest
 edge, feet
weir head, feet
correction for end contractions
 as proposed by Francis
c  Cipolletti weir

   The Cipolletti weir is similar to the
   contracted rectangular weir except
   that the sides of the weir notch are
   inclined outward at a slope of 1 .
   horizontal to 4 vertical.  Discharge
   through a Cipolletti weir occurs as
   though end contractions were absent
   and the standard equation does not
   include a  corresponding factor for
   correction.
  1-2

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                                                                     Flow Measurement
Figure 1.  STANDARD CONTRACTED
      RECTANGULAR WEIR
     The standard equation for discharge
     through a Cipolletti weir is

       Q  = 3.367 LH3/2
     where

       Q =

       L =

       H =
discharge, cfs

length of the level crest edge, feet

weir head, feet
       The discharge of a Cipolletti
       weir exceeds that of a suppressed
       rectangular weir of equal crest
       length by approximately 1 percent.

    d  Triangular weirs

       The crest of a triangular weir  is
       shaped like  a V-notch with sides
       equally inclined from the vertical.
       The central angle of the notch is
       normally 60 or 90 degrees.   Since
       the triangular weir develops more
       head at a given discharge than  does
       a rectangular shape, it is especially
       useful for measurement of small
       or varying flow.  It is preferred for
       discharges less than 1 cfs,  is as
       accurate as other shapes up to 10
       cfs, and  is commonly used in plant
       surveys.
                                                        The standard equation for discharge
                                                        of a 90° triangular weir (Cone
                                                        formula) is

                                                           Q  =  2.49H2'48

                                                        where

                                                           Q  =  discharge, cfs
                                                           H  =  weir head, feet
           Crest height and head
are measured to and from the point
of the notch, respectively.

Accuracy and installation
requirements

Quotations of weir accuracy express
the difference in performance between
two purportedly identical weirs and
do not include the effects of random
error in measurement of head.  Weirs
installed according to the following
specifications should measure dis-
charge withini 5% of the values
observed when the previously cited
standard equations were developed.

1) The upstream face of the bulkhead
   and/or weir plate shall be smooth
   and in a vertical plane perpendicular
   to the axis of the channel.

2) The crest edge shall be level,  shall
   have a square upstream corner,
   and shall not exceed 0. 08 in (2 mm)
   in thickness. If the weir plate is
   thicker than the prescribed crest
   thickness the downstream corner
   of the crest shall be relieved by a
      champfer.
                                              45°
                                            3) The pressure under the nappe
                                              shall be atmospheric.  The maxi-
                                              mum water surface in the down-
                                              stream channel shall be at least
                                              0. 2 ft. below the weir crest.
                                              Vents shall be provided at the
                                              ends of standard  suppressed weirs
                                              to admit air to the space beneath
                                              the nappe.
                                                                                      1-3

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Flow Measurement
        4)  The approach channel shall be
           straight arid of uniform cross
           section for a distance above the
           weir of 15 to 20 times the maximum
           head, or shall be so baffled that a
           normal distribution of velocities
           exists in the flow approaching the
           crest and the water surface at the
           point of head measurement  is free
           of disturbances.   The cross-
           sectional area of the  approach
           channel shall be  at least 6 times
           the maximum area of the nappe at
           the crest.

        5)  The height of the crest above  the
           bottom of the approach channel
           shall be  at least  twice, and
           preferably 3 times, the maximum
           head and not less than 1  foot.
           For the standard suppressed weir
           the crest height shall be 5 times
           the maximum head.  The height of
           triangular weirs  shall be measured
           from the channel bottom to  the
           point of the notch.

        6)  There shall be a  clearance  of at
           least 3 times the maximum head
           between the sides of the  channel
           and the intersection of the maximum
           water surface with the sides of the
           weir notch.

        7)  For standard rectangular suppressed,
           rectangular contracted,  and
           Cipolletti weirs the maximum head
           shall not exceed  1/3 the  length of
           the level crest edge.

        8)  The head on the weir shall be taken
           as the difference in elevation
           between the crest and the water
           surface at a point upstream a
           distance of 4 to 10 times the
           maximum head or a minimum of
           6 feet.
     9)  The head used to compute dis-
        charge shall be the mean of at
        least 10 separate measurements
        taken at equal intervals. The
        head range of the measuring
        device  shall be  0. 2 - 1.5 feet.

  The capacities of weirs which conform
  to these specifications are indicated
  in Table 1.

4 Parshall flume

  The Parshall flume is an open constricted
  channel in which the rate of flow  is
  related to the upstream head or to the
  difference between upstream and down-
  stream heads. It consists of an
  entrance section with converging
  vertical walls and level floor, a throat
  section with parallel walls and floor
  declining downstream, and an exit
  section with diverging walls and floor
  inclining downstream. Plan and
  sectional views are shown in Figure 2.
  Advantages of the Parshall flume include
  a low head requirement,  dependable
  accuracy, large capacity range,  and
  self cleaning capability.  Its primary
  disadvantage is the high  cost of
  fabrication; this cost  may be avoided
  by use of a prefabricated flume.  Use
  of prefabricated flumes during plant
  surveys is becoming increasingly
  popular.

  a  Standard equations

     The dimensions of Parshall flumes
     are specified to insure agreement
     with standard equations.  Table of
     dimensions are available from
     several sources  •  .  For flumes of
     6 inch to 8 foot throat  width the
     following standard  equations have
     been developed.

     1)  6 inch throat width
                        1.58
                                                             Q
               2.06  H
                                                        2) 9 inch throat width
                                                                            i
                                                             Q =  3.07 H    '

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                                                              Flow Measurement
                          TABLE 1 DISCHARGE OF STANDARD WEIRS
Crest Length
(Feet)
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5 '

5.0
6.0
7.0
8.0
9.0
10.0
Contracted Rectangular*
Weir
(discharge-cfs)
Max. Min.
.590 .286
1.65 .435
3.34 .584
5.87 .732
9.32 .881
13.8 1.03
19.1 1.18
25.6 1.33

28.8 1.48
34.9 1.78
41.0 2.07
47.1 2.37
53.2 2.67
59.3 2.97
Suppressed Rectangular*
Weir
(discharge-cfs)
Max. Min.
.631 .298
1.77 .447
3.65 .596
6.30 .744
10.0 .893
14.8 1.04
20.4 1.19
27.5 1.34

30.6 1.49
36.7 1.79
42.8 2.08
48.9 2.38
55.0 2.68
61.1 2.98
Cipolletti*
Weir
(discharge-cfs)
Max. Min.
.638 .301
1.79 .452
3.69 .602
6.37 .753
10.1 .903
15.0 1.05
20.6 1.20
27.8 1.35

30.9 1.51
37. .1 ' 1.81
43.3 2.11
49.5 2.41
55.7 2.71
62.0 3.01
90° Triangular*
Weir
(discharge-cfs)
Max. Min.








6.55 .046






' H- 0.2 ft, H- 1.5 ft, H- 1/3 L
                    Converging
                    1   section
Diverging
  section



Sp^S'^o^^S&g^


FLOW
&Z^-

H~~ —
a


^-y-
~^*!=*'-
^^^5^


*


,_,,-



,. Water surface, s


                                 SECTION

                    FIGURE 2   PARSHALL FLUME
                                                                              1-5

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Flow Measurement
        3) 1 to 8 foot throat width

                        1 522W0'026
             Q  » 4WH  1-ss"w
                      a
        where
             Q  * free-flow discharge,
                  defined as that condition
                  which exists when the
                  elevation of the down-
                  stream water surface
                  above the crest, H,, does
                  not exceed a prescribed
                  percentage of the upstreanl
                  depth above the crest, H .
                  The prescribed percentage
                  of submergence is 60
                  percent for 6 and 9 inch
                  flumes and 70 percent for
                  1 to 8 foot flumes

             W  - throat width,  feet
             H  = upstream head above the
                  flume crest
     b  Mead loss
        The head required by a Parshall
        flume is greater than (H  - IL )
        because Ha is measured at a point
        in the coriverging section where the
water surface has already begun
to decline.  Table 2 indicates the
total head requirements of standard
Parshall flumes.  These losses
should be added to the normal
channel depth to determine the
elevation of the water surface at
the flume entrance.  No head
losses are indicated for discharge-
throat width combinations for which
H& is less than 0.2 ft. or greater
than 2/3 the sidewall depth in the
converging section.

Accuracy and installation require-
ments

A Parshall flume  will measure
discharge within ± 5% of the
standard value if the following
conditions are observed.

1) The dimensions of the flume
   shall conform to standard
   specifications.
                            *

2) The downstream head,  H. , shall
   not exceed the  recommended
   percentage of the upstream head,
   H  .
                  TABLE 2  HEAD LOSS IN STANDARD PARSHALL FLUMES
                                   UNDEft FREE DISCHARGE
Discharge
(cfs)
0.6
1.0
2,5
5.0
10.0
30.0
50.0
Head Loss, Feet, in Flume of Indicated Width
1 foot
.08
.14
.26
.42
.70


2 feet

.09
.16
.27
.45


3 feet

.06
.12
.20
.34
.70

4 feet


.10
.16
.27
.56

5 feet


.08
.13
.22
.47
.68
6 feet


.07
.12
.19
.40
.57
7 feet


.06
.10
. 17
.35
.49
8 feet


.05
.09
.15
.30
.41
    H   > 0.2,    H  <  2.0
     a  —          a -
 1-6

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                                                                      Flow Measurement
     3) The upstream head shall be
        measured in a stilling well
        connected to the flume by a pipe
        approximately 1-1/2 inches in
        diameter.

     4) The flume shall be installed in a
        straight channel with the centerline
        of the flume parallel to the direction
        of flow.

     5) The flume shall be so chosen,
        installed,  or baffled that a normal
        distribution of velocities exists at
        the flume entrance.

5 Tracer materials

  Techniques,  materials,  and instruments
  are presently being refined to permit
  accurate measurement of instantaneous
  or steady flow with several tracer
  materials.  Measurements are made by
  one of two methods:
     tracer material during its passage past
     the sampling point, and  At  = the total
     time of the sampling period.
     Disadvantages of tracer methods include
      incomplete mixing, natural adsorption
     and interference, and high equipment
     costs.

   6 Floats

     Floats may be used to estimate the time
     of travel between two points a known
     distance apart.  The velocity so obtained
     may be multiplied by 0. 85 to give the
     average velocity in the vertical.
     Knowing the mean velocity and the area
     of the flowing stream, the discharge
     may be estimated.  Floats should be
     employed only when other methods are
     impractical.

B  Pipes and Conduits

   1 Weirs and Parshall flumes
  a  Continuous addition of tracer

  b  Slug injection

  With the first method, tracer is injected
  into a  stream at a continuous and uniform
  rate; with the second a single dose of
  tracer material is added.  Both methods
  depend on good transverse mixing and
  uniform dispersion throughout a stream.
  The concentration of tracer material is
  measured downstream from  the point
  of addition.  When continuous addition
  is employed,  flow rates are  calculated
  from the equation:

            q  . C = (Q + q) c

  in which q = rate of tracer addition to
  the stream,  at concentration,  C, Q = the
  stream flow rate, and c = the resulting
  concentration of the stream flow com-
  bined with the tracer.  For the slug
  injection method
            Q '
                 c  At
   in which Q = the stream discharge,
   S = the quantity of tracer added,  c =
   the weighted average concentration of
     Weirs and Parshall flumes are commonly
     installed in manholes and junction boxes '
     and at outfalls to measure flow in pipes.
     All conditions required for measurement
     of open channel flow must be observed.

   2 Tracer materials

     These methods are popular for
     measurement of pipe flow because
     they do not require installation of
     equipment or modification of the flow.
     These are especially convenient for
     measurement of exfiltration and
     infiltration.

   3 Depth-slope

     If the depth of the flowing stream and
     the slope of the sewer invert are known,
     the discharge may be computed by
     means of any one of several formulas.

     a  Manning formula
             1  4RR      9 /<* 1/9
        Q  = ii^££  A RZ/V/-i
               n

     where

        Q = discharge, cfs
                                                                                   1-7

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Flow Measurement
        n  =  roughness coefficient

        A  =  area of flow,  sq. ft.

        R  =  hydraulic radius, ft.

           =  area divided by wetted perimeter,  ft.

        S  =  slope,  ft. per ft.

      b Chezy formula

        Q  «

      where

        Q   ==  discharge,  cfs

        C   s  friction coefficient

        A   ~  area of flow,  sq. ft.

        R   =  hydraulic radius,  ft.

            =  area divided by wetted perimeter, ft.

        S   =  slope,  ft. per ft.

   4  Open end pipe  flow

      The  following methods can be employed
      when other more precise means are not
      practical.  They can be employed,
      however, only when there is  free  dis-
      charge  to the air.

      a Coordinate method
        (Figures 3, 4,  and 5)

        Discharge may be computed by the
        following formula:
         ._  .     .
         Q  (gpm)
                      1800  AX
      where
         A  = cross sectional area of liquid
              in the pipe (sq.  ft. )

         X  = distance between the end of the
              pipe and the vertical gauge in
              ft. ,  measured parallel to the
              pipe.
       Y  =  vertical distance from water
             surface at the end of the pipe
             to the intersection of the water
             surface with the vertical gauge,
             in ft.
                          MJmUblc not so tl»t
                          X ail* Is parsMcl to sevnr
                          tad T sils Is vertical-
b = ( fro» tattoo of pipe to nrfui of talllnf liquid)
   OPEN-PIPE FLOW MEASUREMENT - THIS DEVICE, ADJUSTED TO THE
   SLOPE OF A SEWER AND CALIBRATED, CAN THEN BE CLAMPED TO
   THE SEWER OUTFALL.
                  Figure 3
 OPEN-PIPE FLOW MEASUREMENT REQUIRES TWO DIMENSIONS THAT
 LOCATE THE SURFACE OF STREAM AFTER IT LEAVES THE PIPE.
                                                                        Figure 4
   1-8

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                                                                       Flow Measurement
                 V = Velocity - fps
                 A • Cross-Motional    --
 DlKharce IB CPU
  • «SO AV
                X ((I) to c«nlvr of sire™

   HOW TO MEASURE VELOCITY AND DISCHARGE FROM A PIPE.
                 Figure 5

      b California pipe flow method
        (Figure  6)

        This method may be used only for
        horizontal pipes having free dis-
        charge.  If the pipe is not horizontal,
        a connection must be made to one
        that is.  The horizontal length must
        be not less than 6 times the diameter
        of the  pipe.  Discharge may be com-
        puted by the following formula:

        Q (gpm)  =  T X W

        where T = 3,900 (1  - ^)  '

        w  = d2'48

        where a and d are measured in feet
                     MEASUREMENTS NEEDED FCC
                     CALIFORNIA PIPE FLOW METHOD
Inclined pipes should be connected to
e. horizontal lenitb of pipe bj hose.
                Figure 6
C  Head Measuring Devices

   Several of the above gauging methods re-
   quire the measurement of water level in
   order that discharge may be determined..
   Any device used for this purpose must be
   referenced to some zero elevation.  For;-
   example, the zero elevation for weir
   measurements is the elevation of the weir
   crest.  The choice of method is dependent
   upon the degree  of accuracy and the type
   of record desired.
   1  Hook gauge

      The hook gauge measures water eleva-
      tion from a fixed point.  The hook is
      dropped below the water surface and
      then raised until  the point of the hook,
      just breaks the surface. This method
      probably will give the most precise
      results when properly applied.

   2  Staff gauge

      The staff gauge is merely a graduated
      scale placed in the water so that eleva-
      tion may be read directly.

   3  Plumb line

      This method involves measurement of
      the distance from a fixed reference
      point to the water surface, by dropping
      a plumb line until it just touches the
      water surface.

   4  Water level recorder

      This instrument is used when a continu-
      ous record of water level is desired.  A
      float and counterweight are connected
      by a steel tape which passes over a
      pulley.  The float should be placed irt
      a stilling well.  A change in water level
      causes the pulley to rotate which, through.
      a gearing system,  moves a pen. The pen,
      traces water level on a chart which is
      attached to a drum that is rotated by a
      clock mechanism.  When properly in-
      stalled and maintained, the water level
      recorder will provide an accurate,
      continuous record.
                                                                                      1-9

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Flow Measurement
A CKNOWLEDGMENT:

Certain portions of this outline contains
training material from prior outlines by
P.E.  Langdon, A.E. Becher, and P.P.
Atkins, Jr.

REFERENCES

1  Planning and Making Industrial Waste
     Surveys  - Ohio River Valley Water
     Sanitation Commission.

2  Stream Gauging Procedure. U. S.  Geological
     Survey.  Water Supply Paper 888. (1943)

3  King, H.W. Handbook of Hydraulics.
     4th Edition, McGraw-Hill.  (1954)
   Water Measurement Manual.  United
      States Department of the Interior
      Bureau of Reclamation.  (1967)
This outline was prepared by F.P. Nixon,
formerly Acting Regional Training Officer,
Northeast Regional Training Center,  EPA,
WPO, Edison Water Quality Research
Laboratory, Edison,  NJ  08817
Descriptors:  Chezy Equation,  Discharge
Measurement, Flow, Flow Measurement,
Flow Rates, Flumes,  Mannings Equation,
Open Channel Flow, Pipe Flow, Streamflow,
Venturi Flumes, Water Flow, Water Level
Recorders, Weirs
   1-10

-------
                                WATER  LEVEL RECORDERS
A    Commonly used instruments produce
     a  graphic  record of the  rise and fall
     of a water surface with  respect to
     time.

B    Advantages over non-continuous  staff
     gage  readings;

     1   Continuous record provides most
        accurate means of determining
        daily average gage  height and flow.

     2   Maximum and minimum stages are
        recorded, and time of  occurrence
        can be noted.

     3   Record can  be obtained when ob-
        server not there,  ot not available.

C    Principal elements

     1   Time  element;  clock mechanism
        activated by spring, weight,  or
        electric motor.

     2   Gage height element activated by
        a  float, cable, or tape, and  a
        counterwieght.

        Gear reduction mechanism  is pro-
        vided  in  the height element so that
        the gage-height  reading on  the
        recorder is changed in proportion
        to  the  rise and  fall of  the water
        surface.

D    Common  types  of  recorders

     a   Drum Recorders

        1   Horizontal drum;  clock positions
           pen along drum axis and gage
           height element fotates drum.

        2   Vertical  drum:  clock mechanism
           rotates drum and  water stage a
           function of displacement of pen
           along drum axis.
    b   Strip- chart

        1  Totalizer  -  records flow rates
           with respect to time and auto-
           matically  totalizes  volume.   Strip-
           chart provides graphic record.

E   Installation of recorders;

    1   Gage tape or cable should be long
        enough to accomodate  the  full range
        of  water surface changes.

    2   Recorder  should be installed over
        a stilling  well  and shielded from
        the weather.

    3   Staff gage needed near stilling well
        to  check the  recorder.

F  Stilling Well,  Float Well

    1   Prime  purpose to prevent oscillations
        of  the float caused by surging water
        or wave action.
   •
    2   Must be anchored to prevent movements
        that would introduce oscillations within
        the well.

    3   Outside surges can be  damped out by
        restricting area of the water inlet to
        about  1/1000 of the inside horizontal
        cross-sectional area of the well.

    4   Can be made of galvanized iron  culvert
        pipe, sewer pipe, tongue and groove
        creosoted lumber, stove pipe, or other
        suitable material.

    5   Sometimes built outside the  channel  in
        which the liquid is flowing,  and
        connected to  it by an opening or pipe.
        In  sewage applications, provision must
        be  made for  frequent cleaning or, water
        levels in well will not be representative.

        Inlet to  float well must be  checked fre-
        quently  for clogging by sludge and
        sediment.
 WP.SUR. sg. 7. 3.75
                                                                                     2-1

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Water Level Recorders
G   Errors in Float-Operated Devices

    1  Float  Lag.   If a float performs  any
       mechanical work,  i.e., turns gears,
       moves an index hand or a recorder
       pen,  etc.,  there is always  a lag of
       the index behind the true water level.

    2  Line  Shift.   With every  change  of
       stage,  a portion of the float line
       passes from one side  of the float
       pulley to the  other.   This change of
       weight changes the depth  of flotation
       of the float, hence the stylus deviates
       from the true water height  by a small
       amount, depending on  the change in
       stage  since the last .correct setting,
       and the weight of the line used  be-
       tween the float and counterpoise.

    3  Submergence  of Counterpoise.   When
       the counterpoise and any  portion of
       the line becomes  submerged, the
       pull on the  float is reduced and  its
       depth  of flotation  is increased.

    4  Corrections for errors due  to the
       above are  so small as to be unim-
       portant if instruments are correctly
       made  and properly cared for.   All
       those  errors vary  inversely as  the
       square of the diameter of the float,
       and directly as the weight of line
       and counterpoise.   To minimize them,
       therefore,  use a sufficiently large
       float  with light lines and  counter-
       weights, and keep  the instrument
       properly oiled and free from dust
       to render the force required to move
       the index as low as possible.

    5  Errors from humidity effects on
       charts.  All paper- is  affected by
       humidity changes.   It  expands with
       increasing humidity and contracts as
       humidity decreases.   The result is
       to introduce errors in the graphs
       made  by recording instruments.
       Under ordinary conditions those
       errors are  quite negligible.   In
       extreme cases,  however,  an ex-
       pansion from  excessive humidity of
       as  much as 2% has been  noted.
       The heights, however, may  not  be
      in error by that amount.   The  actual
      error  depends on the height itself and
      the position of the neutral axis of
      expansion.

      By far the  most practicable procedure
      to prevent paper expansion is to  reduce
      the effect of humidity to a minimum,
      by  keeping  excessively humid air from
      the recorder.   The use of a desiccator
      such as silica gel within the recorder
      case is quite effective in keeping the
      interior atmosphere  reasonably dry.
      This turns  pink when its capacity is
      exhausted.   Heating  in an  oven will
      drive  off the moisture and restore it
      to its  normally blue color.

   6  The "Human Equation" is the greatest
      possible source of error,  with  the
      following among the  more  conspicuous
      reasons for erroneous or incomplete
      records:

      a   Incorrect gage  reading

      b   Inaccurate setting of pen
      c   Failure  to wind clock
      d   Failure  to start clock after  winding

      e   Failure  to put stylus  on paper
      f   Wrongly attaching float,  so that pen
          moves up when it  should go down
      g   Failure  to note gage reading and
          time on  record sheet

      h   Failure  to see  that inlet to well is
          open

      i   Failure  to oil bearings  occasionally

      j   Use of gummy  oil

REFERENCES:

   1  Hydrographic Data Book.  Leupold  &
          Stevens,  Inc. Portland,  Oregon,
         8th  ed.

   2  Water Measurement Manual.   U. S. Dept.
         of the  Interior,  Bureau  of Reclama-
         tion,  Denver, CO.  2nd  ed.
 2-2

-------
                                                          Water Level Recorders
3  BUchanari,  T.J. &  Somers, W.P.        This outline was prepared by  Charles E.
      Stage Measurement  at Gaging          Sponagle, Sanitary Engineer, National
      Stations.   Book 3,  Chapter A7.        Training Center,  WPO,  EPA, Cincinnati
      Techniques of Water Resources        OH   45268
      Investigations of the U. S\  Geolog-
      ical Survey.  U. S.  Government
      Printing Office,  Washington,  0. Ci     Descriptors;  Flow Measurement, Open
      1968.                                 Channel Flow, Str'eamflow,  Water Level
                                           Recorders
                                                                              2-3

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                               TRACING NATURAL WATERS  •
 I  INTRODUCTION

 The validity of water pollution studies is de-
 pendent upon an ability to describe the actual
 time and  space situation of a pollutant as it
 blends with the natural receiving water.  This
 understanding is important in order to predict
 the subsequent effects of the pollutant on the
 receiving water and subsequent users or to
 detect the sources of pollution.

 The nature  of this blending is dependent upon
 the physical and  chemical properties of both
 the pollutant and the receiving water as well
 as the mixing and flow characteristics of the
 receiving channel, basin, or aquifer. Tracing
 is an attempt to approximate the actual motion
 and  mixing  of this blend as it moves through
 the channel, basin or aquifer.

 Although  there has been scant  reference to
 tra'cers in texts there has been rapidly in-
 creasing  number of technical articles de-
 scribing their use in recent years.  A com-
 prehensive  compilation of early radioactive
 tracer studies may be found in the report of
 the "Time of Flow Studies,  Ottawa River,
 Lima,  Ohio"* ' one of the early attempts to
 compare  various tracer techniques under
 similar field conditions.  Several reports
 (see Bibliography) have discussed the use Of
 dyes during the early 1960's.

 More refined and extensive tracer investiga-
 tions may be expected as experience is gained
 and  as more complex studies arise.  A water
 quality agency should develop competence in
 at least one tracer technique and would be
 wise to be capable  in some  others as well.
II   PURPOSES

 Tracer applications are still in a highly
 developmental stage.  One finds many words
 or phrases to suggest similar field findings.
In broad terms these measurements are con-
cerned with indications of mass movement,
blend mixing,  and flow direction.  Basically,
tracers are used to determine:

A  Flow Rates

   1  In freshwater channels or lakes such
      terms as passage time, time of travel,
      time of flow, flow quantity or flow
      volume are used.

   2  In estuaries such terms as  rate of re-
      newal, flushing rate, drift velocity,
      mass transfer or net (tidal) drift are
      used. The tidal fluctuation imposes a
      need for consideration of flow reversal
      effects.

   3  In subsurface waters such terms as
      recharge rate, flow rate and residence
      time are used.  Related factors such
      as basin capacity, porosity and per-
      meability are determined.

B  Flow Direction

   1  In estuaries

      a  Direction of flow relative to tidal
        phases  which do not necessarily
        correspond to slack conditions.

      b  Obscure current circulation as in'-
        fluenced by the interlated factbrs of
        tides,  winds,  Coriolis forces,
        topography and density gradients.

   2  In ground water

      a  Aquifer may flow contrary to initial
        superficial impression.

      b  Hydrostatic conditions may change
        and reverse normal flow direction.
 WP.SUR.tr.lb.3.74
                                    3-1

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  Tracing Natural Waters
  C  Mixing Patterns

     1   Short circuit effects

        a  Eddying - primarily the result of
          surface channel and basin
          configuration.

        b  Stratification - primarily due to
          temperature and density differences.

        c  Inter-connections  - between aquifers
          in ground water as well as solution
          channels and open fractures.

     2   Distribution phenomena
                   i
      .  a  Dispersion of colloidal,  soluble and
          suspended substances

        b  Diffusion of temperatures or gaseous
          substances
III  TYPES OF STUDIES

  Tracers are used in many situations,
  including:

  A Treatment Plant Units

  B Closed  Conduits

  C Open Channels

  D Large Water Bodies

  E Hydraulic Models

  F Subsurface Aquifers

  G Subsurface Basins


IV  TYPES  OF TRACERS

  Materials  used for tracers include:

  A Floats

    1  Surface (wooden and plastic devices as
       well  as fruit). Influenced by wind
       action and debris.
   2  Sub-surface "drogues". Apparent di-
      rection must be carefully evaluated.

B  Salts

   1  Common salt - hard to detect when less
      than 1 mg/1.

   2  Brackish and freshwater mixing in
      estuaries and coastal aquifers.

   3  Ammonium chloride.  (See Table 1)

C  Dyes, such as:

   1  Rhodamine series - See section VI

   2  Pontacyl Brilliant Pink B - most stable
      of fluorescent dyes.  Rather expensive
      dye.

   3  Fluorescein - very inexpensive.  Fluo-
      resces very near natural stream back-
      ground level.

   4  Uranine -  fluoresces near stream
      background level.

D  Radioactive substances, such as:  (see
   Table 2) AEC must  approve all experi-
   ments.  AEC has published tables on
   permissible concentrations in unrestricted
   areas.(13'

   1  Rubidium-86

   2  Iodine-131

   3  Tritium (H3) gives best overall perfor-
      mance in subsurface' ' tracing.

   4  Krypton-85 - used recently in gas
      transfer measurement  in laboratory
      and stream studies.'12'

E  Waste return characteristics;  significant
   built-in factors such as:

   1  Silt - understandably dependent upon
      velocity and obstructions.

   2  Foam - ABS will foam  at levels as low
      as  1 - 5 mg/1 depending on hardness.
   3-2

-------
                                                                    Tracing Natural Waters
              Table 1.  SUBSURFACE NONRADIOISOTOPE FLUID TRACERS
   Tracer
                  Form
Test conditions
                                                                          Remarks
Chloride      NaCl
Dextrose      Sugar
Fluorescein
  Dye

Chloride      NaCl
Dextrose      Sugar
Fluorescein
  Dye

Dextrose      Sugar
Ammonia     Chloride
Fluorescein
  Dye
Boron        I^BO,
                              Lab: Columns of sand and
                              sandy loam.
                              Field: 4 feet thick aquifer of
                              sand and gravel, 90 feet below
                              surface.
                              Field: Added in Cone, of 10,000
                              ppm, 500 ppm, 50 ppm, and 200
                              ppm respectively into a 4 foot
                              thick sand horizon at 2,100 feet
                              below surface in  McKean
                              County,  Pennsylvania.
Boron
             Na2B4Cy 10H2O Field: Injection into input wells.
                              Dilution factor ~5.
             H3B°3
Thiocyanate  NH4SCN
             Alkali-Metal
              SCN

Rhodamine B
                              Field: Water flooding.
                              Field: Karst topography.
Oxygen-18
Deuterium
             H2O
             HDD
                                                              Chloride most rapid.  Fluores-
                                                              cein far from satisfactory.
                                                              Fluorescein considered best of
                                                              dyes.  Chloride in the large cone.
                                                              required caused density  currents.
                        Adsorption observed: dextrose -
                        little or none, boron - slight
                        (cone,  dropped 30%) ammonia -
                        moderate, fluorescein - strong
                        (moved at rate about 1/10 that of
                        flood water).  Greenburg*33'found
                        that borax in  Hanford fine sandy
                        loam penetrated only 2 feet.

                        Limit of detection is reached
                        after 103 dilution.

                        Dilution possible 10 .
                        Dye not detected after 6 X 10
                        dilution.

                        Variations resulting from sea-
                        sonal fluctuations are useful to
                        study climate. (Also useful in
                        meteorology and glaciology).
                       Reproduced with permission of Isotopes, Inc.
                                                                                 3-3

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 Tracing Natural Waters
         Table 2.  SUBSURFACE RADIOISOTOPE FLUID TRACERS - FIELD TESTS
                                                                                (6)
Tracer  Half-life
             Form
                    Test conditions
                                          Remarks
  H
12. 5 y.   HTO
              Karst groundwater
                               Useful results up to 30 km
                               distance.
                                  Chalk River soil, undistur-
                                  bed,  but penetrated with
                                  driven piezometers.
                                                      Fluorescein dye traveled only
                                                      about 3/4 as fast as tritium.
  ,35
  ,131
  ,131
  Co
    60
  Br
    82
  Rb

   131
    86
  I
   32
          89. d
          3d. .
8 a.
5.2y.
36 h.
19 d.
8 d.
14 d.
          Iodine,
          carrier free
KI
EDTA - (Co)
   and
K  Co(CN)
 6       b
NH Br
RbCI
Nal
Na2HP04
                        Glaciofluvial sand and gravel,
                        various till soils,  and fis-
                        sures and channels.

                        Chalk River soil at waste
                        disposal site.
 4 feet thick aquifer of sand
 and gravel, 90 feet below
 surface.

 Tracer compounds used with
 carriers KI, EDTA-(Na) and
 KgCo(CN)g. Limestone aqui-
 fer consisted of some argil-
 laceous material and marly
 dolomite. Single-well pulse
 technique.

 Aquifers in alluvium and per-
 meable strata in calcareous
 marl series.

 Groundwater direction at re-
 servoir site in Egypt.

 Single pulse tracing at dam
site for Jesenitz Reservoir
on Odrava River in
Czechoslovakia.
                                                                Chromium complex traveled
                                                                as fast as tritium.
                                            Lags behind tritium indicating
                                            some interaction with the soil.
                                            In soil containing appreciable
                                            amounts of gypsum there would .
                                            • be exchange and loss of S  .

                                            Should be used with several ppm
                                            stable  iodine carrier.
Co   complex unsuitable in this
aquifer.  K3Co60(CN) appeared
to be most suitable of tracers
tested in limestone aquifer.
   Q O
Br  satisfactory under.these
conditions.
Tracer detectable 7 miles from
injection after 5 d.

Detected very high subterranean
water velocity necessitating
sealing of porous layer.
I101
82
Br24
Na
8 d.
36 h.
15 h.
Nal
NaBr
NaCI
Single pulse tracing at dam Determined direction and velo-
site on arges River in city of flow of subsurface water.
Roumania.
                        Reproduced with permission of Isotopes, Inc.

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                                                                    Tracing Natural Waters
    3  Acid - severe effects upon total stream
      ecology.

    4  Temperature - infra-red filming
      techniques reveal 1°C temperature
      differences.

    5  Lignins - "Orzan" is a commercial
      product based upon these non-degrading
      materials.

    6  dyes - prevalent in textile wastes.

 F  Biota, various techniques possible with:

    1  Mammals

    2  Fish

    3  Shellfish


V   DESIRABLE CHARACTERISTICS

 Ideally a tracer should be:

 A  Biologically innocuous to human and
    aquatic life, with special reference to

    1  Acute toxicity

    2  Long-term toxicity

    3  Carcinogenic effects

    4  Genetic effects

 B  Stable or persistant despite the effects of

    1  Stream chemical constituents

    2  Bacteria

    3  Sunlight

    4  Adsorption

    5  Temperature

    6  Wind actiori

    7  Inherent decay
     8  Channel obstructions

     9  Stratification

  C Readily detectable either

     1  Visually and/or

     2  Instrumentally despite dilution or
        background effects;

  D Representative:   coincide with the real
     waste and stream blend under study:  this
     involves miscibility and  specific gravity
     characteristics similar to the blend.

  E Economic: this involves careful evaluation
     of the costs for

     1  Materials

     2  Material preparation, and release -
        ease of handling

     3  Detection equipment

     4  Detection technique and recording -
        convenience in operation

     5  Stream deterioration

  F Esthetically agreeable: inoffensive to:

     1  Taste

     2  Sight - conflict with easy detection
VI  ELEMENTS OF A TRACER STUDY -
    USING RHODAMINE DYES (Currently
    in widespread use for surface studies.)

 A  Rhodamine Dyes

    1  Available frc-m American Cyanamid,
       DuPont, Allied Chemical and General
       Aniline.

    2  Price $4. 95 per pbund except
       Rhodamine  WT which is $10.00 per
       pound.
                                                                                    ,  3-5

-------
Trafcihg Natural Waters
   3  Specific gravity of 0.99 to 1. 12 depending
      on the proportions of Alcohol,  Ethylen-e
      Glycol, Acetic Acid, and Water used as
      solvents in preparing the solution.
          f
   4  Solutions normally 15%, 20%,  30%, or
      40% dye by weight.

   5  pH 1.0 to 9.0.

   6  Peak wavelength of adsorption at  525-556^
      and of fluorescence at 548-585^ are not
      normally found in natural waters.

   7  Detectable to 0'. 5 ng/1 with continuously
      sampling and recording equipment.

   8  Visibly red to approximately 1. 0 mg/1.

   9  Rapid dispersion when  dropped in water.

  10  Subject to some destruction by light, but
      much less than experienced with
      fluorescein.

  11  Destroyed by agents such as hypochlorite.
      Rhodamine WT is more resistant.

  12  Essentially non-toxic.

B  Measuring Equipment

   1  Available from Turner Associates,
      Beckman Instruments,  Inc., American
      Instrument Co.,  Inc.
so two or more runs with temperatures  '_••'•
covering the expected range of the study .  -.
water are desirable.  Also it is advisable  ,»';•
to make a calibration with the study water..':'
This will check for background and-any
difference in fluorescent characteristics..  ..'
A few helpful hints are:             '

1  Pumping a known concentration sample,'.
   from a plastic bag immersed in a • • .  ;  '>"'
   constant temperature water bath,
   through the meter is a convenient •••    . :;.
   calibration procedure.  Continuous. '  '. '
   dousing with a concentrated solution
   into the bag will give  various instrument
   readings  through the detectable ranges.

2  Flush the system with methyl or butyl •
   alcohol solution before each calibration  •„•'
   to remove traces of dye.

3  Bubbles in the flow  cuvette will cause
   erratic readings.           .      '     '

4  The fluorometer door is not light tight,. .'
   so the instrument should be draped and \
   used out of intense light to prevent •
   erroneous high readings..  '    ' ;.   -. -  ' \

5  The sample lines on a continuous'flow
   system will transmit  light through the   .;
   instrument door causing erroneous high
   readings.  Taping these tubes with
   black electrical tape for about one foot
   will alleviate problem.     .         .    •
  <2 Price as low as $1, 000 for photofluorometer  D  Study Procedures
     complete or $500 for unit to modify a
     spectrophotometer. ,
   3 Available for individual grab sample
     analysis or continuous  analysis of
     sample pumped through instrument.

   4 Automatic recorders available.

   5 Will measure several dyes (fluorescein,
     rhodamine, etc. )

C  Fluorometer Calibration

   The instrument should be  calibrated before
   and after a study.  The calibration is a
   function of temperature (about 2.3%/°C)
1  Select objectives which then dictate:

   a  Length of time to be studied

   b  Release point              .   s'  "; '

   c  Monitoring locations and schedules

2  Determine physical properties of'1";..';
   affected area.                :'•   .   ; •
                                • j Vl  /•'•
   a  Probable net flows

   b  Total water volumes       •  ;

   c  Probable water temperature '    ••.••}•
    3-6

-------
                                                                    Tracing Natural Waters
      d  Natural fluorescent background

      e  Salinity

      f  Suspended solids

    3  Estimate  required quantities of dye

    4  Calibrate fluorometers

    5  Conduct mock run to test equipment

    6  Public relations

    7  Release and monitor dye

    8  Process and interpret data

    9  Report
   2  Using these measurements and a two
      dimensional dispersion model com-
      pute D  and D .
            x      y

   3  Two dimensional model
|£ =  D  *_| + D
dt      xdx2     y
                      C   T"r
                             ax " vdy
      U  = velocity in x direction
      V  = velocity in y direction

      Normalized solution:
     	D_
     2inr 
     where f (t) represents concentration
     function and g(t)  = decay function

  C Coastal Water Dispersion Study, Hilo
     Bay, Hawaii*9*

     1  From instantaneous release measure
        concentration profile during slack
        water
D  Channel Studies

   1  From instantaneous release compute
      longitudinal dispersion coefficient

   2  Measure time-concentration profile
      at given station

   3  This C vs. t curve fits equation
        C  =
               M
                   •n exp -
             A(4irDt)2

   4  Take log of both sides
                            (x - ut)'
                              4 Dt
      Ct2  =  log
(x - ut)'
  4 Dt
                                   log e
   5  D can be determined from semi-log

                             .2
        plot of Ct2 vs. (X " Ut)<
                          t
                                                                                        3-7

-------
Tracing Natural Waters
 REFERENCES                         .        9  O'Connell,  R.L., and Walter, C.M. A
                                                       Study of Dispersion in Hilo Bay,
 1  Straub, C.P.,  Ludzack,  F.J., Hagee,              .Hawaii. Report prepared for the
        G.R., and Goldin, A.S.  Time of                 U.S. Army Engineer District,
        Flow Studies.  Ottawa River, Lima,               Honolulu,  Hawaii.  September,  1963.
        Ohio.  Transactions,  Amer.  Geo-
        physical Union,  Vol.  39. pp 420-         10  Tsivoglou,  E.G., et al.  Tracer Measure-
        426.  1958                                      ments of Atmospheric Reaeration.
                                                       I.  Laboratory Studies.  Presented at
 2  Feuerstein, D. L.,  and Selleck,  R.E.                the Water  Pollution Control Feder-
        Flourescent Tracers  for Dispersion               ation Conference, Bal Harbour,
        Measurements.  Journal of the                   Florida.  September,  1964.
        Sanitary Engineering  Division, ASCE,
        Vol. 89, No. SA4, Proc. Paper  3586,     11  Cawley,  W.A.  and Rutledge, W.  C.
        pp 1-21.  August,  1963.                          Application of Radioactive Tracer
                                                       Techniques to Stream Improvement.
 3  Buchanan,  Thomas J.  Time of Travel               Journal, San. Eng. Div., ASCE, Vol.
        of Soluble Contaminants in  Streams.               92,  No. SA1, Proc. Paper 4640, p. 1,
        Journal of the Sanitary Engineering               February,  1966.
        Division,  ASCE,  Vol. 90, No. SA3,
        Proc.  Paper 3932, ppl-12.  June,  1964.  12  Tsivoglou, E.G.,  O'Connell, R.L.,
                                                       Walter, C.M., Godsil, P.J., and
 4  Wright, R.R. and Collings,  M.R.  Appli-            Logsdon, G.S.  Tracer Measurements
        cation of Fluorescent  Tracing Tech-               of Atmospheric Reaeration-1.  Labor-
        niques to Hydrologic Studies.  Hydraulic          atory Studies.  Jour.  WPCF,  Vol,  37,
        Engrs.,  USGS, Atlanta, GA., Jour.               p. 1343.
        AWWA, Vol. 56:748.  June,  1964.
                                               13  U.S. Atomic Energy Commission, Re-
 5  Pritchard, D.W. and Carpenter, J.H.                print from Federal Register,  17915,
        Measurements of Turbulent Diffusion              Part 30. 70,  Dec.  17,  1964,  p. 5.
        in Estuarine and Inshore Waters.  Ches-
        apeake Bay Institute,  Johns Hopkins       14  American Cyanamid Company, A Dis-
        University.  Contribution No. 53.  1960.          cussion of Techniques and Tracer
                                                       Dyes used in Water Tracing, Dyes
 6  Ault,  W.U., and Hardeway, J.E. Surface            and  Textile Chemicals Department-
        Tracing with Radioisotopes.  Isotopics,            Bound Brook, NJ  08805
        Isotodes, Inc. Vol.  2  #1.  January, 1965.
    Diachishin, A.N.  Dye Dispersion Studies.    This outline was prepared by Dale B. Parke,
       Jour. San. Engr. Div.,  ASCE, Vol.  89,   Former Sanitary Engineer, Hudson-Delaware
       No. SA1,  Proc. Paper 3386,  p. 29.       Basins Office,  EPA,  WPO, Edison, NJ
       January,  1963.
                                               Descriptors:  Fluorescence, Fluorescent Dye,
    O'Connell,  R.L.,  and Walter, C.M.          Path of Pollutants, Radioisotopes, Tracers
       Hydraulic Model Tests of Estuarial
       Waste Dispersion.  Jour.  San. Engr.
       Div., ASCE, Vol.  89, No.  SA1, Proc. '
       Paper 3394, January, 1963.  p. 51.
  3-8

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                       SAMPLING IN WATER QUALITY STUDIES
I  INTRODUCTION

A   Objective of Sampling

    1   Water quality characteristics are not
       uniform from one body of water to
       another,  from place to place in a
       given body of water, or even from time
       to time at a fixed location in a given
       body of water.  A sampling program
       should recognize  such variations and
       provide a basis for interpretation of
       their effects.

    2   The purpose of collection of samples is
       the accumulation  of data  which can be
       used to interpret  the quality or condition
       of the water under investigation.  Ideally,
       the sampling program should be so de-
       signed that a statistical confidence
       limit may be associated with each
       element of data.

    3   Water quality surveys are undertaken
       for a great variety of reasons. The
       overall objectives of each survey greatly
       influence the location of sampling
       stations,  sample  type,  scheduling of
       sample collections, and other factors.
       This influence should always be kept in
       mind during planning of the  survey.

    4   The sampling and testing program should
       be established in  accordance with princi-
       ples which will permit valid interpre-
       tation.

       a  The collection, handling, and testing
          of each sample should be scheduled
          and conducted in such a manner as
          to assure that the results will be
          truly  representative of the sou rces
          of the individual samples at the time
          and place of collection;

       b   The locations  of sampling stations
          and the schedule of sample collections
          for the total sampling program should
       be established in such a manner that
       the stated investigational objectives
       will be met; and

    c  Sampling should be sufficiently
       repetitive over a period of time to
       provide valid data about the condition
       or quality of the water.

B  Sample Variations

   Interpretation of survey data is based on
   recognition that variations will occur in
   results from individual samples. While
   it is beyond the scope of this discussion
   to consider the implications of each in
   detail, the following can be identified as
   factors producing variations in data and
   should be  considered in planning the
   sampling program.

   1 Apparent Variations

     a  Variations of a statistical nature,
         due to collection of samples from
         the  whole body of water,  as con-
         trasted with examination of all the
         water in the system.

     b  Variations due  to inherent precision
         of the analytical procedures.

     c  Apparent variations are usually
         amenable to statistical analysis.

   2 True Differences

     a  Variations of a  cyclic nature

         Diurnal variations,  related to alter-
         nating periods of sunlight  and
         darkness.

         Dirunal Variations related to  waste
         discharges from communities.
WP. SUR. sg. Ib. 3.74

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Sampling in Water Quality Studies
         Seasonal variations,  related to
         temperature and its subsequent
         effects on chemical and biological
         processes and interrelationships.

         Variations due to tidal influences,
         in coastal and estuarine waters.

      b  Intermittent variations

         Dilution by  rainfall and runoff.

         Effects of irregular or intermittent
         discharges  of wastewater,  such as
         "slugs" of industrial wastes.

         Irregular release of  water  from
         impoundments,  as from power
         plants.

      c  Continuing changes in water quality

         Effects downstream from points of
         continuous release of wastewater.

         Effects of confluence with other
         bodies of water.

         Effects of passage of the water
         through or over geological  forma-
         tions of such chemical or physical
         nature as to alter the characteristics
         of the water.

         Continuing interactions of biological,
         physical,  and chemical factors in
         the water, such as in the process of
         natural self-purification following
         introduction of organic contaminants
         in a body  of water.
II   LOCATION OF SAMPLING STATIONS

 A  The Influence of Survey Objectives

    Much of the sampling design will be
    governed by the stated purpose of the
    water investigation.  As an example  of
    how different objectives might influence
    sampling design, consider a watercourse
    with points A and B located as indicated
    in Figure 1.
                                                        A
                                                        •y
         flow
             Figure 1
                               B
Point A can be the point of discharge of
wastes from Community A.  Point B can
be any of several things,  such as an intake
of water treatment plant supplying Com-
munity B,  or it might be the place where
the river crosses a political boundary, or
it may be the place where the water is
subject to some legitimate use, such as
for fisheries or for recreational use.

1  Assume that the objective of a water
   quality investigation is to determine
   whether designated standards of water
   quality are met  at a water plant intake
   at Point B.  In this case, the objective
   only is concerned with the quality of the
   water as it is available at Point B.
   Sampling will be conducted only at
   Point B.

2  Alternately,  consider that there is a
   recognized unsatisfactory water quality
   at Point B,  and  it is alleged that this
   is due to discharges of inadequately
   treated wastes,  originating at Point A.
   Assume that the charge is to investigate
   this  allegation.

   In this case the  selected sampling  sites
   will  include at least three elements:

   a  At least one sampling site will be
      located upstream from Point A,  to
      establish base  levels of water quality,
      and to check  the possibility that the
      observed conditions actually originated
      at some point upstream from Point A.

   b  A  site or sites must be located  down-  .
      stream from Point A. Such a site
      should be downstream a sufficient
      distance to permit adequate mixing in
      the receiving water.

   c  Sampling would be necessary at Point
   .   B in order to demonstrate that the
      water  quality is in fact impaired, and
      that the impairment is due  to influences
      traced from  Point A.

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                                                          Sampling in Water Quality Studies_
B  Hydraulic Factors

   1  Flow rate and direction

      a  In a survey of an extended body of
         water it is necessary to determine
         the rate and direction of water move-
         ment influences selection of sam-
         pling sites.  Many workers plan
         sampling stations representing not
         less than the distance water flows
         in a 24-hour period.   Thus,  in
         Figure 1,  intervening sampling
         stations would be selected at points
         representing the distance water
         would flow in about 24 hours.

      b  In a lake or impoundment direction
         of flow is the major problem influenc-
         ing selection of sampling stations.
         Frequently it is necessary to estab-
         lish some sort of grid network of
         stations in the vicinity of the sus-
         pected sources of pollution."

      c  In a tidal estuary, the oscillating
         nature of water movement will re-
         quire establishment of sampling
         stations in both directions from
         suspected sources of pollution.

   2  Introduction of other water

      a  In situations in which a stream being
         studied is joined by another stream
         of significant size and character,
         sampling stations will be located
         immediately above the extraneous
         stream, in the extraneous stream
         above its point of juncture with the
         main stream, and in the main
         stream below the point of juncture,

      b  Similar stations will be needed with
         respect to other water discharges,
         such as from industrial outfalls,
         other communities, or other  instal-
         lations in which water is introduced
         into the main stream.

   3  Mixing

      a  Wherever possible, one sampling
         point at a sample collection site  is
        used in stream surveys.  This
        usually is near the surface of the
        water,  in the main channel of flow.

     b  In some streams mixing does not
        occur quickly, and introduced  water
        moves downstream for considerable
        distances below the point of con-
        fluence with the main streams.
        Example:  Susquehanna River at
        Harrisburg, where 3 such streams
        are recognizable in the main river.
        Preliminary survey operations
        should identify such situations.

        When necessary, collect separate
        samples at two or more points
        across  the body of water.

     c  Similarly, vertical mixing may not
        be rapid.  This is noted particularly
        in tidal estuaries, where it may be
        necessary to make collections both
        from near the bottom and near the
        surface of the water.

     d  Collection of multiple samples from
        a station requires close coordination
        with the laboratory, in terms of the
        number of samples that can be
        examined.   Some types of samples
        may be  composited.   The decision
        must be reached separately for each
        type df sample.

C  Types of Analytical Procedure

   1 Samples collected for physical, chemi-
     cal, and bacteriological tests and
     measurements may be collected from
     the same series of sampling stations.

   2 Sampling stations selected for biological
     (ecological) investigation require
     selection of a series of similar aquatic
     habitats (a series of riffle areas, or a
     series of pool areas, or both).  The
     sites used by the  aquatic biologist may
     or may not be compatible with those
     used for the rest of the survey.  Accord-
     ingly, in a given stream survey,  the
     stations used by the aquatic biologist
     usually are somewhat different from thp
     stations used for other examinations.

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  Sampling in Water Quality Studies
  D Access to Sampling Stations

    For practical reasons, the sampling site
    should be easily reached by automobile if
    a stream survey,  or by boat if the survey
    is on a  large body of water.  Highway
    bridges are particularly useful, if the
    sample collector can operate in safety.
Ill  FACTORS IN SCHEDULING OF SAMPLING
    PROGRAMS

 A Survey Objectives

 B Time of Year

    1  In short-term water quality investiga-
       tions, particularly in pollution
       investigations,  there often is need to
       demonstrate the extremes of pollution
       effects on the aquatic environment.
       For this reason,  many short-term
       surveys are conducted during the
       warmer season of the year, at such
       times  as the water flow rate and
       volume is at a minimum and there is
       minimum likelihood of extensive
       rainfall.

    2   In a long-term investigation, sampling  •
       typically is  conducted at all  seasons
       of the year.

 C  Daily Schedules

    As shown in an introductory paragraph,
    water quality is subject to numerous
    cyclic or  intermittent variations.  Sched-
    uling of sample collections should be de-
    signed to  reveal such variations.

    1   In short-term surveys it is common
       practice to collect samples from each
       sampling site at stated intervals through
       the 24-hour day,  continuing the program
       for 1-3 weeks.  Sampling at 3-hour
      .intervals is preferred by many workers,
       though practical considerations may re-
       quire extension to 4- or even 6-hour
       intervals.
    2  In an extended survey there is a ten-
       dency to collect samples from each
       site at not more than daily intervals,
       or even longer.  In such cases the
       hour of the day should be varied through
       the entire program, in order that the
       final survey show cyclic or intermittent
       variations if they exist.

    3  In addition, sampling in tidal waters
       requires consideration of tidal flows.
       If samples are collected but once daily,
       many workers prefer to make the col-
       lections at low slack tide.

    4  In long-term or any other survey in
       which only once-daily samples are
       collected, it is desirable to have an
       occasional period of around-the-clock
       sampling.


IV  IDENTIFICATION OF SAMPLING SITES

 A River Mile System

    The FWPCA method of identifying points
    on a water course is by counting river
    miles from the mouth (or junction with a
    larger stream) back to the source.  This
    should not be confused with other systems,
    such as those in which the river mile is
    started at the source of the stream and
    proceeds to the mouth of the stream or
    confluence with another body of water.

 B STORET System

    The STORET System is a computer-oriented
    data processing system used by FWPCA for
    storage,  retrieval, and analysis of water
    quality data collected by federal, state,
    local, and private  agencies.

    The system includes a complex system -
    based on the river mile system - for
    identifying sampling locations on all rivers
    and streams in the United States.  A recent
    addition to the system introduces a location
    procedure based on geographic coordinates;
    this procedure is especially adapted to
    location of sampling stations in large bodies
    of water such as lakes and impoundments.
    k-t*

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                                                           Sampling in Water Quality Studies
    Not all locations have been coded at this
    time, although the coding systems have
    been established. The interested worker
    should consult Public  Health Service Publi-
    cation No. 1263, "The Storage and Retrieval
    of Data for Water Quality Control. "  1963.

IV  SAMPLE  COLLECTION

 A Types of Samples                    ,
                                         i
    1  "Grab" sample -  a grab sample is usually
       a manually collected single portion of  the
       wastewater  or stream water.  An analysis
       of a grab sample  shows the concentration
       of the constituents in the water at the time
       the sample was taken.

    2  "Continuous" sample - when  several points
       are to  be sampled at frequent intervals or
       when a continuous record of quality at a
       given sampling station is required, an
       automatic or continuous  sampler may  be
       employed.

       a  Some automatic samplers collect a
          given volume of sample at definite time
          intervals; this is satisfactory when the
          volume of flow is constant.

       b  Other automatic samplers take samples
          at variable rates in proportion to chang-
          ing  rates of flow.  This type of sampler
          requires some type of flow measuring
          device.

     3  "Composite"  sample - a composite
        sample is the collection and mixing
        together of various individual samples
        based  upon the ratio of the volume of
        flow at the time the individual samples
        were taken to the total  cumulative
        volume of flow.  The desired composite
        period will dictate the magnitude of the
        cumulative volume of flow.  The more
        frequently the samples are collected,
        the more representative will be the
        composite sample  to the actual situa-
        tion.  Composite samples may be
        obtained by:

        a  Manual sampling and volume of flow
           determination made when each sam-
           ple is taken.
      b  Constant automatic sampling (equal
         volumes of sample taken each time)
         with flow determinations made as
         each sample is taken.

      c  Automatic sampling which takes
         samples at pre-determined time
         intervals and the volume of sample
         taken is proportional to the volume
         of flow at any given time.

B  Type of Sampling Equipment

   1  Manual sampling

      a  Equipment is specially designed
         for collection of samples from the
         bottom muds, at various depths,
         or at water surfaces.   Special
         designs are related to protection of
         sample integrity in terms of the
         water characteristic or component
         being measured.

      b  For details of typical sampling equip-
         ment used in water quality surveys.
         see  outlines dealing with biological,
         bacteriological, and chemical tests
         in this manual.

      c  Manual sampling equipment has
         very broad application in field work,
         as great mobility of operation is
        possible, at lower cost than may be
        possible with automatic sampling
         equipment.

    2  Automatic sampling equipment

       Automatic sampling equipment has
       several important advantages over
       manual methods.  Probably the most
       important consideration is the  reduction
       in personnel requirements resulting
       from the use of this equipment.  It
       also allows more frequent sampling
       than is practical manually, and elimi-
       nates many of the human  errors in-
       herent in manual sampling.

       Automatic sampling equipment has
       some disadvantages.  Probably the
       most  important of these  is the  tendency
       of many automatic devices to become

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Sampling in Water Quality Studies
      clogged when liquids high in solids are
      being sampled.  Individual portions of
      composite samples are usually quite
      small which may in  some cases be
      disadvantageous.  In using automatic
      samplers, sampling points  are fixed,
      which results in a certain loss of
      mobility as compared to manual
      methods.

      Automatic sampling equipment should
      not be  used indiscriminately; some typec
      of samples - notably bacteriological,
      biological, and DO samples - should
      not be  composited.  In cases of doubt,
      the appropriate analyst should be
      consulted.
a  Compositing samplers

   1)  Jar and tube sampler - this type-
      samples effectively when flow
      is nearly constant.  As water
      drains from the upper carboy,
      the vacuum created syphons
      waste into the lower one.  The
      rate-of-flow is regulated by the
      pinch clamp to fill the lower
      carboy during the sampling
      period.  (See Figure 2 )
                                        ^ WASTE SAMPLE    SCREW CLAMP

                                     -WASTE STREAM

                                        C MUST BE GREATER THAN A+B
                                          Figure 2

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                                                  Sampling in Water Quality Studies
2)  Scoop type

   a)  Rotating scoop

      This device consists of a
      power driven scoop mounted
      upstream from a weir.  The
      scoop is so designed and
      mounted that  the sample vol-
      ume grabbed  on each rotation
      of the scoop is proportional to
      the  flow,  as governed by the
      head on the weir.  The scoop
      may be rotated at a constant
      speed or timed to sample
      at fixed time  intervals.

   b)  Revolving wheel with cups
      (Figure 3)

      This device consists of a
      power driven wheel or disc
      mounted upstream from a
      weir.  A number of freely
      suspended buckets are mount-
      ed at varying distances from
      the  axis so that increased
      flow will cause more buckets
      to be filled, thereby giving a
      sample proportionate to flow.
      Both this device  and the
      rotating scoop sampler can,
      of course, be used for non-
      proportionate sampling.

  c) Bucket elevators

     This device may consist  of a
     single bucket alternately
     lowered into and raised out of
     the waste stream, or it may
     consist of a series of buckets
     on an endless  chain passing
     through the waste stream.  In
     either case, it will include a
     tripping mechanism to cause
     the bucket or buckets to spill
     into a sampling funnel.  Both
     types may be operated contin-
     uously or timed for intermittent
     operation.  This method  is not
     well  adapted to proportionate
     sampling.
                      WEIR CREST
        Figure 3
 WHEEL WITH BUCKETS

3  Pumps

   a) Chemical feed pumps have
     been found useful for sampling,
     because of their ability to
     meter out small doses of
     liquids.  A timing mechanism
     may be used to make the pump
     run for longer periods during
     heavy flow, thereby allowing
     collection of the sample in
     proportion to flow.  These
     pumps are usually provided
     with adjustable stroke and
     variable speed features which
     allow variation of the volume
     of sample being pumped.
     Figure 4 illustrates a battery
     operated pump.

   b) Automatic shift sampler
     (Figure 5 )

     Figure 5 shows the automatic
     shift sampler.  It consists
     first of a Randolph or other
     "squeegee-" type pump unit.

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Sampling in Water Quality Studies
               The 2-rpm gear motor drives
               the pump at between 1 and 2
               rpm through the spring-loaded
               adjustable-pitch pulley and
               adjustable motor-base arrange-
               ment.

               We use 1/8-in. (. 32-cm)  ID
               or 1/4-in. (. 64-cm) ID
               polyethylene  tubing for  sam-
               ple intake from the waste
               stream.  The sample flow is
               delivered to the distributor
               viaa3/16-in.  (. 48-cm) ID
               Tygon  tube which is supported
               loosely by a wire attached to
               the framework.
               Operation of the distributor is
               very simple.  The 1-rpm clock
               motor powers the chain-and-
               sprocket drive which turns a
               threaded bolt.  Rotation of the
               bolt moves the discharge  tube
               down the plastic trough at a
               rate equal to one division
               every eight  hours.   With  the
               10 sample-jar receivers  the
               timer can be set on  Friday,
               and the 9 week-end shift
               samples can be picked up on
               Monday.
   4)  Solenoid-valve arrangements

      A solenoid valve employed in
      connection with a timing device
      may be used for withdrawing
      waste from a pipe under pressure.
      Used in connection with a pump
      such devices  may be employed in
      sampling free flowing streams.
      (See Figures  6 and 7.)
  SOLENOID VU>C
I INSTALL 10 AS NOT
TO ACT AS TRAP)
TM1NO MICH
                                                    •OUS* SGKECN TO
                                                    MOTCCT
                    Figure 4
         BATTERY OPERATED PUMP
                                                                  Figure 6
                                                      PUMP - SOLENOID VALvE - TIMER
                                                                TYPE SAMPLER
   5)  Vacuum operated

      In its simplest form, the vacuum
      is created by a suitably mounted
      siphon.  It collects the sample
      at a uniform rate and is not
      suitable for use  when proportional
      sampling  is required.

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                                                                Sampling in Water Quality Studies
        Pump -
               Sompli I
                                       Tubing Joint
                                  -SOLENOID PUSHES
                                  -Nomol Position of
                                   Sampling TuM fetimMg
                                   to S«w«r
                                                      WASTES
                                                      FROM SEWER
7)  Drip sampler

   Two types of this device are
   illustrated in Figures 9 and 10.
   Both devices are  simple methods
   of obtaining a composite sample
   at a fairly constant rate.
                     WIRE ROD SOLDERED
                     TO FUNNEL AND BENT
                     TO PASS THROUGH
                     WATER  JET


                            FUNNEL WITH
                            NARROW SLOT
                            CUT IN SIDE
                    Figure 7
PUMP  - SOLENOID-DIVERTER - TIMER
               TYPE SAMPLER
         6) Air vent control

            This type of device is illustrated
            in Figure 8.  The rate of sample
         c  collection is controlled by the
            bubbling mechanism.  It is not
            suitable for use when proportion-
            ate  sampling is required.
                            VUVt HANOU-
                  FROM SAMPUH
                  TO BUBBLES •
     mm* INLIT
                                                                        Figure 9
                                                             FUNNEL AND ROD DIVERTER
                                                                                     TUBHM SLOTTED MO
                                                                                  •LID OVCH OLASS TUBE -
      PIPC IS* LONG
                   Figure  8
        AIR RELEASE TYPE SAMPLER
                                                                       SAMPLE COILCCTMO BOTTLES
        Figure 10
 DRIP TUBE SAMPLERS

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    Sampling in Water Quality Studies
VI
  b   Continuous recording equipment

      Instruments have been developed
      which provide direct measurement
      of temperature,  pH, conductivity,
      color, and dissolved oxygen.  Such
      instruments may be equipped for
      continuous recording.  Instruments
      of this type are quite expensive and
      their installation is often difficult.
      They are best adapted to permanent
      installations, although good portable
      non-recording instruments are
      available for the measurement of
      temperature, pH, and conductivity.


SOME CONSIDERATIONS IN SAMPLING
OPERATIONS
 All procedures in care and handling of samples
 between collection and the performance of
 observations  and tests are directed toward
 maintaining the reliability of the sample as an
 indication of the  characteristics of the sample
 source.

 A Sample Quantity

    1   Samples for a series of chemical
        analyses require determination of the
        total sample  volume required for all
        the tests,  and should include enough
        sample in  addition to provide a safety
        factor for  laboratory errors or acci-
        dents.  Many workers collect about
        twice the amount of sample actually
        required for the chemical tests.  As
        a  rule of thumb, this is on the order
        or 2 liters.

    2   Bacteriological samples,  in general,
        are collected in 250  - 300 ml sterile
        bottles; approximately 150 - 200 ml of
        samples is adequate in practically all
        cases.

 B Sample Identification

    1   Sample identification must be main-
        tained throughout any survey.  It is
        vital, therefore, that adequate records
        be made of all information relative  to
        the source of the sample and conditions
      under  which the collection was made.
      All information must be clearly under-
      standable and legible.

   2  Every sample should be identified by
      means of a tag or bottle marking,
      firmly affixed to the sample bottle.
      Any written material should be with
      indelible marking material.

   3  Minimum information on the sample
      label should include identification of
      the sample  site,  date and time of col-
      lection,  and identification of the
      individual collecting the sample.

   4  Supplemental identification of samples
      is strongly recommended, through
      maintenance of a sample collection
      logbook.   If not included on the sample
      tag  (some prefer to duplicate such infor-
      mation) the  logbook can show not only
      the  sample site and date and hour of
      collection, but also the  results of any
      tests made on site (such as temperature,
      pH,  dissolved oxygen).  In addition, the
      logbook should provide for notation of
      any unusual observations made at the
      sampling site, such  as rainfall, direc-
      tion and strength of unusual winds, or
      evidence  of  disturbance of the collection
      site by human or other animal activity.

C  Care and  Handling of Samples

   1  As  a general policy, all observations
      and tests should be  made as soon as
      possible after sample collection.

      a  Some measurements require perfor-
          mance at the sampling site, such as
          temperature, pH, dissolved oxygen,
          chlorine, flow rate, etc.

      b   Some tests are best made at the
          sampling site because the procedures
          are simple,  rapid, and of acceptable
          accuracy. These may include such
          determinations as conductivity.

      c   Some additional  determinations, such
          as  alkalinity, hardness, and turbidity
          may be made in the  field,  provided

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                                                       Sampling in Water Quality Studies
      that ease, convenience, and reliability
      of results are acceptable for the pur-
      poses of the study.

2  Samples to be analyzed in the laboratory
   require special protection to assure that
   the quality measured in the sample repre-
   sents the condition of the source. Many
   samples, especially those subjected to
   biological analysis, require special pre-
   servation,  protection, and handling pro-
   cedures.  In case of doubt, the appropriate
   analyst should be  consulted.  Most com-
   mon procedures for sample protection
   include:

   a  Examination within brief time after
      collection.

   b  Temperature control.

   c  Protection from light.

   d  Addition of preservative chemicals.

   Applications of these sample protective
   procedures are along the following
   lines:

3  Early examination of sample

   Applicable to all types of samples.

4  Temperature control

   a  All biological materials for  examina-
      tion in a living  state should be iced
      between collection and examination.

   b  Bacteriological samples should be
      iced during a maximum transport
      time of  6 hours.  Such samples should
      be refrigerated upon receipt in. the
      laboratory and processed within 2 hours.

   c  Chemical samples often require
      icing

      Preservation by refrigeration at 4°C
      is recommended for acidity, alkalinity,
      BOD,  color,  sulfate,  threshold odor,
      and other samples.  Holding times vary.
      Quick freezing will permit retention
      of many samples for up to several
      months prior to laboratory examina-
      tion.

5  Protection from light

   a  Any constituent of water which may
      be influenced by physiochemical
      reactions involving light should be
      protected. DO samples brought to
      the iodine stage, for example, should
      be protected from light prior to
      titration.

   b  In addition, any water constituent
      (such as  dissolved oxygen) which
      may be influenced by algal activity
      should be protected from light.

6  Addition of chemical preservatives

   a  Bacteriological samples never
      should be "protected" by addition
      of preservative  agents.  The only
      permissible chemical additive  is
      sodium thiosulfate, which is used
      to neutralize free residual chlorine,
      if present.

   b  Samples for biological examination
      should be protected by  chemical
      additives only under specific
      direction of the principal biologist
      in a water quality study.

  c   For chemical tests, preservatives
      are useful for a number of water
      components.  The following examples
      are cited:

      Nitrogen and phosphorus analyses:

      The addition of 40 mg HgCl2  per
      liter of sample and refrigeration at
      4°C will retard biological activity
      which might otherwise alter the
      concentration of these constituents
                                                                                    4-11

-------
Sampling in Water Quality Studies
       Metals :  The addition of 5 ml of
       HNOs acid per liter of sample
       prevents precipitation of the metal
       in the container.

       COD and Organic Carbon: Addition
       of 2  ml sulfuric acid per liter of
       sample  is suggested.

       In general, samples requiring re-
       tardation of biological activity can
       be temporarily preserved by addi-
       tion of chloroform; tests should be
       run as soon as possible, however.

       Cyanide  determinations may be
       delayed temporarily through addition
       of alkali to the sample.  Addition of
       sodium hydroxide to pH 10 is recom-
       mended.

       Sulfide  analysis may be delayed up
       to 7 days by addition of 2 ml/liter of
       sample of 2N solution of zinc acetate.

       Phenol  analysis can be delayed
       temporarily by acidification to pH 4. 0
       with  phosphoric acid and preservation
       with  1 gram  CuSO4- 5H2O per liter
       of sample.
REFERENCES

1  Standard Methods for the Examination
      Water,  Sewage and Industrial Wastes.
      13th Ed.  A.P.H.A.  1971

2  Planning and Making Industrial Waste
      Surveys.  Ohio River  Valley Water
      Sanitation Commission.

3  Industry's  Idea Clinic.  Journal of the
      Water Pollution Control Federation.
      April,  1965.
 This outline was prepared by H. L. Jeter,
 Director,  National Training Center, EPA,
 WPO, Cincinnati, OH  45268 and P.P.
 Atkins,  Jr., formerly Sanitary Engineer,
 Training Activities.
 Descriptors:  Instrumentation, On-Site
 Investigations, Preservation, Samplers,
 Sampling,  Water Sampling, Handling,
 Sample, Surface Waters, Wastewater
 4-12

-------
                   SAMPLE HANDLING - FIELD THROUGH LABORATORY
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


H  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 Compositjng 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 the 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  SAMPLE 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
                                                                                     5-1

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 Sample Handling - Field Through 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 constituent('g) to be determined
       - Cations can adsorb readily on some
       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 constituents) to
    be determined.
   1  Phosphate detergents should not be
      used to clean containers for phosphorus
      samples.

   2  Traces of dichromate cleaning solution
      will interfere with metal analyses.

E  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.
    5-2

-------
                                        Sample Handling - Field Through Laboratory
     . 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 met.*4'

      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
                           ,(2)
    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 (1. 2, 3) as part of tne 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 procedures.  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.
                                                                                      5-3

-------
  Sample Handling - Field Through Laboratory
 A   The allowable holding time for samples
     depends on the nature of the sample, the  •
     stability of the constituents) 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 lor other determinations
        range from 6 hours (BOD) to 7 days
        (COD).  Metals may be held up to 6
         months. ^  '
                                (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 (See
       Part HI)

     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
     5  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
   5-4

-------
                                               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, MPQD
EPA,  WPO, Cincinnati, Ohio  45268.
Descriptors: On-Site Data Collections,
On-Site Investigations, Planning, Handling,
Sample, Sampling, Water Sampling,
Surface Waters, Preservation, Wastewater
                                                                                    5-5

-------
                                  SAMPLE PRESERVATION
Complete and unequivocal preservation of
samples,  either domestic sewage, industrial
wastes or natural waters, is a practical
impossibility.  Regardless of the nature of
the sample, complete stability for every
constituent can never be achieved. At best,
preservation techniques can only retard the
chemical and biological changes that
inevitably continue after the sample is re-
moved from the parent source.

The changes that take place in a sample are
either chemical or biological.  In the former
case, certain changes occur in the chemical
structure of the constituents that are a
function of physical conditions.  Metal cations
may precipitate as hydroxides or form com-
plexes with other constituents; cations or
anions may change valance states under cer-
tain reducing or oxidizing conditions; other
constituents may dissolve or volatilize with
the passage of time.  Metal cations may
also adsorb onto surfaces (glass,  plastic,
quartz,  etc. ), such as,  iron and  lead.
Biological changes taking place in a sample
may change the state of an element or a
radical to a different state.   Soluble con-
stituents may be converted to organically
bound material in cell structures,  or cell
lysis may result in release of cellular
materials into solution.   The well known
nitrogen and phosphorus cycles are examples
of biological influence on sample composition.

Methods of preservation are relatively
limited and are  intended generally to
(1) retard biological action, (2) retard
hydrolysis of chemical compounds and
complexes and (3) reduce volatility of
constituents.

Preservation methods are generally limited
to pH control, chemical  addition,  refrigeration,
and freezing.  Table  1 shows the various  pre-
servatives that may be used to retard changes
in samples.
Preservative
HgCl2
    Table 1

   Action

Bacterial
 Inhibitor
   Applicable to:

Nitrogen forms.
 Phosphorus forms
Acid (HNO3)  Metals sol-   Metals
                vent.prevents
                precipitation
Acid (H2SO4)  Bacterial
                Inhibitor
              Salt forma-
                tion with
                organic
                bases

Alkali (NaOH) Salt forma-
                tion with
                volatile
                compounds

Refrigeration Bacterial
                Inhibitor
              Organic samples
                (COD, oil and
                grease,  organic
                carbon)

              Ammonia, amines
              Cyanides, organic
                acids
              Acidity - alkalinity
               organic materials,
               BOD,  color, odor,
               organic P, organic
               N,  carbon,  etc.,
               biological organisms
               (coliform, etc.)
In summary, refrigeration at temperatures
near freezing or below is the best preservation
technique available, but is not applicable to
all types of samples.

The recommended choice of preservatives for
various constituents is given in Table 2.
These choices are based on the accompanying
references and on information supplied by
various Regional Analytical Quality Control
Coordinators.
WP.SUR. sa. 2a. 3.75
                                      6-1

-------
Sample Preservation
Table 2
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT (D

Measurement
Acidity
Alkalinity
Arsenic
BOD
Bromide
COD
Chloride
Chlorine Req.
Color
Cyanides

Dissolved Oxygen
Probe
Winkler
Vol.
Req.
(ml)
100
100
100
1000
100
50
50
50
50
500


300
300

Container
P.G(2)
P.G
P.G
P.G
P.G
P,G
P.G
P.G
P.G
P,G


G only
G only

Preservative
Cool. 4°C
Cool, 4°C
HNOQ to pH< 2
O
Cool, 4°C
Cool, 4°C
H2SO4to pH< 2
None Req.
Cool, 4°C
Cool, 4°C
Cool, 4°C
NaOH to pH 12

Det. on site
Fix on site

Holding
Time(")
24 Hrs.
24 Hrs.
6 Mos.
6Hrs.(3)
24 Hrs.
7 Days
7 Days
24 Hrs.
24 Hrs.
24 Hrs.


No Holding
No Holding
 6-2

-------
Sample Preservation



Measurement
Fluoride
Hardness
Iodide
MBAS
Metals
Dissolved
Suspended
Total
Mercury
Dissolved



Total



Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT (D
Vol.
Req.
(ml) Container Preservative
300 P, G Cool, 4°C
100 P, G Cool, 4° C
100 P, G Cool,4°C
250 P,G Cool,4°C

200 P, G Filter on site
HNO, to pH< 2
O
Filter on site
100 HNO3topH<2

100 P, G Filter
HNO, topH<2
O


100 P, G HNO3 to PH< 2






Holding
Time (6)
7 Days
7 Days
24 Hrs.
24 Hrs.

6 Mos.
6 Mos.
6 Mos.

38 Days
(Glass)
13 Days
(Hard
Plastic)
38 Days
(Glass)
13 Days
(Hard
Plastic)
               6-3

-------
Sample Preservation



Measurement
Nitrogen
Ammonia

Kjeldahl
Nitrate

Nitrite
NTA
Oil and Grease
Organic Carbon
pH
Phenolic s
Phosphorus
Ortho-
phosphate
Dissolved
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT 
-------
                                                                  Sample Preservation
Table 2 (Continued)
RECOMMENDATION FOR SAMPLING AND PRESERVATION
OF SAMPLES ACCORDING TO MEASUREMENT^
Measurement
Hydrolyzable
Total
Total.
Dissolved
Residue
Filterable
Non-
Filterable
Total
Volatile
Settleable Matter
Selenium
Silica
Specific
Conductance
Vol.
Req.
(ml)
50
50
50

100
100
100
100
1000
50
50
100
Container
P.G
P.G
P.G

P.G
P.G
P.G
P.G
P,G
P.G
P only
P.G
Preservation
Cool, 4° C
H2SO4 to pH< 2
Cool, 4°C
Filter on site
Cool, 4°C

Cool, 4°C
Cool. 4°C
Cool, 4°C
Cool, 4°C
None Req.
HNO3topH<2
Cool, 4°C
Cool, 4°C
Holding
Time W
24 Hrs. (4)
24Hrs.(4)
24Hrs.<4)

7 Days
7 Days
7 Days
7 Days
24 Hrs.
6 Mos.
7 Days
24Hrs.(5)
Sulfate
50
P.G
Cool.  4°C
7 Days
                                                                                 6-5

-------
Sample Preservation
                                   Table 2 (Continued)
                 RECOMMENDATION FOR SAMPLING AND PRESERVATION
                     OF SAMPLES ACCORDING TO MEASUREMENT*1)
Measurement
Sulfide

Sulfite
Temperatue
Threshold
Odor
Turbidity
Vol.
Req.
(ml)
50

50
1000

200
100
Container
P.G

P.G
P,G

G only
P,G
Preservation
2 ml zinc
acetate
Cool, 4°C
Det. on site

Cool, 4°C
Cool, 4°C
Holding
Time (6)
24 Hrs.

24 Hrs.
No Holding

24 Hrs.
7 Days
1  More specific instructions for preservation and sampling are found with each procedure
   as detailed in this manual.  A general discussion on sampling water and industrial
   wastewater may be found in ASTM, Part 23, p.  72-91(1973).

2  Plastic or Glass

3  If samples cannot be returned to the laboratory in less than 6 hours and holding time
   exceeds this limit,  the final reported data should indicate the actual holding time.

4  Mercuric chloride may be used  as an alternate preservative at a concentration of 40
   mg/1,  especially if a longer holding time is required.  However, the use of mercuric
   chloride is discouraged whenever possible.

5  If the sample is stabilized by cooling,  it should be warmed to 25°C for reading, or
   temperature correction made and results  reported at 25°C.

6  It has been shown that samples properly preserved may be held for extended periods
   beyond the recommended holding time.
 6-6

-------
                                                                Sample Preservation
An excerpt from Manual of Chemical Analy-       Descriptors: Preservation,  Sampling,
sis of Water and Wastes, 1974, EPA,             Wastewater(Pollution), Water.
Methods Development and Quality Assur-
ance Research Laboratory, NERC-Cincinnati,
Ohio  45268.

-------
                      BACTERIOLOGICAL SAMPLING IN THE FIELD
 I   INTRODUCTION

 The first step in the examination of a water
 supply for bacteriological examination is
 careful collection and handling of samples.
 Selection of sample sites, frequency of
 sampling, and numbers of samples are all
 directly related to the survey objectives.
II  SELECTION OF SAMPLING LOCATIONS

 The basis for locating sampling points is
 collection of representative samples.

 A Grab samples from streams are frequently
   collected for control data or application of
   regulatory requirements. A grab sample
   can be taken in the stream near the surface.

 B For intensive stream studies on source
   and extent of pollution,  representative
   samples are taken by considering site,
   method,  and time of sampling. The
   sampling sites may be a compromise
   between  physical limitations of the labora-
   tory, detection of pollution peaks, and
   frequency of sample collection in certain
   types of  surveys.  Decisions must be
   made as to the total number of samples
   that can  be processed by laboratory per-
   sonnel and the types of samples desired,
   as, for instance, those measuring
   immediate water quality or those measur-
   ing an averaging collection method.

   A site designed to measure average con-
   ditions should be far enough downstream
   to ensure a complete mixing after the
   source entry of pollutants. Averaging
   does not eliminate variations but tends to
   minimize sharp fluctuations.  Since these
   stations  are removed from the point
   source,  a sampling program should not be
   instituted with excessive sampling.
    Samples from lakes, ponds, or reservoirs
    usually are collected at the same depth
    and on frequent occasions may be collected
    over the entire surface.

    Oceanic or estuarine sample sites can
    often cover large areas and may,
    depending upon the survey objectives,
    necessitate vehicular and/or boat
    transporation.  Frequently the need for
    surface and depth sampling programs are
    necessary especially if tidal or current
    patterns create a stratified environment.
Ill  BOTTLES FOR WATER SAMPLES

 A The sample bottles should have capacity
    for at least 100 ml of sample, plus an air
    space.  The bottle and cap must be of
    bacteriological inert materials.  Resistant
    glass or heat resistant plastic are
    acceptable. Wide mouth ground-glass
    stoppered bottles (Figure 1),  plastic
    bottles of heat-resistant polyethylene, and
    8 ounce glass jars with rubber lined screw
    caps are popular types in many laboratories.
                                                                 Figure 1
 W. BA. sa. 2. 3. 75
                                                                                     7-1

-------
  Bacteriological Sampling in the Field
    All bottles must be properly washed and
    sterilized.  Protect the top of the bottles.
    and cap from contamination by paper or
    metal foil hoods.  Both glass and heat
    resistant plastic bottles may be steri-
    lized in an autoclave.  Hold plastic  at
    121°C for at least 10 minutes.  Hot air
    sterilization, 1 hour at 170°C, may be
    used for glass  bottles.

  B Add sodium thiosulfate to bottles intended
   . for halogenated water  samples.  A quantity
    of 0.1 ml of a  10% solution provides 100
    mgAiter in a 100 ml sample.  This
    level shows no effect upon viability  or
    growth.

  C Supply catalogs list wide mouth ground-
    glass stoppered bottles of borosilicate
    resistance glass, specially for water
    samples.
IV  TECHNIQUE OF SAMPLE COLLECTION

  Follow aseptic technique as nearly as
  possible.  Nothing but sample water must
  touch the inside of the bottle or cap.  To
  avoid loss of sodium thiosulfate, fill the
  bottle  directly and do not rinse.  Always
  remember to leave an air space.

  A In sampling from a distribution system,
    first run the faucet wide open until the
    service line is cleared.  A time of 3-5
    minutes generally is sufficient.  Reduce
    the flow and fill the  sample bottle without
    splashing.  Some authorities stress flam-
    ing the tap before collection.  A chlorine
    determination is often made on the site.

  B The bottle may be dipped into some
    waters by hand.  Avoid introduction of
    bacteria from the human hand and from
    surface debris.  Some suggestions follow:
    Hold the bottle near the base with one
    hand and with the other remove the hood
    and  cap.  Push the bottle rapidly into the
    water  mouth down and tilt up towards the
    current to fill.  A depth of about 6 inches
    is satisfactory.  When there is no current
    move the  bottle through the water
    horizontally and away from the hand.
   Lift the bottle from the water,  spill a
   small amount of sample to provide an
   air space,  and return the uncontaminated
   cap.

C  Samples may be dipped from swimming
   pools.   Determine residual chlorine on
   the pool water at the site.  Test the
   sample  at the laboratory to check chlorine
   neutralization by the thiosulfate.

D  Sample  bathing beach water by wading out
   to the two foot depth and dipping the
   sample  up from  about 6 inches below the
   surface. Use the procedure described in
   III.

E  Wells with pumps are similar to  distri-
   bution systems.   With  a hand pumped well,
   waste water for  about five minutes before
   taking the sample.  Sample a well without
   a pump  by lowering a sterile bottle
   attached to a weight.  A device which
   opened the bottle underneath the water
   would avoid contamination by surface
   debris.

F  Various types of sampling devices are
   available where  the sample point is in-
   accessible or depth samples are  desired.
   The general problem is to put a sample
   bottle in place, open it, close it, and
   return it to the surface. No bacteria but
   those in the sample must enter the bottle.

   1  The J -  Z sampler described by Zobell
      in 1941,. was  designed for deep sea
      sampling but  is useful elsewhere
      (Figure  2). It has a metal frame,
      breaking device for a glass tube, and
      sample bottle.  The heavy metal
      messenger strikes the lever arm
      which breaks  the glass tubing  at a file
      mark.  A bent rubber tube straightens
      and the water is drawn in several inches
      from the apparatus.  Either glass or
      collapsible rubber bottles are sample
      containers.

      Commercial adaptations are available.
    7-2

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                                                    Bacteriological Sampling in the Field
                Figure
Reproduced with permission of the Journal of
Marine Research 4:3,  173-188 (1941) by the
Department of Health,  Education and Welfare.
      Note the vane and lever mechanism on
      the New York State Conservation
      Department sampler in Figure 3.
      When the apparatus is at proper depth
      the suspending line is given a sharp
      pull. Water inertia against the vane
      raises the stopper and water pours
      into the bottle.  Sufficient sample is
      collected prior to the detachment  of the
      stopper from the vane arm allowing a
      closure of the sample bottle.

      A commercial sampler is available
      which is an evacuated sealed tube with
      a capillary tip.  When a lever on the
      support rack breaks the tip,  the tube
                                                                  Figure 3
      fills.  Other samplers exist with a
      lever for pulling the stopper,  while
      another uses an electromagnet.
V  DATA RECORDING

A  Information generally includes:  date, time
   of collection, temperature of water, location
   of sampling point, and name of the sample
   collector.  Codes are often used.  The
   location description must be exact enough
   to guide another person to the site.
   Reference to bridges, roads, distance to
   the nearest town may help.  Use of the
   surveyors' description and maps are
   recommended.  Mark identification on the
   bottles or on securely fastened tags.
   Gummed tags may soak off and are
   inadvisable.

B  While a sanitary survey is an indispensable
   part of the evaluation of a water supply, its
   discussion is not  within the  scope of this
   lecture.  The sample collector could supply
   much  information if desired.
                                                                                    7-3

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  Bacteriological Sampling in the Field
 VI  SHIPPING CONDITIONS

  A  The examination should commence as soon
     as possible, preferably within one hour.
     A maximum elapsed time between collec-
     tion and examination is  30 hours for
     potable water samples and 8 hours for
     other water samples (collection 6 hours
     and laboratory procedures 2 hours).
     Standard Methods (13th Edition) recom-
     mends icing of samples  between collection
     and testing.
VII  PHOTOGRAPHS

  A photograph is a sample in that it is evidence
  representing water quality.  Sample collectors
  and field engineers may carry cameras to
  record what they see.  Pictures help the
  general public and legal courts to better
  understand laboratory data.
  REFERENCES

  1  APHA, AWWA, WPCF,  Standard Methods
        for the Examination of Water and
        Wastewater.  (12 Ed.) 1965.

  2  Prescott,  S. C., Winslow,  C.E.A.,  and
        McCrady,  M. H.  Water Bacteriology.
        6th Ed., 368 pp. John Wiley and Sons,
        Inc., New York.  1946.

  3  Haney, P. D,,  and Schmidt, J.
        Representative Sampling and Analytical
        Methods in Stream Studies.  Oxygen
        Relationships in Streams,  Technical
        Report W58-2 pp.   133-42. U.S.
      Department of Health, Education and
      Welfare, Public Health Service, Robert
      A. Taft Sanitary Engineering Center,
      Cincinnati, Ohio.   1958.

   Velz, C. J.  Sampling for Effective
      Evaluation of Stream Pollution.  Sewage
      and Industrial Wastes,  22:666-84.  1950.

   Bathing Water Quality and Health III
      Coastal Water.  134pp.  U.S.
      Department of Health, Education, and
      Welfare, Public Health Service, Robert
      A. Taft Sanitary Engineering,
      Cincinnati, Ohio.   1961.

   Zobell,  C. E. Apparatus for Collecting
      Water Samples from Different Depths
      for Bacteriological Analysis.  Journal
      of Marine Research, 4:3:173-88.  1941.
This outline was originally prepared by
A. G. Jose, former Microbiologist, FWPCA
Training Activities,  SEC and updated by
the Training Staff, National Training Center,
MPOD,  WPO, EPA, Cincinnati, Ohio
45268.
Descriptors: Equipment,  Microbiology,
Sampling, Water Sampling
    7-4

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                               BIOLOGICAL FIELD METHODS
 I   INTRODUCTION

 A  Due to the variability in habitats and
    modes of living, methods for the collection
    of different types of aquatic organisms
    differ. In general we can recognize those
    that swim or float and them that crawl,
    those  that are big and those that are little.
    Each comprises a part of "the life" at any
    given  survey station and consequently a
    "complete" collection at any given station
    would include all types of organisms that
    live there.

 B  Field  collecting methods in this outline
    are grouped under 4 general categories.

    1   Bottom (or benthic organisms)

    2   All types of plankton will be considered.
       Benthic forms which break off or loose
       their hold to a substrate may become
       temporarily a part of the plankton.

    3   The periphyton or "aufwuchs" com-
       munity,  which resembles both the
       plankton and the benthics.

    4   Fishes represent the fourth major group
       and need no further qualification.

 C  Aquatic mammals and birds, in most
    cases, require still other approaches and
    are not included here.

 D  A definite policy should be  established
    as  to the size range of organisms to be
    collected and counted,  i. e.: microscopic
    only,  microscopic and macroscopic, those
    retained by "30 mesh" screens only or
    #10 nets, invertebrates and/or vertebrates,
    etc.
II   Certain Standard Supplementary Procedures
    are a Part of All Collecting Techniques
A  In order to be interpreted and used, every
   collection must be associated with a record
   of environmental conditions at the time of
   collection.

   1 Data recorded should include the
     following as far as practicable:

     Location

     Station number (particular location of
     which a full description should be on
     record)

     Date

     Time
     Air temperature

     Water temperature (at various depths,
     if applicable)

     Salinity (at various depths, if applicable)
     Tidal flow (ebb or flood)
     Turbidity (or light penetration, etc.)
     Weather

     Wind direction and velocity
     Sky or  cloud cover
     Water color

     Depth

     Type of bottom
     Type of collecting device and
     accessories

     Method of collecting
     Type of sample (quantitative or
     qualitative)

     Number of samples at each station
     Chemical data, e.g.,  dissolved oxygen,
     nutrients,  etc.
     Collector

     Miscellaneous observations (often very
     important!)
 BI.MET.fm.le. 1. 74
                                   8-1

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  Biological Field Methods
III
    2  All collecting containers should be
       identified at least with location, station
       number, sample number, and date.
       Spares are very handy.

    3  Much transcription of data can be
       eliminated by using sheets or cards
       with a uniform arrangement for includ-
       ing the above data.  The same field data
       sheet may include field or laboratory
       analysis.

    Compact kits of field collecting equipment
    and materials greatly increase collecting
    efficiency, especially if collection site is
    remote from transportation.
COLLECTION OF BOTTOM OR BENTHIC
ORGANISMS
 A Shoreline or Wading Depth Collecting

    1  Rakes,  shovels, dipnets,  or dredges
       may be used here depending upon type
       of bottom, area to be sampled, or
       qualitative or  quantitative samples.

    2  Ekman dredges are best used on soft
       bottoms.  This dredge is essentially a
       completely closing clamshell  type
       dredge with spring operated jaws.  Size
       of dredge is usually 6" X 6" or 9" X 9".

    3  For  samples of hard packed clay or
       sand, or gravelly bottoms,  or where
       the current is  moderately swift a
       Petersen dredge may be used.

       a  This consists of two heavily con-
          structed half cylinders hinged
          together, and closed by a strong
          lever action.  The area sampled
          is commonly 0. lm^.

       b  To be effective in cutting into hard
          bottoms they are built to weigh 30 -
          40 Ibs., and may have additional
          weights added td bring the  total
          weight up to 70 Ibs. or more.

       c  Due to their weight,  especially
          when loaded with bottom material
         they are best handled by a small
         winch or crane.  However, they can
         be hand operated in shallow waters
         using a stout line of large enough
         diameter to be handled by wet hands.

   4  Ooze suckers and core samplers are
      other devices for sampling mud  bottom
      organisms.

   5  After bottom samples to obtain marine
      organisms are collected,  they are best
      placed into some type of container and
      then washed and grossly screened and
      picked in the field, conditions permitting.

      a  This removes large stones, etc.,
         and gives one a quick idea of  what
         the sample consists of.

      b  With many bottom samples, weight
         becomes a problem.  Washing,
         therefore, reduces this weight
         making it easier to transport  the
         samples. Also space is saved with
         smaller samples.

   6  Hand picking of readily visible plants
      and animals is valuable for immediate
      qualitative description.

      a  A frame of known dimensions may
         be placed over ah area to be sampled
         and the material within cropped out.
         This is especially good for larger
         plants.  This method yields quanti-
         tative  data.

      b  Underwater swimming or use of
         SCUBA is quite valuable for direct
         observation and collecting.

B  Deep Water  Benthic Collecting

   1  When sampling  from vessels,  a  crane
      and winch,  either hand or power operated,
      is used.   The Petersen dredge is com-
      monly employed.  SCUBA  diving is used
      also.  The general ideas described for
      shallow waters  when applicable,  apply
      to deeper waters.

   2  Since most biological populations are
      not normally distributed, but rather
   8-2

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                                                                    Biological Field Methods
IV
       some form of log normal distribution
       occurs, it is most always best to take
       2 but preferably 3 or more bottom
       samples at a station in order to get a
       true estimate of the population.

    Besides the types  of bottom collecting
    gear mentioned here,  there are many more
    types available, such as the orange-peel
    bucket, the Shipdek sampler,  the plow
    dredge, scallop type dredge, etc.  Each
    of these has  its own advantages and dis-
    advantages and it is up to the worker and
    his operation to decide what is best for his
    particular needs.  The Petersen and
    Eckman dredges are perhaps the most
    commonly used.
THE COLLECTION OR SAMPLING OF
PLANKTON
 A Phytoplankton - Planned Program

    1  A planned program of analysis should
       involve periodic sampling at biweekly,
       weekly or even more frequent intervals.

    2  A well-planned study and analysis of the
       growth pattern of phytoplankton in one
       year can provide a basis for predicting
       conditions for the following year since
       seasonal patterns tend to be repeated
       from year to year.

       a  Since  the seasons and years differ,
          the  more records accumulated, the
          more  useful they become.

       b  As the time for an anticipated bloom
          approaches, the frequency  of collect-
          ing  and analyses may be increased.

 B Field Equipment

    1  Two general aspects of plankton
       analysis  are commonly recognized:
       quantitative and qualitative. Either
       approach is useful, a combination is
       best.

    2  Equipment for collecting samples in the
       field is varied.
      a A Kemmerer type sampler,  a
        Nansen Bottle, or some similar
        vessel of known volume is suggested
        for quantitative sampling.

      b Plankton nets selectively concentrate
        (depending upon mesh size) the
        sample in the act of collecting and
        capturing certain larger forms
        which escape the bottles.  Only the
        more elaborate nets are quantitative,
        however.

      c Other instruments such as the Clark-
        Bumpus,  or the continuous plankton
        recorder may be employed for
        special purposes.

      d For phytoplankton,  #20 or #25 size
        nets are commonly used.  Usually
        a net diameter of 5 - 10 inches is
        sufficient.

C  Zooplankton Collecting

   1  Since zooplankton have the ability to
      swim away from water bottles,  etc.
      nets towed at moderately slow speed
      are used for their capture.

      a Number 12 nets (aperature size
        0.119 mm,  125 meshes 1 inch)
        or smaller numbered net sizes
        are commonly  used.  A net diameter
        greater than 5" is preferred. Fre-
        quently half meter nets or larger
        are employed.   These may be
        equipped with flow measuring de-
        vices for measuring the amount of
        water entering the net.

      b Other instruments such as the Clark-
        Bumpus, Gulf-Stream, Hardy
        continuous plankton recorder, and
        high-speed instruments are used for
        collecting zooplankton, also..

      c The devices  used for collecting
        plankton capture both the plant and
        animal types.  The  mesh size (net
        no.) is a method for selecting which
        category of plankton is to be collected.
                                                                                      8-3

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Biological Field Methods
   2  Plankton is subjected to the force of
      the winds and currents.  As a result,.
      the plankton is often in patches or
      "wind rows. "  For this reason when
      using a net,  it is often desirable to
      collect at right angles to the wind or
      current.

   3  Not only  do plankton occur horizontally
      in patches, but vertically as well.
      Zooplankton, especially are very
      numerous near the bottom in daylight.
      Therefore, a series of tows at different
      depths is necessary  to obtain a com-
      plete sampling.  One technique often
      employed is to take an oblique tow
      from the bottom to the top of the water
      column.

D  The Location of Sampling Points is
   Important

   1  Both  shallow and deep samples are
      suggested.   Sometimes,  the surface
      film itself is significant.

   2  The number of sampling  stations es-
      tablished is  limited only  by the ability
      of the laboratory to analyze the samples.

      a  There should be at least one sample
        station near an outfall.

      b  Every major natural division of
        the bay or estuary should be sampled.

      c  Additional sampling points should be
        established on the basis of experience
        and resources.

      d  Continuous sampling,  or the com-
        positing of periodic samples over a
        24-hour period, for example, is also
        very useful.

      e  For plankton, it is necessary to
        sample different periods in the
        stage of the tide,  otherwise  samples
        would be biased to a given time, or
        type of water carried by the tidal
        currents.
          Pilot studies to indicate sampling
          locations and intervals are often
          mandatory.

          Some.studies require random sam-
          pling points.
 V  COLLECTING PERIPHYTON,  ETC.

 A Hand-picking of forms attached to rocks,
    pilining, etc. is very valuable.

 B Microscopic attached forms or micro-
    benthos may be sampled by scraping
    or otherwise removing all organisms from
    a measured surface area.

 C Another technique used widely is to suspend
    artificial  substrates (slides, cement blocks,
    wood,  rope,  plastic,  etc.) with clean sur-
    faces in the water.  After a designated
    time interval organisms become attached
    which are subsequently removed and
    studied.
VI  FISH COLLECTING

 A Fish must be sought in the obscure and
    unlikely areas as well as the obvious
    locations  in order for the collection to be
    complete.  Several techniques should be
    employed wherever possible (this is
    appropriate for all biota).  It is advisable
    to check with local authorities to inform
    them of the reasons for sampling, because
    many of the techniques are not legal for
    the layman.  In this area, perhaps more
    than any other, professionally trained
    workers are important. Also, there
    must be at least one helper,  as a single
    individual always has difficulty in pulling
    both ends  of a 20 foot seine simultaneously.
    The more common techniques are listed
    below.

 B Seines

    1  Straight seines range from 4-6 feet
       and upwards in length.  "Common
   8-4

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                                                                   Biological Field Methods
      sense" minnow seines with approxi-
      mately 1/4 inch mesh are widely used
      along shore for collecting the' smaller
      fishes.

   2  Bag seines have an extra trap or bag
      tied in the middle which helps trap
      and hold fish  when seining in difficult
      situations.

C  Gill nets are of use in offshore and/or
   deep waters.  They range in length from
   approximately 30 yards upward.  A mesh
   size is designed to catch a specified size
   of fish.  The trammel net is a variation
   of the gill net.

D  Traps range from small wire boxes or
   cylinders with inverted cone entrances to
   Semi permanent weirs a half mile or more
   in length.  All tend to induce fish to swim
   into an inner chamber protected by an
   inverted cone or V-shaped notch to pre-
   vent escape.  Current operated rotating
   fish traps are also very effective (and
   equally  illegal) in suitable situations.

E  Trawls  are submarine nets, usually of
   considerable size, towed by vessels at
   speeds sufficient to overtake and scoop
   in fish,  etc.  The mouth of the  net must
   be held  open by some device such as a
   long beam (beam trawl) or two  or more
   vanes or "otter boards" (otter trawl).

   1  Beam and otter trawls are usually
      fished on the bottom, but otter trawls
      when suitably rigged are now being
      used to fish mid-depths.

   2  The Isaacs-Kidd midwater trawl re-
      sembles a plankton net 10 - 15 feet in
      diameter.  It is proving very effective
      for collecting at mid-depths.

F  Electric seines and screens are widely
   employed by fishery workers in small
   and difficult streams.  They may also be
   used in  shallow water areas with certain
   reservations.

G  Poisoning is much used in fishery studies.
   and management.  Most widely used and
     generally satisfactory is rotenone in
     varying formulations,  although many
     others have been employed from time to
     time.  Under suitable circumstances,
     fish may even be killed selectively
     according to species.

  H Personal  observation by competent
     personnel, and also informal inquiries
     and discussions with local residents will
     often yield information of real use.  Many
     laymen are keen observers, although they
     do not always  understand what they are
     seeing. The organized creel census
     technique yields data on what and how
     many fish are being caught.

  I  Angling remains in its own right a very
     good technique in the hands of the skilled
     practitioner, for determining what fish
     are present.  Spear-fishing also is now
     being used in some studies.

  J  Fish are often tagged to trace their move-
     ments during migration and at other times.
     Miniature radio transmitters can now be
     attached or fed to fish (and other organ-
     isms) which enable them to be tracked
     over considerable distances.  Physiological
     information is often obtained in  this  way.
     This is known as telemetry.
VII  DISPOSITION AND PRESERVATION OF
     COLLECTIONS

  A  The organisms may be picked out on the
     spot,  or the entire mess taken into the
     laboratory for further analysis.

  B  Organisms may be simply counted,
     weighed, or measured volumetrically;
     usually a combination is best.  They may
     be separated and recorded in groups or
     species.

  C  Specimens may be simply observed and
     recorded or they may be preserved as a
     permanent record.

  D  Preservation - provisions should be made
     for the field stabilization of the sample
     until the laboratory examination can be
                                                                                    B-5

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  Biological Field Methods
     made if more than an hour or so (especi-
     ally in warm weather) is to elapse.

     1  Refrigeration or icing is very helpful.

     2  Ninety-five per cent ethanol (ethyl
        alcohol) is highly satisfactory.  A
        final strength of 70% is necessary for
        prolonged storage.

     3  Formaldehyde is more widely available
        and is effective in concentrations of
        3 - 10% of the commercial formulation.
        However, it shrinks and hardens  speci-
        mens,  collector, and laboratory
        analyst without favor!  In order to
        minimize bad effects from formalin,
        commercial neutral formaldehyde is
        recommended.

        a  Phytoplankton is best preserved
           with a 3% formalin solution.

        b  Zooplankton,  benthos, and fish are
           usually preserved in a 5 - 10%
           solution.

     4  Lugol's solution is good for
        phyloplankton.

     5  Other formulations are available.
VIII  SOURCE OF COLLECTING EQUIPMENT

  Many specialized items of biological collect-
  ing equipment are not available from the
usual laboratory supply houses.   Conse-
quently,  the American Society  of  Limnology
and Oceanography has  compiled a list of
companies handling  such items and released
it  as:  "Special Publication No. 1, Sources
of Limnological Apparatus  and  Supplies."
This may be obtained from the Secretary  of
the Society  for  $1. 00.
REFERENCES

1  Barnes, H. (ed.).  Symposium on New
      Advances in Underwater Observations.
      Brit. Assoc. Adv.  Sci., Liverpool.
      pp.  49-64.  1953.

2  Hedgepeth, Joel W.  Obtaining Ecological
      Data in the  Sea Chapter 4 in "Treatise
      on Marine Ecology and Paleoecology."
      Memoir 67.  Geol.  Soc. Am.  1963.

3  Isaacs,  John D. and Columbus, O. D.
      Oceanographic Instrumentation NCR
      Div.  Phys.  Sci. Publ.  309, 1954.
      233 pp.

4  Sverdrup, H.U. et al.  Observations and
      Collections  at Sea.  Chapter X in:  The
      Oceans, Their Physics, Chemistry, and
      Biology.  Prentice-Hall, Inc., N.Y.
      1942.  1087 pp.

5  Welsh, Paul S. Limnological Methods.
      The Blakiston Co.  Pa.  1948.
    8-6

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                                    THE AQUATIC ENVIRONMENT

                              Part 1:' The Nature and Behavior of Water
I  INTRODUCTION

The earth is physically divisible into the
lithosphere or land masses, and the
hydrosphere which includes the oceans,
lakes,  streams,  and subterranean waters.

A Upon the hydrospere are based a number
   of sciences which represent different
   approaches.  Hydrology is the general
   science of water itself with its various
   special fields such as  hydrography,
   hydraulics,  etc.  These in turn merge
   into physical chemistry and chemistry.

B Limnology and oceanography combine
   aspects of all of these, and deal not only
   with the physical liquid water and its
   various naturally occurring solutions and
     forms, but also with living organisms
     and the infinite interactions that occur
     between them and their environment.

  C  Water quality management, including
     pollution control, thus looks to all
     branches of aquatic science in efforts
     to coordinate and improve man's
     relationship  with his aquatic environment.
 H   SOME FACTS ABOUT WATER

  A  Water is the only abundant liquid on our
     planet.   It has many properties most
     unusual for liquids, upon which depend
     most of the familiar aspects of the world
     about us as we know it.   (See Table 1)
                                               TABLE 1

                                      UNIQUE PROPERTIES OF WATER
                           Property
         Significance
                   Highest heat capacity (specific heat) of any
                   solid or liquid (except NH,)
Stabilizes temperatures of organisms and
geographical regions
                   Highest latent heat of fusion (except NH,)
Thermostatlc effect at freezing point
                   Highest heat of evaporation of any substance
Important In heat and water transfer of
atmosphere
                  The only substance that has Its maximum
                  density as a liquid <4<>C)
Fresh and brackish waters have maximum
density above freezing point.  This is
Important In vertical circulation pattern
In lakes.
                   Highest surface tension of any liquid
Controls surface and drop phenomena,
important in cellular physiology
                   Dissolves more substances in greater
                   quantity than any other liquid
Makes complex biological system possible.
Important for transportation of materials
in solution.
                  Pure water has the highest dl-electric
                  constant of any liquid
Leads to high dissociation of inorganic
substances In solution
                   Very little electrolytic dissociation
Neutral, yet contains both H+ and OH  ions
                   Relatively transparent
Absorbs much energy In Infra red and ultra
violet ranges, but little In visible range.
Hence "colorless"
  BI.  2le.l.74
                                           9-1

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The Aquatic Environment
B  Physical Factors of Significance

   1  Water substance

      Water is not simply "H-O" but in
      reality is a mixture of some 33
      different substances involving three
      isotopes each of hydrogen and oxygen
      (ordinary hydrogen H1,  deuterium H2,
      and tritium H3; ordinary oxygen O1",
      oxygen 17, and oxygen 18) plus  15
      known types of ions.   The molecules
      of a water mass tend to associate
      themselves as polymers rather than
      to remain as discrete units.
      (See Figure 1)
     SUBSTANCE OF PURE WATER
                 TABLE 2

 EFFECTS OF TEMPERATURE ON DENSITY
        OF PURE WATER AND ICE*
Temperature (°C) Density
Water Ice**
-10
- 8
- 6
- .4
- 2
0
2
4
6
8
10
20
40
60
80
100
.99815
.99869
.99912
.99945
.99970
.99987 	
.99997
1.00000
.99997
.99988
.99973
.99823
.99225
.98324
.97183
.95838
.9397
.9360
.9020
.9277
.9229
.9168










                   Figure 1
 *  -Tabular values for density,  etc., represent
    estimates by various workers rather than
    absolute values, due to the variability of
    water.

**  Regular ice is known as "ice I".  Four or
    more other "forms" of ice are known to
    exist (ice  II, ice III, etc.), having densities
    at 1 atm.  pressure  ranging from 1.1595
    to 1.67.  These are of extremely restricted
    occurrence and may be ignored in most
    routine operations.
   2  Density

     a  Temperature and density:  Ice.
        Water is the only known substance
        in which the solid state will float
        on the liquid state.  (See Table 2)
           This ensures that ice usually
           forms on top of a body of water
           and tends to insulate the remain-
           ing water mass from further loss
           of heat. Did ice sink, there
           could be little or no carryover of
           aquatic life from season to season
           in the higher latitudes.  Frazil or
           needle ice forms colloidally at a
           few thousandths of a degree
           below 0° c.   It is adhesive and
           may build up on submerged objects
           as "anchor ice", but it is still
           typical ice (ice I).
9-2

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                                                      The Aquatic Environment
1) Seasonal increase in solar
   radiation annually warms
   surface waters in summer
   while other factors  result in
   winter  cooling.  The density
   differences resulting establish
   two classic layers:  the epilimnion**"
   or surface laver, and the
   hypolimnion*or lower layer, and
   in between is the thermocline *x^
   or shear-plane.

2) While for certain theoretical
   purposes a "thermocline" is
   defined as a zone in which the
   temperature changes one
   degree centigrade for each
   meter of depth,  in practice,
   any transitional layer between
   two relatively stable masses
   of water of different temper-
   atures  may be regarded as a
   thermocline.

3) Obviously the greater the
   temperature differences
   between epilimnion  and
   hypolimnion and the sharper
   the  gradient in the thermocline,
   the  more stable will the
   situation be.

4) From information given above,
   it should be evident that while
   the  temperature of the
   hypolimnion rarely  drops
   much below 4° C, the
   epilimnion may range from
   0° C upward.

5) When epilimnion and hypolimnion
   achieve the same temperature,
   stratification no longer exists.
   The entire body of water behaves
   hydrologically as a  unit,  and
   tends to assume uniform chemical
   and physical characteristics.
   Even a light breeze  may then
   cause the entire body of water
   to circulate.  Such events are called
   overturns, and usually result in
   water quality changes of consider-
   able physical,  chemical,  and
   biological significance.
        Mineral-rich water from the
        hypolimnion, for example,
        is mixed with oxygenated
        water from the epilimnion.
        This usually triggers a
        sudden growth or "bloom"
        of plankton organisms.

     6) When stratification is present,
        however,  each  layer behaves
        relatively independently, and
        significant quality differences
        may develop.

     7) Thermal stratification as
        described above has no
        reference to the size of the
        water mass; it  is found in
        oceans and puddles.

  b  The  relative  densities of the
     various isotopes of water
     influence its  molecular com-
     position.  For example,  the
     lighter O16 tends to go off
     first in the process of evaporation,
     leading to the relative enrichment
     of air by O16 and the enrichment
     of water by O17 and O18.   This
     can lead to a measurably higher
     OIQ  content in warmer climates.
     Also, the temperature of water
     in past geologic ages can be
     closely estimated  from the ratio
     °f O^g in the carbonate of mollusc
     shells.

  c  Dissolved and/or suspended solids
     may also affect the density of
     natural water masses (see Table 3)

            TABLE 3
EFFECTS OF DISSOLVED SOLIDS
            ON DENSITY
Dissolved Solids
(Grams per liter)
0
1
2
3
10
Density
(at 40 c)
1.00000
1.00085
1.00169
1.00251
1.00818
                                             35 (mean for sea water)
                              1.02822
                                                                              9-3

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The Aquatic Environment
      d  Types of density stratification

         1) Density differences produce
           stratification which may be
           permanent, transient, or
           seasonal.

         2) Permanent stratification
           exists for example where
           there is a heavy mass of
           brine in the deeper areas of
           a basin which does not respond
           to seasonal or other changing
           conditions.

         3) Transient stratification may
           occur with the recurrent
           influx of tidal water in an
           estuary for example,  or the
           occasional influx of cold
           muddy water into a deep lake
           or reservoir.

         4) Seasonal stratification is
           typically thermal in nature,
           and involves  the annual
           establishment of the epilimnion,
           hypolimnion, and the r mo dine
           as described above.

         5) Density stratification is not
           limited to two-layered systems;
           three, four,  or even more
           layers may be encountered in
           larger bodies of water.

      e   A "plunge line"  (sometimes called
         "thermal line")  may develop at
         the mouth of a stream.  Heavier
         water flowing into a lake or
         reservoir plunges below the
         lighter water mass of the epiliminium
         to flow along at  a lower level.  Such
         a line is usually marked by an
         accumulation of floating debris.

      f   Stratification may be modified
         or entirely suppressed in some
         cases when deemed expedient,  by
         means of a simple air lift.

      The viscosity of water is greater at
      lower temperatures (see Table 4).
        This is important not only in situations
        involving the control of flowing water
        as in a sand filter,  but also since
        overcoming resistance to flow gen-
        erates heat, it is significant in the
        heating of water by internal friction
        from wave and current action.
        Living organisms more easily support
        themselves in the more viscous
        (and also denser) cold waters of the
        arctic  than in  the less viscous warm
        waters of the tropics.   (See Table 4).

                   TABLE 4

VISCOSITY OF WATER (In millipoises at 1 atm)
Temp, o C
-in
- 5
0
5
10
30
100
Dissolved solids in g/L
0
oc n
21.4
17.94
15.19
13.10
8.00
2.84
5



18.1
15.3
13.2
8.1
	
10



18.24
15.5
13.4
8.2
	
30



18.7
16.0
13.8
8.6
----
     4  Surface tension has biological as well
        as physical significance.  Organisms
        whose body surfaces cannot be wet by
        water can either ride on the surface
        film or in some instances may be
        "trapped" on the surface film and be
        unable to re-enter the water.

     5  Heat or energy

        Incident solar  radiation is the prime
        source of energy for virtually all
        organic  and most inorganic processes
        on earth.  For the  earth as a whole,
        the total amount (of energy) received
        annually must  exactly balance that
        lost by reflection and radiation into
        space if climatic and related con-
        ditions are to remain relatively
        constant over geologic time.
 9-4

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                                                            The Aquatic Environment
   a  For a given body of water,
      immediate sources of energy
      include in addition to solar
      irradiation:  terrestrial heat,
      transformation of kinetic energy
      (wave and current action) to heat,
      chemical and biochemical
      reactions, convection from the
      atmosphere, and condensation of
      water vapor.

   b  The proportion of light reflected
      depends on the angle  of incidence,
      the temperature, color, and other
      qualities of the water; and the
      presence or absence  of films
      of lighter liquids such as oil.
      In general,  as the depth increases
      arithmetically, the light tends to
      decrease geometrically.  Blues,
      greens,  and yellows tend to
      penetrate most deeply while ultra
      violet, violets, and orange-reds
      are  most quickly absorbed.  On
      the order of 90% of the total
      illumination which penetrates the
      surface film is absorbed in the
      first 10 meters of even the clearest
      water, thus tending to warm the
      upper layers.

6  Water movements

   a  Waves or rhythmic movement

      1) The best known are traveling
        waves caused by wind.  These are
        effective only against objects  near
        the surface. They have little
        effect on the movement of large
        masses of water.

      2) Seiches

        Standing waves or seiches occur
        in lakes,  estuaries, and other
        enclosed bodies of water, but are
        seldom large enough to be
        observed.   An "internal wave or
        seich" is an oscillation in a
        submersed  mass of water such
        as a hypolimnion,  accompanied
        by compensating oscillation in the
        overlying water so that no
      significant change in surface
      level is detected.  Shifts in
      submerged water masses of
      this type can have severe effects
      on the biota and also on human
      water uses where withdrawals
      are  confined to a given depth.
      Descriptions and analyses of
      many other types and sub-types
      of waves and wave-like movements
      may be found in the literature.

b  Tides

   1) Tides are the longest waves
      known, and are responsible  for
      the once or twice a  day rythmic
      rise and fall of the ocean level
      on most shores around the world.

   2) While part and parcel of the
      same phenomenon,  it is often
      convenient to refer  to the rise
      and  fall of the water level as
      "tide, " and to the resulting
      currents as "tidal currents. "

   3) Tides are basically caused by the
      attraction of the sun and moon on
      water masses, large and small;
      however, it is  only  in the oceans
      and  possibly certain of the larger
      lakes that true tidal action has
      been demonstrated.  The patterns
      of tidal action are enormously
      complicated by local topography,
      interaction with seiches, and other
      factors.  The literature on tides
      is very large.

c  Currents (except tidal  currents)
   are steady arythmic water movements
   which have had major study only in
   oceanography although they are
   most often observed in rivers and
   streams.   They are primarily
   concerned with the translocation of
   water masses.  They may be generated
   internally by virtue of  density changes,
   or externally by wind or terrestrial
   topography.  Turbulence phenomena
   or eddy currents are largely respon-
   sible for lateral mixing in a current.
   These are of far more  importance
   in the economy of a body of water than
   mere laminar flow.
                                                                                   9-5

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The Aquatic Environment
       d Coriolis force is a result of inter-
         action between the rotation of the
         earth, .and the movement of masses •
         or bodies on the earth.  The net
         result is a slight tendency for moving
         objects to veer to the right in the
         northern hemisphere, and to the
         left in the southern hemisphere.
         While the result in fresh waters is
         usually negligible, it may be con-
         siderable in marine  waters. For
         example, other factors permitting,
         there is a tendency in estuaries for
         fresh waters to move toward the
         ocean faster along the right bank,
         while salt tidal waters tend to
         intrude farther inland along the -
         left bank.  Effects are even more
         dramatic in the open oceans.

       e Langmuir circulation (or L. spirals)
         is the interlocking rotation of
         somewhat cylindrical masses of
         surface water under the influence
         of wind action.  The axes of the
         cylinders are parallel to the
         direction of the wind.
                              To somewhat oversimplify the
                              concept,  a series of adjoining cells
                              might be thought of as chains of
                              interlocking gears in which at every
                              other contact the teeth are rising
                              while at the alternate contacts, they
                              are sinking (Figure 2).

                              The result is elongated masses of
                              water rising or sinking together.
                              This produces the familiar "wind
                              rows" of foam, flotsam and jetsam,
                              or plankton often seen streaking
                              windblown lakes or oceans. Certain
                              zoo-plankton struggling to maintain
                              a position near the surface tend to
                              collect in the down current between
                              two Langmuir cells, causing such
                              an area to be called the "red dance",
                              while the clear upwelling water
                              between is the "blue dance".

                              This phenomenon may be important
                              in water or plankton sampling on
                              a windy day.
                                                               t)
                                          WATER
                                         SURFACE
                \
             WATER
             RISING
WATER
SINKING
                  Figure  2.  Langmuire Spirals
                  b. Blue dance,  water  rising, r.  Red
                  dance,  water  sinking,  floating  or
                  swimming objects  concentrated.
  9-6

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                                                               The Aquatic Environment
   6   The pH of pure water has been deter-
       mined between 5. 7 and 7. 01. by various
       workers.  The latter value is most
       widely accepted at the present time.
       Natural waters of course vary widely
       according to circumstances.

C  The elements of hydrology mentioned
   above represent a selection of some of
   the more conspicuous physical factors
   involved in working with water quality.
   Other items not specifically mentioned
   include: molecular structure of waters,
   interaction of water and radiation,
   internal pressure,  acoustical charac-
   teristics, pressure-volume-temperature
   relationships, refractivity, luminescence,
   color, dielectrical characteristics and
   phenomena,' solubility,  action and inter-
   actions of gases, liquids and solids,
   water vapor, phenomena of hydrostatics
   and hydrodynamics in general.
REFERENCES

1  Buswell, A.M. and Rodebush,  W. H.
       Water. Sci. Am.  April 1956.

2  Dorsey, N. Ernest.  Properties of
       Ordinary Water - Substance.
       Reinhold Publ.  Corp.  New York.
       pp. 1-673. 1940.

3  Fowle, Frederick E.  Smithsonian
       Physical Tables.  Smithsonian
       Miscellaneous Collection, 71(1),
       7th revised ed.,  1929.

4  Hutcheson, George E.  A Treatise on
       Limnology.  John Wiley Company.
       1957.

This outline was prepared by H. W.  Jackson,
Chief Biologist,  National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.
                                                Descriptors:
                                                Aquatic Environment, Estuarine Environment,
                                                Lentic Environment,  Lotic Environment,
                                                Currents,  Marshes, Limnology, Water
                                                Properties
                                                                                  9-7

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                              THE AQUATIC ENVIRONMENT

                    Part 2: The Aquatic Environment as an Ecosystem
 I  INTRODUCTION

 Part 1 introduced the lithosphere and the
 hydrosphere.  Part 2 will deal with certain
 general aspects of the biosphere, or the
 sphere of life on this earth, which photo-
 graphs from space have shown is a finite
 globe in infinite space.

 This is the habitat of man and the other
 organisms.  His relationships with the
 aquatic biosphere are our common concern.
II   THE BIOLOGICAL NATURE OF THE
    WORLD WE LIVE IN

 A  We can only imagine what this world
    must have been like before there was life.

 B  The world as we know it is largely shaped
    by the forces of life.

    1   Primitive forms of life created organic
       matter and established soil.

    2   Plants cover the lands and enormously
       influence the forces of erosion.

    3   The nature and rate of erosion affect
       the redistribution of materials
       (and mass) on the surface of the
       earth (topographic changes).

    4   Organisms tie up vast quantities of
       certain chemicals, such as carbon
       and oxygen.

    5   Respiration of plants and animals
       releases carbon dioxide to the
       atmosphere in influential quantities.
       CO. affects the heat transmission of
       the atmosphere.
 C  Organisms respond to and in turn affect
    their environment.  Man is one of the
    most influential.
HI  ECOLOGY IS THE STUDY OF THE
    INTERRELATIONSHIPS BETWEEN
    ORGANISMS, AND BETWEEN ORGA-
    NISMS AND THEIR ENVIRONMENT.

 A  The ecosystem is the basic functional
    unit of ecology.  Any area of nature that
    includes living organisms and nonliving
    substances interacting to produce an
    exchange of materials between the living
    and nonliving parts constitutes an
    ecosystem. (Odum, 1959)

    1  From a structural standpoint, it is
       convenient to recognize four
       constituents as composing an
       ecosystem (Figure 1).

       a  Abiotic NUTRIENT  MINERALS
          which are the physical stuff of
          which living protoplasm will be
          synthesized.

       b  Autotrophic (self-nourishing) or
          PRODUCER organisms.  These
          are largely the green plants
          (holophytes), but other minor
          groups must also be included
          (See Figure 2).  They  assimilate
          the nutrient minerals, by the use
          of considerable energy,  and combine
          them into living organic substance.

       c  Heterotrophic (other-nourishing)
          CONSUMERS (holozoic). are chiefly
          the animals. They ingest (or eat)
          and digest organic matter, releasing
          considerable energy in the process.

       d Heterotrophic REDUCERS are chiefly
          bacteria  and fungi that return
          complex organic compounds back to
          the original abiotic mineral condition,
          thereby releasing the remaining
          chemical energy.

       From a functional standpoint, an
       ecosystem has two parts (Figure 2)
 BI. 21e.l.74
                                                                                  9-9

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The Aquatic Environment
                                CO NSUMERS
        PRO DUCERS
                                                           REDUCERS
                                    NUTRIENT
                                    MINERALS
                                       FIGURE 1
B
     a  The autotrophic or producer
        organisms, which construct
        organic substance.

     b  The heterotrophic or consumer and
        reducer organisms which destroy
        organic substance.

   3  Unless the autotrophic and hetero-
     trophic phases of the cycle approximate
     a dynamic equilibrium,  the ecosystem
     and the environment will change.

   Each of these groups includes simple,
   single- celled representatives, persisting
   at lower levels on the evolutionary stems
   of the higher organisms.  (Figure 2)

   1  These groups span the gaps between the
     higher kingdoms with a multitude of
     transitional forms.  They are  collectively
     caUed the PROTISTA.
   2  Within the protista, two principal sub-
     groups can be defined on the basis of
     relative complexity of structure.

     a  The bacteria and blue-green algae,
        lacking a nuclear membrane may
        be considered as the lower protista
        (or Monera).

     b  The single-celled algae and
        protozoa are best referred to as
        the Higher Protista.

C  Distributed throughout these groups will
   be found most of the traditional "phyla"
   of classic biology.
                                             IV  FUNCTIONING OF THE ECOSYSTEM

                                              A  A food chain is the transfer of food energy
                                                 from plants through a series of organisms
                                                 with repeated eating and being eaten.
                                                 Food chains are not isolated sequences but
                                                 are interconnected.
  9-10

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                                                             The Aquatic Environment
RELATIONSHIPS  BETWEEN  FREE  LIVING  AQUATIC  ORGANISMS

          Energy Flows from Left to Right, General Evolutionary Sequence is Upward

     PRODUCERS     |

 Organic Material Produced,
 Usually by Photosynthesis I
       CONSUMERS
   Organic Material Ingested or
          Consumed
       Digested Internally
     REDUCERS

Organic Material Reduced
by Extracellular Digestion
and Intracellular Metabolism
to Mineral Condition
     ENERGY STORED
                             ENERGY RELEASED
                                                             ENERGY RELEASED
Flowering Plants and
Gymnosperms
Club Mosses, Ferns
Liverworts, Mosses
Multicellular Green
Algae
Red Algae
Brown Algae
Arachnids
Insects
Crustaceans
Mammals
Birds
Reptiles
Segmented Worms Amphibians
Molluscs Fishes
Bryozoa
Rotifers
Roundworms
Flat worms
Sponges
Primitive
Chordates
Echinoderms

Coelenterates
Basidiomycetes

Fungi Imperfect!
Ascomycetes

Higher Phycomycetes
          DEVELOPMENT OF MULTICELLULAR OR COENOCYTIC STRUCTURE

                H  I  G" H  E  R      P  R  0 T  I  S   T  A
  Unicellular Green Algae

  Diatoms


  Pigmented Flagellates
        Protozoa


Amoeboid           Cilliated
Flagellated,
 (non-pigmented)
                   Suctoria
 Lower

   Phycomycetes


   (Chytridiales, et. al. )
                      DEVELOPMENT OF A NUCLEAR MEMBRANE
                       1
                 LOWER      PROTISTA
                                (or:    Monera)
  Blue Green Algae



         Phototropic Bacteria



                Chemotropic Bacteria
                                    Actinomycetes


                               Spirochaetes
                     Saprophytic
                     Bacterial
                     Types
  BI.ECO.pl. 2a. 1. 69
                                 FIGURE 2
                                                                                 9-11

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The Aquatic Environment
B  A food web is the interlocking pattern of
   food chains in an ecosystem.  (Figures  3,4)
   In complex natural communities, organisms
   whose food is obtained by the same number
   of steps are  said to belong to the same
   trophic (feeding) level.

C  Trophic Levels

   1  First - Green plants (producers)
      (Figure 5) fix biochemical energy and
      synthesize basic organic substances.
      This is 'primary production .
   2  Second - Plant eating animals (herbivores)
      depend on the producer organisms for
      food.

   3  Third - Primary carnivores, animals
      which feed on herbivores.

   4  Fourth - Secondary carnivores feed on
      primary carnivores.

   5  Last -  Ultimate carnivores are the last
      or ultimate level of consumers.
D Total Assimilation

   The amount of energy which flows through
   a trophic level is distributed between the
   production of biomass (living substance),
   and the demands of respiration (internal
   energy use by living organisms) in a ratio
   of approximately 1:10.
E Trophic Structure of the Ecosystem

   The interaction of the food chain
   phenomena (with energy loss at each
   transfer) results in various communities
   having definite trophic  structure  or energy
   levels.  Trophic structure may be
   measured and described either in terms
   of the standing crop per unit area or in
   terms of energy fixed per unit area per
   unit time at successive trophic levels.
   Trophic structure and function can be
   shown graphically by means of ecological
   pyramids (Figure 5).
        Figure 3. Diagram rf the pond ecosystem. Basic units are as follows: I, abiotic substances-basic inorganic and
         organic compounds; HA, producers—rooted vegetation; IIB, producers—phytoplankton; III-1A, primary consumers
         (herbivores)—bottom forms; Ill-IB, primary consumers (herbivores)— zooplanlcton; III-2, secondary consumers (car-
         nivorej); III-3, tertiary consumers (secondary carnivores); IV, decomposers-bacteria and fungi of decay.
  9-12

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                                              	The Aquatic Environment
  	u-	
       .-••/; •; Light ..•;-••	

Figure 4. A MARINE ECOSYSTEM (After Clark, 1954 and Patten, 1966)
                                                            9-13

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 The Aquatic Environment

(a)
Decomposers 11
r1
1


Carnivores (Secondar
Carnivores (Primary
1 herbivores
Producers
(b)
1
1
(c)
Pn
..wmi
?{]// / / / / /
l///)v / 1 1 1 1 1 / 1
I
I

rf\
/ i i i\
i / i i / iii
 Figure 5.  HYPOTHETICAL PYRAMIDS of
 (a) Numbers of individuals, (b) Biomass, and
 (c) Energy (Shading Indicates Energy Loss).
V  BIOTIC COMMUNITIES

 A Plankton are the macroscopic and
   microscopic animals,  plants, bacteria,
   etc., floating free in the open water.
   Many clog filters, cause tastes, odors,
   and other troubles in water supplies.
   Eggs and  larvae of larger forms are
   often present.

   1  Phytoplankton are plant-like.  These
      are the dominant  producers of the
      waters, fresh and salt,  "the grass
      of the seas".

   2  Zooplankton are animal-like.
      Includes many different animal types,
      range in size from minute protozoa
      to gigantic marine jellyfishes.

B  Periphyton (or Aufwuchs) - The communities
   of microscopic organisms associated with
   submerged surfaces  of any type or depth.
     Includes bacteria, algae, protozoa,  and
     other microscopic animals, and often the
     young or embryonic stages of algae and
     other organisms that normally grow up
     to become a part of the benthos (see below).
     Many planktonic types will also adhere
     to surfaces as periphyton, and some
     typical periphyton may break off and
     be collected as plankters.

  C  Benthos are the plants and animals living
     on, in, or closely associated with the
     bottom.  They include plants and
     invertebrates.

  D  Nekton are the community of strong
     aggressive swimmers of the open  waters,
     often called pellagic.  Certain fishes,
     whales, and invertebrates such as
     shrimps and squids are included here.

  E  The marsh community is based on larger
     "'higher" plants, floating and emergent.
     Both marine and freshwater marshes are
     areas of  enormous biological production.
     Collectively known as "wetlands",  they
     bridge the  gap between the waters  and the
     dry lands.

VI   PRODUCTIVITY

  A  The biological resultant of all physical
     and chemical factors in the quantity of
     life that may actually be present.  The
     ability to produce this "biomass" is
     often referred to as the "productivity"
     of a body of water.  This is neither good
    nor bad per se.  A water of low pro-
     ductivity is a "poor" water biologically,
     and also a relatively "pure" or "clean"
    water; hence  desirable as a water  supply
     or a bathing beach.  A productive water
    on the other hand may be a nuisance to
    man or highly desirable.  It is a nuisance
    if foul odors and/or weed-chocked
    waterways result,  it is desirable if
    bumper crops of bass, catfish, or
    oysters are produced.  Open oceans have
    a low level of productivity in general.
   9-14

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                                                              The Aquatic Environment
REFERENCES

1  Clarke,  G. L.  Elements of Ecology.
      John Wiley & Sons, New York.   1954.

2  Cooke, W. B.  Trickling Filter Ecology.
      Ecology 40(2):273-291.   1959.

3  Hanson,  E.D.  Animal Diversity.
      Prentice-Hall,  Inc., New Jersey.  1964.

4  Hedgpeth, J.W.  Aspects of the Estuarine
      Ecosystem   Amer. Fish. Soc., Spec.
      Publ. No. 3.   1966.
 5  Odum, E.P.  Fundamentals of Ecology,
      W. B. Saunders Company,
      Philadelphia and London.   1959.

 6  Patten, B.C.   Systems Ecology.
      Bio-Science.  16(9).   1966.

 7  Whittaker, R.H.  New Concepts of
      Kingdoms.  Science 163:150-160.  1969.
This outline was prepared by H. W. Jackson,
Chief Biologist,  National Training Center,
Water Programs Operations, EPA,
 Cincinnati,  OH  45268.
                                                Descriptors:
                                                Aquatic Environment, Estuarine Environment,
                                                Lentic Environment, Lotic Environment,
                                                Currents,  Marshes, Limnology, Water Properties
                                                                                  9-15

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                              THE AQUATIC ENVIRONMENT

                           Part 3.  The Freshwater Environment
 I  INTRODUCTION

 The freshwater environment as considered
 herein refers to those inland waters not
 detectably diluted by ocean waters, although
 the lower portions of rivers  are subject to
 certain tidal flow  effects.

 Certain atypical inland waters such as saline
 or alkaline lakes, springs, etc., are not
 treated, as the main objective here in typical
 inland water.

 All waters have certain basic biological cycles
 and types of interactions most of which have
 already been presented, hence this outline
 will concentrate on aspects essentially
 peculiar to fresh inland waters.
n  PRESENT WATER QUALITY AS A
   FUNCTION OF THE EVOLUTION OF
   FRESH WATERS

 A The history of a body of water  determines
   its present condition.  Natural waters have
   evolved in the course of geologic time
   into what we know today.

 B Streams

   In the course of their evolution, streams
   in general pass through four stages of
   development which may be called: birth,
   youth, maturity, and old age.

   These terms or conditions may be
   employed or considered in two contexts:
   temporal,  or spatial.  In terms of geologic
   time, a given point in a  stream may pass
   through each of the stages described below
   or:  at any given time, these various stages
   of development can be loosely identified
   in successive reaches of a stream traveling
   from its headwaters to base level in ocean
   or major lake.
1  Establishment or birth.  This
   might be a "dry run" or headwater
   stream-bed, before it had eroded
   down to the level  of ground water.

   During periods of run- off after a
   rain or  snow-melt,  such a  gulley
   would have a flow of water  which
   might range from torrential to a
   mere trickle.  Erosion may proceed
   rapidly  as there is no permanent
   aquatic  flora or fauna to  stabilize
   streambed materials.  On the other
   hand,  terrestrial grass or  forest
   growth may retard erosion.  When
   the  run-off has passed, however,
   the  "streambed" is dry.

2  Youthful streams. When the
   streambed is eroded below the
   ground water level, spring or
   seepage water enters,  and  the
   stream becomes permanent.  An
   aquatic  flora and  fauna develops
   and water flows the year round.
   Yout hful streams typically have a
   relatively steep gradient, rocky beds,
   with rapids,  falls, and small pools.

3  Mature  streams.  Mature streams
   have wide valleys, a developed
   flood plain, are deeper,  more
   turbid, and usually have  warmer
   water,  sand,  mud, silt,  or clay
   bottom materials  which shift with
   increase in flow.  In their more
   favorable reaches, streams in this
   condition are at a peak of biological
   productivity.   Gradients  are moderate,
   riffles or rapids are often separated
   by long pools.

4  In old age, streams have approached
   geologic base level, usually the
   ocean.   During flood stage  they scour
   their beds and deposit materials on
   the  flood plain which may be very
   broad and flat.  During normal flow
   the  channel is  refilled and many
   shifting bars are  developed.
 BI.21e. 1.74
                                                                                      9-17

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The Aquatic Environment
         (Under the influence of man this
         pattern may be broken up,  or
         temporarily interrupted.  Thus an  '
         essentially "youthful" stream might
         take on some of the characteristics
         of a "mature" stream following soil
         erosion,  drganic enrichment, and
         increased surface runoff.   Correction
         of these conditions might likewise be
         followed by at least a partial reversion
         to the "original" condition).

C Lakes and Reservoirs

   Geological factors which significantly
   affect the nature of either a stream or
   lake include the following:

   1  The geographical location of the
      drainage basin or watershed.

   2  The size and shape of the drainage
      basin.

   3  The general topography, i.e.,
      mountainous or plains.

   4  The character of the bedrocks and
      soils.

   5  The character,  amount, annual
      distribution, and rate of precipitation.

   6  The natural vegetative cover  of the
      land, is, of course,  responsive to and
      responsible for many  of the above
      factors and is also severely subject
      to the whims of civilization.  This
      is one of the major factors determining
      run-off versus soil absorption, etc.

D Lakes have a developmental history which
   somewhat parallels that of streams.   This
   process is often referred to as natural
   eutrophication.

   1  The methods of formation vary greatly,
      but all influence the character and
      subsequent history of the lake.

      In glaciated areas, for example, a
      huge block of ice may have  been covered
      with till.   The glacier retreated, the
      ice melted, ahd the resulting hole
9-18
   became a lake.  Or, the glacier may
   actually scoop out a hole.   Landslides
   may dam valleys, extinct volcanoes  may
   collapse, etc., etc.

2  Maturing or natural eutrophication of
   lakes.

   a  If not already present shoal areas
      are developed through erosion
      and deposition of the shore material
      by wave action and undertow.

   b  Currents produce bars across bays
      and thus cut off irregular areas.

   c  Silt brought in by tributary streams
      settles out in the quiet lake water

   d  Algae grow attached to surfaces,
      and floating free as plankton.  Dead
      organic matter begins to accumulate
      on the bottom.

   e  Rooted aquatic plants grow on
      shoals and bars, and in doing so
      cut  off bays and  contribute to the
      filling of the lake.

   f  Dissolved carbonates and other
      materials are precipitated in the
      deeper portions  of the lake in part
      through the action  of plants.

   g  When filling is well advanced,
      mats of sphagnum  moss may  extend
      outward from the shore. These
      mats are followed by sedges and
      grasses which finally convert the
      lake into a  marsh.

3  Extinction of lakes.  After lakes reach
   maturity, their progress toward
   filling  up is accelerated.  They become
   extinct through:

   a  The downcutting of the outlet.

   b  Filling with detritus eroded from
      the  shores  or brought in by
      tributary streams.

   c  Filling by the accumulation of the
      remains of vegetable materials
      growing in the lake itself.
      (Often two or three processes may
      act  concurrently)

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                                                                The Aquatic Environment
III  PRODUCTIVITY IN FRESH WATERS

 A Fresh waters in general and under
    natural conditions by definition have a
    lesser supply of dissolved substances
    than marine waters, and thus a lesser
    basic potential for the  growth of aquatic
    organisms.  By the same token, they
    may be said to be more sensitive to the
    addition of extraneous  materials
    (pollutants, nutrients,  etc.) The
    following notes are directed toward
    natural geological and  other environ-
    mental factors as they affect the
    productivity of fresh waters.

 B Factors Affecting Stream Productivity
    (See Table 1)

                TABLE 1

     EFFECT OF SUBSTRATE ON STREAM
                PRODUCTIVITY*

     (The productivity of sand bottoms is
     taken as 1)
         Bottom Material
  Sand
  Marl
  Fine Gravel
  Gravel and silt
  Coarse gravel
  Moss on fine gravel
  Fissidens (moss) on coarse
    ;   gravel
  Ranunculus (water buttercup)
  Watercress
  Anacharis (waterweed)
Relative
Productivity
     1
     6
     9
    14
    32
    89
   111

   194
   301
   452
  *Selected from Tarzwell 1937

    To be productive of aquatic life, a
    stream must provide adequate nutrients,
    light, a  suitable temperature,  and time
    for growth to take place.

    1  Youthful streams, especially on rock
       or sand substrates are low in essential
       nutrients.  Temperatures in moun-
       tainous regions are usually low, and
       due to the steep gradient, time for
       growth is short.  Although ample
       light is available,  growth of true
       plankton is thus greatly limited.
       2 As the stream flows toward a more
         "mature" condition, nutrients tend to
         accumulate, and gradient diminishes
         and so time of flow increases, tem-
         perature tends to increase, and
         plankton flourish.

         Should a heavy load of inert silt
         develop on the other hand, the
         turbidity would reduce the light
         penetration and consequently the
         general plankton production would
         diminish.

       3 As the stream approaches base level
         (old age) and the time available for
         plankton growth increases, the
         balance between turbidity, nutrient
         levels, and temperature and other
         seasonal conditions, determines the
         overall productivity.

  C Factors Affecting the Productivity of
    lakes (See  Table  2)

        1  The size,  shape, and depth of the
         lake  basin.  Shallow water is more
         productive than deeper water since
          more light will reach the bottom to
          stimulate rooted plant growth.  As
          a corollary, lakes with more shore-
          line, having more shallow water,
          are in general more productive.
          Broad shallow lakes and reservoirs
          have the greatest production potential
          (and hence  should be avoided for
          water supplies).

                TABLE 2

        EFFECT OF SUBSTRATE
        ON LAKE PRODUCTIVITY *
(The productivity of sand bottoms is taken as 1)
Bottom Material
Sand
Pebbles
Clay
Flat rubble
Block rubble
Shelving rock
Relative Productivity
1
4
8
9
11
77
                   * Selected from Tarzwell 1937
                                                                                       9-19

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The Aquatic Environment
   2  Hard waters are generally more
      productive than soft waters as there
      are more  plant nutrient minerals
      available.  This is often greatly in-
      fluenced by the character  of the soil
      and rocks in the watershed and the
      quality an'd quantity of ground water
      entering the lake.  In general, pH
      ranges of  6. 8 to 8.2 appear to be
      most productive.

   3  Turbidity  reduces productivity as
      light penetration is reduced.

   4  The presence or absence of thermal
      stratification with its semi-annual
      turnovers  affects productivity by
      distributing nutrients throughout the
      water mass.

   5  Climate, temperature, prevalence of
      ice and snow,  are also of  course
      important.

   Factors Affecting the Productivity of
   Reservoirs

   1  The productivity of reservoirs is
      governed by much the same principles
      as that of lakes,  with the difference
      that the water  level is much more
      under  the control of man.  Fluctuations
      in water level  can be used to de-
      liberately  increase or decrease
      productivity.   This can be demonstrated
      by a comparison of the TVA reservoirs
      which  practice a summer drawdown
      with some of those in the west where
      a winter drawdown is the rule.

   2  The level at which water is removed
      from a reservoir is important to the
      productivity of the stream below.
      The hypolimnion may be anaerobic
      while the epilimnion is aerobic, for
      example, or the epilimnion is poor in
      nutrients while the hypolimnion is
      relatively  rich.

   3  Reservoir  discharges also profoundly
      affect  the DO,  temperature,  and
      turbidity in the stream below a dam.
      Too much fluctuation in flow may
      permit sections of the stream to dry,
      or provide inadequate dilution for
      toxic waste.
IV  CULTURAL EUTROPHICATION

 A  The general processes of natural
    eutrophication, or natural enrichment
    and productivity have been briefly out-
    lined above.

 B  When the activities of man speed up
    these enrichment processes by intro-
    ducing unnatural quantities of nutrients
    (sewage, etc.) the result is often called
    cultural eutrophication.  This term is
    often extended beyond its original usage
    to include the enrichment (pollution) of
    streams, estuaries, and even oceans, as
    well as lakes.
V CLASSIFICATION OF LAKES AND
   RESERVOIRS

A The productivity of lakes and impound-
   ments is such a conspicuous feature that
   it is often used as a convenient means of
   classification.

   1  Oligotrophic lakes are the younger,
      less productive lakes, which are deep,
      have clear water, and usually support
      Salmonoid fishes in their deeper waters.

   2  Eutrophic lakes are more mature,
      more turbid,  and'richer.  They are
      usually shallower.  They are richer
      in dissolved solids; N,  P, and Ca are
      abundant.  Plankton is abundant and
      there is often a rich bottom fauna.

   3  Dystrophic lakes, such as bog lakes,
      are low in Ph, water yellow to brown,
      dissolved solids, N,  P,  and Ca scanty
      but humic materials abundant, bottom
      fauna and plankton poor,  and fish
      species are limited.

B Reservoirs may also be classified as
   storage, and run of the river.

   1  Storage reservoirs have a large
      volume in relation to their inflow.

   2  Run of the river reservoirs have a
      large flow-through in relation to their
      storage value.
9-20

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                                                                The Aquatic Environment
    According to location, lakes and
    reservoirs may be classified as polar,
    temperate, or tropical.   Differences in
    climatic and geographic conditions
    result in differences in their biology.
VI  SUMMARY

 A A body of water such as a lake, stream,
    or estuary represents an intricately
    balanced system in a state of dynamic
    equilibrium.  Modification imposed at
    one point in the system automatically
    results in compensatory adjustments at
    associated points.

 B The more thorough our knowledge of the
    entire system, the better  we can judge
    where to impose control measures to
    achieve a desired result.
 6  Tarzwell,  Clarence M.  Experimental
      Evidence on the Value of Trout 1937
      Stream Improvement in Michigan.
      American Fisheries Society Trans.
      66:177-187.  1936.

 7  U. S. Dept. of Health,  Education, and
      Welfare.  Public Health Service.
      Algae and Metropolitan Wastes.
      Transactions of a seminar held
      April 27-29,  1960 at the Robert A.
      Taft Sanitary Engineering Center.
      Cincinnati, OH.  No. SEC TR W61-3.

 8  Ward and Whipple.  Fresh Water
      Biology. (Introduction). John
      Wiley Company.  1918.
 REFERENCES

 1  Chamberlin,  Thomas C.  and Salisburg,
       Rollin P.  Geological Processes and
       Their Results.  Geology 1: pp i-xix,
       and. 1-654. Henry Holt and Company.
       New York. 1904.

 2  Frey,  David  G.  Limnology in North
       America.   Univ.  Wise. Press.  1963.

 3  Hutcheson, George E. A Treatise on
       Limnology Vol. I  Geography, Physics
       and Chemistry.  1957. Vol. II.
       Introduction to Lake Biology and the
       Limnoplankton.  1115  pp.  1967.
       John Wiley Co.

 4  Hynes, H.B.N.  The Ecology of Running
       Waters.  Univ.  Toronto Press.
       555 pp.  1970.

 5  Ruttner,  Franz. Fundamentals of
       Limnology.  University of Toronto
       Press,  pp.  1-242.  1953.
 This outline was prepared by H. W.  Jackson,
 Chief Biologist, National  Training Center,
 Water Programs Operations, EPA, Cincinnati,
 OH 45268.
 Descriptors:
Aquatic Environment, Estuarine  Environment,
Lentic Environment, Lotic Environment,
 Currents, Marshes, Limnology, Water
 Properties
                                                                                     9-21

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                              THE AQUATIC ENVIRONMENT
     Part 4.  The Marine Environment and its Role in the Total Aquatic Environment
                                                                TABLE 1
I  INTRODUCTION

A  The marine environment is arbitrarily
   defined as the water mass extending
   beyond the continental land masses,
   including the plants and animals harbored
   therein.  This water mass is large and
   deep, covering about 70 percent of the
   earth's surface and being as deep as
   7 miles.  The salt content averages
   about 35 parts per thousand.  Life extends
   to all depths.

B  The general nature of the water cycle on
   earth is well known.  Because the  largest
   portion of the surface area of the earth
   is covered with water,  roughly 70  percent
   of the earth's rainfall is on the seas.
   (Figure 1)
  100* Oceanic
   Evaporation
           iire 1.  THE WATER CICLE
 Since roughly one third of the
 rain which falls on the land is again
 recycled through the  atmosphere
 (see Figure 1 again), the total amount
 of water washing over the earth's surface
 is significantly greater than one third of
 the total world rainfall.  It  is thus not
 surprising to note that the rivers which
 finally empty into the sea carry a
 disproportionate burden of dissolved and
 suspended  solids picked up  from the land.
 The chemical composition of this burden
 depends on the composition  of the rocks
 and soils through which the  river flows,
 the proximity of an ocean, the direction
 of prevailing winds,  and other factors.
 This is the substance of geological erosion.
 (Table 1)

BI.21e.l.74
                                                 PERCENTAGE COMPOSITION OF THE MAJOR IONS
                                                      OF TWO STREAMS AND SEA WATER

                                               (Data from Clark, F.W., 1924, "The Composition of River
                                               and Lake Waters of the United States", U. S. Geol. Surv.,
                                               Prof. Paper No. 135; Harvey, H.W.,  1957, "The Chemistry
                                               and Fertility of Sea Waters", Cambridge University  Press,
                                               Cambridge)
Ion
Na
K
Ca
Mg
Cl
so4
C03
Delaware River
at
Lambertville, N. J.
6.70
1.46
17.49
4.81
4.23
17.49
32.95
Rio Grande
at
Laredo, Texas
14.78
.85
13.73
3.03
21.65
30. 10
11.55
Sea Water
30.4
1.1
1. 16
3.7
55.2
7.7
•t-HCO. 0.35
o
 C  For this presentation,  the marine
    environment will be (1) described using
    an ecological approach, (2) characterized
    ecologically by comparing it with fresh-
    water and estuarine environments, and
    (3) considered as a functional ecological
    system (ecosystem).
H   FRESHWATER,  ESTUARINE, AND
    MARINE ENVIRONMENTS

 Distinct differences are found in physical,
 chemical, and biotic factors in going from
 a freshwater to an oceanic environment.
 In  general, environmental factors are more
 constant in freshwater (rivers) and oceanic
 environments than in the highly variable
 and harsh environments of estuarine and
 coastal waters.  (Figure 2)

 A  Physical and Chemical Factors

    Rivers, estuaries,  and oceans are
    compared in Figure 2 with reference to
    the relative instability (or variation) of
    several important parameters.  In the
    oceans, it will be noted, very little change
    occurs in any parameter.  In rivers, while
    "salinity" (usually referred to as "dissolved
    solids") and temperature (accepting normal
    seasonal variations) change little, the other
    four parameters vary considerably.  In
    estuaries, they all  change.
                                       9-23

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  The Aquatic Environment
Type of environment
and general direction
 of water movement
Salinity
                                     Degree of instability
Temperature
 Water
elevation
vertical
strati-
fication
 Avail-
 ability
   of
nutrients
(degree)
Turbidity
 Riverine
    Oceanic
       Figure 2 .  RELATIVE VALUES OF VARIOUS PHYSICAL AND CHEMICAL FACTORS
                  FOR RIVER, ESTUARINE, AND OCEANIC ENVIRONMENTS
 B  Biotic Factors

    1  A complex of physical and chemical
       factors determine the biotic composi-
       tion of an environment.  In general,
       the number of species in a rigorous,
       highly variable environment tends to be
       less than the number in a more stable
       environment (Hedgpeth,  1966).

    2  The dominant animal species (in
       terms of total biomass) which occur
       in estuaries are often transient,
       spending only a part of their lives in
       the estuaries. This results in better
       utilization of a rich environment.
                           C Zones of the Sea

                             The nearshore environment is often
                             classified in relation to tide level and
                             water depth.  The nearshore and offshore
                             oceanic regions together, are often
                             classified with reference to light penetra-
                             tion and water depth.  (Figure 3)

                             1  Neritic  - Relatively shallow-water
                                zone which extends from the high-
                                tide mark to the edge of the
                                continental shelf.
   9-24

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                                                           The Aquatic Environment
                                  MARINE ECOLOGY
     TN^i
           BENTHIC (Bottom)
            Supro-llltorol
            lillorol (Inl.ftiJol)
            Subliltorol
              Innir
              Oulfr
            AbtlfOl
                                BENTHIC
                       FIGURE 3—Classification of marine environments
a  Stability of physical factors is
   intermediate between estuarine
   and oceanic environments.

b  Phytoplankters are the dominant
   producers but in some locations
   attached algae are also important
   as producers.

c  The animal  consumers are
   zooplankton, nekton, and benthic
   forms.

Oceanic  - The  region of the ocean
beyond the continental shelf.  Divided
into three parts, all relatively
poorly populated compared to the
neritic zone.

a  Euphotic zone -  Waters into which
   sunlight penetrates (often to the
   bottom in the neritic zone).  The
   zone of primary productivity often
   extends to 600 feet below the surface.
   1) Physical factors fluctuate
      less than in the neritic zone.

   2) Producers are the phyto-
      plankton and consumers are
      the zooplankton and nekton.

b  Bathyal zone - From the bottom
   of the euphotic zone to about
   2000 meters.

   1) Physical factors relatively
      constant but light is absent.

   2) Producers are absent and
      consumers are scarce.

c  Abyssal zone - All the sea below
   the bathyal zone.

   1) Physical factors more con-
      stant than in bathyal zone.

   2) Producers absent and consumers
      even less abundant than in the
      bathyal zone.
                                9-25

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 The Aquatic Environment
in  SEA WATER AND THE BODY FLUIDS

 A Sea water is a remarkably suitable
    environment for living cells, as it
    contains all of the chemical elements
    essential to the growth and maintenance
    of plants and animals.  The ratio and
    often the concentration of the major
    salts of sea water are strikingly similar
    in the cytoplasm   and body fluids of
    ,marine organisms.  This similarity is
    also evident, although modified somewhat
    in the body fluids of fresh  water and
    terrestrial animals.  For example,
    sterile sea water may be used in
    emergencies as a substitute for blood
    plasma in man.

 B Since marine organisms have an internal
    salt  content similar to that of their
    surrounding medium (isotonic condition)
    osmoregulation poses no problem.  On  the
    other hand,  fresh water  organisms are
    hypertonic (osmotic pressure of body
    fluids is higher than that of the surround-
    ing water).  Hence,  fresh water animals
    must constantly expend more energy to
    keep water out (i.e., high osmotic
    pressure fluids contain more salts, the
    action being then to dilute this concen-
    tration with more water).

    1  Generally, marine invertebrates are
       narrowly poikilosmotic, i.e., the salt
       concentration of the body fluids changes
       with that of the external medium.  This
       has special^ significance in estuarine
       situations where salt  concentrations
       of the water often vary considerably
       in short periods of time.

    2  Marine bony fish (teleosts) have lower
       salt content internally than the  external
       environment (hypotonic).  In order to
       prevent dehydration,  water is ingested
       and salts are excreted through  special
       cells in the gills.
IV FACTORS AFFECTING THE DISTRI-
   BUTION OF MARINE AND ESTUARINE
   ORGANISMS

 A Salinity.  Salinity is the single most
   constant and controlling factor in the
   marine environment, probably followed
   by temperature.   It ranges around
   35, 000 mg. per liter,  or "35 parts per
   thousand" (symbol: 35%0) in the language
   of the oceanographer.   While variations
   in the open ocean are relatively small,
   salinity decreases rapidly as one
   approaches shore and proceeds through
   the estuary and up into fresh water with
   a salinity of "0 %0 (see Figure 2)

 B Salinity and temperature as  limiting
   factors in ecological distribution.

   1  Organisms  differ in the salinities
      and temperatures in which they
      prefer to live, and in the  variabilities
      of these  parameters which they can
      t olerate. These preferences and
      tolerances often change with successive
      life history  stages, and in turn often
      dictate where  the organisms live:
      their "distribution."

   2  These requirements or preferences
      often lead to extensive migrations
      of various species for breeding,
      feeding,  and growing stages.  One
      very important result of this is that
      an estuarine environment is an
      absolute necessity for over half of
      all coastal commercial and sport
      related species of fishes and invertebrates,
      for either all  or certain portions of their
      life histories.  (Part V,  figure  8)

   3  The Greek word roots "eury"
      (meaning wide) and "steno" (meaning
      narrow)  are customarily  combined
      with such words as "haline" for salt,
      and "thermal" for temperature, to
      give us  "euryhaline" as an adjective
      to characterize an organism able to
      tolerate  a wide range of salinity, for
      example; or "stenothermal" meaning
      one which cannot stand much change
      in temperature. "Meso-" is a prefix
      indicating an intermediate'capacity.
    9-26

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                                                               The Aquatic Environment
C  Marine, estuarine,  and fresh water
   organisms.  (See Figure 4)
Fresh Water
Stenohaline
Marine
Stenohaline
                  Salinity
       ca.35
 Figure 4.  Salinity Tolerance of Organisms

   1  Offshore marine organisms are, in
      general, both stenohaline and
      stenothermal unless, as noted above,
      they have certain life history require-
      ments for estuarine conditions.

   2  Fresh water organisms are also
      stenohaline,  and (except for seasonal
      adaptation) meso- or stenothermal.
      (Figure 2)

   3  Indigenous or native estuarine species
      that normally spend their entire lives
      in the estuary are relatively few in
      number.  (See Figure 5). They are
      generally me so- or euryhaline and
      meso- or euryt.hermal.
              10
                   Salinity
                           25  30  35
     Figure 5.  DISTRIBUTION OF
                ORGANISMS IN AN ESTUARY

        a  Euryhaline,  freshwater
        b  Indigenous,  estuarine, (mesohaline)
        c  Euryhaline,  marine
   4   Some well known and interesting
       examples of migratory species which
       change their environmental preferences
       with the life history stage include the
       shrimp (mentioned above), striped bass,
       many herrings and relatives, the salmons,
       and many others. None are more
       dramatic than the salmon hordes which
       lay their eggs in freshwater streams,
       migrate far out to sea to feed and grow,
       then return to the stream where they
       hatched to lay their own eggs before
       dying.

   5  Among euryhaline animals landlocked
      (trapped), populations living in lowered
      salinities often have a smaller maximum
      size than individuals of the same species
      living in more saline waters.  For
      example, the lamprey (Petromyzon
      marinus)  attains a length of 30 - 36"
      in the sea, while in the Great Lakes
      the length is 18 -  24".

      Usually the larvae of aquatic organisms
      are more sensitive to changes in
      salinity than are the adults. This
      characteristic both limits and dictates
      the distribution and size of populations.

D  The effects of tides on organisms.

   1  Tidal fluctuations probably subject
      the benthic or intertidal populations
      to the most extreme and rapid variations
      of environmental stress  encountered
      in any aquatic habitat.  Highly specialized
      communities have developed in this
      zone,  some adapted to the rocky surf
      zones of the open coast,  others  to the
      muddy inlets of protected estuaries.
      Tidal reaches of fresh water rivers,
      sandy beaches, coral reefs and
      mangrove swamps in the tropics; all
      have their own floras and faunas. All
      must emerge and flourish when whatever
      wateir there is rises and covers or
      tears at them,  all must collapse or
      retract to endure drying,  blazing
      tropical sun, or freezing arctic ice
      during the low tide interval. Such a
      community is depicted in Figure 6.
                                                                                     9-27

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The Aquatic Environment
     SNAILS

 o   Littorina neritoides
 C"   L. rudis
 0   L. obtusata
 Q   L. littorea

     BAK.NACLES

 ®   Chthamalus stellatus
 ®   Balanus balanoides
 ©   B. perforatus
  rt «'s i'ir f-j.^v.^-'v.-.;^'"?,-.. • „• • \ .',Y;K.>-:.N   /..-.."A ••
 «^^fp^ ,• 'W^iV.f^^^jc
ivKf "l/^^^X'-^V^^u: :;^^-:-:ir;'J «
6%> ® « cs ^w&^:m$M&;^
                                                 ^/OT
                                                 * J\ <\S'( 1(\'V • (// v» . 0 \'Y A VA-X'x^AXvv
                                       Figure 6
             Zonation of plants, snails,  and barnacles on a rocky shore.  While
             this diagram is based on the situation on the southwest coast of
             England,  the general idea of zonation may  be applied to any  temper-
             ate rocky ocean shore, though the species  will differ.  The gray
             zone consists largely  of lichens.  At the left is the zonation  of rocks
             with exposure too extreme to support algae; at the right,  on a  less
             exposed situation,  the animals are mostly  obscured by the algae.
             Figures at the right hand margin refer to the percent of time that
             the zone is  exposed to the air, i. e., the time that the tide is out.
             Three major1 zones can be  recognized:  the Littorina zone (above the
             gray zone); the Balanoid zone (between the gray  zone and the
             laminarias); and the Laminaria zone.  a.  Pelvetia canaliculata;
             b. P'ucus spiralis;  c. Ascophyllum nodosum; d. Fucus  serratus;
             e. Laminaria digitata. (Based on Stephenson)
 9-28

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                                                              The Aquatic Environment
V  FACTORS AFFECTING THE
   PRODUCTIVITY OF THE MARINE
   ENVIRONMENT

A  The sea is in continuous circulation.  With-
   out circulation, nutrients of the ocean would
   eventually become a part of the bottom and
   biological production would cease. Generally,
   in all oceans there exists a warm surface
   layer which overlies the colder water and
   forms a two-layer system of persistent
   stability.  Nutrient concentration is usually
   greatest in the lower zone. Wherever a
   mixing or disturbance of these two layers
   occurs biological production is greatest.

B  The estuaries are also a mixing  zone of
   enormous importance. Here the fertility
   washed off the land is mingled with the
   nutrient capacity of seawater,  and many
   of the would1 s most productive waters
   result.

C  When man adds his cultural contributions
   of sewage, fertilizer, silt or toxic waste,
   it is  no wonder that the dynamic  equilibrium
   of the ages is rudely upset, and the
   environmentalist cries,  "See what man
   hath  wrought"!
ACKNOWLEDGEMENT:

This outline contains selected material
from other outlines prepared by C. M.
Tarzwell, Charles L. Brown, Jr.,
C. G. Gunnerson,  W. Lee Trent, W.  B.
Cooke, B. H. Ketchum, J. K. McNulty,
J. L. Taylor,  R. M. Sinclair, and others.
 REFERENCES

 1  Harvey,  H. W.  The Chemistry and
      Fertility of Sea Water (2nd Ed.).
      Cambridge Univ.  Press, New  York.
      234pp.  1957.

 2  Hedgpeth,  J. W.  (Ed.).  Treatise on
      Marine Ecology and Paleoecology.
      Vol. I.  Ecology Mem.  67 Geol.
      Soc. Amer., New York.  1296pp.
      1957.

 3  Hill,  M. N. (Ed.).   The Sea. Vol. II.
      The Composition  of Sea Water
      Comparative and  Descriptive
      Oceanography.  Interscience Publs.
      John Wiley & Sons,  New York.
      554 pp.  1963.

 4  Moore, H. B.  Marine Ecology..  John
      Wiley & Sons,  Inc., New York.
      493 pp.  1958.

 5  Reid, G. K.  Ecology of Inland Waters
      and Estuaries.  Reinhold Publ.
      Corp.  New York. 375 pp.  1961.

 6  Sverdrup,  Johnson, and Fleming.
      The Oceans.  Prentice-Hall, Inc.,
      New York.   1087 pp.   1942.
This outline was prepared by H. W.  Jackson,
Chief Biologist, National Training Center,
Water Programs Operations, EPA, Cincinnati,
OH 45268.

Descriptors:
Aquatic Environment, Estuarine Environment,
Lentic Environment, Lotic  Environment,
Currents, Marshes, Limnology, Water Properties
                                                                                    9-29

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                                THE AQUATIC ENVIRONMENT

                                       Part 5:  Wetlands
 I   INTRODUCTION

 A  Broadly defined, wetlands are areas
    which are "to wet to plough but too
B
    saturated with water, salt or fresh,
    and numerous channels or ponds of
    shallow or  open water are common.
    Due to ecological features too numerous
    and variable to list here, they comprise
    in general a rigorous (highly stressed)
    habitat, occupied by a small relatively
    specialized indigenous (native) flora
    and fauna.

    They are prodigiously productive
    however, and many constitute an
    absolutely essential habitat for some
    portion of the life history of animal
    forms  generally recognized as residents
    of other habitats (Figure 8).  This is
    particularly true of tidal marshes as
    mentioned below.
 C  Wetlands in toto comprise a remarkably
    large proportion of the earth1 s surface,
    and the total organic carbon bound in
    their mass constitutes an enormous
    sink of energy.

 D  Since our main concern here is with
    the "aquatic"  environment,  primary
    emphasis will be  directed toward a
    description of wetlands as the transitional
    zone between the  waters and the land, and
    how their desecration by human culture
    spreads degradation in both directions.
II   TIDAL MARSHES AND THE ESTUARY     •=--=
B  Estuarine pollution studies are usually
   devoted to the dynamics of the circulating
   water, its chemical,  physical,  and
   biological parameters, bottom  deposits, etc.

C  It is easy to overlook the intimate relation-
   ships which exist between the bordering
   marshland,  the moving waters, the tidal
   flats, subtidal deposition, and seston
   whether of local, oceanic, or riverine
   origin.

D  The tidal marsh (some inland   areas also
   have salt marshes) is generally considered
   to be the marginal areas of estuaries and
   coasts in the intertidal zone,  which are
   dominated by emergent vegetation. They
   generally extend  inland to the farthest
   point reached by  the spring tides,  where
   they merge into freshwater swamps and
   marshes (Figure 1).  They may range in
   width from nonexistent on rocky coasts to
   many kilometers.
 A  "There is no other case in nature, save
    in the coral reefs, where the adjustment
    of organic relations to physical condition
    is seen in such a beautiful way as the
    balance between the growing marshes
    and the tidal streams by which they are
    at once nourished and worn away. "
    (Shaler,  1886)
                                              Figure 1.  Zon«lon In a positive New England eatuarjr. 1. Spring tide level. 2. Mean high tide.
                                              1. Mean low tide. 4. Bog hole. 5. Ice cleavage pool. 6. Chunk ot asjtlna turf depo.lled by Ice.
                                              7. Organic ooie with associated community, B. eelgrass (Zoitero). 8. Bibbed rauinll (modlolu.1-
                                              clom Imml - mud snail (Najja) community. 10. Sea lettuce (Uly»)
 BI. 21e.l.74
                                                                                        9-31

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  The Aquatic Environment
III  MARSH ORIGINS AND STRUCTURES

 A In general, marsh substrates are high" in
    organic content, relatively low in minerals
    and trace  elements.  The upper layers
    bound together with living roots called
    turf,  underlaid by more compacted peat
    type material.

    1  Rising  or eroding  coastlines may
       expose  peat from ancient marsh
       growth  to wave action which cuts
       into the soft peat rapidly (Figure 2).
Such banks are likely to be cliff-like,
and are often undercut.  Chunks of
peat are often found lying about on
harder substrate below high tide line.
If face of cliff is well above high water,
overlying vegetation is  likely to be
typically terrestrial of  the area.
Marsh type vegetation is probably
absent.

Low lying deltaic,  or sinking coast-
lines,  or those with low energy wave
action are likely to have active marsh
formation in progress.  Sand dunes
are also common in such areas
(Figure 3).  General coastal
configuration is a factor.
       Figure 2. Diagrammatic section of eroding peat cliff
                                                                      MHW-ig 1300tec
                                                                      MHW tf 19SO J AO
                                               Figure 3
                       Development of a Massachusetts Marsh since 1300 BC, involving an
                       18 foot rise in water level.  Shaded area indicates sand dunes. Note
                       meandering marsh tidal drainage.  A: 1300 BC. B:  1950 AD.
  9-32

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                                                                 The Aquatic Environment
a  Rugged or precipitous coasts  or
   slowly rising coasts, typically
   exhibit narrow shelves, sea cliffs,
   fjords, massive beaches, and
   relatively less marsh area (Figure 4).
   An Alaskan fjord subject to recent
   catastrophic subsidence and rapid
   deposition of glacial flour shows
   evidence of the recent encroachment
   of saline waters in the presence of
   recently buried trees and other
   terrestrial vegetation, exposure
   of layers of salt marsh peat along
   the edges of channels, and a poorly
   compacted young marsh turf developing
   at the new high water level (Figure 5).
                                                Figure 4 * River Mouth on a Slowly Rising Coast. Note absence
                                                       of deltaic development and relatively little marshland,
                                                       although mud flats stippled are extensive.
   Figure 5  Some general relationships in a northern fjord with a rising water level.  1. mean low
            water, 2.  maximum high tide, 3.  Bedrock, 4.  Glacial flour to depths in excess of
            400 meters, 5.  Shifting flats and channels. 6.  Channel against bedrock, 7. Buried
            terrestrial vegetation, 8.  Outcroppings of salt marsh peat.
   Low lying coastal plains tend to be
   fringed by barrier islands, broad
   estuaries and deltas, and broad
   associated marshlands (Figure 3).
Deep tidal channels fan out through
innumerable branching and often
interconnecting rivulets.  The
intervening grassy plains are
essentially at mean high  tide level.
                                                                                       9-33

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  The Aquatic Environment
          Tropical and subtropical regions
          such as Florida,  the Gulf Coast,
          and Central America,  are frequented •
          by mangrove swamps.   This unique
          type of growth is able to establish
          itself in shallow water and move out
          into progressively deeper areas
          (Figure 6).   The  strong deeply
          embedded roots enable the mangrove
          to resist considerable  wave action
          at times,  and the tangle  of roots
          quickly accumulates a  deep layer of
          organic sediment.  Mangroves
          in the south may  be considered to
          be roughly the equivalent of the
          Spartina marsh grass in the north
          as a land builder.  When fully
          developed, a mangrove swamp is an
          impenetrable thicket of roots over
          the tidal flat affording  shelter to an
          assortment of semi-? aquatic organisms
          such as various molluscs and
          crustaceans, and providing access
          from the nearby land to predaceous
          birds, reptiles,  and mammals.
          Mangroves are not restricted to
          estuaries,  but may develop out into
          shallow oceanic lagoons, or upstream
          into relatively fresh waters.
    TOPICAL   CONOCARPtrt     . AVICCNMA
     KWtST  Winston ISSOCIU SU.MUKM USOCIU
  Figure 6   Diagrammatic transect of a mangrove swamp
            showing transition from marine to terrestrial
            habitat.
      tidal marsh is the marsh grass, but very
      little of it is used by man as grass.
      (Table  1)

      The nutritional analysis of several
      marsh grasses as compared to dry land
      hay is  shown in Table 2.
'TABLE 1.  General Orders of Magnitude of Gross Primary Productivity in Terms
             of Dry Weight of Organic Matter Fixed Annually
      Ecosystem
                          gms/M /year
                      (grams/square metere/year)    Ibs/acre/year
  Land deserts* deep oceans        Tens
  Grasslands, forests, cutrophic     Hundreds
   lakes, ordinary agriculture
  Estuaries, deltas, coral reefs.     Thousands
   intensive agriculture (sugar
   cane, rice)
Hundreds
Thousands
Ten-thousands
      TABLE 2. Analyses of Some Tidal Marsh Grasses
                                                         T/A
                                                        Dry Wl.
                 Percentage Composition
           Protein    Fat      Fiber    Water
                                                                                               Ash
                                                N-lree Extract
 DiVicMis spicara (pure stand, dry)
   2.6        5.3      1.7      32.4     8.2      6.7        4S.5
 Short Spirtina alterniflora and Saficornia curopaea (in standing water)
   1.2        7.7      2.5      31.1     8.8     12.0        37.7
 Spartr'na altcrniflora (tall, pure stand m standing water)
   3.5        1.f>      2.0      29.0     8.3     15.5        37.3
 Sparll'na pa":m 'pijr«: slamj, dry)
   3.2        ',.0      '2.2      30.0     8.1      9.0        44.5
 Spurtina allcrnitlura and Spjrtinn paroni (mixed stand, wet)
   3.4        6.8      1.9      29.0     8.1     10.4        42.8
 Spjr/fnj attcrnifltira (short, WIM)
   2.2        0.0      2.4      30.4     8.7     13.3        36.3
Comparable Analyses for Hay
 1%l«iil       li.O      2.0      36.2     6.7      4.2        44.9
 ftuJiut     Tl.U      3.7      21).5     10.4      5.9        30.5

   Analyses performed by Roland W. Gilbert,  Department
   of Agricultural Chemistry, U. R. I.
IV  PRODUCTIVITY OF WETLANDS

 A  Measuring the productivity of grasslands
    is not easy,  because today grass is seldom
    used directly as such by man.  It is thus
    usually expressed as production of meat,
    milk,  or in the case of salt marshes, the
    total crop of animals that obtain food per
    unit of area.  The primary producer in a
  9-34

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                                                                  The Aquatic Environment
B  The actual utilization of marsh grass is
   accomplished primarily by its Decom-
   position and ingestion by micro organisms.
   (Figure 7) A  small quantity of seeds and
   solids is consumed directly by birds.
       Sto
               •PAKTIIU— '-  * otTHtTOt

      Figure  7  The nutritive composition of
      successive stages of decomposition of
      Spartina marsh grass, showing increase
      in protein and decrease in carbohydrate
      with increasing age and decreasing size
      of detritus particles.

      The quantity of micro invertebrates
      which thrive on  this wealth of decaying
      marsh has not been  estimated, nor has
      the actual production of small indigenous
      fishes and invertebrates such as the
      top minnows (Fundulus).  or the mud
      snails (Nassa),  and  others.

      Many forms of oceanic life migrate
      into the estuaries, especially the
      marsh areas,  for important portions
      of their life histories as is mentioned
      elsewhere (Figure 8).   It has been
      estimated that in excess of 60% of the
      marine commercial and sport fisheries
      are estuarine or marsh dependent in
      some way.
                                                                         O0O-
                                                                          EGGS
    Figure  8  Diagram of the life cycle
    of white shrimp (after Anderson and
    Lunz 1965).
3  An effort to make an indirect
   estimate of productivity in a Rhode
   Island marsh was made on a single
   August day by recording the numbers
   and kinds of birds that fed on a
   relatively small area (Figure 9).
   Between 700 and 1000 wild birds of
   12 species,  ranging from  100 least
   sandpipers to uncountable numbers
   of seagulls were  counted.   One food
   requirement  estimate for three-
   pound poultry in the confined inactivity
   of a poultry yard is approximately one
   ounce per pound of bird per day.
                                                         Oreater yellow legs (left)
                                                           and black duck
                                                                       Great blue heron
                                                      Figure 9  Some Common Marsh Birds
                                                                                        9-35

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The Aquatic Environment
      One-hundred black bellied plovers
      at approximately ten ounces each
      would weigh on the order of sixty   '
      pounds.  At the same rate of food
      consumption,  this would indicate
      nearly four pounds of food required
      for this species alone.  The much
      greater activity of the wild birds
      would obviously greatly increase their
      food requirements,  as would their
      relatively  smaller size.

      Considering the range of foods con-
      sumed, the sizes of the birds, and the
      fact that at certain seasons, thousands
      of migrating ducks and others pause
      to feed here, the enormous productivity
      of such a marsh can be better under-
      stood.
V  INLAND BOGS AND MARSHES

A  Much of what has been said of tidal
   marshes also applies to inland wetlands.
   As was mentioned earlier,  not all inland
   swamps are salt-free, any more than all
   marshes affected by tidal rythms are
   saline.

B  The specificity of specialized floras to
   particular types  of wetlands is perhaps
   more spectacular in freshwater wetlands
   than in the marine, where Juncus,
   Spartina, and Mangroves tend to dominate.

   1  Sphagnum,  or peat moss, is
      probably one of the most widespead
      and abundant wetland plants on earth.
      Deevey (1958) quotes an estimate that
      there is probably upwards of 223
      billions (dry weight) of tons of peat
      in the world today, derived during
      recent geologic time from Sphagnum
      bogs.  Particularly in the northern
      regions, peat moss tends to overgrow
      ponds and shallow depressions, eventually
      forming the vast tundra plains and
      moores of the north.

   2  Long lists of other bog and marsh plants
      might be cited, each with its own
      special requirements, topographical,
      and geographic distribution, etc.
      Included would be the familiar cattails,
      spike rushes, cotton grasses,  sedges,
      trefoils, alders, and many, many
      others.

C  Types of inland wetlands.

   1  As noted above (Cf: Figure 1)
      tidal marshes often merge into
      freshwater marshes and bayous.
      Deltaic tidal swamps and marshes
      are often saline in the seaward
      portion, and fresh in the landward
      areas.

   2  River bottom wetlands differ from
      those formed from lakes, since wide
      flood plains subject to periodic
      inundation  are the final stages of
      the erosion of river valleys, whereas
      lakes in general tend to be eliminated
      by the geologic processes of natural
      eutrophication  often involving
      Sphagnum and peat formation.
      Riverbottom marshes in the southern
      United States, with favorable climates,
      have luxurient growths such as the
      canebrake  of the lower Mississippi,
      or a characteristic timber growth
      such as cypress.

   3  Although bird life  is the most
      conspicuous animal element in the
      fauna (Cf:  Figure 9), many mammals,
      such as muskrats,  beavers, otters,
      and others are  also marsh-oriented.
      (Figure 12)
      Figure 12
9-36

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                                                                  The Aquatic Environment
VI  POLLUTION

 A No single statement can summarize the
    effects of pollution on marshlands as
    distinct from effects noted elsewhere on
    other habitats.

 B Reduction of Primary Productivity

    The primary producers in most wetlands
    are the grasses and peat mosses.
    Production may be reduced or eliminated
    by:

    1  Changes in the water level brought
       about by flooding or  drainage.

       a  Marshland areas  are sometimes
          diked .and flooded to produce fresh-
          water ponds.  This may be for
          aesthetic reasons, to suppress the
          growth of noxious marsh inhabitating
          insects such as mosquitoes or biting
          midges, to construct an industrial
          waste holding pond,  a thermal or a
          sewage stabilization pond, a
          "convenient" result of highway
          causeway construction, or other
          reason.  The result is the elim-
          ination of an area of marsh.  A
          small compensating border of
          marsh may or may not develop.

       b  High tidal marshes were often
          ditched and drained in former days
          to stabilize the sod for salt hay or
          "thatch" harvesting which was highly
          sought after in colonial days.  This
          inevitably changed the character
          of the marsh,  but it remained as
          essentially marshland.  Conversion
          to outright agricultural land has
          been less widespread because of the
          necessity of diking to exclude the
          periodic floods or tidal incursions,
          and carefully timed drainage to
          eliminate excess precipitation.
          Mechanical pumping of tidal marshes
          has not been economical in this
          country,  although the success of
          the Dutch and others in this regard
          is well known.
2  Marsh grasses may also be eliminated
   by smothering as, for example,  by
   deposition of dredge  spoils, or the
   spill or discharge of sewage sludge.

3  Considerable marsh  area has been
   eliminated by industrial construction
   activity such as wharf and dock con-
   struction, oil well construction and
   operation, and the discharge of toxic
   brines and other chemicals.

Consumer production (animal  life) has
been drastically reduced by the deliberate
distribution of pesticides.  In  some cases,
this has been aimed  at nearby agricultural
lands for economic crop pest control,  in
other cases the marshes have been sprayed
or dusted directly to control noxious
insects.

1  The results have been universally
   disastrous for the marshes, and the
   benefits to the human community often
   questionable.

2  Pesticides designed to kill nuisance
   insects, are also  toxic to other
   arthropods so that in addition to the
   target species, such forage staples as
   the various scuds (amphipods), fiddler
   crabs, and other macroinvertebrates
   have either been drastically reduced
   or entirely eliminated in many places.
   For example,  one familiar with fiddler
   crabs can traverse miles of marsh
   margins, still riddled with their burrows,
   without seeing a single live crab.

3  DDT and related compounds have been
   "eaten up the food chain" (biological
   magnification effect)  until fish eating
   and other predatory birds such as herons
   and egrets (Figure 9), have been virtually
   eliminated from vast areas, and the
   accumulation of DDT in man himself
   is only too well known.
                                                                                       9-37

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The Aquatic Environment
D  Most serious of the marsh enemies is
   man himself.  In his quest for "lebensraum"
   near the water, he has all but killed the
   water he  strives to approach.  Thus up to
   twenty percent of the marsh—estuarine
   area in various parts of the country has
   already been utterly destroyed by cut and
   fill real estate developments (Figures
   10,  11).
E  Swimming birds such as ducks, loons,
   cormorants, pelicans, and many  others
   are severely jeopardized by floating
   pollutants such as oil.
                     Figure 10.  Diagrammatic representation of cut-and-fill for
                                 real estate development,  mlw = mean low water
                     Figure 11.  Tracing of portion of map of a southern
                                 city showing extent of cut-and-fill real
                                 estate development.
 9-38

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                                                               The Aquatic Environment
VII SUMMARY

 A Wetlands comprise the marshes, swamps,
    bogs, and tundra areas of the world.
    They are essential to the well-being of
    our surface waters and ground waters.
    They are essential to aquatic life of
    all types living in the open waters.  They
    are essential as habitat for all forms of
    wildlife.

 B The tidal marsh is the area of emergent
    vegetation bordering the ocean or an
    estuary.

 C Marshes are highly productive areas,
    essential to the maintenance of a well
    rounded community of aquatic life.-

 D Wetlands may be destroyed by:

    1  Degradation of the life  forms of
       which it is  composed in the name of
       nuisance control.

    2  Physical destruction by cut-and-fill
       to create more  land area.
5  Morgan,  J.P.  Ephemeral Estuaries of
      the Deltaic Environment in: Estuaries,
      pp.  115-120, Publ. No. "83, Am.
      Assoc. Adv. Sci. Washington,  DC. 1967.

6  Odum,  E.P.  and Dela Crug, A. A.
      Particulate Organic Detritus in  a
      Georgia Salt Marsh - Estuarine
      Ecosystem, in:  Estuaries, pp. 383-
      388, Publ.  No.  83,  Am.Assoc.  Adv.
      Sci. Washington, DC.   1957.

7  Redfield, A.C. The Ontogeny of a Salt
      Marsh Estuary,  in:  Estuaries,  pp.
      108-114.  Publ.  No. 83, Am.  Assoc.
      Adv. Sci.  Washington, DC.  1967.

8  Stuckey,  O. H.  Measuring the Productivity
      of Salt Marshes.  Maritimes (Grad
      School of Ocean., U.R.I.) Vol. 14(1)':
      9-11.  February 1970.

9  Williams, R. B.  Compartmental
      Analysis of Production and Decay
      of Juncus reomerianus.  Prog.
      Report,  Radiobiol. Lab., Beaufort,  NC,
      Fiscal Year 1968, USDI, BCF,  pp. 10-
      12.
  REFERENCES

  1  Anderson,  W. W.  The Shrimp and the
        Shrimp Fishery of the Southern
        United States.  USDI,  FWS, BCF.
        Fishery Leaflet 589.   1966.

  2  Deevey,  E.S., Jr.  Bogs. Sci. Am. Vol.
        199(4):115-122.  October 1958.

  3  Emery, K. O. and Stevenson.  Estuaries
        and Lagoons.   Part n, Biological
        Aspects by J. W.  Hedgepeth, pp.  693-
        728. in: Treatise on Marine Ecology
        and Paleoecology.  Geol. Soc.  Am.
        Mem. 67.   Washington,  DC.  1957.

  4  Hesse, R., W. C. Allee, and K. P.
        Schmidt.  Ecological Animal
        Geography.  John Wiley  & Sons.  1937.
This outline was prepared by H.  W.  Jackson,
Chief Biologist, National Training Center,
Water  Programs Operations,  EPA, Cincinnati,
OH   45268.
Descriptors:   Aquatic Environment, Estuarine
Environment,  Lentic Environment  Lotic
Environment,  Currents,  Marshes,  Limnology
                                                                                    9-39

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                      THE IDENTIFICATION OF AQUATIC ORGANISMS
I  INTRODUCTION

A  There are few major groups of organisms
   that are either exclusively terrestrial or
   aquatic.  The following remarks will
   therefore apply in large measure to both,
   but primary attention will be directed to
   aquatic types.

B  One of the first questions usually posed
   about an organism is: "What is it, "
   usually meaning "What is it's name?"
   The naming or classification of biological
   organisms  is a science in itself (taxonomy).
   Some of the principles involved need to be
   understood by anyone working with
   organisms.

   1  Names are the "key number, " "code
      designation, " or "file references"
      which we must have to find information
      about an unknown organism.

   2  Why are they so long and why must they
      be in Latin and Greek?  File references
      in large systems have to be long in order
      to designate the many divisions  and
      subdivisions. There are over a million
      and a half items (or species) included
      in the system of biological nomenclature
      (very few libraries have a million
      books).

   3  The system of biological nomenclature
      is regulated by international congresses.

      a  It is based on a system of groups and
        super groups, of which the foundation
        (which actually exists in nature) is
        the species.  Everything else has
        been  devised by man and is subject
        to change and revision as man's
        knowledge and understanding increase.

      b  The categories employed are as follows:

        Similar species are grouped  into
        genera (genus) .
   Similar genera are grouped into
   families

   Similar families are grouped into
   orders
   Similar orders are grouped into
   classes
                        i
   Similar classes are grouped into
   phyla (phylum)

   Similar phyla are grouped into
   kingdoms

The scientific name of an organism is
its genus name plus its species name.
This is analogous to our  system of
surnames (family names) and given
names (Christian names).

a  The generic (genus) name is always
   capitalized and the species  name
   written with a small letter.  They
   should also be underlined or printed
   in italics when used in a technical
   sense.  For example:

   Homo sapiens - modern man

   Homo neanderthalis - neanderthal
                        man
   Esox niger - chain pickerel

   Esox lucius - northern pike

   Esox masquinongy  - muskellunge

b  Common names do not exist for
   most of the smaller and less familiar
   organisms.  For example, if we wish
   to refer to members of the genus
   Anabaena (an alga), we must simply
   use the generic name, and:
   Anabaena planctonica,
   Anabaena constricta, and
   Anabaena flos-aquae
 BI.AQ.3f. 1.74
                                                                                    10-1

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 The Identification of Aquatic Organisms
         are three distinct species which
         have different significances to
         water treatment plant operations.
 that seems most reasonable and either proceed
 to the couplet indicated, or else accept the
 name of the group as given if the selection
 is a terminal statement.  "Cf:   --" means to
 compare with the statement indicated.  "POL"
 means that members of the group have been
 reported to  be tolerant  of organic pollution.
      A complete list of the various cate-
      gories to which an organism belongs
      is known as its  "classification. " This
      may be written as follows for Phacus       1&  The body Qf the organisms comprising a
      Pyrum a green flagellate,  for example:         single microscopic independent cell, or
                                                    many similar and independently functioning
      Kingdom Plantae                              cells associated in a colony,  with little
                                                  •  or no difference between the  cells
      Phylum  Thallophyta                           (i.e., without forming tissues); or com-
                                                    prising masses of multinucleate proto-
      Class Algae                                  plasm; mostly microscopic.  Single celled
                                                    animals	Phylum Protozoa
      Order Volvocales                                                                 '
                                                Ib  The body of the organism comprising
      Family Phacotaceae                           many cells of different kinds (i. e, form-
                                                    ing tissue)  may be microscopic or
      Genus Phacus                                 macroscopic	2

      Species pyrum                            2a  Body of colony usually forming irregular
                                                    masses or layers sometimes  cylindrical,
      a  It should be emphasized that since           goblet-shaped,  vase-shaped, elongate,
         all categories of above species are          serrate, or tree-like.  Range from
         essentially human concepts,  there           barely visible to large	 3
         is often divergence of opinion in
         regard to how certain organisms        2b  Body or colony shows some type of
         should be grouped.  Changes result.         definite symmetry	15

      b  The most appropriate or correct        3a  Colony forming irregular masses,  clumps,
         name for a given species is also            or aggregations of material growing
         sometimes disputed,  and so species         around, over, or clinging to surface of
         names too are changed.  The  species        rocks,  sticks,  pipes,  etc	  4
         itself, as an entity in nature,  how-
         ever, is relatively timeless and so      3b  Irregular, or not obviously symmetrical,
         does not change to man's eye.               but essentially erect or plant-like individ-
                                                    uals or  colonies	  9
II   KEY TO SELECTED LARGER GROUPS
    OF AQUATIC ANIMALS

 The following key is intended to provide an
 introductory acquaintance with  some of the
 more common aquatic organisms.  While
 most of the groups mentioned are animals,
 a few other groups often confused with them
 are also included.   Technical nomenclature
 is kept to a minimum.

 In the use of the key,  each couplet should
 be considered by itself, without reference to
 any other couplet.   Select the alternative
4a  Freshwater	  8

4b  Marine	  5

5a  Surface of colony usually slightly rough
    or bristly in appearance under the micro-
    scope or hand lens	  7

5b  Surface smooth	  6

5c  Colony or organism erect,  plant-like,
    branched or unbranched	12

6a  Mass very thin,  usually pink or white
    (Coral algae)	  13b
    10-2

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                                                     The Identification of Aquatic Organisms
 6b  Mass gelatinous, either as relatively
     thin sheet or clumps of tongue-like club
     shaped projections.  Transparent or
     colored.   Star shaped groups of individ-
     ual organisms often evident.   Colonial
     tunicates or ascideans. Phylum Chordata,
     Class Tunicata  (Cf:12b)

 7a  Surface webbed or bristly in  appearance,
     stick-like crystals or "spicules" often
     protruding from the general surface.
     Pores numerous, one or more usually
     large and conspicuous.  Sponges.
     	  Phylum  Porifera

 7b  Colonies  very thin,  surface slightly
     roughened.  Composed of minute individ-
     ual cells  or  compartments arranged in
     various lacy or coral-like patterns.  Each
     compartment inhabited by a minute bilater-
     ally symmetrical animal which protrudes
     a crown of delicate tentacles. Moss
     animals,  bryozoans. ... Phylum Bryozoa

 8a  Surface of colony rough or bristly  in
     appearance under the microscope or hand
     lens.  Grey,  green, or brown.  Sponges.
     (Figure 1)	Phylum  Porifera

 8b  Surface colony relatively  smooth,  general
     texture of mass  gelatinous, transparent.
     Clumps of minute individual organisms
     variously distributed.  Moss  animals,
     bryozoans.  (Figure  2). .Phylum Bryozoa
 9a  Freshwater.
10
 9b  Marine	  11

lOa  Surface  of structures rough or bristly
     under the microscope	  8a

lOb  Individuals or branches tubular.  Usually
     covered with brownish cuticle to which
     much sediment  may adhere.  Moss
     animals, bryozoans. . . . Phylum Bryozoa
     (Cf:8b) (Figure  2)

lla  Surface  of structure appears  web-like or
     bristly under the microscope	 7a

lib  Surface  of structure an unbroken skin or
     cuticle,  except  for a limited number of
     functional apertures	 13
      12a  Colonies minute, plant-like or coral-like.
           (Figure 2)	  13

      12b  Solitary, sac-like organism, either stalked
           or growing directly on substrate.  Trans-
           parent, covered with mud or sand, or
           variously colored.  Bilateral symmetry
           revealed on close investigation.  Becomes
           flaccid when exposed and relaxed at low
           tide.  Up to several inches in length.
           Seasquirts or tunicates.. Phylum Chordata,
           Class Tunicata
      13a
      13b
      14a
      14b
      16a
      16b
      17a
     Compartments or apertures for separate
     individual animals evident	  14

     No evidence of the presence of individual
     animals.  Structure profusely jointed.
     Individual segments hard, smooth,  usually
     pink or white. May also grow as hard
     smooth thin sheet over surface of rocks.
     Calcareous algae or coral algae.  A
     common Genus;	 .  Corallina

     Minute organisms protruding from holes
     in colony are radially symmetrical. ..  17b

     Organisms protruding are bilaterally
     symmetrical.  (This may be assumed if
     they are not obviously radial)  Moss
     animals, bryozoans.  (Figure 2) (Cf:7b)
     	  Phylum  Bryozoa
15a  Radially symmetrical (body arranged around
     an axis)	 16

15b  Fundamentally bilaterally symmetrical.   13

15c  May be superficially spiral as Figure 13  35
     Plan involves multiples of two, or else
     rather large numbers of tentacles around
     the margin of an umbrella; saucer-like,
     or flower-like structure	 . 17

     Body plan based on multiples of five.
     Outer surface hard or rough to the touch.
     Starfishes, brittle stars, sea urchins,
     sand dollars, sea cucumbers.
     	  Phylum Echinodermata
     Jelly-like body, massive or ovoid, free
     swimming nearly  transparent in nature.
     Eight rows  comb-like paddle plates:
     Comb Jellies	Phylum Ctenophora
                                                                                     10-3

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  The Identification of Aquatic Organisms
                                                23b
                                                23c
                                                24a
17b  Body tube-like or umbrella-like,  tentacles
     around mouth, margin of disc,  or both.
     Jellyfishes,  corals, hydroids,  sea
     anemones,  etc... Phylum Coelenterata

18a  Microscopic.  Action  of two ciliated lobes
     at anterior end in life often gives appear-
     ance of wheels.  Body often  segmented,
     accordion-like.  Free swimming or
     attached.  Rotifers or wheel animalcules
     (Figure  3).' Phylum Trochelminthes (Rotifera)
18b  Often larger, worm-like or else having
     strong skeleton or shell	19     24b

19a  Skeleton (or shell) present may be internal
     or external	30
19b  Body soft and/or worm-like.  Skin may
     range from soft  to parchment-like ...  20

20 a  Three or more pairs of well-formed
     (though possibly small)  jointed legs
     present.  (Phylum Arthropoda)	  36

20b  Legs or appendages, if present, limited
     to pairs of bumps or hooks. Lobes or
     tentacles if present-soft and fleshy, not
     jointed	   21

21a  Body round, oval or flat in cross section
     	   22

21b  Inverted U-shaped cross section,  with
     flat slimy undersurface or foot tapering
     off behind.  Head with two pairs of
     sensitive retractile tentacles.   Marine
     or terrestrial.   Marine forms have fancy
     gill structures on back.  Slugs.
     Class Gastropoda. (Cf: 35a)
     (Phylum Mollusca)	35

22a  Strongly depressed or flattened	  23

226  Oval or round in cross section, or, if
  -  flat, with single  sucker disc at each end.
     	  25

23a  Parasitic in_bodies of higher animals.
     Extremely long and flat, divided into
     sections (like a Roman girdle). Life
     history may involve intermediate host.
     Tape worms.   (Figure 5) Class Cestoda
                                                25 a
                                                26a
Parasitic on bodies of higher animals,
very muscular,  strong external sucker
discs (Figure  9)	29b

Body a single  unit. Mouth and digestive
system present  (but no anus)	  24

External or internal parasite on higher
animals.  Sucking discs present for attach-
ment.  Life history may involve two or
more intermediate hosts or stages.
Flukes	Class Trematoda

Free-living.  Entire body surface  covered
with locomotor cilia.   Eyed areas in head
often appear "crossed. " Pol.  Free-living
flatworms (Figure 6).  Class Turbellaria

Long, slender,  with snake-like motion in
life.  Covered with glistening cuticle.
Parasitic or free-living.  Microscopic to
six feet in length.  Round worms
(Figure 7).... Phylum Nemathelminthe s
                                                25b Divided into sections or segments
                                                    (Figures 8 and 9)	  26
Head a more or less well-formed hard
capsule with jaws, eyes, and antennae.
Pol.   (Figure 8)  Class Insecta.
(Cf:49a)	Order Diptera
                                                26b  Head structure soft (except jaws,  if
                                                    present) (Figure 9)	  27

                                                27a  Head conical or rounded, lateral appenda^ ,s
                                                    not conspicuous or numerous	 28

                                                27b  Head somewhat broad and blunt.  Retractile
                                                    jaws usually present. Soft fleshy lobes or
                                                    tentacles often present in head region.  Tail
                                                    usually narrower. Lateral lobes or fleshy
                                                    appendages on each segment.   (Figure 9A)
                                                    	Phylum  Annelida..... 29

                                                28a  Minute dark colored retractile jaws present,
                                                    body tapering somewhat  at both ends, pairs
                                                    or rings of bumps or "legs" often present,
                                                    even near tail.  Class Insecta. (Cf:49a)
                                                    	  Order Diptera

                                                28b  No jaws, sides of body generally parallel
                                                    except at ends, (similar to Figure 9).
    10-4

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                                                     The Identification of Aquatic Organisms
29a
29b
30a
30b
31a
Thickened area or ring usually present
part way back on body.  Clumps of
minute bristles on most segments.
	(Phylum Annelida)    29

Segments with bristles, and/or fleshy
lobes, or other extensions.  Tube builder,
borer, or burrower.  Often reddish or
greenish in color.   Pol.  Earthworms,
sludge worms,  clam worms, etc.
	Class Chaetopoda

A sucker disc at each end,  the larger
one posterior.  External bloodsucking
parasites on higher animals, often found
unattached to any host;  Pol.  Leeches.
(Figure 9B)	Class Hirudinea
Skeleton internal,  of true bone
	 (Vertebrates)
                                         58
Body covered with external skeleton or
sheU.  (Figure 4)	  31

External jointed skeleton or shell covers
legs and other appendages, often leathery
in nature.  Jointed legged animals.
(Figures 11,  12,  and 14 through 29)
	(Phylum Arthropoda) 36
31b  Shells limey or calcareous.  (Figures 10
     and 13)	  32

32a  Marine, attached,  shell conical, or purse-
     shaped on fleshy stalk.  Crowded individ-
     uals may be more or less cylindrical.
     Shell composed of several parts, two of
     which spread apart to permit extension
     of "hand" for feeding.  Barnacles.
     Phylum Arthropoda,  Class Cirripedia

32b  Shells one or two, or several arranged
     as transverse plates	33

33a  Half inch or less in length.  Two clam-
     like shells.  Soft parts inside include
     delicate flattened jointed appendages.
     Phyllopods or branchipods (Figures 11
     and 12)  Phylum Arthropoda, Class
     Crustacea, Subclass Branchipoda
     (Cf:39a)

33b  Soft parts covered with thin  skin, often
     slimy	(Phylum Mollusca)  34
34a  Marine, oval in shape, eight shells trans-
     versely jointed.  Chitons.
     	Class Amphineura

34b  Shells one or two in number.  Snails,
     clams, etc.  (Figures 10-13)	35

35a  Shell, single may be a spiral, a flattened
     cone, degenerate, or missing.  Pol.
     Snails and slugs	Class Gastropoda

35b  Shell, double,  right and left halves, hinged
     at one point.  Mussels,  clams,  oysters.
     	Class Pelecypoda

36a  Head, thorax,  and abdominal body regions
     usually distinct. Three pairs of regular
     walking legs, or their rudiments.  Wings
     present in all adults  and rudiments in
     some larvae.  Leglike appendages or gills
     present on abdomens of  some larvae.  No
     marine forms.  (Phylum Arthropoda,
     	Class Insecta)	47

36b  More than three pairs of legs apparently
     present.  (Figures 14-19)	  37

37a  Body elongated,  head broad and flat with
     strong jaws.  Appendages following first
     three pairs of legs are rounded  tapering
     filaments.  Up to 3 inches long.  Dobson
     fly and fish fly larvae.     Class Insecta,
     	 Order Megaloptera
                                           37b  Four or more pairs of true legs.
                                         38
                                           38a Four pairs of legs.  Body rounded, bulbous,
                                               head minute.  Often brown or red.  Water
                                               mites.  (Figure 15)  Phylum Arthropoda,
                                               Class Arachnida, Order Acari

                                           38b Five or more pairs of locomotor appendages
                                               (walking or swimming legs) gills, two pairs
                                               of atenna.  (Figures 16-19,  11-12)
                                               Crustaceans.  Phylum Arthropoda,
                                               Class Crustacea.	 39

                                           39a Ten or more pairs of flattened,  leaf-like
                                               swimming (and respiratory) appendages.
                                               Many species swim constantly in life,
                                               some normally upside down.  (Figure 16)
                                               Fairy shrimps,  phyllopods, or branchipods.
                                               	  Subclass Branchipoda
                                                                                   10-5

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  The Identification of Aquatic Organisms
39b  Less than ten pairs of locomotor
     appendages	
40 a
40b
41a
41b
42a
42b
43a
43b
44a
44b
45 a
Body and legs enclosed in bivalved
(2 halves) shell which may or may not
completely hide them.  (Figures 11-12) 41

Appendages more rounded than leaf-like
in cross section,  or if appendages are
flattened for swimming,  eyes are on
stalks (pelagic shrimps).  May be large
or minute	 43

One pair of branched antennae enlarged
for locomotion, extend outside  of shell
(carapace).  Single eye usually visible.
Cyclops or cladocera.  Subclass Cladocera

Locomotion accomplished by body legs
(not antennae). (Figure 16). . .	42

Appendages leaf-like, flattened, more
than ten pairs. (Branchipods)	 39a

Animal less than three mm in length.
Appendages more or less slender and
jointed, often  used for walking. (Figure 11)
Shells opaque.  Ostracodes.
	  Subclass Ostracoda

Body a series of six or more essentially
similar segments, differing mainly in
size	 44

Front part  of body enlarged into a some-
what separate body unit (cephalothorax)
 often covered with a single piece of shell
(carapace).  Back part (abdomen) may be
relatively small,  even folded underneath
front part.   (Figure  19)	45

Body compressed laterally, i.e., high
and thin.  Scuds,  amphipods. (Figure 17)
	 Subclass Aniphipoda

Body depressed, i. e., low and broad.
Flat gills contained in chamber beneath
tail.  (Figure  18)  Sowbugs.
	  Subclass Isopoda

Minute in size. Often somewhat elongated
and cylindrical, with abdomen extending
straight out behind ending in two small
projections.  Two relatively enormous
           masses of eggs are often attached to
40         female.  Locomotion by means of two
           enlarged, unbranched antennae, the only
           large appendages on the body. (Figure 19)
           Copepods	  Subclass Copepoda
      45b  Larger.  Carapace (shell of cephalothorax,
           the enlarged front section) usually more
           conspicuous.  5 pairs of walking legs
           including claws, if present.  Appendages
           present on abdomen	  46

      46a  Eyes immovable, in broad shell shaped
           like a horseshoe, a mud dweller.  Long
           sharp pointed triangular tail extends out
           behind. Flat "book" gills beneath abdomen.
           Horseshoe crab.  Subclass Arachnoidea,
           	Genus Limulus

      46b  Eyes on movable  stalks.  Abdomen tightly
        ..  folded  back under cephalothorax (crabs)
           or continued out behind  thorax to end in
           an expanded "flipper" or swimming paddle.
           Crabs, shrimps,  crayfishes, lobsters,
           etc	 Subclass De capo da

      47a  Two pairs of functional  wings (one pair
           may be more or less hardened as protection
           for the other pair). Adult insects which
           normally live on or in the water (Figures 22,
           25, 28, 29)	  57

      47b  No functional wings, though "pads" in
           which they are  developing may  be visible.
           Some resemble adults very closely,  others
           differ extremely (Figures 8,  23,  24, 26, 27)
           	48

      48a  External pads or cases  in which wings
           develop clearly visible	53

      48b  More or less worm-like,  no external
           evidence of wing development	49

      49a  No jointed legs  (sucker  discs, hooks,
           prolegs, breathing tubes, and other such
           structures may be present.  (Figures 8, 9D)
           Larvae of two winged flies, midges, etc.
           Pol	Order Diptera

      49b  Three pairs of jointed thoracic legs, head
           capsule well-formed	 50

      50a  Minute (2  - 4mm), living on surface film.
           Tail a  strong organ that can be hooked into
  10-6

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                                                      The Identification of Aquatic Organisms
50b
51a
     a "catch" beneath thorax.  When released,
     animal jumps into the air.  Not usually
     classed as aquatic.  These are adults,
     no wings are ever grown.  (Figure 20)
     Springtails	Order Collembola
Larger (rarely collected under 5 mm),
worm-like, living beneath the surface.
                                         51
Construct fixed or movable cases of
bottom materials.  (Figure 21) Abdominal
segments often with minute gill filaments,
no long lateral filaments.  Generally
cylindric in shape.  Caddisfly larvae.
	Order Trichoptera
51b  Free living, i.e.,  not case-building. 52

52a  Somewhat flattened and relatively massive
     in appearance.  First segment of thorax
     about  same size as head, following  two
     segments smaller.  Each abdominal
     segment  with rather stout,  tapering
     lateral filament about as long as body is
     wide.   (Figure 14)  Dobsonfly, alderfly,
     and fishfly larvae.  Order Megaloptera

52b  Generally rounded  in cross section.
     Lateral filaments if present tend to be
     longer and thinner. (Figure 23)  (A few
     forms extremely flattened,  like a suction
     cup: Psephenidae).  Beetle larvae.
     .•	  Order Coleoptera

53a  Two or three filaments or other  structures
     extending out from end of abdomen.
     (Figures  24B, 24D, 26,  27)	  55

53b  Abdomen ending abruptly,  unless terminal
     segment itself is extended as single
     structure.  (Figure 24C)	  54

54a  Head broad, eyes widely spaced.  Front
     of face covered by  extensible folded mouth-
     parts  often called a "mask. "  Larvae of
     dragonflies or darning needles.  Pol.
     (Figure 24E)	Order Odonata
     	(Suborder Anisoptera)

54b  Mouthparts for piercing and sucking.
     Body form varies extremely.  Water bugs,
     walking sticks, water boatmen, back-
     swimmers, electric light bugs, water
     striders,  and others.  (Figure 25)
     	Order Hemiptera
                                          55a Tail extensions (caudal filaments) two.
                                               Stonefly larvae.   (Figure 26)
                                               	Order Plecoptera
                                           55b
                                               56a
                                               56b
                                          57a
                                          57b
                                          58b
Tail extensions usually three. (Figures 24B,
24D, 27)	56

Tail extensions long and  slender.  Rows
of hairs may give them a feather -like
appearance.  Larvae of mayflies.
	Order Ephemeroptera

Tail extensions flat, elongated plates.
Head broad with widely spaced eyes,
abdomen relatively long and slender.
Larvae of damselflies.  Order Odonata
                                                                      , (Suborder Zygoptera)
External wings or wing covers form a
hard protective dome over the inner wings
folded beneath, and the abdomen.  Beetles.
(Figure 28)	Order
External wings somewhat membranous,
usually relatively flat as compared to wing
covers of beetles.   Mouthparts for piercing
and sucking.  Body form various, (Cf:53b).
True bugs.  (Figure 25)  Order Hemiptera
                                          58a  Appendages present in pairs (fins, legs,
                                               wings)	  60
Mouth a round suction disc.  No paired
appendages	
                                                                                   59
                                          59a  Body long and slender.  Several holes
                                               alongside of head.  Lampreys and hagfishes.
                                               Sub-phylum Vertebrata. Class Cyclostomata

                                          59b  Body plump,  oval,  tail extending out abruptly.
                                               Larvae of frogs and toads.  Legs appear
                                               one at  a time during metamorphosis to
                                               adult form.  Tadpoles... Class Amphibia

                                          60a  Paired appendages  are legs	62

                                          60b  Paired appendages  are fins	61

                                          61a  Gills covered with a flap or "operculum. "
                                               True fishes	Class Pisces

                                          61b  Gill  chambers open directly to the outside.
                                               Skin often rough and scratchy or penetrated
                                               by clumps of short  sharp spines. Essentially
                                               marine.  Sharks, skates,  and rays.
                                               	Class Elasmobranchii
                                                                                   10-7

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  The Identification of Aquatic Organisms
62a  Digits with claws,  nails, or hoofs. ... 63     63b  Body covered with horny scales or plates.
                                                     Reptiles	Class Reptilia
62b  Skin naked, no claws on digits.  Frogs,
     toads, and salamanders. Class Amphibia     64a  Body covered with feathers.  Birds.
                                                     	Class Aves

63a  Warm blooded	  64     64b  Body covered with hair.  Mammals.
                                                     	Class Mammalia
   10-8

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                                          The Identification of Aquatic Organisms
                           APPENDIX A
                     LIST OF INCLUDED GROUPS
Phyla or
Subphyla

Protozoa
Porifera
Coelenterata
Platyhelminthes
Rotifera
Nemathelminthes
Bryozoa
Annelida


Arthropoda
Classes or
 Subphyla
Turbellaria
Trematoda
Cestoda
Chaetopoda
Hirudinea
Crustacea
Classes or
  Orders
Other
                      Arachnoidea
                      Insecta
Mollusca

Enchinodermata

Chordata
Gastropoda
Pelecypoda

Tunicata
Cyclostomata
Pisces
Amphibia
Reptilia
Aves
Mammalia
Branchiopoda
Cladocera
Ostracoda
Amphipoda
Isopoda
Copepoda
Decapoda
Cirripedia

Acari
Xiphosura
Collembola
Plecoptera
Ephemeroptera
Odonata
Hemiptera
Megaloptera
Trichoptera
Coleoptera
Diptera
                                                         Limulus
Algae
                                    Coralllna
                                                                        10-9

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The Identification of Aquatic Organisms
            1.  Spongilla spicules
               Up to .2 mm. long.
    3A. Rotifer, Polyarthra
        Up to . 3 mm.
                       3B.  Rotifer, Keratella
                           Up to . 3 mm.
                                      3C. Rotifer, Philodina
     Up to . 4 mm.
     4A. Jointed leg
          Caddisfly
4B. Jointed leg
    Crayfish
                                2B. Bryozoal mass. Up to
                                    several feet diam.
                                         2A. Bryozoa, Plumatella. Individuals up
                                             to 2 mm.  Intertwined masses maybe
                                             very extensive.
4C. Jointed leg
     Ostracod
                                                                     5. Tapeworm head,
                                                                        Taenia.  Up to
                                                                        25 yds. long
          6A. Turbellaria, Mesostoma
             • Up to 1 cm.
                                        6B.  Turbellaria, Dugesia
                                            Up to 1. 6 cm.
       10-10
                              7. Nematodes. Free living
                                 forms commonly up to
                                 1 mm.,  occasionally
                                 more.

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                                                   The Identification of Aquatic Organisms
                     8B. Diptera, Mosquito
                         pupa.  Up to 5mm.
8A. Diptera, Mosquito larvae
    Up to  15 mm. long.
                                   8C. Diptera,  Tendiped  8E. Diptera,  crane fly
                                       larvae.  Up to 2 cm.      pupa. Up to 2. 5 cm.
                                       9D.  Diptera, Rattailed maggot
                                           Up to 25 mm. without tube.
9A. Annelid,
    segmented
    worm, up to
    1/2 meter
                                                           10B. Alasmidonta, end view.
                         10A. Pelecyopod, Alasmidonta
                              Side view,  up to 18 cm. long.
9B. Annelid,  leech up to 20 cm.
 11A. Ostracod, Cypericus
      Side view, up to 7 mm.
                           11B. Cypericus, end view.
                                  12A. Branchiopod
                                      Daphnia.  Up
                                      to 4 mm.
                                                               12B. Branchiopod
                                                                    Bosmina. Up
                                                                    to 2 mm.
                                                                             10-11

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The Identification of Aquatic Organisms
      13.  Gastropod, Viviparus
          Up to 3 inches.
                                     14.  Megaloptera,
                                         Alderfly larvae
                                         Up to 25 mm.
         16. Fairy Shrimp, Eubranchipus
             Up to 5 cm.
                                                                    15. Water mite,
                                                                       Up to 3 mm.
                       17. Amphupod, Pontoporeia
                          Up to 25 mm.
                                                                 18.  Isopod, Asellus
                                                                     Up to 25 mm.
             20. Collembola, Podura
                 Up to 2 mm. long
19A. Calanoid copepod,   IQB- Copepoid copepod.
      Female                 Female
     Up to 3 mm.             Up to 25 mm.
      10-12

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                                                The Identification of Aquatic Organisms
    21A.
                      21B.
        21C.
       2 ID.
                               2 IE.
21. Tricoptera,  Larvae cases,  mostly 1-2 cm.
                         22.  Megaloptera, alderfly
                             Up to 2 cm.
 23A. Beetle larvae,
      Dytisidae,
      Usually about | cm.
23B. Beetle larvae,
     Hydrophilidae,
     Usually about 1 cm.
24A. Odonata, dragonfly
     nymph up to 3 or 4
     cm.
                          24B. Odonata, tail of
                               damselfly nymph
                               (side view)
                           is^4  24E- Odonata, front view
                                     of dragonfly nymph
                                     showing "mask"
                                     partially extended
              24D. Odonata,  damselfly
                   nymph (top view)
                                                              24C. Odonata, tail of
                                                                   dragonfly nymph
                                                                   (top view)
                                                                                     10-13

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The Identification of Aquatic Organisms
              25A.  Hemiptera,
                   Water Boatman
                   About 1 cm.
             27.Ephemeroptera,
                Mayfly nymph
                Up to i cm.
                                               25B.  Hemiptera,
                                                    Water Scorpion
                                                    About 4 cm.
                                26. Plecoptera,
                                    Stonefly nymph
                                    Up to j cm.
28A. Coleoptera,
     Water scavenger
     beetle. Up to 4 cm.
                                                                     28B. Coleoptera,
                                                                          Dytiscid beetle
                                                                          Usually up to 2^ cm.
                 29A. Diptera, Crane
                     fly.  Up to 2j cm.
                                                       29B.  Diptera, Mosquito
                                                             Up to 20 mm.

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                                                   The Identification of Aquatic Organisms
 REFERENCES - General

 Keeler,  C.R.   Books on Aquatic Biology.
    Freshwater and Marine .Facile Press,
    Tallahassee, Florida.  1965.

 REFERENCES - Invertebrates

 1  Eddy, S. and Hodson, A.C.  Taxonomic
      Keys to the Common Animals of the
      North Central States.  Burgess Publ.
      Co., Minneapolis.   1955..           :.

 2  Edmondson,  W. T., Ed.  Ward and
      Whipple's Freshwater Biology.  John
      Wiley & Sons.  New York.     ,        :
      1959.

 3  Hedgepeth, J.,  and Hinton, X.   Common
      Seashore  Life of Southern California.
      Naturegraph  Company, Heraldsburg,
      California.   1961.

 4  Jahn,  T.L.,  and Jahn,  F. F.  How to
      Know the Protozoa.  Win. C.  Brown
      Co., Dubuque, Iowa. 1949.

 5  Kudo, R.   Protozology.  Charles C.
      Thomas, Publisher,  Springfield, Illinois.
      1950.

 6  Light, S.  F., et al.   Intertidal Inverte-
      brates of the Central California Coast.
      University of California Press,
      Berkeley.   1961.

 7  MacGinitie,  G.E., and  N.  Natural
      History of Marine Animals. McGraw-
      Hill.   1949.

 8  Miner, R.W.  Fieldbook of Seashore Life.
      G.B. Putnam's Sons, New York.   1960.

 9  Pennak, R.W.   Freshwater Invertebrates
      of the United States.  The Ronald
      Press Co., New York.
      1953.

10  Pratt, H.W.   A Manual of the Common
      Invertebrate Animals Exclusive of
      Insects.  The Blakiston Company,
      Philadelphia.   1951.
11  Ricketts,  E., and Calvin, J.  Between
      Pacific Tides.   Revised by J.
      Hedgepeth, Stanford Univ. Press,
      Palo Alto, California.   1952.

12  Smith, R.I.   Keys to Marine Invertebrates
      of the Woods Hole Region, Contribution
      No. 11, MBL, Woods Hole, Mass.
      1964.
 REFERENCES - Fishes

 1 American Fisheries Society.  A List of
      Common and Scientific Names of
      Fishes from the United States and
      Canada.  Special Publication No.  2.
      Am. Fish Soc.  Dr. E.A. Seaman,
      Sec. - Treas. Box 483.   McLean, Va.
      1960. (Price $1.00 paper, $2. 00 cloth)

 2 Anon.  Check List of the Florida Game
      and Commercial Marine  Fishes with
      Common Names.  Florida State Board
      of Cons. Educational Bull. No.  12
      (Free).

 3 Benton, A.H., and Stewart.   Keys to the
      Vertebrates of the Northeastern States.
      Burgess Pub. Co.  1964.

 4 Breder,  C.M.  Fieldbook of Marine Fishes
      of the Atlantic Coast.   G. B. Putnam's
      Sons, New York.

 5 Clemens, W.A., and Wilby, G.V.
      Fishes of the Pacific Coast of Canada.
      Fisheries Research Board of Canada.
      Bulletin No. 68, 2nd Ed.   1961.

 6 Hubbs,  C.L., and Lagler, K. F.   Fishes
      of the Great Lakes Region.   Bull.
      Cranbrook Inst.  Science.   Bloomfield
      Hills, Michigan.   1949.

 7 Perlmutter, A.  Guide to Marine Fishes.
      New York University, NY.  1961.

 8 Schrenkeisen, R.   Fieldbook of Fresh-
      water Fishes of North America,  north
      of Mexico.  Putnam's Sons.   1963.
                                                                                  10-15

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The Identification of Aquatic Organisms
   Zim, H. S., and Shoemaker, H. H.
      Fishes--A guide to Fresh and Salt
      Water Species.   Golden.  1959.
This outline was prepared by H. W. Jackson,
Chief Biologist,  National Training Center,
Environmental Protection Agency, OWP.
Cincinnati, OH  45226

Descriptors:
Aquatic Life; Systematics
 10-16 '

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            AQUATIC ORGANISMS OF SIGNIFICANCE IN POLLUTION SURVEYS
I   INTRODUCTION

A   Any organism encountered in a survey is
    of significance.  Our problem is thus not
    to determine which are of significance
    but rather to decide "what is the signifi-
    cance of each?"

B   The first step in interpretation is
    recognition. "The first exercise in
    ecology is systematics."

C   Recognition implies identification and an
    understanding of general relationships
    (systematics).   The following outline will
    thus review the general  relationships of
    living (as contrasted to fossil) organisms
    and briefly describe the various  types.

D   The species problem

    1  Necessity of identifying species

      Studies of the ecology of any habitat
      require the identification of the
      organisms found in it. One cannot
      come  up with definitive evaluations of
      stress on the biota of a system unless
      we can say what species constitute the
      biota.  Species vary in their responses
      to the impact of the environment.

    2  Solutions to  the problem

      a Evasion

        Treat the ecosystem as a "black
        box"--a unit—while ignoring the
        constitution of the system.  This
        may produce some broad generalizations
        and will certainly yield more questions
        than answers.

      b Compromise
        Work only with those taxonomic
        categories with which one has the
        competence to deal.  Describe the
        biotic component as a taxocenosis
        limited to one or two numerically
        dominant  taxonomic categories,
        bearing in mind that numerically
        taxa which are ignored may be very
        important to the ecology of  the ecosystem.
      c Comprehensive description

        Attempt a comprehensive description
        of the biota.  No one can claim
        competence to  deal with more than
        one or two groups.  The cooperation
        of experts must be obtained.  The
        Smithsonian Institution has a clear-
        inghouse for this sort of thing. *
        Lists of expert taxonomists can be
        obtained.  There will be none for
        some groups.  Also collaboration
        is time consuming.

    3  Fungi: extracellular digestion
      (enzymes  secreted externally.)
      Food material then taken in through
      cell membrane where it is metab-
      olized and reduced to the mineral
      condition.  Ecologically known as
      REDUCERS.

E   Each of these groups includes simple,
    single-celled representatives, persisting
    at lower levels on the evolutionary stems
    of the higher organisms.

    1  These groups span the gaps between
      the higher kingdoms with a multitude
      of transitional forms.  They are
      collectively called the PROTISTA.

    2  Within the protista, two principal
      sub- groups  can be defined on the
      basis of relative  complexity  of
      structure.

      a  The bacteria and blue-green algae,
         lacking a nuclear membrane may
         be considered as the lower
         protista (or Monera).

      b  The single-celled algae and
         protozoa are best referred to as
         the higher protista.

F   Distributed throughout these groups will
    be found most of the traditional ."phyla"
    of classic biology.
 BI.AQ.22c. 1.74
                                                                                    11-1

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Aauatic Organisms of Significance
                 NSUMERS
PRODUCERS                  REDUCERS
                 NUTRIENT
                 MINERALS

    Figure 1. BASIC CYCLES  OF LIFE
11-2

-------
                                                           Aquatic Organisms of Significance
II  PLANTS

A  The vascular plants are usually larger
   and possess roots, stems, and leaves.

   1  Some types emerge above the surface
       (emersed).

   2  Submersed typed typically do not
       extend to the surface.

   3  Floating types may be rooted or free-
      floating.

B  Algae generally smaller, more delicate,
   less complex in structure, possess
   chlorophyll like other green plants.
   For convenience the following artificial
   grouping is used in sanitary science:
   1  "Blue-green algae" are typically small
      and lack an organized nucleus,  pigments
      are dissolved in cell sap. Structure
      very simple.

   2  "Pigmented flagellates"  possess nuclei,
      chloroplasts, flagellae and a red eye
      spot. This is an artificial group con-
      taining several remotely related organ-
      isms, may be green,  red, brown, etc.

   3  "Diatoms" have "pillbox" structure of
      SiC>2 - may move.  Extremely common.
      Many minute in size,  but  colonial forms
      may produce hair-like filaments.
      Golden brown in color.

   4  "Non-motile green algae" have no loco-
      motor structure or ability in mature
      condition.  Another artificial group.

      a  Unicellular representatives may be
         extremely small.

      b  Multicellular forms may produce
         great floating mats of material.

in FUNGI

Lack chlorophyll and consequently most are
dependent on other organisms.  They secrete
extracellular enzymes and reduce complex
organic material to simple compounds which
they can absorb directly through the cell wall.
A   Schizomycetes or bacteria are typically
    very small and do not have an organized
    nucleus.

    1  Autotrophic bacteria utilize basic food
      materials from inorganic substrates.
      They may be photo-synthetic or
      chemosynthetic.

    2  Heterotrophic bacteria are most
      common.  The require organic
      material on which to feed.

B   "True fungi" usually exhibit hyphae as the
    basis of structure.

IV  ANIMALS

A   Lack chlorophyll and consequently feed on
    or consume other organisms.  Typically
    ingest and digest their food.

B   The Animal  Phyla

    1  PROTOZOA are single celled organisms;
      many resembling algae but lacking
      chlorophyll (cf: illustration in "Oxygen"
      lecture).

    2  PORIFERA are the sponges; both marine
      and freshwater representatives.

    3  CNIDARIA (=  COELENTERATA)
      include corals, marine and fresh-
      water jelly fishes, marine and
      freshwater hydroids.

    4  PLATYHELMINTHES are the flat worms
      such as tape worms, flukes and Planeria.

    5  NEMATHELMDSTTHES are the round
      worms and include both free-living
      forms and many dangerous parasites.

    6  ROTIFERS are multicellular micro-
      scopic  predators.

    7  BRYOZOA are small colonial sessile
      forms, marine or freshwater.
                                                                                   11-3

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Aquatic Organisms of Significance
    8  MOLLUSCA include snails and slugs, •
      clams, mussels and oysters,  squids,
      and octopi.

    9  BRACHIOPODS are bivalved marine
      organisms usually observed as fossils.

   10  ANNELIDS are the segmented worms
      such as earthworms, sludge worms and
      many marine  species.

   11  ECHINODERMS include starfish, sea
      urchins and brittle stars.  They are
      exclusively marine.

   12  CTENOPHORES, or comb jellies, are
      delicate jelly-like marine organisms.

   13  ARTHROPODA, the largest of aU
      animal phyla.  They have jointed ap-
      pendages and  a chitinous exoskelton.

      a CRUSTACEA are divided into a
        cephalothoraz and abdomen, and
        have many  pairs of appendages,
        including paired antennae.

        1) CLADOCERA include Daphina
           a common freshwater micro-
           crustacean; swim by means of
           branched antennae.

        2) ANOSTRACA (=PHYLLOPODS)
           are the fairy shrimps, given
           to eruptive appearances in
           temporary pools.

        3) COPEPODES are marine and
           freshwater microcrustacea--
           swim by means of unbranced
           antennae.

        4) OSTRACODS are like micro-
           scopic "clams with legs. "

        5) ISOPODS are dorsoventrally
           compressed; called sowbugs.
           Terrestrial and aquatic, marine
           and freshwater.

        6) AMPHIPODA - known as scuds,
           laterally compressed. Marine
           and freshwater.
        7) DECAPODA -  crabs, shrimp,
           crayfish,  lobsters, etc.
           Marine and freshwater.

      b INSECTA - body divided into head,
        thorax and abdomen; 3 paris of legs;
        adults typically with 2 pairs of
        wings and one pair of antennae.
        No common marine species.  Nine
        of the twenty-odd orders include
        species with freshwater-inhabiting
        stages in their life history as follows:

        1) DIPTERA - two-winged flies

        2) COLEOPTERA - beetles
        3) EPHEMEROPTERA - may flies
        4) TRICHOPTERA - caddis flies

        5) PLECOPTERA - stone  flies

        6) ODONATA - dragon flies and
           damsel flies
        7) NEUROPTERA - alder  flies,
           Dobson flies and fish flies.
        8) HEMIPTERA - true bugs,
           sucking insects such as water
           striders,  electric light bugs
           and water boatman

        9) LEPIDOPTERA - butterflies
           and moths, includes a few
           freshwater moths

      c ARACHINIDA - body divided into
        cephalothorax and abdomen; 4 pairs
        of legs - spiders, scorpions,  ticks
        and mites.  Few aquatic repre-
        sentatives except  for the freshwater
        mites and tardigrades.
C  CHORDATA
      PROTOCHORDATES - primitive marine
      forms such as acorn worms,  sea
      squirts and lancelets

      VERTEBRATES - aU animals which
      have a backbone

      a PICES or fishes: including such
        forms as sharks and rays,
        lampreys, and higher fishes; both
        marine and freshwater

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                                                   Aquatic Organisms of Significance
      b  AMPHIBIA - frogs, toads, and
         salamanders - marine species
         rare.

      c  REPTILA - snakes,  lizards and
         turtles.

      d  MAMMALS - whales and other
         warm-blooded vertebrates with
         hair.

      e  AVES - birds - warm-blooded
         vertebrates with feathers.
REFERENCE

Whittaker, R. H.  New Concepts of
   Kingdoms of Organisms.  Science
   163:150-160.   1969.
This outline was prepared by H. W. Jackson,
formerly Chief Biologist,  National Training
Center, Water Program Operations, EPA,
Cincinnati, OH 45268, and revised by
Ralph M. Sinclair, Aquatic Biologist,
National Training Center.


Descriptors:
Aquatic Life; Systematics, Streams,
Surveys, Stream Pollution
                                                                                 11-5

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                                                         Aquatic Organisms of Significance
                                                FUNGI
                      3/4
                          Sohizorayoetes -  Bacteria, free living representatives
                                   «  »                A

                               f  I
                            •  '   I A*
                              '   '*    *'
                                  +*••
         Aerobaoter
          aerogenes

Rhizobium
radio ioola
                                                                        Azotobacter
                         Phyoonyoetes - Saprolegnia; A,detail of immature reproductive
                       stages; B, mature oogonium and autheridia, with egp;s and fertili-
                       zation tubes; C, dead tadpole with growth of S.
  Phyoomyoete - Leptomitus.
this genus includes pollution
tollerant species.

     H.W.Jaokson
                                                         Asoomyoete - Sao char omyoes.  a
                                                      yeast including poll,  tollerant
                                                      species. A, single  cell; B,budding;
                                                      C, ascospore formation.
11-6
                                           PLATE I

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Aquatic Organisms of Significance
                                     BLUE GREEN ALGAE
                  3/4
                       OBoillatorla  spp.. filaments (triohomes) range from .6 to over
                       60/t  in  diameter. Ubiquitous, pollution tollerant.
                       Lypgbia spp.. similar to Osoillatoria "but has a shaath.
                        A, Lynebla oentorta.  reported to be  generally  intollarant
                       of pollutione B  L. birgei.
                                            B
                       Aphanizomenjn flos-aquae
                        A, oolonyi B,filament
  Anabaena flos-aquae
A, akinete; B,heterooyst
                                      PLATE  II
                                                                H.W.Jaokson
                                                                                             11-7

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                                                      Aquatic Organisms of Significance
         1/67
NON-MOTILE  GREEN ALGAE:   COCCOID
         (CHLOROPHYCEAE)
                                Pediaatruin
                   Species  of the Genus  Scenedesmus
                                                       3. dlmorphuB
                                 Desmids
                                                            Coemarium
                              PLATE  III
11-8

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Aquatic Organisms of Significance
                     NON-MOTILE GREEN ALGAE:   FILAMENTOUS




                                (Chlorophyceae)
                                  PLATE  IV
                                                                                 11-9

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                                                               Aquatic Organisms of Significance
                                   PIGMENTED FLAGELLATES
                3/4
                                                          Dinobrron
                                                A, form of colony) B,o«ll la lorlea.
                                                  I.W. Jaokion
                                    PLATE  V
11-10

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Aquatic Organisms of Significance
                                         DIATOMS
                                          vaiv«
               A discoid  or central
               diatom suoh as
                  Stephanodisous
                                        Girdle views,
                                      stylized to show
                                      basic diatom
                                      structure.
A pennate or navioular
   diatom suoh as
    Praelllarla
                   A colony  of Aeterionella
                       (girdle views)
                                                           A colony of Praglllarla
                                                              (girdle views)
    Qomnhonema
A,valve view; B,girdle
        view.
                 Diagram  showing progressive diminution in the size of certain
             frustules  tnrough  saooesolve cell generations of a diatom.
                                                           H.W. Jackson
                                            PLATE VI
                                                                                           11-11

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                                                                Aquatic Organisms of Significance
                                   FREE LIVING PROTOZOA
                I. Flagellated Protozoa,  Class llastigophora
               Atvthophy sis
           Pollution tolerant
                   6>u
Pollution tolerant
       19/t
                                                              Colony of Poteriodendron
                                                              Pollution tolerant , 35ja
                II. Ameboid Protozoa,  Class   Saroodina
              PImastigamoeha
            Pollution tolerant
                  10-50 ,u
 Nuelearla.reported
to be intolerant of
pollution, 45/1*
                III.  Ciliated Protozoa, Class Ciliophora
                 Colpoda
           Pollution tolerant
               20-120 ja.
  Holophrya.reported
 to be intolerant   of
 pollution,  35 /»

 PLATE VII
        Difflueta
    Pollution tolerant
                    60-5 00 JB
 BplatyllB. pollution
tolerant   Colonies often
maorosoopio.

      H.W.Jaokson
11-12

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Aquatic Organisms of Significance
                                    PLANKTONIC PROTOZOA
                                    Peranema trichophorum
                             Top
                            Side
               Chaos
Arcella
vulgarls
Actinosphaerium
              Vorticella
  Codonella
  cratera
   Tintlnnidium
   fluviatle
                                         PLATE  VIII
                                                                                         11-13

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                                           Aquatic Organisms of Significance
                PLANKTONIC  ROTIFERS
       Various Forms of Keratella cochlearis
Synchaeta
pectinata
Polygarthra
vulgaris~
Brachionus
quadridentata
                    Rotaria sp
                PLATE IX

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Aquatic Organisms of Significance
                        FREE LIVING NEMATHELMINTHES,  OR ROUND WORMS
                           -pharynx
                            a«rv« ring
                           -excrBtory pore
                           •salivary gland
                           •renette
                           . Intestine




                           _te»ti«



                           -•eminal vesicle


                           _vaa deferent
                           -ejaculatory gland
                           rectal gland

                           copulatory Bplcule

                          . gubernaculum
MonhyBtera
            Bhabditii
                                                        Acaromadora
                                           PLATE X
                                                                                        11-15

-------
                                                            Aquatic Organisms of Significance
                      FRESH WATER ANNELID WORMS

                                 Phylum Annelida
                 anus
                                    • mouth.

                  Class?  Oligoohaeta, earthworns

                  Ex:  Tub ifex   ,  the  sludgeworm

                          (After Liebman)
                      I.W.Jackson
                                                               mouth
                                                                    posterior sucker   disc
                                                         Class Hirudinea, leeches
                                                            (After  Hegner)
                                                                                anterior end
                                                         Class Polychaeta ,  polychaet worms

                                                         Ex; Manayunkia,  a minute,  rare, tube
                                                            building worm.
                                                                            (After  Leidy)
                                          PLATE XI
11-16

-------
Aquatic Organisms of Significance
                                  SOME MOLLUSCAN TYPES
           Class: Cephalopoda*

           Squids, octopus,

           cuttlefish.
                                       Exclusively marine.

                                   The giant  squid shown

                                   was captured In the

                                   Atlantic in  the early

                                   ninteenth  century.

                                   (After Hegner)
                         Limax.

                       a slug
                     Lymnaea
Campeloma
              an air breathing  mail  a water breathing
                                           snail
Class«  Gaetuopodao  snails and slugs. (After Buchebanm)
                          Class: Pelecypodaj clans, mussels, oysters.

                 Locomotion  of  a freshwater clam, showing how foot is  extended,  the  tip
                 expanded, and  the animal pulled along to its own anchor.  (After Buchs-
                 baum)                                                  H.W.Jackson

                                          PLATE XII
                                                                                         11-17

-------
                                                             Aquatic Organisms of Significance
                     3/4
                                             Class  CRUSTACEA
                                           Fairy Shrimp;
                                        Eupranohjpus. Order
                                           Phyllqpoda
                                             20-25 "m
           Crayfish,  or orawdadj
           Cambarus.  Order  Deoapoda
                   10-20 em
Sow Bug; Asellus.
 Order Jsopoda
          10-20 mm
                   Sbud; Hyalellq
                  Order Imphipoda
                       10-15 nm
 Pish Louse, Argulus;
 a parasitlo Copepod
        5-6 HOB
                                                                           H.W.Jaokson
                                                 i     "
                                    Copepod;  Cyolops. flrder Copepoda

                                                 2-5 «a

                                    PLATE XIII
11-18

-------
Aquatic Organisms of Significance
                                     Two Winged Flies
                                     Order  DIPTERA
                                           Adult midge
                                           (Chironomid)
                                                      Eat-tailed maggot
                                                         (IristaUs)
                                                     A,  adult; B,larva.
Adult sewage fly
   (Psychoda)
                                             Sewage fly larva
              Midge pupa
                                             Sewage fly pupa
                                                dge larva
                                                              H.W.Jackson
                                                              After various authors
                                         PLATE  XIV
                                                                                         11-19

-------
                                                            Aquatic Organisms of Significance
                                          Beetles
                                      Order COLEOPTERA
              body
            A diving beetle (Dytiseus)
         taking air at the surface.
                                                                                I
                                                Whirligig beetle  (Gyrinus) A, Side view
                                             of head-of adult showing divided eye;
                                             B, Larva; C, Adult.  Carnivorous.
                                                      The riffle beetle (Peephenus);
                                                   A.  adult; B, dorsal side of larva;
                                                   C,  ventral  side of larva.  Predominantly
                                                   herbivorous.
            A diving beeile (Cybiater).  The  div-
         ing beetles include some of the largest
         and most voracious of aquatic insects.
         A, larva; B,adult.
                                        PLATE XV
                                                                            B
    Crawling water beetle;
    A;adult; B,larva.  Predominantly
    herbivorous.

         H.W.Jackson.  After
Needham, Penriak, Morgan,and others.
11-20

-------
Aquatic Organisms of Significance
                                   MINOR PHYLA
               Phylum Coelenterata
                 Hydra with bud;
             extended, and contracted^
                                                               Medusa of
                                                           Crasoedacusta
                Phylum Bryozoa
                           Massive colony on
                                stick
                                                         Creeping colony
                                                           on rock
                                 Single zooid, young statoblasts in tube
                                         PLATE XVI
                                                                                      11-21

-------
                                                                    Aquatic Organisms of Significance
                                          SOME PRIMITIVE FISHES

                   Class Agnatha,  jawless fishes (lampreys and  hagfishes) - Family
                   PETROMYZONTIDAE,   the lampreys.  Lampetra aepyptera,  the
                   Brook Lamprey    A: adult,   B:  larva (enlarged)
                   Class Chondrichthyes - cartilagenous fishes (sharks, skates,  rays)
                   Family DASYATIDAE - stingrays.  Dasyatis centroura,  the Roughtail Stingray
                    Class Osteichthyes - bony fishes  - Family ACIPENSERIDAE, sturgeon.
                    Acipenser fulvescens, the Lake Sturgeon
                     Class Osteichthyes - bony fishes - Family POLYODONTIDAE,  the
                     paddlefishes.  Polyodon spathula, the Paddlefish.  A:side view  B:top view
                      Class Osteichthyes - bony fishes - Family LEP1SOSTEIDAE  - gars
                       Lepisosteus osseus,  the Longnose Gar
                      Class Osteichthyes - bony fishes - Family AMIIDAE, bowfins
                      Aniia calva,  the Bowfin
                    Reproduced with permission; Trautman,  1957.
                                                                BI.AQ.pl. 91. 6. 60
                                                  P1ATE XVII
11-22

-------
                                       TYPES OF BONY FISHES
                       /4
                       fe^—
                                                       male
          SKraP2pP£f"
          V?'KwWM«$><»WvS»»«?»>.,
          lV/>A'£'H#'-i'  '*'»'•'''"^T'Trr".-
          &'H#!^;§B^B''v: ':•;!': -^ ••••.:f-T~T?
f^j^^S^^vr^-'.'^:'''1--:
 '=i^*%^^M:Kife',:--:ari«^'  '^^K^
          . .... ,iJ^^Tv-,.'•'•.•;•'••';• '•;:
          •<^^"-^:,'^;ca^
                                                         v:.^^''-f>te::-:r=>^f^vIi;-.';v>'. .:, •','.'.-•'••  -*  _J~- - r-
             Family CLUPEIDAE - herrings
    Dorosoma cepedianum - the eastern gizzard shad
                                                                                             \' /, \
                                                     female
H
H
                                                            Family POECILIIDAE - livebearers
                                                             Gambusia affinis - the mosquitofish
        Family ANGUILLEDAE - freshwater eels
           Anguilla rostrata - the American eel
                                                     Family GADDIDAE - codfishes, hakes, haddock, burbot
                                                               Lota lota - the eastern burbot
                Family ESOCIDAE - pikes
             Esox lucius - the northern pike

    Reproduced with permission; Trautman,  1957

               BI.AQ.pl. 9m. 6. 60
                                                               Family SCIAENIDAE - drums
                                                        Aplodinotus grunniens - the freshwater drum
                                                                                                          >
                                                                                                         iS
il
'o
                                                                                                         ! 3
                                                                                                         ! n
                                                                                                         i n

-------
                KEY TO SELECTED GROUPS OF FRESHWATER ANIMALS
The following key is intended to provide
an introduction to some of the more
common freshwater animals. Technical
language is kept  to a minimum.

In using this key, start with the first
couplet (la, Ib),  and select the alternative
that seems most reasonable.  If you
selected "la" you have identified the
animal as a member of the group, Phylum
PROTOZOA.  If you selected "lb'\ proceed
to the couplet indicated.  Continue this
process until the selected statement is
terminated with the name of a group.

If you wish more information about the
 group, consult references.  (See reference
 St.)
 BI.AQ.21b.5.71
                                                                                    12-1

-------
Key to Selected Groups of Freshwater Animals
la    The body of the organism comprising
      a single microscopic independent
      cell, or many similar and indepen-
      dently functioning cells associated
      in a colony with little or no differ-
      ence between the cells, i. e. , with-
      out forming tissues; or body com-
      prised of masses of multinucleate
      protoplasm.  Mostly microscopic,
      single celled  animals.

          Phylum PROTOZOA

Ib    The body of the organism com-
      prised of many cells of different
      kinds, i.e., forming tissues.
      May be microscopic or macro-
      scopic.

2a    Body or colony usually forming
      irregular masses or layers some-
      times cylindrical,  goblet shaped,
     -vase shaped,  or tree like.  Size
      range from barely visible to
      large.

2b    Body or colony shows some type
      of definite symmetry.

3a    Colony  surface rough or bristly
      in appearance under microscope
      or hand  lens.   Grey, green, or
      brown.   Sponges.
          Phylum PORIFERA (Fig.  1)

3b    Colony  surface relatively smooth.
      General texture of mass gelatinous,
      transparent.  Clumps of minute
      individual organisms variously
      distributed.   Moss animals,
      bryozoans.
          Phylum BRYOZOA (Fig. 2)

4a    Microscopic.   Action of two
      ciliated  (fringed) lobes at an-
      terior (front)  end in life often
      gives appearance of wheels.
      Body often segmented,  accor'dian-
      like. Free swimming or attached.
      Rotifers or  wheel animalcules.
          Phylum TROCHELMINTHES
          (Rotifera) (Fig. 3)

4b    Larger, wormlike,  or having
      strong skeleton or shell.
5a  Skeleton or shell present. Skel- ,      15
    eton may be external or internal.

5b  Body soft and for wormlike.             6
    Skin may range from soft to
    parchment -like.

6a  Three or more pairs of well           19
    formed jointed legs present.
         Phylum ARTHROPODA (Fig. 4)

6b  Legs or appendages,  if present,         7
    limited to pairs of bumps or hooks.
    Lobes or tenacles, if present,
    soft and fleshy, not jointed.

7a  Body strongly depressed or             8
    flattened in cross section.

7b  Body oval,  round, or shaped like       10
    an inverted "U" in cross section.

8a  Parasitic inside bodies of higher
    animals.  Extremely long and flat,
    divided into sections  like a Roman
    girdle.  Life history  may involve
    an intermediate host. Tape worms.
         Class  CESTODA (Fig.  5)

8b  Body a single unit. Mouth and          9
    digestive system present, but no
    anus.

9a  External or internal parasite  of
    higher animals.  Sucking discs
    present for attachment.  Life his-
    tory may involve two or more in-
    termediate hosts or stages.  Flukes.
         Class TREMATODA

9b  Free living. Entire body covered
    with locomotive cilia.  Eye areas
    in head often appear "crossed".
    Free living flatworms.
         Class TURBELLARIA (Fig. 6)

lOa Long,  slender, with snake-like
    motion in life.  Covered with  glis-
    tening cuticle.  Parasitic or free-
    living.  Microscopic  to six feet in
    length.  Round worms.
         Phylum NEMATHELMINTHES
         (Fig. 7)

lOb Divided into sections  or segments      11
  12-2

-------
                                              Key to Selected Groups of Freshwater Animals
lOc  Unsegmented, head blunt, one        18
     or two retractile tentacles.
     Flat pointed, tail.

lla  Head a more or less well-formed,
     hard, capsule with jaws, eyes,
     and antennae.
          Class INSECTA order DIPTERA
          (Figs. 8A,  8C)

lib  Head structure soft, except          12
     jaws (if present).  Fig. 8E.)

12a  Head conical or rounded, lateral     13
     appendages not conspicuous  or
     numerous.

12b  Head somewhat broad and blunt.      14
     Retractile jaws usually present.
     Soft fleshy lobes or tentacles,
     often somewhat flattened, may be
     present in the head region.  Tail
     usually narrow.  Lateral lobes
     or fleshy appendages on each
     segment unless there is a large
     sucker disc at rear end.
          Phylum  ANNELIDA (Fig. 9)

13a  Minute dark colored retractile
     jaws present, body tapering
     somewhat at both ends, pairs or
     rings of  bumps or "legs" often
     present, even near tail.
          Class INSECTA Order DIPTERA
          (Fig. 8)

13b  No jaws,  sides of body  generally     14
     parallel  except at ends.  Thicken-
     ed area or ring usually present
     if not all the way back on body.
     Clumps of minute bristles on most
     segments. Earthworms, sludge-
     worms .
         Order OLIGOCHAETA

14a  Segments with bristles  and/or fleshy
     lobes or other extensions. Tube
     builders, borers, or burrowers.
     Often reddish or greenish in
     color. Brackish or fresh water.
     Nereid worms.
          Order POLYCHAETA (Fig.  9A)

14b  Sucker disc at each end, the large
     one posterior.  External blood-
    sucking parasites on higher animals,
    often found unattached to host.
    Leaches.
         Class HIRUDINEA (Fig.  9B)

15a Skeleton internal, of true bone.
    (Vertebrates)

15b Body covered with an external
    skeleton or shell.
         (Figs.  10,  13,  17,  18, 24,
         25, 28)
                                        40
                                         16
                                         19
                                         17
16a External skeleton jointed, shell
    covers legs and other appendages,
    often leathery in nature.
        Phylum ARTHROPODA

16b External shell entire, not jointed,
    unless composed of two clam-
    like halves.
        (Figs.  10,  11,  12)

17a Half inch or less in length.  Two
    leathery, clam-like shells.  Soft
    parts  inside include delicate,
    jointed appendages.  Phyllopods
    or branchiopods.
        Class CRUSTACEA,  Subclasses
       BRANCHIOPODA (Fig. 12)
        and OSTRACODA (Fig.  11)
17b     Soft parts covered with thin       18
    skin, mucous produced, no jointed legs.
        Phylum MOLLUSCA

18a Shell single,  may be a spiral cone.
    Snails.
        Class GASTROPODA (Fig. 13)

18b Shell double, two halves, hinged
    at one point.  Mussels,  clams.
        Class BIVALVIA (Fig. 10)

19a Three pairs of regular walking        29
    legs, or their rudiments.  Wings
    present in all adults and rudiments
    in some larvae.
        Class INSECTA (Figs. 22, 24D,
        25, 26,  28,  29)

19b More than three  pairs of legs          20
    apparently present.

20a Body elongated,  head broad and flat
                                                                                     12-3

-------
Key to Selected Groups of Freshwater Animals
     with strong jaws.  Appendages follow-
     ing first three pairs of legs are  round-
     ded tapering filaments.  Up to 3
     inches long.  Dobson fly and fish fly
     larvae.
          Class INSECTA  Order
          MEGALOPTERA (Fig.  14)

20b  Four or more pairs of legs.          21

21a  Four pairs of legs.  Body rounded,
     bulbous, head minute. Often
     brown or red.  Water mites.

          Phylum ARTHROPODA,  Class
          ARACHNIDA,  Order ACARI
          (Fig.  15)

21b  Five or more pairs of walking       22
     or swimming legs; gills, two
     pairs of antennae.  Crustaceans.
          Phylum ARTHROPODA,
          Class CRUSTACEA

22a  Ten or  more pairs of flattened,
     leaflike swimming and respiratory
     appendages.  Many species swim
     constantly in life; some swim
     upside down.  Fairy shrimps,
     phyllopods, or branchipods.
          Subclass BRANCHIOPODA
          (Fig.  16)

22b  Less than ten pairs of swimming     23
     or respiratory appendages.

23a  Body and legs inclosed in bi-         24
     valved (2 halves) shell which may
     or may not completely hide them.

23b  Body and legs not enclosed in        26
     bivalve shell.  May be large or
     minute.
          (Figs. 17,  18, 19)

24a  One pair of branched antennae
     enlarged for locomotion, extend
     outside of shell (carapace).
     Single eye usually visible.
     "Water fleas"
          Subclass CLADOCERA (Fig. 12)

24b  Locomotion accomplished by         25
     body legs, not by antennae.
25a Appendages leaflike, flattened,
    more than ten pairs.
        Subclass  BRANCHIOPODA
        (See 22 a)

25b Animal less than 3 mm, in length.
    Appendages more or less slender
    and jointed, often used for  walking.
    Shells opaque.  Ostracods.
        (Fig. 11)  Subclass OSTRACODA

26a Body  a series of six or more          27
    similar segments, differing
    mainly in size.

26b Front part  of body enlarged into       28
    a somewhat separate body unit
    (cephalothorax) often covered
    with a single piece of shell (cara-
    pace). Back part (abdomen) may be
    relatively small,  even folded
    underneath front part. (Fig. 19b)

27a Body  compressed laterally, i.e.,
    organism is tall and thin.   Scuds.
    amphipods.
        Subclass AMPHIPODA (Fig. 17)

27b Body  compressed dorsoventrally,
    i. e.,  organism low and broad.
    Flat gills contained in chamber
    beneath tail.  Sowbugs.
        Subclass ISOPODA (Fig.  18)

28a Abdomen extending straight out
    behind,  ending in two small pro-
    jections. One or two large masses of
    eggs are often attached to female.
    Locomotion by means of two enlarged,
    unbranched antennae, the only large
    appendages on the body.  Copepods.
        Subclass COPEPODA (Fig. 19)

28b Abdomen extending out behind ending
    in an  expanded "flipper" or swim-
    ming  paddle.   Crayfish or craw fish.
    Eyes  on movable stalks.  Size range
    usually from one to six inches.
        Subclass DECAPODA

29a Two pairs of functional wings,         39
    one pair  may be more or less  har-
    dened as protection for the  other
    pair.  Adult insects which normally
    live on or in the water. (Figs. 25, 28)
  12-4

-------
                                               Key to Selected Groups of Freshwater Animals
29b  No functional wings, though          30
     pads in which wings are develop-
     ing may be visible.  Some may
     resemble adult insects very
     closely, others may differ ex-
     tremely from adults.

30a  External pads or cases in which      35
     wings  develop clearly  visible.(Figs.
     24,26,27)
30b  More or less wormlike, or at        31
     least no external evidence of
     wing development.

31a  No jointed legs present.  Other
     structures such as hooks, sucker
     discs,  breathing tubes may be
     present.  Larvae of flies,
     midges, etc.
         Order DIPTERA (Fig. 8)

31b  Three  pairs  of jointed thoracic       32
     legs,  head capsule well formed.

32a  Minute (2-4mm) living on the
     water  surface film. Tail a
     strong organ that can be hooked
     into a  "catch" beneath the
     thorax.  When released animal
     jumps  into the air.  No wings
     are ever grown.  Adult spring-
     tails.
         Order COLLEMBOLA (Fig.  20)

32b  Larger (usually over 5 mm)          33
     wormlike, living beneath the
     surface.

33a  Live in cases or webs in water.
     Cases  or webs have a  silk
     foundation to which tiny sticks,
     stones, and/or bits of debris
     are attached. Abdominal segments
     often with minute gill filaments.
     Generally cylindric in shape.
     CaddisfLy larvae.
         Order TRICHOPTERA (Fig. 21)

33b  Free living,  build no cases.          34

34a  Somewhat flattened in  cross
     section and massive in appear-
     ance.  Each  abdominal segment
     with rather stout, tapering, lateral
     filaments about as long as body
    is wide.  Alderflies, fishflies,  and
    dobsonflies.
        Order MEGALOPTERA (Fig.  22, 14)

34b Generally rounded in cross section.
    Lateral filaments if present tend
    to be long and thin.  A few forms
    extremely flattened, like a suction
    cup.  Beetle larvae.
        Order COLEOPTERA (Fig. 23)

35a Two or three filaments or other       37
    structures extending out from
    end of abdomen.

35b Abdomen ending abruptly, unless      36
    terminal segment itself is extended   •
    as single structure.(Figs. 24A, 24C)

36a     Mouth parts adopted for chewing.
    Front of face covered by extensible
    folded mouthparts  often called  a
    "mask".   Head broad,  eyes widely
    spaced.  Nymphs of dragonflies
    or darning needles.
        Order ODONATA (Figs.24A. 24C, 24E)

36b Mouthparts for piercing and sucking.
    Legs often adapted for water lo-
    comotion.  Body forms various.
    Water bugs, water scorpions,  water
    boatmen,  backswimmers, electric
    light bugs, water striders, water
    measurers, etc.
        Order HEMIPTERA (Fig.   25)

37a Tail extensions (caudal filaments)
    two.  Stonefly larvae.
        Order PLECOPTERA (Fig. 26)
37b Tail extensions three,  at times
    greatly reduced in size.

38a Tail extensions long and slender.
    Rows of hairs may give extensions
    a feather-like appearance.
    Mayfly larvae.
        Order EPHEMEROPTERA
        (Fig. 27)

38b Tail extensions flat, elongated
    plates.  Head broad with widely
    spaced eyes, abdomen relatively
    long and  slender.  Damselfly
    nymths.
        Order ODONATA (Fig.
38
                                                                                    12-S

-------
 Key to Selected Groups of Freshwater Animals
 39a   External wings or wing covers
      form a hard protective dome
      over the inner wings folded
      beneath, and over the  abdomen.
      Beetles.
          Order COLEOPTERA
          (Fig. 28)

 39b   External wings leathery at base,
      Membranaceous at tip. Wings
      sometimes  very short. Mouth-
      parts for piercing and sucking.
      Body form various.  True bugs.
          Order HEMIPTERA (Fig. 25)

 40a   Appendage present in pairs.
      (fins, legs, wings).

 40b   No paired appendages.  Mouth
      a round suction disc.
4la   Body long and slender.  Several
      holes along side of head.
      Lampreys.
          Sub Phylum VERTEBRATA,
          Class CYCLOSTOMATA

41b   Body plump, oval.  Tail extending
      out abruptly.  Larvae of frogs and
      toads.  Legs appear one at a time
      during metamorphosis to adult
      form.   Tadpoles.
          Class AMPHIBIA
        42a   Paired appendages are legs         43

        42b   Paired appendages are fins,
              gills covered by a flap
              (operculum).  True fishes.
                  Class PISCES
        43a   Digits with claws, nails, or hoofs   44

        43b   Skin naked.   No claws or digits.
              Frogs, toads, and salamanders.
                  Class AMPHIBIA
42      44a   Warm blooded
41      44b   Cold blooded.  Body covered
              with horny scales or plates.
                  Class REPTILIA

        45a   Body covered with feathers.
              Birds.
                  Class AVES

        45b   Body covered with hair.
              Mammals.
                  Class MAMMALIA
45
12-6

-------
                                             Key to Selected Groups of Freshwater Animals
REFERENCES - Invertebrates

1  Eddy, Samuel and Hodson, A.C.
      Taxonomic Keys to the Common
      Animals of the North Central States.
      Burgess Pub. Company,  Minneapolis.
      162 p.  1961.

2  Edmondson,  W. T. (ed.) and Ward and
      Whipple's Freshwater Biology.  John
      Wiley & Sons, New York. pp. 1-1248.
      1959.

3  Jahn, T. L. and Jahn,  F.F.  How to Know
      the Protozoa. Wm. C.  Brown Company,
      Dubuque,  Iowa. pp.  1-234.   1949.

4  Klots, Elsie B.  The New Field Book of
      Freshwater Life.  G.P.  Putnam's Sons.
      398pp.   1966.

5  Kudo, R.  Protozoology.  Charles C.
      Thomas, Publisher, Springfield, Illinois.
      pp. 1-778.   1950.

6  Palmer, E.  Lawrence.  Fieldbook of
      Natural History.  Whittlesey House,
      McGraw-Hill Book Company, Inc.
      New York.  1949.

7  Pennak, R.W.   Freshwater Invertebrates
      of the United States. The Ronald Press
      Company, New York. pp. 1-769.  1953.

8  Pimentel,  Richard A.   Invertebrate
      Identification Manual.  Reinhold
      Publishing Corp.   151 pp.   1967.

9  Pratt, H.W.  A Manual of the  Common
      Invertebrate Animals Exclusive of
      Insects. The Blaikston Company,
      Philadelphia,  Pa. pp. 1-854.  1951.
 REFERENCES - Fishes

 1 American Fisheries Society. A List of
      Common and Scientific Names of Fishes
      from the United States and Canada.
      Special Publication No. 2, Am.  Fish
      Soc.  Executive Secretary AFS.
      Washington Bid.  Suite 1040,  15th &
      New York Avenue,  N.W.  Washington,
      DC 20005.  (Price $4.00 paper,
      $7.00 cloth).  1970.

 2 Bailey, Reeve M.  A Revised List  of
      the Fishes of Iowa with Keys for
      Identification,  IN:  Iowa Fish and
      Fishing.  State of Iowa, Super,  of
      Printing.  1956.  (Excellent color
      pictures).

 3 Eddy,  Samuel.  How to Know the
      Freshwater  Fishes.   Wm. C. Brown
      Company, Dubuque, Iowa.  1957.

 4 Hubbs, C.L. and Lagler,  K.F.   Fishes
      of the Great Lakes Region.  Bull.
      Cranbrook Inst.  Science,  Bloomfield
      Hills, Michigan.  1949.

 5 Lagler, K.F.   Freshwater Fishery
      Biology.   Wm. C.  Brown Company,
      Dubuque, Iowa.  1952.

 6 Trautman, M. B.   The Fishes of Ohio.
      Ohio State University Press,  Columbus.
      1957.   (An outstanding example  of a
      State study).
Descriptors:  Aquatic Life, Systematics.
                                                                                     12-7

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Key to Selected Groups of Freshwater Animals
         1. Spongilla spicules
            Up to .2 mm. long.
 3A. Rotifer, Polyarthra
     Up to . 3 mm.
                    3B. Rotifer, Keratella
                        Up to . 3 mm.
                                   3C. Rotifer, Philodina
     Up to . 4 mm.
  4A.  Jointed leg
       Caddis fly
4B. Jointed leg
    Crayfish
                                2B. Bryozoal mass. Up to
                                    several feet diam.
                                       2A.  Bryozoa,  Plumatella.  Individuals up
                                           to 2 mm.  Intertwined masses maybe
                                          very extensive.
4C. Jointed leg
     Ostracod
                                                                  5. Tapeworm head,
                                                                     Taenia.  Up to
                                                                      25 yds. long
       6A. Turbellaria. Mesostoma
           Up to 1 cm.
                                     6B. Turbellaria, Dugesia
                                          Up to 1.6 cm.
12-8
                              7. Nematodes. Free living
                                 forms commonly up to
                                 1 mm.,  occasionally
                                 more.

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                                     Key to Selected Groups of Freshwater Animals
                  8B.  Diptera, Mosquito
                      pupa. Up to 5mm.
8A. Diptera, Mosquito larvae
   Up to 15 mm. long.
                       8C.  Diptera, chironomid 8E'
                            larvae.  Up to 2 cm.
                                  9D.  Diptera, Rattailed maggot
                                      Up to 25 mm. without tube.
 9A. Annelid,
    segmented
    worm, up to
    1/2 meter
                                                    10B. Alasmidonta, end view.
                      10A. Pelecyopod, Alasmidonta
                          Side view, up to 18 cm. long.
9B. Annelid, leech up to 20 cm.
       i\\\
in
 11A. Ostracod, Cypericus
     Side view, up to 7 mm.
12A. Branchiopod,
      Daphnia.  Up
      to 4mm.
                       HB. Cypericus, end view.
                                                         12B.   Branchiopod,
                                                                Bosmina.  Up
                                                                to  2mm.
                                                                            12-9

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 Key to Selected Groups of Freshwater Animals
13. Gastropod, Campeloma
    Up to'3 inches.
                                            15. Water mite,
                                                up.to 3 mm.
                     14.  Megaloptera, Sialis
                         Alderfly larvae
                         Up to 25 mm.
16. Fairy  Shrimp,  Eubranchjpus
    Up to  5  cm.
            17.  Amphipod, Pomtoporeia
                Up to 25 mm.
                                         18.  Isopod,  Asellus
                                              Up  to  25 mm.
20. Collembola,  Podura
    Up  to  2  mm,  long

  12-10
\
19A. Calanoid copepod,
     Female       19B. C'yclopoid  copepod
     Up to 3 mm.       Female
                       Up  to 25 mm.

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                                 Key to Selected Groups of Freshwater Animals
     21A.
21B.
21C.
       21D.            21E.
    21. Trichoptera, l-arval cases,
       mostly 1-2 cm.
                   22. Megaloptera,  alderfly
                       Up  to 2 cm.
23A. Beetle larvae,    23B.  Beetle  larvae, 24A. Odohata, dragonfly
     Dytisidae,             Hydrophilidae       nymph up to 3 or
     Usually about 2  cm.    Usually about       4 cm
         t  .               1  cm.
                   24B. Odonata, tail
                        of damselfly
                        nymph
                        (side view)
                    Suborder
                     Zygoptera
                     (24B, D)
           4 24D.  Odonata, damselfly
                  nymph (top view)
                            24E,  Odonata, front view
                       •(//"„ I       of  dragonfly nymph
                                 showing "mask"
                                 partially extended
                              Suborder
                              Anisoptera
                                  (24A,  E, C)

                          24C. Odonata,  tail of
                               dragonfly nymph
                               (top view)
                                                12-11

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Key to Selected Groups of Freshwater Animals
            2SA. Hemiptera,
                 Water Boatman
                 About 1 cm.
            27 .Epheme ropte ra.
              Mayfly nymph
              Up to 3cm.
                                       25B.  Hemiptera,
                                             Water Scorpion
                                             About 4 cm.
28A.  Coleoptera,
     Water scavenger
     beetle. Up to 4 cm.
                              26. Plecoptera,
                                 Stone fly nymph
                                 Up to 5cm.
                                                               28B.  Coleoptera,
                                                                    Dytiscid beetle
                                                                    Usually up to 4  cm,
    12-12
               29A. Diptera. Crane
                   fly. Up to 2| cm.
                                                  29B. Diptera. Mosquito
                                                       Up to 20 mm.

-------
                   USING BENTfflC BIOTA IN WATER QUALITY EVALUATIONS
  I   BENTHOS ARE ORGANISMS GROWING
      ON OR ASSOCIATED PRINCIPALLY
      WITH THE BOTTOM OF WATERWAYS

      Benthos is the noun.

      Benthonic,  benthal and benthic are
      adjectives.

  II  THE BENTHIC COMMUNITY

  A  Composed of a wide variety of life
      forms that are related because they
      occupy "common ground"--the water-
      ways bottom substrates.  Usually
      they are attached or have relatively
      weak powers of locomotion.  These
      life forms are:

      1  Bacteria

         A wide variety of decomposers work
         on organic materials, breaking them
         down to elemental or simple com-
         pounds (heterotrophic).  Other forms
         grow on basic nutrient compounds or
         form more  complex chemical com-
         pounds (autotrophic).

      2  Algae

         Photo synthetic plants having no true
         roots,  stems,  and leaves.  The basic
         producers of food that nurtures the
         animal components of the community.

      3  Flowering Aquatic Plants (Pondweeds)

         The largest flora, composed of
         complex and differentiated tissues.
         Many are rooted.

      4  Microfauna

         Animals that pass through  a U. S.
         Standard Series No. 30 sieve, but
         are retained on a No.  100 sieve.
         Examples are rotifers and micro-
         crustaceans.  Some forms have
         organs for attachment to substrates,
      while others burrow into soft
      materials or occupy the interstices
      between rocks, floral or faunal
      materials.

    5  Meiofauna

      Animals, mostly metazoans, that
      can pass a 1.0 mm to 0. 5 mm
      screen.  Examples are naiad
      worms and flatworms.

    6  Macrofauna

      Animals that are retained on a
      No. 30 sieve.  This group includes
      the insects, worms, molluscs,  and
      occasionally fish.  Fish are not
      normally considered as  benthos,
      though there are bottom dwellers
      such as sculpins and darters.

B   It is a self-contained community, though
    there is interchange with other commun-
    ities.  For example:  Plankton settles
    to it, fish prey on it and lay their eggs
    there, terrestrial detritus leaves are
    added to it, and many aquatic insects
    migrate from it to the terrestrial en-
    vironment for their mating cycles.

C   It is a stationary water quality monitor.
    The low motility of the biotic. compon-
    ents requires that they "live with" the
    quality changes of the over-passing
    waters.   Changes imposed in the long-
    lived components remain visible for
    extended periods, even after the cause
    has been eliminated.   Only time will
    allow a cure for the community by drift
    and reproduction.

Ill  HISTORY OF BENTHIC OBSERVATIONS

A   Ancient literature records the vermin
    associated with fouled waters.

B   500-year-old fishing literature refers
    to animal forms that are fish food and
    used as bait.
BI. MET. fm.Se. 1.74
                                                                                     13-1

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Using Benthic Biota in Water Quality Evaluations
 C  The scientific literature associating
    biota to water pollution problems is
    over 100 years old (Mackenthun and
    Ingram,  1964).

 D  Early this century, applied biological
    investigations were initiated.

    1  The entrance of State boards of Health
       into water pollution control activities.

    2  Creation of state conservation agencies.

    3  Industrialization and urbanization.

    4  Growth of limnological programs
       at universities.

 E  A decided increase in benthic studies
    occurred in the  1950 decade, and much
    of today's activities are strongly influenced
    by developmental work conducted during
    this period.  Some of the  reasons for this
    are:

    1  Movement of the universities from
       "academic biology" to applied
       pollution programs.

    2  Entrance of the federal government
       into enforcement aspects of water
       pollution control.

    3  A rising economy and the development
       of federal grant systems.

    4  Environmental Protection Programs
       are a current stimulus.

 IV  WHY THE BENTHOS?

 A  It is a natural monitor

 B  The community contains all of the
    components of an ecosystem.

    1  Reducers

    2  Producers

    3  Consumers

       a  Detritivores and bacterial feeders
      b  Herbivores

      c  Predators

C  Economy of Survey

    1 Manpower

    2 Time

    3 Equipment

D  Extensive Supporting Literature

E  Advantages of the Macrobenthos

    1 Relatively sessile

    2 Life history length

    3 Fish food organisms

    4 Reliability of Sampling

    5 Dollars/information

    6 Predictability

    7 Universality

V  REACTIONS OF THE COMMUNITY TO
    POLLUTANTS

A  Destruction of Organism Types

    1 Beginning with the most sensitive forms,
      pollutants kill in order of sensitivity
      until the most tolerant form is the last
      survivor.  This results in a reduction
      of variety or diversity of  organisms.

    2 The usual order of macroinvertebrate
      disappearance on a sensitivity scale
      below pollution sources is shown in
      Figure 2.
Water
Quality
Deteriorati
\
Stoneflies /
Mayflies
ng Caddisffies
Amphipods
Isopods
Midges
^ Oligochaetes
k Water
Quality
Improving
  13-2

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                                            Using Benthic Biota in Water Quality Evaluations
       As water quality improves, these
       reappear in the same order.

B  The Number of Survivors Increase

    1  Competition and predation are reduced
       between forms.

    2  When the pollutant is a food (plants,
       fertilizers, animals, organic materials)

C  The Number of Survivors Decrease

    1  The material added is  toxic or has no
       food value.

    2  The material added produces toxic
       conditions as  a byproduct of decom-
       position (e.g., large organic loadings
       produce an anaerobic environment
       resulting in the production of toxic
       sulfides,  methanes,  etc.)

D  The Effects  May be Manifest in Com-
    binations

    1  Of pollutants and their effects.

    2  Vary with longitudinal  distribution
       in a stream.  (Figure  1)

E  Tolerance to Enrichment Grouping
    (Figure 2)

    Flexibility must be maintained in the
    establishment of tolerance lists based
    on the response  of organisms to the
    environment because of complex relation-
    ships among varying environmental
    conditions.   Some general tolerance
    patterns can be established.  Stonefly
    nymphs, mayfly naiads, hellgrammites,
    and caddisfly larvae represent a grouping
    (sensitive or intolerant) that is general-
    ised quite sensitive to environmental
    changes.  Blackfly larvae, scuds, sow-
    bugs,  snails, fingernail clams, dragon-
    fly nymphs,  damselfly nymphs, and most
    kinds  of midge larve are  intermediate
    (facultative or intermediate) in tolerance.
    Sludge-worms, some kinds of midge
    larvae (bloodworms), and some leeches
  cc
  in
  ID
  s
  I-
                DIRECTION OF FLOW •
     A.
       S /
       fe/
       i;
      8.
Ill
3
5
1
                      in
                      in
                      
                            I
                  TIME OR DISTANCE

         ..NUMBER OF  KINDS
         ..NUMBER OF  ORGANISMS
          .SLUDGE  DEPOSITS
      Four basic responses of bottom animals to pollution.
A. Organic wastes eliminate  the sensitive bottom animals
and provide food in the form of sludges for the surviving toler-
ant forms. B. Large quantities of decomposing organic wastes
eliminate sensitive bottom animals and the excessive quanti-
ties of byproducts of organic decomposition inhibit the tolerant
forms; in time, with natural stream purification, water quality
improves so that the tolerant forms can flourish, utilizing the
sludges as  food. C. Toxic materials eliminate the  sensitive
bottom rtnimalg; sludge is absent and food is restricted to that
naturally occurring in the stream, which limits the number of
tolerant surviving forms. Very  toxic materials may eliminate
all organisms below a waste source. D. Organic sludges with
toxic materials reduce the number of  kinds by  eliminating
sensitive forms. Tolerant survivors do not utilize  the organic
sludges because  the toxicity restricts their growth.
                     Figure 1
     are tolerant to comparatively heavy loads
     of organic pollutants.  Sewage mosquitoes
     and rat-tailed maggots are tolerant of
     anaerobic environments.
                                                                                          13-3

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Using Benthic Biota in Water Quality Evaluations
 F  Structural Limitations

     1 The morphological structure of a
       species limits the type of environment
       it may occupy.

       a  Species with complex appendages
          and exposed complicated respiratory
          structures, such as stonefly
          nymphs, mayfly nymphs, and
          caddisfly larvae, that are subjected
          to a'constant deluge of setteable
          particulate matter soon abandon
          the polluted area because of the
          constant preening required to main-
          tain mobility or  respirotory func-
          tions; otherwise, they are soon
          smothered.

       b  Benthic animals in depositing zones
          may also be burdened by "sewage
          fungus" growths including stalked
          protozoans. Many of these stalked
          protozoans are host specific.

     2 Species without complicated external
       structures,  such as bloodworms and
       sludgeworms, are  not so limited in
       adaptability.

       a  A sludgeworm, for example, can
          burrow in a deludge of particulate
                                 organic matter and flourish on the
                                 abundance of "manna."

                              b  Morphology also determines the
                                 species that are found in riffles, on
                                 vegetation, on the bottom of pools,
                                 or in bottom deposits.

                        VI  SAMPLING PROCEDURES

                        A  Fauna

                            1 Qualitative sampling determines the
                              variety of species occupying an area.
                              Samples may be taken by any method
                              that  will capture representatives of .the
                              species present.  Collections from such
                              samplings indicate changes in the
                              environment, but generally do not
                              accurately reflect the degree of
                              change.  Mayflies,  for example,  may
                              be reduced from 100 to 1 per square
                              foot. Qualitative data would indicate
                              the presence of both species, but might
                              not necessarily delineate the change in
                              predominance from mayflies to sludge-
                              worms .  The stop net or kick sampling
                              technique is often used.

                            2 Quantitative  sampling is performed to
                              observe changes in predominance.  The
                              most common quantitative  sampling
                              tools are the Petersen and Elkman
              REPRESENTATIVE EOT TOM-DWELLING MACROANIMALS

          Drawings from Geckler, j.,  K. M. Mackenthun and W. M. Ingram, 1963.
          Glossary of Commonly Used Biological and Related Terms in Water and
          Waste Water Control,  DHEW.  PHS, Cincinnati, Ohio, Pub. No. 999-WP-2.
    A
    B
    C

    D
    E
    F
    G
    H
(Plecoptera)
(Ephemeroptera)
Stonefly nymph
Mayfly nymph
Hellgrammite or
 Dobsonfly larvae (Megaloptera)
Caddisfly larvae  (Trichoptera)
Black fly larvae  (Simuliidae)
Scud             (Amphipoda)
Aquatic sowbug   (Isopoda)
Snail             (Gastropoda)
I  Fingernail clam
J  Damselfly nymph
K  Dragonfly nymph
L  Bloodworm or mi
   fly larvae
M  Leech
N  Sludgeworm
O  Sewage fly larvae
P  Rat-tailed maggot
  (Sphaeriidae)
  (Zygoptera)
  (Anisoptera)
dge
  (Chironomidae)
  (Hirudinea)
  (Tubificidae)
  (Psychodidae)
  (Tubifera-Eristalis)
                                    KEY TO  FIGURE 2

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                        Using Benthic Biota in Water Quality Evaluations
                   B       X^  C
                   SENSITIVE
              F             G
               INTERMEDIATE
H
M
                  TOLERANT
                                                           13-5

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Using Benthic Biota in Water Quality Evaluations
        grabs and the Surber stream bottom
        or square-foot sampler.  Of these,
        the Petersen grab samples the widest
        variety of substrates. The  Ekman
        grab is limited to fine-textured and
        soft substrates, such as silt and sludge.

        The Surber sampler is designed for
        sampling riffle areas; it requires
        moving water to transport dislodged
        organisms into its net and is limited
        to depths of two feet or less.

        The collected sample is screened with
        a standard sieve to concentrate the
        organisms; these are sorted from
        the retained material, and the number
        of each kind determined. Data are then
        adjusted to number per unit area,
        usually to number of bottom per
        square meter.

        Independently, neither qualitative not
        quantitative data suffice for thorough
        analyses of environmental conditions.
        A cursory  examination to detect damage
        may be made  with either method,  but
        a combination of the two gives a more
        precise determination.  If a choice must
        be made, quantitative sampling would
        be best, because it incorporates a
        partial qualitative sample.
  B   Flora
        Direct quantitative sampling of natu-
        rally growing bottom algae is difficult.
        It is basically one of collecting algae
        from a standardor uniform area of the
        bottom substrates without disturbing
        the delicate growths and thereby dis-
        tort the sample.  Indirect quantitative
        sampling is the best available method.

        Artificial substrates, such as wood
        blocks,  glass or plexiglass slides,
        bricks,  etc., are placed in a stream.
        Bottom-attached algae will grow on
        these artificial substrates.   After two
        or more  weeks,  the artificial sub-
        strates are removed for analysis.
        Algal growths are scraped from the
        substrates and the qunatity measured.
Since the exposed substrate area and
exposure periods are equal at all of
the sampling sites, differences in the
quantity of algae can be related to
changes in the quality of water flowing
over the substrates.

The quantity of algae on artificial sub-
strates can be measured in several
ways.   Microscopic counts of algal
cells and dry weight of algal material
are long established methods.

Microscopic counts involve thorough
scraping, mixing, and suspension of
the algal cells.  From this mixture
an aliquot of cells is withdrawn for
enumeration under a microscope.  Dry
weight is determined by drying and
weighing the algal sample, then ig-
niting the sample to burn off the algal
materials, leaving inert inorganic
materials that are again weighed.
The difference between initial weight
and weight after ignition is attributed
to algae.

Any organic sediments, however, that
settle on the artificial substrate along
with the algae are processed also.
Thus,  if organic wastes are present
appreciable errors may enter into
this method.

During the past decade, chlorophyll
analysis has become a popular method
for estimating algal growth.  Chloro-
phyll is extracted from the algae and
is used as an index of the quantity of
algae present.  The advantages of
chlorophyll analysis are rapidity,
simplicity,  and vivid pictorial results.

The algae are scrubbed from the
artificial substrate samples, ground,
then each sample is steeped in equal
volumes, 90% aqueous acetone,  which
extracts the chlorophyll from the algal
cells.   The  chlorophyll extracts may
be compared visually.
   13-6

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                                          Using Benthic Biota in Water Quality Evaluations
      Because the chlorophyll extracts fade
      with time,  colorimentry should be used
      for permanent records.  For routine
      records, simple colorimeters will
      suffice.  At very high chlorophyll
      densities, interference with colori-
      metry occurs, which must be corrected
      through serial dilution of the sample
      or with  a nomograph.

    4  Autotrophic Index

      The chlorophyll content of the periphyton
      is used  to estimate the algal biomass and
      as an indicator of the nutrient content
      (or trophic status) or toxicity of the
      water and the taxonomic composition
      of the community.   Periphyton growing
      in surface water relatively free  of
      organic pollution consists largely of
      algae, which contain approximately
      1 to 2 percent  chlorophyll a by dry
      weight.  If dissolved or participate
      organic matter is present in high con-
      centrations, large populations of
      filamentous bacteria, stalked protozoa,
      and other nonchlorophyll bearing micro-
      organisms develop and the percentage
      of chlorophyll a is then reduced. If the
      biomass-chlorophyll a relationship is
      expressed as a ratio (the autotrophic
      index),  values greater than 100 may
      result from organic pollution (Weber
      and McFarland,  1969; Weber, 1973).
    ...    . .  T .     Ash-free Wgt (mg/m  )
    Autotrophic Index = -75^	.  ,,& .—^T- o(
           r           Chlorophyll a (mg/m^)
VII FACTORS INVOLVED IN DATA INTER-
    PRETATION

Two very important factors in data evalua-
tion are a thorough knowledge of conditions
under which the data were collected and a
critical assessment of the reliability of the
data's representation of the situation.

A   Maximum-Minimum Values

    The evaluation of physical and chemical
    data to determine their effects on aquatic
    organisms is  primarily  dependent on
    maximum and minimum observed values.
    The mean is useful only when the data are
    relatively uniform.  The minimum or
    maximum values usually create acute
    conditions in the environment.

B   Identification

    Precise identification of organisms to
    species requires a specialist in limited
    taxonomic groups. Many immature
    aquatic forms have not been associated
    with the adult species.  Therefore, one
    who is certain of the  genus but not the
    species should utilize the generic name,
    not a potentially incorrect species name.
    The method of interpreting biological
    data on the basis of numbers of kinds
    and numbers of organisms will typically
    suffice.

C  Lake and Stream Influence

    Physical characteristics of a body of
    water also affect animal populations.
    Lakes or impounded bodies of water
    support different faunal associations
    from rivers. The number of kinds
    present in a lake may be less than that
    found in a stream because of a more
    unifrom habitat.  A lake is all pool,
    but a river is composed of both pools
    and riffles.  The nonflowing water of
    lake exhibits a more  complete set-
    tling of particulate organic matter that
    naturally supports a higher population
    of detritus consumers.  For these
    reasons,  the bottom fauna of a lake
    or impoundment cannot be directly
    compared with that of a flowing stream.

D   Extrapolation

    How can bottom-dwelling macrofauna
    data be extrapolated to other environ-
    mental components?  It must be borne
    in mind that  a component of the total
    environment is being sampled.  If the
    sampled component exhibits changes,
    then so must the other interdependent
    components of the environment.  For
    example, a clean stream with a wide
    variety of desirable bottom organisms
                                                                                    13-7

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Using Benthic Biota in Water Quality Evaluations
    would be expected to have a wide vari-
    ety of desirable bottom fishes; when
    pollution reduces the number of bottom
    organisms,  a comparable reduction
    would be expected in the number of
    fishes.  Moreover,  it would be logical
    to conclude that any factor that elim-
    inates all bottom  organisms would
    eliminate most other aquatic forms
    of life.

 VII IMPORTANT ASSOCIATED ANALYSES

 A  The Chemical Environment

    1  Dissolved oxygen

    2  Nutrients

    3  Toxic materials

    4  Acidity and alkalinity

    5  Etc.

 B  The Physical Environment

    1  Suspended solids

    2  Temperature

    3  Light penetration

    4  Sediment composition

    5  Etc.

 IX AREAS IN WHICH BENTHIC STUDIES
    CAN BEST BE APPLIED

 A Damage Assessment

    If a stream is suffering from pollutants,
    the biota will so indicate.   A biologist
    can determine damages by looking at the
    "critter" assemblage in a matter of hours.
    Usually, if damages are not found, it  will
    not be necessary  to alert the remainder
    of the agency's staff, pack all the equip-
    ment, pay travel  and per diem, and then
    wait five days before enough data can  be
    assembled to begin  evaluation.
B  By determining what damages have been
   done, the potential cause "list" can be
   reduced to a few items for emphasis and
   the entire "wonderful worlds" of science
   and engineering need not be practiced with
   the result that much data are discarded
   later because they were not applicable to
   the problem being investigated.

C  Good benthic data  associated with  chemical,
   physical,  and engineering can be data
   used to predict the direction of future
   changes and to estimate the amount of
   pollutants that need to be removed from
   the waterways.
REFERENCES

1 Hynes,  H. B.N.  The Ecology of Running
   Waters.  Univ. Toronto Press.  1970.

2 Keup,  L. E., Ingram, W.M. and
   Mackenthun, K. M.  The Role of
   Bottom Dwelling Macrofauna in Water
   Pollution Investigations.  USPHS
   Environmental Health Series Publ.
   No. 999-WP-38,  23 pp.  1966.

3 Keup,  L. E., Ingram, W.M. and
   Mackenthun, K.M.  Biology of Water
   Population:  A  Collection of Selected
   Papers on Stream Pollution,  Waste
   Water, and  Water Treatment.
   Federal Water Pollution Control
   Administration Pub. No. CWA-3,
   290pp.   1967.

4 Mackenthun,  K. M.   The Practice of
   Water Pollution Biology.  FWQA.
   281 pp.  1969.

5 Stewart, R.K.,  Ingram, W.M. and
   Mackenthun, K.M.  Water Pollution
   Control, Waste Treatment and Water
   Treatment:  Selected Biological Ref-
   erences on Fresh and Marine Waters.
   FWPCA Pub. No.  WP-23,  126 pp. 1966.
  13-8

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                                         Using Benthic Biota in Water Quality Evaluations
Weber,  Cornelins I.,  Biological Field         	
 and Laboratory Methods for Measuring         This outline was prepared by Lowell E.
 the Quality of Surface Waters and              Keup, Chief, Technical Studies Branch,
 Effluents.  U. S.  Environmental Pro-           Div.  of Technical Support,  EPA,  Wash-
 tection Agency,  NERC, Cincinnati,             ington, D. C. 20242, and revised by
 OH .  Environmental Monitoring Series         Ralph M. Sinclair, Aquatic Biologist,
 670/4.73.001 July 1973                       National Training Center, EPA, WPO,
                                               Cincinnati, OH  45268.

                                               Descriptors:
                                               Aquatic Life, Benthos, Water Quality,
                                               Degradation, Environmental Effects,
                                               Trophic Level,  Biological Communities,
                                               Ecological Distributions
                                                                                 13-9

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             AUTOMATIC INSTRUMENTS FOR WATER QUALITY MEASUREMENTS
I  NEED FOR CONTINUOUS MONITORING
   OF WATER QUALITY

Satisfactory evaluation of the quality of water
depends upon  the availability of adequate data.
Such data must be not only as accurate as
economically  practical but must also be of
sufficient quantity to support reliable con-
clusions.  In general, the more information
available,  the more reliable will be the
interpretations.

In the  past, information on the chemical,
physical, and bacteriological quality of most
surface waters has been obtained by periodic
stream surveys or infrequent "spot" analyses.
Although raw  water supplies are sampled
daily,  the data obtained are restricted to
those tests of importance in water  treatment
and do not include other pollutional param-
eters or water resources not at present being
used for water supply.

It is apparent that in many situations signifi-
cant changes in water quality may occur often
and abruptly.   Seasonal changes in flow, the
occurrence of unpredictable industrial dis-
charges or spills, and the changes in flow
from impoundments may alter the concen-
tration of many of the substances of interest
to the  water user.  In some cases these
changes may occur within a few hours:  for
example, the  diurnal fluctuations in dissolved
oxygen and the salinity changes in tidal
estuaries.  Thus considerable advantage is
gained if continuous monitoring of water
quality can be accomplished.  The  use of
manual sampling and standard laboratory
analyses would be far too expensive; therefore
a wide variety of automatic instruments have
been developed.
H  ELECTRICAL INSTRUMENTS

 The diagram below illustrates a typical
 system for monitoring instruments which
 utilize electrical sensors.
The sensor produces an electrical signal
representative of concentration, the signal
is converted,  and amplified,  then passed to
the recorder to develop a permanent record
of concentration levels.  For a clear
understanding of the operational charac-
teristics, the parts of the system are
discussed separately.

ANALYZER



>
RECORDER
                 DRAIN
             'SENSOR
      SAMPLE

A  Sensors
                Figure 1
   The sensor is the part of the system in
   contact with the sample.  Sensing ele- '
   ments may be immersed directly in the
   stream, or placed in flow cells through
   which the  sample  is pumped.  Both
   methods have advantages and limitations.

   When the sensor is placed in the stream
   the determination is made "in situ. "
   Therefore the sample is not affected by
   pumping, temperature changes, or time
   of travel through the instrument.  Such
   an installation,  however, presents
   certain problems.  The sensing elements
   must be protected  from floating debris
   and must be mounted so as to remain  in
   a fixed position in  spite of changes in
   velocity or direction of current.   In bodies
   of water which fluctuate in surface level,
   such as impoundments, or estuaries,  the
   relative depth of the sensor may change.
   In addition, frequent inspection of the
   sensing elements for attached growths or
   physical damage is difficult and apt to be
   neglected.
 CH.MET. 18b.3.74
                                                                                     14-1

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Automatic Instruments for Water Quality Measurements
   The use of the shore-based system, where
   the stream sample is pumped through flow
   cells within the instrument housing, is
   free from some of the difficulties men-
   tioned previously.  Inspection of the
   sensors is easy,  cleaning is simplified,
   and replacement of sensing elements is
   readily accomplished.  It should be
   recognized, however,  that the precautions
   required for satisfactory mounting and
   protection of sensor units in the stream
   apply equally well to the pump intake.
   Further, it is essential that the sample
   being tested in the flow cell is truly
   representative of the stream water.  If
   dissolved oxygen is included in the
   parameters, a submersible pump is
   required, to avoid cavitation and prevent
   suction removal of dissolved gases.  The
   intake screen must be carefully designed,
   since a fine screen will quickly  clog, while
   a coarse screen may permit floating
   material to enter the system.

   Electrical sensors may be conductimetric,
   potentiometric,  polarographic,  or
   coulimetric.  The sensor may directly
   measure a constituent or property of the
   sample or  it may be used as an  indicating
   mechanism in automatic titration.

B Analyzer-Amplifier

   The function of the analyzer is to convert
   the signal from the sensor to a standard
   EMF, usually ranging from 0-50 millivolts.
   Often bridge circuits are employed.  The
   analyzer must be rugged to avoid shock
   damage and the electronic  circuitry must
   be stable over a wide range of environmental
   conditions.  Considerable advantage is
   gained by the use of standard, readily
   available components.  Provision should
   be made for a standard signal to permit
   internal checking of the circuits.

   In most cases the  signal must be amplified
   to provide  sufficient voltage to drive a
   recorder.  Amplification is generally
   built into the analyzer circuitry.
C  Recorder

   Although read-out meters can be used,  a
   permanent record is desirable..  Strip-
   chart recorders are the most popular
   even though they require line voltage.
   A slow chart speed is necessary, since
   observation periods of several days
   are common.  Difficulties with pens are
   often encountered due to varying con-
.   ditions of humidity and temperature.
   The use of pressure-sensitive paper
   may offer distinct advantage.

   Circular charts have long been used but
   they have certain disadvantages.  Unless
   the chart is inconveniently large the chart
   divisions are very close together making
   hourly fluctuations difficult to interpret.
   In addition, the circular chart does not
   lend itself to mechanical handling of data
   for computer processing.  Chart-drive by
   clock mechanism is possible, however,
   eliminating the need for power connection.

   A preferable system for the recording of
   data is the use of digital output.  Water
   quality instruments can be equipped with
   analog-to-digital converters which change
   the amplitude of the signal to a digital
   value.  By the use of a punch-tape readout
   the data is recorded on paper tape rather
   than on a chart.  The punched tape may
   then be transferred to a computer for data
   processing.  Therefore, a monitor equipped
   with digital tape readout permits the re-
   cording of original signal and the per-
   formance of statistical computations without
   hand transcription.

  When the Surveillance  Program includes
  several automatic monitors on a  river
  system , consideration should be given to
  a central data logging facility. By
  transmitting the data from the monitors
  directly to a small computer (by  leased
  phone lines or radio),  the information on
  water quality of the entire river basin is
  immediately available.  The central
  facility also provides for the detection of
  unusual discharges which may affect
  downstream water users.
   14-2

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                                   Automatic Instruments for Water Quality Measurements
  D  Parameters Measured Electrically

     The following water quality parameters
     can be measured by the  use of electrodes:
     Temperature

     Conductivity

     PH


     Oxygen
Chloride

Residual chlorine

Oxidation-reduc-
tion potential
     A prominent instrument manufacturer is
     now conducting research into the develop-
     ment of a number of glass electrodes
     specifically designed for the measurement
     of other ions.  It appears that the list above
     may be considerably expanded in the future.

    In most cases precision and accuracy is
    not as good as  with standard laboratory
    tests,  but the advantages of continuous
    recording outweigh the limitations in
    performance.

Ill  PHOTOMETRIC INSTRUMENTS

 Because of the importance of colorimetric
 analysis in the water laboratory, considerable
 attention has been given to the  development of
 continuous monitoring instruments employing
 this principle.  A  typical system is shown
 in Figure 2.
                       It should be noted that the sample must be
                       pumped  through the instrument with the
                       attendant problems described above.
A  Measurement of Turbidity and Color


   In the simplest photometric instrument,
   a property of the sample, such as
   turbidity or color, is measured directly.
   These relatively simple parameters,
   however,  are rather difficult to determine.
   Turbidity measurements are affected by
   particle size "and by the true color of the
   sample.  Conversely,  color determinations
   are subject to errors caused by turbidity
   and by the fact that the wave length of the
   color in the sample may vary widely.
   Instruments of this type currently
   available do not satisfactorily compensate
   for  these interferences and the data
   obtained from them does not correlate
   well with standard methods values.
                                               ANALYZER
                                               COLORIMETER
           SAMPLE
                        DRAIN
                                                                                       14-3

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Automatic Instruments for Water Quality Measurements
 B Colorimetric Analyzers

    A second type of photometric instrument
    is designed to reproduce laboratory
    colorimetric procedures.  That is, re-
    agents are added to the sample to produce
    a color change proportional to the con-
    centration of the material being determined.
    Since the sample is flowing continuously
    the reagents must  be metered accurately
    and  mixed thoroughly before photometric
    measurement.  In  a properly designed
    system, almost any colorimetric procedure
    can be duplicated and therefore the potential
    range of determinations is much wider
    than in the electrometric instruments.

    A modification of the colorimetric instru-
    ment is the continuous  titrator.  In this
    system an indicator is  added to the flowing
    sample and the reagent is added at a vari-
    able  rate to maintain a constant color.
    The amount of reagent  required is pro-
    portional to the concentration of the re-
    actant in the sample, and the current used
    by the metering pump acts  as a signal for
    the analyzer.

    In spite of the apparent advantages of the
    photometric  systems, certain special
    problems are inherent.  The color and
    turbidity of the sample  may interfere, the
    accumulation of slime in the cells may
    seriously reduce the sensitivity, and the
    limitations of the filter photometer (wide
    band pass) must be considered.  Further,
    in most colorimetric procedures the
    amount of  reagent  required is proportional
    to the concentration range of the sample,
    a factor which would limit the applicability
    of the instrument.

 C  Parameters  Measured  Photometrically

    Continuous analytical procedures have
    been developed for:
    Turbidity

    Color
    Hardness
    Residual Chlorine
    Alkalinity
Fluoride

Silica
Phosphate
Phenols
Chemical Oxygen
Demand
IV  PERFORMANCE

 To illustrate the quality of the data which may
 be obtained from an integrated water quality
 monitor, the following table shows the results
 of a performance test conducted on a proto-
 type instrument supplied by one  manufacturer.
 The "Acceptable" tolerances were selected as
 representative of the usual requirements for
 continuous data acquisition and may be too
 high or too low, depending upon  the accuracy
 deemed necessary.


  V  CALIBRATION AND MAINTENANCE

  A  Calibration

     In the parlance of the instrument manu-
     facturer, calibration involves two steps;
     (1) the setting of the readout to zero value
     in the absence of sensor signal, and
     (2) setting of the readout to some standard
     value,  such as 5mv. when the sensor
     signal is replaced by a standard signal.
     It is readily apparent that a  calibration
     of this type adjusts  the meter and amplifier
     to reproduce correctly the signal received
     from the sensing elements.  It does not,
     however, "calibrate" the total instrument
     in terms of the concentration of measured
     substance in the sample.

     For proper calibration, it is essential
     that the final readout of the instrument be
     adjusted to correspond to a true value for
     the measured material.  Thus the only
     adequate calibration must involve the
     measurement of a standard solution, such
     as a buffer of known pH or a salt solution
     of known conductance.  Since instrument
     systems may lack linear response to a
     wide signal range,  the instrument should
     be calibrated at several points over the
     range of values anticipated.  In some
     cases current instrument design has not
     included adequate means  of replacing the
     sample with standard solutions for calibra-
     tion purposes.

     The frequency of recalibration depends
     upon both the stability of the analyzer and
     the sensor. In general, analyzer-amplifiers
     are more stable than the sensing systems
     now in use. The Basic Data Program,
  14-4

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                                    Automatic Instruments for Water Quality Measurements
           Test

     PH

     Temperature

     Conductance

     Dissolved Oxygen
        PERFORMANCE DATA

Acceptable deviation      Mean deviation found

     + 0.1 unit               0.1 unit

     + 1°                    0. 2°

     + 5%                    17  (imhos

     + 0.5 mg/1              0.3 mg/1
                              Acceptable

                                80%

                                85%

                                99%

                                73%
  FWPCA, in performance specifications
  for monitoring instruments,has established
  a two-week period for unattended per-
  formance.  While such instrument stability
  is desirable, circumstances  of site location
  and sample characteristics,  often
  necessitate more frequent checking and
  recalib ration.
B  Maintenance

   It is unwise to assume that any stream
   monitoring instrument, no matter how
   well designed and built, can function
   adequately for extended periods of time
   without maintenance.   Sensing elements
   are subject to physical, chemical, and
   biological actions, analyzer components
   may fail or perform erratically, and
   recorders may stop or fail to print.   The
   frequency of maintenance depends upon a
   large number of factors both controllable
   and accidental and can be determined
   only by long-term testing and actual field
   experience. Ease of maintenance can be
   designed into the instrument by  the use of
   replaceable electronic components and
   accessibility for cleaning of flow cells
   and sample lines.
    The availability of trained personnel for
    checking and maintaining the instruments
    in the field is a key factor in the successful
    stream monitoring program.  Such per-
    sonnel need not be either electronic
    specialists or experienced analysts but
    they should have a working knowledge of
    the instrument system and a familiarity
    with the chemical principles involved in
    the use of standard solutions for calibration.

VI  APPLICATIONS

  Stream monitoring systems are now in use
  in a variety of locations throughout the United
  States.  In most cases the instruments
  measure a single parameter or condition of
  the stream rather than a number of para-
  meters.  Some of the instruments employ
  well established measurement systems, for
  example pH and conductance,  while others,
  are still largely experimental.

  Because of the need for continuous stream
  data,  the intensive interest on the part of
  regulatory agencies, and the development
  programs by instrument makers,  it is safe
  to assume that the use of water quality
  monitoring instruments  will increase
  significantly in the near future.

  This outline was prepared by D. G. Ballinger,
  Director,  EPA, WPO, Methods Development
  & Quality Assurance Laboratory, Cincinnati,
  OH  45268

  Descriptors:  Analytical Techniques,  Instru-
  mentation, Testing Monitoring,  Control
  Systems,  Data Collections,  Data Trans-
  mission, Remote Sensing
                                                                                    14-.5

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                                  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 Azide 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.
MnSO + 2
2 Mn(OHL
A
MnO(OH)0
£
Mn(SO.)0 H
4 2
I2 + 2 Na2!
KOH ~* Mn(OH)2 + K2SO4
+ O0 -*2 MnO(OH)
^ ^
+ 2 H SO -* Mn(SO ) + 3H
£> rt *± ^ £
h 2 KI -» MnSO + K SO + I
4 ^ 4 Z
3 O ~* Na SO + 2 Na I
(a)
(b)
,0(c)
(d)

(e)
     2   Reaction sequence

         The series of reactions involves
         five different operational steps in
         converting dissolved oxygen in the
         water into a form in which it can
         be measured.
                              Mn(S04)2
             I -+ Thiosulfate (thio) or
              £t
             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 —*•
MnO(OH)   (reactions a, B) is an
oxygen transfer operation where
the dissolved oxygen in the water
combines with manganous
hydroxide to form an oxygenated
manganic hydroxide.

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

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

c   Transfer of oxygen from the
    dissolved state to the pre-
    cipitate form  involves a two
    phase system of solution and
    precipitate requiring effective
    mixing for quantitative
    transfer.  Normally a gross
    excess of reagents 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.3.74
                                                                                    15-1

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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 MnSO  reagent (2 ml)

2)  Add KOH,  KI, NaN  reagent
    (2 ml)          .  d
    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.
15-2

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                                                    Dissolved Oxygen Determination
              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 NaN3  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 MnO(OH>2.
          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.

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

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

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

       Addition of DO reagents results in
       reaction with dissolved or entrained
       oxygen.  Results for DO are invalid
       if there is any evidence of gas
       bubbles in the sample bottle.

C  The DO reagents respond  to any oxidant
   or reductant in the sample capable of
   reacting within the time allotted.  HOC1
   or H2O2 may raise the DO titration
   while  t^S, & SH  may react with sample
   oxygen to lower the sample titration.
   The items mentioned react rapidly and
   raise  or lower the DO result promptly.
   Other items such as Fe    or SOl 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
                                                                                    15-3

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Dissolved Oxygen Determination - II
    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.
 E
        These reactions may be repre-
        sented as  follows:
2HN02 + 2 HI -J2 + 4H20 + N.O2
                            .
           1/202+
                                      (a)

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

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

3   Sodium azide added to fresh
    alkaline KI reagent is adequate  to
    control interference up to about
    20 mg of  NO2"  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.

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.
                                                 This outline was prepared by F. J. Ludzack,
                                                 Chemist,  National Training Center, EPA,
                                                 WPO,  Cincinnati,  OH 45268, and revised
                                                 by C.  R.  Feldmann,  Chemist, National
                                                 Training  Center.


                                                  Descriptors:  Chemical Analysis, Dissolved
                                                  Oxygen,  Oxygen, Water Analysis
    15-4

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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 differences
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 ion, 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 Zn°  - 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:
2H
+02  -
-4e  3±
°2+4e
2H20

4H    hydrogen oxidized

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 Does
             Not Imply Endorsement by the Environmental Protection
             Agency.
  CH.O. do.32a. 3.74
                                16-1

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

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

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

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

                    Figure 2
Anode
                                     Cathode
              GALVANIC-CELL

                  Figure 1
D  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.

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

   1 The galvanic  cell depends upon
     measurement of electrical energy
     produced as a result of oxygen
 16-2

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

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

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

 A  Reduction of oxygen takes place in two
    steps as shown in the following equations:
    1  O0 + 2H0O + 2e -* HO  + 2OH
        ft     ci           2i  &
    2  H2O2 + 2e -+ 2OH


    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).  Lead
   is commonly used as the anode because
   its decomposition potential favors
   spontaneous reduction of oxygen. The
   process is continuous as long as lead
   and oxygen are in contact in the electrolyte
   and the electrical energy released at
   the  cathode may be dissipated by an
   outside circuit.  The anode may be
   conserved by limiting oxygen availability.
   Interrupting the outside circuit may
   produce erratic behavior for a time
   after reconnection. The resulting
                                                                                      16-3

-------
 Dissolved Oxygen Determination - II
       current produced by oxygen reduction
       may be converted to oxygen concen-
       tration by use of a sensitivity coefficient'
       obtained during calibration.  Provision
       of a pulsed or interrupted signal makes
       it possible to amplify or control the
       signal and adjust  it for direct reading
       in terms of oxygen concentration or to
       compensate for temperature effects.
Ill  E LE CTRONIC DO A NA LY ZER
    APPLICATION FACTORS

 A Polarographic or galvanic DO instruments
    operate as a result of oxygen partial
    pressure at the sensor surface to produce
    a signal characteristic of oxygen reduced
    at the cathode of some electrode pair.
    This signal is conveyed to an indicating
    device with or without modification for
    sensitivity and temperature or other
    influences  depending upon the  instrument
    capabilities and intended use.

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

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

 B Variables Affecting Electronic DO
    Measurement

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

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.

Electrode sensitivity changes occur as
a result of the nature and concentration
of contaminants at the  electrode sur-
faces and possible physical chemical or
electronic side reactions produced.
These may take the form of a physical
barrier,  internal short, high residual
current,  or  chemical changes in the
metal surface.  The  membrane is
intended to allow dissolved gas pene-
tration but to exclude passage of ions
or particulates. Apparently  some ions
or materials producing extraneous ions
within the electrode vicinity are able
to pass in limited amounts which
    16-4

-------
                                                    Dissolved Oxygen Determination
   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 H.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
                                                                                  16-5

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

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

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

      b Substandard  or unsteady amplifier
        or resistor components.

      c Undependable contacts or junctions
        in the sensor, connecting cables, or
        instrument control circuits.

      d Inadequately shielded electronic
        components.

      e Excessive exposure to moisture,
        fumes or chemicals 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 maintain  good working characteristics.

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

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

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

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

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

   9  Major load batteries should be
      rechargeable or readily replaceable.
      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
   16-6

-------
                                                     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.
       a  Adjust the instrument calibration
          control if necessary to compare
          with the titrated DO.

       b  If sensitivity adjustment is not
          possible,  note the instrument
          stabilization point and designate
          it as ua.   A sensitivity coefficient,

           <)> is equal to trrr where DO is the
          titrated value for the sample on
          which ua was obtained.  An unknown
                                ua
          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 specific instrument does not
  imply  EPA   endorsement or recommendation.
  No attempt has been made to include all the
  available  instruments; those described are
  used to indicate the approach used at one
  stage of development which may or may not
  represent the current available model.

  A  The electrode described by Carrit and
    Kanwisher (1) is illustrated in Figure 3.
    This electrode was an early example of
    those using a membrane.  The anode was
    a silver  -  silver oxide reference cell with
    a platinum disc cathode (1-3 cm diameter).
    The salt bridge consisted of N/2 KC1 and
                                                                                  16-7

-------
Dissolved Oxygen Determination - II
   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.
                        — Silver Ring


           'latinum Disk t	Electrolyte Layer
                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
                      AMPUFIK
              -GOIO CATMOOf
   The YSI Model 51 (3) is illustrated in
   Figure  5.  This is another form of
   polarographic DO analyzer.  The  cell
   consists of a silver anode coil,  a  gold
   ring cathode and a KC1 electrolyte with
   a teflon membrane.  The instrument has
   a sensitivity adjustment, temperature and
   DO readout.  The model 51 A has temp-
   erature compensation via manual  preset
   dial.  A field probe and bottle probe are
   available.

               YSI Model 51 DO Sen»or
      'O' Ring

   Membrane

  KCL Solution
       ^^
  Anode  Coil


  Cathode Ring—-tt
                                                                        Figure 5
D  The Model 54 YSI DO analyzer (4) is based
   upon the same electrode configuration but
   modified to include automatic temperature
   compensation, DO readout,  and recorder
   jacks.  A motorized agitator bottle probe
   is available for the Model 54 (Figure  6).
   Figure 4,  THE BECKMAN OXYGEN
                 SENSOR
                                                                            .Mimbran* ••teln*r
                                                                                Ring
                                                                           Agitator Ring
  16-8

-------
                                                       Dissolved Oxygen Determination -
E  The Galvanic Cell Oxygen Analyzer (7, 8)
   employs an indicator for proportional DO
   signal but does not include thermistor
   compensation or signal adjustment.
   Temperature readout is provided.  The
   sensor includes a lead anode ring, and
   a silver cathode with KOH electrolyte
   (4 molar) covered by a membrane film
   (Figure 7).
        Precision Galvanic Call Oxygen Probe
                                       adl
                               Thermistor Cable
                                  Retainer
                               Tapered Section
                              to fit BOD Bottle!
Plaitic Membrane
 Retainer Ring

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

                   This outline contains certain materials from
                   previous outlines by D. G. Ballinger,
                   N. C. Malof,  and J.W. Mandia. Additional
                   information was provided by C.R. Hirth,
                   C.N. Shadix,  D.F. Krawczyk, J.  Woods,
                   and others.
                                                                                       16-9

-------
Dissolved Oxygen Determination
      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
  16-10

-------
       Cable Sealing
           Nut
          A 15017
                       Cable
                     Connection
                       Cover
                      A15016A
                  'O'JRmg
                   R524
                                             Model A15A ELECTRODE COMPONENT PARTS
               Lead Anode
                Complete
                         'O' Ring
                           R389
                                       (A15024A)
                       Membrane Securing
                              ' Rin   "
                              R317
           'O' Ring
            R385
Membrane— Securing
      'O' Ring
       R317
                                  Silver Cathode
                                     A15013A
                                     Filler Screw
                                       Z471
                                                                               mimnirmT
    Anode
    Contact
    A -15 0140
(With Sleeve S24)
'O' Ring
  R612
'O' Ring
 R622
                                     Anode
                                    Contact
                                     Holder
                                    A15015A
                                                                                                 End Cap
                                                                                                 AlSOllA
                                        '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.
OJ
i
                                                        Figure 9

-------
 Dissolved Oxygen Determination
                                         TABLE 1

             CHARACTERISTICS OF VARIOUS LABORATORY DO INSTRUMENTS
                Anode
Cathode   Elec
              DO
              Sig.
Type  Membr  Adj.
Temp.
Comp.      Accessories for
Temp. Rdg. which designed
Carrit &
Kanwisher
Beckman
Yellow Springs
51
Yellow Springs
54
Precision
Sci
Weston &
Stack
300
EIL
Delta
75
Delta
85
silver- Pt
silver ox. disc
ring
Aq
ring
Ag
coil
II
Pb
ring
Pb
coil
Pb
Lead
Lead
Au
disc
Au
ring
11
silver
disc
Pt
disc
Ag
Silver
disc
Silver
disc
KC1
KOH
N/2
KC1
gel
KC1
soln
sat.
II
KOH
4N
KI
40%
KHCOg
KOH
IN
KOH
IN
pol* polyeth
pol tenon
pol teflon

galv**polyeth
galv tenon
galv tenon
galv teflon
galv teflon
no
yes
yes
yes
no
yes
yes
yes
yes
no
yes
yes
no*
yes
yes
yes
no
yes
yes
yes
yes
yes
yes
no
yes
yes
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
 *Pol - Polarographic (or amperometric)
**Galv - Galvanic (or voltametric)
 REFERENCES

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

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

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

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

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

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

                      8  Briggs, R.  and Viney, M.   Design and
                           Performance of Temperature Com-
                           pensated Electrodes  for Oxygen
                           Measurements.  Jour, of Sci.
                           Instruments 41:78-83.  1964.
   16-12

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                                                       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-AQCL,  Cincinnati,
     Pollution Control, pp.  537-539.  1967.              OH, July. 1971.

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


                                                Descriptors ;  Chemical Analysis, Dissolved
                                                Oxygen, Dissolved Oxygen Analyzers,
                                                Instrumentation, On-Site Tests, Water Analysis,
                                                Analysis,  Wastewater
                                                                                   16-13

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                                    DISSOLVED OXYGEN
                       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, or
       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
           20°C.  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. 3. 74
                                                                                      17-1

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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% than  to increase
          oxygen saturation from 0 to about
   95%.   For example:  ^n oxygen-
   depleted sample often will pick up
   significant DO during DO testing;
   changes are unlikely with a  sample
   containing equilibrium amounts
   of DO.
   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.

   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 IQOC is about two
times that at 30o C.  Temperature
often contributes to DO variations
much greater than anticipated by
17-2

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                                               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 loss  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 20° C.   BOD dilutions
should be'adjusted to 20° C + or -
1  1/20 before filling and testing.

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

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/1
under one atmospheric pressure of
        air, about 42 mg DO/liter in
        contact with pure oxygen and 0 mg
        DO/liter in contact with pure
        nitrogen (@  20°  C).

B   Biological or Bio-Chemical Factors

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

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

        a    Photosynthesis occurs only
             under the influence of adequate
             light intensity.

        b    Respiration  of alga is
             continuous.

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

    3   High rate deoxygenation commonly
        accompanies assimilation of
        readily available nutrients and
        conversion into cell mass or
        storage products.  Deoxygenation
        due to cell mass  respiration
        commonly occurs at some lower
        rate dependent upon the nature of
        the organisms present,  the  stage
        of decomposition and the degree
        of predation, lysis, mixing and
        regrowth.  Relatively high
                                                                         17-3

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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 benthlc 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-AQCL,
   Cincinnati, OH, July 1971.
This outline was prepared by F. J.  Ludzack,
Chemist, National Training Center, MDS,
WPO,  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
17-4

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                 CHEMICAL OXYGEN DEMAND AND COD/BOD RELATIONSHIPS
    DEFINITION

    The Chemical Oxygen  Demand (COD)  test
    is a measure  of the oxygen  equivalent of
    that portion of the  organic matter in  a
    sample that is susceptible to oxidation
    under  specific conditions  of  oxidizing
    agent, temperature and time.
B  A variety of terms have been and are used
   for the test described here as COD:

   1 Oxygen absorbed (OA) primarily in
     British practice.

   2 Oxygen consumed (OC) preferred by
     some, but unpopular.

   3 Chemical oxygen demand (COD) current
     preference.

   4 Complete oxygen demand (COD)
     misnomer.

   5 Dichromate oxygen demand (DOC)
     earlier distinction of the current pre-
     ference for COD by dichromate or a
     specified analysis such as Standard
     Methods.

   6 Others have been and are being used.
     Since  1960, terms have been generally
     agreed upon within most professional
     groups as indicated in I-A and B-3 and
     the explanation in B-5.

C  The concept of the COD is almost as old
   as the BOD.  Many oxidants and varia-
   tions in procedure have been proposed,
   but none have been completely
   satisfactory.

   1 Ceric  sulfate  has been investigated,
     but in  general it is not a strong
     oxidant.
2  Potassium permanganate was one of
   the earliest oxidants proposed and
   until recently appeared in Standard
   Methods (9th ed.) as a standard pro-
   cedure.  It is currently used in
   British practice as a 4-hr, test at
   room temperature.

   a  The results obtained with perman-
      ganate  were dependent upon concen-
      tration of reagent, time of oxidation,
      temperature, etc.,  so that results-
      were not reproducible.

3  Potassium iodate or iodic acid is an
   excellent oxidant but methods employing
   this reaction are time-consuming and
   require a  very close control.

4  A number of investigators have  used
   potassium dichromate  under a variety
   of  conditions.  The method proposed
   by Moore at SEC  is the basis of the
   standard procedure.^1'2'  Statistical
   comparisons with  other  methods are
   described. '•*)

5  Effective determination of elemental
   carbon in  wastewater was sought by
   Buswell as a water quality criteria.

   a  Van Slyke* ' described a carbon
      determination based on anhydrous
      samples and mixed oxidizing agents
      including sulfuric,  chromic, iodic
      and phosphoric acids to obtain a
      yield comparable to the theoretical
      on a wide  spectrum  of components.

   b  Van Hall,  et al., '^) used a heated
      combustion tube  with infrared
      detection to determine carbon quickly
      and effectively by wet sample
      injection.

6  Current development shows a trend to
   instrumental methods automating
 CH.O. oc. 10e.3.75
                                                                                      18-1

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 Chemical Oxygen Demand and COD/BOD Relationships
II
      conventional procedures or to seek
      elemental or more specific group
      determination.
RELATIONSHIP OF THE COD TEST WITH
OTHER OXIDATION CRITERIA IS
INDICATED IN TABLE 1.
A Table 1
Test
BOD
COD
IDOD
Van Slyke
Carbon detn.
Carbon by
combustion
+IR
Chlorine
Demand
Test
Temp. °C
20
145
20
400+
950
20
Reaction
time
days
2 hrs.
15'
1 hr.
minutes
20 min.
Oxidation
system
Biol. prod.
Enz. Oxidn.
50% H2SO4
K2Cr207
May be cata-
lyzed
Diss. oxyg.
H3P°4
HI03*
H2S04
K2Cr2°7
Anhydrous
Oxygen atm.
catalyzed
HOC1 soln.
Variables
Compound, environ-
ment, biota, time,
numbers. Metabolic
acceptability, etc.
Susceptibility of
the test sample to
the specified
oxidation
Includes materials
rapidly oxidized by
direct action,
Fe , SH.
Excellent approach
to theoretical oxi-
dation for most
compounds (N-nil)
Comparable to
theoretical for
carbon only.
Good NH3 oxidn.
Variable for other
compounds.
 B  From Table 1 it is apparent that oxidation
    is the only common item of this series of
    separate tests.

    1  Any  relationships among COD & BOD
      or any other tests are fortuitous be-
      cause the tests measure
                                                    the oxidizability of a given sample
                                                    under specified conditions,  which are
                                                    different for each test.

                                                    If the  sample is primarily composed
                                                    of compounds that are oxidized by
                                                    both procedures (BOD and COD) a
                                                    relationship may be established.
       18-2

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                                         Chemical Oxygen Demand and COD/BOD Relationships
          The COD procedure may be sub-
          stituted (with proper qualifications)
          for BOD or the COD maybe used
          as an indication of the dilution
          required for setting up BOD
          analysis.

          If the sample is characterized by a
          predominance of material that can
          be chemically, but  not biochemi-
          cally oxidized, the  COD will be
          greater than the BOD. Textile
          wastes, paper mill wastes, and
          other wastes containing high con-
          centrations of cellulose have a
          high COD, low BOD.

          If the situation in item b is reversed
          the BOD will be higher than the
          COD.  Distillery wastes or refinery
          wastes may have a  high BOD,  low
          COD, unless catalyzed by silver
          sulfate.

          Any relationship established as  in
          2a will change in response to
          sample history and environment.
          The BOD tends to decrease more
          rapidly than the COD.  Biological
          cell mass or detritus produced by
          biological action has a low BOD
          but a relatively high COD.  The
          COD/BOD ratio tends to increase
          with time, treatment,  or conditions
          favoring stabilization.
Ill   ADVANTAGES AND LIMITATIONS OF
     THE COD TEST*2) AS RELATED TO BOD

 A Advantages

     1  Time, manipulation, and equipment
       costs are lower for  the COD test.

     2  COD oxidation conditions are effective
       for a wider spectrum of chemical
       compounds.

     3  COD test conditions can be standardized
       more readily to give more precise
       results.
     4  COD results are available while the
       waste is in the plant, not several
       days later, hence, plant control is
       facilitated.

     5  COD results are useful to indicate
       downstream damage potential in the
       form of sludge deposition.

     6  The COD result plus the oxygen equiva-
       lent for ammonia and organic nitrogen
       is a good estimate of the ultimate BOD
       for many municipal  wastewaters.

 B  Limitations

     1  Results are not  applicable for estimating
       BOD except as a result of experimental
       evidence by both methods on a given
       sample type.

     2  Certain compounds are not susceptible
       to oxidation under COD conditions or
       are too volatile  to remain in the oxida-
       tion flask long enough to be oxidized.

       Ammonia, aromatic hydrocarbons,
       saturated hydrocarbons, pyridine, and
       toluene are examples of materials with
       a low analytical response in the COD
       test.

     3  Dichromate in hot 50% sulfuric  acid
       requires  close control to maintain
       safety during manipulation.

     4  Oxidation of chloride to  chlorine is not
       closely related to BOD but may affect
       COD results.

     5  It is not advisable to expect precise
       COD results on  saline water.
IV  BACKGROUND OF THE STANDARD
    METHODS COD PROCEDURE

 A The COD procedure    considered dichro-
    mate oxidation in 33 and 50 percent sul-
    furic acid.  Results indicated preference
    of the 50 percent acid concentration for
    oxidation of sample components.  This is
    the basis for the present standard
    procedure.
                                                                                   18-3

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Chemical Oxygen Demand and COD/BOD Relationships
B  Muers*' suggested addition of silver
   sulfate to catalyze oxidation of certain
   low molecular weight aliphatic acids and
   alcohols.  The catalyst also improves
   oxidation of most other organic components
   to some extent but does not make the COD
   test universally applicable for all chemical
   pollutants.

C  The unmodified COD test result (A) includes
   oxidation of chloride to chlorine.  Each mg
   of chloride will have a COD  equivalent of
   0. 23 mg.  Chlorides must be determined
   in the sample and the COD result corrected
   accordingly.

   1 For example,  if a sample  shows 300
     mg of  COD  per liter and 200  mg Cl~
     per liter the corrected COD result will
     be 300 -(200 x  0.23)or 300 -  46 =  254
     mg COD/1 on a  chloride corrected basis.

   2 Silver sulfate addition as  a catalyst
     tends to cause partial precipitation of
     silver chloride .even in the hot acid- solu-
    : tion. Chloride corrections are ques-
     tionable unless the chloride is oxidized
     before addition of silver sulfate,  i.e.,
     .reflux for 15 minutes for chloride ox-
     idation, add Ag.SO.,  and continue the
     reflux or use of HgSO4(D).

                      tn\
D  Dobbs and Williams V" proposed prior
   complexation of chlorides with HgSO4 to
   prevent chloride oxidation during the test.
   A ratio of about 10 of Hg++ to 1 of Cl~ (wt.
   basis) appears essential.  The Cl~  must
   be complexed in acid solution before addi-
   tion of dichromate and silver sulfate.

   1 For unexplained reasons the HgSO4
     complexation does not completely
     prevent chloride oxidation in the
     presence of high chloride concentrations.

   2 Factors have been developed to provide
     some estimate of error in the result
     due to incomplete control of chloride
     behavior.  These tend to vary with the
     sample and technique employed.

E  It is not likely that COD results will be
   precise for samples containing high
   chlorides.  Sea water contains 18000 to
   21000 mgCr/1 normally.  Equivalent
   chloride correction for COD exceeds
                                                    4000 mg/ 1.  The error in chloride
                                                    determination may give negative COD
                                                    results upon application of the correction.
                                                    Incomplete control of chloride oxidation
                                                    with HgSO4 may give equally confusing
                                                    results.

                                                      HgSO4 appears to give precise results
                                                      for COD when chlorides do not exceed
                                                      about 2000 mg/1.  Interference in-
                                                      creases with increasing chlorides at
                                                      higher levels.

                                                 F The 12th edition of Standard Methods re-
                                                    duced the amount of sample and  reagents
                                                    to 40% of amounts utilized in previous
                                                    editions.  There has been no change in
                                                    the  relative proportions in the test.  This
                                                    step was taken to reduce the cost of pro-
                                                    viding expensive mercury and silver sul-
                                                    fates required.  Results are comparable
                                                    as long as the  proportions are identical.
                                                    Smaller aliquots of sample and reagents
                                                    require more  care during manipulation
                                                    to promote precision.

                                                 G The EPA Methods for COD

                                                    1  For routine level COD (samples having
                                                       an organic carbon concentration
                                                       greater than 15  mg/liter and a chloride
                                                       concentration less than 2000  mg/liter),
                                                       the EPA specifies the procedures found
                                                       in  Standard Methods ^and in ASTM<8\
                                                    2  For low level COD (samples with less
                                                       than 15 mg/liter organic carbon and
                                                       chloride concentration less than 2000
                                                       mg/liter),  EPA provides an analytical
                                                       procedure ^)_  The  difference from
                                                       the routine procedure primarily in-
                                                       volves a greater sample volume and
                                                       more dilute solutions of dichromate
                                                       and ferrous ammonium sulfate.

                                                    3  For saline samples (chloride level
                                                       exceeds 2000 mg/liter), EPA provides
                                                       an analytical procedure* ' involving
                                                       preparation of a standard curve of COD
                                                       versus mg/liter chloride to correct
                                                       the calculations.  Volumes and concen-
                                                       trations for the sample and reagents
                                                       are adjusted for this type of determination.

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                                     Chemical Oxygen Demand and  COD/BOD Relationships
                                /Q\
V  PRECISION AND ACCURACYV

 Eighty six analysts in 58 laboratories
 analyzed a distilled water solution contain-
 ing oxidizable organic material equivalent
 to 270 mg/1 COD.  The  standard deviation
 was ± 17. 76 mg/1 COD with an accuracy
 as percent relative error (bias) of —4. 7%.
 For a solution equivalent to 12. 3  mg/1
 COD (low level), the standard deviation
 was i 4. 15 mg/1 with an accuracy as percent
 relative error (bias) of  0. 3%.  (EPA Method
 Research Study 3)
VI
REMARKS PERTINENT TO EFFECTIVE
COD DETERMINATIONS INCLUDE:

Sample size and COD  limits for 0. 250 N
reagents are  approximately as given.
For 0. 025 N  reagents  multiply  COD by
0. 1.   Use the weak reagent for COD's
in the range  of 5-50 mg/1, (low level).
        Sample  Size

          20  ml
          10  ml
            5  ml
                         mg  COD/1
                           2000
                           4000
                           8000
 D
Most organic materials oxidize rela-
tively  rapidly under COD test  condi-
tions.   A  significant fraction of
oxidation occurs during the heating upon
addition of acid but the orange color of
dichromate should remain.  If the
sample color changes from orange to
green  after acid addition the sample was
too large.   Discard without reflux and
repeat with a smaller aliquot until the
color after mixing does not go  beyond
a brownish hue.   The dichromate color
change  is  less rapid with sample com-
ponents that are slowly oxidized under
COD reaction conditions.

Chloride concentrations should be known
for all test samples so appropriate
analytical techniques can be used.

Special precautions advisable for the
regular COD procdure and essential
when using 0. 025 N reagents include:
           Keep the  apparatus assembled
           when not  in use.

           Plug the  condenser breather tube
           with glass wool to minimze dust
           entrance.

           Wipe the  upper part of the  flask
           and  lower part of  the  condenser
           with a wet towel before  disassembly
           to minimize  sample  contamination.

           Steam out the  condenser after use
           for high concentration samples  and
           periodically for regular  samples.
           Use  the  regular blank reagent mix
           and  heat, without use  of condenser
           water, to clean the apparatus of
           residual oxidizable components.

           Distilled  water and sulfuric acid
           must be of very high quality to
           maintain  low blanks  on the  refluxed
           samples for  the 0. 025 N oxidant.
VII   NPDES METHODOLOGY

  Under the National Pollutant Discharge
  Elimination System, the accepted method
  (Federal Register, Tuesday, October  16,
  1973, Volume 38,  number  199, Part II)
  for doing the Chemical Oxygen Demand
  Test is given in Standard Methods, ^2^ p. 495;
  ASTM,(8) p. 219 and the EPA  Manual, <9>
  p. 20.
                                                  ACKNOWLEDGEMENT:

                                                  Certain  portions of this  outline contain
                                                  training material from prior  outlines by
                                                  R.  C. Kroner, R.  J.  Lishka, and
                                                  J.  W. Mandia.
  REFERENCES:

  1   Moore,  W.  A., Kroner, R.  C. and
          Ruchhoft, C.  C.  Anal.  Chem.
          21:953.   1949.
                                                                                      18-5

-------
Chemical Oxygen Demand and COD/ BOD Relationships
 2    Standard Methods,  13th Edition, APHA-
          AWWA-WPCF.  1971.

 3    Moore, W. A.,  Ludzack,  F.  J.  and
          Ruchhoft,  C. C.   Anal.  Chem.
          23:1297,  1951.

 4    Van Slyke,  D. D.  and Folch,  J.  J.
          Biol.  Chem.  136:509,  1940.

 5    Van Hall,  C. E., Safranko, J. and
          Stenger,  V.  A., Anal.  Chem.
          35:315,  1963.

 6    Muers, M. M.  J. Soc. Chem.
         Ind.  (London) 55:711,  1936.

 7    Dobbs, R.  A. and Williams, R. T.,
         Anal.  Chem.  35:1064.  1963.
    ASTM Standards,  Part 23,  Water:
        Atmospheric Analysis,  1972.

    Methods for Chemical Analysis
        of Water and Wastes, EPA-
        MDQARL,  Cincinnati,  Ohio
        45268, 1974.
This outline was prepared by F.  J.
Ludzack,  Chemist, EPA, WPO,  National
Training Center, Cincinnati, OH  45268.
Descriptors:  Analysis,  Biochemical
Oxygen Demand, Chemical Analysis,
Chemical Oxygen Demand, Chlorides,
Oxygen Demand, Wastewater, Water
Analysis
  18-6

-------
                          LABORATORY PROCEDURE FOR ROUTINE
                            LEVEL CHEMICAL OXYGEN DEMAND
 I   REAGENTS

 A  Standard Potassium Dichromate (0.250 N):

    Dissolve 12. 259 g of primary standard
    grade K Cr  O^, which had been dried at
    103° C for two nours,  in distilled water and
    dilute to one liter.
 B  Ferrous Ammonium Sulfate (0. IN):
    Dissolve 39 g of Fe (NH4>2
    in distilled water.
6H20
    Carefully add 20 ml of concentrated H SO .
    Cool and dilute to one liter.

 C  Ferroin Indicator:
    Dissolve 1.485 g  1,  10-phenanthroline
    monohydrate and 0.695 g FeSO  • 7H O in
    water and dilute to 100 ml. This indicator
    may be purchased already prepared.

 D  Concentrated Sulfuric Acid (36 N):

 E  Mercuric Sulfate: Analytical Grade

 F  Silver Sulfate:  Analytical Grade

 G  Concentrated Sulfuric Acid - Silver Sulfate:
    Dissolve 22 g of silver sulfate in a 9 Ib
    bottle of concentrated sulfuric acid.
    (1-2 days required for dissolution)
H   EQUIPMENT PREPARATION

 Before use, the Erlenmeyer flask (500 ml,
 24/40 standard taper joint) and reflux con-
 denser (Friedrichs, 24/40 standard taper
 joint) should be steamed out to remove trace
 organic contaminants.  Add  10 ml of
 0. 250 N K Cr2O?,  50 ml of  distilled water,
 and several bumping stones to the flask.
 Carefully add 20 ml of concentrated HgSO
 and mix thoroughly.  Connect the flask
 and condenser, but do not turn on the water
 to the condenser.  Boil the mixture so that
              steam emerges from the top of the con-
              denser for several minutes.  Cool the
              mixture,  carefully discard the acid, and
              rinse the condenser and flask with distilled
              water.  In order to prevent contamination
              from air-borne particles,  the top of the
              condenser should be lightly plugged with
              glass wool during storage  and use.
IH  STANDARDIZATION OF FERROUS
    AMMONIUM SULFATE

 A  Dilute 10. 0  ml of the standard
    potassium dichromate  to about  100 ml
    with distilled water.
                Add 30 ml of concentrated H SO  and
                allow to  cool.
              C Add 2-3  drops of ferroin indicator and
                titrate to a reddish-brown end point
                with the  ferrous ammonium sulfate.
                Calculate the normality, N, of the
                ferrous ammonium sulfate.

              D Calculation
                N of Fe (NH4)2
                ml K2Cr20? XN of
                                                         mlFe(NH4)2
            IV  PROCEDURE

             A Place 0. 4 g HgSO in a 500 ml 24/40
                standard taper Erlenmeyer flask.
                Add 20 ml of sample, or an aliquot
                diluted to 20 ml with distilled water.

             B Add 3 ml of concentrated H SO  and
                swirl to dissolve the HgSO..
 CH.O.oc.lab.3b.3.75
                                                                                    19-1

-------
Laboratory Procedure for Routine Level Chemical Oxygen Demand
C  Add 10. 0 ml of the 0. 250 N
   and mix.
D  Carefully add 27 ml of the sulfuric acid
   silver sulfate reagent.

E  Add several pumice granules or glass
   beads to prevent bumping, and then
   swirl the mixture to insure complete
   mixing.

F  Reflux the mixture for two hours.
J  Calculation
                                                  mg COD/1
               (A-B> NX 8X 1000
                  ml of sample
   COD = chemical oxygen demand

   A • ml Fe(NH,), (SO.L used for blank
                4 £t    4 6t
   B » ml Fe(NH ) (SO.), used for sample
                4 £t    TC £
   N -Nof Fe(NH.L (SO.L
                  4 Cl    4 &
   8 B equivalent weight of oxygen
G  Allow the solution to cool,  wash down
   the condenser with distilled water, and
   add about 75 ml water to bring the
   volume to  about 150 ml.
H  Add 2-3 drops of the ferroin indicator
   and titrate the solution to a reddish -
   brown end point with the ferrous
   ammonium sulfate.

I  A blank consisting of 20  ml of distilled
   water and containing all  reagents is
   refluxed and titrated in the same manner
   as the sample.
A CKNOWLEDGEMENT:

Portions of this outline were taken from
an outline prepared by R. J. Liska.


REFERENCES

1  Methods for Chemical Analysis of
   Water and Wastes, EPA-MDQARL,
   Cincinnati, Ohio,  1974.

2  Standard Methods, APHA-AWWA-
   WPCF,  13th Edition, 1971.
                                                This outline was prepared by Charles R.
                                                Feldmann, Chemist, EPA, WPO, National
                                                Training Center,  Cincinnati, OH 45268.
                                                Descriptors ;   Chemical Analysis, Chemical
                                                Oxygen Demand,  Organic Compounds,
                                                Oxidation,  Oxygen, Oxygen Demand,  Oxygen
                                                Requirements, Water Analysis
 19-2

-------
                        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),  IT1".

    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 discussing wastewater
    topics however.

II   DEFINITIONS OF ACIDITY,
    ALKALINITY AND 'NEUTRALITY

A   A cidity
    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
 D
III
         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 and pH
 values are listed in Table I.
CH.ALK. 3.3.75
                                                                                   20-1

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 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
 20-2
    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-i
    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 HgSO^  X N of standard
    H2SO4 X 50 X 1000/ml sample.
VI   INDICATORS

The term  "suitable indicator" was used
above.  At the end of a titration, the pH of
the solution will not necessarily be 7.   It
may be above or below 7.  A suitable indi-
cator, 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

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                                                         Acidity, Alkalinity, pH and Buffers
VII  BUFFERS

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

                                9.2 - 11.0
Boric Acid + Borax

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

    In many instances, the buffer is composed
    of a weak acid and a salt of the weak acid;
    e.g., acetic acid and sodium acetate.
    1    In water, acetic acid  ionizes or
         "breaks down" into hydrogen ions
         and acetate ions.
      HC2H302  =
                      H+
     (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
         HC2H3C>2; 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 not are "weak".
                                                         Sodium hydroxide is a relatively
                                                         strong base,  while ammonium
                                                         hydroxide is  realtively weak.

                                                     5   Sodium acetate (a salt of acetic acid)
                                                         dissociates or "breaks down" into
                                                         sodium ions and acetate ions when
                                                         placed in water.

                                                     NaC2H302    «   Na+    +    C2H3O2-
                                                    (sodiumacetate) (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  -      H+
                                                                             3   H2C03
                                                                            (carbonic acid)
                                                                    H+    +     HCO3~
                                                               (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+
                                                     (in the
                                                      buffer)
                                                               (hydroxide ion
                                                                 "spilled")
                                                                                H20
                                                     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.
                                                                                     20-3

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Acidity. Alkalinity, pH and Buffers
E   Buffering action is not identical with
    a process in which acid wastes are            This outline was prepared by C. R. Feldmann,
    "neutralized" with alkali wastes, or           Chemist,  National Training Center, MDS,
    conversely.  The desired effect is             WPO, 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.
  20-4

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                          OPERATING CHARACTERISTICS AND USE
                                    OF THE pH METER
 I   INTRODUCTION

 pH is a term used to describe the intensity
 of the acid or alkaline condition of a
 solution.  The concept of pH evolved from
 a series of developments that led to a
 fuller understanding of acids and alkaline
 solutions (bases).  Acids and bases were
 originally distinguished by their difference
 in physical characteristics (acids -sour,
 bases-soapy feel).  In the 18th century it
 was recognized that acids have a sour taste
 (vinegar-acetic acid), that they react with
 limestone with the liberation of a gaseous
 substance (carbon dioxide) and that neutral
 substances result from their interaction
 with  alkaline solutions.

 Acids are also described as compounds
 that yield hydrogen ions when dissolved
 in water.  And that bases yield hydroxide
 ions  when dissolved in water.   The process
 of neutralization is then considered to be
 the union of hydrogen (H ) ions and hydroxyl
 (OH~) ions to form neutral water
(H+ + OH
               H20).
 It has been determined that there are
 1/10, 000, 000 grams of hydrogen ions and
 17/ 10, 000,000 grams 'of hydroxyl ions in
 one liter of pure water.   The product of the
 H+ and OH" ions equal a constant value.
 Therefore, if the concentration of the H+
 ions is increased there is a corresponding
 decrease in OH" ions.  The acidity or
 alkalinity, hydrogen ion concentration of a
 solution is given in terms of pH.  The pH
 scale  extends from 0 to  14 with the neutral
 point at 7. 0.
II   INSTRUMENTATION

 A  General

    Because of the differences between the
    many makes and model of pH meters
   which are available commercially,  it is
   impossible to provide detailed instruc-
   tions for the correct operation of every
   instrument. In each case, follow the
   manufacturer's instructions.  Thoroughly
   wet the glass electrode and the calomel
   electrode and prepare  for use in accor-
   dance with the instructions given.
   Standardize the instrument against  a
   buffer solution with a pH  approaching
   that of the sample,  and then check the
   linearity of electrode response against
   at least one additional  buffer of a
   different pH.  The readings with the
   additional buffers will  afford a rough
   idea of the limits of accuracy to be
   expected of the  instrument and the technic
   of operation.

B  Electrode Design

   About 1925 it was discovered that an
   electrode could be constructed of glass
   which would develop a  potential related
   to the hydrogen-ion concentration with-
   out interference from most other ions.
   The glass pH electrode is the nearest
   approach to a universal pH indicator
   known at present.  It works on the
   principle of establishing  a potential
   across a pH-sensitive, glass membrane
   whose magnitude is proportional to the
   difference in pH of the  solution
   separated by this membrane.

   All glass pH indicating electrodes have
   a similar basic design.  Contained on
   one side of an appropriate glass membrane
   is a solution of constant pH.  In contact
   with the other side of this pH sensitive
   glass is the solution of unknown pH.
   Between the surfaces of the glass mem-
   brane,  a potential is established which
   is proportional  to the pH difference of
   these solutions.  As the pH of one
   solution is constant, this developed
   potential is a measure  of the pH of the
   other.
  CH.pH. 2.3.75
                                                                                      21-1

-------
Operating Characteristics and Use of the pH Meter
   To measure this potential,  a half-cell
   is introduced into both the constant,
   internal solution and into the unknown,
   external solution.  These half-cells are
   in turn connected to your pH meter.  The
   internal reversible half-cell sealed
   within the chamber of constant pH is
   almost exclusively a wire of silver-
   silver chloride.  The external
   reversible half-cell is often silver-
   silver chloride.  If both the internal and
   external electrodes are  combined in a
   common pH  measuring device, the
   electrode  is a combination  pH electrode.

   As the function of these  half-cells is to
   provide a  steady reference  voltage
   against which voltage changes at the
   glass pH sensitive membrane can be
   referred,  they must be protected from
   contamination and dilution  by the unknown
   solutions.  This  is accomplished by
   permanently sealing the  internal half-
   cell in a separate chamber  which makes
   electrical contact to the  unknown
   solution through  a porous ceramic plug.
   This ceramic plug allows current to flow,
   but  does not permit exchange of solution
   to this chamber.  Graduallythe KC1
   solution is slowly lost, therefore a filling
   port is placed in this electrode so that
   additional  saturated potassium chloride
   can be added.

C  Instrument Calibration

   The pH balance control,  by adding a
   voltage in series with the pH electrode
   system, allows the operator to adjust
   the meter readout to conform to the pH
   of the  calibrating buffer. In general,
   calibrate the meter in the general range
   of the  unknown solution.   Appropriate
   buffers can be selected (pH 4. 0,  6. 8,
   7.4 and  10. 0). Always set  the tem-
   perature compensator on the instrument
   to the  temperature of the standard buffer
   solution.

   For most  accurate analysis the pH of the
   sample should be determined, and then
   buffered solutions of a pH above and
   below  the determined pH should be
     selected to re-calibrate the instrument
     and the  determination of the pH of the
     sample  repeated for a final reading.

  D  Precision and Accuracy

     The precision and accuracy attainable
     with a given pH meter will depend upon
     the type and condition of the instrument
     employed and the technique of standard-
     ization and operation.  With the proper
     care,  a precision of t 0.02 pH unit and
     an accuracy of t 0. 05 pH unit can be
     achieved with many of the new and
     improved models.  However, 1 0. 1  pH
     unit represents the limit of accuracy
     under normal conditions.  For this
     reason, pH values generally should be
     reported to the nearest 0. 1 pH unit.

  E  Maintenance  Practices

     The reference chamber of the pH  .
     electrode system should always be kept
     nearly full of saturated KC1 solution.
     Routinely check the level and saturation
     of potassium chloride in this reference
     chamber and add saturated KC1 if
     necessary.

     The pH  sensitive glass membrane
     dehydrates when removed from water,
     and thus it is imperative that dry
     electrodes be soaked in buffer or water
     for several hours before use. To
     avoid this break-in period always keep
     the glass pH  sensitive membrane wet
     between periods of use.

     The buffers are pH standards; do not
     contaminate them.

     If the meter is a battery operated
     instrument.  To conserve the battery
     life, the instrument should be turned
     off when not in use.
Ill  PROCEDURE

 In the measurement of pH values of industrial
 wastes,  effluents, sludges and similar
  21-2

-------
                                         Operating Characteristics and Use of the pH Meter
samples, the electrodes must be thoroughly
rinsed with buffer solution between samples
and after calibrating.  Buffer solutions can
be prepared by use of the formulations shown
in Table I.

The electrodes should be kept free of oil
and grease and stored in water when not in
use.  In testing samples containing gaseous
or volatile components which affect the pH
value, any handling technic such as stirring
or heating may cause loss of such components
and thereby introduce error. For example,
the loss of carbon dioxide from an anaerobic
sludge digester sample due to stirring will
result in an observed pH value which is too
                                         Table I

                           Preparation of pH Standard Solutions
Standard Solution (Molality)              pH at 25 C

Primary standards
   Potassium hydrogen tartrate
      (saturated at 25°C)                  3. 557

   0. 05 potassium dihydrogen citrate       3. 776

   0. 05 potassium hydrogen phthalate      4.008

   0. 025 potassium dihydrogen
      phosphate + 0. 025 disodium
      hydrogen phosphate                  6. 865

   0.008695 potassium dihydrogen
      phosphate + 0. 03043 disodium
      hydrogen phosphate                  7.413

   0.01 sodium borate decahydrate
      (borax)                             9.180

   0. 025 sodium bicarbonate + 0. 025
      sodium carbonate                    10.012

Secondary Standards

   0. 05 potassium tetroxalate dihydrate    1. 679

   Calcium hydroxide (saturated at  25°C)   12. 454
        Weight of Chemicals Needed per 1, 000
           ml of Aqueous Solution at 25 C	
                   11.41gKH2C6H507

                   10. 12gKHC8H404
        3. 388gKH2PO4t +  3.533gNa2HPO4tt
           179gKH2P04t + 4. 302gNa2HPO4tt
           2. 092gNaHCO3 +  2.
               12.61gKH3C408-2H20
                     1.5gCa(OH)2*
*Approximate solubility

tDry chemical at 110-130 C for 2 hr.

tPrepare with freshly boiled and cooled distilled water (carbon dioxide-free)
                                                                                    21-3

-------
Operating Characteristics and Use of the pH Meter
high.  If a sample of sludge or mud is highly
buffered, a small amount of water may be
added but the result cannot be considered
valid unless further dilutions yield the same
pH value.  All dilutions should be reported
along with the result.
3  Instruction - Manual IL 175 Porto-matte
      pH meter,  Instrumentation Laboratory,
      Inc.  Lexington,  Massachusetts,
      Standard Methods, APHA-AWWA-WPCF.,
      13th Ed., 1971.
REFERENCES

1  Nebergall, W. H., Schmidt, F. C. and
      Holtzclaw, Jr., H. F.  College Chem.,
      2nd Ed. Heath and Co., Boston,  1963.

2  Sawyer, C. N.,  and McCarty.  P. L.
      Chem. for San. Eng.  2nd Ed.
      McGraw-Hill, New York 1967
This outline was prepared by P. F.  Hallbach,
Chemist, National Training Center,  DTTB,
MDS, WPO.EPA, Cincinnati, Ohio  45268

Descriptors: Hydrogen Ion Concentration,
Instrumentation, Chemical Analysis
  21-4

-------
                                 SPECIFIC CONDUCTANCE
I  INTRODUCTION

An electrical conductivity measurement of a
solution determines the ability of the solution
to conduct an electrical current.  Very
concentrated solutions have a large population
of ions and transmit current easily or with
small resistance.  Since  resistance is   ^
inversely related to conductivity  K  =  —,
a very concentrated solution has a  very
high electrical conductivity.

Electrical conductivity is determined by
transmitting an electrical current through
a given solution,  using two electrodes.  The
resistance measured is dependent principally
upon the ionic concentration,  ionic charge,
and temperature  of the solution although
electrode characteristics (surface  area  and
spacing of electrodes) is also critical.   Early
experiments in standardizing the measurement
led to construction of a "standard cell" in
which the electrodes were spaced exactly 1 cm
and each had a surface area of 1 cm .  Using
this cell,  electrical conductivity is expressed
as "Specific Conductance".  Modern specific
conductance cells do not have the same
electrode dimensions as the early standard
cell but have a characteristic electrode  spacing/
area ratio known as the "cell constant".
K
  sp
1
7^
R
distance (cm)
      ~T —  9 T — ,
      (cm'')
              area
sp
      1
      —  A
      R
               k  =  cell constant

Specific conductance units are Mhos/cm or
reciprocal ohms/cm.  Most natural,  fresh
waters in the United States have specific
conductances ranging from 10 to 1, 000
micromhos/cm.  (1 micromho  = 10   mho).
                   II  CONDUCTIVITY INSTRUMENTS

                   Nearly all of the commercial specific con-
                   ductance instruments are of a bridge circuit
                   design,  similar to a Wheatstone Bridge.
                   Null or balance is  detected either by meter
                   movement, electron "ray eye" tubes,  or
                   headphones.  Since resistance is directly
                   related to  temperature, some instruments
                   have automatic temperature compensators,
                   although inexpensive models generally have
                   manual temperature compensation.

                   Conductivity instruments offer direct  specific
                   conductance readout when used with a cell
                    matched  to  that particular instrument.

                    Electrodes within  the cell may become
                    damaged or dirty and accuracy may be
                   affected; therefore,  it  is advisable to
                   frequently check the instrument readings
                   with a standard KC1 solution having a known
                   specific conductance.
Ill  CONDUCTIVITY CELLS

  Several types of conductivity cells are
  available,  each having general applications.
  Dip cells are generally used for field
  measurement,  flow cells for measurement
  within a  closed system,  and pipet cells for
  laboratory use. Many modifications of the
  above types are available for specialized
  laboratory applications; the Jones cells and
  inductive capacitance cells are perhaps the
  most common.

  Examples of various cell ranges for the RB3
  - Industrial Instruments model (0-50
  micromhos/cm scale range) are in Table 1.
                            Relative
                 Cell    Conductivity
                Number      Value
         Maximum range
          micromhos / cm
                                           Most accurate range
                                             micromhos / cm
Cell VSO2
Cell VS2
Cell VS20

1
10
100

0 - 50
0 - 500
0 - 5000
Table 1
2 - 30
20 - 300
200 - 3000

CH. COND. 2d.3.75
                                                                                      22-1

-------
 Specific Conductance
IV  Computation of Calibration Constant

    A calibration constant is a factor to which
    scale readings must be multiplied by com-
    pute specific  conductance.
V
    K   =  cM
     sp
    where  K
            sp
            =  actual specific conductance

            =  calibration constant
           M    = meter reading

    For example, a 0. 001 N KC1 solution
    (147 micromhos/cm standard) may show
    a scale reading of 147.
       147 =  c!47, C=TT£
                     147
                     147
1.00
    In this case the cell is perfectly "matched"
    to the instrument,  the  calibration constant
    is 1.00, and the scale  reading represents
    actual specific conductance.  A variety of
    cells, each covering a specific range,
    may be used with any one instrument.
    However,  a calibration constant for each
    cell must  be computed before solutions of
    unknown specific conductance can be
    determined.
RELATIONSHIP OF SPECIFIC CON-
DUCTANCE TO IONIC CONCENTRATION
 Natural water consists of many chemical
 constituents,  each of which may differ
 widely in ionic size, mobility, and solubility.
 Also,  total constituent concentration and
 proportions of certain ions in various natural
 waters range considerably.  However,  it
 is surprising that for most natural waters
 having less than 2, 000 mg/1. dissolved
 solids, dissolved solids values are closely
 related to  specific conductance values,
 ranging in a ratio of . 62 to . 70.  Of course
 this does not  hold true for certain waters
 having considerable amounts of nonionized
 soluble materials, such as organic com-
 pounds and nonionized, colloidal inorganics.
                                             Properties of some inorganic ions in regard
                                             to electrical conductivity are shown below:
                       Ion
                    Calcium
                    Magnesium
                    Sodium
                    Potassium
                    Bicarbonate
                    Carbonate
                    Chloride
                                                                     Micromhos/cm
                                                                     per meq/1 cone.
                                                                            ,0
                                                                            ,6
                             52.
                             46.
                             48.9
                             72.0
                             43.6
                             84.6
                             75.9
 VI ESTIMATION OF CONSTITUENT
    CONCENTRATIONS
Generally speaking, for waters having a
dissolved solids concentration of less than
1, 000 mg/1,  calcium and magnesium (total
hardness), sodium, bicarbonate and
carbonate (total alkalinity), and sulfate are
the principal or most abundant ions,
representing perhaps 90-99% of the total
ionic concentration of the water.  Specific
conductance, total hardness and total
alkalinity are all simple and expedient
measurements which can be performed in
the field. Therefore,  the remaining principal
ions are sodium and sulfate,  and concentrations
of these can be estimated by  empirical
methods.  For example, we find that a certain
water has:

   KSp = 500 micromhos/cm
   Total Hardness • 160 mg/1 or 3. 20 meq/1
   Total Alkalinity = 200 mg/1  or 3.28 meq/1,
   as bicarbonate.

Next we multiply the specific conductance by
*0.011 (500X0.011 = 5.50) to estimate the
total ionic concentration in meq/1.
                                                This factor may vary slightly for
                                                different waters
                                                Cations (meq/1)
                                                         3.20
                Calcium
                Magnesium
                Sodium 5.50-3.20
                                                               2.30
                                               Total Cations 5. 50
                           Anions (meq/1)

                        Carbonate 0. 00
                        Bicarbonate 3.28
                        Sulfate 5.50-3.28  »
2.22
                                                                     Total Anions 5.50
 22-2

-------
                                                                       Specific Conductance
 Realizing that several variables are involved
 in empirical analysis, application rests
 entirely upon testing the formula with previous
 complete laboratory analyses for that
 particular water.  If correlation is within
 acceptable limits,  analytical costs may be
 substantially  reduced.  Empirical analysis
 can also be used in determination of proper
 aliquots (dilution factor) necessary for
 laboratory analysis.

 Records of laboratory chemical analysis may
 indicate that a particular stream or lake
 shows a characteristic response to various
 streamflow rates or lake water levels.  If
 the water's environment has not been altered
 and water composition responds solely  to
 natural causes, a specific  conductivity
 measurement may be occasionally used in
 substitution for laboratory analyses to
 determine water quality.  Concentration of
 individual constituents can thus be estimated
 from a specific conductance value.

VII APPLICATIONS FOR SPECIFIC
    CONDUCTANCE MEASUREMENTS
                           (2)
 A   Laboratory Operations

     1  Checking purity  of distilled and  de-
        ionized water

     2  Estimation of dilution factors for
        samples

     3  Quality control check on analytical
        accuracy

     4  An electrical indicator

 B   Agriculture

     1  Evaluating salinity

     2  Estimating Sodium Adsorption Ratio
            (1)
 C   Industry* '

     1  Estimating corrosiveness of water in
        steam boilers

     2 Efficiency check of boiler operation
D  Geology

   1  Stratigraphic identification and
      characterization

      a   geological mapping
      b   oil explorations

E  Oceanography

   1  Mapping ocean currents

   2  Estuary studies

F  Hydrology

   1  Locating new water supplies

      a   buried stream channels (See Fig. 1)
      b   springs in lakes and
          streams (See Fig. 2)

   2  Detection and regulation of sea water
      encroachment on shore wells

G  Water Quality Studies
                                   /o)
   1  Estimation of dissolved solids
      (See Section V, also Fig.  3)

   2  Empirical analysis of constituent
      concentrations  (See Section VI, also
      reference  2)

   3  Quality control check for salt  water
      conversion studies

   4  Determination of mixing efficiency
      of streams (See Fig. 4)

   5  Determination of flow pattern  of
      polluted currents (See Fig. 4)

   6  Identification of significant fluctuations
      in industrial wastewater effluents

   7  Signal of significant changes in the
      composition of influents to waste
      treatment plants
                                                                                        22-3

-------
Specific Conductance
                                       FIGURE  1

                    DETECTION OF BURIED STREAM CHANNELS
                        TEST WELLS
                                                    X-SECT
                                         FIGURE 2
                   DETECTION OF SPRINGS IN LAKES AND STREAMS
   MAN LOWERING
CONDUCTIVITY CELL
                                                            LAND SURFACE

-------
                                                                Specific Conductance
                          POLLUTION STUOffS
                                         FIGURE 3
                           INDUSTIIAL
                         WASTi [FFLUENT
VIII   EPA METHODOLOGY

A     The current EPA Methods Manual
      specifies that specific conductance be
      measured with a self-contained con-
      ductivity meter,  Wheatstone bridge-'
      type or equivalent.
      Samples should preferably be analyzed
      at 25 C.  If not, temperature corrections
      should be made and results reported
      at 25°C.
      1   The instrument should be standard-
         ized using KC1 solutions

      2   It is essential to keep the conductivity
         cell clean

      The EPA manual specifies using the
      procedure as described in Standard
      Methods*2) or in ASTM Standards*3*.
      These are approved in 40 CFR 136
      for NPDES Report purposes.

      Precision and Accuracy

      Forty-one analysts in 17 laboratories
      analyzed 6 synthetic water samples
      containing the following Kgp increments
      of inorganic salts:  100, 1Q6, 808,  848,
      1640 and 1710 micromhos/cm.
   The standard deviation of the reported
   values was 7.55,  8.14,  66.1,  79.6,  106
   and 119/n mhos/cm respectively.

   The accuracy of the reported values  was
   -2.0,  -0.8,  -29.3,  -38.5,  -87.9 and
 — 86. 9 n mhos /cm bias respectively.

REFERENCES

1  Methods for Chemical Analysis of Water
   and Wastes, MDQARL,    Cincinnati,
   OH   45268.  1974.

2  Standard Methods for the Examination
   of Water and Wastewater, APHA-AWWA-
   WPCF,  13th edition,  1971.

3  ASTM Standards,  Part 23,  1973.
This outline was prepared by John R.
Tilstra,  Chemist, National Eutrophication
Research Program,  Corvallis. Oregon
with additions by Audrey E.  Donahue,  Chemist,
MPOD, WPO, EPA, Cincinnati,
OH  45268.
                                                                                      22-5

-------
                    CALIBRATION AND USE OF A CONDUCTIVITY METER
 I  EQUIPMENT AND REAGENTS

 A Equipment

    1  Solu Bridge conductivity meters

    2  Probes

       a  Cell VSO2

       b  Cell VS2

       c  Cell VS2O

    3  Thermometers

    4  400 ml beakers

 B Reagents

    1  Standard KC1  solutions

      Normality of   Specific Conductance
      KC1 Solution   micromhos/cm.
          0.0001           14.9
          0.001           147.0
          0.01           1413.0
          0.1           12900.0

    2  Distilled water
II   CHECKING THE INSTRUMENT

 A  The  measurement of specific conductivity
    as presented in sections II and  m is
    written for one type of  conductivity meter
    and probe.

 B  A battery  check is  made by depressing
    the  Battery Check  switch,  and  at  the
    same time pressing the on-off  button.
    The  meter needle should deflect to the
    right (positive)  and come to rest in the
    green  zone.

 C  Place  a  10, 000 ohm resistor in the holes
    of the electrical contacts on the meter.
    Turn the temperature knob  to read 25°C.
    Depress the  on-off  button and bring the
    meter needle to a  reading  of 0  by
     turning the specific conductance switch.
     The specific conductance reading  should
     be  approximately 200 micromhos/cm.
Ill   DETERMINATION OF THE CALIBRATION
     CONSTANT

 A  Determine the temperature of the
     standard KC1 solutions and move the
     temperature  knob to that value.

 B  Connect probe Cell VS02 to the  con-
     ductivity meter.

 C  Rinse the probe  in the beaker of
     distilled water,  wipe the excess  water
     with a kimwipe  and  place probe in the
     first beaker  of KC1 solution  (0.0001  N).

 D  Make certain the cell is  submerged to
     a point  at  least 1/2  inch  above the air
     hole and that no  entrapped air remains.
     The cell should also be at least  1/2
     inch from  the inside walls of the flask.

 E  Press and hold down the ON-OFF
     button,  simultaneously rotating the main
     scale knob until the meter reads zero.
     Release the button. (If the meter needle
     remains off scale or cannot be nulled,
     discontinue testing in that solution.)

 F  Record the scale reading  in Table 1  and
     proceed to KC1 solutions  0. 001N,
     0. 01N,  0. 1 N using Steps C, D and E.

 G  Repeat steps C through F using the VS2,
     then the VS20 probe.

 H  Compute the cell calibration constant-a
     factor by which scale readings must be
     multiplied to compute  specific conductance:
     K   = cM
       sp
     where K   = actual specific conductance,
     c = calibration constant
     M= meter  reading
                 (continued next page)
  CH. COND.lab.3c. 3.75
                                                                                        23-1

-------
   Calibration and Use of a Conductivity Meter
                       TABLE 1   DATA FOR CALIBRATION CONSTANTS
Probe
KC1
Solutions
Test #
1


Cell
Constant
Cell VSO2
0.0001N




0.001N




0.01N




0. IN




Cell VS2
0.0001N




0. COIN




0.01N




0. IN




: Cell VS20
0.0001N




0. 001N




0.0 IN




0. IN




 For each cell, calculate the cell constant
 by using the meter reading closest to the
 400  - 600 range.  The known specific
 conductance for the corresponding KC1
 solution can be found in IB Reagents.  Record
 the cell constants on Table I.
IV DETERMINATION OF Ksp FOR SAMPLES

   Obtain meter readings, M, for samples
   A,  B and C using Section III C, D and E.
   Record M in Table 2.  See Table I for
   the appropriate cell constant,  c,  to
   calculate Kgp for each sample where
   K0   = cM.  Record results in Table  2.
     sp
 V EPA METHODOLOGY

   The current EPA Manual^1' specifies
   using the procedures found in References
   2 and 3. These procedures have been
   adapted for this laboratory session and
   all  are  approved in 40CFR136 for NPDES
   report purposes.
A CKNOWLEDGMENT

This outline contains certain portions of
previous outlines by Messrs. J. W.  Mandia,
and J. R. Tilstra.

REFERENCES

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

2   Standard Methods for the Examination
    of Water and Wastewater, 13th
    Edition.   1971.

3   Book of ASTM Standards,  Part 23,  1973

 This outline was  prepared by C. R. Feldmann,
 Chemist, National Training Center,  and
 revised by Audrey E. Donahue,  Chemist,
 National Training Center,  MPOD,  WPO, .•
 USEPA,  Cincinnati, OH 45268.

 Descriptors:  Analytical Techniques,
 Conductivity,  Electrical Conductance,
 Specific  Conductivity, Water Analysis
                            TABLE 2.   SPECIFIC CONDUCTIVITY TESTS
Sample
Probe
1


Cell
Constant
Sp. Cond.
u mhos /cm
A
Cell
VSO2





Cell
VS2





Cell
VS2O





B
Cell
VSO2





Cell
VS2





Cell
VS2O





C
Cell
VSO2





Cell
VS2





Cell
VS2O





    23-2

-------
             CHLORINE DETERMINATIONS AND THEIR INTERPRETATION
    INTRODUCTION

 Chlorine normally is applied to water as a
 bactericidal agent; it reacts with water con-
 taminants to form, a variety of products
 containing chlorine.  The difference between
 applied and residual chlorine represents the
 chlorine demand of the water under conditions
 specified.  Wastewater chlorination is parti-
 cularly difficult because the concentration of
 organisms and components susceptible to
 interaction with chlorine are high and variable.
 Interferences with the chlorine determination
 in wastewater confuse interpretation with
 respect to the chlorine residual at a given
 time and condition, its bactericidal potency,
 or its future behavior.
II   CHEMISTRY OF CHLORINATION

 A  Chlorine compounds (C12) dissolve in water,
    and hydrolyze immediately  according to the
    reaction.
C1
      H2O
HOC1 + H1" + Cl
    The products of this reaction are hypo-
    chlorous and hydrochloric acid.  The
    reaction is reversible, but at pH values
    above 3. 0 and concentrations of chlorine
    below 1000 mg/1 the shift is predominantly
    to the right leading to hypochlorous acid
    (HOC1).

    Hypochlorous acid is a weak acid and con-
    sequently ionizes in water according to the
    equation:
         HOC1
                    H  + OCl
    This reaction is reversible.  At a pH value
    of 5. 0 or below almost all of the chlorine
    is present as hypochlorous acid (HOCl)
    whereas above pH 10. 0 nearly all of it
                                                    exists as hypochlorite ion (OCl").  The pH
                                                    value that will control is the  pH value
                                                    reached after the addition of chlorine.
                                                    Chlorine addition tends to lower the pH
                                                    and the addition of alkali hypochlorites
                                                    tends to raise the pH.

                                                    The initial reactions on adding chlorine to
                                                    wastewaters may be assumed to be funda-
                                                    mentally the same as when chlorine is
                                                    added to water except for the additional
                                                    complications due to contaminants and
                                                    their concentration.

                                                    Hypochlorous acid (HOCl) reacts with
                                                    ammonia and with many other complex
                                                    derivatives of ammonia to produce com-
                                                    pounds known as chloramines.  Formation
                                                    of the simple ammonia chloramines includes:
                                                    1  NH3 + HOCl
                     NH2C1   +

                     monochloramine
                                                                        H20
2  NH2C1  + HOCl— *NHC12  +  H

                      dichloramine

              NHC1  -
                                                    3  NH2C1
                                                              N2  +   3  HC1
                                                    The distribution of the ammonia chloramines
                                                    is dependent on pH, as illustrated below:
Ph
5
6
7
8
9
Percentage of Chlorine Present as
Monochloramine Dichloramine
16
38
65
85
94
84
62
35
15
6
 PC. lib. 9.74
                                                                                        24-1

-------
 Chlorine Determinations and Their Interpretation
    The formation of the ammonia chloramines
    are dependent on pH,  temperature, and
    chlorine-ammonia ratio.  Chlorine re-
    actions with amino acids are likely;
    product disinfecting powers are lower
    than those of chlorine or of ammonia
    chloramines.
Ill  TERMINOLOGY

 A Terms used with Respect to Application
    Site

    1  Pre-chlorination - chlorine added
       prior to any other treatment.

    2  Post-chlorination - chlorine added
       after other treatment.

    3  Split chlorination - chlorine added at
       different points in the plant - may
       include pre- and post-chlorination.

 B Terms Used in Designating Chlorine
    Fractions

    1  Free available residual chlorine - the
       residual chlorine present as hypo-
       chlorous acid  and hypochlorite ion.

    2  Combined available residual chlorine -
       the residual chlorine  present as
       chloramines and organic chlorine con-
       taining compounds.

    3  Total available residual chlorine - the
       free available residual chlorine + the
       combined available residual chlorine -
       may represent total amount of chlorine
       residual present without regard to type.

       In ordinary usage these terms are
       shortened to free residual  chlorine,
       combined residual chlorine and total
       residual chlorine. In the chlorination
       of wastewaters only combined residual
       chlorine is ordinarily present and is
       often improperly termed chlorine
       residual.

 C Breakpoint chlorination specifically refers
    to the ammonia-chlorine reaction where
    applied chlorine hydrolyzes and reacts to
    form chloramines and HC1 with the
    chloramines eventually forming N2 + HC1
    as in II. B. 3.  Assuming no other chlorine
    demand, the total chlorine residual will
    rise, decrease to zero and rise again with
    increasing increments of applied chlorine.
    Other substances may produce humps in
    the applied chlorine vs residual chlorine
    plot due to oxidation of materials other
    than ammonia.  Sometimes these are
    erroneously considered as a breakpoint.
IV  ANALYTICAL METHODS

 lodometric and amperometric titration
 methods are the basis for deter-
 mining chlorine residuals  in water.
 The  relative advantages of a specific
 determination depend upon the form in which
 the reactable chlorine exists and the amount
 and nature of interferences in the water.

 A Amperometric Titration - Direct Method

    1  Scope and application

       This method is applicable to the
       determination of free, combined or
       total residual  chlorine in all types of
       water and wastewaters that  do not
       contain substantial amounts of organic
       matter.

    2  Summary of Method

       When the cell  of the titrator is immersed
       in a sample at pH 7. 0, the cell unit pro-
       duces a small direct current if free
       chlorine  (an oxidizing agent) is present.
       As phenylarsine oxide (PAO) solution
       is added, it reduces the free chlorine.
       When all the chlorine is neutralized,  the
       generation of current ceases.   At  this
       end point, the microammeter  pointer on
       the apparatus no longer deflects
       down-scale.

       To determine  combined chlorine,  pH 4. 0
       buffer and potassium  iodide are then
       added to  the sample.  Free  iodine
       released by the combined chlorine also
       causes the cell to produce a small direct
       current.  Addition of PAO reduces the
       free  iodine and the generation of current
       ceases.   Again,  the end point  occurs
 24-2

-------
                                           Chlorine Determinations and Their Interpretation
      when the microammeter pointer no
      longer deflects down-scale.

      In either titration, the amount of PAO
      reducing agent used to reach the end
      point is  ultimately stoichiometrically
      proportional to chlorine present in the
      sample.  The sum of the free and
      combined chlorine is the total residual
      chlorine in the sample.

      Total  residual chlorine can be deter-
      mined directly by adding pH 4. 0 buffer
      and potassium iodide to the sample
      before beginning the titration.  Any
      free or  combined chlorine present will
      stoichiometrically liberate free iodine
      which is then reduced  with PAO
      titrant.  The amount of PAO titrant
      used measures the total amount of free
      and combined chlorine originally
      present  in the  sample.

   3  Interferences

      a  Organic matter reacts with liberated
         iodine.

      b  Cupric  ions may cause erratic
         behavior of the apparatus.

      c  Cuprous and silver ions tend to
         poison the electrode by plating out
         on  it.

B  Amperometric Titration - Indirect Method

   1  Scope and Application

      This method is applicable to  the
      determination of total chlorine residual
      in  all  types of water and wastewaters.
      In  contrast to the direct amperometric
      titration, this  back-titration  procedure
      minimizes interferences in waters
      containing substantial amounts  of
      organic matter.

   2  Summary of Method

      A sample is treated with a measured
      excess of standard phenylarsine oxide
      (PAO) solution followed by addition
      of  potassium iodide and a buffer to
      maintain the pH between 3. 5  and 4. 2.
   Any form of chlorine present will
   stoichiometrically liberate iodine
   which immediately reacts with the
   PAO before significant amounts are
   lost to reactions with organic  matter
   in the sample.

   When the cell of the amperometric
   titrator  is immersed in a sample so
   treated, no current is generated since
   neither free chlorine nor free iodine
   is present.

   The amount of PAO used to reduce  the
   liberated iodine is then determined by
   titrating the excess with a standard
   iodine solution.  No current is generated
   until all the excess PAO has been
   oxidized by the iodine.   At this end
   point the next small addition of iodine
   causes current to be generated and the
   microammeter pointer permanently
   deflects up-scale.

   The excess PAO thus measured is
   subtracted from the original amount
   of PAO added.  The difference is the
   PAO used to reduce the liberated iodine
   and is ultimately a measure of the total
   chlorine originally present in  the sample.

   NOTE:  Sodium thiosulfate solution may
   be used  instead of PAO, but PAO is
   more stable and is to be preferred.

3  Interferences

   a Cupric ions may cause erratic
     behavior of the apparatus.

   b Cuprous and silver ions tend to
     poison  the electrode by plating out
     on it.

Colorimetric Starch-Iodide Titration -
Indirect  Method

1  Scope and Application

   This method is applicable to the
   determination of total chlorine residual
   in all types of water and wastewater.
   A back-titration procedure is  used  to
   minimize interferences in waters con-
   taining substantial amounts of organic
   matter.
                                                                                       24-3

-------
Chlorine Determinations and Their Interpretation
    2  Summary of Method

       A sample is treated with a measured
       excess of standard phenylarsine oxide
       (PAO) solution followed by addition
       of potassium iodide and a buffer to
       maintain the pH between 3. 5 and 4. 2.
       Any form of chlorine present will
       stoichiometrically liberate iodine
       which immediately reacts with PAO
       before significant amounts are lost to
       reactions with organic matter in the
       sample.

       The amount of PAO used to reduce the
       liberated iodine is then determined by
       titrating the excess with a standard
       iodine solution in the presence of starch
       until the PAO is  completely oxidized.
       At this end point, the  next small addition
       of iodine causes  a faint blue color to
       persist in the sample.

       The excess PAO thus  measured is
       subtracted from the original amount
       of PAO added.  The difference is the
       PAO used to reduce the liberated
       iodine and is ultimately a measure of
       the total chlorine originally present
       in the sample.

       NOTE:  Sodium thiosulfate solution may
       be used instead of PAO, but PAO is
       more stable and  is to be preferred.

    3  Interferences

       a An unusually high content of organic
         matter may cause some uncertainty
         in the end point.  If manganese,
         iron and nitrite are definitely absent,
         this uncertainty can be reduced by
         acidification to pH  1.0.

       b Color and turbidity in the sample
         cause difficulty with end-point
         detection.
V  EVALUATION OF METHODS

 lodometric titration using amperometric
 end point detection appears to be the most
 accurate residual chlorine method
 available.
          1,2
Besides being inherently
 more accurate than color detection methods,
 this electrical end point is free of interfer-
 ence from color and turbidity.

 The accuracy of the colorimetric starch-
 iodide end point is improved by employing
 the indirect titration method described
 above.   By adding an excess of the standard
 reducing agent and back-titrating, contact
 between the liberated iodine and organic
 matter in the sample is minimized.
  1  Standard Methods for the Examination of
       Water and Wastewaters, 13th Ed.,
       APHA,  AWWA, WPCF.  1971.

  2  Book of ASTM Standards, Part 23 -
       Industrial Water; Atmospheric
       Analysis. American Society for
       Testing and Materials.
       Philadelphia,  PA.  1972.

  3  Sawyer,  C. N.  Chemistry for Sanitary
       Engineers.  McGraw-Hill Book
       Company.  New York.  1960.

  4  Moore, E. W.   Fundamentals of
       Chlorination of Sewage and. Wastes.
       Water and Sewage Works.  Vol. 98.
       No. 3.  March 1951.

  5  Day, R.  V., Horchler, D.  H.,  and
       Marks,  H. C.   Residual Chlorine
       Methods and Disinfection of Sewage.
       Industrial and Engineering Chemistry.
       May 1953.
 24-4

-------
                                      Chlorine Determinations and Their Interpretation
Marks,  H.  C.,  Joiner, R.  R.,  and
   Strandskov,  F.  B.  Amperometric
   Titration of Residual Chlorine in
   Sewage.  Water and Sewage Works.
   May 1948.
This outline was prepared by J.  L.  Holdaway,
Chemist, Technical Program, EPA, Region III,
Charlottesville,  VA  22901 (1971); and revised
by Audrey E. Donahue,  Chemist, National
Training Center, MPOD,   WPO, USEPA,
Cincinnati, OH 45268.
                                             Descriptors:  Chemical Analysis,  Chlorination,
                                             Chlorine, Disinfection, Sewage Treatment,
                                             Wastewater Treatment, Water Analysis.
                                                                               24-5

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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 algal blooms,
(b) its sequestering action, which causes
interference in coagulation, (c) the difficulty
of removing phosphorus from water to some
desirable low  concentration,  and (d)  its
characteristic of converting from one to
another of many possible forms.

A  Phosphorus is one of the primary nutrients
   such as hydrogen (H), carbon (C),
   nitrogen (N),  sulfur (S) and phosphorus (P).

   1  Phosphorus is unique among nutrients
      in that its oxidation does not contribute
      significant energy because it commonly
      exists in oxidized form.

   2  Phosphorus is intimately involved in
      oxidative energy  release from and
      synthesis of other nutrients into cell
      mass via:

      a  Transport of nutrients across
         membranes into cell protoplasm is
         likely to include phosphorylation.

      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 10 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  Wastewaters 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. 3.75
                                25-1

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Phosphorus  in the Aqueous Environment
A  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.

B  Chemically,  P exists in several mineral
   and organic forms that may be converted
   from one to another under favorable
   conditions. Analytical estimates are
   based upon physical or chemical techniques
   necessary to convert various forms of P
   into  ortho phosphates which alone can be
   quantitated in terms of the molybdenum
   blue colorimetric test.

C  Engineering interest in phosphorus is
   related to the prediction, treatment, or
   control of aqueous systems to favor
   acceptable water quality objectives.
   Phosphorus removal is associated with
   solids removal.
D  Solubility and temperature are major
   physical factors in phosphorus behavior.
   Soluble P is much more available than
   insoluble P for chemical or biological
   transformations and the rate of conversion
   from one to another is strongly influenced
   by temperature.

E  Table 1 includes a  classification of the
   four main types of  chemical P and some
   of the relationships controlling solubility
   of each group. It is apparent that no
   clear-cut separation can be made on a
   solubility basis as  molecular weight,
   substituent and other factors affect
   solubility.
   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 be significantly
      different for:
      1  Membrane filter separation
         (0. 5 micron pore size)

      2  Centrifugation (at some specified
         rpm and time)

      3  Paper filtration (specify paper
         identification)

      4  Subsidence (specify time and
         conditions)
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.
 25-2

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                                                    Phosphorus  in the Aqueous Environment
                                           Table 1


                        PHOSPHORUS COMPOUNDS CLASSIFIED BY
                          CHEMICAL  AND SOLUBILITY  RELATIONS
              Form
1.  Ortho phosphates
       ' "4(P3010)"5(P3°9)
    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, Prepresents P, PO , 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.
                                                                                    25-3

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Phosphorus  in the Aqueous Environment
                                          Table  2
                       PHOSPHORUS COMPOUNDS CLASSIFIED  BY
                              ANALYTICAL  METHODOLOGY

1.
2.

Desired P Components
Ortho phosphates
Polyphosphates
(2)-(l) = poly P
(hydrolyzable)

Technique
No treatment on clear
samples
acid hydrolysis on clear
samples, dilute
(a) H2S04
(b) HC1
heated
(2)
Incidental P Included '
Easily hydrolyzed
(a) poly phosphates -
(b) organic -P, -
(c) Mineral -P, + or -
(a) ortho-P +
(b) organic -P + or -
(c) mineral -P + or'-

     3. Organic phosphorus
          (3) - (2) + orgP
          (hydrolyzable)
acid + oxidizing hydrolysis
on whole sample,  dilute

 (a) H2S04

 (b) HS0
(a) ortho P   +
(b) poly P     +
(c) mineral P + or -
                                    heated
     4. Soluble phosphorus
        (preferably classified
        by clarification method)
clarified liquid following
filtration, centrifugation
or subsidence
generally includes
(a) 1, 2. or 3

(b) particulates not
    completely separated
     5. Insoluble phosphorus
        (residue from clari-
        fication)
Retained residues separated
during clarification
       See  (6)
(a) generally includes
    sorbed or complexed
    solubles.
     6. Total phosphorus
Strong acid + oxidant
digestion
 (a)
                                            ^  + HNO,
                                            4        3
                                    (b) H.SO .  + HNO, + HCK)
                                        ^   4        3        4
 (c)
                                            + Mg(N03)2 fusion
all components in
1,  2, 3, 4, 5 in the
whole sample
     (1)  Determinative step by phospho molybdate colorimetric method.

     (2)  Coding: + quantitative yield
                 - a small fraction of the amount present
                 + or  - depends upon the individual chemical and sample history

-------
                                                     Phosphorus  in the Aqueous Environment
IV   Polyphosphates are of major interest in
 cleaning agent formulation,  as dispersants,
 and for corrosion control.

 A  They are prepared by dehydration of ortho
     phosphates to form products having two or
     more phosphate derivatives per molecule.

     1  The simplest polyphosphate may be
       prepared as follows:
                            NaO
                                    = O
                                 P  = O
                                          H2°
                                7
 mono sodium ortho
    phosphate  (2)
         disodium dihydrogen
           polyphosphate
                            insoluble polyphosphate than the same
                            cation in the form of insoluble ortho
                            phosphate. Insolubility increases with
                            the number of P atoms in the
                            polyphosphate.  The  "glassy" poly-
                            phosphates are  a special group with
                            limited solubility that are used to aid
                            corrosion resistance in pipe distribu-
                            tion systems and similar uses.

                      B  Polyphosphates tend to hydrolyze or
                          "revert" to the ortho P  form by addition
                          of water.  This occurs whenever
                          polyphosphates are found 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
       The general form for producing
       polyphosphates from mono substituted
       orthophosphates is:
                     heat
                   200-4000C
  (NaPO )  + n HO
        o n     £
       Di-substituted ortho phosphates or
       mixtures of substituted ortho phosphates
       lead to other polyphosphates:
                                         c)  Enzymes, hydrolase enzymes
                                            increase rate

                                         d)  Colloidal gels,  increase rate

                                         e)  Complexing cations and ionic
                                            concentration increase rate

                                         f)  Concentration of the polyphosphate
                                            increases rate
 disodium hydrogen
  ortho phosphate
                       heat ,
mono sodium
 di hydrogen
ortho phosphate
 Na5P3°10
• pentasodium
 tri-phosphatc
        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
Items a, b and c have a large effect
upon reversion rate compared with
other factors listed.  The actual
reversion rate is a combination of
listed items and  other conditions or
characteristics.

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.
                                                                                         25-5

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Phosphorus  in the Aqueous Environment
 V  SAMPLING AND PRESERVATION
    TECHNIQUES

 A  Sampling

    1  Great care should be exercised to
      exclude any benthic deposits from
      water samples.

    2  Glass containers should be acid rinsed
      before use.

    3  Certain plastic containers may be
      used.  Possible adsorption of low con-
      centrations of phosphorus should be
      checked.

   4  If a differentiation of phosphorus forms
      is to be made, filtration should be
      carried out immediately upon sample
      collection.  A membrane filter of
      0.45M pore size  is recommended for
      reproducible  separations.

B  Preservation

   1  If at all possible,  samples should be
      analyzed on the day of collection.  At
      best, preservation measures only
      retard possible changes in the  sample.

      a  Possible physical  changes include
        solubilization,  precipitation,
        absorption on  or desorption from
        suspended  matter.

     b  Possible chemical changes include
        reversion of poly to ortho P and
        decomposition of organic or min-
        eral P.

      c    Possible  biological changes
          include microbial decomposition
          of organic P and algal or
          bacterial growth forming organic
          P.

   2  Refrigeration at 4 C  is recommended
      if samples are to be stored up to
      24 hours.  This decreases hydrolysis
      and reaction  rates and also losses due
      to volatility.
         Addition of 40 mg HgCl2/liter is
         recommended for longer storage
         periods.  This chemical limits
         biological changes.

         a   HgCL  is an interference in the
             analytical procedure if the
             chloride level is low (less than
             50 mg.  Cl /I).  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 Pig.  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 using a standard curve.

       Reagent preparation and the detailed
       procedure can be found in  the EPA
       manual.

       The methods described there are
       usable in the 0. 01 to 0. 5 mg/liter
       phosphorus range.
  25-6

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                                                Phosphorus  in the Aqueous Environment
SAMPLE i Totnl Sjirnolo CAe, VJ If i-.nrlfln) ,

1
1

Direct
Coloriaetry
Total
Orthophosphate
1
H2SOi 1 Persulfato
liydrolystj & 1 Digestion
Colorinetry 1 4 Colorioetry
Total Hydrolyzeble Totnl
& Orthophosphate Phosphorus
Filter 0.45 Micron Membrane Filter
v'- A
! Residue Filtrate j
*
il
Direct | H2SO^
Colo rime try I Hydrolysis
J, & Colorimetry
Ortho?
.
hosphote & Orthophosphate
inorganic polyphosphate
phosphorus + some organic
c expounds phosphorus

Persulfata
Digestion
6 Colorimetry

Phosphorus
inorganic +
organic
phosphorus
                                       Compounds       compounds
                                              FIGURE 1

                             ANALYTICAL SCHEME FOR DIFFERENTIATION 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.
                           I C\
C   Precision and Accuracy

    1  Organic phosphate - 33 analysts in
       19 laboratories analyzed natural
       water samples containing exact in-
       crements of organic phosphate of
       0.110,  0.132,  0.772,  and 0.882 mg
       P/liter.

       Standard deviations obtained were
       0.033,  0.051,  0.130 and 0.128
       respectively.

       Accuracy results as bias, mg P/liter
       were: +0.003, +0.016, +0.023  and
       -  0.008, respectively.

    2   Orthophosphate was determined by
       26 analysts in  16 laboratories using
       samples containing orthophosphate
       in amounts of 0.029, 0.038, 0.335
       and 0.383  mg P/liter.

       Standard deviations obtained were
       0.010,  0.008,  0.018 and 0.023
       respectively.
                                                                                     25-7

-------
 Ph
in the Aqueous Environment
         Accuracy results as bias, mg P/liter
         were-0.001, -0.002, -0.009  and
         -0.007 respectively.

  D 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
     H2SO  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.
                                                        1.0
                                        u
                                        8
                                        on
                                      D
                                                         0.05 mg P
                                             BLANK
       0.1   0.2  0.3   0.4  0.5   0.6
            H2SO  NORMALITY

            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  (^ 1°C), (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.
    25-8

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                                                    Phosphorus  in the Aqueous Environment
VIII DETERMINATION OF TOTAL
     PHOSPHORUS

 A   Determination of total phosphorus
     content involves omission of any
     filtration procedure and using the acid-
     hydrolysis and persulfate treatments
     to convert  all phosphorus forms to the
     test-sensitive orthophosphate form.

 B   Determining total phosphorus content
     yields the most meaningful data since
     the various forms of phosphorus may
     change from one  form to another in a
     short period of time.  (See part V, Bl)
 IX   DEVELOPMENT OF A STANDARD
     PROCEDURE

     Phosphorus analysis received intensive
     investigation; coordination and validation of
     methods is more difficult than  changing
     technique.

 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.  Eacn has a "favorite" scheme that
   may be quite effective but progress
   toward widespread application of "one"
   method has been slow.  Consequently,
   many methods are available.  Reagent
   acidity,  Mo content,  reductant and
   separation techniques are the major
   variables.

 C At the present  time there is not sufficient
   data to warrant EPA endorsement of the
   P procedure for sediment-type samples,
   sludges, algal  blooms,  etc.  Following
   is a procedure (not included in the EPA
   manual) which  is useful when solids
   are present in samples:

   1  If sample contains large particles,
      grind and emulsify solids in a blender.

   2  Transfer 50 ml sample, or aliquot
      diluted to 50 ml, into a 250 ml
      Erlenmeyer flask.

   3  Add 6. 0 ml of 18N H2SO4,  5 ml
      concentrated HNO~o,  2 berl saddles
      and digest on hot plate.

   4  Digest until  the disappearance of nitric
      acid fumes and the appearance of white
      803 fumes.  Continue digestion for
      approximately 5 minutes.   Cool before
      proceeding with Step 5.

   5 Add 2 ml of HNO3-HC1O4 mixture
     and 5 ml concentrated HNOg.  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.
                                                                                     25-9

-------
Phosphorus  in the Aqueous Environment
   8
     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.  Earth, 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   Earth,  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,  WPO,  EPA,    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.
 25-10

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           CHEMICAL TESTS,OBSERVATIONS,AND MEASUREMENTS IN THE FIELD
 I   INTRODUCTION

 A  Laboratory determinations with approved
    equipment and convenient facilities by
    experienced specialists generally are
    easier, faster,  and more reliable.  It is
    advisable to transport the  samples to the
    laboratory whenever  it is feasible to do so.

 B  Field tests are essential because:

    1  Certain sample components are
      inherently unstable with respect to
      biological, chemical, or physical
      changes.  Any test result performed in
      the laboratory may not  represent true
      conditions on site  at the time because
      of delay,  displacement, or changed
      conditions.

    2  Subsequent operations generally may be
      coordinated and made more meaningful
      by preliminary on site field investigation
      to identify and evaluate problems, locate
      critical areas, and minimize surprises.
II   FIELD TEST CONDITIONAL
    CONSIDERATIONS

 A  Moving the laboratory into the field means
    improvisation and adaptation to more
    "primitive" conditions.

    1  Rugged construction of field equipment
      is a first  consideration.   Sturdy and
      convenient cases are required; the case
      often may be the only available work-
      bench.  Polyethylene bottles, beakers,
      burets, and pipets generally are
      necessary to eliminate breakage of
      fragile glass.

    2  Portability is essential,  particularly
      when the site  is not accessible by boat
      or car. Ideally, the field kit should
      be small enough and light enough to be
      carried by one man for extended periods.
Procedures for field use are restricted
in equipment and manipulation.  In
general, it is not possible to use long,
tedious routines or highly precise
measurements.  Numerous  reagents
generally cannot be carried.  Quick
positive reactions are essential.  Small
visual comparators, test papers or spot
tests are popular.  Titration assemblies
must be modified for  field use.

Instrumentation increases objectivity
of the measurements  but the instruments
must be adapted to the items outlined in
II. A. 1, 2 and 3.

The instruments commonly  used must
be simplified yet rugged enough to keep
working in spite of temperature changes,
moisture, bumps, etc. A meaningful
validation procedure must be worked out
for calibration on site with simple
adjustment or repair  for emergencies.

Personnel engaged in field testing often
are unaccustomed to laboratory
procedures.  Advance training is
essential in procedures and instrumentation
used.  Field testing is a multi-
disciplinarian operation:

a    The individuals must be good
     observers arid recognize what may
     be  significant later.

b    They must be good interpreters
     of a variety of observed and
     derived test information.

c    They must be good technicians to
     follow prescribed procedures
     diligently and recognize anomalous
     behavior when it  occurs.

d    They must be good reporters  to
     describe what happened, where
     it happened,  and  when.   Failure
     to report anomalous behavior
   CH.4b.3.74
                                                                                       26-1

-------
 Chemical Tests Observations and Measurements in the Field
          and related events in an understandable
          and consistent fashion may render the
          entire effort meaningless because  of
          some doubt or inconsistency.
          Subsequent operations also may be
          mislead by the early observer.

 B Use of field data imposes certain con-
    straints relatable to the item, the methods,
    and to the personnel involved:

    1  How valid is  it?

    2  How consistent is it  in line with other
       observed or recorded information?

    3  What backup  information is required
       to  verify suggested information?

 C Decisions must be made on all field
    data applications:

    1  Where "in-place" and "now"
       measurements are required how much
       of  the laboratory must be moved out
       to  the site to obtain data reliable
       enough to meet objectives?

    2  Which items  are to be determined on
       site?  Which items in a central
       laboratory?

    3  What use is to be made of the
       preliminary field estimates?

    4  What do you "need" to know?

    5  What would be "nice" to know?

    6  In  line with facilities, time, cost,
       manpower and objectives, what
       can you determine?
Ill  COMPONENT CHARACTERISTICS
    FAVORING FIELD TESTING:
  \ Any item that changes rapidly during a
    holding period as a result of temperature
    or pressure variations, or is highly
    reactive  from a biological,  chemical,
    or physical standpoint,  usually means
    that it must be determined on site and in
    place with  minimum time lapse and
    manipulation.

   26-2
      1  Dissolved gases, such as O_, CO9,
         H2S,  C12, are sensitive to pressure
         or temperature changes and may
         react with other sample components
         readily.  These require in place and
         now readout.

      2  The collective analysis of all forms
         of a given item may be preserved
         and analyzed later.  Estimates of
         different forms of the same substance
         are not subject to delay unless they
         can be separated promptly.  Examples
         of this include the relative ratios of
         oxidized or reduced substances, such
         as Cu+| and Cu+2,  Fe   and Fe+3,  Cr
         and Cr  ; hydrolysis is the principal
         factor in the ratios of organic and
         NHg-N and in  changes among organic,
         poly and ortho-P,

      3  Reactive substances contributing to
         "oxygen demand" tests,  such as BOD
         or other respiratory tests generally
         require a  minimum time delay before
         testing and are not  amenable to
         preservation without altering results.

      4  Soluble/insoluble ratios  commonly
         change with time and conditions due
        to complexation, hydrolysis
         precipitation,  agglomeration, or
         other factors.  Particulate size may
        increase or decrease. Solubles may
        besorbed  on or desorbed from solid
         surfaces.   Settleability or turbidity
         are transient phenomena subject to
         change with time or conditions.

      5  Biological progressions are dynamic
        entities that shift among "critters"
        in relation to predominence in
        variety, numbers,  growth, and decay.
        Whatever  is, will change in response
        to nutrition and conditions.
IV   SITUATIONS FAVORING  FIELD TESTING

 A   Preliminary information often is needed
     to guide situation evaluation, problem
     identification, on site variation with
     respect to cross section,  depth,  or
     time.  These data are useful to determine
     whether there is a problem or not and
     for planning of subsequent operations.

-------
                         Chemical Tests Observations and Measurements in the Field
1  It may be necessary to locate suspected
   inflows,  channels, or sources of items
   affecting water quality.

2  Definition of mixing zones often is
   required.

3  Laboratory time may be reduced if
   approximate concentrations of items
   sought are known.

4  Field tests or observations  may
   suggest other tests that are more
   critical in evaluation of the  given
   situation.

5  Stratification may be evaluated for
   guidance of subsequent operations.

6  Knowledge of the distribution of
   components  is useful for selecting
   meaningful future  sampling  sites.

The field test may be used to check
compliance with regulations prior to
more rigorous  backup testing.

1  An established operation may be in
   control or out of control.

2  The field test may reveal which of
   multiple discharges are in conformance.

3  Undisclosed discharges may become
   apparent.

4  It may be necessary to "track" some
   hazardous discharge down stream
   to guide subsequent users on the
   choice of intake or storage water.

5  A complaint or inquiry may be
   evaluated by field tests.

In-plant field type tests are essential
to guide operations toward the  production
of continuous high quality effluents.
Record tests commonly are provided too
late to do anything about the situation;
quick test results are emphasized for
process control because time,  manpower,
and change are critical.
    1  Surprises in the form of wastewater
       changes in flow, concentration, or
       significant components are common
       for treatment process operators.  They
       need quick numbers  rather than
       impressions to distinguish real from
       apparent problems.

    2  Process upsets are minimized or
       prevented by regular and meaningful
       tests from which trends  may be
       established.  Small imbalances may
       be corrected before they become
       major upsets.

    3  Backup  or  supplementary treatment
       such as coagulation, adsorption,
       neutralization,  may prevent serious
       process disruption if tests indicate
       the problem and its magnitude in
       time to  do  something about it.

V   TYPICAL FIELD TESTS - INSTRUMENTAL

A   pH is one of the simplest and most
    valuable field tests.  If the  result is due
    to mineral acids or alkalies in stable
    form,  color indicators or comparators,
    impregnated paper or other devices
    will  serve.   When free CO_ in solution
    is a  major variable such as in the
    vicinity of benthic deposits  or in active
    biological systems, the application of
    an electronic  sensor  in-place and
    extension wire to an indicator instrument
    is essential.  Any delay, pressure change
    or manipulation will change free CC"
    and alter pH response.  Electronic pH
    instruments are produced for field use
    by many apparatus supply firms.  With
    care, they will function effectively.

B   Conductivity is a very useful index of
    ionic materials and usually may be
    correlated with wet chemical data to
    give a reasonable correlation with total
    dissolved solids for a given mixture.
    This test is very useful to detect saline
    intrustions or discharges,  springs,
    hidden channels of different salinity
    from the main body of water.  Several
    reliable instruments  are marketed.
                                                                                 26-3

-------
 Chemical Tests,Observations, and Measurements in the Field
VI
      DO analyzers are available for field
      use application from several manufacturers.
      They differ in portability accessory
      equipment and in versatility so that it is
      possible to obtain one or more fitting almost
      any requirement.   These units are based
      upon reduction of  oxygen at the cathode
      surface to convert chemical to electrical
      energy.  Direct measurement of electrical
      energy produced or the change in some
      carrier current or voltage may be used
      for readout.  The signal may be  amplified
      in line with sensitivity to give direct
      concentration readout. Temperature
      compensation is useful and available.
      Membrane covered sensors  selective
      for dissolved gases protect sensor
      surfaces from most interfering components.
      The analyst must  learn to use his
      particular instrument effectively to enable
      him to obtain valid results under conditions
      in which a wet chemical procedure would
      be misleading.
TYPICAL FIELD TESTS -
WET CHEMICAL

DO titrations for clean water samples
may be adapted for field use by shifting
to dry  reagents encased in plastic
pillows,  substituting phenyl arsene
oxide for -thio and using plastic burets,
sample containers, etc.  Powdered
starch substitutes also are available.
Sample sizes may be altered along
with reagent concentration to maintain
the 1 to 1 ratio of DO to titrant volume.
Hach,  La Motte and probably others,
provide DO test kits packaged for field
use. "Home made" kits are common.

Alkalinity and hardness titrations are
commonly  sought under field conditions
using standard acid and EDTA  solutions.
Methyl purple or mixed indicators
commonly  are used in place of methyl
orange indicator because of better
                                                 visibility in the field.  Powder pillows
                                                 for buffer and indicator are available
                                                 for the hardness test.

                                                 Chlorine tests using ortho tolidine in a
                                                 comparator,  such as those by Hilger
                                                 or Taylor and others,  frequently are
                                                 used for rapid on site  measurements.
                                                 The comparators, reagents and comparator
                                                 tubes commonly are available from
                                                 apparatus, equipment, or chlorine
                                                 suppliers.
VII   SUMMARY

      Simple and rapid field test kits are
      available from several manufacturers,
      suppliers and scientific apparatus
      houses for individual tests, as well
      as combination test kits for the items
      mentioned and others.

      It is possible for the analyst to devise
      one suited to his particular requirements
      with available or improvised materials.
      Effectiveness of the field operation
      depends on the knowledge  and care
      exercised in using the equipment  and
      procedure.

      CAUTION:  Field test results are
                 useful, but not always valid
                 enough for the record.

 ACKNOWLEDGEMENT:

      This outline contains significant
      material from a previous  outline  by
      D. G.  Ballinger.
                                                    This outline was prepared by F. J.  Ludzack,
                                                    Chemist, National Training Center,  WPO,
                                                    Cincinnati,  OH  45268.
                                                    Descriptors :  Chemical Analysis, On-Site
                                                    Tests, Water Analysis
      26-4

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                  BACTERIOLOGICAL INDICATORS OF WATER POLLUTION

                                 Part 1.  General Concepts
I  INTRODUCTION

A  Bacterial Indication of Pollution

   1  In the broadest sense,  a bacterial
      indicator of pollution is any organism
      which, by its presence, would demon-
      strate that pollution has occurred, and
      often suggest the source of the pollution.

   2  In a more restrictive sense, bacterial
      indicators of pollution are associated
      primarily with demonstration of con-
      tamination of water, originating from
      excreta  of warm-blooded animals
      (including man,  domestic and wild
      animals, and birds).

B  Implications of Pollution of Intestinal
   Origin

   1  Intestinal wastes from warm-blooded
      animals regularly include a wide
      variety of genera and species of
      bacteria.  Among these the coliform
      group may be listed, and species of
      the genera Streptococcus,  Lactobacillus,
      Staphylo coccus,  Proteus,  Pseudomonas,
      certain spore-forming bacteria, and
      others.

   2  In addition, many kinds of pathogenic
      bacteria and other microorganisms
      may be released in wastes on an inter-
      mittent basis, varying with the  geo-
      graphic area,  state of community
      health, nature and degree of waste
      treatment,  and other factors.  These
      may include the following:

      a Bacteria:  Species of Salmonella,
        Shigella,  Leptospira, Brucella,
        Mycobacterium, and Vibrio comma.
      b  Viruses:  A wide variety, including
         that of infectious hepatitis,  Po}io-
         viruses, Coxsackie virus, ECHO
         viruses (enteric cytopathogenic
         human orphan -- "viruses in search
         of a disease"), and unspecified
         viruses postulated to account for
         outbreaks of diarrheal and upper
         respiratory diseases of unknown
         etiology, apparently infective by
         the water-borne route.

      c  Protozoa:  Endamoeba histolytica

      As routinely practiced, bacterial
      evidence of water pollution is a test
      for the presence and numbers of
      bacteria in wastes which, by their
      presence,  indicate that intestinal
      pollution has occurred.  In this con-
      text,  indicator groups discussed in
      subsequent parts of this outline are
      as follows:

      a  Coliform group and certain  sub-
         groupings

      b  Fecal streptococci  and certain
         sub groupings

      c  Miscellaneous indicators  of pollution

      Evidence of water contamination by
      intestinal wastes of warm-blooded
      animals is regarded as evidence of
      health hazard in the water being tested.
II  PROPERTIES OF AN IDEAL INDICATOR
   OF POLLUTION

 A An "ideal" bacterial indicator of pollution
   should:

   1  Be applicable in all types of water
  W.BA.48g. 2. 75
                                     27-1

-------
  Bacteriological Indicators of Water Pollution
in
       Always be present in water when
       pathogenic bacterial constituents of
       fecal contamination are present.
       Ramifications of this include —
          a Its density should have some direct
            relationship to the degree of fecal
            pollution.
          b It should have greater survival time
            in  water than enteric pathogens,
            throughout its course of natural
            disappearance from the water body.
          c It should disappear rapidly from
            water  following the disappearance
            of pathogens, either through natural
            or  man-made processes.
          d It always should be absent in a
            bacteriologically safe water.
    3  Lend itself to routine quantitative
       testing procedures without interference
       or confusion of results due to extra-
       neous bacteria

    4  Be harmless to man and other animals

    In all probability, an "ideal" bacterial
    indicator does not exist.  The discussion
    of bacterial indicators of pollution in the
    following parts of this outline include
    consideration of the merits and limitations
    of each group,  with their applications in
    evaluating bacterial quality of water.
APPLICATIONS OF TESTS FOR
POLLUTION INDICATORS
 A Tests for Compliance with Bacterial
    Water Quality Standards

    1  Potability tests on drinking water to
       meet Interstate Quarantine or other
       standards of regulatory agencies.

    2  Determination of bacterial quality of
       environmental water for which quality
       standards may exist,  such as shellfish
       waters, recreational waters,  water
       resources for municipal or other
       supplies.
    3  Tests for compliance with established
       standards in cases involving the pro-
       tection or prosecution of municipalities,
       industries,  etc.

  B Treatment Plant Process Control

    1  Water treatment plants

    2  Wastewater treatment plants

  C Water Quality and Pollutant Source Monitoring

    1  Determination of intestinal pollution
       in surface water to determine type and
       extent of treatment required for com-
       pliance with standards.

    2  Tracing sources of pollution

    3  Determination of effects on bacterial
       flora, due to addition of organic or
       other wastes

  D Special Studies,  such as

    1  Tracing sources of intestinal pathogens
       in epidemiological investigations

    2  Investigations of problems due to the
       Sphaerotilus group

    3  Investigations of bacterial interference
       to certain industrial processes, with
       respect to such  organisms as Pseudo-
       monas,  A chromobacter, or others


IV  SANITARY SURVEY

  The laboratory bacteriologist is not alone in
  evaluation of indication of water pollution of
  intestinal origin.  On-site study (Sanitary
  Survey) of the aquatic environment and
  adjacent areas,  by a qualified person, is a
  necessary collateral study with the laboratory
  work and frequently will reveal information
  regarding potential bacteriological hazard
  which may or may not be demonstrated
  through laboratory findings from a single
  sample or short series of samples.
     27-2

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                                              Bacteriological Indicators of Water Pollution
                       Part 2.  The Coliform Group and Its Constituents
I  ORIGINS AND DEFINITION

A  Background

   1 In 1885, Escherich, a pioneer bacteri-
     ologist,  recovered certain bacteria from
     human feces, which he found in such
     numbers and consistency as to lead him
     to term  these organisms "the charac-
     teristic  organism of human feces. "

     He named these organisms  Bacterium
     coli- commune and _B.  lactis aerogenes.
     In 1895, another bacteriologist,
     Migula,  rename dE5. coli commune as
     Escherichia coli. which today is the
     official name for the type species.

   2 Later work has substantiated much of
     the original concept of Escherich, but
     has shown that the above species are
     in fact a heterogeneous complex of
     bacterial species and species variants.

     a  This  heterogeneous group occurs not
         only in human feces but representatives
         also are to  be found in many environ-
         mental media, including sewage,
        . surface freshwaters of all categories,
         in and on soils, vegetation, etc.

     b  The group may be subdivided into
         various categories on the basis of
         numerous biochemical and other
         differential tests that may be applied.

B  Composition of the Coliform Group

   1 Current definition

     As defined in "Standard Methods for the
     Examination of Water and Wastewater"
     (13th ed):  "The coliform group includes
     all of the aerobic and facultative
     anaerobic,  Gram-negative, nonspore-
     forming rod-shaped bacteria which
     ferment lactose with gas formation
     within 48 hours at 35°C. "
n
   !   The term "coliforms" or "coliform
      group" is an inclusive one, including
      the following bacteria which may
      meet the definition above:

      a Escherichia coli,  E. aurescens,
        JE.  freundii, E. intermedia

      b Aerobacter aerogenes, _A. cloacae

      c Biochemical intermediates between
        the genera Escherichia and Aero-
        bacter

    The above terminology is in accordance
    with the current editions of Standard
    Methods and Bergey's Manual of Deter-
    minative Bacteriology and will be
    consistent throughout this  manual until
    these sources are modified.

    There is no provision in the definition
    of coliform bacteria for "atypical" or
    "aberrant" coliform strains.
    a An individual strain of any of the above
       species may fail to meet one of  the
       criteria of the coliform group.
    b Such an organism,  by definition, is
       not a member of the coliform group,
       even though a taxonomic bacteriologist
       may be perfectly correct in classifying
        the strain in one of the above species.
SUBDIVISION OF COLIFORMS INTO
"FECAL" AND "NONFECAL"
CATEGORIES
 A  Need

    Single-test differentiations between
    coliforms of "fecal" origin and those of
    "nonfecal" origin are based on the
    assumption that typical E_. coli and
    closely related strains are of fecal
    origin while _A. aerogenes and its close
    relatives are not of direct fecal origin.
    (The latter assumption is not fully borne
    out by investigations at this Center.
    See Table 1, IMViC Type --++).   A
    number of single differential tests have
    been proposed to differentiate between
    "fecal" and "nonfecal" coliforms.
                                                                                      27-3

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Bacteriological Indicators of Water Pollution
   Without discussion of their relative merits,
   several may be cited here:

B  Types of Single-Test Differentiation

   1  Determination of gas ratio

      Fermentation of glucose by E3. coli
      results in gas production, with
      hydrogen and carbon dioxide being
      produced in equal amounts.
      Fermentation of glucose by A.
      aerogenes results in generation of
      twice as much carbon dioxide as
      hydrogen.

      Further studies suggested absolute
      correlation between H2/CO_ ratios
      and the terminal pH resulting from
      glucose fermentation.  This led to the
      substitution of the methyl red test.

   2  Methyl red test

      Glucose fermentation by E_. coli
      typically results in a culture medium
      having terminal pH in the range 4.2-
      4. 6 (red color a positive test with the
      addition of methyl red indicator).
      A_. aerogenes typically results in a
      culture medium having pH 5. 6 or
      greater (yellow color,  a negative test).

   3  Indole

      When tryptophane, an amino acid, is
      incorporated in a nutrient broth,
      typical E_.  coli strains are capable of
      producing indole (positive test) among
      the end products,  whereas A_.  aerogenes
      does not (negative test).

      In reviewing technical literature, the
      worker should be  alert to the method
      used to detect indole formation,  as the
      results may be greatly influenced by
      the analytical procedure.

   4  Voges-Proskauer test (acetylmethyl
      carbinol test)

      The test is for detection of acetylmethyl
      carbinol, a derivative of 2, 3, butylene-
   glycol, as a result of glucose
   fermentation in the presence of
   peptone.   A. aerogenes produces
   this end product (positive test)
   whereas E.  coli gives a negative test.

   a  Experience with coliform cultures
      giving a positive test has shown a
      loss of this ability with storage on
      laboratory media for 6 months to
      2iyears, in 20 -  25% of cultures
      (105 out of 458 cultures).

   b  Some workers consider that all
      coliform bacteria produce acetyl-
      methyl carbinol in glucose metab-
      olism.  These workers regard
      acetylmethyl carbinol-negative
      cultures as those  which have
      enzyme systems capable of further
      degradation of acetylmethyl
      carbinol to other end products
      which do not give  a positive test
      with the analytical procedure.
      Cultures giving a  positive test for
      acetylmethyl carbinol lack this
      enzyme system.

   c  This reasoning leads to a hypothesis
      (not experimentally proven) that the
      change  of reaction noted in certain
      cultures in 4. a above is due to the
      activation of a latent enzyme system.

5  Citrate utilization

   Cultures of E_. coli are unable to use
   the carbon of citrates (negative test)
   in their metabolism, whereas cultures
   of _A_. aerogenes are  capable of using
   the carbon of citrates in their metab-
   olism (positive  test).

   Some workers (using Simmons Citrate
   Agar) incorporate a pH indicator
   (brom thymol blue) in the  culture
   medium in order to demonstrate the
   typical alkaline reaction (pH 8.4  - 9.0)
   resulting with citrate utilization.

6  Elevated temperature (Eijkman) test
   a  The test is based  on evidence that
      E.  coli and other  coliforms of fecal
    27-4

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                                         Bacteriological Indicators of Water Pollution
   origin are capable of growing and
   fermenting carbohydrates- (glucose
   or lactose) at temperatures signif-
   icantly higher than the body tem-
   perature of warm-blooded animals.
   Organisms not associated with direct
   fecal origin would give a negative
   test result, through their inability
   to grow at the elevated temperature.

b  While many media and techniques
   have been proposed,  EC Broth, a
   medium developed by Perry and
   Hajna, used as a confirmatory
   medium for 24 hours at 44. 5 ±
   0.2oc are the current standard
   medium and method.
   While the "EC" terminology of the
   medium suggests "_E. coli" the
   worker should not regard this as  a
   specific procedure for isolation of
   E. coli.
c  A similar medium,  Boric Acid
   Lactose Broth,   has developed
   by Levine and his associates.  This
   medium gives results virtually
   identical with those obtained from
   EC Broth,  but requires 48 hours  of
   incubation.

d  Elevated temperature tests require
   incubation in a water bath.  Standard
   Methods  13th Ed. requires this
   temperature  to be 44. 5+0. 2°C.
   Various workers have urged use of
   temperatures ranging between
   43. 00C and 46.00C.  Most of these
   recommendations have provided a
   tolerance of + 0. 5° C from the rec-
   ommended levels.  However, some
   workers, notably in the Shellfish
   Program of the Public Health Service,
   stipulate a temperature of 44. 5 +
   0. 2°C.  This requires use of a water
   bath with forced circulation to main-
   tain this close tolerance.  This
   tolerance range   was instituted
   in the 13th Edition of Standard Methods
   and the laboratory worker should
   conform to these new limits.
      e The reliability of elevated temper-
        ature tests is influenced by the
        time required for the newly-
        inoculated cultures to reach the
        designated incubation temperature.
        Critical workers insist on place-
        ment of the  cultures in the water
        bath within 30 minutes, at most,
        after inoculation.

   7  Other tests

      Numerous other tests for differentiation
      between coliforms of fecal vs. nonfecal
      origin have been proposed.  Current
      studies suggest little promise for the
      following tests in this application:
      uric acid test,  cellobiose fermentation,
      gelatin liquefaction, production of
      hydrogen sulfide, sucrose fermentation,
      and others.

C  IMViC Classification

   1  In 1938, Parr reported on a review of
      a literature survey on biochemical tests
      used to differentiate between coliforms
      of fecal vs. nonfecal origin.  A summary
      follows:
        Test
No. of times
used for dif-
ferentiation
   Voges-Proskauer             22
      reaction
   Methyl red test               20
   Citrate utilization             20
   Indole test                    15
   Uric acid test                 6
   Cellobiose fermentation        4
   Gelatin liquefaction            3
   Eijkman test                   2

   Hydrogen sulfide               1
      production
   Sucrose fermentation           1

   a-Methyl-d-glucoside          1
      fermentation
                                                                           27-5

-------
Bacteriological Indicators of Water Pollution
   2  Based on this summary and on his own
      studies. Parr recommended utilization
      of a combination of tests, the indole,
      methyl red,  Voges-Proskauer,  and the
      citrate utilization tests for this differ-
      entiation.  This series of reactions is
      designated by the mnemonic "iMViC".
      Using this scheme, any coliform culture
      can be described by an "IMViC  Code"
      according to the reactions for each
      culture.  Thus,  a typical culture of
      E. coli would have a code ++—, and a
      typical A. aerogenes culture would
      have a code --++.

   3  Groupings of coliforms into fecal,
      non-fecal, and intermediate groups,
      as shown in "Standard Methods for the
      Examination of Water and Wastewater"
      are shown at the bottom of this page.

D  Need for Study of Multiple Cultures

   All the systems used for differentiation
   between coliforms of fecal vs. those of
   nonfecal origin require isolation and study
   of numerous pure cultures.  Many  workers
   prefer to study at least 100 cultures from
   any environmental source before attempting
   to categorize the probable source of the
   coliforms.
                   NATURAL DISTRIBUTION OF COLIFORM
                   BACTERIA

                A Sources of Background Information

                   Details of the voluminous background of
                   technical information on coliform bacteria
                   recovered from one or more environ-
                   mental media are beyond the scope of
                   this discussion. References of this
                   outline are suggested routes of entry
                   for workers seeking to explore this
                   topic.

                B Studies on Coliform Distribution

                   1  Since 1960  numerous workers
                      have engaged in a continuing study of
                      the  natural distribution of coliform
                      bacteria and an evaluation of pro-
                      cedures for differentiation between
                      coliforms of fecal vs. probable non-
                      fecal origin.  Results of this work
                      have special significance because:

                      a  Rigid uniformity of laboratory
                        methods have been applied through-
                        out the series of studies

                      b  Studies are based on massive
                        numbers of cultures, far beyond
                        any similar studies heretofore
                        reported
              Groupings of Coliforms into Fecal, Nonfecal and Intermediate Groups
            Organism
Indole
Methyl
 red
 Voges-
Proskauer
Citrate
        E. coli.  Variety I
                 Variety n

        E_. freundii
           (Intermediates)
                 Variety I
                 Variety n

        A_. aerogenes
                 Variety I
                 Variety n
                                             ±
                                             +



                                             +
                                             +
  27-6

-------
                                            Bacteriological Indicators of Water Pollution
   c A wider variety of environmental and
     biological sources is being gtudied
     than in any previous series of reports.

   d All studies are based on freshly
     recovered pure culture isolates
     from the designated sources.

   e All studies are based on cultures
     recovered from the widest feasible
     geographic range,  collected at all
     seasons of the year.  It is believed
     that no more representative series
     of studies has been made or is in
     progress.

2  Distribution of coliform types

   Table 1 shows the consolidated results
   of coliform distributions from various
   biological and environmental sources.

   a The results of these studies show a
     high order of correlation between
     known or probable fecal origin and
     the typical J3. coli IMViC  code
     (++--).  On the other hand,
     human feces also includes
     numbers of_A. aerogenes and other
     IMViC types,  which some regard as
     "nonfecal" segments of the coliform
     group.  (Figure 1)

   b The majority of coliforms attributable
     to excretal origin tend to be limited
     to a relatively small number of the
     possible IMViC codes; on the other
     hand,  coliform bacteria recovered
     from undisturbed soil,  vegetation,
     and insect life represent a wider
     range of IMViC codes than fecal
     sources, without clear dominance of
     any one type.  (Figure 2)

   c The most prominant IMViC code
     from nonfecal sources is the inter-
     mediate type, -+-+, which accounts
     for almost half the coliform cultures
     recovered from soils, and a high
     percentage of those recovered from
     vegetation and from insects.  It
     would appear that if any coliform
     segment could be termed a "soil
     type" it would be IMViC code -+-+.
d  It should not be surprising that
   cultures of typical E_. coli are
   recovered in relatively smaller
   numbers from sources judged,
   on the basis of sanitary survey,
   to be unpolluted.  There  is no
   known way  to exclude the influence
   of limited fecal pollution from small
   animals and birds in such environ-
   ments .

e  The distribution of coliform types
   from human sources should be
   regarded as a representative value
   for large numbers of sources.
   Investigations have shown that there
   can be large differences  in the
   distribution of IMViC types from
   person to person,  or even from an
   individual.

Differentiation between  coliforms  of
fecal vs. nonfecal origin

Table 2 is a summary of findings
based on a number of different criteria
for differentiating between coliforms
of fecal origin and those from other
sources.

a  IMViC type ++- - is a measurement
   of E.  coli,  Variety I, and appears
   to give reasonably good correlation
   between known or highly  probable
   fecal origin and doubtful  fecal origin.

b  The combination of IMViC types,
   ++--,  +	, and -+--,  gives
   improved identification of probable
   fecal origin,  and appears also to
   exclude most of the coliforms not
   found in excreta of warm-blooded
   animals in  large numbers.

c  While the indole,  methyl red,
   Voges Proskauer,  and citrate
   utilization tests, each used alone,
   appear to give useful answers when
   applied only to samples of known
   pollution from fecal sources, the
   interpretation is not  as clear when
   applied to coliforms  from sources
   believed to be remote from direct
   fecal pollution.
                                                                                  27-7

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to

-J
I

CO
Table 1.  COLIFORM DISTRIBUTION BY IMViC TYPES AND ELEVATED TEMPERATURE

              TEST  FROM ENVIRONMENTAL AND BIOLOGICAL SOURCES
                   *120 of these   *129 of these

                   were -H—,     were ++--,

                   15 --++,        27 -+-+,

                   11 -+--        5  ++-+
IMViC
type
Vegetation
No.
strains

4-+--
--++
-+--
+++-
-+- +
++- +
-+++
++++
+-++
	 h
--H--
--+-
+-+-
+ — +
+ 	
Total
No. EC +
%EC +
128
237
23
2
168
116
32
291
88
87
5
19
2
5
0
1203
169*
14. 1*
% of
total

10.6
19. 7
1. 9
0. 2
14.0
9. 6
2. 7
24.2
7. 3
7. 2
0.4
1. 6
0. 2
0.4
<0. 1



Insects
No.
strains
.
134
113
0
0
332
118
28
254
46
42
0
0
0
8
9
1084
162*
14.9*
% of
total

12.4
10.4
<0. 1
<0. 1
30. 6
Soil
Undisturbed
No.
strains

131
443
78
7
1131
10.9 | 87
2.6
23.4
4.2
3.9
<0. 1
<0. 1
<0. 1
0. 7
0.8



181
159
67
4
1
53
6
0
0
2348
216
9.2
% of
total

5.6
18. 8
3.3
0.3
48. 1
3. 7
7. 7
6. 8
2.9
0.2
<0. 1
2.3
0. 3
<0. 1
<0. 1



Polluted
No.
strains

536
13
1
0
87
22
5
0
0
1
0
0
0
0
0
665
551
82.9
% of
total

80. 6
2. 0
0.2
<0. 1
13.0
3. 3
0. 7
<0. 1
<0. 1
0.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1



Fecal sources
Human
No.
strains

3932
245
99
106
50
35
21
6
14
2
0
0
0
0
2
4512
4349
96.4
% of
total

87.2
5.4
2. 2
2.4
1. 1
0. 8
0.5
0. 1
0.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1



Livestock
No.
strains

2237
0
14
59
1
27
0
0
0
0
0
0
0
0
0
2339
2309
98.7
%of
total

95.6
<0. 1
0.6
2.5
<0. 1
1.2
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1



Poultry
No.
strains

1857
1
20
0
5
% of
total

97.9
0. 1
1. 1
<0. 1
0. 3
i
11 i 0.6
0
0 '
0
0
0
0
0
0
2
1896
1765
93.0
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1
<0. 1



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-------
                         Bacteriological Indicators of Water Pollution
          HUMAN
         EC ® 96.4
        BALB® 94.7
SUM/MARY
Type
n- —
-+ —

-------
Bacteriological Indicators of Water Pollution
Table 2.  COMPARISON OF COLIFORM STRAINS ISOLATED FROM WARM-BLOODED ANIMAL
      FECES, FROM UNPOLLUTED SOILS AND POLLUTED SOILS .WITH USE OF THE
      IMViC REACTIONS AND THE ELEVATED TEMPERATURE TEST IN EC MEDIUM
      AT 44. 50  C ( +0. 50) (12th ed. 1965; Standard Methods for the Examination of Water
      and Wastewater)
Test
+ + - -
+ + - -,
+ 	 and
- + - -
Indole positive
Methyl red positive
Voges-Proskauer positive
Citrate utilizers
Elevated temperature (EC).
positive .
Number of cultures
studied
Warm-blooded
animal feces
91.8%
93. 3%
94. 0%
96. 9%
5. 1%
3.6%
96. 4%
8, 747
Soil:
Unpolluted
5.6%
8.9%
19.4%
75. 6%
40. 7%
88. 2%
9.2%
2,348
Soil:
Polluted
80. 6%
80. 7%
82. 7%
97. 9%
97. 3%
19. 2%
82. 9%
665
Vege-
tation
10. 6%
12. 5%
52. 5%
63. 6%
56. 3%
85. 1%
14. 1%
1,203
Insects
12. 4%
13. 2%
52.4%
79. 9%
40. 6%
86. 7%
14. 9%
1,084
                         Total Pure Cultures Studied:  14, 047
      d  The elevated temperature test gives
         excellent correlation with samples
         of known or highly probable fecal
         origin.  The presence of smaller,
         but demonstrable, percentages of
         such organisms in environmental
         sources not interpreted as being
         polluted could be attributed largely
         to the  warm-blooded wildlife in the
         area,  including birds, rodents, and
         other  small mammals.

      e  The elevated temperature test yields
         results equal to those obtained from
         the total IMViC code.  It has marked
         advantages in speed, ease and
         simplicity of performance,  and yields
         quantitative results  for each water
         sample.  Therefore, it is
         the official standard method for
         differentiation  coliforms of
         probable direct fecal origin from those
         which  may have become established
         in the  bacterial flora of the aquatic
         or terrestrial habitat.
IV  EVALUATION OF COLIFORMS AS
    POLLUTION INDICATORS

 A The Coliform Group as a Whole

    1  Merits

       a  The absence of coliform bacteria is
          evidence of a bacteriologically safe
          water.

       b  The density of coliforms is roughly
          proportional to the amount of
          excretal pollution present.

       c  If pathogenic bacteria of intestinal
          origin are present, coliform
          bacteria also are present, in much
          greater numbers.

       d  Coliforms are always present in the
          intestines  of humans  and other warm-
          blooded animals,  and are eliminated
          in large numbers in fecal wastes.
   27-10

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                                            Bacteriological Indicators of Water Pollution
   e  Coliforms are more persistent in
      the aquatic environment than are
      pathogenic bacteria of intestinal
      origin.

   f  Coliforms are generally harmless
      to humans and can be determined
      quantitatively by routine laboratory
      procedures.

2  Limitations

   a  Some of the constituents of the
      coliform group have a wide environ-
      mental distribution in addition to
      their occurrence in the intestines
      of warm-blooded animals.

   b  Some strains of the coliform group
      may  multiply in certain polluted
      waters ("aftergrowth"),  of high
      nutritive values thereby adding to
      the difficulty of evaluating a pollution
      situation in the aquatic environment.
      Members of the _A.  aerogenes section
      of the coliform are commonly
      involved in this kind of problem.

   c  Because of occasional aftergrowth
      problems, the age of the pollution
      may be difficult to evaluate under
      some circumstances.

   d  Tests for coliforms are subject to
      interferences due to other kinds of
      bacteria.  False negative results
      sometimes occur when species of
      Pseudomonas are present.   False
      positive results sometimes occur
      when two or more kinds of non-
      coliforms produce gas from lactose,
      when neither can do so alone
      (synergism).

The Fecal  Coliform  Component of the
Coliform Group (as determined by elevated
temperature test)

1  Merits

   a  The majority (over 95% of the coli-
      form bacteria  from intestines of
      warm-blooded animals grow at the
      elevated temperature.
   b  These organisms are of relatively
      infrequent occurrence except in
      association with fecal pollution.

   c  Survival of the fecal coliform group
      is shorter in environmental waters
      than for the coliform group as
      whole.  It follows, then, that high
      densities of fecal coliforms is
      indicative of relatively recent
      pollution.

   d  Fecal coliforms generally do not
      multiply outside the  intestines of
      warm-blooded animals. In certain
      high-carbohydrate wastes, such as
      from the sugar beet  refineries,
      exceptions have been noted.

   e  In some wastes, notably those from
      pulp and paper mills, Klebsiella has
      been  found in large  numbers
      utilizing the elevated temperature
      testi  There has been much contro-
      versy about whether  the occurrence
      of Klebsiella is due to aftergrowth
      due to  soluble carbohydrates  in such
      wastes.  The significance of
      Klebsiella as an indicator of  direct
      discharge of intestinal wastes thus
      is under challenge.   The issue is
      still further complicated by questions
      over whether Klebsiella is in and of
      itself a pathogenic organism or is
      potentially pathogenic.  This  is a
      serious problem which is the subject
      of current intensive research by this
      Agency.


2  Limitations

   a  Feces from warm-blooded animals
      include some (though proportionately
      low) numbers of coliforms which do
      not yield a positive fecal coliform
      test when the elevated temperature
      test is used as the criterion of
      differentiation. These organisms
      are E. coli varieties by present
      taxonomic classification.

   b  There  is at present no established
      and consistent correlation between
                                                                                    27-11

-------
Bacteriological Indicators of Water Pollution
         ratios of total coliforms/fecal
         coliforms in interpreting sanitary-
         quality of environmental waters.

         In domestic sewage,  the fecal
         coliform density commonly is
         greater than 90% of the total
         coliform density.  In environmental
         waters relatively free from recent
         pollution, the fecal coliform density
         may range from 10-30% of the total
         coliforms.  There are,  however,
         too many variables relating to
         water-borne wastes and surface
         water runoff to permit sweeping
         generalization on the numerical
         relationships between fecal- and
         total coliforms.

      c  Studies have been made
         regarding the  survival of fecal
         coliforms in polluted waters
         compared with that of enteric
         pathogenic bacteria.   In recent
         pollution studies, species of
         Salmonella have been found in the
         presence of 220 fecal coliforms per
         100 ml (Spino), and 110 fecal
         coliforms per 100  ml (Brezenski,
         Raritan Bay Project).

       The issue of the Klebsiella problem
       described in an earlier paragraph
       may ultimately be resolved as a
       merit or as a limitation of the value
       of the fecal coliform test.
V  APPLICATIONS OF COLIFORM TESTS

 A Current Status in Official Tests

   1  The coliform group is designated,  in
      "Standard Methods for the Examination
      of Water and Wastewater" (13th ed.,
      1971), through the  Completed Test
      MPN procedure as the official test
      for bacteriological potability of water.
     The Confirmed Test MPN procedure
     is accepted where it has been demon-
     strated,  through comparative tests,
     to yield results equivalent to the
     Completed Test.  The membrane filter
     method also is accepted for examination
     of waters subject to interstate regulation.

  2  The 12th edition of Standard Methods
     introduced  a  standard test for fecal
     coliform bacteria. It is emphasized
     that this is to be used in pollution
     studies,  and does not apply to the
     evaluation of water for potability.
     This procedure has been continued in
     the  13th Edition.

B  Applications

   1  Tests for the coliform group  as  a
     whole are used in official tests to
     comply with interstate drinking water
     standards,  state standards for shell-
     fish waters, and in most, if not  all,
     cases where bacterial standards of
     water quality have been established
     for such use as in recreational or
     bathing waters, water supplies,  or
     industrial supplies.  Laboratory
     personnel should be aware of possible
     implementation of the fecal coliform
     group as the official test for recreational
     and bathing waters.

   2  The fecal coliform test has application
     in water quality surveys, as an adjunct
     to determination of total coliform
     density.   The fecal coliform test is
     being used increasingly in all water
     quality surveys.

   3  It is emphasized that no responsible
     worker advocates substitution of a
     fecal coliform test for total coliforms
     in evaluating drinking water quality.
  27-12

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                                              Bacteriological Indicators of Water Pollution
                             Part 3.  The Fecal Streptococci
 I  INTRODUCTION
Investigations regarding streptococci
progressed from the streptococci of medical
concern to those which were distributed in
differing environmental conditions which,
again, related to the welfare of man.  The
streptococci were originally reported by Laws
and Andrews (1894),  and Houston (1899, 1900)
considered those streptococci, which we now
call "fecal streptococci, "as ...  "indicative
of dangerous pollution, since they are readily
demonstrable in waters recently polluted and
seemingly altogether absent from waters above
suspicion of contamination.

From their discovery to the present time  the
fecal streptococci appear characteristic of
fecal pollution,  being consistently present in
both the feces of all warm-blooded animals
and in the environment associated with animal
discharges.  As early as 1910 fecal strepto-
cocci were proposed as indicators to the
Metropolitan Water Board of London.
However, little progress resulted in the
United States until improved methods of
detection and enumeration appeared after
World War II.

Renewed interest in the group as indicators
began with the introduction of azide dextrose
broth in 1950, (Mallmann & Seligmann,  1950).
The method which is in the current edition
of Standard Methods appeared soon after.
(Litsky, et al. 1955).

With the advent of improved methods for
detection and enumeration of fecal strep-
tococci, significant body of technical
literature has appeared.

This outline will consider the findings of
various investigators regarding the fecal
streptococci and the significance of discharges
of these organisms into the aquatic environment.
H  FECAL MATERIALS

 A Definition
The terms "enterococci, " "fecal
streptococci, " "Group D streptococci, "
"Streptococcus fecalis, " and even
' streptococci  have been used in a loose
and interchangeable manner to indicate
the streptococci present in the enteric
tract of warm-blooded animals or of the
fresh fecal material excreted therefrom.

Enterococci are characterized by specific
taxonomic biochemistry.   Serological
procedures differentiate the Group D
streptococci from the various groups.
Although they overlap,  the three groups,
fecal streptococcus, enterococcus, and
Group  D streptococcus, are not synonymous.
Because our emphasis is on indicators of
unsanitary origin, fecal streptococcus is
the more  appropriate term and will include
the enterococcus as well as other groups.

A rigid definition of the fecal  streptococcus
group is not possible with our present
knowledge.  The British Ministry of Health
(1956)  defines the  organisms as "Gram-
positive"  cocci, generally occurring in
pairs or short chains,  growing in the
presence  of bile salt, usually capable of
development at 45° C,  producing acid but
not gas in mannitol and lactose, failing to
attack  raffinose,  failing to reduce nitrate
to nitrite, producing acid in litmus milk
and precipitating the casein in the form of
a loose,  but solid curd, and exhibiting a
greater resistance to heat, to alkaline
conditions and to high concentrations of
salt than most vegetative bacteria. "
However, it is pointed out that "streptococci
departing in one or more particulars from
the type species cannot be disregarded
in water."

For the proposes of this outline, and in line
with the consensus of most water micro-
biologists in this country,  the definition
of the fecal streptococci is:

.  . . "The group composed of Group D
     species consistently present in
     significant numbers in fresh fecal
     excreta of warm-blooded animals,
     which includes all of the enterococcus
     group in addition to other  groups of
     streptococci."
                                                                                   27-13

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Bacteriological Indicators of Water Pollution
  B Species Isolated

     1  Findings

        a  Human feces

           Examination of human fecal specimens
           yields a high percentage of the
           enterococcus group and usually
           demonstration of the S. salivarius
           which is generally considered a
           member of the human throat flora
           and to be surviving in human fecal
           materials rather than actively
           multiplying in the enteric tract.
           Also present would be a small
           percentage of variants or biotypes
           of the enterococcus group.

        b  Nonhuman Feces

           1) Fecal material which are from
              nonhuman or not from fowl will
              yield high percentages of the
              S. bovis and/or S. equinus
              organisms with a concomitantly
              reduced percentage of the
              enterococcus group.

           2) Fowl excreta

              Excrement from fowl characteris-
              tically yields a large percentage
              of enterococcal biotypes as well
              as a significant percentage of
              enterococcus group.

     2  Significance

        Species associations with particular
        animal hosts is an established fact and
        leads to the important laboratory
        technique of partition counting of colonies
        from the membrane filter  or agar
        pour plates in order  to establish or
        confirm the source of excretal
        pollution in certain aquatic investi-
        gations .

        It is important to realize that a suitable
        medium is necessary in order to
        allow all of the streptococci which
        we consider to be fecal streptococci
HI
    to grow in order to give credence to
    the derived opinions.  Use of liquid
    growth media into which direct
    inoculations from the sample are
    made have not proven to be successful
    for partition  counting due to the differing
    growth rates of the various species of
    streptococci  altering the original
    percentage relationships.  Due to the
    limited survival capabilities of some
    of the fecal streptococci it is necessary
    to sample fresh fecal material or water
    samples in close proximity to the
    pollution source especially when
    multiple sources are contributing to a
    reach of water.  Also the pH range
    must be within the range of 4. 0-9. 0.
FECAL STREPTOCOCCI IN THE
AQUATIC ENVIRONMENT
 A General

    From the foregoing it is appears that
    the preponderant human fecal streptococci
    are composed of the enterococcus group
    and, as this is the case, several media are
    presently available which will detect only
    the enterococcal group will be suitable
    for use with aquatic samples which are
    known to be contaminated or potentially
    contaminated with purely  domestic
    (human) wastes.  On the other hand,
    when it is known or suspected that other-
    than-human wastes have potential egress
    to the aquatic environment under investi-
    gation,  it is necessary to utilize those
    media which are capable of quantitating
    the whole of the fecal streptococci group.

 B Stormwaters and Combined Sewers

    1   General

       Storm sewers are a series of pipes
       and conduits which receive surface
       runoffs from the action of rainstorms
       and do not include sewage which are
       borne by a system of sanitary sewers.
       Combined sewers receive both the storm
       runoff and the water-borne
       wastes of the sanitary system.
    27-14

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                                              Bacteriological Indicators of Water Pollution
      Both storm water and combined
      sewer flows have been found to
      usually contain large quantities of
      fecal streptococci in numbers which
      generally are larger than those of
      the fecal coliform indicator organisms.

   2  Bacteriological Findings

      Table 1 represents,  in a modified form,
      some of the findings  of Geldreich and
      Kenner (1969) with respect to the
      densities of fecal streptococci when
      considering Domestic sewage in contrast
      to Stormwaters:
              Table 1

DISTRIBUTION OF FECAL STREPTOCOCCI
IN DOMESTIC SEWAGES AND STORMWATER
                RUNOFFS
               Fecal Streptococci
                 per 100 ml       Ratio
Water Source    median values     FC/FS
                                   5.3
                                   4.5
                                   4.9
                                   4.4
                                   4.0
                                  27.9
                                  16.9
Domestic Sewage
Preston, ID
Fargo, ND
Moorehead, MN
Cincinnati, OH 2,
Lawrence, MA 4,
Monroe, MI
Denver, CO 2,
Stormwater
Business District
Residential
Rural
64, 000
290, 000
330, 000
470, 000
500, 000
700, 000
900, 000
51,000
150, 000
58, 000
                                                                                     0.26
                                                                                     0.04
                                                                                     0.05

                                                        The Ratio FC/FS is that of the
                                                        Fecal coliform and Fecal streptococci
                                                        and it will be noted that in each case,
                                                        when considering the Domestic
                                                        Sewage,  it is 4. 0 or greater while
                                                        it is less than 0. 7 for stormwaters.
                                                        The use  of this ratio is useful to
                                                        identify the source of pollution as

   Table 2.  ESTIMATED PER CAPITA CONTRIBUTION OF INDICATOR MICROORGANISMS
                                   FROM SOME ANIMALS*
                           Average indicator
                           density per gram
                                of feces
    Average contribution
    per capita per 24 hr
Animals
Man
Duck
Sheep
Chicken
Cow
Turkey
Pig
Avg wt of
Feces/24 hr,
wet wt, g
150
336
1, 130
182
23,600
448
2,700
Fecal
coliform,
million
13.0
33.0
16.0
1.3
0.23
0.29
3.3
Fecal
streptococci,
million
3.0
54.0
38.0
3.4
1.3
2.8
84.0
Fecal
coliform,
million
2,000
11,000
18, 000
240
5,400
130
8,900
Fecal
streptococci,
million
450
18,000
43, 000
620
31,000
1,300
230, 000
Ratio
FC/FS
4.4
0.6
0.4
0.4
0.2
0.1
0.04
*Publication WP-20-3, P. 102
                                                                                   27-15

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Bacteriological Indicators of Water Pollution
       being human or nonhuman warm-
       blooded animal polluted.  When the ratio
       is greater than 4. 0 it is considered to be
       human waste contaminated while a ratio
       of less than 0.7 is considered to be
       nonhuman. • It is evident that the storm-
       waters have been primarily polluted by
       excreta of rats and other rodents and
       possibly domestic and/or farm animals.

       Species differences are the main cause
       of different fecal coliform-fecal
       streptococci ratios.  Table 2 compares
       fecal streptococcus and fecal coliform
       counts for different species.  Even
       though individuals vary widely, masses
       of individuals in a species  have charac-
       teristic proportion of indicators.

  C Surface Waters

    In general, the occurrence of fecal
    streptococci indicates fecal pollution and
    its absence indicates that little or no
    warm-blooded fecal contribution.  In
    studies of remote surface waters the fecal
    streptococci are infrequently isolated and
    occurrences of small numbers can be
    attributed to wild life and/or snow melts
    and resultant drainage flows.

    Various examples of fecal streptococcal
    occurrences are shown in Table 3 in
    relation to surface  waters of widely varying
    quality.  (Geldreich and Kenner 1969)
IV  FECAL STREPTOCOCCI:
    AND LIMITATIONS
ADVANTAGES
 A  General

    Serious studies concerning the streptococci
    were instituted when it became apparent
    that they were the agents responsible or
    suspected for a wide variety of human
    diseases.  Natural priority then focused
    itself to the taxonomy of these organisms
    and this study is still causing consternation
    as more and more microbiological techniques
    have been brought to bear on these questions.
    The sanitary microbiologist is concerned
    with those streptococci which inhabit the
    enteric tract of warm-blooded animals,
    their detection, and utilization in develop-
    ing a criterium for water quality standards.
                                    Table 3

                      INDICATOR ORGANISMS IN SURFACE
                                     WATERS
                                       Densities/100 ml
                                       Fecal       Fecal
                        Water Source  coliform streptococci

                      Prairie Watersheds

                      Cherry Creek, WY     90        83
                      Saline River,  KS       95       180
                      Cub River, ID        110       160
                      Clear Creek,  CO      170       110

                      Recreational Waters

                      Lake Mead            2        444
                      Lake Moovalaya        9        170
                      Colorado River         4        256
                      Whitman River         32        88
                      Merrimack River      100        96

                      Public Water Intakes

                      Missouri River (1959)
                        Mile 470.5      11,500   39,500
                        Mile 434.5      22,000   79,000
                        Mile 408.8      14,000   59,  000
Kabler (1962) discussed the slow acceptance
of the fecal streptococci as indicators of
pollution resulting from:

1  Multiplicity and difficulty of laboratory
   procedures

2  Poor agreement between methods of
   quantitative enumeration

3  Lack of systematic studies of ....

   a  sources

   b  survival, and

   c  interpretations, and

4  Undue attention to the S. faecalis group.
  27-16

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                                             Bacteriological Indicators of Water Pollution
   Increased attention to the fecal streptococci,
   especially during the last decade, have
   clarified many of the earlier cloudy issues
   and have elevated the stature of these
   organisms as indicators of pollution.
   Court precedents establishing legal status
   and recommendations of various technical
   advisory boards have placed the fecal
   coliform group in a position of primacy
   in many water quality applications.  The
   fecal streptococci have evolved from a
   position of a theoretically useful indicator
   to one which was ancillary to the coliforms
   to one which was useful when discrepancies
   or questions evolved as to the validity of
   the  coliform data to one where an equality
   status was achieved in certain applications.
   In the future it is anticipated that, for
   certain applications, the fecal streptococci
   will achieve a position of primacy for
   useful data,  and, as indicated by Litsky
   (1955) "be taken out of the realm of step-
   children and given their legitimate place
   in the field of santiary bacteriology as
   indicators of sewage pollution. "

B   Advantages and Limitations

   1  Survival

      In general, the fecal streptococci have
      been observed to have a more limited
      survival time in the aquatic environment
      when compared to the coliform group.
      They are rivaled in this respect only
      by the fecal coliforms.  Except for cases
      of persistence in waters of high electro-
      lytic content, as may be common to
      irrigation waters, the fecal streptococci
      have not been observed to multiply in
     polluted waters as may sometimes be
      observed for some of the coliforms.
      Fecal streptococci usually require a
      greater abundance of nutrients for sur-
     vival as compared to the  coliforms and
     the coliforms are more dependent upon
     the oxygen tension in the  waterbody.
      In a number of situations it was concluded
     that the fecal streptococci reached an
      extinction point more rapidly in warmer
     waters while the reverse was  true in the
      colder situations as the coliforms now
     were totally eliminated sooner.
2  Resistance to Disinfection

   In artificial pools the source of
   contamination by the bathers is
   usually limited to throat and skin
   flora and thus increasing attention
   has been paid to indicators other
   than those traditionally from the
   enteric tract.   Thus,  one of the
   organisms  considered to be a fecal
   streptococci, namely, S. salivarius,
   can be a more reliable indicator
   when detected along with the  other
   fecal streptococci especially since
   studies have confirmed the greater
   resistance  of the fecal streptococci
   to chlorination.  This greater
   resistance  to chlorination, when
   compared to the fecal coliforms, is
   important since the dieoff curve
   differences are insignificant  when
   the  curves  of the fecal coliforms
   are compared to various Gram
   negative pathogenic bacteria  which
   reduces their effectiveness as
   indicators.

3  Ubiquitous  Strains

   Among the  fecal streptococcus are
   two organisms, one a biotype and
   the  other a variety of the S. faecalis,
   which, being ubiquitous (omnipresent)
   have limited sanitary significance.
   The biotype, or atypical,  S.  faecalis
   is characterized by its ability to
   hydrolyze starch while the varietal
   form, liquefaciens, is nonbeta
   haemolytic  and  capable  of liquefying
   gelatin. Quantitation of these organisms
   in anomalous conditions is due to their
   capability of survival in soil  or high
   electrolytic waters and in waters with
   a temperature of less than 12 Degrees C.

   Samples have been encountered which
   have been devoid of fecal coliforms
   and yet contain  a substantial number of
   "fecal streptococci" of which these
   ubiquitous strains  constitute the majority
   or all of the isolations when analyzed
   biochemically.
                                                                                    27-17

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Bacteriological Indicators of Water Pollution
  V STANDARDS AND CRITERIA

  Acceptance and utilization of Total Coliform
  criteria,  which must now be considered a
  pioneering effort, has largely been supplanted
  in concept and in fact by the fecal coliforms
  in establishing standards for recreational
  waters.

  .The first significant approach to the utiliza-
  tion of the fecal streptococci as a criterium
  for recreational water standards occurred in
  1966 when a technical committee recommended
  the utilization of the fecal streptococci with the
  total coliforms as criteria for standards
  pertaining to the  Calumet River and lower
  Lake Michigan waters.   Several sets of
  criteria were established to fit the intended
  uses for this area.  The use of the fecal
  streptococci as a criterium is  indicated to
  be tentative pending the accumulation of
  existing densities and could be modified in
  future standards.

  With the existing state-of-the-art knowledge
  of the presence of the fecal streptococci in
  waters containing low numbers of fecal
  coliforms it is difficult to establish a specific
  fecal streptococcus density limit of below
  100 organisms/100 ml when used alone or
  in con junction .with the total coliforms.
 27-18

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                                                Bacteriological Indicators of Water Pollution
                     Part 4.  Other Bacterial Indicators of Pollution
I  TOTAL BACTERIAL COUNTS

A  Historical

   1  The early studies of Robert Koch led
      him to develop tentative standards of
      water quality based on a limitation of
      not more than 100 bacterial colonies
      per ml on a gelatin plating medium
      incubated 3 days at 2QQ C.

   2  Later developments led to inoculation
      of samples on duplicate plating media,
      with one set incubated at 37° C and the
      other at 20° C.

      a  Results were used to develop a ratio
         between the 37° C counts and the
         20°C counts.
      b  Waters having a predominant
         count at 37° C were regarded as
         being of probable sanitary  signifi-
         cance, while those giving
         predominant counts at 20°  C were
         considered to be of probable soil
         origin, or natural inhabitants  of
         the water being examined.


B  Groups Tested

   There is no such thing as "total" bacterial
   count in terms of a laboratory determination.

   1  Direct microscopic counts  do not
      differentiate between living and dead
      cells.

   2  Plate counting methods enumerate only
      the bacteria which are capable of using
      the culture medium provided,  under the
      temperature and other growth conditions
      used as a standard procedure.  No one
      culture medium and set of growth
      conditions can provide,  simultaneously,
      an acceptable environment  for all the
      heterogeneous, often conflicting,
      requirements of the total range of
      bacteria which may be recovered from
      waters.
C  Utilization of Total Counts

   1  Total bacterial counts, using plating
      methods, are useful for;

      a  Detection of changes in the bacterial
        composition of a water source

      b  Process control procedures in
        treatment plant operations

      c  Determination of sanitary conditions
        in plant equipment or distributional
        systems

   2  Serious limitations in total bacterial
      counts exist because:
      a No information is given  regarding
         possible or probable fecal origin
         of bacterial changes.  Large numbers
         of bacteria can sometimes be
         cultivated from waters known to be
         free of fecal pollution.

      b  No information of any kind is given
         about the species of bacteria
         cultivated.

      c  There is no differentiation between
         harmless or potentially dangerous
         forms.

    3  Status of total counts

      a  There is no total bacterial count
         standard for any of the following:

            Interstate Quarantine  Drinking
              Water Standards

             PHS regulations for  water
              potability (as shown in
              "Standard Methods" Public
              Health Service Drinking
              Water Standards of  1962.)

      b  The most widely used current
         application of total bacterial counts
         in water bacteriology today is in
                                                                                     27-19

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Bacteriological Indicators of Water Pollution
          water treatment plants, where some
          workers use standard plate counts
          for process control and for deter-
          mination of the bacterial quality of
          distribution systems and equipment.

       c  Total bacterial counts are not used
          in PHS water quality studies, though
          extensively used until the 1940's.

  B  Spore-Forming Bacteria (Clostridium
     perfringens, or C. welchii)

     1  Distribution

       This is one of the most widely distributed
       species of bacteria.  It is regularly
       present in the intestinal tract of warm-
       blooded animals.

     2  Nature of organism

       C. perfringens is a Gram-positive,
       spore-forming rod. The spores cause
       a distinct swelling of the cell when
       formed.  The organism is extremely
       active in fermentation of carbohydrates,
       and produces the well-known "stormy
       fermentation" of milk.

     3  Status

       The organism, when present,  indicates
       that pollution has occurred at some
       time.  However, because of the ex-
       tremely extended viability of the spores,
       it is impossible to  obtain even an
       approximation of the recency of pollution
       based only on the presence of
       C!. perfringens.

       The presence of the organism does not
       necessarily indicate an unsafe water.

  C  Tests for Pathogenic Bacteria of Intestinal
     Origin

     1  Groups considered include Salmonella
       sp, Shigella sp, Vibrio comma,
       Mycobacterium sp, Pasteurella sp,
       Leptospira sp, and others.
   2  Merits of direct tests:

      Demonstration of any pathogenic
      species would demonstrate an
      unsatisfactory water quality, hazardous
      to persons consuming or coming into
      contact with that water.

   3  Limitations

      a  There is no available routine pro-
         cedure for detection of the full
         range of pathogenic bacteria cited
         above.

      b  Quantitative methods are not avail-
         able for routine application  to any
         of the above.

      c  The intermittent release of  these
         pathogens makes it impossible to
         regard water as safe, even  in the
         absence of pathogens.

      d  After  detection, the public already
         would have been exposed to the
         organism; thus, there is no  built-in
         margin of safety,  as exists with
         tests for the coliform group.


   4  Applications

      a  In tracing the source of pathogenic
         bacteria in epidemiological investi-
         gations

      b  In special research projects

      c  In water quality studies concerned
         with enforcement actions against
         pollution, increasing attention is
         being given to the demonstration of
         enteric pathogenic bacteria in the
         presence of the bacterial indicators
         of pollution.

D  Miscellaneous Indicators

   It is beyond this  discussion to explore the
   total range of microbiological indicators
   of pollution that have been proposed and
   27-20

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                                              Bacteriological Indicators of Water Pollution
   investigated to some extent. Mention can
   be made, however,  of consideration of
   tests for the following.

   1 Bacteriophages specific for any of a
     number of kinds of bacteria

   2 Serological procedures for detection
     of coliforms and  other indicators; a
     certain amount of recent attention has
     been given to applications of fluorescent
     antibodies in such tests

   3 Tests for Pseudomonas aeruginosa

   4 Tests for viruses,  which may persist
     in waters even longer than members
     of the coliform group.

REFERENCES

1  Standard Methods for the Examination of
     Water and Wastewater,  13th ed.,
     APHA,  AWWA, WPCF.  Published by
     American Public Health Association,
      1790 Broadway, New York,  N.Y.  1971.

2  Prescott,  S.C., Winslow,  C.E.A., and
     McCrady,  M.  Water Bacteriology.
     John Wiley & Sons, Inc.  1946.

3  Parr, L.W.  Coliform Intermediates in
     Human Feces.   Jour. Bact. 36:1.
      1938.

4  Clark,  H. F. and Kabler, P.W.  The
     Physiology of the Coliform Group.
     Proceedings of the Rudolfs Research
     Conference on Principles and Appli-
     cations in Aquatic Microbiology.  1963.

5  Geldreich,  E.E., Bordner, R.H., Huff,
     C.B., Clark, H.F. ,  and Kabler,  P.W.
     Type Distribution of Coliform Bacteria
     in the Feces of Warm-Blooded Animals.
     JWPCF.   34:295-301.   1962.

6  Geldreich et al.   The Fecal Coli-Aerogenes
     Flora of Soils from Various Geographic
     Areas.   Journal of Applied Bacteriology
     25:87-93.   1962.
 7  Geldreich, E.E., Kenner, B.A., and
      Kabler, P.W.   Occurrence of
      Coliforms, Fecal Coliforms,  and
      Streptococci on Vegetation and Insects.
      Applied Microbiology.  12:63-69. 1964.

 8  Kabler,  P.W.,  Clark, H.F., and
      Geldreich, E.E.  Sanitary Significance
      of Coliform and Fecal Coliform
      Organisms in Surface Water.   Public
      Health Reports.  79:58-60.   1964.

 9  Clark, H.F. and Kabler, P.W.
      Re-evaluation of the Significance of the
      Coliform  Bacteria.   Journal AWWA.
      56:931-936.   1964.

10  Kenner,  B. S.,  Clark, H.F., and
      Kabler, P.W.   Fecal Streptococci.
      EL.  Quantification in Feces.  Am. J.
      Public Health.   50:1553-59.   1960.

11  Litsky,  W.,  Mailman, W.L., and Fifield,
      C.W.   Comparison of MPN of
      Escherichia  coli and Enterococci in
      River Water. Am. Jour.  Public Health.
      45:1949.  1955.

12  Medrek, T. F.  and Litsky, W.
      Comparative Incidence of Coliform
      Bacteria and Enterococci in
      Undisturbed  Soil.   Applied Micro-
      biology.   8:60-63.  1960.

13  Mailman, W.L., and Litsky, W.
      Survival of Selected Enteric Organisms
      in Various Types of Soil.  Am. J.
      Public Health.  41:38-44.  1950.

14  Mailman, W. L., and Seligman,  E.B.,  Jr.
      A Comparative Study  of Media for
      Detection of  Streptococci  in Water and
      Sewage.   Am. J. Public Health.
      40:286-89.   1950.

15  Ministry of Health (London).   The
      Bacterial Examination of  Water Supplies.
      Reports on Public Health and Medical
      Subjects.  71:34.
                                                                                    27-21

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Bacteriological Indicators of Water Pollution
16  Morris. W. and Weaver, R.H.
       Streptococci as Indices of Pollution
       in Well Water.   Applied Microbiology,
       2:282-285.  1954.

17  Mundt, J.O., Goggin,  J.H., Jr.,  and
       Johnson, L. F.   Growth of
       Streptococcus fecalis var. liquefaciens
       on Plants.  AppliedMicrobiology.
       10:552-555.  1962.

18  Geldreich, E.E.  Sanitary Significance
       of Fecal Coliforms  in the Environment.
       U. S.  Department of the Interior.
       FWPCA Publ. WP-20-3.   1966.

19  Geldreich, E.E. and Kenner,  B.A.
       Concepts of Fecal Streptococci  in
       Stream Pollution.  J. WPCF. 41:R336.
       1969.

20  Kabler, P.W.  Purification and Sanitary
       Control of Water (Potable and Waste)
       Ann. Rev.  of Microbiol.  16:127.  1962.

21  Litsky, W., Mailman, W. L., and Fifield,
       C. W.   Comparison  of the Most  Probable
       Numbers of Escherichia coli and
       Enterococci in River Waters.  A. J, P. H.
       45:1049.   1955.
22  Geldreich, E.E.  Applying Bacteriological
       Parameters to Recreational Water
       Quality.  J. AWWA.  62:113.  1970.

23  Geldreich, E.E., Best,  L. C., Kenner,  B.A.
       and Van Donsel, D. J.  The Bacteriolog-
       ical Aspects of Stormwater Pollution.
       J. WPCF.  40:1860.  1968.

24  FWPCA  Report of Water Quality Criteria
       Calumet Area - Lower Lake Michigan,
       Chicago, IL.  Jan.  1966.
This outline was prepared by H. L. Jeter,
Director, National Training Center and
revised by R.  Russomanno,  Microbiologist.
National Training Center, WPO,  EPA,
Cincinnati, OH 45268.
Descriptors;   Coliforms, Escherichia coli
Fecal Goliforms,  Fecal Streptococci,  Indicator
Bacteria,  Microbiology, Sewage Bacteria,
Water Pollution
 27-22

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                      EXAMINATION OF WATER FOR COLIFORM AND
                             FECAL STREPTOCOCCUS GROUPS
                          (Multiple Dilution Tube [MPN]  Methods)
I  INTRODUCTION

The subject matter of this outline is contained
in three parts, as follows:

A  Part 1

   1  Fundamental aspects of multiple dilution
      tube ("most probable numbers") tests,
      both from a qualitative and a  quantitative
      viewpoint.

   2 • Laboratory bench records.

   3  Useful techniques in multiple dilution
      tube methods.

   4  Standard supplies, equipment, and
      media in multiple dilution tube tests.
B  Part 2

   Detailed, day-by-day,  procedures in tests
   for the coliform group and subgroups
   within the coliform group.

C  Part 3

   Detailed, day-by-day,  procedures in tests
   for members of the fecal streptococci.

D  Application of Tests to Routine Examinations

   The following considerations (Table 1) apply
   to the selection of the Presumptive Test,
   the Confirmed Test,  and the Completed
   Test.  Termination of testing at the
   Presumptive Test level is not practiced
   by laboratories of this agency.  It must
   be realized that the Presumptive Test alone
   has limited use when water quality is to
   be determined.
                                           TABLE 1
                              Examination Terminated at -
Type of Receiving
Water
Sewage Receiving
Treatment Plant - Raw
Chlorinated
Bathing
Drinking

Other Information
Presumptive
Test
Applicable
Applicable
Not Done
Not Done
Not Done


Confirmed Test
Applicable
Applicable
Applicable
Applicable
Applicable

Applicable in all
cases where Pre-
sumptive Test alone
is unreliable.
Completed Test
Important where results
are to be used for control
of raw or finished water .
Application to a statis-
tically valid number of
samples from the
Confirmed Test to estab-
lish its validity in
determining the sanitary
quality.
NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
        Environmental Protection Agency.
 W. BA. 3L. 2.75
                                 28-1

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 MPN Methods
H   BASIS OF MULTIPLE TUBE TESTS

 A  Qualitative Aspects

    1  For purely qualitative aspects of testing
      for indicator organisms, it is convenient
      to consider the tests applied to one
      sample portion, inoculated into a tube
      of culture medium,  and the follow-up
      examinations and tests on results of the
      original inoculation.  Results of testing
      procedures are definite: positive
      (presence of the organism-group is
      demonstrated) or negative (presence of
      the organism-group is not demonstrated.)

    2  Test procedures are based on certain
      fundamental assumptions:

      a  First,  even if only one living cell of
         the test organism is present in the
         sample, it will be able to grow when
         introduced into the primary inoculation
         medium;

      b  Second, growth of the test organism
         in  the culture medium will produce
         a result which indicates presence of
         the test organism; and,

      c  Third,  extraneous organisms will
         not grow, or if they do grow, they
         will not limit growth of the test
         organism; nor will they produce
         growth effects that will be confused
         with those of the bacterial group for
         which the test is  designed.

    3  Meeting these assumptions usually
      makes it necessary to conduct the tests
      in a series of stages (for example, the
      Presumptive,  Confirmed, and Completed
      Test stages, respectively,  of standard
      tests  for the coliform group).

    4  Features of a full, multi-stage test

      a  First stage:  The culture medium
         usually serves primarily as an
         enrichment medium for the group
         tested.  A good first-stage growth
         medium should support growth of all
         the living cells  of the group tested,
         and it should include provision for
         indicating the presence of the test
   organism being studied.  A first-
   stage medium may include some
   component which inhibits growth
   of extraneous bacteria, but this
   feature never should be included
   if it also inhibits growth of any
   cells of the group for which the
   test is designed.  The Presumptive
   Test for the coliform group is a
   good example.  The medium
   supports growth, presumably, of
   all living cells of the coliform
   group; the culture container has a
   fermentation vial for demonstration
   of gas production resulting from
   lactose fermentation by coliform
   bacteria, if present; and sodium
   lauryl sulfate may be included in
   one of the approved media for
   suppression of growth of certain
   noncoliform bacteria.  This
   additive apparently has no adverse
   effect on growth of members  of the
   coliform group in the concentration
   used.  If the result of the first-stage
   test is negative,  the  study of  the
   culture is terminated, and the result
   is recorded as a negative test.  No
   further study is made of negative
   tests. If the result of the  first-
   stage test is positive, the  culture
   may be subjected to further study
   to verify the findings of the first
   stage.

b  Second stage:  A transfer  is made
   from positive cultures  of the  first-
   stage test to a second culture medium.
   This test stage emphasizes provision
   to reduce confusion of results due to
   growth effects of extraneous  bacteria,
   commonly achieved by addition of
   selective inhibitory agents.   (The
   Confirmed Test for coliforms meets
   these requirements. Lactose and
   fermentation vials are  provided for
   demonstration of coliforms in the
   medium.  Brilliant green  dye and
   bile salts are included as  inhibitory
   agents which tend to suppress growth
   of practically all kinds of noncoliform
   bacteria, but do not  suppress growth
   of coliform bacteria when used as
   directed).
   28-2

-------
                                                                               MFN Methods
       If result of the second- stange test is
       negative, the study of the culture is
       terminated,  and the result is recorded
       as a negative test. A negative test here
       means that the positive results of the
       first-stage test were "false positive, "
       due to one or more kinds of extraneous
       bacteria.  A positive second-stage test
       is partial confirmation of the positive
       results obtained in the first-stage test;
       the culture may be subjected to final
       identification through application of still
       further testing procedures.  In routine
       practice, most  sample examinations
       are terminated  at the end of the second
       stage, on the assumption that the result
       would be positive  if carried to the third,
       and final stage.  This practice  should be
       followed only if adequate testing is done
       to demonstrate  that the assumption is
       valid.  Some workers recommend contin-
       uing at least 5% of all sample examina-
       tions to the third stage to demonstrate
       the reliability of the second-stage results.
B  Quantitative Aspects of Tests

   1  These methods for determining bacterial
      numbers are based on the assumption
      that the  bacteria can be separated from
      one another (by shaking or other means)
      resulting in a suspension of individual
      bacterial cells, uniformly distributed
      through  the original sample when the
      primary inoculation is made.

   2  Multiple dilution tube tests for quantita-
      tive determinations apply a Most Probable
      Number (MPN)  technique.  In this  pro-
      cedure one or more measured portions
      of each of a stipulated series of de-
      creasing sample volumes is inoculated
      into the  first-stage culture medium.
      Through decreasing the sample incre-
      ments, eventually a volume is reached
      where only one  cell is introduced into
   some tubes, and no cells are introduced
   into other tubes.  Each of the several
   tubes of sample-inoculated first-stage
   medium is tested independently,
   according to the principles previously
   described, in the qualitative aspects
   of testing procedures.

3  The combination of positive and
   negative results is used in an application
   of probability mathematics to secure
   a single MPN value for the sample.

4  To obtain MPN values,  the following
   conditions must be met:

   a  The testing procedure must result
      in one or more tubes in which the
      test organism ^s demonstrated to
      be present; and

   b  The testing procedure must result
      in one or more tubes in which the
      test organism is not  demonstrated
      to be present.

5  The MPN value for a given sample  is
   obtained through the use of MPN Tables.
   It is emphasized that the precision  of
   an individual MPN value is not  great
   when compared with most physical  or
   chemical determinations.

6  Standard practice in water pollution
   surveys conducted by this organization,
   is to plant five tubes in  each of a series
   of sample  increments,  in sample
   volumes decreasing at decimal intervals.
   For example,  in testing known  polluted
   waters,  the initial sample inoculations
   might consist of 5 tubes each in volumes
   of 0.1,  0.01,0.001, and 0.0001ml,
   respectively.  This series of sample
   volumes will yield determinate results
   from a low of 200 to a high of 1, 600, 000
   organisms per 100 ml.
                                                                                      28-3

-------
   MPN Methods
IH  LABORATORY BENCH RECORDS

 A Features of a Good Bench Record Sheet

    1  Provides complete identification of the
       sample.

    2  Provides for full, day-by-day informa-
       tion about all tests performed on the
       sample.

    3  Provides easy step-by-step record
       applicable to any portion of the sample.

    4  Provides for recording of the quantitative
       result which will be transcribed to sub-
       sequent reports.

    5  Minimizes the amount of writing by the
       analyst.

    6  Identifies the analyst(s).
     There is no such thing as "standard"
     bench sheet for multiple tube tests; there
     are many versions of bench sheets. Some
     are prescribed by administrative authority
     (such as the Office of a State Sanitary
     Engineer); others are devised by laboratory
     or project personnel to meet specific needs.
     It is not the purpose of this discussion to
     recommend an "ideal" bench form; however,
     the form used in this training course
     manual is essentially similar to that used
     in  certain research laboratories of this
     organization.  The student enrolled in the
     course for which this manual is written
     should make himself thoroughly familiar
     with the bench sheet and its proper use.
     See Figure 1.
IV  NOTES ABOUT WORKING PROCEDURES
    IN THE LABORATORY

 A Each bacteriological examination of water
    by multiple dilution tube methods requires
    a considerable amount of manipulation;
    much is quite repetitious.  Laboratory
    workers must develop and maintain good
    routine working habits, with constant
    alertness to guard against lapses into
    careless, slip-shod laboratory procedures
    and "short cuts" which only can lead to
    lowered quality of laboratory work.


    The student reader is urged to review  the
    form for laboratory surveys (PHS-875,
    Rev. 1966) used by Public Health Service
    personnel charged with responsibility for
    accreditation of laboratories for examination
    of water under Interstate Quarantine
    regulations.

 B Specific attention is brought to the following
    by no means exhaustive,  critical aspects of
    laboratory procedures in multiple  dilution
    tube tests:

    1  Original sample

       a Follow  prescribed care and handling
         procedures before  testing.

       b Maintain absolute identification of
         sample at all stages in testing.

       c Vigorously shake samples (and
         sample dilutions) before planting
         in culture media.

    2  Sample measurement  into primary
       culture medium

       a Sample portions must be measured
         accurately into the culture medium
         for reliable quantitative tests to be
         made.  Standard Methods prescribes
         that calibration errors should not
         exceed + 2. 5%.
     28-4

-------
                Project
                Sample Station
Collection Data

Date £./(f/G, 7   Time
TempeVature___^°C
Other Observations
                                                                  Analytical Record
By
Bench Number of Sample	_,
Analyst ^&^g^
Test started at
  ^Coliform MPN/100 ml
       Confirmed:
       Completed:
  Fecal Coliform MPN:
                               Figure 1.  SAMPLE BENCH SHEET
                                     Fecal Streptococcus MPN/100^ml
                                              A-D - EVA:
                                                                                               28-5

-------
    Methods
      Suggested sample measuring practices
      are as follows: Mohr measuring
      .pipets are recommended.  10 ml
      samples are delivered at the top of
      the culture tube, using 10 ml pipets.
      1. 0 ml samples are delivered down
      into the culture tube,  near the sur-
      face of the medium, and "touched
      off" at the  side of the tube when the
      desired amount of sample has been
      delivered.  1. 0 ml or 2. 0 ml pipets
      are used for measurement of this
      volume.  0.1 ml samples are
      delivered in the same manner as 1. 0
      ml samples,  using, great care that
      the sample actually gets into the
      culture medium.   Only 1. 0 ml pipets
      are used for this sample volume.
      After delivery of all sample incre-
      ments into the culture tubes, the
      entire rack of culture tubes may be
      shaken gently  to carry down any of
      the sample adhering to the wall of
      the tube above the  medium.
      Workers should demonstrate by actual
      tests that the pipets and the technique
      in use actually delivers the rated volumes
      within the prescribed limits of error.
      Volumes as small as  0.1 ml routinely
      can be delivered directly from the
      sample with suitable pipets.  Lesser
      sample volumes first should be diluted,
      with subsequent delivery of suitable
      volumes of diluted sample into the
      culture medium.  A diagrammatic
      scheme for making dilutions is shown
      in Figure 2.
 b  Gas in any quantity is a positive test.
    It is necessary to work in conditions
    of suitable lighting for easy recog-
    nition of the extremely small amounts
    of gas inside the tops of some
    fermentation vials.

 Reading of liquid  culture tubes for
 growth as  indication of a positive test
 requires good lighting.  Growth  is
 shown by any amount of increased
 turbidity or opalescence in the culture
 medium, with or  without deposit of
 sediment at the bottom of the tube.

 Transfer of cultures with inoculation
 loops and needles

 a  Always sterilize inoculation loops
    and needles in  flame immediately
    before transfer of culture; do not
    lay it down or touch it to any non-
    sterile object before making the
    transfer.

 b  After sterilization, allow sufficient
    time for cooling,  in the air, to avoid
    heat-killing bacterial cells on the
    hot wire.

 c  Loops should be 3 mm in inside
    diameter, with a capability of holding
    a drop of water or culture.

    For routine standard transfers
    requiring transfer of 3 loopsful of
    culture, many workers form three
    3-mm loops on the same length of
   wire.
   Reading of culture tubes for gas
   production

   a  On removal from the incubator.
      shake culture rack gently,  to
      encourage release of gas which
      may be supersaturated in the culture
      medium.
As an alternative to use of standard
inoculation loops, the use of
"applicator sticks"  have now been
sanctioned by the 13th Edition of
Standard Methods.
28-6

-------
                                                                             MPN Methods
                         Figure 2.   PREPARATION OF DILUTIONS
            Dilution Ratios:
Delivery volume
                                                                             1:10000
                                                             1ml
                  Tubes «.
                                          Petri Dishes or Culture Tubes
Actual volume
of sample in tube
1ml     O.lml  0.01 ml 0.001ml
                                                0. 0001 ml  o. 00001 ml
      The applicator sticks are dry heat
      sterilized (autoclave sterilization is
      not acceptable because of possible
      release of phenols if the wood is
      steamed) and are used on a single-
      service basis.  Thus, for every culture
      tube transferred, a new applicator
      stick is used.

      This use of applicator sticks is
      particularly attractive in field
      situations where it is inconvenient or
      impossible to provide a gas burner
      suitable for  sterilization of the
      inoculation loop.  In addition, use of
      applicator sticks is favored in
      laboratories where room temperatures
      are significantly elevated by use of
      gas burners.
                         7  Streaking cultures on agar surfaces

                            a  All streak-inoculations should be
                               made without breaking the surface
                               of ifhe agar.   Learn to use a light
                               touch with the needle; however,
                               many inoculation needles are so
                               sharp that they are virtually useless
                               in this respect.   When the needle is
                               platinum or platinum- iridium wire,
                               it sometimes is beneficial to fuse
                               the working tip into a small sphere.
                               This  can be done by momentary
                               insertion of a well-insulated (against
                               electricity) wire into a carbon arc,
                              or some other extremely hot environ-
                              ment.  The sphere should not be more
                              than twice the diameter of the wire
                              from which it is formed, otherwise
                              it will be entirely too heat-retentive
                              to be useful.
                                                                                    28-7

-------
MPN Methods
        When the needle is nichrome
        resistance wire, it cannot be heat-
        fused; the writer prefers to bend
        the terminal 1/16 -  1/8" of the wire
        at a slight angle to the overall axis
        of the needle.  The side of the
        terminal bent portion of the needle
        then is used for inoculation of agar
        surfaces.

     b  When streaking for colony isolation,
        avoid using too much inoculum. The
        streaking pattern is somewhat
        variable according to individual
        preference.  The procedure favored
        by the writer is shown in the
        accompanying figures.  Note
        particularly that when going from
        any one stage of the streaking to the
        next, the inoculation needle is heat-
        sterilized.

      Preparation of cultures for Gram
      stain

      a  The  Gram stain always should be
        made from a culture grown on a
        nutrient  agar surface (nutrient agar
        slants are used here) or from nutrient
        broth.
         The culture should be young, and
         should be actively growing.  Many
         workers doubt the validity of the
         Gram stain made on a culture more
         than 24 hours old.
         Prepare a thin smear for the staining
         procedure.  Most beginning workers
         tend to use too much bacterial sus-
         pension in preparing the dried smear
         for staining.   The amount of bacteria
         should be so small that the dried film
         is barely visible to the naked eye.
V   EQUIPMENT AND SUPPLIES

 Consolidated lists of equipment,  supplies,
 and culture media required for all multiple
 dilution tube tests described in this outline
 are shown in Table 2.;.  Quantitative infor-
 mation is not presented; this is variable-
 according to the extent of the testing pro-
 cedure, the number of dilutions used, and
 the number of replicate tubes per dilution.
 It is noted that requirements for alternate
 procedures are fully listed and choices are
 made in accordance to laboratory preference.
  28-8

-------
                                                         MPN Methods
                      a Flame-sterilize an inoculation needle and air-cool.

                      b Dip the tip of the inoculation needle into the bac-
                        terial culture being studied.

                      c Streak the inoculation needle tip lightly back and
                        forth over half the agar surface,  as in (1),  avoid-
                        ing scratching or breaking the  agar surface.

                      d Flame-sterilize the inoculation needle and air-cool.
                      a  Turn the Petri dish one-quarter-turn and streak the
                        inoculation needle tip lightly back and forth over one-
                        half the agar surface,  working from area (1) into one-
                        half the unstreaked area of the agar.
                      b  Flame-sterilize the inoculation needle and air-cool.
                     a  Turn the Petri dish one-quarter-turn and streak the
                        inoculation needle tip lightly back and forth over one-
                        half the agar  surface, working from area (2) into
                        area (3), the remaining unstreaked area.
                     b  Flame-sterilize the inoculation needle and set it aside.

                     c  Close the culture container and incubate as prescribed.
   Figure 3.  A SUGGESTED PROCEDURE FOR COLONY ISOLATION BY A
                       STREAK-PLATE TECHNIQUE
AREA 1 (Heavy inoculum)
AREA  3  (Isolated colonies]
                                                AREA  2
                                               (Moderate  growth)
                              APPEARANCE OF  STREAK -  PLATE
                               AFTER  INCUBATION  INTERVAL
                                                                        28-9

-------
MPN Methods
         TABLE 2. APPARATUS AND SUPPLIES FOR STANDARD
                      FERMENTATION TUBE TESTS
Description of Item
Lauryl tryptose broth or Lactose
broth. 20 ml amounts of 1. 5 X
concentration medium, in 25 X 150 mm
culture tubes with inverted fermen-
tation vials, suitable caps.
Lauryl tryptose broth or Lactose
broth. 10 ml amounts of single
strength medium in 20 X 150 mm
culture tubes with inverted fermen-
tation vials, suitable caps.
Brilliant green lactose bile broth, 2%
in 10 ml amounts, single strength,
in 20 X 150 mm culture tubes with
inverted fermentation vials,
suitable caps.
Eosin methylene blue agar, poured
in 100 X 15 mm Petri dishes
Endo Agar. poured in 100 X 15 mm
dishes
Nutrient agar slant, screw cap tube
Boric actd lactose broth, 10 ml
amounts of single strength medium
in fermentation tubes.
EC Broth, 10 ml amounts of single
strength medium in fermentation
tubes:
Formate ricinoleate broth
(provisional)
Culture tube racks, 10 X 5 openings;
each opening to accept 25 mm dia-
meter tubes.
Pipettes, 10 ml, Mohr type, sterile,
in suitable cans.
Pipettes, 2 ml (optional), Morh type,
sterile, in suitable cans
Pipettes, 1 ml, Mohr type, sterile
in metal suitable cans
Standard buffered dilution water,
sterile, 99-ml amounts in screw-
capped bottles.
Gas burner, Bunsen type
Inoculation loop, loop 3mm dia-
meter, of nichrome or platinum-
iridium wire, 26 B & S gauge, in
suitable holder, (or sterile applicator.
stick)
Inoculation needle, nichrome, or
platinum -iridium wire, 26 B & S
gauge, in suitable holder.
Incubator, adjusted to 35 + 0. 50 C
Waterbath incubator, adjusted to
43+0.2°C
Waterbath incubator, adjusted to
44.5+ 0.2°C.
Glass microscopic slides, . 1 " X 3 "
Slide racks (optional)
Gram-stain solutions, complete set
Compound microscope, oil immer-
sion lens. Abbe' condenser
Basket for discarded cultures
Container for discarded pipettes
Total Coliform Group
Presumptive
Test
X
X





X
X
X
X
X



X






X
X
Confirmed
Test


X
X
X


X




X
X
X
X






X

Completed
Test

X
X
X
X

X
X




X
X
X
X


X
X
X
X
X

Fecal Coliforms
(BALE)




X


X




X
X


X





X

(EC broth)





X

X




X
X



X




X

28-10

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                                           Part 2

                 DETAILED TESTING PROCEDURES FOR MEMBERS OF THE
                COLIFORM GROUP BY MULTIPLE DILUTION TUBE  METHODS
I  SCOPE

A  Tests Described

   1  Presumptive Test

   2  Confirmed Test

   3  Completed Test

   4  Fecal Coliform Test

B  Form of Presentation

   The Presumptive, Confirmed,  and
   Completed Tests are presented as total,
   independent procedures.  It is  recognized
   that this form of presentation is somewhat
   repetitious,  inasmuch as the Presumptive
   Test is preliminary to the Confirmed
   Test, and both the Presumptive Test and
   the Confirmed Test  are preliminary to the
   Completed Test for  total coliforms.

   In using these procedures, the worker
   must know at the outset what is to be the
   stage at which the test  is to be ended,  and
   the details of the procedures throughout,
   in order to prevent the possibility of
   discarding gas-positive tubes before
   proper transfer procedures have been
   followed.

   Thus, if the worker knows that the test will
   be  ended at the Confirmed Test, he will
   turn at once to Section HI, TESTING TO
   THE CONFIRMED TEST STAGE, and will
   ignore Sections II and IV.

   The Fecal Coliform Test is described
   separately,  in Section V, as an
   adjunct to the Confirmed Test and to the
   Completed Test.
H   TESTING TO PRESUMPTIVE TEST
    STAGE

 A  First-Day Procedures

    1  Prepare a laboratory data sheet for
      the sample.  Record the following
      information:  assigned laboratory
      number,  source of sample, date and
      time of collection, temperature of the
      source, name of sample collector,
      date and time of receipt of sample in
      the laboratory.  Also show the  date
      and time of starting tests in the
      laboratory, name(s)  of worker(s) per-
      forming the laboratory tests,  and the
      sample volumes planted.

    2  Label the tubes of lauryl tryptose broth
      required for .the initial planting of the
      sample (Table 3).  The label should
      bear three  identifying marks.  The
      upper number is the  identification of
      the worker(s) performing the test
      (applicable to personnel in training
      courses), the number immediately
      below is the assigned laboratory num-
      ber, corresponding with the laboratory
      record sheet. The lower number is the
      code to designate the sample volume
      and which tube of a replicate series is
      indicated.
 NOTE: Be sure to use tubes containing
 the correct concentrations of culture medium
 for the inoculum/tube volumes. (See the
 chapter on media and solutions for multiple
 dilution tube methods or refer to the current
 edition of  Standard Methods for Water and
 Wastewater).
                                                                                    28-11

-------
 MPN Methods
Tabla 3. SUGGESTED LABELING SCHEME FOR ORIGINAL CULTURES AND
        ' SUBCULTURES IN MULTIPLE DILUTION TUBE TESTS

Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Bench number
Volume & tube
Tube
1
312
A
312
a
312
a
312
la
312
2a
Tube
2
312
B
312
b
312
t±
312
Ib
312
2b
Tube
3
312
C
312
c
312
c
312
Ic
312
2c
Tube
4
312
D
312
d
312
d
312
Id
312
2d
Tube
5
312
E
312
e
312
e
312
le
312
2e
Sample volume
represented
Tubes with 10 ml
of sample
Tubes with 1 ml
of sample
Tubes with 0. 1 ml
of sample
Tubes with 0.01 ml
of sample
Tubes with 0.001 ml
of sample
                                                                     Typical Example

RB
312-


A ,
^x
                                                                                 Lab. Worker
                                                                               Side ntif ication
                                                                                -Bench Number

                                                                                "Sample Volume
                                                                  Tube of Culture Medium
  The labeling of cultures can be reduced by labeling  only the first tube of
  each series of identical sample volumes in the initial planting of the sample.
  All subcultures  from initial plantings should be labeled completely.
       Place the labeled culture tubes in an
       orderly arrangement in a culture tube
       rack, with the tubes intended for the
       largest sample volumes in  the front
       row, and those intended for smaller
       volumes in the succeeding  rows.
       Shake the sample vigorously, approxi-
       mately  25 times,  in an arc  of one foot
       within seven seconds and withdraw the
       sample  portion at once.

       Measure the predetermined sample
       volumes into the  labeled tubes of lauryl
       tryptose broth, using care to avoid
       introduction of any bacteria into the
       culture  medium except those in the
       sample.

       a  Use a 10 ml pipet for 10 ml sample
         portions,  and  1 ml pipets for portions
         of 1 ml or less.  Handle sterile pipets
         only  near the mouthpiece, and protect
         the delivery end from external con-
         tamination.  Do not remove the cotton
         plug  in the mouthpiece as this is
         intended to protect the user from
         ingesting any sample.
      b  When using the pipet to withdraw
         sample portions, do not dip the
         pipet more than 1/2 inch into the
         sample; otherwise sample running
         down the outside of the pipet will
         make measurements inaccurate.

   6  After measuring all portions of the
      sample into their respective tubes  of
      medium, gently shake the rack of
      inoculated tubes to insure good mixing
      of sample with the culture  medium.
      Avoid vigorous shaking,  as air bubbles
      may be shaken into the fermentation
      vials and thereby invalidate the test.

   7  Place the rack of inoculated tubes in the
      incubator at 35° + 0.50C for 24 +
      2 hours.

B  24-hour Procedures

   1  Remove the rack of lauryl tryptose
      broth cultures from the incubator,  and
      shake gently.  If gas is about to appear
      in the fermentation vials, the shaking
      will speed the process.
     28-12

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                                                                             MPN Methods
     2   Examine each tube carefully.  Record,
        in the column "24" under LST on the
        laboratory data sheet,  each tube showing
        gas in the fermentation vial as a positive
        (+) test  and each tube not showing gas
        as a negative (-) test.  GAS IN ANY      2
        QUANTITY IS A POSITIVE TEST.

     3   Discard all gas-positive tubes of lauryl
        tryptose broth,  and return all the gas-
        negative tubes to the 35°C incubator for
        an additional 24 i 2 hours.

 C   48-hour Procedures

     1   Remove the rack of culture tubes from
        the incubator, read and record gas
        production for each tube.

     2   Be sure to record all results under the
        48-hour LTB column on the data sheet.
        Discard all tubes.  The Presumptive
        Test is  concluded at this point, and
        Presumptive coliforms per  100 ml can
        be computed according to the methods
        described elsewhere in this manual.
Ill   TESTING TO CONFIRMED TEST STAGE

 Note that the description starts with the
 sample inoculation and includes the
 Presumptive Test stage.  The Confirmed
 Test preferred in Laboratories of this agency
 is accomplished by means of the brilliant
 green lactose bile broth (BGLB) and the
 acceptable alternate tests are mentioned in
 HI F.  In addition, the Fecal Coliform Test is
 included as an optional adjunct to the procedure.

 A   First-Day Procedures

     1   Prepare a laboratory data sheet for the
        sample.  Record the following infor-
        mation:  assigned laboratory number,
        source of sample, date and time of
        collection, temperature of the source,
        name  of sample collector, date and
        time of receipt of sample in the
        laboratory.  Also show the date and
time of starting tests in the laboratory.
name(s) of worker(s) performing the
laboratory tests,  and the sample
volumes planted.

Label the  tubes of lauryl tryptose broth
required for the initial planting of the
sample.   The label should bear three
identifying marks.  The upper number
is the identification of the worker(s)
performing the test (applicable to
personnel in training courses), the
number immediately below is the
assigned laboratory number, corres-
ponding with the laboratory record
sheet.  The lower number is the  code
to designate the sample volume and
which tube of a replicate series is indicated.
NOTE:  If 10-ml samples are being
planted,  it is necessary to use tubes
containing the correct concentration
of culture medium,-  This has previously
been noted in II  A-2.

Place the labeled culture tubes in an
orderly arrangement in a culture tube
rack, with the tubes intended for the
largest sample volumes in the front
row, and those intended for smaller
volumes in the succeeding rows.

Shake the sample vigorously, approxi-
mately 25 times,  in  an up-and-down
motion.

Measure the predetermined sample
volumes into the labeled tubes of lauryl
tryptose broth, using care to avoid
introduction of any bacteria into the
culture medium except those in the sample.

a  Use a 10-ml pipet for  10 ml sample
   portions,  and 1-ml pipets for portions
   of 1 ml or less.   Handle sterile pipets
   only near the mouthpiece, and protect
   the delivery end from external con-
   tamination. Do not remove the cotton
   plug in the mouthpiece as this is intended
   to protect the user from ingesting any
   sample.
                                                                                     28-13

-------
 MPN Methods
     b  When using the pipet to withdraw
        sample portions, do not dip the
        pipet more than 1/2 inch into the
        sample; otherwise sample running
        down the outside of the pipet will
        make measurements inaccurate.

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

     d  Prepare preliminary dilutions of
        samples for portions of 0. 01 ml or
        less before delivery into the culture
        medium.  See Table  1 for preparation
        of dilutions.  NOTE: Always deliver
        diluted sample portions into the
        culture medium as soon as possible
        after preparation.  The interval
        between preparation of dilution and
        introduction of sample into the
        medium never should be as  much
        as 30 minutes.

   6 After measuring all portions of the
     sample into their respective tubes of
     medium,  gently shake the rack of
     inoculated tubes to insure good mixing
     of sample with the culture medium.
     Avoid vigorous shaking, as air bubbles
     may be shaken into the fermentation
     vials and thereby invalidate the test.

   7 Place the rack of inoculated tubes in
     the incubator at 35° + 0. 5° C for 24 +
     2 hours.

B  24-hour Procedures

   1 Remove the rack of lauryl tryptose
     broth cultures from the incubator, and
     shake gently. If gas is about to appear
     in the fermentation vials, the shaking
     will speed the process.
2  Examine each tube carefully.  Record,
   in the column  "24" under LST on the
   laboratory data sheet,  each tube showing
   gas in the fermentation vial as a
   positive (+) test and each tube not
   showing gas as a negative (-) test.
   GAS IN ANY QUANTITY IS A POSITIVE
   TEST.

3  Retain all gas-positive tubes  of lauryl
   tryptose broth culture in their place
   in the rack,  and proceed.

4  Select the gas-positive tubes  of lauryl
   tryptose broth culture for Confirmed
   Test procedures.   Confirmed Test
   procedures may not be required for all
   gas-positive cultures.  If, after 24-hours
   of incubation,  all five replicate cultures
   are gas-positive for two or more con-
   secutive sample volumes,  then select
   the set of five  cultures representing
   the smallest volume of sample in which
   all tubes were gas-positive.  Apply
   Confirmed Test procedures to all these
   cultures and to any other gas-positive
   cultures representing smaller volumes
   of sample, in which some  tubes were
   gas-positive and some were gas-negative.

5  Label one tube of brilliant green lactose
   bile broth (BGLB) to correspond with
   each tube of lauryl tryptose broth
   selected for Confirmed Test procedures.

6  Gently shake the rack of Presumptive
   Test cultures. With a flame-sterilized
   inoculation loop transfer one  loopful of
   culture from each gas-positive tube to
   the corresponding tube of BGLB.  Place
   each newly inoculated culture into BGLB
   in the position of the original gas-positive
   tube.

7  After making the transfers, the rack
   should contain some 24-hour  gas-
   negative tubes of lauryl tryptose broth
   and the newly inoculated BGLB.

8  If the Fecal Coliform Test is  included
   in the testing procedures,  consult
   Section V of this part of the outline of
   testing procedures.
    28-14

-------
                                                                            MPN Methods
   9 Incubate the 24-hour gas-negative
     BGLB tubes and any newly-in.oculated
     tubes of BGLB an additional 24 ft- 2
     hours  at 35°+ 0.5°C.         "~

C  48-hour Procedures

   1 Remove the rack of culture tubes from
     the incubator,  read and record gas
     production for each tube.

   2 Some tubes will be lauryl tryptose broth
     and some will be brilliant green lactose
     bile broth (BGLB).   Be sure to record
     results from LTB under the 48-hour
     LTB column and the BGLB results under
     the 24-hour column of the data sheet.

   3 Label tubes of BGLB to-correspond with
     all (if any) 48-hour gas-positive cultures
     in lauryl tryptose broth.  Transfer one
     loopful of culture from each gas-positive
     LTB culture to the correspondingly-
     labeled tube of BGLB.  NOTE: All
     tubes  of LTB  culture which were
     negative at 24 hours and became
     positive at 48 hours are to be transferred.
     The option described above for 24-hour
     cultures does not apply at 48 hours.

   4 If the  Fecal Coliform Test is  included
     in the testing  procedure,  consult
     Section V of the part of the outline
     of testing procedures.

   5 Incubate the 24-hour gas-negative
      BGLB tubes and any newly-inoculated
     tubes  of BGLB 24 + 2 hours at 35°  +
      0.5QC.

   6  Discard all tubes of LTB and all 24-hour
      gas-positive BGLB cultures.

 D 72-hour  Procedures

    1  If any cultures remain to be examined,
      all will be BGLB.  Some  may be 24
      hours old and some may be 48 hours
      old. Remove such cultures from the
      Incubator, examine each tube for gas
      production, and record results on the
      data sheet.
    2  Be sure to record the results of 24-hour
      BGLB cultures in the "24" column under
      BGLB and the 48-hour results under the
      "48" column of the data sheet.

    3  Return any 24-hour gas-negative cultures
      for incubation 24  + 2 hours at 35 +
      0.5°c.

    4  Discard all gas-positive BGLB cultures
      and all 48-hour gas-negative cultures
      from BGLB.

    5  It is possible that all cultural work and
      results for the Confirmed Test have
      been finished at this point.  If so, codify
      results and determine Confirmed Test
      coliforms  per  100 ml as described in
      the outline on use of MPN Tables.

E  96-hour Procedures

   At most only  a few 48-hour cultures in
   BGLB may be present.  Read and record
   gas production of such cultures in the "48"
   column under BGLB on the data sheet.
   Codify results and determine Confirmed
   Test coliforms per 100 ml.

F  Streak-plate methods for the Confirmed
   Test,  using eosin methylene blue agar or
   Endo agar plates,  are accepted procedures
   in Standard Methods.  The worker who
   prefers to use one of these media in
   preference to BGLB (also approved in
   Standard Methods) is advised to refer to
   the current edition of "Standard Methods-
   for the Examination of Water and Waste-
   water" for procedures.
                                                                                    28-15

-------
  MPN Methods
IV  TESTING TO COMPLETED TEST STAGE

 (Note that this description starts with the
 sample inoculation and proceeds through the
 Presumptive and the Confirmed Test stages.
 In addition, the Fecal  Coliform Test is
 referred to as an optional adjunct to the
 procedure.)

 A First-Day Procedures

    1  Prepare a laboratory data sheet for the
       sample.  Record the following information:
       assigned laboratory number,  source of
       sample, date and time of collection,
       temperature of the  source, name of
       sample collector, date and time of
       receipt of sample in the laboratory.
       Also show the date  and time of starting
       tests in the laboratory,  name(s) of
       worker(s) performing the laboratory
       tests,  and the sample volumes planted.

    2  Label the tubes  of lauryl tryptose broth
       required for the initial planting 'of the
       sample.  The label should bear three
       identifying marks.  The upper number
       is the identification of the worker(s)
       performing the test (applicable to
       personnel in training  courses),
       the number immediately below is the-
       assigned laboratory number, corres-
       ponding with the laboratory record
       sheet. The lower number is the code
       to designate the sample volume and
       which tube of a replicate  series is
       indicated.  Guidance  on labeling for
       laboratory data'number and identification
       of individual tubes is  described else-
       where ;in this outline.
   NOTE:  If 10-ml samples are being
   plated, it is necessary to use tubes
   containing the correct concentration
   of culture medium.  This has previously
   been noted elsewhere in this outline"
   and referral is made to tables.

3  Place the labeled culture tubes in an
   orderly arrangement in a culture tube
   rack,  with the tubes intended for the
   largest sample volumes in the front
   row, and those intended for smaller
   volumes in the succeeding rows.

4  Shake the sample vigorously, approxi-
   mately 25 times,  in an up-and-down
   motion.

5  Measure the predetermined sample
   volumes into the labeled tubes of lauryl
   tryptose broth, using care to avoid
   introduction of any bacteria into the
   culture medium except those in the
   sample.

   a  Use a 10-ml pipet for 10 ml sample
     portions, and 1-ml pipets for portions
     of 1 ml or less.  Handle sterile
     pipets only near the mouthpiece,
     and protect the delivery end from
     external contamination.  Do not move
     the cotton plug in the mouthpiece
     as this is intended to protect the
     user from ingesting any sample.
     When using the pipet to withdraw
     sample portions, do not dip the
     pipet more than 1/2 inch into the
     sample; otherwise sample running
     down the outside of the pipet will
     make measurements inaccurate.

     When delivering the sample into the
     culture medium, deliver sample
     portions of 1 ml or less down into
     28-16

-------
                                                                             MPN Methods
        the culture tube near the surface of
        the medium.  Do not deliver small
        sample volumes at the top of the
        tube and allow them to run down
        inside the tube; too much of the
        sample will fail to reach the culture
        medium.

      d Prepare preliminary dilutions of
        samples for portions of 0.01 ml or
        less before delivery into the culture
        medium.  See-Table 2 for preparation
        of dilutions.   NOTE: Always  deliver
        diluted sample portions into the
        culture medium as soon as possible
        after preparation.  The interval
        between preparation of dilution and
        introduction of sample into the
        medium never should be as much as
        3 0 minutes.

   6  After measuring all portions of the
      sample into their respective tubes of
      medium, gently shake the rack of
      inoculated tubes  to insure good mixing
      of sample with the culture medium.
      Avoid vigorous shaking, as air bubbles
      may be shaken into the fermentation
      vials and thereby invalidate the test.

   7  Place the rack of inoculated tubes in
      the incubator at 35O +  0. 5OC for 24 +
      2 hours.

B  24-hour Procedures

   1  Remove the rack of lauryl tryptose broth
      cultures from the incubator, and shake
      gently.   If gas is about to appear in the
      fermentation vials, the shaking will
      speed the process.

   2  Examine each tube  carefully.  Record,
      in the column "24"  under LST on-the
      laboratory data sheet, each tube showing
      gas in the fermentation vial as a positive
      (+) test and each tube not showing gas
      as a negative (-) test.  GAS IN ANY
      QUANTITY IS A  POSITIVE TEST.

   3  Retain all gas-positive tubes of lauryl
      tryptose broth culture in their place in
      the rack, and proceed.
   4  Select the gas-positive tubes of lauryl
      tryptose broth culture for the  Confirmed
      Test procedures.  Confirmed Test
      procedures may not be required for
      all gas-positive cultures.  If, after
      2 4-hours of incubation, all five
      replicate cultures are gas-positive for
      two or more consecutive sample
      volumes, then select the set of five
      cultures representing the smallest
      volume of sample in which all tubes
      were gas-positive.  Apply Confirmed
      Test procedures to all these cultures
      and to any other gas-positive cultures
      representing smaller volumes  of
      sample, in which some tubes were
      gas-positive and some were gas-
      negative .

   5  Label one tube of brilliant green lactose
      bile broth (BGLB) to correspond with
      each tube of lauryl tryptose broth
      selected for Confirmed Test procedures.

   6  Gently shake the rack of Presumptive
      Test cultures.  With a flame-sterilized
      inoculation loop transfer one loopful of
      culture from each gas-positive tube to
      the corresponding tube of BGLB.  Place
      each newly inoculated culture into
      BGLB in the position of the original
      gas-positive tube.

   7  If the Fecal Coliform Test is included
      in the testing procedure,  consult
      Section V of this outline for details of
      the testing procedure.

   8  After making the transfer, the rack
      should contain some 24-hour gas-
      negative tubes of lauryl tryptose borth
      and the newly inoculated BGLB.
      Incubate the rack of cultures at 35° C
      + O.SOCfor 24 + 2 hours.

C  48-hour Procedures

   1  Remove the rack of culture tubes from
      the incubator,  read and record gas
      production for each tube.

   2  Some tubes will be lauryl tryptose broth
      and  some will be brilliant green lactose
                                                                                   28-17

-------
MPN Methods
     bile broth (BGLB).  Be sure to record
     results from LTB under the 48-hour .
     LTB column and the BGLB results
     under the 2 4-hour column of the data
     sheet.

   3 Label tubes of BGLB to correspond with
     all (if any) 48-hour gas-positive cultures
     in lauryl tryptose broth.  Transfer one
     loopful of culture from each gas-positive
     LTB culture to the correspondingly-
     labeled tube of BGLB.  NOTE: All tubes
     of LTB culture which were negative at
     24 hours and became positive at 48 hours
     are to be transferred.  The Option
     described above  for 24-hour LTB
     cultures does not apply at 48 hours.

   4 Incubate the 24-hour gas-negative BGLB
     tubes and any newly-inoculated tubes of
     BGLB 24 + 2 hours at 35° + 0. 5° C.
     Retain all~24-hour gas-positive cultures
     in BGLB for further test  procedures.

   5 Label a Petri dish preparation of eosin
     methylene blue agar (EMB agar) to
     correspond with  each gas-positive
     culture in BGLB.

   6 Prepare a streak plate for colony
     isolation from each gas-positive culture
     in BGLB on the correspondingly-labeled
     EMB agar plate.

     Incubate the EMB agar plates 24 + 2
     hours at 35°+ 0.5° C.

D  72-hour Procedures

   1 Remove the cultures  from the incubator.
     Some may be on  BGLB; several EMB
     agar plates also  can be expected.

   2 Examine and record gas production
     results for any cultures in BGLB.

   3 Retain any gas-positive BGLB cultures
     and prepare streak plate  inoculations
     for colony isolation in EMB agar.
     Incubate the EMB agar plates 24 +
     2 hours at 35 + 0.5° C.  Discard the
     gas-positive BGLB cultures after
     transfer.
   4  Reincubate any gas-negative BGLB
      cultures 24 + 2 hours at 35° + 0.50 C.

   5  Discard all 48-hour gas-negative BGLB
      cultures.

   6  Examine the EMB agar plates for the
      type of colonies developed thereon.
      Well-isolated colonies having a dark
      center (when viewed from the lower
      side, held toward a light) are termed
      "nucleated or fisheye" colonies, and
      are regarded as "typical" coliform
      colonies.  A surface sheen may or may
      not be present on  "typical" colonies.
      Colonies which are pink or opaque but
      are not nucleated are regarded as
      "atypical colonies. "  Other colony
      types are considered "noncoliform. "
      Read and record results as + for
      "typical" (nucleated) colonies + for
      "atypical" (non-nucleated pink or
      opaque colonies),  and - for other types
      of colonies which might develop.

   7  With plates bearing "typical" colonies,
      select at least one well-isolated colony
      and transfer it to a correspondingly-
      labeled tube of lactose broth and to an
      agar slant.  As a second choice, select
      at least two  "atypical" colonies (if
      typical colonies are not present) and
      transfer them to labeled tubes of
      lactose broth and to agar slants.  As a
      third choice, in the absence of typical
      or atypical coliform-like colonies,
      select at least two well-isolated
      colonies representative of those
      appearing on the EMB plate, and trans-
      fer them to lactose broth and to agar
      slants.

   8  Incubate all cultures transfered from
      EMB agar plates 24+2 hours at 35 +
      0.50C.

E  96-hour Procedures

   1  Subcultures from the samples being
      studied may  include:  48-hour tubes
      of BGLB,  EMB agar plates, lactose
      broth tubes,  and agar slant cultures.
   28-18

-------
                                                                         MPN Methods
If any 48-hour tubes of BGLB are
present, read and record gas production
in the "48" column under BGLB. From
any gas-positive BGLB cultures pre-
pare streak plate inoculations for colony
isolation on EMB agar.  Discard all
tubes of BGLB, and incubate EMB agar
plates 24 + 2 hours at 35 + 0. 5O C.

If any EMB plates are  present,  examine
and record results in the "EMB" column
of the data sheet.  Make transfers to
agar slants and to lactose broth from
all EMB agar plate cultures.  In
decreasing order of preference,  transfer
at least one typical colony,  or at least
two atypical colonies,  or at least two
colonies representative of those on the
plate.

Examine and record results from the
lactose  broth cultures.

Prepare a Gram-stained smear from
each of  the agar slant cultures,  as
follows:

NOTE:  Always prepare  Gram stain
from an actively growing culture,
preferably about 18 hours old, and
never more than 24 hours old.  Failure
to observe this precaution often results
in irregular staining reactions.

a  Thoroughly clean a  glass slide to
   free  it of any trace  of oily film.

b  Place one drop of distilled water on
   the slide.

c  Use  the inoculation needle to suspend
   a tiny amount of growth from the
   nutrient agar slant  culture in the
   drop of water.

d  Mix  the thin suspension of cells  with
   the tip of the inoculation needle, and
   allow the water to evaporate.

e  "Fix" the smear by gently warming
   the slide over a flame.

f  Stain the smear by flooding it for 1
   minute with crystal violet  solution.
g  Flush the excess crystal violet
   solution off in gently running water,
   and gently blot dry with filter
   paper or with other clean absorbent
   paper.

h  Flood the smear with Lugol's
   iodine for 1 minute.

i  Wash the  slide in gently running
   water and blot dry with filter paper.

j  Decolorize the smear with 95%
   alcohol solution with gentle
   agitation for 10-30 seconds,
   depending upon extent of removal
   of crystal violet dye,  then blot dry.

k  Counterstain for 10 seconds with
   safranin solution,  then wash in
   running water and blot dry.

1  Examine the slide under the
   microscope,  using the oil
   immersion lens. Goliform
   bacteria are  Gram-negative,
   nonspore-forming, rod-shaped
   cells, occurring singly,  in pairs,
   or rarely in short chains.

m If typical coliform staining reaction
   and morphology are observed,
   record + in the appropriate space
   under the "Gram Stain" column of
   the data sheet.  If typical morphology
   and staining reaction are not
   observed, then mark it + or -,  and
   make suitable comment in the
   "remarks" column at the right-hand
   side of the data sheet.

n  If spore-forming bacteria are
   observed, it will be necessary to
   repurify the culture from which
   the observations were made.
   Consult the instructor, or refer
   to Standard Methods,  for procedures.

At this point,  it is possible that all
cultural work for the  Completed Test
has been finished.  If so, codify results
and determine Completed Test coliforms
per 100 ml.
                                                                                28-19

-------
 MPN Methods
 F  120-hour Procedures and following:

    1  Any procedures to be undertaken from
      this point are "straggler" cultures on
      media already described, and requiring
      step-by-step methodology already given
      in detail.  Such cultures may be on:
      EMB plates, agar slants, or lactose
      broth.  The same time-and-temperature
      of incubation required for earlier studies
      applies  to the "stragglers" as do the
      observations, staining reactions,  and
      interpretation of results. On con-
      clusion  of all cultural procedures,
      codify results and determine Completed
      Test coliforms per 100 ml.
V  FECAL COLIFORM TEST

A General Information

   1  The procedure described is an elevated
      temperature test for fecal coliform
      bacteria.

   2  Equipment required for the tests are
      those required for the Presumptive
      Test of Standard Methods, a water-bath
      incubator,  and the appropriate culture
      media.

B Fecal  Coliform Test with EC Broth

   1  Sample:  The test is applied to gas-
      positive tubes from the Standard
      Methods Presumptive Test (lauryl
      tryptose broth),  in parallel with
      Confirmed Test  procedures.

   2  24-hour Operations.  Initial procedures
      are the planting  procedures described
      for  the Standard Methods Presumptive
      Coliform test.

      a After reading and recording gas-
        production on lauryl tryptose broth,
        temporarily retain all gas-positive
        tubes.

      b  Label a tube of EC broth to corre-
         spond with each gas-positive  tube
        of lauryl tryptose broth.  The option
        regarding transfer of only a limited
      number..of tubes to the Confirmed
      Test sometimes can be applied here.
      However, the worker  is urged to
      avoid exercise of this option until
      he has assured the applicability of
      the option by preliminary tests on
      the sample source.
   c  Transfer one loopful of culture from
      each gas-positive culture in lauryl
      tryptose broth to the correspondingly
      labeled tube of EC broth.

   d  Incubate EC broth tubes 24 i 2 hours
      at 44. 5 t 0. 2°C in a waterbath
      with water depth sufficient to come
      up at least as high as the top of the
      culture medium in the tubes. Place
      in waterbath as soon as possible
      after inoculation and always within
      30 minutes  after inoculation.

3  48-hour operations

   a  Remove the rack of EC cultures
      from the waterbath,  shake gently,
      and record  gas production for each
      tube.  Gas in any quantity is a
      positive test.

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

   c  Transfer any additional 48-hour
      gas-positive tubes of lauryl tryptose
      broth to correspondingly labeled
      tubes of EC broth.   Incubate 24 +
      2 hours at 44. 5 ±  0.2°C.

4  72-hour operations

   a  Read and record gas production for
      each  tube.  Discard all cultures.

   b  Codify results and determine fecal
      coliform count per 100 ml of sample.
    28-20

-------
                                                                 MPN Methods
                              TESTS FOR COUfOKM GROUP
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LACTOSE OR LAURYl TRYPTOSE BROTH AMD ARE MCUBATCD AT 35 DEC Ct
FERMENTATION TUBES (SERIAL DILUTION) OJ DEC C.
yX" 'V GAS POsrnvK TUBES (ANY AMOUNT
/ ^\ Of GAS) CONSTITUTE A POSIT ATE
1 1 PRESUMPTIVE TEST
,. GAS POSITIVE GAS NEGATIVE
| (24 HR.t 2 HR.) | TOTAL INC UB A TON TIME FOR LACTOSE
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! COIIFORM GROUP ABSENT
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1 / V INfllBATF BfilB TIIBFS FOR 18 HffS
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tNDO AGAR / \
"ATES GAsrosmvr GASNtGATnrf INCUBATE EMB OR ENDO AGAR
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COIFORM GROUP COIIFORM GROUP or nrG r+ OJ DEG C
CONFIRMED NOTCONFKMED ~
	 _> 	 / 	
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^^JEMB PLATES 1
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^NUTRIENT AGAR SLANT LACTOSE BROTH TUBE
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RODS AND/O* RODS ^X'
SPOREFORMERS NO SPORES ^X^ COUFORM GROUP ABSENT
ifOJH COMflcFED TEST
GAS POSITIVE GAS NEGATIVE
•^ COIIFORM GROUP ABSENT
TRANSFER TO EMB PLATE
  AND REPEAT PROCESS
                                                                               28-21

-------
                                           Part 3
                   LABORATORY METHODS FOR FECAL STREPTOCOCCUS
                                  (Day-By-Day Procedures)
 I  GENERAL INFORMATION

 A The same sampling and holding procedures
   apply as for the coliform test.

 B The number of fecal streptococci in water
   generally is lower than the number of
   coliform bacteria. It is good practice
   in multiple  dilution tube tests to start the
   sample planting series with one sample
   increment larger than for the coliform
   test. For example: If a  sample planting
   series of 1.0,  0.1, 0.01, and 0.001 ml
   is planned for the coliform test, it is
   suggested that  a series of 10, 1.0, 0. 1,
   and 0.01 ml be planted for the fecal
   streptococcus test.

 C Equipment required for the test is the same
   as required for the Standard Methods
   Presumptive and Confirmed Tests, except
   for the  differences in culture media.
H  STANDARD METHODS (Tentative)
   PROCEDURES

 A First-Day Operations

   1  Prepare the sample data sheet and
      labeled tubes of azide dextrose broth
      in the same manner as for the
      Presumptive Test.  NOTE: If 10-ml
      samples are included in the series,  be
      sure to use a special concentration
      (ordinarily double-strength) of azide
      dextrose broth for these sample
      portions.

   2  Shake the sample vigorously,  approxi-
      mately 25 times, in an up-and-down
      motion.

   3  Measure the predetermined sample
      volumes into the labeled tubes of azide
      dextrose broth, using the sample
      measurement and  delivery techniques
      used for the Presumptive Test.
   4  Shake the rack of tubes of inoculated
      culture media, to insure good mixing
      of sample with medium.

   5  Place the rack of inoculated tubes in
      the incubator at 35° + 0. 5° C for 24 +
      2 hours.

B  2 4-hour Operations

   1  Remove the rack of tubes from the
      incubator.   Read and record the results
      from each tube.  Growth is a positive
      test with this test.  Evidence of growth
      consists either of turbidity  of the
      medium, a  "button" of sediment at the
      bottom  of the culture tube,  or both.

   2  Label a tube of ethyl violet  azide broth
      to correspond with each positive culture
      of azide dextrose broth.  It may be
      permissible to use the same confirmatory
      option as described for the  coliform
      Confirmed Test, in this outline.

   3  Shake the rack of cultures gently,  to
      resuspend any living cells which have
      settled  out to the bottom of  the culture
      tubes.

   4  Transfer three loopfuls of culture from
      each growth-positive tube of azide
      dextrose broth to the correspondingly
      labeled tube of ethyl violet azide broth.

   5  As transfers are made,  place the newly
      inoculated tubes of ethyl violet azide
      broth in the positions in the rack
      formerly occupied by the growth-
      positive tubes of azide dextrose broth.
      Discard the tubes of azide dextrose
      broth culture.

   6  Return  the rack, containing 24-hour
      growth-negative azide dextrose broth
      tubes and newly-inoculated  tubes of
      ethyl violet azide broth,  to the incubator.
      Incubate 24+2 hours at 35° + 0. 5°C.
                                                                                      28-23

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MPN Methods
C 48-hour Operations

   1  Remove the rack of tubes from the
      incubator.  Read and report results.
      Growth, either in azide dextrose broth
      or in ethyl violet azide broth,  is a
      positive test.  Be sure to report the
      results of the azide dextrose broth
      medium under the "48" column for that
      medium and the results of the  ethyl
      violet azide broth cultures under the
      "24" column for that medium.

   2  Any 48-hour growth-positive cultures
      of azide dextrose broth are to  be
      transferred (three loopfulls) to ethyl
      violet azide broth. Discard all 48-hour
      growth-negative tubes of azide dextrose
      broth and all 24-hour growth-positive
      tubes of ethyl violet azide broth.

   3  Incubate the 24-hour growth-negative
      and the newly-inoculated tubes of ethyl
      violet azide broth 24 + 2 hours at 35°
      + 0.5QC.

D 72-hour Operations

   1  Read and report growth results of all
      tubes of ethyl violet azide broth.

   2  Discard all growth-positive cultures
      and all 48-hour growth-negative
      cultures.

   3  Reincubate any 24-hour growth-negative
      cultures in ethyl violet azide broth 24
      + 2 hours at 35O + 0.5QC.

E 96-hour Operations

   1  Read and report growth results of any
      remaining tubes of ethyl violet azide
      broth.
      Codify results and determine fecal
      streptococci per 100 ml.
REFERENCES

1  Standard Methods for the Examination of
      Water and Wastewater (13th Ed)".
      Prepared and published jointly by
      American Public Health Association,
      American Water Works Association,
      and Water Pollution Control
      Federation.   1971.

2  Geldreich,  E.E., Clark, H.F.,  Kabler,
      P.W., Huff, C.B. andBordner, R.H.
      The Coliform Group.  II.  Reactions
      in EC Medium at 45° C.  Appl.
      Microbiol.  8:347-348.  1958.

3  Geldreich,  E.E., Bordner, R.H., Huff,
      C.B., Clark, H.F.  and Kabler, P.W.
      Type Distribution of Coliform Bacteria
      in the Feces of Warm-Blooded Animals.
      J. Water Pollution Control Federation.
      34:295-301.   1962.

4  Recommend Proc.  for the Bacteriological
      Examination of Sea Water and Shellfish.
      3rd Edition,  American Public Health
      Association.  1962.
This outline was prepared by H. L. Jeter,
Director, National Training Center, Water
Programs Operations, Environmental
Protection Agency, Cincinnati, OH 45268.
Descriptors:  Coliforms,  Fecal Coliforms,
Fecal Streptococci, Indicator Bacteria,
Laboratory Equipment,  Laboratory Tests,
Microbiology, Most Probable Number, MPN,
Sewage Bacteria, Water Analysis
  28-24

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                        EXAMINATION OF WATER FOR COLIFORM
                          AND FECAL STREPTOCOCCUS GROUPS
                                (Membrane Filter Methods)
                                    Part  1 - Overview
I  INTRODUCTION

A  Scope

   1  The membrane filter consists of a
      porous disk of cellulose esters 47 mm
      in diameter and approximately 0. 15 mm
      thick.  Filters used in water bacter-
      iology have pores 0. 45 microns in
      diameter. The basic  principles  of use
      in water bacteriology  are as follows:
      Currently, active research is under-
      way to reevaluate the  pore size in an
      effort thereby to improve colony
      productivity.

      a A sterile membrane filter is
        fastened into a suitable filter  holding
        device and a predetermined volume
        of a water sample is filtered through
        the membrane filter.

      b The filter is placed in a culture con-
        tainer either on a paper pad
        impregnated with moist culture
        medium or upon an agar medium.

      c The inoculated filter is incubated
        under prescribed conditions of
        temperature and humidity for a
        designated time.

      d After incubation, the resulting
        culture is examined,  and the neces-
        sary interpretations and/or
        additional tests are made.

   2  Through variations in such factors as
      the composition of the culture medium,
      incubation time and temperature, and
      combinations with other cultural and
      biochemical tests, several kinds of
      bacterial determinations are possible.
    Basic Principles and Origins of
    Membrane Filter Techniques

    The purpose of this discussion is to
    provide orientation in the types and
    amount of equipment and supplies
    needed in membrane filter analyses
    in water quality surveys, the basic
    methodology, limitations of membrane
    filter procedures,  and the status of
    membrane filter methods in water
    quality testing. Finally, it considers
    the level of technical skill and the
    workloads of personnel assigned to
    perform tests by this method.
II   REQUIREMENTS FOR EQUIPMENT AND
    SUPPLIES

 A  Laboratory Supplies and Equipment

    1  Membrane filters

      a  Membrane filters are available
         from  several different commercial
         sources, representing the United
         States,  Britain,  and Germany.
         Each  source applies a  distinctive
         tradename to its MF product; such
         as:

         "Millipore Filters" - Millipore
         Filter Corp., U. S. A.

         "Bac-T-Flex Filters"  - Schleicher
         and Schuell, U.S.A. representatives
         for Sartorius - Werke, Germany.

         "Oxoid Filter" - Oxo Limited,
         Britain

      b  Membrane filters are available in
         a wide variety of controlled pore
 W.BA. mem. 86.2.75
                                    29-1

-------
Examination of Water
         sizes.  Each commercial source
         will designate the filter most suitable
         for water bacteriological tests upon  •
         request.

   2  Absorbent pads for nutrients

      Absorbent paper disks for mechanical
      support of the filters during incubation
      and for maintaining an adequate supply
      of  culture medium in contact with the
      filters, ordinarily are sold with the
      membrane filters. Additional supplies
      are obtainable from filter manufacturers.
      These pads may be the source of cult-
      ural problems due to chemical
      impurities.

   3  Filter-holding units

      a  The filter-holding unit is a device
         for supporting the membrane filter
         and  for holding the sample until  it
         passes through the filter. During
         the filtration, the sample passes
         through a circular area,  usually
         about 35 mm in diameter, in the
         center of the filter.  The outer part
         of the filter disk is clamped between
         the two essential components of  the
         filter-holding unit.

         1) The lower element, called the
           filter base,  or receptable,
           supports the membrane filter on
           a plate about 50 mm in diameter.
           The central part of this plate is
           porous,  to allow free passage of
           liquid.   The outer part of the
           plate is  a smooth nonporous sur-
           face.   The lower element includes
           fittings for mounting the unit  in
           a suction flask or other container
           suitable for filtration with vacuum.

         2) The upper element, usually called
           the funnel, holds the sample until
           it is drawn through the filter. Its
           lower portion is a flat ring that
           rests firectly over the nonporous
           part of the filter support  plate.
      3) When assembled, the two
        elements of the filter-holding
        unit are joined by a locking ring
        or by one or more clamps or
        springs.

   b  Filter-holding units are either
      metal or glass.  Metal units are
      available from at least six commer-
      cial sources, and glass units are
      available from at least two sources.
      Most workers prefer to use metal
      filter-holding units, due to their
      greater durability.  Most of the use
      of glass units seems to be dictated
      by economic considerations, as
      these are much lower in initial cost.

4  Culture media

   a  Culture media are commercially
      available for use with membrane
      filters in the determination of
      bacterial plate counts, for coliform
      counts, fecal coliform counts,  fecal
      streptococcus counts and, in addition,
      for several special applications.

   b  These media can be obtained in
      several forms, including:

      1) Dehydrated media, requiring
        weighing,  addition of a suitable
        amount of distilled water, and
        some form of sterilization prior
        to use.   This form of media is
        preferred as it appears to give
        the most satisfactory combina-
        tion of performance quality,
        stability and economy.

      2) Ready-to-use culture media,
        sealed in glass ampoules.

5  Culture containers

   Several forms of culture containers are
   suitable for membrane filter works.
   These include small glass Petri dishes,
   and plastic Petri dishes.  The plastic
   Petri dishes may be preferred, as they
   are sufficiently cheap to encourage
   single-service use.
 29-2

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                                                                  Examination of Water
   6  Vacuum sources

      In the fixed laboratory,  central vacuum
      service, laboratory-scale electric
      Vacuum pumps, or water pumps
      ("aspirators") are suitable for vacuum
      filtration of samples.

   7  Glassware

      Conventional laboratory glassware is
      used in fixed laboratories for  measure-
      ment and delivery of samples  for filtra-
      tion, sample dilutions, and the like.
      Under field conditions it sometimes is
      necessary to use a limited amount of
      laboratory glassware, with some form
      of field application of sterilization
      methods.

   8  General laboratory facilities

      Such facilities as autoclave, incubators,
      weighing equipment,  and the like are
      required in conventional bacteriology
      laboratories,  and are needed equally
      when membrane filter methods are
      used.

B  Field Equipment

   Field operations cannot be  carried  on
   indefinitely without support from a  central
   laboratory.  Functions such as preparation
   of distilled water, pre-weighing of  culture
   media, sterilization of membrane filters
   and culture containers,  and other general
   preparations are best performed in a
   central laboratory facility.

   1  With the introduction of  membrane
      filters to the bacteriological analysis
      of water, a number of investigators
      gave attention to the development of '
      membrane filter equipment which
      can  be used in the field in easily
      portable, self-contained water labora-
      tories.  Such portable laboratory kits
      would have three major  fields of
      usefulness:
   a  They would be useful in certain
      routine water quality control
      operations.  Examples include such
      places as on board ships,  in some
      national parks, on airlines, etc.

   b  They could be used in certain types
      of stream surveys, where there is
      an excessive delay between sample
      collection and the time when
      examination can be started, due to
      time required for transporation of
      samples.

   c  Finally, such units would be invalu-
      able in emergencies, when existing
      laboratories are overburdened or
      inoperative.  Portable kits already
      have proven extremely useful in
      testing many small water supplies
      in a short period of time.   Further,
      there is a predictable need for such
      equipment in the event of natural or
      wartime civil defense disaster.

2  Availability of field equipment

   Commercially developed field equipment
   is  currently available from only one
   source.

   Equipment of this type includes
   provision for sample filtration,
   field sterilization (where necessary)
   of  filtration equipment, limited
   capacity for reserves of culture
   media and other supplies, and
   provision for incubation of cultures.
   Such equipment does not include
   satisfactory visual aid equipment
   (a  low powered microscope is needed
   for streptococcus counts,  and at
   least a simple lens is needed for
   coliform work),  and provision for
   many of the supplies is not included.
                                                                                    29-3

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 Examination of Water
III  MEMBRANE FILTER PROCEDURES IN
    WATER QUALITY AND POLLUTANT
    SOURCE MONITORING

 A Standard Coliform Test

    1  Basic procedures

       a  Sample filtration volumes are
          selected (usually 3 volumes) to give
          at least one volume which will con-
          tain 20-80 typical coliform colonies.

       b  The sample volumes  are filtered
          through membrane filters,  and
          incubated at 35°C for 20-22 hours
          in an atmosphere at or near
          saturation.

       c  Following incubation, the typical
          coliform colonies are counted and
          reported as coliforms per 100 ml
          of sample.

    2  Applications

       The total coliform count has been used
       most widely of all procedures  when
       bacteriological data are collected in a
       survey. Applications of membrane
       filter methods in surveys necessarily
       have a  short history.  The method has
       been used in certain studies of the
       Potomac River System,  in the Illinois -
       Great Lakes studies, and in certain
       surveys in the West.

       The Standard MF coliform test is
       applicable when it can be  initiated
       within the recommended period between
       sample collection and receipt at  the
       laboratory.  The procedure  is applicable
       with MF field units.

 B  "Fecal Coliform" Test with Membrane
    Filters

    1   Procedures

       a  Sample filtration procedures are the
          same as for the Standard Membrane
          Filter coliform test.  Sample
        filtration volumes should be
        selected so that at least one filter
        will produce 20-60 fecal coliform
        colonies.

     b The culture medium is M-FC
        Broth.  The medium requires
        addition of a rosolic acid solution
        prior to use.

     c Within 20 minutes after sample
        filtration, the membrane filters
        are placed in small plastic bags,
        which are sealed, and immersed
        in a forced circulation water bath
        at 44.5°C ± 0. 2°C for a 24 hour
        incubation time.

     d Fecal coliform colonies are blue or
        blue-hues, generally 1-3 mm in
        diameter.  The fecal coliform
        density is reported as fecal coliforms
        per 100 ml of sample.

   2 Application

     The fecal coliform test is being used
     increasingly in water quality studies.
     Fecal coliform criteria are being
     established for compliance with
     effluent limits for NPDES Permit
     Compliance.

C  Fecal Streptococcus Tests

   1 Basic procedures

     a The filtration procedures are the
        same as with the coliform tests.
        The sample volumes should be such
        that at least one membrane filter
        will produce 20 - 100 fecal strepto-
        coccus colonies.

     b The culture medium can be the
        M-Enterococcus Agar developed  by
        Slanetz and his associates, or it  can
        be KF-Broth, developed by Kenner
        and his associates.  In either case,
        incubation is 48 hours at 35 C.
   29-4

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                                                                     Examination of Water
IV
       c  With both media, fecal streptococcus
          colonies are pale pink to deep red,
          up to 2 mm in diameter.

       d  Because the density of fecal
          streptococci in most waters is
          lower than coliform density,  it is
          necessary to filter greater volumes
          of water. Further, the number of
          colonies that can be counted effec-
          tively is greater than the number of
          coliform colonies.  Sample volumes
          often can be 10 to  100-fold greater
          for fecal streptococcus counts than
          for coliform counts.

    2  Application

       Fecal streptococcus counts are
       especially useful when supplementary
       data will support information from the
       coliform counts.   Fecal  streptococcus
       counts are used in many states in
       addition to coliform tests in examina-
       tion of bathing waters as well as in
       stream surveys.
LIMITATIONS OF MEMBRANE FILTER
METHODS
  Most of the recognized limitations of the
  membrane filter method have been related
  to tests for members of the coliform group.
  Extension of these recognized limitations to
  tests for other pollution indicators requires
  special interpretation.

  A Need for Selection of Suitable Filtration
    Volume

    1  In tests for coliform bacteria, the
       sample filtration volumes should be
       so selected that one of volumes
       yields 20-80 coliform colonies. With
       fewer than 20 colonies, the random
       variation of colony counts becomes
       great enough to cause some difficulty
       in statistical analysis; with more  than
       80 coliform colonies, the proportionality
       between number of coliform colonies
       differentiated and sample volume be-
       gins to fail. This is especially trouble-
       some in waters containing large numbers
       of noncoliform bacteria capable of
       growing on the filters.

   2  In tests for the fecal streptococci,
      fewer nonstreptococcus interference
      colonies are capable of growing on
      the medium.  For this reason and
      because fecal streptococcus colonies
      are small, the sample filtration
      volumes are so selected as to yield
      20-100 colonies on a membrane filter.

B  Influence of Turbidity

   When the water being studied  contains
   large amounts of suspended matter, such
   as clay, and has relatively low counts of
   the indicator organisms being studied,
   cultural difficulties often result.   The
   particulate matter suspended in the water
   is deposited on the surface of the membrane
   filter in sample filtration,  with any
   bacteria contained in the sample.  Culture
   medium diffusing through the membrane
   filter forms a capillary layer around all
   the particles of matter deposited on the
   filter.  This results in development of
   spreading,  poorly defined bacterial
   colonies.

   The solutions to problems of this type are:
   1) to filter the sample in several smaller
   increments so that the  combined sample
   filtration volume is spread over several
   membrane filters, each of which has a
   lesser amount of particulate matter
   deposited on the surface; or, 2) perform
   the analysis by means of the dilution tube
   (MPN) procedure.

C  Influence of large numbers of bacteria
   representing forms other than the
   indicator group being studied.
                                                                                       29-5

-------
Examination of Water
   1 In colif orm testing,  this ^can-he A
     serious problem.  The difficulty arises
     when appreciable numbers of non-
     coliform bacteria are present and are
     capable of growing on the Endo-type
     culture medium used.  When large
     numbers of noncoliform bacteria grow
     on the medium, and few coliform
     bacteria are present, the sheen-
     producing capacity of many of the
     coliform colonies is impaired, and
     the result indicates the presence of
     fewer coliform bacteria than actually
     are present.

     A solution for this type of problem
     is to  filter several smaller sample
     increments, or else to use the MPN
     method.

   2 In tests for fecal streptococci, the
     presence of large numbers of bacteria
     of other groups has not proven trouble-
     some.  The medium is extremely
     selective,  and the few forms that do
     seem to grow on the medium produce
     extremely small colonies which have
     not been shown to have any adverse
     effect on the qualitative results obtained
     in tests for fecal streptococci.

   Increasingly, problems in sensitivity
   of the test are being recognized in
   tests on chlorinated effluents from
   wastewater treatment facilities.
   OFFICIAL  STATUS OF MEMBRANE
   FILTER METHODS

   American Public Health Association,
   American Water Works Association,
   and Water Pollution Control Federation,
   in "Standard Methods for the Exam-
   ination of Water and Wastewater. "
   (13th Edition,  1971)

   1 A single-stage procedure, using
      M-Endo Broth MF for tests for
      members of the coliform group,  is
      described as an official testing
     procedure.
   2 A new two-stage procedure has been
     added.

   3 A delayed incubation procedure, using
     benzoated M-Endo Broth MF, is
     designated a Tentative Method.

   4 A test for enterococci (fecal strepto-
     cocci),  using M Enterococcus Agar
     or KF broth,  is designated a
     Tentative Method.

B  The Technical Committee on  Methods for
   the Bacteriological Examination of
   Boundary Waters Recommended use of
   membrane filter methods for the
   examination of boundary waters.  The
   Advisory Committee adopted  the recom-
   mendation in January,  1957.

C  The U. S.  Government, in Interstate
   Quarantine Drinking Water  Standards

   1 (Federal Register, October 23, 1956,
     pp.  8110-11 and March 1,  1957,
     Vol. 22, p. 1271). Preliminary and
     final authorization was granted to use
     membrane filter coliform  tests for
     the examination of potable waters at
     interstate carrier water points  and
     other waters under water quality
     control regulations of the Public
     Health Service or other agencies of
     the U. S. Government.

D  The U. S.  Government, in test procedures
   for compliance with water quality criteria.
   Federal Register, October  jj>,  1973,
   pp.  28758-60,  Volume  38, No.  199.  The
   same information also  appears in the
   Code of Federal Regulations, Title 40,
   Part 136, pp. Ill ff, state  July 1, 1974.
 29-6

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                        DETAILED MEMBRANE FILTER METHODS
                                          Part 2
I  BASIC PROCEDURES

A  Introduction

   Successful application of membrane filter
   methods requires development of good
   routine operational practices.  The
   detailed basic procedures described in
   this Section are applicable to all mem-
   brane filter methods in water bacteriology
   for filtration,  incubation, colony counting,
   and reporting of results.  In addition,
   equipment and supplies used in membrane
   filter procedures described here are not
   repeated elsewhere in this text in such detail.

   Workers using membrane filter methods
   for the first time are urged to become
   thoroughly familiar with these basic
   procedures and precautions.

B  General Supplies and Equipment List

   Table 1 is a check list of materials.

C  "Sterilizing" Media

   Set tubes of freshly prepared medium in a
   boiling waterbath for 10 minutes.  This
   method suffices for medium in tubes up to
   25X150 mm. Frequent agitation is needed
   with media containing agar.

   Alternately, coliform media can be
   directly heated on a hotplate to the first
   bubble of boiling.  Stir the medium
   frequently if direct heat is used,  to avoid
   charring the medium.

   Do not sterilize in the autoclave.
D  General Laboratory Procedures with
   Membrane Filters

   1  Prepare data sheet

      Minimum data required are:  sample
      identification,  test performed including
      media and methods, sample filtration
      volumes, and the bench numbers
      assigned to individual membrane filters.

   2  Disinfect the laboratory bench surface.

      Use a suitable disinfectant solution and
      allow the surface to dry before
      proceeding.

   3  Set out sterile culture containers in an
      orderly arrangement.

   4  Label the culture containers.

      Numbers correspond with the filter
      numbers shown on the data sheet.

   5  Place one  sterile absorbent pad* in
      each culture container, unless an agar
      medium is being used.

      Use sterile forceps for all manipulations
      of absorbent pads and membrane filters.
      Forceps sterility is maintained by
      storing the working tips in about 1  inch
      of methanol or ethanol.  Because the
      alcohol deteriorates the filter,  dissipate
      it by burning before using the forceps.
      Avoid heating the forceps in the burner
      as hot metal chars the filter.
*When an agar medium is used, absorbent pads are not used.  The amount of medium should be
sufficient to make a layer approximately 1/8" deep in the culture container.  In the 50 mm
plastic culture containers this corresponds to approximately 6-8 ml of culture medium.

NOTE: Mention of commercial products and manufacturers does not imply endorsement by the
        Office Of Water Programs, Environmental Protection Agency.
                                                                                 29-7

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Detailed Membrane Filter Methods
                            Table 1. EQUIPMENT. SUPPLIES AND MEDIA

Item
Funnel unit assemblies
Ring stand, with about a 3" split ring, to
support the filtration funnel
Forceps, smooth tips, type for
MF work
Methanol, in small wide-mouthed bottles.
about 20 ml for sterilizing forceps
Suction flasks, glass, 1 liter, mouth to
fit No. 8 stopper
Rubber tubing, 2-3 feet, to connect
suction flask to vacuum services, latex
rubber 3/16" I.D. by 3/32" wall
Pinch clamps strong enough for tight
compression of rubber tubing above
Pipettes, 10 ml, graduated, Mohr type.
sterile, dispense 10 per can per working
space per day. (Resterilize daily to
meet need).
Pipettes, 1 ml, graduated, Mohr type.
sterile, dispense 24 per can per working
space per day. (Resterilize daily to
meet need).
Pipette boxes, sterile, for 1 ml and
10 ml pipettes (sterilize above pipettes
in these boxes).
Cylinders, 100 ml graduated, sterile.
(resterllize daily to meet need).
Jars, to receive used pipettes
Gas burner, Bunsen or similar
laboratory type
Wax pencils, red, suitable for writing
on glass
Sponge in dilute iodine, to disinfect the
desk tops
Membrane filters (white, grid marked.
sterile, and suitable pore size for
microbiological analysts of water)
Absorbent pads for nutrient, (47 mm in
diameter), sterile, in units of 10 pads
per package. Not required if medium
•contains agar.
Fetri dishes,' disposable, plastic.
50 X 12 mm, sterile
Waterbath incubator 44.5 + 0.2°C
Vegetable crispers, or cake boxes.
plastic, with tight fitting covers, for
membrane filter incubations
Fluorescent lamp, with extension cord.
Ring stand, with clamps, utility type
Tot
M-Endo
Broth
X
X

X

X

X

X


X

X



X



X


X

X
X

X

X

X


X

••

X


X


X
X
1 Conforms
L.E.S.
Coliform
X
X

X

X

X

X


X

X



X



X


X

X
X

X

X

X


X



X


X


X
X
Delayed
Coliform
X
X

X

X

X

X


X

X



X



X


X

X
X

x

X

X


X



X


X


X
X

Fecal
Coliform
X
X

X

X

X

X


X

X



X



X


X

X
X

X

X

X


X



X

X



X
X

Fecal
Streptococcus
X
X

X

X

X

X


X

X



X



X


X

X
x.

X

X

X


X



X


X


X
X

Verified
Tests




























X


















X

    29-8

-------
                                Detailed Membrane Filter Methods
Table 1.  EQUIPMENT, SUPPLIES AND MEDIA (Cont'd)

Item
Half-round glass paper weights for
colony counting, with lower half of a
2-oz metal ointment box
Hand tally, single unit acceptable,
hand or desk type
Stereoscopic (dissection) microscope,
magnification of 10X or 15X, prefer-
able binocular wide field type
Bacteriological inoculating needle
Wire racks for culture tubes,
10 openings by five openings pre-
ferred, dimensions overall approxi-
mately 6" X 12"
Phenol Red Lactose Broth in 16 X
150 mm fermentation tubes with
metal caps, 10 ml per tube
Eosin Methylene Blue Agar
(Levine) in petri plates, prepared
ready for use
Nutrient agar slants, in screw
capped tubes, 16 X 126 mm
Gram stain solutions, 4 solutions
per complete set
Microscope, compound, binocular,
with oil immersion lens, micro-
scope lamp and immersion oil
Microscope slides, new, clean,
l"X3"size
Water proof plastic bags
for fecal coliform culture
dish incubation
M-Endo medium, MF dehydrated
medium in 25 X 95 mm flat bottomed
screw- capped glass vials, 1.44g
per tube, sufficient for 30 ml of
medium
Ethanol, 95% in small bottles or
screw-capped tubes, about 20 ml
per tube
Sodium benzoate solution, 12%
aqueous, in 25 X 150 mm screw-
capped tubes, about 10 ml per tube
L. E. S. Endo Agar MF, dehydrated
M-Endo medium, 0. 36 g per 25 X
95 mm flat bottomed screw-capped
glass vial, plus 0.45 g agar, for 30 ml
Lactose Lauryl Sulfate Tryptose Broth
in 25 X 150 mm test tube without
included gas tube, about 25 ml, for
enrichment in L. E. S. method
Total Coliform s
M-Endo
Broth
X
X
X












X

X




L.E.S.
Coliform
X
X
X














X

X
X

Delayed
Coliform
X
X
X












X

X
X




Fecal
Coliform
X
X
X










X









Fecal
Streptococcus
X
X
X




















Verified
Tests


X
X
X
X

X
X
X
X

X









                                                                29-9

-------
Detailed Membrane Filter Methods
                        Table 1. EQUIPMENT. SUPPUES AND MEDIA (Cont'd)

Item
M-FC Broth for fecal collform.
dehydrated medium in 25 X 95 mm
flat bottomed screw-capped glass
vials, 1. 11 g per tube, sufficient
for 30 ml of culture medium
Rosolic acid, 1% solution, in
0. 2N NaOH, in 25 X 150 mm flat
bottomed screw-capped tubes.
about 5 ml per tube, freshly
prepared
M-Enterococcus Agar. dehydrated
medium In 25 X 150 mm screw-
capped tubes, sufficient for 30 ml,
1.26 g per tube
Dilution bottles, 6-oz, preferable
boro- silicate glass, with screw-
cap (or rubber stopper protected
by paper) . each containing 99 ml
of sterile phosphate buffered
distilled water
Electric hot plate surface
Beakers, 400 - 600 ml (for water-
bath in preparation of membrane
filter culture media)
Crucible tongs, to be used at
electric hot plates, for removal
of hot tubes of culture media for
boiling waterbath
Total Coliforms
M-Endo
Broth














X





X
X


X



L. E.S.
Collform














X





X
X


X



Delayed
Collform














X





X
X


X




Fecal
Collform
X




X








X





X
X


X




Fecal
Streptococcus










X



X





X
X


X




Verified
Test




























29-10

-------
                                                      Detailed Membrane Filter Methods
 6  Deliver enough culture medium to
   saturate each absorbent pad,   using
   a sterile pipette.

   Exact quantities cannot be stated
   because pads and culture containers vary.
   Sufficient medium should be applied so
   that when the culture container is tipped,
   a good-sized drop of culture medium
   freely drains cut of the absorbent pad.

 7  Organize  supplies and equipment for
   convenient sample filtration.  In
   training courses, laboratory instructors
   will suggest useful arrangements;
   eventually the individual will select a
   system of bench-top organization most
   suited to his own needs.  The important
   point  in any arrangement is to have all
   needed equipment and supplies con-
   veniently  at hand,  in such a pattern as
   to minimize lost time in useless motions.

 8  Lay a sterile membrane filter on the
   filter holder, grid-side up, centered
   over the porous part of the filter
   support plate.

   Membrane filters are extremely
   delicate and easily damaged.  For
   manipulation, the sterile forceps
   should always grasp the outer part
   of the filter disk, outside the part
   of the filter through which the sample
   passes.

 9  Attach the funnel element to the base
   of the filtration unit.

   To avoid damage to  the membrane
   filter, locking forces should only be
   applied at the locking arrangement.
   The funnel element never should be
   turned or twisted while being seated
   and locked to the lower element of the
   filter holding unit.   Filter holding units
   featuring a bayonet joint and locking
   ring to join the upper element to the
   lower element require special care on
   the part of the operator.  The locking
   ring should be turned sufficiently to
   give a snug fit, but should not be
   tightened excessively.
10  Shake the sample thoroughly.

11  Measure sample into the funnel with
    vacuum turned off.

    The primary objectives here are:
    1) accurate measurement of sample;
    and  2) optimum distribution of colonies
    on the filter after incubation.  To
    meet these objectives, methods of
    measurement and dispensation to the
    filtration assembly are varied with
    different sample filtration volumes.

    a With samples greater than 20 ml,
      measure the sample with a sterile
      graduated cylinder and pour it into
      the funnel.  It is important to rinse
      this graduate with sterile buffered
      distilled water to preclude the loss
      of excessive sample volume.  This
      should be poured into the funnel.

    b With samples of  10 ml to 20 ml,
      measure the sample with a sterile
      10 ml or 20 ml pipette, and pipette
      on a dry membrane in the filtration
      assembly.

    c With samples of 2 ml to 10 ml,  pour
      about 20 ml of sterile dilution water
      into the filtration assembly, then
      measure the sample into the sterile
      buffered dilution water with a  10 ml
      sterile pipette.

    d With samples of 0. 5 to 2 ml, pour
      about 20 ml of sterile dilution water
      into the funnel assembly,  then
      measure the sample into the sterile
      dilution water in the funnel with a
      1 ml or a 2 ml pipette.

    e If a sample of less than 0. 5 ml is to
      be filtered,  prepare appropriate
      dilutions in sterile dilution water,
      and proceed as applicable in item c
      or d above.

      When dilutions of samples are needed,
      always make the filtrations as soon
      as possible after dilution of the
      sample; this  never should exceed
NOTE:  Mention of commercial products and manufacturers does not imply endorsement
        by the Office of Water Programs,  Environmental Protection Agency.
                                                                                      29-11

-------
 Detailed Membrane Filter Methods
       30 minutes.  Always shake sample •
       dilutions thoroughly before delivering
       measured volumes.

12  Turn on the vacuum.

    Open the appropriate spring clamp or
    valve, and filter the sample.

    After sample filtration a few droplets
    of sample usually remain adhered to
    the funnel walls.  Unless these droplets
    are removed, the bacteria contained in
    them will be a source of contamination
    of later  samples. (In laboratory
    practice the funnel unit is not routinely
    sterilized between successive filtrations
    of a series).  The purpose of the funnel
    rinse is to flush all droplets of a sample
    from the funnel walls to the membrane
    filter.  Extensive tests have shown that
    with proper rinsing technique, bacterial
    retention on the funnel walls is negligible.

13  Rinse the sample through the filter.

    After all the sample has passed through
    the membrane filter, rinse down the
    sides of the funnel walls with at least
    20 ml of sterile dilution water.  Repeat
    the rinse twice after all the first rinse
    has passed through the filter.  Cut off
    suction on the filtration assembly.

14  Remove the funnel element of the filter
    holding unit.

    If a ring stand with  split ring is used,
    hang the funnel element on the ring;
    otherwise, place the inverted funnel
    element on the inner surface of the
    wrapping material.  This requires
    care  in opening the  sterilized package,
    but it is effective as a protection of the
    funnel ring from contamination.

15  Take the membrane filter from the
    filter holder and carefully place it.
    grid-side up on the medium.

    Check that no air bubbles have been
    trapped between the membrane filter
    and the underlying absorbent pad or
    agar.  Relay the membrane if necessary.
16  Place in incubator after
    filtration series.
    Invert the containers.  The immediate
    atmosphere of the incubating membrane
    filter must be at or very near 100%
    relative humidity.

17  Count colonies which have appeared
    after incubating for the prescribed
    time.

    A stereoscopic microscope magnifying
    10- 15 times and  careful illumination
    give best counts.

    For reporting results,  the computation
    is:

      bacteria/ 100 ml =

      No. colonies  counted X 100
      Sample volume filtered in ml

    Example:

      A total of 36 colonies grew after
      filtering a 10 ml sample.  The
      number reported is:


      36lCOm"ieS X 10° = 36° Per 10° ml


   Report results to two significant figures.

   Example:

      A total of 40 colonies grew after
      filtering a 3 ml sample.

      This calculation gives:
      40
                     10°  •  1333. 33 per 100 ml
      But the number reported should be
      1300 per 100 ml.
    29-12

-------
                                                        Detailed Membrane Filter Methods
H  MF LABORATORY TESTS FOR
   COLIFORM GROUP

 A Standard Coliform Test (Based on M-Endo
   Broth MF)

   1   Culture medium

      a  M-Endo Broth MF Difco 0749-02
         or the equivalent BBL M- Coliform
         Broth 01-494

         Preparation of Culture Medium
         (M-Endo Broth)  for Standard MF
         Coliform Test
 Yeast extract
 Casitone or equivalent
 Thiopeptone or equivalent
 Tryptose
 Lactose
 Sodium desoxycholate
 Dipotassium phosphate
 Monopotassium phosphate
 Sodium chloride
.Sodium lauryl  sulf ate
 Basic fuchsin (bacteriological)
 Sodium sulfite

 Distilled water (containing
 20.0 ml ethanol)
 1.5
 5.0
 5.0
10.0
12.5
 0.1
 4.375 g
 1.375g
 5.0   g
       g
       g
       g
                                   0.05
                                   1 . 05
                                   2.1
                                         g
                                         g
                                         g
                                         g
                                         g
                                         g
                                   1000 ml
         This medium is available in
         dehydrated form and it is rec-
         ommended that the commercially
         available medium be used in
         preference to compounding the
         medium of its individual constituents.

         To prepare the medium for use,
         suspend the dehydrated medium at
         the rate of 48 grams per liter of
         water containing ethyl alcohol at
         the rate of 20 ml per liter.

         As a time-saving convenience,  it is
         recommended that the laboratory
         worker preweigh the dehydrated
         medium in closed tubes for several
         days, or even weeks, at one operation.
b
With this system,  a large number
of increments of dehydrated medium
(e.g.,  1.44 grams), sufficient for
some convenient (e.g., 30 ml)
volume of finished culture medium
are weighed and dispensed into
screw-capped culture tubes,  and
stored until needed. Storage should
preferably be in a darkened disiccator.

A supply of distilled water containing
20 ml stock ethanol per liter is
maintained.

When the medium is to be used, it
is reconstituted by adding 30 ml of   •
the distilled water-ethanol mixture
per tube of pre-weighed dehydrated
culture medium.

Medium is "sterilized" as directed
in I, C.
c  Finished medium can be retained
   up to 96 hours if kept in a cool,
   dark place.  Many workers prefer
   to reconstitute fresh medium daily.
                    Filtration and incubation procedures
                    are as given in I,  D.

                    Special instructions:

                    a  For counting,  use the wide field
                        binocular dissecting microscope, or
                        simple lens.  For illumination, use
                        a light source  perpendicular to the
                        plane of the membrane filter.  A
                        small fluorescent lamp is ideal for
                        the purpose.

                    b  Coliform colonies have a "metallic"
                        surface sheen  under reflected light
                        which may cover the entire colony,  or
                        it may appear  only in the center.  Non-
                        coliform colonies range from
                        colorless to pink, but do not have
                        the characteristic sheen.

                    c  Record the colony counts on the
                        data sheet, and compute the coliform
                        count per 100  ml of sample.
                                                                                      29-13

-------
    Detailed Membrane Filter Methods
B  Standard Coliform Tests (Based on L. E. S.
   Endo Agar)

   The distinction of the L. E. S. count is a
   two hour enrichment incubation on LST
   broth.  M-Endo L.E.S. medium is used
   as agar rather than the broth.

   1  Preparation of culture medium
      (L.E.S. Endo Agar) for L.E.S.
      coliform test

      a Formula from McCarthy, Delaney,
        and Grasso  &)
Bacto-Yeast Extract
Bacto- Casitone
Bacto-Thiopeptone
Bacto-Tryptose
Bacto-Lactose
Dipotassium phosphate
Monopotassium phosphate
Sodium chloride
Sodium desoxycholate
Sodium lauryl sulfate
Sodium sulfite
Bacto-Basic fuchsin
Agar

Distilled water (containing
20 ml ethyl alcohol)
1.
3.
3.
7.
9.
3.
1.
3.
0.
2
7
7
5
4
3
0
7
1
g
g
g
g
g
g
g
g
g
 0.05 g
 1.6  g
 0.8  g
15    g

1000 ml
      b  To rehydrate the medium,  suspend
        51 grams in the water-ethyl alcohol
        solution.   ;

      c  Medium is "sterilized" as directed
        in I, C.

      d  Pour 4-6 ml of freshly prepared Agar
        into the smaller half of the container.
        Allow the medium to cool and solidify.


   2  Procedures for filtration and incubation

      a  Lay out the culture dishes in a row
        or series of rows as usual.  Place
        these with the upper (lid) or top
        side down.

      b  Place one sterile absorbent pad in
        the larger half of each container
        (lid).  Use sterile forceps for all
    29-14
        manipulations of the pads.
        (Agar occupies smaller half or
        bottom).

      c  Using a sterile pipette, deliver
        enough single strength lauryl
        sulfate tryptose broth to saturate
        the pad only. Avoid excess medium.

      d  Follow general procedures for
        filtering in I,  D. Place filters on
        pad with lauryl sulfate tryptose
        broth.

      e  Upon completion of the filtrations,
        invert the culture containers and
        incubate at 350 C for 1  1/2 to 2
        hours.

   3  2-hour procedures

      a  Transfer the membrane filter from
        the enrichment pad in the upper  half
        to the agar medium in  the lower
        half of the container.  Carefully
        roll the membrane onto the agar
        surface to avoid trapping air
        bubbles beneath the membrane.

      b  Removal of the used absorbent pad
        is optional.

      c  The container is inverted and
        incubated 22 hours + 2 hours + 0.5  C.

   4  Counting procedures are as in I,  D.

   5  L.E.S.  Endo Agar may be used as  a
      single-stage medium (no enrichment
      step) in the same manner as M-Endo
      Broth, MF.

C  Delayed Incubation Coliform Test

   This  technique is applicable  in situations
   where there is an excessive  delay between
   sample collection and plating.  The  procedure
   is  unnecessary when the interval be-
   tween sample collection and  plating  is
   within acceptable limits.
                      Preparation of culture media for
                      delayed incubation coliform test

                      a  Preservative media M-Endo Broth
                         base

-------
                                                    Detailed Membrane Filter Methods
     To 30 ml of M-Endo Broth MF
     prepared in accordance with
     .directions in n, A, 1 of this
     ' outline,  add 1. 0 ml of a sterile
     12% aqueous solution of sodium
     benzoate.

     L. E. S. MF Holding Medium-
     Coliform:  Dissolve 12.7 grams in
     1 liter of distilled water.  No
     heating is necessary.  Final pH
     7.1  + 0.1. .This medium contains
     .sodium benzoate.

   b Growth media

     M-Endo Broth MF  is used, prepared
     as described in II,  A, 1 earlier in
     this outline. Alternately,  L. E. S.
     Endo Medium may  be used.

2  General filtration followed is in I, D.

   Special procedures are:

   a Transfer the membrane filter from
     the filtration apparatus to a pad
     saturated with benzoated M-Endo
     Broth.

   b Close the culture dishes and hold
     in a container at ambient temperature.
     This may be mailed or transported
     to a central laboratory. The mailing
     or transporting tube  should contain
     accurate transmittal data sheets which
     correspond to properly labeled dishes.

     Transportation time, in the case of
     mailed containers, should not exceed
     three days to the time of reception
     by the testing laboratory.

   c On receipt in the central laboratory,
     unpack mailing carton, and lay out
     the  culture  containers on the labora-
     tory bench.

    d Remove the tops from the culture
      containers. Using sterile forceps,
      remove each membrane and its
      absorbent pad to the other half of
      the  culture container.
      e With a sterile pipette or sterile
        absorbent pad, remove preservative
        medium from the culture container.

      f Place a sterile absorbent pad in
        each culture container, and deliver
        enough freshly prepared M-Endo
        Broth to saturate each pad.

      g Using sterile forceps, transfer the
        membrane to the new absorbent pad
        containing M-Endo Broth.  Place
        the membrane carefully to avoid
        entrapment of air between  the
        membrane and the underlying
        absorbent pad.  Discard the
        absorbent pad containing pre-
        servative medium.

      h After incubation of 20 + 2 hours
        at 35° C, count colonies as in the
        above section A,  2.

      i If L. E.S. Endo Agar is used, the
        steps beginning with (e) above are
        omitted; and the membrane filter is
        removed from the preservative
        medium and transferred to a fresh
        culture container with L.E.S.  Endo
        Agar, incubated, and colonies
        counted in the usual  way.

D  Verified Membrane Filter Coliform Test

   This procedure applies to  identification
   of colonies growing on Endo-type media
   used for determination of total coliform
   counts.  Isolates from these colonies are
   studied for gas production from lactose
   and typical coliform morphology.  In
   effect,  the procedure corresponds  with
   the  Completed Test stage of the multiple
   fermentation tube test for  coliforms.

          Procedure:

          1  Select a membrane filter bearing
             several well-isolated  coliform-type
             colonies.

          2  Using sterile technique, pick all
             colonies in a selected area with the
             inoculation needle, making transfers
             into tubes of phenol red  lactose broth
             (or lauryl sulfate tryptose lactose
                                                                                 29-15

-------
 Detailed Membrane Filter Methods
         broth). Using an appropriate data
         sheet record the interpretation of
         each colony, using,  for instance,  .
         "C" for colonies having the typical
         color and sheen of coliforms; "NCr
         for colonies not conforming to
         coliform colony appearance on
         Endotype media..

3  Incubate the broth tubes at 35° C+ 0.5°C,

4  At 24 hours:

   a  Read and record the results from
      the lactose broth fermentation tubes.
      The following code is suggested:
Code
 O
   No indication of acid or gas
   production, either with or
   without evidence of growth.

   Evidence of acid but not gas
   (applies only when a pH indicator
   is included in the broth medium)

   Growth with production of gas.
   If pH indicator is used, use
 .  symbol AG to show evidence of
   acid.  Gas  in any quantity is a
   positive test.

Tubes not showing gas production are
returned to the 35° C incubator.
   c  Gas-positive tubes are transferred
      as follows:

      1) Prepare a streak inoculation on
        EMB agar for colony Isolation, and
        using the same culture.

      2) Inoculate a nutrient agar slant.'

      3) Incubate the  EMB agar plates and
        slants at 35° C' + o. 5°C.
 5  At 48 hours:

    a  Read and record results of lactose
       broth tubes which were negative at
       24 hours and were returned for
       further incubation.
   b  Gas-positive cultures are subjected
      to further transfers as in 4c.
      Gas-negative cultures are discarded
      without further study; they are
      coliform- negative.

   c  Examine the cultures transferred
      to EMB agar plates and to nutrient
      agar slants, as follows:

      1) Examine the EMB agar plate for
        evidence of purity of culture; if
        the culture represents more than
        one colony type, discard the
        nutrient agar culture and  reisolate
        each of the representative colonial
        types on the EMB plate and  resume
        as with 4c for each isolation.
        ft purity of culture appears  evident,
        continue with c (2) below.

      2) Prepare a smear and Gram stain
        from each nutrient agar slant
        culture.  The Gram stain should
        be made on a culture not more
        than 24 hours old.  Examine under
        oil immersion for typical coliform
        morphology, and record results.

6  At 72 hours:

   Perform procedures  described  in 5c
   above,  and record results.

7  Coliform colonies are considered
   verified if the procedures  demonstrate
   a pure  culture of bacteria  which are
   gram negative nonspore-forming rods
   and produce  gas from lactose at 35° C
   within 48 hours.
                                        E Fecal Coliform Count (Based on M-FC
                                           Broth Base)

                                           The count depends upon growth on a
                                           special medium at 44. 5+0. 2°C.

                                           1  Preparation of Culture Medium
                                              (M-FC Broth Base) for Fecal
                                              Coliform Count
29-16

-------
                                                        Detailed Membrane Filter Methods
     a  Composition

     Tryptose                      10. 0 g
     Proteose Peptone No. 3         5.0g
     Yeast extract                   3.0 g
     Sodium chloride                5. 0 g
     Lactose                       12.5 g
     Bile salts No.  3                1.5 g
     Rosolic acid* (Allied           10. 0 ml
       Chemical)
     Aniline blue (Allied Chemical)   0.1 g
     Distilled water
1000 ml
  Filter membranes for fecal coliform
  counts consecutively and immediately
  place them in their culture containers.
  Insert as many as six culture containers
  all oriented in the same way (i. e., all
  grid sides facing the same direction)
  into the sacks and seal.  Tear off the
  perforated top, grasp the side wires,
  and twirl the sack to roll the open end
  inside the folds of sack. Then submerge
  the sacks with culture containers in-
  verted beneath the surface of a 44. 5
  + 0. 2 C waterbath.
     b  To prepare the medium dissolve
        37.1 grams in a liter of distilled
        water which contains 10 ml of 1%
        rosolic acid (prepared  in 0.2 N
        NaOH).

        Fresh solutions of rosolic acid give
        best results.  Discard  solutions
        which have changed from dark red
        to orange.

     c  To sterilize,  heat to boiling as
        directed in I,  C.

     d  Prepared medium may be retained
        up to 4 days in the dark at 2-8° C.

  2  Special supplies

     Small water proof plastic  sacks capable
     of being sealed against water with
     capacity of 3 to 6 culture containers.

   3  Filtration procedures are as given in
     I, D.

   4  Elevated temperature incubation
     a  Place fecal coliform count mem-
        branes at 44.5 + 0. 2°C as rapidly
        as possible.
              Ill
                     b  Incubate for 22+2 hours.

                  5  Counting procedures

                     Examine and count colonies as follows:

                     a  Use a wide field binocular dissecting
                        microscope with 5 -  10X magnification.

                     b  Low angle lighting from the side is
                        advantageous.

                     c  Fecal coliform colonies are blue,
                        generally  1-3 mm in diameter.

                     d  Record the colony counts on.the
                        data sheet, and report the fecal
                        coliform count per 100 ml of  sample.
                        (I, D, 17 illustrates  method)
TESTS FOR FECAL STREPTOCOCCAL
GROUP-MEMBRANE FILTER METHOD
               A 48 hour incubation period on a choice of
               two different media, giving high selectivity
               for fecal streptococci, are the distinctive
               features of the tests.
^Prepare 1% solution of rosolic acid in 0. 2 N NaOH.  This dye is practically insoluble in water.
                                                                                    29-17

-------
Detailed Membrane Filter Methods
 Test for Members of Fecal Streptococcal
 Group (Tentative, Standard Methods) M-
 Enterococcus Agar Medium

 1 Preparation of the culture medium

   a  Formula (The Difco formula is shown,
      but equivalent constituents from
      other sources are equally acceptable).
   Bacto tryptose             20. 0 g
   Bacto yeast extract          5. 0 g
   Bacto dextrose              2. 0 g
   Dipotassium phosphate       4. 0 g
   Sodium Azide                0.4 g
   Bacto agar                 10.0 g
   2, 3, 5.  Triphenyl          0.1 g
      tetrazolium chloride


   b  The medium is prepared by
      rehydration at the rate of 42 grams
      per 1000 ml of distilled water. It
      is recommended that the medium in
      dehydrated form be preweighed and
      dispensed into culture containers
      (about 25 X 150 mm) in quantities
      sufficient for preparation of 30 ml
      of culture medium (1. 26 g per tube).

   c  Follow I, C, for "sterilizing" medium
      and dispense while hot into culture
      containers.  Allow plates to harden
      before use.

2  List of apparatus, materials, as given
   in Table  I.

3  Procedure,  in general,  as  given in I.

   Special instructions

   a  Incubate for 48 hours, inverted,
       with  100% relative humidity, after
       filtrations are completed. If the
       entire incubator  does not have
       saturated humidity,  acceptable
       conditions can be secured by placing
       the cultures in a tightly closed
       container with wet paper, towels,
       or other moist material.
        After incubation, remove the
        cultures from the incubator,  and
        count all colonies under wide field
        binocular dissecting microscope
        with magnification set at 10X or
        2OX.  Fecal streptococcus colonies
        are 0. 5 - 2 mm in diameter, and
        flat to raised smooth, and vary
        from pale pink to dark red in color.

        Report  count per
        100 ml of sample.  This  is con-
        veniently computed:
        No. fecal streptococci per 100 ml =

No. fecal streptococcus colonies counted
     Sample filtration volume in ml
                                       X100
   Test for Members of Fecal Streptococcal
   Group based on KF-Agar

   1 Preparation of the culture medium

     a  Formula: (The dehydrated formula
        of Bacto 0496 is shown, but
        equivalent constituents from other
        sources are acceptable).  Formula
        is in grams per liter of reconstituted
        medium.
   Bacto proteose peptone #3      10. 0
   Bacto yeast extract            10. 0
   Sodium chloride (reagent grade)  5. 0
g
g
g
g
g
g
g
   Sodium glycerphosphate        10.0
   Maltose (CP)                  20.0
   Lactose (CP)                   1. 0
   Sodium azide (Eastman)         0.4
   Sodium carbonate               0.636 g
     (Na2CO  reagent grade)
   Brom cresol purple             0. 015 g
     (water soluble)
   Bacto agar                     20.0   g

     b  Reagent

         2, 3,  5-Triphenyl tetrazolium
         chloride reagent (TPTC)

         This reagent is prepared by making
         a 1% aqueous solution of the above
         chemical passing it through a Seitz
         filter or membrane filter. It can
    29-18

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                                                    Detailed Membrane Filter Methods
     be kept in the refrigerator in a
     screw-capped tube until used.

     The dehydrated medium described
     above is prepared for laboratory
     use  as follows:

     Suspend 7. 64 grams of the dehydrated
     medium in 100 ml of distilled water
     in a flask with an aluminum foil
     cover.

     Place the flask in a boiling water-
     bath, melt the dehydrated medium,
     and  leave in the boiling waterbath
     an addional 5 minutes.

     Cool the medium to 50°-60OC, add
     1. 0  ml of the TPTC reagent,  and
     mix.

     For membrane filter studies,  pour
     5-8  ml in each 50 mm glass or
     plastic culture dish or enough to
     make a layer approximately 1/8"
     thick.  Be sure  to pour plates before
     agar cools and solidifies.

     For plate counts, pour as for standard
     agar plate counts.


     NOTE:  Plastic dishes containing
     media may be stored in a dark,  cool
     place up to 30 days without change
     in productivity of the medium, pro-
     vided that no dehydration occurs.
     Plastic dishes may be incubated in
     an ordinary air incubator.  Glass
     dishes must be incubated in an
     atmosphere with saturated humidity.
   Apparatus,
   Table 1.
and materials as given in
3  General procedure is as given in I.
     Special instructions

    a  Incubate 48 hours, inverted with
       100% relative humidity after
       filtration.
                                     b  After incubation,  remove the
                                        cultures from the incubator, and
                                        count colonies under wide field
                                        binocular dissecting microscope,
                                        with magnification set at 10X or
                                        2 OX.  Fecal streptococcus colonies
                                        are pale pink to dark wine- color.
                                        In size they range from barely
                                        visible to approximately 2mm in
                                        diameter.  Colorless colonies are
                                        not  counted.

                                     c  Report fecal streptococcus count
                                        per 100 ml of sample .   This is
                                        computed as follows:

                                     No.  fecal streptococci per 100 ml =
                                No. fecal streptococcus colonies
                                 Sample filtration volume in ml
C Verification of Streptococcus Colonies

   1  Verification of colony identification
      may be required in waters containing
      large numbers of Micrococcus orga-
      nisms.  This has been noted
      particularly with bathing waters,  but
      the problem is by no means  limited to
      such waters.

   2  A verification procedure is  described
      in "Standard Methods for the Examination
      of Water and Wastewater]' 13th ed,
      .(1971).  The worker should  use
      this reference for the step-by-
      step procedure.

IV  PROCEDURES FOR USE OF MEMBRANE
    FILTER FIELD UNITS

  A Culture Media

    1  The  standard coliform media used with
       laboratory tests are used.

    2  To simplify field operations, it is
       suggested that the medium be sent to
       the field,  pre weighed, in vials or
       capped culture tubes.  The medium
       then requires only the addition of a
       suitable volume of distilled water-
       ethanol prior to sterilization.
                                                                                 29-19

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 Detailed Membrane Filter Methods
  3  Sterilization procedures in the field
     are the same as for laboratory methods.

  4  Laboratory preparation of the media,
     ready for use, would be permissible
     provided that the required limitations
     on time and conditions of  storage are
     met.

B Operation of Millipore Water Testing Kit,
  Bacteriological

  1  Supporting supplies and equipment are
     the same as for the laboratory
     procedures.

  2  Set the incubator voltage selector
     switch to the voltage of the available
     supply,  turn on the unit and adjust as
     necessary to establish operating
     incubator  temperature at 35+ 0.5°C.

  3  Sterilize the funnel unit assembly by
     exposure to formaldehyde  or by
     immersion in boiling water.  If a
     laboratory autoclave is available, this
     is preferred.

     Formaldehyde is produced by soaking
     an asbestos ring (in the funnel base)
     with methanol, igniting, and after a
     few seconds of burning, closing the
     unit by placing the stainless steel
     flask over the funnel and base. This
     results in  incomplete combustion of
     the methanol, whereby formaldehyde
     is produced.  Leave the unit closed
     for 15 minutes to allow adequate
     exposure to formaldehyde.

  4  Filtration  and incubation procedures
     correspond with laboratory methods.

  5  The unit is supplied with a booklet
     containing detailed step-by-step
     operational procedures. The  worker
     using the equipment should become
     completely versed in its contents and
     application.
C  Other commercially available field kits
   should be used according to manu-
   facturer's instructions.  It is emphasized
   that the standards of performance are
   required for field devices as for laboratory
   equipment.

 D .Counting of Colonies on Membrane Filters

   1  Equipment and materials

      Membrane filter cultures to be
      examined

      Illumination source

      Simple lens,  2X to 6X magnification

      Hand tally (optional)

   2  Procedure

      a  Remove the cultures from the
         incubator and arrange them in
         numerical sequence.

      b  Set up illumination source as that
         light will originate from an area
         perpendicular to the plane of
         membrane filters being examined.
         A small fluorescent lamp is ideal
         for the purpose.  It is highly
         desirable that a simple lens be
         attached to the light source.

      c  Examine results.   Count  all coliform
         and noncoliform colonies.  Coliform
         and noncoliform colonies.  Coliform
         colonies have a "metallic" surface
         sheen .under reflected light,  which
         may cover the entire colony or may
         appear only on the center.
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                                                         Detailed Membrane Filter Methods
        Noncoliform colonies range from
        colorless to pink or red, but do not
        have the characteristic "metallic""
        sheen.

        Enter the colony counts in the data
        sheets.

        Enter the coliform count per 100 ml
        of sample for each membrane having
        a countable number of coliform
        colonies. Computation is as follows:

        No. coliform per 100 ml =
  No.  coliform colonies on MF
  No.  milliliters sample filtered
X100
                     McCarthy,  J.A.,  Delaney, J.E. and
                        Grasso, R.J.   Measuring Coliforms
                        in Water.  Water and Sewage Works.
                        1961: R-426-31.   1961.
                 This outline was prepared by H. L. Jeter,
                 Director,  National Training Center,
                 EPA, WPO, Cincinnati, OH   45268.
Descriptors:  Biological Membranes,
Coliforms, Fecal Coliforms, Fecal
Streptococci, Filters,  Indicator Bacteria,
Laboratory Equipment, Laboratory Tests,
Membranes, Microbiology, Water Analysis
REFERENCES

 1   Standard Methods for the Examination of
       Water and Wastewater.  APHA,
       AWWA, WPCF.  12th Edition.   1965.
                                                                                    29-21

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