c/EPA
              United States
              Environmental Protection
              Agency
National Training
and Opera'tional
Technology Center
Cincinnati OH 45268
EPA-430/1-80-005
May 1980
             Water
             Plankton and
             Periphyton Analyses
             Training Manual

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                                                  EPA-430/ 1-80-005
                                                  May 1980
     Plankton and Periphyton Analyses
This course is offered for personnel concerned with
the evaluation of surface water by means of direct
observation and chemical and instrumental
measurements of microorganisms.

After successfully completing the course, the
student will be able to carry out basic laboratory
procedures in identification and  counting; recognize
common types of  organisms; calibrate a microscope;
carry counting and group identification to the point
of obtaining results which are qualitatively and
quantitatively accurate.

The training activities consists of laboratory and
classroom sessions.
   U. S. ENVIRONMENTAL PROTECTION AGENCY
           Office of Water Program Operations
  National Training and Operational Technology Center

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                       DISCLAIMER









Reference to commercial products, trade names,  or




manufacturers is for purposes of example and illustration.




Such references do not constitute endorsement by the




Office of Water Program Operations, U. S. Environmental




Protection  Agency.

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                                      CONTENTS
Title or Description




The Aquatic Environment




Classification of Communities, Ecosystems, and Trophic Levels




Limnology and Ecology of Plankton




Biology of Zooplankton Communities




Optics and the Microscope




Structure and Function of Cells




Types of Algae




Blue-Green Algae




Green and Other Pigmented Flagellates




Filamentous  Green Algae



Coccoid Green Algae




Diatoms




Filamentous  Bacteria




Fungi and the "Sewage Fungus" Community




Protozoa,  Nematodes, and Rotifers




Activated Sludge Protozoa




Free-Living Amoebae and Nematodes




Animal Plankton




Techniques of Plankton Sampling Programs




Preparation and Enumeration of Plankton in the Laboratory




Attached Growths (Periphyton or Aufwuchs)




Determination of Plankton Productivity




Algal Growth Potential Test




Algae and Actinomycetes in Water Supplies




Algae as Indicators of Pollution
Outline Number




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      25

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Title or Description                                                    Outline Number

Odor Production by Algae and Other Organisms                                26

Plankton in Oligotrophic Lakes                                                27

Using Benthic Biota in Water Quality Evaluation                                28

Ecology Primer                                                              29

Global Environmental Quality                                                 30

The Effects of Pollution on Lakes                                              31

Application of Biological Data                                                 32

Significance of "Limiting Factors" to Population Variation                      33

Algae and Cultural Eutrophication                                             34

Control of Plankton in Surface Waters                                          35

Control of Interference  Organisms in Water Supplies                            36

The Biology of Pipes, Conduits, and Canals                                    37

San Francisco Experience with Nuisance Organisms                            38

Laboratory:  Identification of Diatoms                                          39

Preparation of Permanent Diatom Mounts                                      40

Laboratory:  Identification of Animal Plankton                                  41

Laboratory:  Proportional Counting of Plankton                                 42

Calibration and Use of Plankton Counting Equipment                            43

Laboratory:  Fundamentals of Quantitative Counting                             44

Key to Selected Groups  of Freshwater Animals                                 45

Key to Freshwater Algae Common in Water Supplies and                        46
in Pollutated Water

A Key for the Initial Separation of Some Common Plankton Organisms            47

Classification - Finder  for Names of Aquatic Organisms in                      48
Water Supplies and Polluted Waters

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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;
and the atmosphere.

 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.
 II   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	
                   Highest heat capacity (specific heat) of any
                   solid or liquid (except NH^)
         Significance
Stabilizes temperatures of organisms and
geographical regions
                   Highest latent heat of fusion (except NH )
                                                o
Thermostatic effect at freezing point
                   Highest heat of evaporation of any substance
Important In heat and water transfer of
atmosphere
                   The only substance that has its maximum
                   density as a liquid (4°C)
Fresh and brackish waters have maximum
density above freezing point.  This is
important in vertical circulation pattern
in lakes.
                   Highest surface tension of any liquid
Controls surface and drop phenomena,
important 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 di-electric
                   constant of any liquid
Leads to high dissociation of inorganic
substances In solution
                   Very little electrolytic dissociation
Neutral, yet contains both H+ and OH  ions
                   Relatively transparent
Absorbs much energy in infra red and ultra
violet ranges, but little in visible range.
Hence "colorless"
 BI.21g. 3.80
                                                                                                   1-1

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The Aquatic Environment
B  Physical Factors of Significance

   1  Water substance

      Water is not simply "H,,C)" 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 H^; ordinary  oxygen O  ,
      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*
                '•)   (o18   7o">1   o")   (o
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).
1-2

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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 layer,  and the
   hypolimnion or lower layer, and
   in between is the thermocline
   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 Olg.   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
     of 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
                                                                               1-3

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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 thermocline
           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 miUipoises at 1  atm)
Temp, o c
-10
- 5
0
5
10
30
100
Dissolved solids in g/L
0
26.0
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.
 1-4

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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.
                                                                                    1-5

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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 Langmuire spirals (or Langmuire
         circulation) are a relatively
         massive cylindrical motion imparted
         to surface waters under the influence
         of wind.  The axes of the  cylinders
         are parallel to the direction of the
         wind, and their depth and velocity
                              depend on the depth of the water,
                              the velocity and duration of the
                              wind, and other factors.  The net
                              result is that adjacent cylinders
                              tend to rotate in opposite directions
                              like meshing cog wheels.  Thus
                              the water between two given spirals
                              may be meeting and sinking, while
                              that between spirals on either side
                              will be meeting and rising.  Water
                              over the  sinking, while that between
                              spirals on either side will be meet-
                              ing and rising.  Water over the
                              sinking areas tends to accumulate
                              flotsam and jetsam on the surface
                              in long conspicuous lines.

                              a  This phenomenon is of consider-
                                able importance to those sampling
                                for plankton (or even chemicals)
                                near the surface when the wind
                                is blowing.  Grab samples from
                                either dance might obviously
                                differ considerably,  and if
                                a plankton tow is contemplated
                                it should be made across the
                                wind in order that the net
                                may pass through a succession
                                of both dances.
             WATER
             RISING
                                                                   WATER
                                                                  SURFACE
WATER
SINKING
                  Figure  2.  Langmuire Spirals
                  b. Blue dance,  water  rising, r.  Red
                  dance,  water  sinking,  floating or
                  swimming ofojects  concentrated.
  1-6

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                                                             The Aquatic Environment
     b  Langmuire spirals are not
        usually established until the
        wind has either been blowing
        for an extended period,  or
        else is blowing rather hard.
        Their presence can be detected
        by the lines of foam and
        other floating material which
        coincide with the direction
        of the wind.

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.

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
arid 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.
                                                                                   1-7

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                    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.

    6   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.
Ill  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.

    2  From a functional standpoint,  an
       ecosystem has two parts (Figure 2)
                                                                                 1-9

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The Aquatic Environment
                                CO  NSUMERS
        PRO DUCERS
                                                       REDUCERS
                                    NUTRIENT
                                    MINERALS
                                      FIGURE 1
 B
  a  The autotrophic or producter
     organisms, which utilize light
     energy or the oxidation of in-
     organic compounds as their
     sole energy source.

  b  The heterotropic or consumer
     and reducer organisms which
     utilizes organic compounds for
     its energy and carbon requirements.

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
   called the PROTISTA  and MONERA.
                                                   These two groups can be defined on
                                                   the basis of relative complexity of
                                                   structure.

                                                   a  The bacteria and blue-green algae,
                                                      lacking a nuclear membrane are
                                                      the Monera.

                                                   b  The single-celled algae and
                                                      protozoa are 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.
 1-10

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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
Segmented Worms
Molluscs
Bryozoa
Rotifers
Roundworms
Flatworms
Mammals
Birds
Reptiles
Amphibians
Fishes
Primitive
Chordates
Echinoderms

Coelenterates
Sponges
Basidiomycetes

Fungi Imperfect!
Ascomycetes

Higher Phycomycetes
         DEVELOPMENT OF MULTICELLULAR OR COENOCYTIC STRUCTURE

                               PROTISTA
                               Protozoa
 Unicellular Green Algae

 Diatoms

 Pigmented Flagellates
Amoeboid

Flagellated,
 (non-pigmented)
Cilliated

Suctoria
Lower

  Phycomycetes

  (Chytridiales, et. al. )
                     DEVELOPMENT OF A NUCLEAR MEMBRANE
                                                   J
                                MONERA
 Blue Green Algae


         Phototropic Bacteria


                Cheiriotropic Bacteria
                                   Actinomycetes

                               Spirociiaotes
                     Saprophyt :c
                     Bacterial
                     Types
                                    I  I
 BI. ECO. pi. 2a. 1. 69
                                FIGURE 2
                                                                               1-11

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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 cf the pond ecosystem. Basic units are as follows: I, abiotic substances—basic inorganic and
         organic compounds; IIA, producers—rooted vegetation; IIB, producers-phytoplankton; III-1A, primary consumers
         (herbivores)-bottom forms; Ill-IB, primary consumers (herbivores)— zooplankton; III-2, secondary consumers (car-
         nivores); III-3, tertiary consumers (secondary carnivores); IV, decomposers-bacteria and fungi of decay.
 1-12

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                                        	   The Aquatic Environment
Figure 4.  A MARINE ECOSYSTEM (After Clark, 1954 and Patten, 1966)
                                                                13

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 The Aquatic Environment

(a)
Decomposers fl Carnivores (Secondar
M Carnivores (Primary
| | Herbivores
| Producers |


(b)
1
1 1
(c)
Pn
("UvA/// / 1
?{]// /////////
[///>/ / 1 1 1 1 1 I 1 1 1 1 1 1



///I
 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.
   1-14

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                                                                  The Aquatic Environment
I  PERSISTENT CHEMICALS IN THE
   ENVIRONMENT

Increasingly complex manufacturing processes,
coupled with rising industrialization, create
health hazards for humans and aquatic life.

Compounds besides being toxic (acutely or
chronic) may produce mutagenic effects
including cancer,  tumors, and teratogenicity
(embryo defects).  Fortunately there are tests,
such as the Amis test, to screen chemical
compounds for these effects.


A   Metals - current levels of cadmium, lead
     and other substances constitute a mount-
     ing concern.   Mercury pollution, as at
     Minimata, Japan has been fully documented.

B Pesticides

   1  A pesticide and its metabolites may
      move through an ecosystem in many
      ways.  Hard (pesticides which are
      persistent, having a long half-life  in
      the environment includes the organo-
      chlorines, ex., DDT) pesticides
      ingested or otherwise borne by the
      target species will stay in the
      environment, possibly to be recycled
      or concentrated further through the
      natural action of food chains if the
      species is eaten.  Most of the volume
      of pesticides do not reach their target
      at all.

   2  Biological magnification

      Initially, low levels of persistent
      pesticides in air, soil,  and water  may
      be concentrated at every step up the
      food chain.  Minute aquatic organisms
      and scavengers, which screen water and
      bottom mud  having pesticide levels of a
      few parts per billion, can accumulate
      levels measured in parts per million—a
      thousandfold increase.  The sediments
      including fecal deposits are continuously
      recycled by the bottom animals.
D
  a Oysters,  for instance, will con-
    centrate DDT 70, 000 times higher
    in their tissues than it's concentration
    in surrounding water.  They can
    also partially cleanse themselves
    in water free of DDT.

  b Fish feeding on lower organisms
    build up concentrations in their
    visceral fat which may reach several
    thousand parts per million and levels
    in their edible flesh of hundreds of
    parts per million.

  c Larger animals, such as fish-eating
    gulls and other birds, can further
    concentrate the chemicals.  A survey
    on organochlorine residues in aquatic
    birds in the Canadian prairie provinces
    showed that California and ring-billed
    gulls were  among the most contaminated.
    Since gulls breed in colonies,  breeding
    population changes can be detected and
    related to levels of  chemical con-
    tamination.  Ecological research on
    colonial birds to monitor the effects
    of chemical pollution  on the environ-
    ment is useful.

"Polychlorinated biphenyls'1 (PCB's).
PCB's were used in plasticizers, asphalt,
ink, paper, and a host of other products.
Action was taken to curtail their release
to the environment, since  their effects
are similar to hard pesticides.  However
this doesn't solve the problems of con-
taminated sediments and ecosystems and
final fate  of the  PCB's still circulating.

There are numerous other compounds
which are toxic and accumulated in the
ecosystem.
                                                                                        1-15

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The Aquatic Environment
REFERENCES                                  5  Odum,  E.P.  Fundamentals of Ecology.
                                                     W. B. Saunders Company,
1  Clarke,  G. L.  Elements of Ecology.                 Philadelphia and London.   1959.
      John Wiley & Sons,  New York.   1954.
                                               6  Patten, B.C.  Systems Ecology.
2  Cooke  W.B.  Trickling Filter Ecology.             Bio-Science.   16(9).   1966.
      Ecology 4
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                            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 is 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.
II  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.  Meanders
   and  ox-bow lakes are often formed.
                                                                                       1-17

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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,  organic 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, and the resulting hole
1-18
   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
   lake s.

   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
  Elodea (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
                                                                                       1-19

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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 and 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.

 D 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.
1-20

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                                                                The Aquatic Environment
  C 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.
 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  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.

 3  Hynes, H.B.N.  The Ecology of Running
       Waters.  Univ. Toronto Press.
       555 pp.  1970.
    De Santo, Robert S.
      Applied Ecology.
      Verlag.  1978.
Concepts of
Springer-
                                                                                      1-21

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       Part 4.   The Marine Environment and its Role in the Total Aquatic Environment
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)
                  1.  THE WATER CYCLE
   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)
                  TABLE 1

  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
S°4
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
<-HCO, 0.35
0
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).
                                                  E  FRESHWATER, ESTUARINE,
                                                     MARINE ENVIRONMENTS
                                 AND
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.
                                       23

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  The Aquatic Environment
Type of environment
and general direction
 of water movement
                                    Degree of instability
Salinity
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.
  1-24

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                                                 The Aquatic Environment
                       MARINE ECOLOGY

                             PEL  A  G I C-
                                        -OCEANIC—

                                        € f i f » I at I e
                                                               ApC'Ot
                                                               dOIN


                                                              Jl
                             ////S/J'S/J'SS/J'J'SSSSm
PCLMIC
  Nirilie
  Octonic
    Cpiptlaga
    Uetopetigie
BENTHIC (BoMom)
  Supro-liMorol
  Litlorol (IMtrliJol)
  Sublilloral

    OuKi
  Bolhyol
  Abyliol
  Hotel
                                                        U i i a p a lag it
                                                  *bSSJ'SJSSSJSSS
                     BENTHIC
             FIGURE 3—ClassifittitioH 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.

2  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 nsritic 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.

                                       k  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.
                                                                        1-25

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 The Aquatic Environment
III  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%o) 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 %a (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
      tolerate.  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.
   1-26

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                                                               The Aquatic Environment
C  Marine, estuarine,  and fresh water
   organisms.  (See Figure  4)
Fresh Water
Stenohal ine
             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 meso-  or euryhaline and
     meso- or eurythermal.
              10
15   20
Sa
                           25   30  35
                   Salinity

     Figures.  DISTRIBUTION OF
                ORGANISMS IN AN ESTUARY

        a  Euryhaline,  freshwater
        b  Indigenous, estuarine, (mesohaline)
        c  Euryhaline,  marine
   4   Some will 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 hatch in headwater streams,
       migrate far out to feed and grow,
       then return to the mountain 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
      water 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.
                                                                                     1-27

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The Aquatic Environment
                                                                         dfr
                                                                          A'"'  '
     SNAILS
 a   Littorina neritoides
 C>   L. rudis
 0   L. obtusata
 Q   L. littorea

     RAitNACLES

 *   Chthamalus stellatus
 ®   Balanus balanoides
 (fy   B. perforatus
                                                               , ''^Sfer -'*»
                                                              /^,V>>*
                                                              '.'.:.••;.:••••••,.•.• •-•i.»,^a  fl  o
                                                              'l-;''^ '•^.'•.•'.•••••"^-.•':-'--.:f'"f
                                                           M^^'a'^-'g-t&Z
                                                         ^fPSS^% -
                                                                              •'
                                      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 major 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. Fucus spiralis; c. Ascophyllum nodosum; d.  Fucus serratus;
            e. Laminaria digitata. (Based on Stephenson)
    28

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                                                               The Aquatic Environment
V FACTORS AFFECTING THE                    REFERENCES
   PRODUCTIVITY OF THE MARINE
   ENVIRONMENT                               1  Harvey,  H. W.  The Chemistry and
                                                      Fertility of Sea Water (2nd Ed.).
A The sea is in continuous circulation. With-           Cambridge Univ.  Press, New  York.
   out circulation, nutrients of the ocean would          234 pp.  1957.
   eventually become a part of the bottom and
   biological production would cease.  Generally,   2  Wickstead, John H.  Marine Zooplankton
   in all oceans there exists a warm surface             Studies in Biology no. 62.   The Institute
   layer which  overlies the colder water and             of Biology. 1976.
   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 would'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"!


A CKNOWLEDGEMENT:

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.
                                                                                     1-29

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                                        Part 5:  Wetlands
  I   INTRODUCTION

  A  Broadly defined, wetlands are areas
     which are "to wet to plough but too
     thick to flow. "   The soil tends to be
     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.
 B
They are prodigiously productive
however, and many constitute an
absjolutely 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 earth'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     L-rr	-.^H--r=lI^Er-^=
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.
    "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.  Zonatlon in a positive Now England estuary. 1. Spring tide level. 2. Mean Ugh tide,
                                          3. Mean low tide, 4. Bog hole. 5. Ico cleavage pool, 6. Chunk of Spartina turf deposited by ice.
                                          T, Organic ooze with associated community, 6. eolgrass (Zoatera), 9. Ribbed mussels (modiolus)-
                                          clom (mm) - mud snail (Nassa) community. 10. Sea lettuce (Ulva)
                                                                                         1-31

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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-IB iioo >ec
                                                                            0
                                                                     MHW 0' 1930 ! 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.
  1-32

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                                                               The Aquatic Environment
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 A River Mouth on a Slowly Rising Coast.  Note absence
                                                     of deltaic development and relatively little marshland,
                                                     although mud flats stippled are extensive.
                       Shifting flats
                                                                     o I Terrestria
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.
                                                                                    1-33

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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.
     TROPICU.   CONOCAHPUS     . AWCENMA
     fOBlST  TRANSITION ASSOCICS SALT-MARSH ASSOOCS
RHIZOPMORA
CONiOCIES
                               MOOUM not
  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
                                      ((trams/ square meters/year)
Ibs/acre/year
                 Land deserts, deep oceans        Tens
                 Grasslands, forests, cutrophlc     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            Percentage Composition
                                                        Dry Wt.    Protein   Fat     Fiber    Water
                                                         Ash
                                                                N-free Extract
                 Dis'ichiis spicara (pure stand, dry)
                  2.8       5.3     1.7      32.4      8.2      6.7        45.5
                 Short Spartina altcrnillora and Saficornia curopaea (in standing water)
                  1.2       7.7     2.5      31.1      8.8      12.0        37.7
                 Spartina alternillora (tall, pure stand in standing water)
                  3.5       7.f>     2.0      29.0      8.3      15.5        37.3
                 Sparfina pa":m 'pur': Mand, dry)
                  3.2       Ci.0     2.2      30.0      8.1      9.0        44.5
                 Spwiina altcrnillura and Spjrt'nu porens (mixed stand, wet)
                  3.4       6.8     1.9      29.11      8.1      10.4        42.8
                 SfMrtinj altcrniflura (short, wrt)
                  2.2       0.0     2.4      30.4      8.7      13.3        36.3
                Comparable Analyses lor Hay
                 Isi r in      fi.O     2.0      36.2      6.7      4.2        44.9
                 2nd < ill     11.0     3.7      2U.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
   1-34

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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.
      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.
                                                                          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.
                                                         Greater yellow legs (left)
                                                           and black duck
                                                                       Great blue heron  <"


                                                      Figure Q  Some Common Marsh Birds
                                                                                        1-35

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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
      eutrophi cation  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
 1-36

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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.
                                                                                      1-37

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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.
 1-38

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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.   19S7.

                          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.
     Deevey, E.S., Jr.
        199(4):115-122.
Bogs.  Sci. Am. Vol.
October 1958.
                          This outline was prepared by H.  W.  Jackson,
                          former Chief Biologist,  National Training
                          Center,  and revised by R. M. Sinclair,  Aquatic
                          Biologist, National Training Center,  MOTD,
                          OWPO, EPA, Cincinnati, OH 45268.
  3  Emery, K. O. and Stevenson.  Estuaries
        and Lagoons.  Part n, Biological
        Aspects by J.W. Hedgepsth, 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.
Descriptors: Aquatic Environment, Biological
Estuarine Environment, Lentic Environment,
Lotic Environment, Currents, Marshes,
Limnology,  Magnification,  Water Properties
                                                                                     1-39

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        CLASSIFICATION OF COMMUNITIES, ECOSYSTEMS, AND TROPHIC LEVELS
 I  A COMMUNITY is an assemblage of
    populations of plants, animals, bacteria,
    arid fungi that live in an environmental and
    interact with one  another, forming together
    a destinctive living system with its  own
    composition, structure, environmental
    relations, development, and function.

II   An ECOSYSTEM is a community and its
    environment treated together as a functional
    system of complementary relationships,
    and transfer and circulation of energy and
    matter.  (A delightful litte essay on the
    odyssey of atoms X and Y through an
    ecosystem is in Leopold's, A Sand County
    Almanac.)

III  TROPHIC levels are a convenient means
    of classify ing organisms according to
    nutrition,  or food and feeding.  (See
    Figure 1. )

A  PRODUCER, the  photo synthetic plant or
    first organism on the food chain sequence.
    Fossil fuels were produced photosynthe-
    ticallyl

B  Herbivore or primary CONSUMER,  the
    first animal which feeds on plant food.

C  First carnivore or secondary CONSUMER,
    an animal feeding on a plant-eating  animal.

D  Second carnivore or tertiary CONSUMER
    feeding on the preceding.

E  Tertiary carnivore.

F  Quaternary carnivore.

G  DECOMPOSERS OR REDUCERS, bacteria
    which break down the above organisms.
    Often called the middlemen or stokers of
    the furnace of photosynthesis.

H  Saprovores or DETRITIVORES which feed
    on bacteria and/or fungi.

I   Macroinvertebrates have been subdivided
    into trophic  levels according to feeding
    habits (See Figure 1 from Cummin's).
     1  Collectors strain, filter, or otherwise
        collect fine particulate organic matter
        from the passing current.

     2  Shredders feed on leaves,  detritus,
        and coarse particulate organic matter.

     3  Grazers feed on attached growths.

     4  Predators feed on other organisms.

IV   Taxonomic Groupings

 A  TAXOCENES, a specific group of organisms.
     Ex. midges.  For obvious reasons most
     systematists  (taxonomists) can specialize
     in only one group of organisms.  This fact
     is difficult for the non-biologist to graspl

 B  Size,  which is often dictated by the inves-
     tigator's sampling equipment and specific
     interests.

 V   Arbitrary due to organism habitat prefer-
     ences, available sampling devices,
     personal preference of the investigator,
     and mesh sizes of nets and sieves.

 A  PLANKTON,  organisms suspended in a
     body of water and at the mercy of currents.
     This group has  been subject to numerous
     divisional schemes.  Plants are PHYTO-
     PLANKTON,  and animals,  ZOOPLANKTON.
     Those retained  by nets are obviously, MET
     PLANKTON.  Those passing thru even the
     finest meshed nets are NANNOPLANKTON.

 B  PERIPHYTON, the community of micro-
     organisms which grow on  submerged
     objects (substrates).  Literal meaning
     "to grow around plants", however
     standard glass  microslides are sub-
     mersed in the aquatic habitat to
     standardize results.

  C  BENTHOS, is often used to mean
     MACROINVERTEBRATES, although there
     are benthic organisms  in other plant,
     animal, and protist groups.   Benthic
     refers strictly to the bottom substrates of
     lakes, streams, and other water bodies.
  BI. ECO. 25a. 3. 80
                                       2-1

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Classification of Communities. Ecosystems and Trophic Levels
                                        PRODUCERS
                                        (PHYTOPLANKTON)
                                            OLLECTORS
                                         (ZOOPLANKTON)
                                                                                 EDATORS
                                                                                    •QMS
                                          FIGURE  1
  2-2

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                                Classification of Communities. Ecosystems and Trophic Levels
D   MACROINVERTEBRATES, are animals
    retained on a No.  30 mesh screen (approx-
    imately 0. 5 mm) and thus visible to the
    naked eye.

E   MACROPHYTES,  the larger aquatic plants
    which are divided into emersed, floating,
    and submersed communities.  Usually
    vascular plants but may include the larger
    algae and "primitive" plants.  These have
    posed tremendous economic problems in
    the large man-made lakes, especially
    in tropical areas.

F   NEKTON, in freshwater,  essentially fish,
    salamanders, and the larger Crustacea.
    In contrast to PLANKTON, these organisms
    are not at the mercy of the current.

G   NEUSTON,  or PLEUSTON, are inhabitants
    of the surface film (meniscus organisms),
    either supported by it, hanging from,  or
    breaking through it. Other organisms
    are trapped by this neat little barrier of
    nature.  The micro members of this are
    easily sampled by placing a clean cover
    slip on top of the surface film then either
    leaving it a  specified time or examining
    it immediately under the microscope.

H   DRIFT, macroinvertebrates which drift
    with the streams current either periodically
    (diel or 24 hour),  behaviorally, catastro-
    phically or incidentally.

I    BIOLOCIAL FLOGS, are suspended
    microorganisms that are formed by
    various means.  In wastewater treatment
    plants they are encouraged in concrete aer
    aeration basins using diffused air or
    oxygen (the  heart of the activated sludge
    process).
J  MANIPULATED SUBSTRATE COMMUNI-
   TIES.  Like the preceding community,
   theses are manipulated by man.   Placing
   artificial or natural substrates in a body
   of water will cause these communities
   to appear thereon.

K  We will again emphasize ARBITRARY,
   because  organisms confound our neat
   little schemes to classify them.   Many
   move from one community to another
   for various reasons.  However,  all
   these basic scheme do have intrinsic
   value,  provided they are used with
   reasonl
 REFERENCES

 1 Cummins,  Kenneth W.  The Ecology
      of Running Waters, Theory and
      Practice in Proc.  Sandusky River
      Symposium.  International Joint
      Commission.  1975.
 2 Leopold, Aldo.  A Sand County Almanac.
      Oxford University Press.   1966.

 3 Peters, Robert Henry.  The Unpredictable
      Problems of Tropho-dynamics.
      Env. Biol. Fish 2:97-101.  1977.
 This outline was prepared by R. M. Sinclair,
 Aquatic Biologist, National Training and
 Operational Technology Center, MOTD,
 OWPO, USEPA, Cincinnati, Ohio  45268.

 Descriptors:  Biological Communities
                                                                                      2-3

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                    LIMNOLOGY AND ECOLOGY OF PLANKTON
  INTRODUCTION

  A  Most Interference Organisms are
      Small.

  B  Small Organisms generally have
      Short Life Histories.

  C  Populations of Organisms with
      Short Life Histories may Fluctuate
      Rapidly in Response to Key Environ-
      mental Changes.

  D  Small Organisms are Relatively
      at the Mercy of the Elements

  E  The Following Discussion will
      Analyze the Nature of These Ele-
      ments with Reference to the Res-
      ponse of Important Organisms.
  PHYSICAL FACTORS OF THE ENVIRON-
  MENT

  A   Light is a Fundamental Source of
       Energy for Life and Heat.

       1    Insolation is affected by geo-
           graphical location and mete-
           orological factors.

       2    Light penetration in water is
           affected by angle of incidence
           (geographical),  turbidity,  and
           color.  The proportion of light
           reflected depends on the angle
           of incidence,  the temperature,
           color, and other qualities  of
           the water.  In general, as the
           depth increases arithmetically,
           the light tends to decrease geo-
           metrically.  Blues, greens, and
           yellows tend to penetrate most
           deeply while  ultra violet,  vio-
           lets, 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.

       3    Turbidity may originate within
        or outside of a lake.

        a    That which comes in from
             outside (allochthonous) is
             predominately inert solids
             (tripton).

        b    That of internal origin (auto-
             chthonous) tends to be bio-
             logical in nature.

B   Heat and Temperature Phenomena
    are Important in Aquatic Ecology.

    1   The total quantity of heat avail-
        able to a body of water per year
        can be calculated and is known
        as the heat budget.

    2   Heat is derived directly from in-
        solation; also by transfer from
        air, internal friction, and other
        sources.

C   Density Phenomena

    1   Density and viscosity affect the
        floatation and locomotion of
        microorganisms.

        a    Pure fresh water achieves
             its maximum density at 4  C
             and its maximum viscosity
             at 0°C.

        b    The rate of change of density
             increases with the temperature.

    2   Density stratification affects
        aquatic life and water uses.

        a    In summer,  a mass of warm
             surface water, the epilimnion,
             is usually present and  separated
             from a cool  deeper mass,  the
             hypolimnion, by a relatively
             thin layer known as the
             thermocline.

        b    Ice  cover and annual spring
             and fall overturns are  due to
             successive seasonal changes
             in the relative densities of
             the  epilimnion and the hypo-
BI. MIC. eco. 4e.3.80
                                                                                   3-1

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 Limnology and Ecology of Plankton
               limnion, profoundly influ-
               enced by prevailing meteoro-
               logical conditions.

               The sudden exchange of
               water masses having differ-
               ent chemical characteris-
               tics may have catastrophic
               effects on certain biota,  may
               cause others to bloom.
D  Shore development, depth,  inflow -
   outflow pattern,  and topographic
   features affect the behavior of the water.

E  Water movements that may affect organ-
   isms include such phenomena as waves,
   currents, tides,  seiches, floods, and
   others.
           d   Silt laden waters may seek
               certain levels, depending
               on their own specific gravity
               in relation to existing layers
               already present.

           e   Saline waters will also
               stratify according to the
               relative densities of the
               various layers.

   3  The viscosity of water is greater
     at lower temperatures.

           a   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 generates heat, it is
               significant in the heating
               of water by internal friction
               from wave and current ac-
               tion and many delay the
               establishment of anchor
               ice under  critical conditions.
        Waves or rhythmic movement

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

        b  Standing waves or seiches
           occur in all lakes but are
           seldom large enough to be
           observed.  An "internal seich"
           is  an oscillation in a density
           mass  within a lake with no
           surface  manifestation may
           cause considerable water
           movement.
               It is easier for plankton
               to remain suspended in cold
               viscous (and also dense)
               water than in less viscous
               warm water.   This  is re-
               flected in differences in the
               appearance of winter vs
               summer forms of life (also
               arctic vs tropical).
3-2

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                                           Limnology and Ecology of Plankton
Currents

a   Currents are arhythmic
    water movements which have
    had major  study only in ocean-
    ography. They primarily
    are concerned with the trans-
    location of water masses.
    They may be generated inter-
    nally by virtue  of density
    changes, or externally by
    wind or runoff.

b   Turbulence phenomena or
    eddy currents are largely re-
    sponsible for lateral mixing
    in a current.  These are of
    far more importance in the
    economy of a body of water
    than mere  laminar flow.

c   Tides, or rather tidal
    currents, are reversible
    (or oscillatory) on a relative-
    ly long and predictable period.
    They are closely allied to
    seiches. For all practical
    purposes,  they are restricted
    to oceanic  (especially coastal)
    waters.

    If there is  no freshwater
    inflow involved, tidal currents
    are basically "in and out;"
    if a significant amount of
    freshwater  is added to the
    system at a constant rate, the
    outflowing current will in general
    exceed the inflow  by the amount
    of freshwater input.
           There are typically two tidal
           cycles per lunar day (approx-
           imately 25 hours),  but there is
           continuous gradation from this
           to only one cycle per (lunar) day
           in some places.

           Estuarine plankton populations
           are extremely influenced by local
           tidal patterns.

         d Flood waters range from torren-
           tial velocities which tear away
           and transport vast masses of
           substrate to quiet backwaters
           which may inundate normally dry
           land areas for extended periods
           of time.  In the former  case,
           planktonic life is flushed away
            completely; in the  latter,  a local
            plankton  bloom may develop which
            may be of immediate significance,
            or which may serve as an inoculum
            for receding  waters.

 F  Surface Tension and the Surface Film

    1      The surface film is the habitat of
          the "neuston", a group of special
          significance.

    2     Surface tension lowered by surfactants
          may eliminate the  neuston.  This can
          be a significant biological observation.

Ill  DISSOLVED SUBSTANCES

 A Carbon dioxide is released by plants and
    animals in respiration, but taken in by
    plants in photosynthesis.

 B Oxygen is the biological complement of
    carbon dioxide, and necessary for all
    animal life.

 C Nitrogen and phosphorus are fundamental
    nutrients for plant life.

     1     Occur in great dilution, concentrated
          by plants.
                                                                           3-3

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 Limnology and Ecology of Plankton
            The distribution.of nitrogen
            compounds is generally correlat-
            ed with the oxygen curve, espe-
            cially in oceans.
 D  Iron, manganese, sulphur, and silicon
    are other minerals important to aquatic
    life which exhibit biological stratification.

 E  Many other minerals are present  but their
    biological distribution in waters is less
    well known, fluorine,  tin, and vanadium
    have recently been added to the "essential"
    list, and more may well follow.

 F  Dissolved organic matter is present in
    even the purest of lakes.

IV  BIOLOGICAL FACTORS

 A  Nutritional Classification of Organisms

    1        Holophyt ic or independent or-
            ganisms, like green plants,  pro-
            duce their own basic food elements
            from the physical environment.

    2        Holozoic or dependent organisms,
            like animals, ingest and  digest
            solid food particles of organic
            origin.

    3        Saprophytic or carrion eating
            organisms, like many fungi and
            bacteria, digest and assimilate
            the dead bodies of other organ-
            isms or their products.

 B  The Prey-Predator Relationship is
    Simply one Organism Eating Another.

 C  Toxic and Hormonic Relationships

    1        Some organisms such as  certain
            blue green algae and  some ar-
            mored flagellages produce sub-
            stances poisonous to  others.

    2        Antibiotic action in nature is
            not well understood but has been
            shown to play a very  influential
            role in the economy of nature.
V  BIOTIC COMMUNITIES (OR ECOSYSTEMS)

A  A biotic community will be defined here
   as an assemblage of organisms living in
   a given ecological niche (as defined
   below). Producer (plant-like), consumer
   (animal-like) and reducer (bacteria and
   fungi) organisms are usually included.
   A source of energy (nutrient, food) must
   also be present.  The essential concept
   in that each so-called community is a
   relatively independent entity.  Actually
   this position is only tenable at any given
   instant, as individuals are constantly
   shifting from one community to another in
   response to stages in their life cycles,
   physical conditions, etc.  The only one
   to be considered in detail here is the
   plankton.

B  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.

   1     Those that pass through a plankton
        net (No. 25 silk bolting cloth or
         equivalent) or sand filter are often
        known as nannoplankton (they
        usually greatly exceed the "net"
        plankton in actual quantity).

   2     Those less than four microns in
         length are sometimes called
         ultraplankton.

   3     There are many ways in which
        plankton may be classified:  taxo-
        nomic, ecological, industrial.

   4     The concentration  of plankton varies
         markedly in space and time.

         a   Depth, light, currents,  and
             water quality profoundly affect
             plankton distribution.

         b   The relative abundance  of
             plankton in the various sea-
             sons is generally:
                                                               1 spring,
                                                               4 winter
                       2 fall, 3 summer,
   3-4

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                                             Limnology and Ecology of Plankton
    5        Marine plankton include many
            larger animal forms than are
            found in fresh waters.

 C  The benthic community is generally
    considered to be the macroscopic life
    living in or on the bottom.

 D  The jDeripJivton community might be
    defined as the microscopic benthos,
    except that they are by no means confined
    to the bottom.  Any surface, floating, or
    not, is usually covered by film of living
    organisms. There is frequent exchange
    between the periphyton and plankton
    communities.

 E  The_ngktqn is the community of larger,
    free-swimming animals (fishes,  shrimps,
    etc.), and so is dependent on the other
    communities for basic plant foods.

 F  Neuston or Pleuston

    This community inhabits  the air/water
    interface, and may be suspended above
    or below  it or break it.  Naturally this
    interface is a very critical one,  it being
    micro molecular and allowing interchange
    between atmospheric contaminants and
    the water medium.  Rich in bacteria,
    metals,  protozoa,  pesticides etc.

VI   THE EVOLUTION OF WATERS

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

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

     1       Establishment of birth. In an
             extant stream,  this might be
             a "dry run" or  headwater
             streambed,  before it had eroded
             down to the level of ground water.

     2       Youthful streams; when *he
             stream bed is eroded below the
             ground water level, spring water
             enters  and the stream becomes
             permanent.

     3       Mature streams; have wide
             valleys,  a developed flood plain.
         deeper, more turbid,  and usually
         warmer water,  sand,  mud, silt,
         or clay bottom materials which
         shift with increase in flow.

   4     In old age, streams have approa-
         ched base level.  During flood
         stage they scour their bed and de-
         posit 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.

         (Under  the influence of man this
         pattern may be broken  up, or tem-
         porarily interrupted.  Thus as essen-
         tially "youthful" stream might take
         on some of the characteristics of a
         "mature" stream following soil
         erosion, organic enrichment,  and
         increased surface runoff.  Correction
         of these conditions might likewise be
         followed by at least a partial rever-
         sion to  the "original" condition.)

C  Lakes have a developmental history
   which somewhat parallels that of streams.

   1     The method of formation greatly
         influences the character and sub-
         sequent history of lakes.

   2     Maturing or natural eutrophication
         of lakes

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

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

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

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

         e  Dissolved carbonates and other
           materials are precipitated in the
           deeper portions of the lake in
           part through the action of plants.
                                                                                 3-5

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  Limnology and Ecology of Plankton
             f    When filling is well advanced
                 sphagnum mats extend out-
                 ward 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 out-
                 let.

             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 pro-
                 cesses may act concurrently)

                 When man hastens the above
                 process,  it is often called
                 "cultural eutrophication. "

VII PRODUCTIVITY

  A The biological resultant of all physical
    and chemical factors is 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 producti-
    vity is a "poor" water biologically, and
    also a relatively "pure" or "clean" water;
    hence  desirable as a water supply.  A
    productive water on the other hand may
    be  a nuisance to man or highly desirable.
    Some 01 the factors which influence the
    productivity of waters are as  follows:
                             i
  B Factors affecting  stream productivity.
    To be  productive of plankton,  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.  Tempera-
      tures in mountainous 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, temperature 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 avail-
      able for plankton growth increases,
      the balance between turbidity,
      nutrient levels, and temperature
      and other seasonal conditions,
      determines the overall produc-
      tivity.

Factors Affecting the Productivity
of Lakes

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 shoreline, 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).

2     Hard waters are generally more
      productive than soft waters as
      there are more plant nutrient
      minerals  available.  This is often
  3-6

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                       FACTORS AFFECTING PRODUCTIVITY
                                 Geographic Location
   Human
   Influence
                                           Latitude
                                                  Longitude
                                                         Altitude
                     Geological
                     Formation
Topography
Composition
of Substrate
                                            Shape of Basin
                                               Area    Bottom   \   Precipitation // \ Insolation
                                                     Conformation \      ^,     / / \
 Drainage
  Area
   Sewage
 Agriculture
   Mining
Primary
Nutritive
Materials
Nature oŁ    Inflow of —^- Trans-—^^-Lighft      Heaf Penetration ^D? Penetra  Develop of^*> Seasonal Cycle
 Bottom  ^Allochthonous  parency   Penetration  and Stratification  ^  and       Littoral   Circulat. Stagnation
 Deposit*    Materials                 , .  /         /        _j^ Utilization   Region     Growing Season
                 -T?
                              Trophic Nature of a Lake
                                                                                             o
                                                                                             I—'
                                                                                             o
                                                                                                                 a
                                                                                                                 H
                                                                                                                 o
                                                                                                                 o

-------
 Limnology and Ecology of Plankton
            greatly influenced by the
            character of the soil and rocks
            in the watershed,  and the quality
            and 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 affect
            productivity by distributing
            nutrients throughout the water
            mass.

   5        Climate,  temperature, pre-
            valance of ice and snow,  are
            also important.

D 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
            deliberately increase of decrease
            productivity.  This can be dem-
            onstrated 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 re-
            moved from the reservoir is also
            important.  The upper epilimnion
            may have a high plankton turbi-
            dity while lower down the plankton
            count may be less,  but a taste
           and odor causer (such as Mallpj-
           monas) may be present. There
           may be two thermoclines,  with
           a mass of muddy water flowing
           between a clear upper epilimnion
           and a clear hypolimnion.   Other
           combinations ad infinitum may
           occur.
      3     Reservoir discharges also pro-
           foundly 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 periodically.

VIII 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 means of
      classification.
      1
         Oligotrophic lakes are the
         geologically younger, less produc-
         tive lakes, which are deep, have
         clear water, and usually support
         Salmonoid fishes.

  2       Mesotropic lakes are generally
         intermediate between oligotrophlc
         and eutrophic lakes.  They are
         moderately productive, yet
         pleasant to be around.

   3    Jlu^oghic_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.  Nuisance
         conditions often appear.

   4     Dystrophic lakes - bog lakes -
         low in pH, water yellow to brown,
         dissolved solids; N, P,  and Ca
         scanty but humic materials abun-
         dant; bottom fauna and plankton
        poor, and fish species are limited.

B  Reservoirs may be classified as storage,
   or 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.
3-8

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                                                        Limnology and Ecology of Plankton
 C 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.

DC THE MANAGEMENT OR CONTROL OF
    ENVIRONMENTAL FACTORS

 A Liebig's Law of the Minimum states
    that productivity is limited by the
    nutrient present in the least amoung
    at any given time relative to the
    assimilative capacity of the organism.

 B  Shelford's Law of Toleration:
Minimum Limit
of toleration
Absent
Decreasing
Abundance
Range of Optimum
of factor
Greatest abundance
Maximum limit of
toleration
Decreasing
Abundance
Absent
   The artificial introduction of nutrients
   (sewage pollution or fertilizer) thus
   tends to eliminate existing limiting
   minimums for some species and create
   intolerable maximums for other species.
   1
            Known limiting minimums may
            sometimes be deliberately
            maintained.

            As the total available energy
            supply is increased, productivity
            tends to  increase.

            As productivity increases, the
            whole character of the water
            may be changed from a meagerly
            productive clear water lake
            (oligotrophic) to a highly pro-
            ductive and usually turbid lake
            (eutrophic).

            Eutrophication leads to treatment
           troubles.
D  Control of e utrophication may be accom-
   plished by various means

   1       Watershed management, ade-
           quate preparation of reservoir
           sites, and pollution control tend
         to maintain minimum limiting nu-
         tritional factors.

   2     Shading out the energy of insola-
         tion by roofing or inert turbidity;
         suppresses photosynthesis.

   3     Introduction of substances toxic
         to some fundamental part of the
         food chain (such as copper sul-
         phate) tends to temporarily inhibit
         productivity.

X SUMMARY

A A body of water such as a lake rep-
   resents 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.

REFERENCES

1  Chamberlin,  Thomas C.,  and Salisburg,
         RollinP., Geology Vol. 1,  "Geolo-
         gical Processes and Their Results",
         pp i-xix,  and 1-654, Henry Holt and
         Company, New York, 1904.
2  Dorsey, N. Ernest.  Properties of
         Ordinary Water - Substance.
         Reinhold Publ.  Corp., New York.
         pp. 1-673. 1940.

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

4  Ruttner, Franz. Fundamentals of
         Limnology.   University of Toronto
         Press,  pp.  1-242.  1953.

5  Tarzwell,  Clarence M.  Experimental
         Evidence on the Value of Trout 1937
         Stream  Improvement in Michigan.
         American Fisheries Society Trans.
         66:177-187.  1936.
                                                                                   3-9

-------
 Limnology and Ecology of Plankton
 6  US DHEW. PHS.  Algae and Metropolitan
         Wastes, transactions of a seminar
         held April 27-29,  1960 at the
         Robert A. Taft Sanitary Engineer-
         ing  Center, Cincinnati, Ohio.
         No. SEC TR W61-3.

 7  Ward and Whipple.   Freshwater Biology
         (Introduction).  John Wiley
         Company.  1918.

 8  Whittaker, R. H.  Communities and
         Ecosystems.   Macmillan,
         New York.  162 pp.  1970.

 9  Zhadin, V. I. and Gerd, Sr.   Fauna
         and Flora of the lakes and
         Reservoirs of the USSR. Avail-
         able from the Office of Technical
         Services, U. S. Dept.  Commerce,
         Washington, DC.

10  Josephs,  Melvin and Sanders, Howard J.
         Chemistry and the Environment
         ACS Publications, Washington,  DC.
         1967.
This outline was prepared by H. W. Jackson,
former Chief Biologist, National Training
Center, and revised by R. M. Sinclair,
Aquatic Biologist, National Training Center,
MOTD, OWPO. USEPA, Cincinnati, Ohio
45268.

Descriptors:  Plankton, Ecology, Limnology
 3-10

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                       BIOLOGY OF ZOOPLANKTON COMMUNITIES
I  CLASSIFICATION

A  The planktonic community is composed of
   organisms that are relatively independent
   of the bottom to complete their life history.
   They inhabit the open water of lakes
   (pelagic zone).  Some species have inactive
   or resting stages that lie on the bottom
   and carry the species through periods of
   stress; e. g. , winter.  A few burrow in
   the mud and enter the pelagic zone at night,
   but most live in the open water all the
   time that the species is present in an active
   form.

B  Compared to the bottom fauna and flora,
   the plankton consists of relatively few
   kinds  of organisms that are consistently
   and abundantly present.  Two major cat-
   egories are often called phytoplankton
   (plants) and zooplankton (animals), but
   this is based on an outmoded classification
   of living things.   The modern tendency is
   to identify groupings according to their
   function in the ecosystem:  Primary pro-
   ducers (photosynthetic organisms), consumers
   (zooplankton),  and decomposers (hetero-
   trophic bacteria and fungi).

C  The primary difference then is nutritional;
   phytoplankton use inorganic nutrient
   elements and solar radiation. Zooplankton
   feed on particles, much of which can be
   phytoplankton cells,  but can be bacteria or
   particles of dead organisms (detritus)
   originating in the plankton, the shore
   region,  or the land surrounding the lake.

D  The swimming powers of planktonic
   organisms is so limited that their hori-
   zontal distribution is determined mostly
   by movements of water.  Some of the
   animals are able to swim fast enough that
   they can migrate vertically tens of meters
   each day,  but they are capable of little
   horizontal navigation.  At most,  some
   species of crustaceans show a general
   avoidance of the shore areas during calm
   weather when the water is moving more
   slowly than the animals can swim.  By
   definition,  animals that are able to control
   their horizontal location are nekton, not
   plankton.
 E  In this presentation, a .minimum of clas-
    sification and taxonomy is used, but it
    should be realized that each group is
    typified by adaptations of structure on
    physiology that are related to the plank-
    tonic mode of existence.  These adapta-
    tions are reflected in the classification.
II   FRESHWATER ZOOPLANKTON

 A  The freshwater zooplankton is dominated
    by representatives of three groups of
    animals,  two of them crustaceans:
    Copepoda, Cladocera, Rotifera.  All have
    feeding mechanisms that permit a high
    degree of selectivity of food,  and two can
    produce resting eggs that can withstand
    severe environmental conditions. In
    general the food of usual zooplankton pop-
    ulations ranges from bacteria and small
    algae to small animals.
 B  The Copepoda reproduce by a normal
    biparental process, and the females lay
    fertilized eggs in groups which are carried
    around in sacs until they hatch.  The
    immature animals go through an elaborate
    development with many stages.  The later
    stages have mouthparts that permit them
    to collect particles.  In many cases, these
    are in the form of combs which remove
    small particles by a sort of filtration
    process.   In others, they are modified to
    form grasping organs by which small
    animals or large  algae are captured
    individually.

 C  The Cladocera (represented by Daphnia)
    reproduce much of the time by partheno-
    genesis,  so that only females are present.
    Eggs are held by  the mother in a brood
    chamber  until the young are developed far
    enough to fend for themselves.  The newborn
    animals look  like miniature adults, and do
    not go through an elaborate series of
    developmental stages in the water as do
    the copepods.  Daphnia has comb-formed
    filtering structures on some of its legs
    that act as filters.
  BI.AQ. 29. 6. 76
                                        4-1

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 Biology of Zooplankton Communities
    Under some environmental conditions the
    development of eggs is affected and males
    are produced.  Fertilized eggs  are produced
    that can resist freezing and drying, and
    these carry the population through
    unsatisfactory conditions.

    The Rotifera are small animals with a
    ciliated area on the head  which  creates
    currents used both for locomotion and for
    bringing food particles to the mouth.  They
    too reproduce by parthenogenesis during
    much of the year, but production of males
    results in fertilized,  resistant resting eggs.
    Most rotifers lay eggs one at a  time and
    carry them until they hatch.
Ill  ZOOPLANKTON POPULATION DYNAMICS

 A In general,  zooplankton populations are at
    a minimum in the cold seasons, although
    some species flourish in cold water. Species
    with  similar food requirements seem to
    reproduce at different times of the year or
    are segregated in different layers of lakes.

 B There is no single, simple measurement
    of activity for the zooplankton  as a whole
    that can be used as an index of production
    as can the uptake of radioactive carbon for
    the phytoplankton. However, it is possible
    to find the rate of reproduction of the species
    that carry their eggs.  The basis of the
    method is that the number of eggs in a
    sample taken at a given time represents
    the number of animals that will be  added
    to the population during an interval that
    is equal to the length of time it takes the
    eggs to develop.  Thus the potential growth
    rate of the populations can be determined.
    The actual growth rate, determined by
    successive samplings and counting, is less
    than the potential, and the difference is a
    measure of the death rate.

 C Such measurements of birth and death rates
    permits a more penetrating analysis to be
    made of the causes of population change
    than if data were available for  population
    size alone.

 D Following is an indication of the major
    environmental factors in the control of
    zooplankton.

    1  Temperature has an obvious effect in
       its general control of rates.  In addition,
       the production and hatching  of resting
       eggs may be affected.
2  Inorganic materials

   Freshwater lakes vary in the content
   of dissolved solids  according to the
   geological situation.  The total salinity
   and proportion of different dissolved
   materials in water  can affect the pop-
   ulation.  Some species are limited to
   soft water, others to saline waters, as
   the brine shrimp.   The maximum pop-
   ulation size developed maybe related
   to salinity, but this is probably an
   indirect effect working through the
   abundance of nutrients and production
   of food.

3  Food supply

   Very strong correlations have been
   found between reproduction and food
   supply as measured by abundance of
   phytoplankton.  The rate of food supply
   can affect almost all aspects of pop-
   ulation biology including rate of indi-
   vidual growth, time of maturity,  rate
   of reproduction and length of life.

4  Apparently in freshwater, dissolved
   organic materials are of little nutri-
   tional significance,  although some
   species can be kept if the concentration
   of dissolved material is high enough.
   Some species require definite vitamins
   in the food.

5  Effect of  predation on populations

   The kind,  quantity and relative pro-
   portions of species  strongly affected
   by grazing by vertebrate and. inverte-
   brate predators.  The death rate  of
   Daphnia is correlated with the abun-
   dance of a predator.  Planktivorous
   fish (alewives) selectively feed on
   larger  species, so a lake with alewives
   is dominated by the smaller species of
   crustaceans and  large ones are scarce
   or absent.

6  Other aspects of zooplankton

   Many species migrate vertically  con-
   siderable distances each day.  Typically,
   migrating species spend the daylight
   hours deep in the lake and rise toward
   the surface in late afternoon and  early
   evening.

   Some species go through a seasonal
   change of form (cyclomorphosis)  which
   is not fully understood. It may have an
   effect in reducing predation.
   4-2

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                                                        Biology of Zooplankton Communities
REFERENCES
1  Baker, A. L.  An Inexpensive Micro-
      sampler.   Limnol. and Oceanogr.
      15(5):  158-160.  1970.

2  Brooks, J.  L. and Dodson, S. I.
      Predation, Body,  Size, and Com-
      position of Plankton.  Science 150:
      28-35.  1965.

3  Dodson, Stanley I.  Complementary
      Feeding Niches Sustained by Size-
      Selective  Predation, Limnology
      and Oceanography 15(1):  131-137.

4  Hutchinson,  G.  E.  1967.  A Treatise
      on Limnology.  Vol. II.  Introduction
      to Lake Biology and the Limnoplankton.
      xi+ 1115.  John Wiley & Sons, Inc.,
      New York.

5  Jossi, JackW.  Annotated Bibliography
      of Zooplankton Sampling Devices.
      USFWS.   Spec. Sci. :  Rep. -Fisheries.
      609.  90 pp.  1970.

6  Likens, Gene E. and Gilbert, John J.
      Notes on Quantitative Sampling of
      Natural Populations of Planktonic
      Rotifers.   Limnol. and Oceanogr.
      15(5):  816-820.
 7  Lund, J. W.  G.   1965.  The Ecology of
      the Freshwater Plankton.  Biological
      Reviews, 40:231-293.

 8  UNESCO.  Zoolplankton Sampling.
      UNESCO Monogr. Oceanogr.  Methodol.
      2.  174 pp.  1968.  (UNESCO. Place
      de Fortenoy,  75, Paris 7e France).
This outline was prepared by W. T.  Edmondson,
Professor of Zoology,  University of
Washington,  Seattle, Washington.

Descriptor:   Zooplankton
                                                                                     4-3

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Biology of Zooplankton Communities
       FIGURE  1   SEASONAL CHANGES OF ZOOPLANKTON IN LAKE ERKEN,  SWEDEN
                  Diaptomus  gracixoides
                  Ceriodaphnia quadraneula
               Each panel  shows  the abundance of a species of animal.   Each
               mark on the vertical axis represents 10 individuals/liter.
               Nauwerck, A.  1963.  Die Beziehungen zwlschen Zooplankton und
               Phytoplankton im  See Erken.  Symbolae Botanicae Upsaliensis, 17:1-163.

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FIGURE  2  REPRODUCTIVE RATE OF ZOOPLANKTON ASA FUNCTION OF ABUNDANCE OF FOOD
                                                 Young per  -
                                                    Brood  _

                                                        20 -
                                                        10
Cells/ml
of food
             100,000
                                                                          Days
                                                                                           \
                                                                                          4O
Total young
  223
  177
  132


   -76
               200
                           400
                                        600
                                                    Number of young  produced in each brood  by Daphnia living in
                                                    four different concentrations  of food organisms, renewed
                                                    daily.  The total number produced during the life of a
                                                    mother is shown  by the numbers at the right.  The Daphnia
                                                    at the two lowest concentrations produced their first batch
                                                    of eggs on the same day as  the others,  but the eggs degen-
                                                    erated, and the  first  viable eggs were  released two days
                                                    later.  Richman, S.   1958.   The transformation of energy by
                                                    Daphnia pulex.   Ecol.  Monogr.   28:  273-291.
     Abundance of food organisms ygm/1, dry weight

     Mean  rate of laying eggs by the planktonic
     rotifer Keratella cochlearis in natural
     populations as a function of abundance of
     food  organisms and temperature.  W. T.
     Edmonson. 1965.  Reproductive rate of
     planktonic rotifers as related to food
     and temperature in nature.  Ecol. Mmogr.
     35:   61-111.
                                                                 IN
                                                                 O
                                                                 n
                                                                 O
                                                                 3
                                                                 3
                                                                                                                              0>
                                                                                                                              en

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Biology of Zooplankton Communities
                                         PROTOZOA
                                         Difflugia
                                        Amoebae
                                         Codonella
                Stentor
Epistylis
                                         Ciliates

-------
                                      Biology of Zooplankton Communities
                         ROTIFERA
   Synchaeta
Polygarthra
Brachionus
Cladocera
                        ARTHROPODA
                         Crustacea
                                                Copepoda
                   Nauplius larva of copepod
                     Insecta - Chaoborus

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Biology of Zooplankton Communities
                           PLANKTON1C BIVALVE LARVAE
                          380M,
377|i
                  spined  (fin attached)
   simple  (gill  attached)
                     Glochidia (Unionidae) Fish Parasites
                                    (1-3)
             veliger
  pediveliger
                Veliger Larvae (Corbiculidae) Free  Living Planktonic
                                    (4-5)
                       Pediveliger attaches byssus lines)

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                              OPTICS AND THE MICROSCOPE
I  OPTICS

An understanding of elementary optics is
essential to the proper use of the microscope.
The  microscopist will find that unusual pro-
blems in illumination and photomicrography
can be handled much more effectively once
the underlying ideas in physical optics are
understood.

A Reflection

   A good place  to begin is with reflection at
   a surface or interface.  Specular (or
   regular) reflection  results when a beam
   of light leaves a surface at the same angle
   at which it  reached it.  This type of
   reflection occurs with highly polished
   smooth surfaces.  It is stated more pre-
   cisely as Snell's Law, i. e., the angle of
   incidence,  i,  is equal to the angle of
   reflection,  r  (Figure  l). Diffuse (or
   scattered)  reflection results when a beam
   of light strikes a rough or irregular sur-
   face and different portions of the  incident
   light are reflected from the surface at
   different angles.  The light reflected from
   a piece of white paper or  a ground glass is
   an example of diffuse reflection.
                 Figure 1
SPECULAR REFLECTION -  SNELL'S
LAW
BI. MIC.  18. 2. 79
   Strictly speaking, of course, all reflected
   light, even diffuse,  obeys Snell's Law.
   Diffuse reflected light is made up of many
   specularly reflected rays, each from a
   a tiny element of surface, and appears
   diffuse when the reflecting elements are
   very numerous and  very small.  The terms
   diffuse and .specular, referring to reflection,
   describe not so much a  difference in the
   nature of the reflection  but rather a differ-
   ence in the type of surface.  A polished sur-
   face gives specular reflection; a rough
   surface gives diffuse reflection.

   It is also important to note and remember
   that specularly reflected light tends to be
   strongly polarized in the plane of the  reflect-
   ing surface. This is due to the fact that
   those rays  whose  vibration directions lie
   closest to the plane of the reflection surface
   are most strongly reflected.  This  effect  is
   strongest when the angle of incidence is
   such that the tangent of  the angle  is equal
   to the refractive index of the reflecting sur-
   face.  This particular angle of incidence is
   called the Brewster angle.

B  Image Formation  on Reflection

   Considering reflection by mirrors,  we find
   (Figure 2) that a plane mirror forms  a
   virtual image behind the mirror,  reversed
   right to left but of the same size as the
   object.  The word virtual means that the
   image appears to  be in a given plane but
   that a ground glass  screen or a photographic
   film placed in that plane would show no
   image.  The converse of a virtual image is
   a real image.

   Spherical mirrors are either convex or con-
   cave with the surface of the  mirror repre-
   senting a portion of the  surface of a sphere.
   The center of curvature is the center of the
   sphere, part of whose surface forms  the
   mirror.  The focus lies halfway between the
   center of curvature  and  the mirror  surface.
                                                                                    5-1

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Optics and the Microscope
   Object
                               1
Virtual
Image
                  Mirror
                 Figure 2

 IMAGE FORMATION BY PLANE MIRROR


   Construction of an image by a concave
   mirror follows from the two premises
   given below  (Figure 3):
                 Figure 3
IMAGE FORMATION BY CONCAVE MIRROR

   1 A ray of light parallel to the axis of
     the mirror must pass through the
     focus after reflection.

   2 A ray of light which passes through the
     center of curvature  must return along
     the  same path.

A corollary of the  first premise is:
                                                  3 A ray of light which passes through the
                                                    focus is reflected parallel to the axis
                                                    of the mirror.

                                                  The image from an object can be located
                                                  using the familiar lens formula:
                        _t
                        P
I
q
                   where p = distance from the object to
                            the mirror
                         q = distance from the image to
                            the mirror
                         f  = focal length

             C  Spherical Aberration

                No spherical surface can be perfect in its
                image-forming ability. The most serious
                of the imperfections, spherical aberration,
                occurs in spherical mirrors of large
                aperture (Figure 4).  The rays of light
                making up an image point from the outer
                zone of a spherical mirror do not pass
                through  the same point as the more central
                rays. This type of aberration is reduced by
                blocking the outer zone rays from the  image
                area or by using aspheric surfaces.
                               Figure 4

                    SPHERICAL ABERRATION BY
                         SPHERICAL  MIRROR
 5-2

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                                                                     Optics and the Microscope
 D  Refraction of Light

    Turning now to lenses rather than mirrors
    we find that the most important charade r-
    istic is refraction.  Refraction  ivIVrs to
    the change of direction and/or velocity of
    light as it passes from one  m.-dium to
    another.   The ratio of the velocity in air
    (or more correctly in a vacuum) to tin-
    velocity in the medium is called the
    refractive  index.  Some typical values of
    refractive  index measured with mono-
    chromatic  light (sodium D line) are listed
    in Table  1.

    Refraction causes an object immersed in
    a medium of higher refractive index than
    air to appear closer to the surface than  it
    actually is (Figure 5). This effect may
                                          into focus and the now micrometer reading
                                          is taken.  KinaUy, the microscope is re-
                                          focused until the surface of the liquid appears
                                          in sharp focus.  The micrometer reading
                                          is taken again and, with this information,
                                          the refractive index miy be calculated from
                                          the simplified equation:

                                                  ..   ,.   .  .      actual depth
                                                rclruclive index = 	f-—77-
                                                                  apparent depth
                                       Table  1.  RKKRACTIVK  INDICES OF COMMON
                                       MATERIALS MKASURED  WITH SODIUM LIGHT
Vacuum
Air
CO.;
Water
1 . 0000000
1. 0002918
1. 0004498
1. 3330
Crown glass
Rock salt
Diamond
Lead sulfide
1.48 to
1.5443
2.417
3.912
1.61



Actual
depth
Apparent I
 depth  |
                                      Air
                                     Medium
                     Image

                     Object
When the situation is reversed, and a ray
of light from a medium of high refractive
index passes through the interface of a
medium of lower index,  the ray is refracted
until a critical angle is reached beyond which
all of the light  is reflected from the interface
(Figure 6).  This critical angle,  C, has the
following relationship to the refractive indices
of the two media:                       {
                                                          sin C =  — 2
                                                                       where
                                                                                
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  Optics and the Microscope
 E Dispersion

    Dispersion is another important property
    of transparent materials.  This is the
    variation of refractive index with color
    (or wavelength) of light.  When white light
    passes through a glass prism,  the light
    rays are refracted by different amounts
    and separated into the colors of the
    spectrum.  This spreading of light into
    its component colors is due to dispersion
    which, in turn, is due to the fact that the
    refractive index of transparent substances,
    liquids and solids, is lower for long wave-
    lengths than for short wavelengths.

    Because of dispersion, determination of
    the refractive index  of a  substance re-
    quires designation of the particular wave-
    length used.  Light from a  sodium lamp
    has a strong,  closely spaced doublet with
    an average wavelength of 5893A, called
    the D line,  which is  commonly used as a
    reference wavelength.  Table 2 illustrates
    the change of refractive index with wave-
    length for a few common substances.
                                              F  Lenses

                                                 There are two classes of  lenses, con-
                                                 verging and diverging, called also convex
                                                 and concave,  respectively.  The focal
                                                 point of a converging lens is  defined as
                                                 the point at which a bundle of light rays
                                                 parallel to the axis of the  lens appears to
                                                 converge after passing through the lens.
                                                 The focal length of the lens is the distance
                                                 from the lens to the focal  point (Figure 7).
 Table 2.  DISPERSION OF REFRACTIVE
INDICES OF SEVERAL COMMON MATERIALS

                       Refractive index
                    F line   D line    C line
                    blue    (yellow)   (red)
                    4861A   5893A    6563A
Carbon disulfide
Crown glass
Flint glass
Water
1. 6523
1.5240
1. 6391
1.3372
1.6276
1.5172
1.6270
1.3330
1.6182
1. 5145
1. 6221
1. 3312
   The dispersion of a material can be defined
   quantitatively as:
   _,.     .
v = dispersion =

    n (593mn) - 1
                    n (yellow)  - 1
                 =
                                 _
                           . n (red)
       n (486m|i) - n(656mn)

    where n is the refractive index of the
    material at the particular wavelength
    noted in the parentheses.
                                                                Figure  7

                                              CONVERGENCE OF LIGHT AT FOCAL POINT
G  Image Formation by Refraction

   Image formation by lenses (Figure 8)
   follows rules analogous to those already
   given above for mirrors:

   1  Light traveling parallel to the axis of
     the lens will be refracted so as to pass
     through the focus of the lens.

   2  Light traveling through the geometrical
     center  of the lens will be unrefracted.

   The position of the image can be determined
   by remembering that a light ray passing
   through the focus. F, will be parallel to
   the axis of the lens on the  opposite side of
   the lens and  that a ray passing through the
   geometrical  center of the lens will be
   unrefracted.
5-4

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                                                                   Optics and the Microscope
                 Figure? 8

IMAGE FORMATION BY A CONVEX LENS

   The magnification,  M, of an image of an
   object produced by a lens is given by the
   relationship:
           image size _  image distance  _ q
           object size    object distance    p

   where q = distance from image to lens
     and p = distance from object to lens.

H  Aberrations of Lenses

   Lenses have aberrations of several types
   which,  unless corrected,  cause loss of
   detail in the image.  Spherical aberration
   appears in lenses with spherical surfaces.
   Reduction of spherical aberration can be
   accomplished by diaphragming the outer
   zones of the lens or by designing special
   aspherical surfaces in the lens system.

   Chromatic aberration is a phenomenon
   caused by the variation of refractive index
   with wavelength (dispersion).  Thus a lens
   receiving  white light from an object will
   form a violet image closer to the lens and
   a red one  farther away.  Achromatic
   lenses are employed to minimize this
   effect.   The lenses are combinations of
   two or more lens elements made up of
   materials having different dispersive
   powers.  The use of monochromatic light
   is another obvious  way of eliminating
   chromatic aberration.

   Astigmatism is a third aberration of
   spherical  lens systems.  It occurs when
object points are not located on the optical
axis of the lens and  results in the formation
of an indistim.-t image.  The simplest
remedy for astigmatism is to place the
object dose to the axis of the lens system.

Interference Phenomena

Interference and diffraction are two phe-
nomena which are due to  the wave character-
istics of light.  The  superposition of two
light rays arriving simultaneously at a given
point will give  rise to interference effects,
whereby the intensity at that point will vary
from dark to bright depending on the phase
differences between  the two light rays.

The first requirement for interference is
that the light must come from a single
source.  The light may be split into any
number of paths but  must originate from
the same point (or coherent source).   Two
light waves from a coherent source arriv-
ing at a point in phase agreement will
reinforce each other (Figure 9a).  Two
light waves from a coherent source arriv-
ing at a point in opposite phase will cancel
each other (Figure 9b).
Figure 9a.  Two light rays,  1 and  2, of
           the same frequency but dif-
           ferent amplitudes, are in phase
           in the upper diagram.   In the
           lower diagram,  rays 1 and 2
           interfere constructively to give
           a single wave of the  same fre-
           quency and with an amplitude
           equal to the summation of the
           two former waves.
                                                                                    5-5

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Optics and the Microscope
                                  o+b
  Figure .9b.  Rays 1 and 2 are IKKV 180°
             out of phase and interfere
             destructively.  The resultant,
             in the bottom diagram,  is of
             the same frequency but is of
             reduced amplitude (a is
             negative and is subtracted
             from b).
  The reflection of a monochromatic light
  beam by a thin film results in two beams,
  one reflected from the top surface and one
  from the bottom surface.  The distance
  traveled by the latter  beam in excess of
  the first is twice the thickness of the film
  and its equivalent air  path is:

                 2 nt

     where n is the refractive index and
     t is the thickness of the film.

  The second beam,  however, upon reflection
  at the bottom surface, undergoes a half
  wavelength shift and now the total retard-
  ation of the second beam with respect to
  the first is given as:
        retardation = 2 nt + -5-
     where X is the wavelength of the light
     beam.

  When retardation is exactly an odd number
  of half wavelengths, destructive interfer-
  ence takes place resulting in darkness.
  When it is zero or an even number of half
  wavelengths,  constructive interference
  results in brightness (Figure 10).
5-6
                Figure 10

    INTERFERENCE IN A THIN FILM

A simple interferometer can be made by
partially silvering a microscope slide and
cover slip.  A preparation between the two
partially silvered surfaces will show inter-
ference  fringes when viewed with mono-
chromatic light, either transmitted or by
vertical illuminator.  The fringes will be
close together with a wedge-shaped prep-
aration and will reflect refractive index
differences due to temperature variations,
concentration differences, different solid
phases,  etc.  The method has been used to
measure quantitatively the concentration of
solute around a growing crystal^ '(Figure 11).

              50% Mirror/
                        N
                          \
J-	Cover  slip-  - f_
      Specimen
                             W"
               Figure 11
MICROSCOPICAL METHOD OF VIEWING
          INTERFERENCE IMAGES
 a  Examination ia by transmitted light.
      Light ray  undergoes multiple
      reflections and produces dark and
      light fringes  in the field.  A speci-
      men introduces a phase shift and
      changes the fringe  pattern.
 b  Illumination is from the top.  The
      principle is the same but fringes
      show greater contrast.

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                                                                    Optics and the Microscope
Each dark band represents an equivalent
air -thickness of an odd number of half
wavelengths.   Conversely, each bright
band is the  result of an even number of
half wavelengths.

With interference illumination,  the effect
of a transparent  object of different re-
fractive index  than the medium  in the
microscope field is:

1  a change of light intensity of  the object
   if the  background  is  uniformly illumi-
   nated (parallel cover slip), or

2  a shift of the interference bands within
   the  object if the background consists
   of bands  (tilted cover slip).

The relationship of refractive indices of
the surrounding medium and the object is
as  follows:
                 .  ex   ,
                                                          d =
                                                              2.44 fx
              m*
                     360t
      where ns = refractive index of the
                 specimen
            nm = refractive index of the
                 surrounding medium
            9  = phase shift of the two
                 beams, degrees
            X  = wavelength of the light
            t  = thickness of the specimen.

J Diffraction
  In geometrical optics,  it is assumed that
  light travels in straight lines.  This is not
  always true.   We note that a beam passing
  through a slit toward a screen creates a
  bright band wider than the slit with alter-
  nate  bright and dark bands appearing on
  either side  of the central bright band,
  decreasing in intensity as a function of
  the distance from the center.  Diffraction
  describes this phenomenon and, as one of
  its practical consequences, limits the
  lens  in it.= ability to reproduce an image.
  For example, the image  of a pin point of
  light produced by a lens  is not a pin point
  but is revealed to be a somewhat larger
  patch of light surrounded by  dark  and
  bright rings.  The diameter, d, of this
  diffraction disc (to the  first dark  ring)
  is given as:
                                                       where f is the focal length of  the lens,
                                                       X the wavelength,  and D the diameter
                                                       of the lens.

                                                    It is seen that in order to maintain a
                                                    small diffraction disc at a given wave-
                                                    length,  the diameter of  the lens should
                                                    be as large as possible with respect to
                                                    the focal length.  It should be noted,
                                                    also, that a shorter wavelength produces
                                                    a  smaller  disc.

                                                    If two pin points of light are to be distin-
                                                    guished in an image,  their diffraction discs
                                                    must not overlap more than one half their
                                                    diameters.  The ability to distinguish such
                                                    image points is  called resolving power and
                                                    is expressed as  one half of the preceding
                                                    expression:
                                                          resolving power =
                             1. 22 f X
                               D
II   THE COMPOUND MICROSCOPE

 The compound microscope is an extension in
 principle of the simple magnifying glass;
 hence it is essential to understand fully the
 properties of this simple lens system.

 A  Image Formation by the Simple Magnifier

    The apparent size of an object is  determined
    by the angle that is  formed at the eye by the
    extreme rays of the object.  By bringing the
    object closer to the  eye, that angle (called
    the visual angle) is  increased.  This also
    increases the apparent size.  However a
    limit of accommodation of the eye is reached,
    at which distance the eye can no longer focus.
    This limiting distance  is about  10 inches or 25
    centimeters.  It is at this distance that the
    magnification of an  object observed by the
    unaided eye is  said  to  be unity.   The eye can,
    of course, be focused at shorter distances but
    not usually  in a relaxed condition.

    A  positive,  or  converging,  lens can be used
    to permit placing an object closer than 10
    inches to the eye (Figure 12).  By this means
    the visual angle of the object  is increased
    (as is its apparent size) while the image of
                                                                                      5-7

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Optics and the  Microscope
                                                                          Eye
                                L«ns   Rtllno
                                   Ey«
    VIRTUAL IMAGE FORMATION BY
             CONVEX LENS

   the object appears to be 10 inches from
   the eye,  where it is best accommodated.

B  Magnification by a Single Lens System

   The magnification, M,  of a simple magni-
   fying glass is given by:
      where f = focal length of the lens in
                centimeters.

   Theoretically the magnification can be
   increased with shorter focal length lenses.
   However such lenses require placing the
   eye very close to the lens surface and
   have much image distortion and other
   optical aberrations.  The practical limit
   for a simple magnifying glass is about
   20X.

   In order to go to magnifications higher
   than 20X,  the compound microscope is
   required.   Two lens systems are used
   to form an enlarged image of an object
   (Figure 13).  This is accomplished in
   two steps,  the first by a lens called the
   objective and the second by a lens known
   as the eyepiece (or ocular).

C  The Objective

   The objective is the lens (or lens system)
   closest  to the object.  Its function is to
   reproduce an enlarged image of the object
   in the body tube of the microscope.
   Objectives are available  in various focal
                                                                             Eyepiece
         Objective
         V rural Imoge
                 Figure 13

         IMAGE FORMATION IN
        COMPOUND MICROSCOPE

lengths to give different magnifications
(Table 3).  The magnification is calculated
from the focal length by dividing the latter
into  the tube length, usually 160 mm.

The  numerical aperture (N. A.) is a measure
of the ability of an objective to resolve detail.
This is more fully discussed in the next
section.  The working distance  is in the free
space between the objective and the cover
slip  and varies slightly for objectives of the
same focal length depending upon the degree
of correction and the manufacturer.

There are  three basic classifications of
objectives:  achromats,  fluorites and
apochromats, listed in the order of their
complexity.  The achromats are good for
routine work while  the fluorites and apo-
chromats offer additional optical corrections
to compensate for spherical, chromatic and
other aberrations.
  5-8

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                                                                Optics and the Microscope
Table 3. NOMINAL CHARACTERISTICS OF USUAL
Nominal
focal length
mm
56
32
16
8
4
4
1.8
Nominal
magnif.
2. 5X
5
10
20
43
45
90
N.A.
0.08
0. 10
0.25
0.50
0. 66
0. 85
1. 30
Working
distance
mm
40
25
7
1.3
0. 7
0.5
0.2
Depth
focus
V-
50
16
8
2
1
1
0.4
Diam. of
field
mm.
8.5
5
2
1
0.5
0.4
0.2
MICROSCOPE OBJECTIVES
Resolving
power, white
light, ji
4.4
3.9
1.4
0.7
0.4
0.35
0. 21
Maximum
useful
magnif.
80X
90X
250X
500X
660X
850X
1250X
Eyepiece
for max.
useful magnif
3 OX
20X
25X
25X
15X
2 OX
12X
Another system of objectives employs  •
reflecting surfaces in the shape of concave
and convex mirrors.  Reflection optics,
because they have no refracting elements,
do not suffer from chromatic aberrations
as ordinary refraction objectives do.  Based
entirely on reflection,  reflecting objectives
are extremely useful in the infrared and
ultraviolet regions of the  spectrum.  They
also have a much longer working distance
than the refracting objectives.

The body tube of the microscope supports
the objective at  the bottom (over the object)
and the eyepiece at the top. The tube
length is maintained at 160 mm except for
Leitz instruments, which have a 170-mm
tube length.

The objective support may be of two kinds,
an objective clutch changer or  a rotating
nosepiece:

1  The objective clutch changer ("quick-
   change" holder) permits the  mounting   ,
   of only one objective at a time on the
   microscope.  It has a centering arrange-
   ment, so that each objective need be
   centered only once with respect to the
   stage  rotation.  The changing of objec-
   tives with this system is somewhat
   awkward compared with the rotating
   nosepiece.

2  The revolving nosepiece allows mounting
   three  or four objectives on the microscope
      at one time (there are some nosepieces
      that accept five and even six objectives).
      In this system, the  objectives are
      usually noncenterable and the  stage is
      centerable.  Several manufacturers pro-
      vide centerable objective mounts so that
      each objective on the nosepiece need be
      centered only:once to the fixed rotating
      stage.  The insides of objectives are
      better protected from dust by  the rotating
      nosepiece.  This, as well as the incon-
      venience of the so-called "quick-change"
      objective holder, makes it worthwhile
      to have one's microscope  fitted with
      rotating nosepiece.

D  The Ocular

   The eyepiece, or ocular,  is necessary in
   the second  step of the magnification process.
   The eyepiece functions as a simple magni-
   fier viewing the  image formed by the
   objective.

   There are three classes of eyepieces in
   common use:  huyghenian,  compensating
   and flat-field. The huyghenian (or huyghens)
   eyepiece is designed to be used with
   achromats  while the compensating type  is
   used with fluorite and apochromatic
   objectives.   Flat-field eyepieces, as the
   name  implies, are employed in photo-
   micrography or  projection and can be used
   with most objectives.   It is best to follow
   the recommendations  of the manufacturer
   as to the proper combination of objective
   and eyepiece.
                                                                                 5-9

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Optics and the Microscope
   The usual magnifications available in
   oculars run from about 6X up to 25 or
   SOX.  The 6X is generally too low to be of
   any real value while the 25 and  SOX oculars
   have slightly poorer imagery than medium
   powers and have a very low eyepoint.  The
   most useful eyepieces lie in the 10 to 20X
   magnification range.

E  Magnification of the Microscope

   The total magnification of the objective-
   eyepiece combination is simply the product
   of the two individual magnifications.  A
   convenient working rule to assist in the
   proper choice  of eyepieces states that the
   maximum useful magnification  (MUM) for
   the  microscope is 1, 000 times  the  numeri-
   cal  aperture (N. A.) of the objective.
   The MUM is related to resolving power
   in that magnification in excess  of MUM
   gives little or no additional resolving
   power and results in what is termed empty
   magnification.  Table 4 shows the results
   of such combinations and a comparison
   with the 1000XN. A. rule.  The under-
   lined figure shows the magnification near-
   est to the MUM and the eyepiece required
   with each objective to achieve the MUM.
   From this table it is apparent that only
   higher power eyepieces can  give full use
   of the resolving power of the objectives.
   It is obvious that a 10X, or even a 15X,
   eyepiece gives insufficient magnification
   for the eye to see detail actually resolved
   by the objective.

F  Focusing the  Microscope

   The coarse adjustment is used to roughly
   position the body tube (in some newer
   microscopes,  the stage) to bring the image
   into focus.  The fine adjustment  is used
   after the coarse adjustment to bring the
   image into perfect focus and to maintain
   the focus as the slide is moved across the
   stage.  Most  microscope objectives are
   parfocal so that once they are focused any
   other objective can be swung into position
   without the necessity of refocusing except
   with the fine adjustment.

   The student of the microscope should first
   learn to focus in the  following fashion,  to
   prevent damage to a  specimen or objective:

   1   Raise the body tube and place the speci-
      men on the stage.

   2   Never focus the body tube down (or the
      stage up) while observing the  field
      through the eyepiece.

   3   Lower the  body tube (or raise the stage)
      with the coarse adjustment while care-
      fully observing the space between the
                   Table 4.  MICROSCOPE MAGNIFICATION CALCULATED
                   FOR VARIOUS OBJECTIVE-EYEPIECE COMBINATIONS
Objective
Focal Magni-
length
56mm
32
16
8
4
1.8
fication
3X
5
10
20
40
90
5X
15X
25X
SOX
100X
200X
450X
10X
SOX
SOX
100X
200X
400X
900X
Eyepiece
15X
45X
75X
150X
300X
600X
135 OX

2 OX
60X
100X
200X
40 OX
80 OX
1800X

25X
75X
125X
25 OX
500X
1000X
2250X
MUMa
(1000 NA)
80X
100X
250X
500X
660X
1250X
            aMUM = maximum useful magnification
       5-10

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                                                                    Optics and the Microscope
      objective and slide and permitting the
      two to come close together without
      touching.

   4  Looking through the microscope and
      turning the fine adjustment in such a
      way as to move the objective away from
     .the specimen, bring the image into
      sharp focus.

   The fine adjustment is usually calibrated
   in one-  or two-micron steps to indicate
   the vertical movement of the body tube.
   This feature  is useful in  making depth
   measurements but should not be relied
   upon for accuracy.

G  The Substage Condenser

   The substage holds the condenser and
   polarizer.  It can usually be focused in a
   vertical direction so that the condenser can
   be brought into the correct position with
   respect to the specimen for proper
   illumination.  In some models, the conden-
   ser is center-able so that it may be set
   exactly  in the axis of rotation of the stage;
   otherwise it will have been precentered at
   the factory and should be permanent.

H  The Microscope Stage

   The stage of the microscope supports the
   specimen between the condenser and
   objective, and may offer a  mechanical stage
   as an attachment to provide a means of
   moving the slide methodically during  obser-
   vation.  The polarizing microscope is
   fitted with a circular rotating stage to
   which a mechanical stage may be added.
   The rotating stage,  which is used for object
   orientation to observe optical effects, will
   have centering screws  if the objectives  are
   not centerable, or vice versa.  It is un-
   desirable to have both objectives and  stage
   centerable as this does not provide a fixed
   reference axis.

I  The Polarizing Elements

   A polarizer is fitted to the  condenser of all
   polarizing microscopes.  In routine instru-
   ments,  the polarizer is fixed with its
   vibration direction oriented north-south
   (east-w°st for most European instruments)
   while in research microscopes,  the
   polarizer can be rotated.  Modern instru-
   ments have polarizing filters (such as
   Polaroid) replacing the older calcite
   prisms.  Polarizing filters are preferred
   because they:

   1  are low-cost;

   2  require no maintenance;

   3  permit use of the full condenser
      aperture.

   An analyzer, of the same construction as
   the polarizer, is fitted in the body tube of
   the microscope  on a slider so that it may
   be easily removed from the optical path.
   It is  oriented with its plane of vibration
   perpendicular to the corresponding direction
   of the polarizer.

J  The Bertrand Len&

   The Bertrand lens is  usually found only on
   the polarizing microscope although some
   manufacturers are beginning to include it
   on phase microscopes.   It is located in the
   body tube  above  the analyzer on a slider
   (or pivot)  to permit quick removal from
   the optical path.  The Bertrand lens is used
   to observe the back focal plane of the objective.
   It is  convenient for checking quickly the  type
   and quality of illumination, for observing
   interference figures of crystals, for adjust-
   ing the phase annuli in phase  microscopy
   and for adjusting the annular and central
   stops in dispersion staining.

K  The Compensator Slot

   The compensator slot receives compensators
   (quarter-wave, first-order red and quartz-
   wedge) for observation of the  optical prop-
   erties of crystalline materials.   It is usually
   placed at the lower end of the body tube just
   above the objective mount, and is oriented
   45° from the vibration directions of the
   polarizer and analyzer.

L  The Stereoscopic Microscope

   The stereoscopic microscope, also called
   the binocular, wide-fie Id, dissecting or
                                                                                       5-11

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  Optics and the Microscope
    Greenough binocular microscope, is in
    reality a combination of two separate
    compound microscopes.  The two micro-
    scopes, usually mounted in one body, have
    their optical axes inclined from the vertical
    by about 7° and from each other by twice
    this angle.  When an object is placed on the
    stage of a stereoscopic microscope,  the
    optical systems view it from slightly
    different angles, presenting a stereoscopic
    pair of images to the eyes, which fuse the
    two into a single three-dimensional image.
                           .1
    The objectives are supplied  in pairs,  either
    as separate units to be mounted on the
    microscope or, as in the new instruments,
    built into a rotating drum.  Bausch and
    Lomb was the first manufacturer  to have a
    zoom lens system which gives a continuous
    change in magnification over the full range.
    Objectives for the stereomicroscope run
    from about 0. 4X to 12X,  well below the
    magnification range of objectives  available
    for single-objective microscopes.

    The eyepieces supplied with stereoscopic
    microscopes run from 10 to 25X and have
    wider fields than their counterparts in the
    single-objective microscopes.

    Because of mechanical limitations, the
    stereomicroscope is limited to about 200X
    magnification and usually does not permit
    more than about 120X.  It is most useful
    at relatively low powers in observing
    shape and surface texture, relegating the
    study of greater detail to the monocular
    microscope.  The stereomicroscope is
    also helpful in manipulating  small samples,
    separating ingredients of mixtures,  pre-
    paring specimens for detailed study at
    higher magnifications and performing
    various mechanical operations under micro-
    scopical observation, e. g. micromanipulation.
Ill  ILLUMINATION AND RESOLVING POWER

 Good resolving power and optimum specimen
 contrast are prerequisites for good microscopy.
 Assuming the availability of suitable  optics
 (ocular, objectives and substage condenser)
 it is still of paramount importance to use
 proper illumination.  The requirement for a
good illumination system for the microscope
is to have uniform intensity of illumination
over the entire field of view with independent
control of intensity and of the angular aperture
of the illuminating cone.

A  Basic Types of Illumination

   There are three types of illumination
   (Table 5) used generally:

   1  Critical.  This is used when high levels
      of illumination intensity are necessary
      for oil immersion, darkfield,  fluores-
      cence,  low birefringence or photo-
      micrographic studies.  Since the lamp
      filament is imaged in the plane of the
      specimen, a ribbon filament or arc
      lamp is required.  The lamp must be
      focusable and have an iris diaphragm;
      the position of the filament must also
      be adjustable in all directions.

   2  Ko'hler.  Also useful for intense illumi-
      nation,  Ko'hler illumination may be
      obtained with any lamp not fitted with a
      ground  glass.  The illuminator must,
      however, be focusable, it must have an
      adjustable field diaphragm (iris) and the
      lamp filament position must be adjust-
      able in all directions.

   3  "Poor man's".  So-called because a low-
      priced illuminator  may be used, this
      method gives illumination of high quality
      although of lower intensity because of the
      presence of a ground glass in the system.
      No adjustments are necessary on the
      illuminator or lamp filament although
      an adjustable diaphragm on the illuminator
      is helpful.

   All three types of illumination require that
   the microscope  substage condenser focus
   the image of the  illuminator aperture in the
   plane of the specimen. In each case, then,
   the lamp iris acts as a field diaphragm and
   should be closed to just illuminate the field
   of view. The differences in these three
   types of illumination lie in the adjustment
   of the lamp condensing lens.  With poor
   man's illumination there is no lamp conden-
   ser,  hence no adjustment.  The lamp should
   be placed close  to the  microscope so that
  5-12

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                                                                 Optics and the Microscope
                         Table 5.  COMPARISON OF CRITICAL,
                      KOHLER AND i'OOR MAN'S ILLUMINATION

Lamp filament
Lamp condensing lens
Lamp iris
Ground glass at lamp
Image of light source
Image of field iris
Image of substage iris
Critical
ribbon filament
required
required
none
in object plane
near object
plane
back focal plane
of objective
Kohler
any type
required
required
none
at substage
iris
in object •
plane
back focal plane
of objective
Poor man's
any type
none
useful
present
none
near object
plane
back focal plane
of objective
the entire field of view is always
illuminated.  If the surface structure of the
ground glass becomes apparent in the field
of view the substage condenser  is very
slightly defocused.

Critical Illumination

V/ith critical illumination the lamp conden-
ser is focused to give parallel rays;  focus-
ing the lamp filament on a far wall is
sufficient.  Aimed, then, at the substage
mirror, the substage condenser will focus
the lamp filament in the object plane. The
substage condenser iris will now be found
imaged in the back focal plane of the ob-
jective; it serves as a control over con-
vergence of the illumination.  Although the
substage iris also affects the light intensity
over the field of view it should most  decid-
edly not be used for this purpose.  The
intensity of illumination may be varied by
the use of neutral density filters and, unless
color photomicrography is anticipated, by
the use of variable voltage on the lamp
filament.
  it
Kohler illumination (Figure 14)  differs
from critical illumination in the use  of the
lamp condenser.   With critical  illumination
the lamp condenser focuses, the  lamp
filament at infinity; with Kohler illumination
the lamp filament is focused in  the plane  of
the substage condenser iris (also coincident
with the anterior focal plane of the substage
condenser).  The functions of the lamp
condenser iris and the substage condenser
iris in controlling, respectively,  the area
of the illuminated field of view and the
angular aperture of the illuminating cone
are precisely alike for all three types of
illumination.

Critical illumination  is seldom used because
it requires a special  lamp filament and be-
cause, when used,  it shows no advantage
over well-adjusted Kohler illumination.
  ii
Kohler Illumination

To arrange the microscope and illuminator
for Kohler illumination it is well to proceed
through the following steps:

   a  Remove the diffusers and filters
      from the lamp.

   b  Turn the lamp on and aim at a con-
      venient wall or vertical screen about
      19 inches away. Open the lamp
      diaphragm.

   c  By moving the lamp  condenser,  focus
      a  sharp image of the filament.   It
      should be of such a size as to fill,
      not necessarily evenly,  the microscope
                                                                                       5-13

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Optics and the Microscope
                Critical
Kohler
    Eye

  Eyepoint


    Ocular



  Focal plane
  Focal plane
  Objective
  Preparation


  Substage
    condenser


  Substage  —,
     iris
  Lamp iris  —
  Lamp
    condenser

  Light source
Poor man's
5-14

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                                                              Optics and the  Microscope
   substage condenser opening.  If it
   does not,  move the lamp away from
   the wall to enlarge the filament image;
   refocus.

d  Turn the lamp anil aim it at the micro-
   scopc mirror so as to maintain the
   same 18  inches (or adjusted  lamp
   distance).

e  Place a specimen on Hie microscope
   stage and focus sharply with a Hi-mm
   (1GX) objective.  Open fully the
   aperture diaphragm  in the substage
   condenser.  If the  light is loo bright,
   temporarily place  a neutral density
   filter or  a diffuser in the  lamp.

f  Close the lamp diaphragm, or field
   diaphragm,  to about a 1-cm opening.
   Rack the microscope substage con-
   denser up and down to focus the
   field diaphragm sharply in the same
   plane as  the specimen.

g  Adjust  the mirror  to center the field
   diaphragm in the field of view.

h  Remove the 16-mm objective  and
   replace with a 4-mm objective.  Move
   the specimen so that a clear area is
   under observation.  Place the
   Bertrand lens in the optical path, or
   remove the eyepiece and insert an
   auxiliary telescope (sold with phase
   contrast accessories) in its place,
   or remove the eyepiece and observe
   the back aperture of the objective
   directly.   Remove any ground glass
   diffusers from the lamp.  Now
   observe the lamp filament through
   the microscope.

i  If the filament does not appear to be
   centered,  swing the lamp housing in
   a horizontal arc whose center is at
   the field diaphragm.   The purpose
   is  to maintain the field diaphragm on
   the lamp  in its centered position.  If
   a vertical movement of the filament
   is  required, loosen the buib base and
   slide it up or down.  If the base is
   fixed, tilt the lamp housing in a
   vertical arc with the field diaphragm
      as the center of movement (again
      endeavoring to keep the lamp dia-
      phragm in the centered position).
      If you have  mastered this  step, you
      have accomplished the  most difficult
      portion.   (Belter microscope lamps
      have adjustments to move  the bulb
      independently of the lamp  housing to
      simplify this step. )

   j  Put. the specimen in place, replace
      the eyepiece and the desired objec-
      tive and  rofocus.

   k  Open  or close the field diaphragm
      until it just disappears  from the field.

   1  Observe the back aperture of the
      objective, preferably with the Bertrand
      lens or the ..auxiliary telescope, and
      close  the  aperture diaphragm on the
      substage condenser until it is about
      four-fifths the diameter of the back
      aperture.  This is the best position
      for the aperture diaphragm,  a posi-
      tion which minimizes glare and maxi-
      mizes the resolving power.  It is
      instructive to vary  the aperture dia-
      phragm and observe the image criti-
      cally during the manipulation.

   m If the  illumination is too great,
      insert an  appropriate neutral density
      filter  between the illuminator and
      the condenser.  Do not  use the con-
      denser aperture diaphragm or the
      lamp field diaphragm to control the
      intensity of illumination.

Poor Man's Illumination

Both critical and Kohler illumination  re-
quire expensive illuminators  with adjust-
able focus,  lamp iris and adjustable lamp
mounts. Poor man's illumination  requires
a cheap illuminator although an expensive
illuminator  may be used if its expensive
features are negated by inserting a ground
glass diffuser or by using a frosted bulb.
Admittedly an iris diaphragm on the lamp
would be a help though it  is not necessary.

   a  The illuminator must have a frosted
      bulb or a ground glass diffuser.
                                                                                5-15

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Optics and the Microscope
         It should bo possible to direct it in
         the general direction of the substage
         mirror, very close thereto or in
         place thereof.

      b  Focus on any preparation after
         tilting the mirror to illuminate the
         field.

      c  Remove the top lens of the condenser
         and, by  racking the condenser up or,
         more often, down, bring into focus
         (in the same plane as the specimen)
         a finger, pencil or other object placed
         in the same general region as the
         ground  glass diffuser on the lamp.
         The glass surface itself can then be
         focused  in the plane of  the specimen.

      d  Ideally  the ground glass surface will
         just fill  the field of view when centered
         by the substage mirror; adjustment
         may be made by moving the lamp
         closer to or farther from the micro-
         scope (the position might be marked
         for each objective used) or by cutting
         paper diaphragms of fixed aperture
         (one for each objective used).  In this
         instance a lamp iris would be useful.

      e  Lower the condenser just sufficiently
         to defocus the ground glass surface
         and render the field of  illumination
         even.

      f  Observe the back aperture of the
         objective and open the substage con-
         denser  iris about  75 percent of the
         way.  The final adjustment of the
         substage iris is made while observing
         the preparation; the iris should be
         open as  far as possible, still giving
         good contrast.

      g  The intensity of illumination should
         be adjusted only with neutral density
         filters or by changing the lamp voltage.

   Proper illumination is one of the most im-
   portant operations in microscopy.   It is
   easy to.judge a microscopist's ability by
   a glance at his field of view and the objec-
   tive back lens.
  5-16
H  Ill-solving Power

   The resolving power of the microscope is
   its ability to distinguish separate details
   of «:lo.s
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                                                                Optics and the Microscope
1  Maximum useful magnification

   A helpful rule of thumb is that the use-
   ful magnification will not exceed 1,000
   times the numerical aperture of the
   objective (see Tables 3 and  4).  Although
   somewhat higher magnification may be
   used in specific cases,  no additional
   detail will be resolved.

   It is curious, considering the figures
   in the table, that most, if not all, manu-
   facturers of microscopes furnish a 10X
   eyepiece as the highest power.  A 10X
   eyepiece is useful  but anyone interested
   in critical work should use a 15-25X eye-
   piece; the  5-10X eyepieces are best for
   scanning purposes.

2  Abbe's theory of resolution

   One of the most cogent theories of
   resolution is due to Ernst Abbe, who
   suggested  that microscopic  objects act
   like diffraction gratings (Figure 16) and
   that the angle of diffraction, therefore,
   increases  with  the fineness  of the detail.
   He proposed that a given microscope
   objective would resolve a particular
   detail if at least two or three transmitted
   rays (one direct and two diffracted  rays)
   entered the objective.  In Figure 16 the
   detail shown would be resolved in A and
   C but not in B.   This theory, which can
   be borne out by simple experiment,  is
   useful in showing how to improve resolu-
   tion.  Since shorter wavelengths will
   give a smaller diffraction angle,  there
   is more chance of  resolving fine detail
   with short wavelengths.  Also,  since
   only two of the transmitted rays are
   needed, oblique  light and a high N.A.
   condenser will aid in resolving fine detail.

3  Improving resolving power

   The following list summarizes  the
   practical approaches to higher  resolu-
   tion with the light microscope:

   a The specimen should be  illuminated
     by either critical or Kohler
     illumination.
V
                Figure 16

    ABBE THEORY OF RESOLUTION
   b  The condenser should be well-
      corrected and have a numerical
      aperture as high as the objective to
      be used.

   c  An apochrornatic oil-immersion
      objective should be used with a com-
      pensating eyepiece of at least 15X
      magnification.  The immersion oil
      should have an index  close to 1. 515
      and have proper dispersion for the
      objective being used.

   d  Immersion oil should be placed
      between the condenser and slide and
      between cover slip and  objective.
      The  preparation itself should be
      surrounded by a liquid having a
      refractive index of 1. 515 or more.

   e  The  illumination  should be reasonably
      monochromatic and as short in wave-
      length as possible. An interference
      filter transmitting a wavelength of
      about 480-500 millimicrons is a
      suitable answer to this  problem.
      Ideally, of course, ultraviolet light
      should be used to decrease the wave-
      length still further.

   The practical effect of many of these
   factors is critically discussed  by
   Loveland^2) in a paper on the optics of
   object space.
                                                                                    5-17

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 Optics and the Microscope
IV  PHOTOMICROGRAPHY

 A Introduction

    Photomicrography, as distinct from micro-
    photography,  is the art of taking pictures
    through the microscope.  A microphoto-
    graph is a small photograph; a photomicro-
    graph is a photograph of a small object.
    Photomicrography  is a valuable tool in
    recording the results of microscopical
    study.  It enables the microscopist to:

    1  describe a microscopic field objectively
       without resorting to written descriptions,

    2  record a particular field for future
       reference,

    3  make particle size counts and counting
       analyses easily  and without tying up a
       microscope,

    4  enhance or exaggerate the visual micro-
       scopic field to bring out or emphasize
       certain details not readily apparent
       visually,

    5  record images in ultraviolet and infra-
       red microscopy  which are otherwise
       invisible to the unaided eye.

    There are two general approaches to photo-
    micrography; one requires only a plate or
    film holder supported above the eyepiece
    of the microscope with a light-tight bellows;
    the other utilizes any ordinary camera with
    its own lens system,  supported with a light-
    tight adaptor above the eyepiece.  It is
    best, in the latter case,  to use a reflex
    camera so that the  image can be carefully
    focused on the ground glass.   Photomi-
    crography of this type can be regarded
    simply as replacing the eye with the camera
    lens system.  The camera should be focused
    at infinity, just as the eye is for visual
    observation, and it should be positioned
    close to and over the  eyepiece.

    The  requirements for photomicrography,
    however, are more rigorous than those
    for visual work.  The eye can normally
    compensate for varying light intensities,
   curvature of field and depth of field.  The
   photographic plate, however,  lies in one
   plane; hence the greatest care must be
   used to  focus sharply on the subject plane
   of interest and  to select optics to give
   minimum amounts of field curvature and
   chromatic aberrations.

   With black and  white film, color filters
   may be  used to enhance the contrast of
   some portions of the specimen while mini-
   mizing chromatic aberrations of the lenses.
   In color work,  however, filters cannot
   usually  be used for this purpose and better
   optics may be  required.

   Photomicrographic cameras which fit
   directly onto the microscope are available
   in 35-mm or up to 3-1/4 X 4-1/4 inch sizes.
   Others are made which  accommodate larger
   film sizes and which have their own support
   independent of the  microscope.  The former,
   however, are preferred for ease of handling
   and lower cost.  The latter system is pre-
   ferred for greater flexibility and versatility
   and lack of vibration. The Polaroid camera
   has many applications in microscopy and
   can be used on the microscope directly but,
   because of its weight, only when the micro-
   scope has a vertically moving stage for
   focusing rather than a focusing body tube.

B  Determination of Correct Exposure

   Correct exposure determination can be
   accomplished by trial and error, by relating
   new conditions to previously used successful
   conditions and by photometry.

   With the trial and error method a series of
   trial exposures is  made, noting the type of
   subject, illumination, filters, objective,
   eyepiece,  magnification, film and  shutter
   speed.   The best exposure is selected.  The
   following parameters can be changed and
   the exposure time adjusted accordingly:

   1   Magnification.   Exposure time varies
      as the square of the magnification.

      Example:   Good exposure was  obtained
                 with a I/10-second  exposure
                 and  a magnification  of 100X.
                 If'(he magnification  is now
      5-18

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                                                            Optics and the Microscope
           200X,  the correct exposure
           is calculated as follows:
new
    exposure time = old exposure time

   new magnification.2 _ ,/10 /200>2 _
   \>ld magnification '     '   1100;

4/10 or,  say,  1/2 second.
           Kodachrome II Type A
           Professional is 40.

new exposure time = old exposure time

X A.S.A. of old film  = 1/100(400/40) =
  A. S. A. of new film

10/100 or 1/10 second.
It should be noted, however,  that the
above calculation can be made only when
there has been no change in the illumi-
nation system including the condenser
or the objective.  Only changes in magni-
fication due to changing eyepieces or
bellows extension distance can be hand-
led in the above manner.

Numerical aperture.  Exposure time
varies inversely as the  square of the
smallest working numerical aperture
of the condenser and objective.

Example:   Good exposure was obtained
            at 1/10 second with the 10X
            objective, N.A. 0.25, at
            full aperture.  With a 20X
            objective, N.A. 0. 25, at
            full aperture and the same
            final magnification, what  is
            the correct  exposure  time?

new exposure time = old exposure time
      _
   new N.A.
say, 1/50 second.
                                   or,
It is seen that more light reaches the
photographic film with higher numeri-
cal apertures at the same magnification.

Film.  Exposure time varies inversely
with the American Standards Association
speed index of the film.

Example:   A good picture was obtained
            with Eastman Tri-X film at
            I/ 100 second.  What is the
            correct exposure for
            Eastman Kodachrome II
            Type A.   The A. S. A. speed
            for Tri-X is 400 and for
                                              4  Other parameters may be varied but the
                                                 prediction of exposure time cannot be
                                                 made readily.  Experience and photo-
                                                 electric devices are the best guides to
                                                 the proper exposure.

                                              Photoelectric devices are excellent for
                                              determining correct exposure.  Since
                                              ordinary photographic exposure meters
                                              are not  sensitive enough for photomi-
                                              crography, more sensitive instruments,
                                              having a galvanometer or electronic
                                              amplifying circuit, are required.   Some
                                              photosensitive cells are inserted in the
                                              body tube in place of the eyepiece for
                                              light intensity readings. This has the
                                              advantage of detecting the light level at a
                                              point  of high intensity but does not take
                                              into account the eyepiece, the distance to
                                              the film or the  film speed.

                                              The cell may be placed just above the eye-
                                              piece so that it registers the total amount
                                              of light  leaving the eyepiece.  Again, the
                                              effects of film speed and the projection
                                              distance are not accounted for.  The  prin-
                                              cipal  drawback with the total light
                                              measuring photometer is the difficulty of
                                              taking into account the area of field covered.
                                              Take, for example,  a bright field in which
                                              only a few crystals appear; perhaps 1 per-
                                              cent of the light entering the field of  view is
                                              scattered by the crystals and  the photometer
                                              shows close to  a maximum reading.  Now
                                              assume that everything remains constant
                                              except the number of crystals and, conse-
                                              quently,  the amount of light scattered.
                                              The photometer reading could easily  drop
                                              by 50 percent,  yet the proper  exposure is
                                              unchanged.  The situation is similar  for
                                              photomicrography with crossed polars since
                                              the photometer reading depends on the
                                              intensity of illumination, on the bire-
                                              fringence and thickness of the crystals and
                                                                                5-19

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Optics and the Microscope
   on the number and size of the crystals in
   the field or,  alternatively, on the art-a of
   the field covered by birefringent crystals.
   One of the best solutions to this problem
   is to measure the photometer reading with
   no preparation on the stage.  A first-order
   red compensator or a quartz wedge is  in-
   serted when crossed polars are being used
   to illuminate the entire field.

   An alternative is to place the cell on the
   ground glass where the film will be
   located.  However, although all variables
   except film speed are now taken into
   account, measurements in the image plane
   have the disadvantage of  requiring a more
   sensitive electronic photoelectric apparatus.

   No matter what method is used for placing
   the photocell, the exposure time can be
   determined by the  general formula:

      exposure time = meter.kreading  •

   The constant k will depend on the physical
   arrangement and film used. To determine
   k for any particular system, first set up
   the microscope to  take a picture.  Record
   the meter reading  and take a series of
   trial exposures.  Pick out the best exposure
   and calculate k.  Then the k which was
   determined holds as long as no change is
   made in the  light path beyond the  photocell,
   e.g.  changing to a faster film or changing
   the projection distance.   Thus the objective,
   condenser position or illuminator may be
   changed without affecting k if the  cell is
   used as described  above.

   Example:  With one particular arrange-
              ment of photocell and film.
              the meter reading is found to
              be 40.  A series of photographs
              are taken at  1/2, 1/5, 1/10,
               1/25 and 1/50 seconds.  The
              photomicrograph taken at 1/5
              second is judged to be the best;
              hence k is calculated as follows:

              k = meter reading X exposure
              time = 40 X 1/5  = 8.

              Assume now that a new picture
               is to be taken at another
               magnification (but with the
               same film and projection
               distance) and that the new
               meter reading is 16; therefore:

               exposure time = k/meter
               reading = 8/16 =  1/2 second.
V   MICROMETRY

 A  Particle Size Determination

    Linear distances and areas can be
    measured with the microscope. This
    permits  determination of particle size
    and quantitative analysis of physical
    mixtures.  The usual unit of length for
    microscopical  measurements is the  micron
    (1 X 10-3mm or about 4 X lO'Sinch).
    Measuring particles in electron microscopy
    requires an even smaller unit, the  milli-
    micron (1 X 10~3 micron or 10 Angstrom
    units).  Table 6 shows the approximate
    average' size of a few common airborne
    materials.
 Table 6.  APPROXIMATE PARTICLE SIZE OF
      SEVERAL COMMON PARTICULATES
 Ragweed pollen

 Fog droplets

 Power plant flyash
 (after precipitators)

 Tobacco smoke


 Foundry fumes
   25 microns

   20 microns

 2-5 microns


   0. 2 micron
   (200 millimicrons)

   0.1 - 1 micron
(100-1000 millimicrons)
    The practical lower limit of accurate
    particle size measurement with the light
    microscope is about 0. 5 micron.  The
    measurement of a particle smaller than
    this with the light microscope leads to
    errors which,  under the best circum-
    stances, increase to about + 100 percent
    (usually +).

    One of the principal uses of high resolving
    power is in the precise measurement of
  5-20

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                                                                Optics and the Microscope
particle size.  There are, however,  a
variety of approximate and useful proce-
dures as well.

1  Methods of particle size measurement

   a  Knowing the magnification of the
      microscope (product of the magni-
      fication of objective and eyepiece),
      the size of particles can be esti-
      mated.  For example,  with a 10X
      eyepiece  and a 16-mm  (or 10X)
      objective, the total magnification
      is 100X.  A particle that appears to
      be  10-mm at 10 inches from the eye
      has an  actual size of 10 mm divided
      by  100  or 0. 10 mm or  100 microns.
      This  is in no sense an accurate
      method, but it does  permit quick
      estimation of particle size; the error
      in this  estimation is usually 10-25
      percent.

   b  Another approximate method is also
      based on  the use of known data.  If
      we  know approximately the  diameter
      of the microscope field, we can
      estimate the percentage of the
      diameter occupied by the object to
      be measured and calculate from
      these figures the approximate size
      of the object. The size of the micro-
      scope field depends  on  both the  objec-
      tive and the ocular although the latter
      is a minor influence.  The size of
      the field should be determined with
      a millimeter scale for each objective
      and ocular.  If this is done,  esti-
      mation of sizes by comparison with
      the entire field diameter can be quite
      accurate (5-10%).

   c  The movement of a graduated mechan-
      ical stage can also be used for rough
      measurement of diameters of large
      particles.  Stages are usually gradu-
      ated (with vernier) to read to 0. 1
      millimeter,  or 100 microns.  In
      practice,  the leading edge of the
      particle is brought to one of the lines
      of the cross hair in the eyepiece and
      a reading is taken of the stage position.
      Then  the particle is  moved across  the
      field by moving the mechanical stage
   in an appropriate direction until the
   second trailing edge just touches the
   cross-hair line.  A second reading is
   taken and the difference in the two
   readings is the distance moved or the
   size of the particle.   This method is
   especially useful when the particle
   is larger than the field, or when the
   optics give a distorted image near the
   edge of the field.

d  The above method can be  extended to
   projection or photography.  The image
   of the particles can be projected on a
   screen with a suitable light source or
   they may be photographed.  The final
   magnification, M, on the  projection
   surface (or film plane) is given approxi-
   mately by

        M = DXO. M.  XE. M. /25

      where O. M.  = objective magnification
            E. M.  = eyepiece magnification
               D  = projection distance
                    from the eyepiece  in
                    centimeters.

   The image detail can then be measured
   in centimeters and the actual  size com-
   puted by  dividing by M. This method
   is usually accurate to within  2-5 percent
   depending on  the size range of the detail
   measured.

e  The stated magnifications and/or focal
   lengths of the microscope optics are
   nominal and vary a bit from objective
   to objective or eyepiece to eyepiece.
   To obtain accurate measurements, a
   stage micrometer is used to  calibrate
   each combination of eyepiece and
   objective. The stage micrometer is
   a  glass microscope slide that has,
   accurately engraved  in the center, a
   scale, usually 2  millimeters long,
   divided into 200 parts, each part repre-
   senting 0. 01 millimeter.  Thus when
   this scale is observed, projected or
   photographed, the exact image magni-
   fication can be determined.  For
   example,  if 5 spaces of the stage micro-
   meter measure  6 millimeters when
   projected, the actual magnification is
                                                                                    5-21

-------
Optics and the Microscope
              5(0.01)
                       = 120 times.
        This magnification figure can be
        used to improve the accuracy of
        method 4 above.

      f  The simplest procedure and the most
        accurate is based on the use of a
        micrometer eyepiece.  Since  the
        eyepiece magnifies a real image
        from the objective,  it is possible
        to place a transparent scale in the
        same plane as the image from the
        objective and thus have a scale
        superimposed over the  image.  This
        is done by  first placing an eyepiece
        micrometer scale disc  in the  eyepiece.
        The eyepiece micrometer has an
        arbitrary scale and  must be cali-
        brated with each objective used.  The
        simplest way to do this is to place
        the stage micrometer on the stage
        and note a  convenient whole number
        of eyepiece micrometer divisions.
        The value in microns for each eye-
        piece micrometer division  is then
        easily  computed.   When the stage
        micrometer is removed and replaced
        by the  specimen,  the superimposed
        eyepiece scale can be used for accu-
        rate measurement of any feature in
        the specimen by direct observation,
        photography or projection.

   2  Calibration of eyepiece micrometer

     Each micrometer stage scale has
     divisions  lOOjj (0. 1 mm) apart; one
     or two of  these are usually subdivided
     into  10\i (0. 01-mm) divisions.   These
     form the standard against which the
     arbitrary  divisions in the micrometer
     eyepiece are to be calibrated.   Each
     objective  must be calibrated separately
     by noting  the correspondence between
     the stage  scale and the eyepiece scale.
     Starting witti the lowest power objective
     focus on the stage scale, arrange the
     two scales parallel and in good focus.
     It should be possible to determine the
     number of eyepiece divisions exactly
     equal to some  whole number of
     divisions of the stage scale, a distance
     readily, expressed in microns.
       The calibration consists,  then, of
       calculating the number of microns per
       eyepiece scale division.  To make the
       comparison as accurate as possible,  a
       large part of each scale must be used
       (see  Figure 17).   Let's assume that
       with  the low power 16-mm objective
       6 large divisions of the stage  scale
       (s. m. d.) are equal to 38 divisions of
       the eyepiece scale.  This means that
       38 eyepiece micrometer divisions  (e.m. d.)
       are equivalent to 600 microns.  Hence:

            1 e. m. d. =  600/38
                     =  15. 8n.
                  Figure 17

    COMPARISON OF STAGE MICROMETER
SCALE WITH EYEPIECE  MICROMETER SCALE

       Thus when that micrometer eyepiece
       is used with that 16-mm objective each
       division of the eyepiece scale is equivalent
       to 15. 8u,  and it can be used to make an
       accurate measurement of any object on
       the microscope stage.  A particle,  for
       example,  observed with the 16-mm objec-
       tive and measuring 8. 5 divisions on the
       eyepiece scale  is 8. 5 (15. 8) or  135n in
       diameter.

       Each objective  on your microscope  must
       be calibrated in this manner.

       A convenient way to record the  necessary
       data and to calculate n/emd is by means
       of a table.
    5-22

-------
                                                            Optics and theJVIicroscope

Objective
32-mm
16-mm
4-mm

No.
no.
18
6
1
Table
smd =
emd
= 44
= 38
= 30
7
V-
no.
1800
600
100

emd
= 44
= 38
= 30


H =
1 emd
40.
15.
3.
9n
8n
33(1
Determination of particle size
distribution

The measurement of particle size can
vary in complexity depending on parti-
cle shape.  The size of a sphere  may be
denoted by its diameter.  The size of a
cube may be  expressed by the length of
an edge or diagonal.  Beyond these two
configurations, the particle "size" must
include information about the shape of
the particle in question, and the
expression of this shape takes a more
complicated form.

Martin's diameter is the simplest means
of measuring and expressing the  dia-
meters of irregular particles and is
sufficiently accurate when averaged for
a large number of particles.  In this
method,  the horizontal or east-west
dimension of each particle which divides
the projected area into halves is  taken as
Martin's diameter (Figure 18).
P — |
          I- P -j
                    "9
            Figure 18

      MARTIN'S DIAMETER
   The more particles counted,  the more
   accurate will be the average particle
   size.  Platelike and needlelike particles
   should have a correction factor applied
   to account for the third dimension since
   all such particles are restricted in  their
   orientation on the microscope slide.
   When particle size is reported, the
   general shape of the particles as well as
   the method used to determine the
   "diameter" should be noted.

   Particle  size distribution is determined
   routinely by  moving a preparation of
   particles past an eyepiece micrometer
   scale in such a way that their Martin's
   diameter can be tallied. All particles
   whose centers fall within two  fixed
   divisions on  the scale are tallied.  Move-
   ment of the preparation is usually
   accomplished by means of a mechanical
   stage but may be carried out by rotation
   of an off-center rotating stage.  A sample
   tabulation appears in Table 8.  The  eye-
   piece and objective are chosen so that
   at least six,  but not more than twelve,
   size classes  are required and sufficient
   particles are counted to give a smooth
   curve. The actual number tallied (200 -
   2, 000) depends on particle shape
   regularity and the range of sizes.  The
   size tallied for each particle is  that
   number of eyepiece micrometer divisions
   most closely approximating Martin's
   diameter for that particle.

4  Calculation of size averages

   The size  data may be treated in a variety
   of ways; one  simple, straightforward
   treatment is  shown in Table 9.  For a
   more complete  discussion of the treat-
   ment of particle size data see Chamot
   and Mason's Handbook of Chemical
   Microscopy^3',  page 26.

   The averages^ with respect to number,
   d_i; surface, d3; and weight or volume,
   d,},  are calculated as follows for the
   data in Table 9.
                                                                                   5-23

-------
Optics and the Microscope
                 Table 8. PARTICLE SIZE TALLY FOR A SAMPLE OF STARCH GRAINS
          Size claM
            (emd»)
        Number of particles
                                                  Total
                                    M-4J  1
       n-wi
rt-+4  ri-w
                                 r-*-u
             rr-w  «-*4  rt-w  rt-u
             rr-»u  rv*4  111
rt-w  r-t-w  r*4.i  rt-u  r-r-w  r-r-u
rt-w  M-*J  rt-w  rt-u  I-*-AJ  rt-w
                    rt-u
                                                                          16
                                                                          as



                                                                         no
             i-t-w  r*-*j
rt-w  «-*j  rt-w  r-w-i
       r-t-*a
                                                                         107
                                                              rt-w.
                      1 1
                                          r*-i-a  rt-*~i
                                          rn-i
                      rt-w
                             fl-*4
                      1 1
                                                                          45
             *emd • ejceniece mlcrometeE.dlKialaa«
            d1 =  Łnd/En = 1758/470

              = 3.74 emdX 2.82*= 10. 5^

            d3 = End3/ 2nd2 = 37440/7662
              = 4. 89 emdX 2. 82 = 13. 8p

            d4 =  2nd4/Znd3 =  199194/37440

              = 5. 32 emdX 2.82 =  15. OJJL

            *2. 82 microns per emd
            (determined by calibration of the
            eyepiece-objective combination
            used for the determination).

      Cumulative percents by number,
      surface and weight (or volume) may be
      plotted from the data in Table 9.  The
      calculated percentages, e. g.
                             d = 1
                                             d = 15
                                               nd4 X 100
                                                   d = 1
                          for the cumulative weight or volume
                          curve, are plotted against d.   Finally,
                          the specific surface, Sm,  in square
                          meters per gram,  m, may be calculated
                          if the density, D, is known; the surface
                          average d3, is used.

                          If D = 1. 1,  Sm = 6/d3D =  6/13. 8(1. 1)

                                         = 0. 395m2/g.
   5-24

-------
                                                                   Optics and the Microscope
                 Table 9.  CALCULATIONS FOR PARTICLE SIZE AVERAGE
d
(Aver. diam.
in emd)








1
2
3
4
5
6
7
8
n
16
98
110
107
71
45
21
2
nd
16
196
330
423
355
270
147
16
nd2
16
392
990
1712
1775
1620
1029
128
nd3
16
784
2970
6848
8875
9720
7203
1024
nd4
16
1568
8910
27392
44375
58320
50421
8192
                                     470   1758  7662   37440   199194
B  Counting Analysis

   Mixtures of particulates can often be
   quantitatively analyzed by counting the
   total number of particulates from each
   component in a representative sample.
   The calculations are, however, compli-
   cated by three factors:  average particle
   size, particle  shape and the density
   of the components.  If all of the compon-
   ents were equivalent in  particle size,
   shape and density then the weight per-
   centage would  be  identical to the number
   percentage. Usually, however, it is
   necessary to determine  correction factors
   to account for  the differences.

   When properly applied,  this method can
   be accurate to within +_ 1 percent and,
   in special cases,  even better.  It is often
   applied to the analysis of fiber mixtures
   and is then usually called a dot-count
   because the tally  of fibers is kept as the
   preparation is moved past a point or dot
   in the eyepiece.

   A variety of methods can be used  to
   simplify recognition of the different
   components.  These include chemical
   stains or dyes and enhancement of optical
   differences such as refractive indices,
   dispersion or color.  Often, however, one
   relies on the differences in morphology.
e. g.  counting the percent of rayon fibers
in a sample of "silk".

Example 1: A dot-count of a mixture of
           fiberglass and nylon shows:
                  nylon
              fiberglass
             262
             168
Therefore:
              % nylon = 262/(262 + 168)X 100
                      = 60. 9% by number.
However, although both fibers are smooth
cylinders,  they do have different densities
and usually different diameters.  To
correct for diameter one must measure
the average diameter of each type of fiber
and calculate the  volume of a unit length
of each.
             aver. diam.    volume of
                  M.        l-(i slice, n3
   nylon
 fiberglass
18.5
13.2
268

117
The percent by volume is,  then:

                     262 X 268
      % nylon
               (262 X 268)+(168X 117)

             = 78. 1% by volume.
                     X 100
Still we must take into account the density of
each in order to calculate the weight percent.
                                                                                      5-25

-------
Optics and the Microscope
   If the densities are 1. 6 for nylon and 2. 2
   for glass then the percent by weight is:
        nylon =•
                         262 X 268 X 1.6
                (262 X 268 X 1.6)+(168X 117X2.2)
              = 72% by weight.

      Example 2:  A count of quartz and
                  gypsum shows:
                    quartz
                   gypsum
                283
                467
   To calculate the percent by weight we must
   take into account the average particle size,
   the  shape and the density of each.

   The average particle size with respect to
   weight,  64, must be measured for each
   and the  shape factor must be determined.
   Since gypsum is more platelike than quartz
   each particle of gypsum is thinner.  The
   shape factor can be approximated or can be
   roughly calculated by measuring the actual
   thickness of a number of particles.  We
   might find, for example, that gypsum parti-
   cles average 80% of the  volume of the aver-
   age quartz particle; this is our shape factor.
   The final equation for the weight percent is:
        quartz =
                          283 X ird4/6X Dq
                                                 X 100
238
                              Dq + 467 X IT dj/ 6 X 0. 80 X Dg
                                                            X 100
   where Dq and Dg are the densities of quartz
   and gypsum_ respeŁtively; 0.80 is the shape
   factor and d4 and d4 are the average parti-
   cle sizes with respect to weight for quartz
   and gypsum respectively.
 ACKNOWLEDGMENT:  This outline was
 prepared by the U. S. Public Health Service,
 Department of Health. Education and Welfare,
 for use in ita Training Program.

 REFERENCES

 1   Bunn,  C.  W.   Crystal Growth from
        Solution.  Discussions of the Faraday
        Society No. 5. 132.  Gunery and Jackson.
        London. (1949).
                             2  Loveland.  R. P.,  J.  Roy.  Micros. Soc.
                                  79, ?9.  (1980).

                             3  Chamot. Smile Monnin,  and Maaon,
                                  Clyde Walter.  Handbook of Chemical
                                  Microscopy, VoL  1.  third ed.
                                  John Wiley and Sons,  New York (1959).
                             DESCRIPTORS:
                             properties
Microscope and Optical
 5-26

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                           STRUCTURE AND FUNCTION OF CELLS
 I   INTRODUCTION

 What are cells?  Cells may be defined as the
 basic structural units of life.  The cell has
 many different parts which carry on the
 various functions of cell life.  These are
 called organelles ("little organs").

 A  The branch of biology which deals with the
    form and structure of plants and animals
    is called "Morphology. "  The study of the
    arrangement of their several parts  is
    called "anatomy", and the study of cells
    is called "cytology".
 B  There is no "typical" cell,  for cells differ
    from each other in detail, and these
    differences are in part responsible  for the
    variety of life that exists on the earth.
H   FUNDAMENTALS OF CELL STRUCTURE

 A How do we recognize a structure as a cell?
    We must look for certain characteristics
    and/or structures which have been found
    to occur in cells. The cell is composed
    of a variety of substances and structures,
    some of which result from cellular
    activities.  These include both living and
    non-living materials.

    1  Non-living components include:

      a  A "cell wall" composed of cellulose
         may be found as the outermost
         covering of many plant cells.

      b  "Vacuoles" are chambers in the
         protoplasm which contain fluids of
         different densities (i.e., different
         from the surrounding protoplasm).

    2  The "living" parts of the cell are called
      "protoplasm. " The following structures
      are included:

      a  A thin "cell membrane" is located
         just  inside the cell wall.  This
membrane may be thought of as the
outermost layer of protoplasm.

In plant cells the most conspicuous
protoplasmic structures are the
"chloroplasts",  which contain
highly organized membrane systems
bearing the photosynthetic pigments
(chlorophylls, carotenoids, and
xanthophylls) and enzymes.

The "nucleus" is a spherical body
which regulates cell function by
controlling enzyme synthesis.

"Granules" are  structures of small
size and may be "living" or
non-living" material.

"Flagella" are whip-like structures
found in both plant and animal cells.-
The flagella are used for locomotion,
or to circulate the surrounding
medium.

"Cilia"resemble short flagella, found
almost exclusively on animal cells.
In the lower animals, cilia are used
for locomotion and food gathering.

The "pseudopod",  or false foot, is
an extension of the protoplasm of
certain protozoa, in which the
colloidal state of the protoplasm
alternates from a "sol" to a "jel"
condition from time to time to
facilitate cell movement.

"Ribosomes" are protoplasmic bodies
which are the site of protein
synthesis. They are too small
(150 A in diameter)to be seen with
a light microscope.

"Mitochondria" are small mem-
branous structures containing
enzymes that oxidize food to produce
energy transfer compounds (ATP).
 BI.CEL. la. 6.76
                          6-1

-------
Structure and Function of Cells
B  How basic structure is expressed in some
   major types of organisms.

   We  can better visualize the variety of cell
   structure by considering several specific
   cells.

   1 Bacteria have few organelles, and are
     so minute that under the light
     microscope only general morphological
     types (i.e., the three basic shapes;
     rods,  spheres,  and spirals) can be
     recognized.   The following structures
     have been defined:

     a The "capsule" is a thick protective
        covering of the cell exterior, con-
        sisting of polysaccharide or
        polyp eptide.

     b The cell wall and plasma membrane
        are present.

     c Although no well defined nucleus is
        visible in bacterial cells, the
        electron microscope has revealed
        areas of deoxyribose nucleic acid
        (DNA) concentration. This sub-
        stance is present within the nucleus of
        of higher cells,  and is the genetic
        or hereditary material.

     d Some types of bacteria contain a
        special type of chlorophyll
        (bacteriochlorophyll) and carry on
        photosynthesis.

   2 The blue-green algae are similar to the
     bacteria in structure, but contain the
     photo synthetic pigment chlorophyll a.

     a Like the bacteria, these forms also
        lack an organized nucleus (the
        nuclear region is not bounded by a
        membrane).

     b The chlorophyll-bearing membranes
        are not localized in distinct bodies
        (chloroplasts), but are dispersed
        throughout the cell.

     c Gas-filled  structures called
        "pseudovacuoles" are found in some
        types of blue-greens.
The green algae as a group include a
great variety of structural types,
ranging from single-celled non-motile
forms to large motile colonies.   Some
types are large enough to resemble
higher aquatic plants.

a  The chloroplasts are modified into
   a variety of shapes and are located
   in different positions.  Examples
   of chloroplast shape and position are:


   1) Parietal  - located on the
      periphery of the cell; usually
      cup-shaped and may extend
      completely around the inner
      surface of the plasma membrane.

   2) Discoid - also located on the
      periphery of the cell, but are
      plate-shaped; usually many per
      cell.

   3) Axial - lying in the  central axis
      of the cell; may be ribbon-like
      or  star-shaped.

   4) Radial  - have arms  or processes
      that extend outward from the
      center  of the cell (radiate),
      reaching the plasma membrane.

   5) Reticulate - a mesh-like network
      that extends throughout volume
      of the cell.

b  Located in the chloroplasts may be
   dense, proteinaceous,  starch-
   forming bodies called "pyrenoids".

The flagellated algae  possess one-to-
eight flagella per cell.  The chloro-
plasts may contain brown  and/or red
pigments in addition to chlorophyll.

a  Reserve food may  be stored as
   starch (Chlamydomonas) paramylon
   (Euglena), or as oil.

The protozoa are single-celled
animals which exhibit a variety of
cell structure.
   6-2

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                                                           Structure and Function of Cells
          The amoebae move by means of
          pseudopodia,  as described
          previously.

          The flagellated protozoa
          (Mastigophora) possess one or more
          flagella.

          The ciliates are the most highly
          modified protozoans.  The cilia may
          be more or less evenly  distributed
          over the entire surface  of the cell,
          or may be localized.
IE  FUNCTIONS OF CELLS

 What are the functions of cells and their
 structural components?   Cellular function
 is called "life", and life is difficult to define.
 Life is characterized by processes commonly
 referred to as reproduction, growth, photo-
 synthesis, etc.

 A Microorganisms living in surface waters
    are subjected to constant fluctuations in
    the physical and chemical characteristic
    of the environment, and must constantly
    modify their activities.

    1  The cell requires a source of chemical
       energy to carry on  life processes and
       successfully compete with other
       organisms.  Plant cells may obtain
       this energy from light, which is
       absorbed by chlorophyll and converted
       into ATP or food reserves,  or from
       the oxidation of food stuffs.  Animal
       cells obtain energy only from the
       oxidation of food.

    2  Cells must obtain raw materials from
       the environment in  order to grow and
       carry out other life functions.  Inorganic
       and organic materials may be taken up
       by passive diffusion or by "active
       transport".  In the  later process,
       energy is used to build up and maintain
       a higher concentration of a substance
       (such as phosphate) inside the cell than
       is found outside.  Algae are able to
       synthesis organic matter from inorganic
       raw materials (carbon dioxide and
       water),  with the aid of energy derived
       from light, whereas animal cells must
       obtain their organic matter  "ready-
       made" by consuming other organisms,
       organic debris, or dissolved organics.
IV  SUMMARY

 The cell is made up of many highly special-
 ized substructures. The types of sub-
 structures present, and their appearance
 (shape, color,  etc,) are very important in
 understanding the role of the organism in
 the aquatic community, and in classification.
 REFERENCES

 1  Bold, H. C.   Cytology of algae.  In:  G.M.
       Smith, (ed.), Manual of Phycology.
       Ronald Press.   1951.
    Bourne,  GeoffryH.,
       Cell Physiology.
       Press.   1964.
ed.  Cytology and
3rd ed.  Academic
 3  Brachet, Jean.   The Living Cell.
       Scientific American. 205(3).  1961.

 4  Corliss, John O.  Ciliated Protozoa.
       Pegamon.   1961.

 5  Fritsch, F. E.   The structure and
       reproduction of the algae.   Cambridge
       Univ. Press.   1965.

 6  Frobisher,  M.  Fundamentals of
       microbiology.  7th edition.   W. B.
       Saunders Co., Philadelphia.  1962.

 7  Round,  F.E.   The biology of the algae.
       St. Martin's Press.   New York.  1965.
                                                  This outline originally prepared by Michael
                                                  E. Bender, Biologist, formerly with
                                                  Training Activities, FWPCA, SEC. and
                                                  revised by Cornelius I. Weber,  March 1970.

                                                  Descriptor: Cytological Studies
                                                                                      6-3

-------
                                      TYPES OF ALGAE
 I   INTRODUCTION

 A  Algae  in general may be  defined as
     small  pigmented plant-like organisms
     of relatively simple  structure.  Actually
     the size range  is extreme: from only a
     few microns to  over  three hundred feet
     in length.   Commonly  observed examples
     include the greenish  pond scum or frog
     spittle of freshwater ponds,  much  of the
     golden brown slime covering rocks in  a
     trout stream, and  the  great  marine kelps
     and  seaweeds.   Large freshwater forms
     as Nitella  and Chara  or  stonewort are
     also included.

 B  Algae  approach ubiquity in distribution.
     In addition  to the commonly  observed
     bodies of water, certain algae also live
     in such unlikely places  as thermal springs,
     the surface of melting snow,  on the hair
     of the  three toed sloth in Central America,
     and  in conjunction  with  certain fungi to
     form lichens.
Ill   ALGAE WILL  BE GROUPED FOR THE
     SAKE OF CONVENIENCE INTO FOUR
     GENERAL TYPES:

 A  Blue-greens (See  plate: Blue-Green
     Algae,  Cyanophyceae).  This is a valid
     technical  group.   The size range  is not
     very great,   some being so small  as  to
     approach  the size range of the bacteria.

     1  These  are the only algae  in which
        the pigments are not localized in
        definite bodies but dissolved through-
        out the cell.   Blue,  red,  or other
        pigments are  present in addition to
        chlorophyll  thus  giving the cells a
        bluish  green,  yellow, or red color,
        at least  enmasse.

     2  The  nucleus lacks a nuclear membrane.

     3  Tend to  achieve  nuisance  concentrations
        more frequently  in the warm summer
        months and in the richer  waters.
4  Vegetative reproduction, in addition
   to cell division, includes the forma-
   tion of "hormogones, " or short specif-
   ically delimited sections of trichomes
   (filaments).

5  Spores of three types are  encountered:

   a  Akinetes  are usually  larger, thick
      walled resting spores.

   b  Heterocysts appear like  empty
      cell walls,  but are actually filled
      with protoplasm,  have occasionally
      been observed to germinate.

   c  Endospores, also called  "gonidia"
      or conidia,   are formed by repeated
      division  of  the protoplast within a
      given cell wall.   Present in only a
      few genera.

6  Some common  examples  of blue-
   green  algae are:

      Anacystis (Microcystis or
      Polycystis), Anabaena,  Aphani-
      zomenon, and Oscillatoria
The  Pigmented flagellates (in contrast
to the non-pigmented or  animal-like
flagellates)  are  a heterogeneous
collection of motile forms from several
different algal groups (See plate:
Flagellated  algae).

1  There may be  one,  two,  four,  or
   more flagella per cell.

2  There is a well organized nucleus.

3  A light-sensitive red eyespot usually
   present.

4  The  chlorophyll is contained in one
   or more  distinctive  bodies called
   plastids.
   Bl.MIC.cla. 19a. 6. 76
                                                                                       7-1

-------
Types  of Algae
    5  Two or  more cells may be associated
       in a colony.

    6  Non-motile life history stages may
       be encountered.

    7  Masses  of stored starch called  •
       pyrenoid bodies are  often conspicuous.

    8  Some examples of pigmented  flag-
       ellates are:  Euglena, Phacus,
       Chlamydomonas,  Gonium, Volvox,
       Peridinium,  Ceratium Mallomonas,
       Synura and Dinobryon.
   The Non-motile  green algae constitute
   another heterogeneous assembly  of un-
   related forms (See plate: Non-Motile
   Green Algae)

   1   Like the  flagellates they have well
       organized nuclei and chloroplasts.
       The  shape of the chloroplast is often
       distinctive.

   2   They lack flagella or any other loco-
       motor device.

   3   There is  extreme  structural variation
       among the group.

   4   Some types tend to occur as a general
       planktonic mass or bloom, " often in
       combinations of two  or more species.

       Some examples are:  Sphaerocystis,
       Pediastrum,  Scenedesmus, and the
       desmid Cosmarium.

   5   Threadlike (filamentous)  green algae
       may  form masses  or blankets, cutting
       off light,  and reducing water circula-
       tion.   They also add considerably to
       the total mass of organic matter.
       Some examples of  this type are:
       Spirogyra, Hydrodictyon, Cladophora,
       Oedogonium,  and  Chara.
D  The Diatoms constitute another valid
   technical group (See plate: Diatoms-
   Bacillariophyceae).
        In appearance,  they are geometrically
        regular in  shape.   The presence of a
        brownish pigment  in addition to  the
        chlorophyll gives them a  golden to
        greenish color.

        Motile  forms have a  distinctive
        hesitating progression.

        The most distinctive  structural
        feature is the two-part shell
        (frustule) composed of silicon
        dioxide (glass).

        a One part fits inside the other as
           the  two halves of a pill box,  or a
           petri dish.

        b The surface of these shells are
          sculptured with minute pits and
          lines arrange'd with geometrical
          perfection.

        c The view  from the side is called
          the  "girdle view, "  that from above
          or below,  the  "valve view. "

        There are two general shapes of
        diatoms,  circular  (centric) and
        elongate (pennate).   The elongate
        forms may be motile,  the circular
        ones are not.

        Diatoms may associate in colonies
        in various ways.

        Examples  of  diatoms  frequently  en-
        countered  are:  Stephanodiscus
        Cyclotella,  Asterionella.  Fragilaria,
        Tabellaria, Synedra.  and  Nitzschia.
This outline was prepared by H. W. JACKSON,
former Chief Biologist. National Training
Center, and revised by R. M. SINCLAIR,
Aquatic Biologist, National Training Center,
MOTD. OWPO,  USEPA. Cincinnati, Ohio
45268.

Descriptor: Algae
 7-2

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                                                                          Types of Algae
           KEY FOR IDENTIFICATION OF GROUPS OF FRESHWATER ALGAE
              Beginning with "la" and "ib",  choose one of the two contrasting
              statements and follow this procedure with the "a" and "b" state-
              ments of the number given at the end of the chosen statement.
              Continue until the name of the algal group is given instead of
              another key number.
               la.   Plastid (separate color body) absent; complete protoplast
                    pigmented; generally blue-green; iodine starch test*
                    negative			Blue-green algae

               Ib.   Plastid or plastids present; parts of protoplast free of some
                    or all pigments; generally green, brown,  red,  etc., but not
                    blue-green; iodine starch test* positive or negative	2
              2a.   Cell wall permanently rigid (never showing evidence of
                    collapse), and with regular pattern of fine markings
                    (striations, etc.); plastids brown to green; iodine starch test*
                    negative; flagella absent; wall of two essentially similar halves,
                    one placed over the other as a cover	Diatoms

              2b.   Cell wall, if present,  capable of sagging, wrinkling, bulging
                    or  rigidity, depending on existing turgor pressure of cell
                    protoplast; regular pattern of fine markings on wall generally
                    absent; plastids green, red, brown, etc.; iodine starch test*
                    positive or negative;  flagella present or absent; cell wall
                    continuous and generally not of two parts	3
              3a.   Cell or colony motile; flagella present (often not readily visible);
                    anterior and posterior ends of cell different from one another in
                    contents and often in shape	Flagellate algae

              3b.   Non-motile; true flagella absent; ends of cells often not
                    differentiated	Green algae and associated forms
*Add one drop Lugol's (iodine) solution,  diluted 1-1 with distilled water.  In about 1 minute,
 if positive, starch is stained blue and,  later black.  Other structures (such as nucleus,
 plastids, cell wall) may also stain, but turn brown to yellow.


                                                                                  7-3

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Types of Algae
CMP
               COMPARISON OF FOUR MAJOR GROUPS OF ALGAE

Color
Location
of pigment
Starch
Slimy
coating
Nucleus
Flagellum
Cell Wall
"Eye "spot
Blue-Green
Blue-Green
(Brown)
Throughout
cell
Absent
Present
Absent
Absent
Inseparable
from slimy
coating
Absent
Pigmented
flagellates
Green
Brown
In plastids
Present or
Absent
Absent
in most
Present
Present
Thin or
Absent
Present
Greens
Green
In plastids
Present
Absent
in most
Present
Absent
Semi-rigid
smooth or
with spines
Absent
Diatoms
Brown
(Light-Green)
In plastids
Absent
Absent
in most
Present
Absent
Very rigid,
with regular
markings
Absent

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                                   BLUE-GREEN ALGAE
I   WHAT ARE THE BLUE-GREEN ALGAE?

The blue-green algae (Myxophyceae) comprise
that large group of microscopic organisms
living in aquatic or moist habitats, carrying
on photosynthesis and having differentiation
of cells which is  a little more complex than
bacteria,  and simpler than all of the other
plants called algae.
II   WHY ARE THEY CALLED BLUE-GREEN:

In addition to the green photosynthetic pigment
(chlorophyll-a) they always have a blue pig-
ment (phyocyanin-c) which tends to give the
cushions or mats they may form a blue-green
tinge.
Ill   WHERE ARE THE BLUE-GREENS FOUND?

Some are free floating (pelagic and planktonic),
others grow from submerged or moist soil,
rocks, wood and other objects in both fresh-
wa.ter and marine habitats.
IV   WHAT ARE SOME OF THEIR GENERAL
     CHARACTERISTICS?

Some are gelatinous masses of various shapes
floating in water.  Others, microscopic in
size, grow in great numbers so as to color
the water in which they live.  Structurally
their cells are similar to bacteria.  Their
protoplasts may be sheathed or imbedded in
gelatin, making them slimy.  Cells of blue -
green algae are without organized nuclei,
central vacuoles, or cilia and flagella.
No sexual reproduction is known.  Asexual
reproduction may be effected by fragmentation,
in which case special separation devices are
formed (dead cells, and heterocysts).  Some
species are preserved over unfavorable
periods by special spores (akinetes and endo-
spores).
V   OF WHAT IMPORTANCE ARE BLUE-
     GREEN ALGAE?

They have both positive and negative economic
significance.  Because they can convert
radient energy into chemical energy, they
are producers forming a first link at the base
of the food chain.  Because many very in-
tricate nutritional relationships exist among
the myraids of organisms it is difficult to
know the value of the blue-greens.  However,
people who know what the blue-greens can dp
to drinking and recreational water classify
them as of negative economic importance,
because they are often nuisances when they
impart color, bad odors, and fishy tastes,
or toxins.  Some of them can foul pipes
and clog filters.


VI  WHEN ARE THEY MOST COMMON?

They are widely distributed in time and space,
but tend to reach nuisance concentrations more
frequently in the late summer and in eutrophic
waters.
VII WHAT DO BLUE-GREEN ALGAE DO
    FOR A LIVING?

The pioneer-forms are of great ecological
importance because they live in habitats fre-
quented by few other forms of live,  synthesiz-
ing organic substances and building substrata
that can support other kinds of life.

    A   Some blue-greens live in association
        with other organisms as symbionts.
        Still others are  found in polluted
        waters, because they are able to
        exist in habitats poor in oxygen.  The
        growth of these  kinds of algae under
        such conditions  tends to make a pol-
        luted condition worse.

    B   On the other hand some species
        should be promoted because they
        provide oxygen and food through photo-
        synthesis.  The  first evident  product
        of photosynthesis is  glycogen, and
        is the cause of the brown coloration
        with the iodine test.   Some of the
        glycogen is used to produce glycopro-
        teins.  The gelatinous sheath is com-
        posed of pectic substances,  cellulose
        and related compounds.
BI.MIC.cla. 16a. 6.76
                                   8-1

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Blue-Green Algae
        When blue-greens mat at the surface
        of the water the increased lighting
        may be too strong, resulting in a
        kill.  At this time they may turn
        from a blue-green to a yellow-green
        color.  Here they decompose in
        mass.  The resulting intermediate
        products of decomposition may be
        highly undesirable, because of bad
        looks, four odors,  bad tastes and
        toxins.  Under these conditions the
        BOD may produce conditions not
        unlike raw sewage.
VHI  WHAT DO BLUE-GREEN ALGAE LOOK
     LIKE UNDER THE MICROSCOPE?

    A   A cross section of a typical cell
        would show an outside nonliving
        gelatinous layer surrounding a woody
        cell wall,  which is bulging from
        turgor pressure from the cell (plasma)
        membrane, pushing the wall outward-
        ly.  The protoplasm, contained with-
        in the plasma membrane, ie divided
        into two regions.  The peripheral
        pigmented portion called chroma-
        toplasm, and an inner centroplas'm,
        the centroplasm contains chromatins,
        which is also known as in incipient
        nucleus or central body, containing
        chromosomes and genes.  Structures
        (chromatophores or plastids) con-
        taining pigments have not been found
        in the blue-greens.  The photosyn-
        thetic pigments are dissolved in the
        peripheral cytoplasm, which is known
        as the chromatoplasm.

    B   A simple way to understand the cross
        section would be to compare it with
        a doughnut, with the hole represent-
        ing the colorless central body or
        incipient nucleus,  which houses the
        chromatoplasm, having the charac-
        teristic blue-green color from its
        dissolved photosynthetic pigments.
DC  WHAT CAUSES THESE FOUL-TO-SMELL
    UNSIGHTLY BLOOMS?

When the protoplasts become sick or old they
may develop a great number of "pseudovac-
uoles" filled with gas.  These gas bubbles make
the algae buoyant in such a way that they may
"flower" or bloom by rising to the surface
(planktonic, healthy blue-greens normally
possess pseudovacuoles,  which are here
excepted).  Soon they begin to stink because
of the odors produced from putrefaction.
The lack of dissolved oxygen during this
period may affect other organisms.
X   ARE ALL BLOOMS PUTREFACTIVE?

No.  Healthy blooms are produced by myraids
of cells living near the surface of the water
at times when environmental conditions are
especially favorable for them.  Putrefactive
blooms are usually from masses of algae
undergoing degradation.
XI  WHAT ARE SOME OF THE MAJOR
    KINDS OF BLUE-GREENS?

Most species of blue-greens may be placed
into two major groups: the nonfilamentous
(coccoid) forms,  and the filamentous forms.
See the set of drawings following this treat-
ment to get a graphic concept of the two
groups.
XH WHAT ARE SOME OF THE MORE
    DISTINCTIVE FEATURES OF BLUE-
    GREENS?

    A   In comparing the blue-greens with
        other algae it is easier to tell what
        they do not possess than what they
        do.   They do not have chromatophores
        or plastids,  cilia,  flagella, organized
        nuclei, gametes, central vacuoles,
        chlorophyll-b,  or true starch.

    B   Many of the  filamentous forms, es-
        pecially the Oscillatoriaceae, exhibit
        an unexplained movement.  When the
        filamentous forms  are surrounded
        by a gelatinous  sheath the row of cells
        inside is called a trichome. and the
        trichome with its enclosing sheath is
        called a filament.  There may be more
        than one trichome within a sheath.
 8-2

-------
                                                                          Blue-Green Algae
         True branching occurs when a cell
         of the series divides lengthwise and
         the outer-formed cell adds cells to
         form a true branch.  However, two
         or more trichomes within a single
         sheath may be so arranged that though
         they appear to be branches, their cells
         actually have all divided in the same
         plane, and the trichomes have pushed
         out from growth to form false branch-
         ing, as in Tolypothrix.

    C   An occasional  reticulated or bubbly
         appearance is  referred to as  pseudov-
         acuolation,  and en mass imparts a
         pale, yellowish color to the algae.
         Under low powers these vacuoles
         appear dark, under higher  magnifi-
         cations they are reddish.

    D   Vegetative reproduction in addition
         to cell division for the unicellular
         forms, is by special kinds  of frag-
         mentation.  This includes the for-
         mation of hormogones, which are
         specifically delimited sections of
         trichomes,  and are characteristic
         of some taxonomic entities.

    E   Spores of three types are encountered.

         1  Akinetes are usually larger, non-
           motile, thick-walled resting spores.
         2  Heterocysts appear like empty cell
           walls, but are filled with colorless
           protoplasm and have been  occasion-
           ally observed to germinate.
         3  Endospores, also called gonidia,
           are formed by a repeated division
           of the protoplast within a cell wall
           container.
XIII   WHAT ARE SOME EXAMPLES OF BLUE-
      GREEN ALGAE?

    A  Anacystis (Microcystis) is common
        in hard waters.

        1   Colonies are always free floating.

        2   Their shapes may be roughly
            spherical or irregular,  micro-
            scopic or macroscopic.

        3   The gelatinous matrix may be
    extremely transparent,  easily
    broken up on preservation.

4   They frequently contain pseudov-
    acuoles.

Anabaena is an example of a fila-
mentous form.

1   Filaments may occur singly or
    in irregular colonies,  and free
    floating or in a delicate nucous
    matrix.

2   Trichomes have practically
    the same  diameter throughout;
    may be straight,  spiral, or
    irregularly contorted.

3   Cells are usually spherical,
    or barrel shaped,  rarely cy-
    lindrical and never discoid.

4   Heterocysts are usually the same
    shape but are  slightly larger
    than the vegetative cells.

5   Akinetes are always larger than
    the vegetative cells, roughly
    cylindrical, and with rounded
    ends.

6   It may be readily distinguished
    from Nostoc by the lack of  a
    firm gelatinous envelope.

7   It may produce an undesirable
    grassy, moldy or other odor.

Aphanizomenon is  a strictly plank-
tonic filamentous form.

1   Trichomes are relatively straight,
    and laterally joined into loose
    macroscopic free-floating flake-
    like colonies.

2   Cells are cylindrical or barrel
    shaped, longer than broad.

3   Heterocysts occur within the
    filament (i. e., not terminal).

4   Akinetes are cylindrical and
    relatively long.
                                                                                        8-3

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Blue-Green Algae
                      SOME   BLUE-GREEN   ALGAE

           Anacystis (Chroococcus) X600.
                                          Agmenellum
                                         (Merismopediurn.)-..X600.
             Coccochloris  (Gloeocapaa)  X600.
                                                                     (X825)
         II.  Filamentous  blue-green algae:
                   Trichotnea  of Spirulina. (X600).
Trichomes of Arthrospira
          (X60TJT:
    j^)}vegetative cell

        neterocyst
                       akinete
                      I (spore)

                       Anabaena
                        (X825).

                                                         Phortnidium (with sheath)
                                                                      (X825).
                                                      Oscillatoria  (without sheath)
                                                                     (X825)
                                           False branching
                     True branching         Tolypothrix  (X375)
                     Hapatf^phon  Prepared by Louis  G. Williams
                                   Aquatic : Biologist, Basic Data, SEC.
  8-4

-------
                                                                     Blue-Green Algae
    5   Often imparts grassy or nastur-
        tium-like odors to water.

D  Oscillatoria is a large and ubiquitous
    genus.

    1   Filaments may occur singly or
        interwoven to form mats of
        indefinite extent.

    2   Trichomes are unbranched, cy-
        lindrical, and practically with-
        out sheaths.

    3   Species with narrow trichomes
        have long cylindrical cells
        while those with broader tri-
        chomes have short broad cells.

    4   No heterocysts or akinetes are
        known in Oscillatoria.   It re -
        produces  by fragmentation from
        hormongonia only.

    5   Live species exhibit "oscillatoria"
        movements, which are oscillating.

    6   Species of Oscillatoria may be
        readily distinguished from
        Lyngbya by the absence of a
        sheath.

E   Nodularia is an occasional producer
    of blooms.
             Trichomes are practically the
             same diameter throughout.

             Sheaths are usually distinct,
             fairly firm, and with a single
             trichome.
REFERENCES

1  Bartsch, A. F.  (ed.)  Environmental
      Requirements of Blue-Green Algae.
      FWPCA. Pacific Northwest Water
      Laboratory, Corvallis, Oregon.
      lllpp.  1967.

2  Desikachary, T. V.  Cyanophyta, Indian
      Council Agric. Res.  New Delhi.   1959.

3  Drouet, Francis.   Mxyophyceae.  Chapter
      5 in Edmondson.  Freshwater Biology.
      p.  95-114.  Wiley.  1959.

4  Drouet, Francis.   Revision of the Classifi-
      cation of the Oscillariaceae.  Monograph
      15.  Acad.  Nat.  Sci.  Phil. 370 pp.   1968.

5  Jackson, Daniel F.  (ed.) Algae, Man, and
      the Environment. Univ. Syracuse Press.
      554 pp.  1968.
This outline was prepared by L. G. Williams,
Formerly Aquatic Biologist, Aquatic Biology
Activities, Research and Development,
Cincinnati Water  Research Laboratory, FWPCA.
    1   Vegetative cells, heterocysts,
        and even the akinetes are broader
        than long.
 Descriptor: Cyanophyta
                                                                                  8-5

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                       GREEN AND OTHER PIGMENTED FLAGELLATES
  I  INTRODUCTION

  A A flagellate is a free swimming cell
    (or colony) with one or more flagella.

  B Motile flagellated cells occur in most
    (not all) great groups of plants and animals.

  C Out main concern will be with "mature"
    flagellated algae.
 E  THE STRUCTURE OF A PIGMENTED OR
    PLANT-LIKE FLAGELLATE

 A There is a well organized nucleus.

 B The flagellum is  a long whip-like process
    which acts as a propeller.

    1  It has a distinctive structure.

    2  There may be one or several per cell.

 C The chlorophyll is contained in one or
    more chloroplasts.

 D Two or more cells may be associated in
    a colony.

 E Non-Motile Life history stages may be
    en countered.

 F Size is of little use in identification.

 G Pyrenoid bodies are often conspicuous.
in  The Euglenophyta or Euglena-like algae
 (Figures 1-4) are almost exclusively single
 celled free swimming flagellates.  Nutrition
 may be holophytic, holozoic,  or saprophytic,
 even within the same species.  Referred to
 by zoologists as mastigophora;  many animal
 like forms are parasitic or commensalistic.
 Food reserves of plant-like forms are as
 paramylin (an insoluble carbohydrate) and
 fats (do not respond to starch test).  Thick
 walled resting stages (cysts) are common.
"Metabolic movement" characteristics of
some genera (Euglena).

Eyespot usually present in anterior end,
rarely more than one flagellum.

A  Euglena is a large genus with pronounced
   metabolic movement (Figure 1).

   1  Cells  spindle shaped

   2  Single flagellum

   3  Eyespot usually present

   4  Chloroplasts numerous, discoid
      to band shaped

   5  E_.  sanguinea has red pigment.

   6  E.  viridis generally favors water
      rich in organic  matter.

   7  E_.  gracilis is less tolerant of pollution.

B  Phacus cells maintain a rigid shape
   (Figure 2).

   1  Often  flattened and twisted, with
      pointed tip or tail end.

   2  Cell wall (periplast) often marked
      with fine ridges.

   3  P.  pyrum favored by polluted water.

   4  P.  pleuronectes relatively intolerant
      of pollution.

C  Trachelomonas cells surrounded by a
   distinct shell (lorica) with flagellum
   sticking through hole or collar (Figure 4).

   1  Surface may be smooth or rough

   2  Usually brown in color

   3  Some  species such as T. cerebea
      known to clog filters
  BI.MIC.cla.6c. 6.76
                                                                                     9-1

-------
   Green and Other Pigmented Flagellates
  D Lepocinclis has rigid naked cells with
    longitudinal or spiral ridges (Figure 3).

    1  Cells uncompressed,  elipsoidal to oval
       (in contrast to phacus)

    2  Only two species with pointed tails

    3  L. texta often associated with waters
       of high organic content
IV  The Chlorophyta or grass green algae
 (Figures 5-9) are the largest and most varied
 group.  Non-flagellated forms predominate but
 many conspicuous flagellates are included.
 Food reserves are usually stored as starch
 which is readily identified with iodine.
 Usually two flagella of equal length are
 present.  More planktonic forms are included
 than in any other group,  predominating in the
 late spring and early autumn.

 The cell is typically surrounded by a definite
 wall and usually has a definite shape. Cell
 pigments closely resemble those of higher
 plants,  but some have accessory pigments
 and a few forms have little or none.  The
 chloroplasts always have a shape charac-
 teristic of the genus.

 The flagellated chlorophyta are contained in
 the Order  Volvocales, the Volcocine algae.
 All are actively motile during vegetative
 phases. May be unicellular or colonial.  All
 have an eyespot near the base of the flagella.
 Colonies may range from a simple  plate
 (Gonium sociale) to a complete hollow sphere
 (Volvox spp  ).

 A  Chlamydomonas is a solitary free swimming
    genus (Figure 5).

    1  Species range from cylindrical to
       pearshaped.

    2  Some species have a gelatinous sheath.

    3  There  are two flagella  inserted close
       together.

    4  Generally favored by polluted waters.
B  Carteria resembles Chlamydomonas very
   closely except that it has four flagella
   instead of two.  Generally favored by
   polluted water (Figure 7).

C  Phacotus usually has free swimming
   biflagellate cells surrounded by biconcave
   envelopes resembling two clam shells.
   These are usually sculptured, dark
   colored,  and impregnated with calcium
   carbonate.

   1  The eyespot ranges from anterior
      to posterior.

   2  Several daughter cells may be retained
      within the old envelopes of the parent
      cell.

   3  A clean water indicator.

D  Chlorogonium is a distinctive genus in
   which the cell is fusiform,  the tail end
   pointed, and the anterior end slightly
   blunt (Figure 6).

   1  The two flagella only about half as
      long as the cell.

   2  The cell wall is rather delicate.

   3  An eyespot usually present near the
      anterior end.

   4  Favored by pollution.

E  Gonium colonies typically have 4  to 32
   cells arranged  in a plate (Figure  8).

   1  The cells are imbedded in a gelatinous
      matrix.

   2  Sixteen celled colonies move through
      the water with a somersault-like
      motion.

   3  Four and eight celled colonies swim
      flagella end first.

   4  Gonium pectorale is typically a
      plankton form.

F  Pandorina colonies range up  to 32 cells,
   usually roughly spherical (Figure 9).
  9-2

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                                                   Green and Other Pigmented Flagellates
   1  Cells arranged in a hollow sphere
      within a gelatinous matrix.

   2  Often encountered especially in hard-
      water lakes, but seldom abundant.

   3  P. morurn may cause a faintly fishy
      odor.

G  Eudorina has up to 64 cells in roughly
   spherical colonies.

   1  The cells may be deeply imbedded in
      a gelatinous matrix.

   2  Common in the plankton of soft water
      lakes.

   ^  !?• elegans is widely distributed.

   4  May cause faintly fishy odor.

H  Pleodorina has up to 128 cells located
   near the surface of the gelatinous matrix.
   It is widespread in the United States.

I  Volvox rarely has less than 500 cells
   per colony.

   1  Central portion of the mature colony
      may contain only water.

   2  Daughter colonies form inside the
      parent colony.

   3  V. aureus imparts a fishy odor to the
      water when present in abundance.

J  Chlamydobotrys has "mulberry shaped"
   colonies,  with biflagellate cells alternately
   arranged in tiers of four each.
   (Spondylomorum has quadriflagellate cells).

   1  There is no enveloping sheath.

   2  C. stellata is favored by pollution.
V   The Pyrrhophyta includes principally the
 armored or dinoflagellates (Dinophyceae)
 (Figures 14-16).  This group is almost
 exclusively flagellated and is characterized
 by chromatophores which are yellow-brown
 in color.  Food reserves are stored as
 starch or oil.  Naked,  holozoic,  and
 saprozoic representatives are found.
 Both "unarmored", and "armored" forms
 with chromatophores are found to ingest
 solid food readily, and holozoic nutrition
 may be as important as holophytic.

 The great majority have walls of cellulose
 consisting of a definite number of articulated
 plates which may be very elaborate in
 structure.  There is always a groove
 girdling the cell in which one flagellum
 operates, the other extends backward from
 the point of origin.

 Most of the dino-flagellates are marine and
 some are parasitic.  There are six fresh
 water genera of importance in this country.

 A  Gymnodinum species are generally naked
    except for a few freshwater species.

    G.  brevis (marine) is a toxic form
    considered to be responsible for the
    "red tide" episodes in Florida and
    elsewhere.

 B  Species of Gonyaulax (catanella and
    tamarensis) are responsible for the
    paralytic shellfish poisoning.

 C  Ceratium is distinctive in that the
    anterior and posterior ends are con-
    tinued as long horns (Figure 16).

    1  Seasonal temperature changes have  a
      pronounced effect on the shape of the
      cells of this species.

    2  C.  hirudinella in high concentration is
      reported to produce a  "vile stench".
                                                                                9-3

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  Green and Other Pigmented Flagellates
 D Peridinium is a circular, oval,  or
    angular form, depending on the view
    (Figure 15).

    1  Cell wall is thick and heavy.

    2  Plates are usually much ornamented.

    3  I\ cinctum has been charged with a
       fishy odor.
VI  The Division Chrysophyta contains two
 classes which include flagellates,  the
 Xanthophyceae or Heterokontae (yellow-
 green algae) and the Chrysophyceae (golden-
 green algae) (Figures 10-13). The third
 class, the diatoms  (Bacillarieae or
 Bacillariophyceae),  is not flagellated.

 A  None of the Xanthophyceae are  included
    in the present discussion.

 B  The Chrysophyceae possess chroma-
    tophores of a golden brown color, usually
    without pyrenoids.  Food reserves are
    stored as fats and leucosin. One or two
    flagella; if two,  they may be of equal or
    unequal length.  Internal silicious cysts
    may be formed.   Tend to occur in
    relatively pure water.  Both holozoic and
    holophytic types of nutrition are found.
    Certain minute forms considered to be
    highly  sensitive  to pollution.

    1  Mallomonas is a solitary,  free
       swimming genus with one flagellum
       (Figure 13).

       a Covered with silicious plates,  many
         of which bear long silicious spines.

       b Tends to inhabit  clear water lakes
         at moderate depths.

       c M. caudata imparts a fishy odor
         to the water.

    2  Chrysococcus cells are minute, with
       two  yellowish brown chromatophores
       and  one flagellum.

       a Droplets of stored oil present

       b Lorica distinct
   c  C. rufesceus a clean water form

3  Chromulina has a single flagellum,
   may accumulate single large granule
   of leucosin at posterior end of cell
   (Figure 10).

   C.  rosanoffii is a clean water indicator.

4  Synura is a biflageUate form growing
   in radially arranged, naked colonies
   (Figure 11).

   a  Flagella equal in length

   b  Cells pyriform or egg shaped

   c  Łi. uvella produces a cucumber or
      muskmelon odor

5  Uroglenopsis forms free swimming
   colonies of approximately spherical
   biflagellate cells embedded near the
   periphery of a roughly spherical
   gelatinous matrix.

   a  Flagella are unequal in length.

   b  U. americana may range up to
      . 5 mm in diameter, and contain
      1000 or more cells.

   c  U. am. also causes strong fishy
      odor.

6  Dinobryon may be solitary or colonial,
   free floating or attached.  Colonies
   are arborescent (Figure 12).

   a  Cells attached to bottom of open
      roughly cylindrical lorica or sheath.

   b  Two flagella of unequal length.

   c  Conspicuous eyespot usually present.

   d  Taxonomy of the group is involved.

   e  D_. sertularia may clog filters.

   f  D. divergens may cause a fishy odor.
   9-4

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                                              Green juid Other Pigmented Flagellates
(fig 1 - 13 from Lackey and Callaway)
    Euglena
                  Phacus
                                  Lepocinclis
Trachelomonas
                                                            GREEN    EUGLENOIDS
     Chlamydomonas
                      Chlorogonium
                                                           GREEN   PHYTOMONADS
                  11
                                      Dinobryon
                                                        YELLOW    CHRYSOMONADS
                                  Peridinium
                                                         16
                                                               Ceratium
                                                 YELLOW-BROWN    DINOFLAGELLATES

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  Green and Other Pigmented Flagellates	
                                          FLAGELLATES
                                         (MASTIGOPHORA)
                           PLANT  FLAGELLATES
                            (PHYTOMASTIGINA)
ANIMAL FLAGELLATES
  (ZOOMASTIGINA)
1
SOMONADI
CRYPT
YA
3MONADIN


1

PHYTOMONADINA
RmZOli
IASTIGINA




PROTOMONADINA

                                          EUGLENOIDINA
         POLYMASTIGINA
                      Figure 17 Phylogenetic Family Tree of the Plagellates
                                   (from Calaway and Lackey)
VII  There are two distinctive groups whose
  systematic position is uncertain, the chloro-
  monads and the cryptomonads.  Only one
  genus of the latter group is included here.

  A  Rhodomonas may range from bright red
     through pale brown to olive green.

     1  Cells compressed, narrow at the
        posterior end

     2  Two flagella of unequal length

     3  R. lacustris a small form intolerant
        of pollution
 REFERENCES

 1  Calaway, Wilson T. and Lackey, James
       B.   Waste Treatment Protozoa
       Flagellata.   Series No. 3. Univ. Fla.
       140 pp.   1962.

 2  Gojdics,  M.   The Genus Euglena.
       Univ.  of Wisconsin Press,  Madison.
       1953.
 This outline was prepared by H. W. Jackson,
 former Chief Biologist, National Training
 Center,  MOTD, OWPO, USEPA, Cincinnati,
 Ohio 45268.
                                                    Descriptor:  Algae, Flagellates
   9-6

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                               FILAMENTOUS GREEN ALGAE
 I  MANY OF THESE FORMS ARE VISIBLE
   TO THE UNAIDED EYE

 A They may be several inches or even a foot
   or more in length.  In many cases they are
   not found as isolated filaments but develop
   in large aggregations to form floating or
   attached mats or tufts. The attached
   forms are generally capable of remaining
   alive after being broken away from the
   substrate.

 B Included in the group are  some of the most
   common and most conspicuous algae in
   freshwater habitats.  A few of them have
   been given common names such as pond
   silk, green felt,  frog-spawn algae, and
   stoneworts.
  C Specialized structures are present in
    some filaments.

    1  Some filaments break up into "H"
       sections.

    2  Apical caps are present in others.

    3  Replicate  end walls are present in
       some.

    4  Some filaments are overgrown with a
       cortex.

    5  Attached filaments have the basal cell
       developed into a "hold fast cell"
       (hapteron).
II   CHARACTERISTICS OF FILAMENTOUS
    ALGAE

 A  These algae are in the form of cylindrical
    cells held together as a thread ("filament"),
    which may be in large clusters or growing
    separately.  Some are attached to rocks
    or other materials while others are free.
    They may be unbranched ("simple") or
    branched; the tips are gradually narrowed
    ("attenuated") to a point.  Some are
    surrounded by a mucilaginous envelope.

 B  Each cell is a short or long cylinder with
    a distinct wall.  The protoplast contains
    a nucleus which is generally inconspicuous.

    1  The plastid or chloroplast is the
      prominent  structure. It contains
      chlorophyll and starch centers
      ("pyrenoids"), and varies in size,
      shape, and number per cell.  It may
      be pressed against the wall  ("parietal")
      or extend through the central axis of
      the cell ("axial").

    2  Clear areas of cell sap ("vacuoles") are
      generally present in the cell.

 1  Including a few yellow-brown and red algae.
Ill  REPRODUCTION MAY TAKE PLACE
    BY SEVERAL METHODS

 A Cell division may  occur in all cells or
    in certain selected ones.

 B Spores called akinetes may be formed.

 C Zoospores (motile) and aplanospores
    (non-motile) are common.

 D Fragmentation of filaments may occur.

 E Many kinds reproduce sexually, often
    with specialized gamete forming cells.
IV  EXAMPLES OF FILAMENTOUS GREEN
    ALGAE ARE:

 A Unbranched forms

    *Spirogyra
    *Mougeotia
     Zygnema
     Ulothrix
     Microspora
     Tribonema
     Desmidium
     Oedogonium
  -i'Planktonic or occasionally planktonic
 BI. MIC.cla. 14b.6.76
                                                                                      10-1

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  Filamentous Green Algae
  B Branched forms

    Cladophora
    Pithopora
    Stigeoclonium
    Chaetophora
    Draparnaldia
    Rhizoclonium
    Audouinella
    Bulbochaete
    Nitella

  C Specialized and related forms

    Schizomeris
    Comsopogon
    Batrachospermum
    Chara
    Lemanea
    Vaucheria
 V  Habitats include the planktonic growths as
  well as surface mats or blankets and benthic
  attached forms on rocks in riffles of streams,
  at the shoreline of lakes and reservoirs,
  concrete walls, etc.

  A Attached forms may break loose to
    become mixed with plankton or to form
    floating mats.

  B Cladophora mats are a nuisance on many
    beaches on the Great Lakes.
VI  IMPORTANCE OF FILAMENTOUS
    GREEN ALGAE

 A  They may cause  clogging of sand filters,
    intake screens, and canals.

 B  They may produce tastes and odors in
    water or putrid odor (also producing
    H9S which damage painted surfaces) when
    washed ashore around lakes and reservoirs.

 C  They may cause  unsightly growths or
    interfere with fishing and swimming in
    recreation areas.

 D  Some are useful  as indicators of water
    quality in relation to pollution.
  E  Together with other algae, they release
     oxygen required by fish, and for self-
     purification of streams.

  F  They may produce a slime which inter-
     feres with some industrial uses of water
     such as in paper manufacture and in
     cooling towers.
VII  CLASSIFICATION

  A  Ulotrichaceae

     Ulothrix, Microspora. Hormidium

  B  Cladophoraceae

     Cladophora.  Pithophora. Rhizoclonium

  C  Chaetophoraceae

     Chaetophora, Stigeoclonium. Draparnaldia

  D  Oedogeniaceae

     Oedogonium. Bulbochaete

  E  Schizomeridaceae

     1  Schizomeris

  F  Ulvaceae

     Enteromorpha.  Monostroma

  G  Zygnemataceae

     Zygnema.  Spirogyra. Mougeotia

  H  Desmidiaceae

     Desmidium.  Hyalotheca

  I  Tribonemataceae

     Tribonema.  Bumilleria

  J  Characeae

     Chara. Nitella, Tolypella
  10-2

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                                             Filamentous Green Algae
13.  GREENS, FILAMENTOUS

-------
   Filamentous Green Algae
Vin  IDENTIFICATION

   A Branching and attenuation are of primary
     importance.

   B Plastids:  shape,  location and number per
     cell are essential.

   C Other characteristics include grouping
     of filaments, gelatinous envelope and
     special features such as "H"  shaped
     fragments.
   REFERENCES

   1  Collins,  F.S.   1909.  The green algae
        of North America.  Tufts College
        Studies, Scientific Series  2:79-480.
        Reprinted Hafner Publ. Co.,   1928
        (Reprinted, 1968) Lew's Books,
        San Francisco.

   2  Faridi, M.  A monograph of the fresh-
        water species  of Cladophora and
        Rhizoclonium.  Ph.D.  Thesis.
        University Microfilms, Ann Arbor.

   3  Hirn, K. E.  Monograph of the
        Oedogoniaceae.   Hafner Publ.,
        New York.   1960.
4  Pal,  B.P., Kundu, B.C.,  Sundaralingam,
      V. S.,  and Venkataraman, G. S.
      Charophyta.   Indian Coun. Agric.
      Res.,  New Delhi.   1962.

5  Soderstrom, J.  Studies in Cladophora.
      Almquist, Uppsala.   1963.

6  Tilden, J.   The Myxophyceae of North
      America.  Minn. Geol.  Surv.
      (Reprinted 1967, J. Cramer,  Lehre,
      Germany)  1910.

7  Transeau, E.N.  The Zygnemataceae.
      Ohio State Univ. Press.   1951.
8  Van der Hoek, C.  Revision of the
      European species of Cladophora.
      Brill Publ,  Leiden, Netherlands.
1963.
9  Wood, R. D. and Imahari, K.   A revision
      of the Characeae.   Volume I.
      Monograph (by Wood).  Vol. II,
      Iconograph (by Wood & Imahari).  1964.
This outline was prepared by C.M. Palmer,
Former Aquatic Biologist, In Charge,
Interference Organisms Studies,  Micro-
biology Activities, Research and
Development, Cincinnati Water Research
Laboratory, FWPCA.

Descriptor;  Green Algae
    10-4

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                                  COCCOID GREEN ALGAE
 I   INTRODUCTION

 For the sake of convenience,  the non-motile
 green algae  are to be discussed in two
 sections: those that tend to live as relatively
 discrete or free floating planktonic units,
 and those that tend to grow in masses or
 mats of material, often filamentous in nature,
 attached or free floating.

n   The green or "grass green" algae is one
 of the most varied and  conspicuous groups
 with which we have to deal.  The forms
 mentioned below have been artificially grouped
 for convenience according to  cell shape.
 Botanists would list these genera in several
 different categories in  the family "Chloro-
 phyceae. "

 These algae typically have a relatively high
 chlorophyll content, and the food reserves
 accumulated are typically starch.  Thus
 these forms will usually give a typical black
 or  deep purple color when treated with iodine.

 A  Individual cells of the following genera are
    perfectly round,  or  nearly so.   The first
    does not form organized colonies.  In the
    next two the colonies themselves tend to
    be round, and in the last, the colonies are
    triangular or irregular, and the cells bear
    long slender spines.

    1  Chlorella cells are small and spherical
      to broadly elliptical.  They have a
      single parietal chloroplast.   This is a
      very large genus  with an unknown
      number of similar appearing species,
      living  in a great variety of habitats.
      Although often accumulating in great
      numbers,  organized colonies are not
      formed.
   a  Chlorella ellipsoides is reported to
      be a common plankton form.

   b  Chlorella pyrenoidosa and Chlorella
      vulgaris are often found in
      organically enriched waters.
      Indeed a dominance of Chlorella
      species is considered in some
      places to be an indication that a
      sewage stabilization pond is func-
      tioning to maximum capacity.

   c  Chlorella pyrenoidosa is reported
      as a filter clogger in water treat-
      ment plants.

2  Sphaerocystis colonies are free floating
   and almost always with a perfectly
   spherical, homogeneous gelatinous
   envelope. Up to 32 spherical cells
   may be included. Sphaerocystis
   scheoeteri, the only species, is of
   wide occurrence in  the plankton of
   lakes and reservoirs.

3  Coelastrum forms coenobial* colonies
   of up to  128 cells.  Generally spherical
   or polygonal in shape--both cells and
   colony.   Cells connected by protoplasmic
   processes of varying length.
   Coelastrum microporum is often
   reported in the plankton of water
   supplies.  Not surrounded by gelatinous
   envelope as in Sphaerocystis.

4  Micractinium.  The cells of this alga
   are spherical to broadly ellipsoidal and
   are usually united in irregular 4-celled
   coenobes. These in turn are almost
   always united with other coenobes to
   form multiple associations of up to
   100 or more cells.  The free face of
 1  Including miscellaneous yellow-brown algae.

 *A coenobe is a colony in which the number of cells does not increase during the life of the
 colony.  It was established by the union of several independent swimming cells which simply
 stick together and increase in size.
Bt.MIC.cla. 9c. 6.76
                                11-1

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Coccoid Green Algae
      each cell in a coenobe bears from one
      to seven very long slender setae or
      hairs.

      Micractinium pusillum.   This is a
      strictly planktonic genus.
   Individual cells of the following genera
   are elongate.  In the first two they are
   relatively straight or irregular and pointed.
   The next two are also long and pointed,
   but bent into a tight  "C" shape (one in a
   gelatinous envelope,  one naked).  The last
   one (Actinastrum) is long and straight,
   but with blunt ends,  and with the cells of
   a coenobe attached at a point.

   1  Ankistrodesmus cells are usually long
      and slender, tapering to sharp point at
      both ends.  They may be  straight,
      curved,  or twisted into loose aggregations.
      Ankistrodesmus falcatus  is often found
      in the plankton in water supplies and is
      considered to be one  of the forms
      indicative of clean water.

   2  Schroederia is a solitary, free floating
      alga.  Cells are long and pointed at
      both ends.  May be bent in various ways.
      Terminal points are continued as long
      slender spines which may be forked and
      bent back, or end as  a plate.  Of the
      three species reported in this country,
      Schroederia setigera has  been reported
      in water supplies.

   3  Selenastrum cells are pointed at both
      ends, and bent so that their tips approach
      each other.  They tend to occur in groups
      of 4,  8,  or  16, which may be associated
      with other groups  to form masses of a
      hundred or more  cells.  There is  no
      gelatinous envelope.  Selenastrum
      gracile occurs in the plankton of water
      supplies.

   4  Kirchneriella.  The cells of this genus
      are generally relatively broad, tapering
      to a sharp or rounded point at each end,
      and the whole cell bent into a C-shape.
   They usually occur in groups of four
   to eight in a broad, homogeneous,
   gelatinous matrix. Kirchneriella
   lunaris is known principally from the
   plankton.

5  Actinastrum colonies or "coenobes"
   are composed of 4, 8,  or 16 elongate
   cells that radiate in all directions from
   a common center.

   Actinastrum is  a widely distributed
   plankton organism.   There are two
   species:

   Actinastrum gracillimum and
   Actinastrum Hantzschii differ only
   in the sharpness of the taper toward
   the tips of the cells.  The former has
   relatively little taper,  and the latter,
 .  more.

Cells of the  following genera are
associated in simple naked colonies.
The first has elongate cells arranged
with their long axes parallel (although
some cells may be curved).  The last
two are flat  plate-like coenobes.
Crucigenia has four-celled coenobes
while Pediastrum  coenobes may be
larger,  appear plate-like, and are much
more ornate.

1  Scenedesmus is a flat plate of elliptical
   to double ended pointed cells arranged
   with their long axes parallel.  Coenobes
   consist of up to 32, but usually 4 to 8
   cells.  The number of  cells in a
   coenobe may vary from mother to
   daughter  colony.  The  appearance of
   cells may vary  considerably with the
   species.

   a Scenedesmus bijuga, S.  dimorphus,
     and Ł>. quadricauda are common
     p]anktonic forms.

   b Scenedesmus quadricauda is also
     common in organically enriched
     water,  and may become  dominant.

   c Scenedemus  abundans is reported
     to impart a grassy odor to drinking
     water.
 11-2

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                                                                   Coccoid Green Algae
   2  Crucigenia forms free floating four-
      celled coenobes that are solitary or
      joined to one another to form plate-
      like multiple coenobes of 16 or more
      cells.  The cells may be elliptical,
      triangular, trapezoidal, or semi-
      circular in surface view.  Crucigenia
      quadrata is a species  often reported
      from water supplies.

   3  Pediastrum.  Colonies are free floating
      with up to 128 polygonal cells arranged
      in a single plane.  There may or may
      not be open spaces between the cells.
      The exact arrangement of the cells
      seems to depend largely on the chance
      distribution of the original motile
      swarming zoaspores at the time  the
      coenobe was formed.  Peripheral cells
      may differ in shape from interior cells.

      a Pediastrum boryanum and P.  duplex
        are frequently found in the plankton,
        but seldom dominate.

      b Pediastrum tetras  has been reported
        to impart a grassy odor to water
        supplies.

D  Cells of the following Genera are slightly
   elongated.

   1  Oocystis.  The cells of Oocystis may
      be solitary,  or up to 16 cells may be
      surrounded by a partially gellatinized
      and greatly expanded mother cell wall.
      Cells may be ellipsoidal or almost
      cylindrical,  cell wall  thin,  no spines
      or other ornamentation.  Oocystis
      borgei. for example,  is of frequent
      occurrence in the plankton.

   2  Dimorphococcus cells are arranged in
      groups  of four, and these tetrads are
      united to one another in irregularly
      shaped free floating colonies by the
      branching remains of  old mother-cell
      walls.  Two shapes of cell are normally
      found in each tetrad (hence the name),  two
      longer ovate cells end to end, and a
      pair of  slightly shorter, C-shaped cells
      on either side.  Dimorphococcus
      lunatus is a widely distributed plankton
      organism, sometimes reported in
      considerable numbers.
E  A distinctive group of green algae
   characterized by a median constriction
   dividing the  cell into two geometrically
   similar halves is known generally as the
   "desmids. "  (Closterium and Penium do
   not have this construction). Each half
   of the cell is known as a  "semicell. "
   The nucleus lies in the "isthmus. "
   Extremes of ornamentation and structural
   variety exist.  Most are unicellular, but
   a few are filamentous or have the  cells
   associated in shapeless colonies.  They
   are found sparingly in the plankton almost
   everywhere, but predominate  in acid
   waters.

   1  Closterium is one of the exceptional
      genera without a median constriction.
      The cells are elongate,  attenuated
      toward the tips but not sharply  pointed,
      usually somewhat bent.

      a  Closterium aciculare is a planktonic
        species.

      b  Closterium moniliforme is reported
        as a filter clogging organism.

   2  Cosmarium is a large, poorly defined
      genus of over 280 species,  many of
      which apparently  intergrade with other
      genera such as  Staurastrum. In
      general,  it can be said that Cosmarium
      species are relatively small, with a
      length only slightly greater than the
      width, and with a deep median con-
      striction. Shapes of the semicells
      may vary greatly. Although shallow
      surface ornamentation may occur,
      long spines do not occur.

      a  Cosmarium botrytis is reported in
        plankton from water supply
        reservoirs.

     b  Cosmarium portianum is said to
        impart a grassy odor to water.

      c  Other  species have been reported
        to be sufficiently resistant to
        chlorine to penetrate rapid sand
        filters and occur in distribution
        systems  in considerable numbers.
                                                                                      11-3

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  Coccoid Green Algae
    3  Micrasterias is relatively common,
       ornate.

    4  Euastrum cells tend to be at least twice
       as long as broad, with a deeply con-
       stricted isthmus, and a dip or incision
       at the tip of each semicell.  The cell
       wall may be smooth,  granulate, or
       spine d.

       Euastrum oblongum is reported as a
       planktonic species from water reser-
       voirs.   It has also been noted as
       intolerant of pollution, and hence an
       indicator of clean water.

    5  Staurastrum is the  commonest of the
       desmids in the  plankton of fresh waters;
       the genus contains upwards of 245 species
       in the United States alone.  Inter-
       gradation with other genera such as
       Cosmarium make it a difficult group
       to define.  Most of the species are
       radially symmetrical, and almost all
       have a deeply constricted isthmus.
       The cell wall may be  smooth, orna-
       mented, or spined in  a variety of ways.
       Relatively long truncated processes
       extending from the cell body in
       symmetrical patterns are common.

       a  Staurastrum polymorphum is a
          typical planktonic form.

       b  Staurastrum punctulatum is reported
          as an indicator of clean water.

       c  Staurastrum paradoxicum causes a
          grassy odor in water.
   1  The plant body is a free floating colony
      of indefinite shape, with a cartilag-
      inous and hyaline or orange-colored
      envelope; surface greatly wrinkled
      and folded.

   2  Individual cells lie close together, in
      several aggregates connected in
      reticular fashion by strands of the
      colonial envelope.

   3  The envelope structure tends to
      obscure cell structure.  Considerable
      deep orange colored oil may collect
      within the envelope, outside of the
      cells,  obscuring cell structure.

B  Ophiocytium capitatum like Botryococcus,
   is widely distributed, but seldom abundant.

   1  Both ends of cylindrical cell are
      rounded,  with a sharp spine extending
      therefrom.

   2  Many nuclei and several chloroplasts
      are present.
REFERENCES

1  Palmer,  C.M.   Algae in Water Supplies.
      Government Printing Office.   PHS
      Publication No. 657.  1959.

2  Smith, G.S.  Phytoplankton of the
      Inland Lakes of Wisconsin.  Part I.
      Bulletin No. 57, Scientific Series
      No. 12.  1920.
IE  A type of "green" alga known as "golden
 green" (Xanthophyceae) is represented in the
 plankton by two genera.  In these algae there
 is a predominance of yellow over green pig-
 ments, hence frequently imparting a yellowish
 or golden tint to the cell.  Reserve food
 material is stored as oil and leucosin,  rather
 than as starch,  hence giving a negative test
 with iodine in most cases.

 A Botryococcus braunii is a widely dis-
    tributed plankton alga, though it is
    rarely abundant.
This outline was prepared by H. W. Jackson,
former Chief Biologist,  National Training
Center, and revised by R. M. Sinclair,
Aquatic Biologist, National  Training Center,
MOTD, OWPO, USEPA.  Cincinnati, Ohio
45268.	

Descriptor: CMorophyta
  11-4

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                                          DIATOMS
 I  GENERAL CHARACTERISTICS

 A  Diatoms have cells of very rigid form due
    to the presence of silica in the wall.  They
    contain a brown pigment in addition to the
    chlorophyll.  Their walls are ornamented
    with markings which have a specific pattern
    for  each kind.

    1 The cells often are isolated but others
      a.re in filaments or other shapes of
      colonies.

    2 The protoplast contains normal cell
      parts, the most conspicuous being the
      plastids. No starch  is present.
 B  Cell shapes include the elongate ("pennate")
    and the short cylindric ("centric") one view
    of which is circular.

    1  Pennate diatoms may be symmetrical,
       transversely unsymetrical,  or longitudi-
       nally unsymmetrical.
       Internal shelves ("septae") extending
       longitudinally or transversely.
 II  REPRODUCTION

 A The common method is by cell division.
    Two new half cells are formed between the
    halves of the parent cell.

 B Auxospores and gametes may also be
    formed.
Ill  EXAMPLES OF COMMON DIATOMS:

 A Pennate,  symmetrical:

          Navicula
          Pinnularia
          Synedra
          Nitzschia
          Diatom a
          Fragilaria
          Tabellaria
          Cocconeis
 C  Wall is formed like a box with a flanged
    cover fitting over it.

    1  "Valve" view is that of the top of the
       cover or the bottom of the box.

    2  "Girdle" view is that of the side where
       flange of cover fits over the box.

    3  End view is also possible for pennate
       types.
 D  Cell markings include:

    1  Raphe or false raphe extending
       longitudinally.

    2  Striations which are lines of pores
       extending from the area of the raphe to
       the margin.  Coarse ones are "costae".

    3  Nodules which may be terminal and
       central.
  B Pennate,  unsymmetrical:

          Gomphonema
          Surirella
          Cymbella
          Achnanthes
          Asterionella
          Meridion


  C Centric:

          Cyclotella
          Stephanodiscus
          Melosira
IV  Habitats include fresh and salt water. Both
 planktonic and attached forms occur,  the latter
 often are broken loose.  They may be attached
 by stalks or by their slimy surface.
BI.MlC.cla. lOa. 6. 76
                                                                                     12-1

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  Diatoms
 A Many diatoms are more abundant in late
    autumn,  winter,  and early spring than in
    the warmer season.
              Fragilaria
              Synedra
              Asterionella
  B The walls of dead diatoms generally remain
    undecomposed and may be common in water.
    Many deposits of fossil diatoms exist.
  V Importance of diatoms is in part due to
  their great abundance and their rigid walls.

  A They are the most important group of
    organisms causing clogging of sand filters.

  B Several produce tastes and odors in water,
    including the obnoxious fishy flavor.


  C Mats of growth may cause floors or steps
    of swimming pools to be slippery.

  D They may be significant  in determining
    water quality in relation to pollution.

  E They release oxygen into the water.
     2  Achnanthineae.  Group with cells
        having one false and one true raphe.

        a  Representative genera:

              Cocconeis
              Achnanthes
     3  Naviculineae.  True raphe group with
        raphe in center of valve.

        a  Representative genera:

           Navicula
           Pinnularia
           Stauroneis
           Pleurosigma
           Amphiprora
           Gomphonema
           Cymbella
           Epithemia
VI  Classification.  There are several thou-
 sand species of diatoms.  Only the most com-
 mon of the freshwater forms are considered
 here.

 A Centrales Group

    1  Representative genera:

          Cyclotella
          Stephanodiscus
          Melosira
          Rhizosolenia
          Biddulphia


 B  Pennals Group

    1  Fragilarineae.  The  false raphe group.

       Representative genera:

          Tabellaria
          Me rid ion
          Diatoma
     4  Surirellineae.  True raphe group with
        raphe near  one  side of valve.

        a  Representative genera:

           Nitzschia
           Cymatopleura
           Surirella
           Campylodiscus
VII  IDENTIFICATION OF DIATOMS

  A  Some genera are easily recognized by their
     distinctive shape.

  B  Many genera and most species can be
     determined only after diatoms are freed
     of their contents and observed under the
     high magnification of an oil immersion
     lens of the compound microscope.

  C  Contents  of the cell  are generally not
     used in identification.  Only the char-
     aracteristics of the  wall are used.
  12-2

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                                                                                   Diatoms
 D  For identification of genera, most im-
    portant features include:

    1  Cell shape,  and form of colony

    2  Raphe and false raphe

    3  Striations

   4  Septa
   For identification of species,  measure-
   ments involving the number of striae per
   10 microns,  the direction of the striae
   and many other characteristics may be
   needed.
REFERENCES

1  Boyer, C.S.  The Diatomaceae of
      Philadelphia and Vicinity. J. B. Lippin-
      cott Co.  Philadelphia. 1916.  p 143.

2  Boyer, C.S.  Synopsis of North America
      Diatomaceae.  Parts 1(1927)  and II
      (1928).  Proceedings of the Academy
      of Natural Sciences.  Philadelphia.

3  Elmore,  C. J. The Diatoms of Nebraska.
      University of Nebraska Studies.  21:
      22-215.   1921.

4  Hohn, M. H.  A Study of the  Distribution
      of Diatoms in Western New York
      State.   Cornell University Agricultural
      Experimental Station. Memoir 308.
      pp 1-39.   1951.

5  Pascher, A.  Bacillariophyta (Diatomeae).
      Heft 10 in Die Susswasser-Flora
      Mitteleuropas, Jena.  1930. p 466.

6  Patrick,  R.   A Taxonomic and Ecological
      Study of Some Diatoms from the
      Pocono Plateau  and Adjacent Regions.
      Farlowia. 2:143-221.  1945.
 7  Patrick,  Ruth and Reimer, Charles W.
       The Diatoms of the United States.
       Vol.  1 Fragilariaceae, Eunotiaceae,
       Achnanthaceae, Naviculaceae.
       Monog.  13.  Acad. Nat. Sci.
       Philadelphia.  688 pp.  1966.

 8  Smith, G.M.   Class Bacillariophyceae.
       Freshwater Algae of the United
       States,  pp 440-510,  2nd Edition.
       McGraw Hill Book Co.  New York.
       1950.

 9  Tiffany,  L. H. and Britton, M.E.  Class
       Bacillariophyceae.  The Algae of
       Illinois,  pp 214-296.  University
       of Chicago Press.  1952.

10  Ward, H. B. and Whipple, G. C.  Class
       I, Bacillariaceae (Diatoms).  Fresh-
       water Biology,  pp  171-189.  John
       Wiley & Sons.   New York.  1948.

11  Weber, C.  I.  A Guide to the  Common
       Diatoms at Water  Pollution
       Surveillance System Stations.
       FWPCA.  Cincinnati.  101 pp.  1966.

12  Whipple, G.C., Fair, G. M.,  and
       Whipple, M.C. Diatomaceae.
       Microscopy of Drinking Water.
       Chapter 21. 4th Edition.   John Wiley
       & Sons. New York.  1948.
 This outline was prepared by C.M. Palmer,
 Former Aquatic Biologist,  In Charge,
 Interference Organisms Studies, Microbiology
 Activities,  Research and Development,
 Cincinnati Water Research Laboratory,
 FWPCA.

 Descriptor: Diatoms
                                                                                      12-3

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                                    FILAMENTOUS BACTERIA
 I  INTRODUCTION

 There are a number of types of filamentous
 bacteria that occur in the aquatic environment.
 They include the sheathed sulfur and iron
 bacteria such as Beggiatoa,  Crenothrix and
 Sphaerotilus, the actinomycetes which are
 unicellular microorganisms that form chains
 of cells with special branchings, and
 Gallionella,  a unicellular organism that
 secretes a long twisted ribbon-like stalk.
 These filamentous  forms have at times
 created serious problems in rivers,
 reservoirs,  wells,  and water distribution
 systems.
 II  BEGGIATOA

 Beggiatoa is a sheathed bacterium that grows
 as a long filamentous form.   The flexible
 filaments may be as  large as 25 microns wide
 and 100 microns long.  (Figure  1)
Figure 1
                           Beggiatoa alba
                                 up to 1 .OOO/J
Transverse separations within the sheath
indicate that a row of cells is included in
one sheath.  The sheath may be clearly
visible  or so slight that only special staining
will indicate that it is present.

The organism grows as a white slimy or
felted cover on the surface of various objects
undergoing decomposition or on the surface
of stagnant areas of a stream receiving
sewage. It has also been observed on the
base of a trickling filter and in contact
aerators.

It is most commonly found in sulfur springs
or polluted waters where H^S is present.
Beggiatoa is distinguished by its ability to
deposit sulfur within its cells; the sulfur
deposits appear as large refractile globule's.
(Figure 2)
                                                                      Figure 2

                                                                 Filaments of Beggiatoa
                                                                 containing granules of
                                                                 sulphur.
When H S is no longer present in the environ-
ment, tne sulfur  deposits disappear.
Dr. Pringsheim of Germany has recently
proved that the organism can grow as a true
autotroph obtaining all its energy from the
oxidation of H?S and using this energy to fix
CO  into all material.  It can also use
certain organic materials if they are present
along with the H0S.
               Łt
Faust and Wolfe, and Scotten and Stokes
have grown the organism in pure culture in
this country.  Beggiatoa exhibits a motility
that is quite different from the typical
flagellated motility of most bacteria; the
filaments have a flexible gliding motion.
   BA. 8a. 6.76
                                     13-1

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 Filamentous Bacteria
 The only major nuisance effect of Beggiatoa
 known has been overgrowth on trickling filters
 receiving waste waters rich in H2S.  The
 normal microflora of the filter was suppressed
 and the filter failed to give  good treatment.
 Removal of the H  S from the water by blowing
 air through the water before it reached the
 filters caused the slow decline of the
 Beggiatoa and a recovery of the normal
 microflora.  Beggiatoa usually indicates
 polluted conditions with the presence of tLS
 rather than being a direct nuisance.
Ill  ACTINOMYCETES AND EARTHY ODORS
    IN WATER

 Actinomycetes are unicellular microorganisms,
 1 micron in diameter, filamentous,  non-
 sheathed, branching monopodially, and
 reproduced by fission or by means of special
                             Figure 4

                            Egg albumin isolation plate.
                           'A' an actinomycete colony,
                           and 'B1 a bacterial colony
  Appearance:
Appearance:
 conidia.  (Figure 3)
       Figure 3    Filaments  of Actinomycetes
 Their filamentous habit and method of
 sporulation is reminiscent of fungi.  However,
 their size, chemical composition, and other
 characteristics are more similar to bacteria.
 (Figure 4)
dull and powdery   smooth and mucoid

     These organisms may be considered as a
     group intermediate between the fungi and
     the bacteria.  They require organic matter
     for growth but can use a wide variety of
     substances and are widely distributed.

     Actinomycetes have been implicated as the
     cause of earthy odors in some drinking
     waters (Romano and Safferman, Silvey and
     Roach) and in earthy smelling substance has
     been isolated from several members of the
     group by Gerber and Lechevalier.  Safferman
     and Morris have reported on a method for the
     "isolation  and Enumeration of Actinomycetes
     Related to Water Supplies. ". But the actino-
     mycetes are primarily soil microorganisms
     and often grow in fields or on the banks of a
     river or lake used for the water supply.
     Although residual chlorination will kill the
     organisms in the treatment plant or distribution
   13-2

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                                                                      Filamentous Bacteria
 system, the odors often are present before
 the waier enters the plant.  Use of perman-
 ganate oxidation and activated carbon filters
 have been most successful of the methods
 tried to remove the odors from the water.
 Control procedures to prevent the odorous
 material from being washed into the water
 supply by rains or to prevent possible develop-
 ment of the actinomycetes in water rich in
 decaying organic matter is  still needed.
IV  FILAMENTOUS IRON BACTERIA

 The filamentous iron bacteria of the
 Sphaerotilus- Leptothrix group,  Crenothrix,
 and  Gallionella have the ability to either
 oxidize manganous or ferrous ions to  manganic
 or ferric salts or are able to accumulate
 precipitates of these  compounds within the
 sheaths of the organisms.  Extensive  growths
 or accumulations of the empty, metallic
 encrusted sheaths devoid of cells,  have
 created much trouble in -wells or water dis-
 tribution  systems.  Pumps and back surge
 valves have been clogged with masses of
 material,  taste and odor problems have
 occurred, and rust colored masses of
 material have spoiled products in contact
 with water.

 Crenothrix polyspora has only been examined
 under the microscope as we have never been
 able to grow it in the laboratory.  The orga-
 nism is easily recognized by its  special
 morphology.  Dr.  Wolfe of the University of
 Illinois has published photomicrographs of
 the organism. (Figure 5)

 Organisms of the Sphaerotilus- Leptothrix
 group have been extensively studied by many
 investigators (Dondero ^t.  al., Dondero,
 Stokes, Waitz and Lackey, Mulder and van
 Veen, and Amberg and Cormack.)  Under
 different environmental conditions the mor-
 phological appearance of the organism varies.
 The  usual form found in polluted  streams or
 bulked activated sludge is Sphaerotilus natans.
 (Figure 6)
       Figure 5
Crenothrix polyspora

  cells  are very variable In
  size  from small cocci or
  polyspores to cells 3X12^
       Figure 6

   Sphaerotilus natans


    3-8 X 1.2 - 1.8/U
        cells
                                                                                         13-3

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Filamentous Bacteria
This is a sheathed bacterium consisting of
long,  unbranched filaments, whereby individual
rod-shaped bacterial cells are enclosed in a
linear order within the sheath.  The individual
cells  are 3-8 microns long and 1.2-1.8
microns wide.  Sphaerotilus grows in great
masses; at times in streams or rivers that
receive wastes from pulp mills, sugar
refineries, distilleries,  slaughterhouses,
or milk processing plants.  In these conditions,
it appears  as large masses or tufts attached
to rocks, twigs, or other projections and the
masses may vary in color from light grey to
reddish brown.  In some rivers large masses
of Sphaerotilus break loose and clog water
intake pipes or foul fishing nets. When the
cells  die, taste and odor problems may also
occur in the water.

Amberg, Cormack,  and Rivers and McKeown
have reported on methods to try to limit the
development of Sphaerotilus in rivers by
intermittant discharge of wastes.  Adequate
control will probably only be achieved once
the wastes are treated before discharge to
such an extent that the growth of Sphaerotilus
is no  longer favored in the river. Sphaerotilus
grows well at cool temperatures and slightly
low DO levels in streams receiving these
wastes and domestic sewage.  Growth is slow
where the only nitrogen present is inorganic
nitrogen; peptones and proteins are utilized
preferentially.

Gallionella is an iron bacterium which appears
as a kidney-shaped cell with a. twisted ribbon-
like stalk emanating from the concavity of the
cell.  Gallionella obtains its energy by
oxidizing ferrous iron to ferric iron and uses
only CO9 and inorganic salts to form all of
the cell material; it is an autotroph.  Large
masses of  Gallionella may cause problems
in wells  or accumulate in low-flow low-
pressure water mains.  Super chlorination
(up to 100 ppm of sodium hypochlorite for
48 hours) followed by flushing will often
remove the masses of growth and periodic
treatment will prevent the  nuisance effects
of the extensive masses of Gallionella.
(Figure 7)
/ A
\iA

Figure 7
!• "\j Galionella furruginea
/
i
i

O.5 X O.7 - 1.1/4
Cefls
REFERENCES

Beggiatoa

1  Faust, L. and Wolfe, R.S.  Enrichment
      and Cultivation of Beggiatoa Alba.
      Jour.  Bact.,  81:99-106.   1961.

2  Scotten,  H. L. and Stokes, J. L.
      Isolation and Properties of Beggiatoa.
      Arch Fur. Microbiol.  42:353-368.
      1962.

3  Kowallik, U.  and Pringsheim,  E.G.
      The Oxidation of Hydrogen Sulfide by
      Beggiatoa.  Amer. Jour, of Botany.
      53:801-805.    1966.

Actinomycetes and Earthy Odors

4  Silvey, J.K. G.  etaL Actinomycetes and
      Common Tastes and Odors.  JAWWA,
      42:1018-1026.   1950.

5  Safferman, R. S.  and Morris,  M. E.
      A Method for the Isolation and
      Enumeration of Actinomycetes Related
      to Water Supplies. Robert A. Taft
      Sanitary Engineering Center Tech.
      Report W-62-10.   1962.
13-4

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                                                                     Filamentous Bacteria
 6  Gerber, N.N. and Lechevalier,  H.A.
       Geosmin,  an Earthy-Smelling Substance
       Isolated from Actinomycetes.  Appl.
       Microbiol.  13:935-938.   1965.

 Filamentous Iron Bacteria
 7  Wolfe, R.S,,   Cultivation, Morphology, and
       Classification of the Iron Bacteria.
       JAWWA.  50:1241-1249.   1958.

 8  Kucera, S. and Wolfe, R.S.   A Selective
       Enrichment Method for Gallionella
       ferruginea.   Jour. Bacteriol.  74:344-
       349.   1957.

 9  Wolfe, R.S.  Observations and Studies
       of Crenothrix polyspora.   JAWWA,
       52:915-918.   1960.

10  Wolfe, R.S.  Microbiol.   Concentration
       of Iron and Manganese in Water with
       Low Concentrations of these Elements.
       JAWWA.  52:1335-1337.   1960.

11  Stokes, J. L.   Studies on the Filamentous
       Sheathed Iron Bacterium Sphaerotilus
       natans.  Jour. Bacteriol.   67:278-291.
       1954.

12  Waitz, S.  and Lackey,  J. B.  Morphological
       and Biochemical Studies on the
       Organism  Sphaerotilus natans.  Quart.
       Jour. Fla. Acad. Sci.  21(4):335-340.
       1958.

13  Doridero,  N.C.,  Philips, R.A. and
       Henkelkian, H.   Isolation and
       Preservation of Cultures of Sphaerotilus.
       Appl. Microbiol. 9:219-227.  1961.
14  Dondero, N. C.  Sphaerotilus. Its Nature
       and Economic Significance. Advances
       Appl. Microbiol. 3:77-107.   1961.

15  Mulder, E.G.  and van Veen,  W. L.
       Investigations on the Sphaerotilus-
       Leptothrix Group.  Antonie van
       Leewenhoek.   29:121-153.  1963.

16  Amberg,  H.R. and Cor mack,  J. F.
       Factors Affecting Slime Growth in
       the Lower Columbia River and
       Evaluation of Some Possible Control
       Measures.  Pulp and Paper Mag.  of
       Canada.  61:T70-T80.   1960.

17  Amberg,  H.R., Cormack,  J. F.  and
       Rivers, M.R.  Slime Growth  Control
       by Intermittant Discharge of Spent
       Sulfite Liquor.  Tappi.   45:770-779.
       1962.

18  McKeown, J.J.  The  Control of
       Sphaerotilus natans.  Ind. Water
       and Wastes.   8:(3) 19-22  and
       8:(4)30-33.   1963.

19  Curtis, E. J. C.,  Sewage Funrus; Its
      Nature and Effects.  Wat. Res.
      3:289-311. 1969.

20  Lechevalier, Hubert A., Actinomycetes
      of Sewage Treatment Plants.  Envir.
      Prot. Tech.  Series USEPA,
      600/1-75-031.  1975.

  This outline was prepared by R. F.  Lewis
  Bacteriologist, Advanced Waste Treatment
  Research Laboratory, NERC, USEPA,
  Cincinnati, Ohio 45268.

  Descriptors: Aquatic Bacteria,  Sphaerotilus
  Actinomycetes, Nocardia
                                                                                      13-5

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                      FUNGI AND THE "SEWAGE FUNGUS" COMMUNITY
 I    INTRODUCTION

A    Description

     Fungi are heterotrophicachylorophyllous
     plant-like organisms  which possess true
     nuclei  with nuclear membranes and nu-
     cleoli.   Dependent upon the species and
     in some  instances  the  environmental
     conditions, the body of  the fungus,  the
     thallus,  varies from a microscopic
     single  cell to an extensive plasmodium
     or mycelium.  Numerous forms produce
     macroscopic fruiting bodies.

 B   Life Cycle

     The life cycles of fungi  vary from simple
     to complex and may include sexual  and
     asexual stages  with varying spore  types
     as the  reproductive units.

 C   Classification

     Traditionally,  true fungi are classified
     within  the Division Eumycotina of the
     Phylum Mycota of  the plant  kingdom.
     Some authorities consider the  fungi an
     essentially monophyletic group distinct
     from the  classical plant and animal
     kingdoms.
                                            Ill   ECOLOGY


                                             A   Distribution


                                                 Fungi are ubiquitous in nature and mem-
                                                 bers of all classes may occur in large
                                                 numbers  in aquatic habitats.  Sparrow
                                                 (1968) has briefly reviewed the ecology
                                                of fungi in i'reshwaters with particular
                                                 emphasis on the zoosporic phycomycetes.
                                                 The occurrence and ecology of fungi in
                                                 marine and estuarine waters  has  been
                                                 examined recently by a number of in-
                                                 vestigators (Johnson and  Sparrow, 1961;
                                                 Johnson,  1968;  Myers,  1968; van Uden
                                                 and Fell,  1968).
                                            B   Relation to Pollution


                                                 Wm. Bridge Cooke,  in a  series  of in-
                                                 vestigations  (Cooke,  1965), has  estab-
                                                 lished that fungi other than phycomycetes
                                                 occur in  high  numbers  in sewage and
                                                 polluted waters.  His reports on organic
                                                 pollution  of streams (Cooke, 1961; 1967)
                                                 show that the variety of the  Deuteromy-
                                                 cete flora is decreased at the immediate
                                                 sites of pollution, but dramatically  in-
                                                 creased downstream from these regions.
II
ACTIVITY
 In general, fungi possess broad enzymatic
 capacities.  Various species are able to
 actively degrade  such  compounds as
 complex polysaccharides (e.g., cellulose,
 chitin,  and glycogen), proteins (casein,
 albumin,  keratin), hydrocarbons (kerosene)
 and pesticides. Most species possess an
 oxidative or microaerophilic metabolism,
 but anaerobic catabolism is  not uncommon.
 A few species  show  anaerobic metabolism
 and growth.
                                                 Yeasts,  in particular, have  been found
                                                 in large numbers in organically enriched
                                                 waters (Cooke, et al., 1960;  Cooke and
                                                 Matsuura, 1963; Cooke, 1965b; Ahearn,
                                                 et al.,  1968).   Certain yeasts are of
                                                 special interest  due to their potential
                                                 use as  "indicator" organisms and their
                                                 ability  to  degrade or utilize proteins,
                                                 various hydrocarbons,  straight and
                                                 branch chained alkyl-benzene sulfonates,
                                                 fats,  metaphosphates, and wood sugars.
  BI.FU. 6a. 6. 76
                                                                                    14-1

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 Fungi
C   "Sewage Fungus" Community  (Plate I)

    A few microorganisms have long been
    termed "sewage fungi. "  The most
    common microorganisms included in
    this  group are the iron bacterium
    Sphaerotilus natans and the phycomy-
    cete Leptomitus  lacteus.

    1  Sphaerotilus natans  is not a fungus;
       rather it is a sheath  bacterium of
       the  order  chlamydobacteriales.
       This polymorphic bacterium occurs
       commonly in organically enriched
       streams where it  may produce
       extensive slimes.

       a Morphology

         Characteristically, S. natans
         forms chains of rod shaped
         cells (1. 1 -2. On x 2.5- l?n)
         within a clear sheath or tri-
         chome composed ofaprotein-
         polysaccharidae-lipid complex.
         The rod cells are  frequently
         motile upon release from the
         sheath;.the flagella are lopho-
         trichous.  Occasionally two
         rows of cells may be present
         in a single sheath. Single tri-
         chomes may be  several mm
         in length and bent  at various
         angles.  Empty  sheaths, ap-
         pearing like thin cellophane
         straws, may be present.


       b  Attached growths

         The trichomes are cemented
         at one end to solid substrata
         such as stone or metal, and
         their cross attachment and
         bending gives a  superficial
         similarity to true fungal hyphae.
         The ability to attach firmly to
         solid substrates gives S. natans
         a  selective advantage in  the
         population of flowing streams.
         For more thorough reviews of
         S. natans see Prigsheim( 1949)
         and Stokes (1954).
Leptomitus  lacteus also produces
extensive slimes and fouling floes
in fresh waters.  This species forms
thalli typified by regular constrictions.

a  Morphology

   Cellulin plugs may be present
   near the constrictions and there
   may be numerous granules in
   the  cytoplasm. The basal cell
   of the thallus may possess
   rhizoids.

b  Reproduction

   The segments delimited by. the
   partial constrictions are  con-
   verted basipetally to sporangia.
   The zoospores are diplanetic
   (i. e., dimorphic)  and each
   possesses one whiplash and one
   tinsel flagellum.  No sexual
   stage  has been demonstrated
   for this species.

c  Distribution

   For further information on the
   distribution and  systematics
   of_L_. lacteus  see Sparrow (1960),
   Yerkes (1966) and Emerson and
   Weston (1967). Both S. natans
   and L_. lacteus appear to thrive
   in organically enriched cold
   waters (5°-22°C) andbothseem
   incapable of extensive growth at
   temperatures of about 30°C.

d  Gross morphology

   Their metabolism is oxidative
   and growth of both species may
   appear as reddish brown floes
   or stringy slimes of 30 cm or
   more  in length.

e  Nutritive requirements
   Sphaerotilus natans  is able to
   utilize a wide variety of organic
   compounds, whereas L. lacteus
   does not assimilate simple
  14-2

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                             PLATE I


          "SEWAGE FUNGUS" COMMUNITY OR "SLIME GROWTHS"

               (Attached "filamentous" and slime growths)
                                                                Fungi
  Zoogloea
                               Sphaerotilus natans
      ' >»'—•* *-*"^
      i»!
   38^
         ^ssg^"
                          Beggiatoa alba
                                            BACTERIA
Fusarium aqueductum
                                             Leptomitus lacteus
                          Geotrichum candidum
                                                  FUNGI
Eplstylia  8
                                                             10

                                                       Opercularia




                                                        PROTOZOA
                                                                   14-3

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Fungi
                                                            PLATE  II
                                                 REPRESENTATIVE  FUNGI
       Figure       •*•
       Fusarium aquaeductuum
       (Radlmacher and
       Rabenhorst) Saccardo
         Microconidia (A)  produced
       from phialides as in Cephalo-
       sporium, remaining  in slime
       balls. Macroconidia  (B), with
       one  to  several  cross  walls,
       produced from collared phial-
       ides. Drawn from culture.


             Figure   3
             Geotrichum candidum
             Lank ex Persoon
               Mycelium  with  short cells
             and  arthrospores.  Young hy-
             pha  (A) ; and mature arthro-
             spores (B).  Drawn from cul-
             ture.
   Figure  :
   Achlya americana Humphrey
     Ooogonium  with  three oo-
   spores  (A);  young zoospor-
   angium  with  delimited zoo-
   spores (B); and zoosporangia
   (C)  with  released  zoospores
   that remain encysted in clus-
   ters at the mouth of the dis-
   charge tube. Drawn from cul-
   ture.
Figure
Leptomitiu lacteus (Roth)
Agardh
  Cells of the hyphae show-
ing constrictions with cellulin
plugs. In  one cell large  zoo-
spores have  been delimited.
Redrawn  from Coker, 1923.
          Zoophagus insidians
          Sommerstorff
            Mycelium with hyphal pegs
           (A)  on  which rotifers will
           become impaled; gemmae (B)
           produced as conidia on short
           hyphal branches;  and rotifer
           impaled on hyphal  peg (C)
           from   which   hyphae   have
           grown into the rotifer  whose
           shell  will  be discarded  after
           the  contents  are consumed.
           Drawn from culture.
                                                                                  FIGURE   /   -i-faplosporidium costnlc.   A—mature .spoiv;
                                                                                    B—early  pln$modium.
  •Figures  1  through 5 from  Cooke; Figures  6 and 7  from Galtsoff.
        1

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                                                                                    Fungi
      sugars and grows most luxuriantly in
      the presence of organic nitrogenous
      wastes.

   3  Ecological roles

      Although the "sewage fungi" on
      occasion attain visually noticeable
      concentrations,  the less obvious
      populations of deuteromycetes may
      be more important in the ecology of
      the aquatic habitat.  Investigations of
      the past decade  indicate that numerous
      fungi are of primary importance in the
      mineralization of organic wastes; the
      overall significance and exact roles of
      fungi in this process are yet to be
      established.

D  Predacious Fungi

   1  Zoophagus insidians

      (Plate II,  Figure 4) has been observed
      to impair functioning of laboratory
      activated sludge units (see Cooke and
      Ludzack).

   2  Arthrobotrys is usually found along
      with Zoophagus  in laboratory activated
      sludge units.  This fungus is predacious
      upon nematodes.  Loops rather  than
      "pegs" are used in snaring nematodes.
IV  CLASSIFICATION

 In recent classification schemes, classes
 of fungi are distinguished primarily on the
 basis of the morphology of the sexual and
 zoosporic stages. In practical schematics,
 however, numerous  fungi do not demonstrate
 these stages.  Classification must therefore
 be based on the sum total of the morphological
 and/or physiological characteristics.  The
 extensive review by  Cooke (1963) on methods
 of isolation and classification of fungi  from
 sewage and polluted  waters precludes  the
 need herein of extensive' keys and species
 illustrations.  A  brief synopsis key of the
 fungi adapted  in part from Alexopholous
 (1962) is presented on the following pages.
 This outline was prepared by Dr. Donald G.
 Ahearn,  Professor of Biology, Georgia State
 College, Atlanta,  Georgia 30303.

 Descriptor:  Aquatic Fungi
           PLATE II (Figure 4)
                                                                                     14-5

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Fungi
      KEY TO THE MAJOR TAXA OF FUNGI

      1        Definite cell walls lacking,  somatic phase a free living Plasmodium	
              	Sub-phylum Myxomycotina  . . (true slime molds). .Class Myxomycetes
      I1       Cell walls usually well defined,  somatic phase not a  free-living Plasmodium	
              	(true fungi)	Sub-phylum Eumycotina	2

      2        Hyphal filaments  usually coenoctytic,  rarely septate, sex cells when  present  forming
              oospores or zygospores, aquatic species propagating asexually by zoospores,  terrestrial
              species by zoospores,  sporangiospores conidia or conidia-like sporangia .'.'Phycomycetes". . .  3

                   The phycomycetes are generally considered to include the most primitive of the true
              fungi.  As a whole,  they encompass a wide diversity of forms with some showing relation-
              ships to the flagellates, while others closely resemble colorless algae, and still others
              are true molds.   The vegetative body (thallus)  may be non-specialized and entirely con-
              verted into a reproductive organ (holocarpic),  or it may bear  tapering rhizoids,  or be
              mycelial and very extensive.  The outstanding  characteristics of the thallus is a tendency
              to be nonseptate and, in most groups, multinucliate;  cross walls are  laid down in vigorously
              growing material  only to delimit the reporductive organs.  The spore unit of nonsexual re-
              production is borne in a sporangium, and, in aquatic and semiaquatic orders,  is provided
              with a  single posterior or anterior  flagellum or two laterally attached ones. Sexual activity
              in the phycomycetes  characteristically results in the formation of resting spores.

      2' (I1)   Hyphal filaments when present septate, without zoospores, with or without  sporangia,
              usually with conida; sexual reproduction absent or  culminating in the  formation of asci
              or basidia	8

      3 (2)    Flagellated cells characteristically produced	4
      3'       Flagellated cells lacking or  rarely produced	7

      4 (3)    Motile cells uniflagellate	5
      4'       Motile cells biflagellate	6

      5 (4)    Zoospores posteriorly uniflagellate, formed inside the sporangium. . . class. . .Chytridiomycetes

                  The Chytridiomycetes produce  asexual zoospores with a single posterior whiplash
              flagellum.   The thallus is highly variable; the most primitive forms are unicellular and
              holocarpic and in  their  early stages of development are plasmodial (lack cell walls),  more
              advanced forms develop rhizoids and  with further evolutionary progress develop mycelium.
              The principle chemical component of the cell wall is  chitin, but cellulose  is also present.
              Chytrids are typically aquatic organisms but may be  found in other habitats. Some species
              are chitinolytic and/or  keratinolytic.   Chytrids may be isolated from  nature by baiting (e.g.
              hemp seeds or pine pollen) Chytrids occur both in marine and  fresh water habitats and are
              of some economic importance due to their parasitism of algae and animals.  The genus
              Dermocystidium may be provisionally grouped  with the chytrids.  Species of this genus
              cause serious epidemics of oysters and marine and fresh water fish.

      5'       Zoospores anteriorly uniflagellate,  formed inside or outside the sporangium	class
              	Hyphochytridiomycetes

                  These fungi are aquatic  (fresh water or  marine)  chytrid-like fungi whose  motile cells
              possess a single anterior flagellum of the tinsel type (feather-like).  They are parasitic on
              algae and fungi or may be saprobic.   Cell walls contain chitin  with some species also demon-
              strating cellulose  content.  Little information is available on the biology of this class  and
              at present  it is limited to less than  20 species.

      6 (4')    Flagella .nearly equal, one whiplash the other tinsel	class	Oomycetes

                  A number of representatives of the Oomycetes have been shown to have cellulosic cell
              walls.   The mycelium is coenocytic,  branched  and "well developed in most cases. The sexual
              process results in the formation of  a  resting spore of the oogamous type,  i. e. , a type of
              fertilization in which two heterogametangia come in contact and fuse their contents through
             .a pore  or tube.  The  thalli in this class range from unicellular to profusely branched
             •filamentous types. Most forms are eucarpic; zoospores  are produced throughout the class
              except  in the more highly advanced  species.  Certain species are of economic  importance due
              to their destruction of food crops (potatoes and grapes) while others cause serious diseases of
              fish (e.g. Saprolegina parasitica).  Members of the family Saprolegniaceae are the common

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                                                                                                         Fungi
         "water molds1' and are among the  most ubiquitous fungi in nature.   The order Lagenidiales
         includes only a few species which  are parasitic on algae, small animals,  and other aquatic
         life.   The somatic structures of this taxon are holocarpic and endobiotic.   The sewage  fungi
         are classified in the order Leptomitales.  Fungi of this order are characterized by the
         formation of refractile constrictions; ''cellulin plugs" occur throughout the thalli or,  at least,
         at the bases of hyphae or to cut off reproductive  structures.  Leptomitus lacteus  may
         produce rather extensive fouling floes  or slimes in organically enriched waters.

6'        Flagella of unequal size, both whiplash	class. . . Plasmodiophoromycetes

             Members of this class are obligate endoparasites of  vascular plants,  algae,  and  fungi.
         The thallus consists of a plasmodium which develops within the host cells.  Nuclear division
         at some stages of the life cycle is of a type found in no other  fungi but  known to occur in
         protozoa.  Zoosporangia which arise directly from the plasmodium bear zoospores with two
         unequal anterior falgella.  The cell walls of these fungi apparently lack cellulose.

7(3')    Mainly 'saprobic,  sex cell when present a zygospo re	class....  Zveomycetes

             This class has well developed mycelium with septa developed in portions  of the
         older  hyphae; actively growing hyphae  are normally non-septate.  The asexual spores are
         non-motile sporangiospores (aplanospores).  Such spores lack flagella and are usually
         aerialy disseminated.  Sexual reproduction is initiated by the fusion of two gametangia
         with resultant formation of a thick-walled, resting  spore, the zygospore.   In the  more
         advanced species,  the sporangia or the sporangiospores are conidia-like.  Many  of the
         Zygomycetes are of economic importance due to their ability to synthesize commercially
         valuable organic acids and alcohols, to transform steroids  such as  cortisone, and to
         parasitize and destroy food crops.  A few species are capable of causing disease in man
         and animals (zygomycosis).

7'        Obligate commensals of arthropods, zygospores usually lacking	class. . . . Trichomycetes

             The Trichomycetes are an ill-studied group of fungi which appear to be obligate
         commensals of arthropods.   The trichomycetes are associated with a wide variety of insecta,
         diplopods, and Crustacea of terrestrial and aquatic (fresh and marine) habitats.   None of
         the members of this class have been cultured in vitro for continued periods of times with any
         success.   Asexual reproduction is by means of sporangiospores.  Zygospores have been
         observed in species of several orders.

8 (2')    Sexual spores borne in  asci	class	Ascomycetes

             In the Ascomycetes the products of meiosis, the ascospores, are borne in sac
         like structures termed  asci.  The ascus usually contains eight ascospores, but the number
         produced  may vary with the species or strain.  Most species produce  extensive septate
         mycelium.  This large  class is divided into two  subclasses on the presence or absence
         of an  ascocarp.  The Hemiascomycetidae lack an ascocarp and do not  produce ascogenous
         hyphae; this subclass includes the true yeasts.  The Euascomycetidae usually are divided
         into three series (Plectomycetes,  Pyrenomycetes,  and Discomycetes) on the  basis of
         ascocarp  structure.

8'       Sexual spores borne on  basidia	class	Basidiomycetes

             The Basidiomycetes generally are considered the  most highly evolved of the fungi.
         Karyogamy and meiosis occur in the basidium which bears sexual exogenous spores,
         basidiospores.   The mushrooms,  toadstools, rusts, and smuts'are included in this class.

8"       Sexual stage  lacking	'.	Form class.(Fungi Imperfecti).,Peuteromycetes

             The Deuteromycetes is a form class for those fungi  (with morphological affinities
         to the Ascomycetes or Basidiomycetes) which have  not demonstrated a sexual stage.
         The generally employed classification scheme for these fungi is based on the  morphology
         and color  of the asexual reproductive stages.  This  scheme is briefly  outlined below.
         Newer concepts of the classification based on conidium development after the classical
         work  of S. J. Hughes (1953) may eventually replace the gross morphology  system (see
         Barren 1968).

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Fungi
       KEY TO THE FORM-ORDERS OF THE FUNGI IMPERFECT!

       1        Reproduction by means of conidia, oidia,  or by budding	2
       1'        No reproductive structures present	Mycelia Sterilia

       2 (1)     Reproduction by means of conidia borne in pycnidia	Sphaeropsidales
       2'        Conidia, when formed, not in cycnidia	3

       3 (2')    Conidia borne in acervuli	Melanconiales
       3'       Conidia borne otherwise, or reproduction by oidia or by budding	Moniliales

       KEY TO THE FORM-FAMILIES OF THE MONILIALES

       1        Reproduction mainly by unicellular budding, yeast-like;  mycelial phase,  if present,
               secondary,  arthrospores occasionally produced,  manifest melanin pigmentation lacking	2
       1"       Thallus mainly filamentous; dark melanin pigments sometimes produced	3

       2 (1)    Ballistospores produced	Sporobolomycetaceae
       2'       No ballistospores	Cryptococcaceae

       3       Conidiophores,  if present,  not united into sporodochia or synnemata	4
       3'       Sporodochia present	Tuberculariaceae
       3"      Synnemata present                                                              Stilbellaceae

       4 (3)    Conidia and Conidiophores or oidia hyaline or brightly colored	Moniliaceae
       4'       Conidia and/or Conidiophores, containing dark melanin pigment	Dematiaceae

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                                                                              Fungi Fungi
 SELECTED REFERENCES

 Ahearn, D.G.,  Roth, F.J. Jr.,  Meyers,  S.P.
     Ecology and Characterization of Yeasts
     from Aquatic Regions of South Florida.
     Marine Biology 1:291-308.  1968

 Alexopoulos, J. C.  Introductory Mycology.
     2nd ed.  John Wileyand Sons, New York,
     613pp.  1962

 Barren, G. L.  The Genera of Hyphomycetes
     from Soil.  Williams and Wilkins Co.,
     Baltimore. 364 pp. 1968

 Cooke, W. B.   Population Effects on the
     Fungus Population of a Stream.
     Ecology 42:1-18.  1961

 	.  A Laboratory Guide to Fungi in
      Stagnant waters.  I.  Morphology and
      Occurrence in Nature.  Amer. J.
      Botany 54:702-719.  1967

Hughes,  S.J.  Conidiophores, Conidia and
      Classification.  Can. J. Bot. 31:577-
      659.   1953

Johnson, T.W., Jr.  Saprobic Marine Fungi.
      pp. 95-104.  InAinsworth,  G.C. and
      Sussman, A.S. The Fungi,  III.
      Academic Press,  New York. 1968

            and Sparrow,  F.K., Jr. .  Fungi
    Polluted Waters, Sewage, and Sewage
    Treatment Systems.  U. S.  Dept.  of
    Health, Education and Welfare,  Cincinnati,
    132 pp.  1963

    	.  Fungi in Sludge Digesters.
    Purdue Univ. Proc. 20th Industrial
    Waste Conference,  pp 6-17.  1965a

    	.  The Enumeration of Yeast
    Populations in a Sewage Treatment Plant.
    Mycologia  57:696-703.  1965b

    	.  Fungal Populations in Relation
    to Pollution of the Bear River,  Idaho-Utah.
    UtahAcad.  Proc. 44(1):298-315.  1967

     	and Matsuura, George S.  A Study
    of Yeast Populations in a Waste Stabilization
    Pond System. Protoplasma 57:163-187.
    1963

	, Phaff,  H.J., Miller,  M.W.,
    Shifrine, M., and Khapp,  E.  Yeasts
    in Polluted Water and Sewage.
    Mycoiogia 52:210-230.  1960

Emerson, Ralph and Weston, W.H.
    Aqualinderella  fermentatis Gen.  et Sp.
    Nov., A Phycomycete Adapted to
      in Oceans and Estuaries. Weinheim,
      Germany.  668 pp.  1961

Meyers, \S. P.  Observations on the Physio-
      logical Ecology of Marine Fungi. Bull.
      Misaki Mr. Biol.  Inst. 12:207-225.  1968

Prigsheim,  E.G.  Iron Bacteria.  Biol. Revs.
      Cambridge Phil.  Soc. 24:200-245.  1949

Sparrow,  F. K., Jr. Aquatic Phycomycetes.
      2nd ed.  Univ. Mich. Press, AnnArbor.
      1187 pp.  1960.

	.  Ecology of Freshwater Fungi.
      pp.  41-93.  InAinsworth, G.C. and
      Sussman, A.~S.  The Fungi, III.  Acad.
      Press, New York.   1968

Stokes,  J. L.  Studies on the Filamentous
      Sheathed Iron Bacterium  Sphaerotilus
      natans.  J.  Bacteriol. 67:278-291.  1954

van Uden, N. and Fell, J.W.  Marine Yeasts.
      pp.  167-201.  In Droop, M.R.  and Wood,
      E. J.F.  Advances in Microbiology of
      the Sea, I.  Academic Press, New York.
      1968

Yerkes, W. D.  Observations on an Occurrence
      of Leptomitus lacteus in Wisconsin.
      Mycologia 58:976-978.  1966

Cooke, William B.  and Ludzack,  F.J.
      Predacious Fungus Behavior in
      Activated Sludge Systems.  Jour. Water
      Poll. Cont. Fed. 30(12): 1490-1495.  1958.
                                                                                     14-9

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                       PROTOZOA,  NEMATODES, AND ROTIFERS
I  GENERAL CONSIDERATIONS

A  Mierobial quality constitutes only one
   aspect of water sanitation; microchemicals
   and. radionuclides are attracting increasing
   amount of attention lately.

B  Microbes  considered here include bacteria,
   protozoa,  and microscopic metazoa; algae
   and. fungi  excluded.

C  Of the free-living forms,  some are
   members  of the flora and fauna of surface
   waters; others washed into the water from
   air and soil; still others of wastewater
   origin; nematodes most commonly from
   sewage effluent.

D  Hard to separate  "native" from "foreign"
   free-living microbes, due to close
   association of water with  soil and other
   environments; generally speaking, bacteria
   adapted to water are those that can  grow
   on very low concentrations of nutrient
   and zoomicrobes adapted to water are
   those that feed on algae, and nsmatodes,
   especially bacteria eaters, are uncommon
   in water but in large numbers in sewage
   effluent.

E  More species and lower densities of
   microbes in clean water and fewer species
   and higher densities in polluted water.

F  Pollution-tolerance or nontolerance of
   microbes closely related to the DO  level
   required in respiration.

G  From pollution viewpoint, the following
   groups of microbes are of importance:
   Bacteria,  Protozoa, Nematoda,  and
   Rotifera.

II  BACTERIA

A  No ideal method for studying distribution
   and ecology of bacteria in freshwater.

                       (9)
B  According to Collins,   Pseudomonas,
   Achrombacter, Alcaligenes,  Chromobac-
   terium, Flavobacterium,  and  Micrococcus
   are the most widely distributed and may be
    considered as indigenous to natural
    waters.  Sulfur  and iron bacteria are
    more common in the bottom mud.

 C Actinomycetes,  Bacillus sp. Aerogenes
    sp., and nitrogen-fixation bacteria are
    primarily soil dwellers and may be washed
    into the water by runoffs.

 E Nematodes are usually of aerobic sewage
    treatment origin.

 D E.  coji,  streptococci, and _C1.  perfringens
    are true indicators of fecal pollution.

IE  PROTOZOA

 A Classification

    1  Single- cell animals in the most
       primitive phylum (Protozoa) in the
       animal kingdom.

    2  A separate kingdom, Protista, to in-
       clude protozoa, algae, fungi,  and
       bacteria proposed in the 2nd edition
       of Ward-Whipple's Fresh-Water
       Biology .(10)

    3  Four subphyla or classes:

       a  Mastigophora (flagellates)-Subclass
          phytomastigina dealt with under
          algae; only subclass Zoomastigina
          included here; 4 orders:

          1)  Rhizomastigina - with flagellum
             or flagella and pseudopodia

          2)  Protomonadina - with 1 to 2
             flagella mostly free-living many
            parasitic
          3)  Polymastigina - with 3 to 8
             flagella; mostly parasitic in
             elementary tract of animals
             and man

          4)  Hypsrmastigina - all inhabitants
             of alimentary tract of insects.
W.BA. 45c.6. 76
                                 15-1

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Protozoa, Nematodes, and Rotifers
         Ciliophora or Infusoria (ciliates)  -
         no pigmented members; 2 classes:

         1)  Ciliata - cilia present during the
            whole trophic life; containing
            majority of the ciliates

         2)  Suctoria - cilia present while
            young and tentacles during trophic
            life.
         Sarcodina (amoebae) - Pseudopodia
         (false feet) for locomotion and food-
         capturing; 2 subclasses:

         1)  Rhizopoda - Pseudopodia without
            axial filaments; 5 orders:

            a)  Proteoniyxa - with radiating
               pseudopodia; without test or
               shell

            b)  Mycetozoa - forming plasmodium;
               resembling fungi in sporangium
               formation

            c)  Amoebina - true amoeba -
               forming lobopodia

            d)  Testacea - amoeba with single
               test or shell of chitinous
               material

            e)  Foraminifera -  amoeba with 1
               or more shells of calcareous
               nature; practically all marine
               forms
        Sporozoa - no organ of locomotion;
        amoeboid in asexual phase; all
        parasitic
B  General Morphology

   1  Zoomastigina:

      Relatively small size (5 to 40 n); with
      the exception of Rhizomastigina, the
      body has a definite shape (oval,  leaf-
      like, pear-like,  etc.);  common members
      with 1 or 2 flagella and some with  3, 4,
      or more; few forming colonies; cyi .:'tome
      present in many for feeding.
   2  Ciliophora:

      Most highly developed protozoa; with
      few exceptions,  a macro and a micro-
      nucleus; adoral zone of membranellae,
      mouth,  and groove  usually present in
      swimming and crawling forms, some
      with conspicuous ciliation of a disc-like
      anterior region and little  or no body
      cilia (stalked and shelled  forms);
      Suctoria nonmotile (attached) and with-
      out cytostome cysts formed in most.
   3  Sarcodina:

      Cytoplasmic membrane but no cell wall;
      endoplasm  and ectoplasm distinct or in-
      distinct; nucleus with small or large
      nucleolus; some with test or shell;
      moving by protruding pseudopodia; few
      capable of flagella transformation; fresh-
      water actinopods usually sperical with
      many radiating axopodia; some Testacea
      containing symbiotic algae and mistaken
      for pigmented amoebae; cysts with single
      or double wall and 1 or 2 nuclei.
   4  Sporozoa: to be mentioned later.
C  General Physiology

   1 Zoomastigina:
     Free-living forms normally holozic;
     food supply mostly bacteria in growth
     film on surfaces or  clumps relatively
     aerobic, therefore the first protozoa to
     disappear in anaerobic conditions and
     re-appearing at recovery; reproduction
     by simple fission or occasionally by
     budding.
   2  Ciliophora:

      Holozoic; true ciliates concentrating
      food particles by ciliary movement
      around the mouth part; suctoria sucking
      through tenacles; bacteriaand small
 15-2

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                                                          Protozoa, Nematodes, and Rotifers
       algae and protozoa constitute main
       food under natural conditions; some
       shown in laboratory to thrive on dead
       organic matter and serum protein; not as
       aerobic as flagellates - some surviving
       under highly anaerobic conditions, such
       as Metopus; reproduction by simple
       fission, conjugation or  encystment.
    3  Sarcodina:

       Holozoic; feeding through engulfing by
       pseudopodia; food essentially same as
       for ciliates;  DO requirement somewhat
       similar to ciliates - the small amoebae
       and Testacea frequently present in large
       numbers in sewage effluent and polluted
       water; reproduction by simple fission
       and encystation.
IV  NEMATODES

 A Classification

    1  All in the phylum Nemata (nonsegment-
       ed round worms); subdivided by some
       authors into two classes:

       Secernentea - 3 orders:
        (phasmids)
          Tylenchida,  Rhabditida, Strongylida,
          and Teratocephalida; with papillae on
          male tail, caudal glands absent.
       Adenophora - 6 orders:
        (aphasmids)
         Araeolaimida, Dorylaimida,
         Chromdorida,  Monhysterida, Enoplida,
         and Trichosyringida no papillae on
         male caudal glands absent.
       Orders encountered in water and sewage
       treatment - Free-living forms  inhabitat-
       ing sewage treatment plants are usually
       bacteria-feeders and those feeding on
       other nematodes; those inhabitating clean
       waters feeding on plant matters; they
       fall into the following orders:
      Tylenchida - Stylet in mouth; mostly
      plant parasites; some feed on
      nematodes, such as Aphelenchoides.

      Rhabditida - No stylet in mouth or caudal
      glands in tail; mostly bacteria-feeders;
      common genera: Rhabiditis,  Diplogaster,
      DiplogasteroideSj  Monochoides^ Pelodera,
      Panagrellus, and Turbatrix.

      Dorylaimida - Relatively large nematodes;
      stylet in mouth; feeding on other nematodes,
      algae and probably zoomicrobes; Dorylaimus
      common genus.

      Chromadorida -  Many marine forms;
      some freshwater dwellers feeding on
      algae; characterized by strong orna-
      mentation of knobs, bristles or
      punctations in cuticle.

      Monhysterida - Freshwater dwellers;
      esophago-intestinal valve spherical to
      elongated;  ovaries single or paired,
      usually straight; common genus  in
      water - Monhystera.

      Enoplida  -  Head  usually with  a number
      of setae;  Cobb reported one genus,
      Mononchulus. in sand filters  in
      Washington, D.C.
B  General Morphology

   Round, slender, nonsegmented (transverse
   markings in cuticle of some) worms;
   some small (about | mm long, as Tri-
   cephalobus),  many 1 to 2 mm long
  (Rhabditis.  Diplogaster. and Diplogasteriodes
   for instance),  and some large (2 to 7 mm,
   such as Dorylaimus); sex separated but few
   parthenogenetic;  complete alimentary canal;
   with  elaborate mouth parts with or  without
   stylet; complete reproductive system in
   each sex; no circulatory or respiratory
   system; complex nervous system with
   conspicuous nerve ring across oesophagus.
C  General Physiology

   1  Feeding - Most sewage treatment plant
      dwellers feeding on bacteria; others
      preying on protozoa, nematodes, rotifers,
                                                                                    15-3

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Protozoa, Nematodes, and Rotifers
      etc.,  clean-water species apparently
      vegetarians; those with stylet in mouth
      use the latter to pierce the body of animal
      or plant and suck contents; metabolic
      waste mostly liquid containing ammonium
      carbonate or bicarbonate;  enteric
      pathogens swallowed randomly with
      suspending fluid, hence remote possi-
      bility of sewage effluent-borne nematodes
      being pathogen-carriers.
   2  Oxygen requirement - DO apparently
      diffused through cuticle into body; DO
      requirement somewhat similar to
      protozoa; Khabditis tolerating reduced
      DO better than other Rhabditida members;
      all disappear under sepsis in liquid; some
      thrive in drying sludge.
   3  Reproduction - Normal life cycle requires
      mating, egg with embryo formation,
      hatching of  eggs inside or outside femais,
      4 larval stages, and adult; few repro-
      duce in the absence of males.
V ROTIFERS

A Classification:

   1  Classified either as a class of the phylum
      Aschelminth.es (various forms of worms)
      or as a separate phylum (Rotifera); com-
      monly called wheel animalcules, on
      account of apparent circular movement of
      cilia around head (corona); corona con-
      tracted when crawling or swimming and
      expanded when attached to catch food.

   2  Of the 3 classes, 2 (Seisonidea and
      Bdelloidea) grouped by some authors
      under Digononta (2 ovaries) and the
      other being Monogononta (1 ovary);
      Seisonidea containing mostly marine
      forms.

   3  Class Digononta containing 1 order
      (Bdelloida) with 4  families, Philodinedae
      being the  most important.
   4  Class Monogononta comprising 3 orders:
      Notommatida (mouth not near center of
      corona) with  14 families,  Floscularida
      Melicertida(corona  with two  wreaths of
      cilia and furrow between them) with 3
      families;  most import genera included
      in the order Notommatida: Brachionus,
      Keratella,  Monostyla,  Trichocerca,
      Asplanchna, Polyarthra, Synchaeta,
      Microcodon; common genera under the
      order Flosculariaceae: Floscularia,
      and Atrochus.  Common genera under
      order Melicertida:  Limnias and
      Conochilus.

   5  Unfortunately orders and families of
      rotifers partly  based on character of
      coi'jna and trophi (chewing organ),
      which are difficult, to study, esp. the
      latter; the foot  and cuticle  much easier
      to study.

B  General Morphology and Physiology

   1  Body weakly differentiated  into head,
      neck,  trunk, and foot,  separated by
      folds;  in some,  these regions are
      merely gradual changes in  diameter
      of body and without a separate neck;
      segmentation external only.

   2  Head with corona, dosal antenna, and
      ventral mouth; mastax,  a chewing organ,
      located in head  and neck, connected to
      mouth anteriorly by a ciliated gullet  and
      posteriorly to a large stomach occupying
      much of the trunk.

   3  Common rotifers reproducing partheno-
      genetically by diploid eggs; eggs laid in
      water,  cemented to plants,  or carried
      on  female until  hatching.

   4  Foot,  a prolongation of body, usually
      with 2 toes; some with  one  toe;  some
      with one toe and an extra toe-like
      structure (dorsal spur).

   5  Some,  like Phllodina, concentrating
     bacteria and other microbes and minute
     particulate organic matter by ciliary
     movement on corona  larger microbes
     chewed by mastax; some  such as
     Monostyla feeding on clumped matter,
     such as bacterial growth, fungal masses,
     etc. at bottom; virus generally not
     ingested - apparently undetected by
     cilia.
   6  DO requirement somewhat  similar to
      protozoa; some  disappearing under
      reduced DO, others, like Philodina,
      surviving at as  little as 2 ppm DO.
  15-4

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                                                         Protozoa, Nematodesf and Rotifers
  VI SANITARY SIGNIFICANCE

  A Pollution tolerant and pollution non-
     tolerant  species - hard to differentiate -
     requiring specialist training in protozoa,
     nematocles,  and rotifers.

  B Significant quantitative  difference in clean
     and polluted waters - clean waters con-
     taining large variety of genera and species
     but quite low in densities.

  C Aerobic  sewage treatment processes
     (trickling filters and activated sludge
     processes, even primary settling) ideal
     breeding grounds for those that feed on
     bacteria, fungi, and minute protozoa and
     present in very large numbers; effluents
     from such processes carrying large num-
     bers of these zoomicrobes; natural waters
     receiving such effluents showing significant
     increase in all 3 categories.

  D Possible Pathogen arid Pathogen  Carriers

     1  Naeg_le_ria causing swimming associated
        meningoi'iK-rplial ilis and Acanthann:oba:
        causing nonswimrning associated cases.

     2  Amoebae and nematodes grown on
        pathogenic enteric bacteria in lab; none
        alive in amoebic cysts; very few alive
        in nematodes after 2 days after ingestion;
        virus  demonstrated in nematodes only
        when very high virus concentrations
        present; some freeliving amoebae
        parasitizing humans.

     3  Swimming ciliates and some rotifers
        (concentrating food by corona) ingesting
        large  numbers of pathogenic enertic
        bacteria, but digestion rapid;  no
        evidence of concentrating virus; crawling
        ciliates and flagellates feeding on clumped
        organisms.

    . 4  Nematodes concentrated from sewage
        effluent in Cincinnati area showing
        live_E_.  cpli and streptococci, but no
        human enertic pathogens.

VII   EXAMINATION OF WATER FOR MICROBES

  A Bacteria - not dealt here.
B  Protozoa and rotifers - should be included
   in examination for planktonic microbes.
C  Nematodes
                        (3)
D  Laboratory Apparatus

   1  Sampte, Bottles - One-gallon glass or
      plastic bottles with metal or plastic
      screw caps, thoroughly washed and
      rinsed three times with distilled water.

   2  C_up_illarv Pipettes and (lubber Bulbs -
      \ , ('.) in. ) Pasteur capillary pipettes
      and rubber bulbs of 2 rnl capacity,

   '*  Zil!rŁ?iiPn ^LnJJi " ^ny filter holder
      assembly use/1 in bacteriological
      examination.   The funnel should be
      at  ieast 650 ml and the filter flask at
      least 2 liter capacity.

   4  Filter_MernbŁane_s - Millepore SS 'SS
      047 MM) type  membranes or equivalent.

   5  Microscope -  Binocular microscope
      with 10X eyepiece,  4X, 10X, and 43X
      objectives, and mechanical stage.

E  Collection of Water Samples

   Samples  are collected in the same manner.,
   as those  for bacteriological examination,
   except that a dechlorinating agent is not
   needed.  One-half to one gallon samples are
   collected from raw water and one-gallon
   samples  from, tap water.  Refrigeration is
   not essential and samples may be transported
   without it unless  examination is to be delayed
   for more than five days.

F  Concentration of  Samples

   1  One gallon of tap water can usually bs
      filtered through a single  8-u membrane
      within 15 minutes unless the water  has
      high turbidity.  At least one gallon  of
      sample should be used in a single examina-
      tion.  Immediately after  the last of the
      water is disappearing from the membrane,
      the saction line is disconnected and the
      membrane  placed on the wall of a clean
      50 to  100 ml beaker and flushed repeatedly
      with about 2-5 ml of sterile distilled water
                                                                                       15-5

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Protozoa, Nematodes, and Rotifers
       with the aid of a capillary pipette and a
       rubber bulb.  The concentrate is then
       pipetted into a clean Sedgewick-Rafter
       Counting Cell and is ready for examina-
       tion.

    2  In concentration of raw water samples
       having visible turbidity, two to four
       8-mic.ron membranes may be required
       per sample, with filtration through each
       membrane being limited to not more
       than 30 minutes.  Samples ranging from
       500 ml to 2 liters may be  filtered with
       ons membrane, depending on degree of
       turbidity. After filtration the membranes
       are placed on the walls of separated
       beakers and washed as above.  To
       prevent the particulates from obscuring
       the nematodes, the  washing from each
       filter  is examined in a separate counting
       chamber.

G Direct Microscopic Examination

   Each counting chamber containing the
   filter concentrate is first examined under
   a 4X objective.  Unless the concentrate
   contains more than 100 worms, the  whole
   cell area  is surveyed for nematodes, with
   respect to number, developmental stage,
   and motility.  When an  object  having an
   outline resembling that of a nematode  is
   observed,  it  is re-examined under  a 10X
   objective  for anatomical structures, unless
   the object exhibits typical nematode move-
   ment, which  is sufficient for identifying the
   object as  a nematode.   When the concentrate
   contains more than 100  worms, the worm
   density can be estimated by counting the
   number of worms in representative micro-
   scopic fields and multiplying the average
   number of worms per field by the number
   of fields in the  cell area. The nematode
   density may be expressed as number of
   worms per gallon with or without differenti-
   ation as to adult or larval stages  or as to
   viability.                                   VIII
H General Identification of Nematodes

   1  While actively motile nematodes can be
      readily recognized by any person who
      has some  general concept of micro-
      scopic animals,  the nonmotile or
   sluggishly motile nematodes may be
   confused with root fibers,  plant fila-
   ments of various types,  elongated
   ciliates such as Homalozopn vermi-
   culare. or segments of appendages of
   small Crustacea.  To facilitate a
   general identification of nematodes, the
   gross morphology of three of the free-
   living nematodes that are frequently
   found in water supplies is shown in the
   attached drawing.  The drawing provides
   not only the general anatomy for recogni-
   tion of nematodes but also most of the
   essential structures for guidance to those
   who want to use the "Key to Genera"  in
   chapter No. 15 on Nemata by B.  G.
   Chitwood and M. W. Allen in the book,
   Fresh Water Biology. (1°)

2  Under normal conditions, practically
   all nematodes seen in samples of
   finished water are in various larval
   stages and will range from 100 to 500
   microns in length and 10 to 40 microns
   in width.  Except in the fourth (last)
   stage, the larvae have no sexual organs
   but show other structural characteristics.

3  If identification of genera is desired,
   the filter washings are centrifuged  at
   500 rpm for a few minutes.  The
   supernate is discarded, except a few
   drops, and the sediment is resuspended
   in the remaining water.  A drop of the
   final suspension is examined under both
   10X and 43X objectives  for anatomical
   characteristics without  staining,  and  for
   supplementary study of structures the
   rest is fixed in 5% formalin or  other
   fixation fluid and stained according to
   instructions given in Chitwood and
   Allen's Chapter on Nemata, (?)
   Goodey's Soil  and Freshwater Nema-
   todes' 11) or other books on nematology.
USE OF ZOOMICROBES AS
POLLUTION INDEX

Idea not new, protozoa suggested long ago;
many considered impractical because of
the need of identifying pollution-intolerant
and pollution-tolerant species - proto-
zoologist required.  Method also time
consuming.
   15-6

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                                                           Protozoa, Nematodes. and Rotifers
 B Can use them on a quantitative basis -
    nematodes,  and nonpigmented
    protozoa present in small numbers in
    clean water.  Numbers greatly increased
    when polluted with effluent from aerobic
    treatment plant or recovering from sewage
    pollution; no significant error introduced
    when clean-water members included in the
    enumeration if a suitable method of com-
    puting the pollution index developed.
  C Most practical method involves the
    equation:  A +B + 1000 C = Z.P.I.,
                     "A
    where
    A = number ol pigmented protozoa,
    B = non pigmented protozoa,  and
    C = nematodes in a unit volume of sample,
    and Z.P.I. = zoological pollution index.
    For relatively clean water, the value of
    Z.P.I, close to 1; the larger the  value
    above 1, the greater the pollution by aerobic
    effluent (see attached report on zoomicrobial
    indicator of water pollution).
IX  CONTROL .
 A Chlorination of effluent
 B Prolongation of detention time of effluent
 C Elimination of slow sand filters in
    nematode control.
  LIST OF  COMMON ZOOLOGICAL ORGANISMS
  FOUND IN AEROBIC SEWAGE TREATMENT
  PROCESS
 PROTOZOA
    Sarcodina - Amoebae
       Arnoe_ba proteus; A radiosa
       Hartmannella
       Arcella Vulgaris
       Noegleria gruberi
       Actinophryjs 	
FLAGELLATA
   Bodo caudatus
   Pleuromonas  jaculans
   Oikomonas termo
   Cercomonas longicauda
   Peranema trichophorum

   Swimming type
      Ciliophora:
         Colpidium colpoda
         Colpoda cuculus
         Glaucoma pyriformis
         Paramecium candatum; P bursaria
   Stalked type
      Opercularia sp. (short stalk dichotomous)
      Vor_ticella_sp.  (stalk single and contractile)
      Episjy lis pli cat ill s (like opercularia,  more
                colonial,  stalk not contractile)
      Carchesium sp.  (like vorticella bat colonial,
                individual zooids contractile)
      _Zopthamnium sp. (entire colony contracts)
   Crawling type
      Aspicisca costata
      Euplotes patella
      Stylonychia mylitus
      Urostyla sp.
      Oxytricha sp.
NEMATODA
      pipjogaster sp.      Dorylamus sp.
      Moiiochoides sp.      Chlindrocorpus sp.
      Diplogasteroides sp.  Cephalobus sp.
      Rhabditis sp.         5^^it°j2-HH}iS. SP•
      Pelodera_sp.          Monhystera  sp.
                     sp.    Trilobus sp.
                                                                                     15-7

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Protozoa, Nematodes^  and Rotifers	
ROTATORIA

   Diglena

   Monostyla

   Polyarthra

   Philodina,

   Keratella

   BrachJLomis_

OLIGOCHAETA (bristle worms)

   Aelosoma he_mŁŁichi_

   Aulophorus limosa

   Tubjfex tubifex

   Lumbricillus lineatus
INSECT LARVAE

   Chironomus

   Psychoda sp. (trickling filter fly)

ARTHROPODA

   JLessertia sp.

   Pgrrhgmma sp,

   Achoratus subuiaticus (collembola)

   Folsomia sp. (collembola)

   Tomocerus sp.  (collembola)

REFERENCES

1  American Public Health Association,
      American Water Works Association and
      Water Pollution Control Federation.
      Standard Methods for the Examination
      of Water and Wastewater,  13th ed.
      New York".  1971.~

2  Chang, S. L.,  et al.  Survey  of Free-
      Living Nematodes and Amoabas in
      Municipal Supplies.  J.A.W.W.A. 52:
      613-618.
3  Chang, S.  L.  Interactions between Animal
     Viruses and Higher Forms of Microbes.
     Proc. Am. Soc. Civ. Eng. Jl. San. Eng.
     96:151.  1970

 4  Chang,  S. L.  Zoomicrobial Indicators
      of Water Pollution presented at the
      Annual Meeting of Am. Soc. Microbial,
      Philadelphia, April 23-28,  1972.

 5  Chang,  S, L.  Pathogenic Free-Living
      Amoebae and Recreational Waters.
      Presented at 6th International Confer-
      ence of Water Pollution Research
      Association,  Jerusalem,  Israel,
       June 19-24,  1972.

 6  Chang,  S. L.  Proposed Method for
      Examination of Water for Free-Living
      Nematodes.   J.A.W.W.A.  52:695-698.
      1960.

 7  Chang,  S. L., et al.  Survival and Protection
      Against Chlorination of Human Enteric
      Pathogens in Free-Living Nematodes
      Isolated from Water Supplies.  Am. Jour.
      Trop.  Med.  and Hyg. 9:136-142.  1960.

 8  Chang,  S. L. Growth of Small Free-Living
      Amoebae in Bacterial and Bacteria-Free
      Cultures. Can,  J.  Microbial.
      6:397-405.   1960.

 9  Chang,  S. L. and Kabler,  P. W.  Free-Living
      Nematodes in Aerobic Treatment Plant
      Effluents. J.W.P.C. F.  34:1256-2161.
      1963.

10  Chitwood, B. G. and Chitwood,  M. B.  An
      Introduction to Nematology.  Section I:
      Anatomy. 1st ed. Monumental Printing
      Co. Baltimore.  1950.  pp 8-9.

11  Cobb, N.A.  Contributions to the  Science of
      Nematology VII.  Williams and Wilkins Co.
      Baltimore.  1918.

12  Collins, V.G.  The Distribution and Ecology
      of Bacteria in Freshwater, Pts. I & II,
      Proc.  Soc. for Water Treatment and Exam.
      12:40-73. 1963.  (England)

13  Edmondson, W.T.,  et al. Ward-Whipple's
      Fresh  Water Biology.  2nd ed. John
      Wiley  & Sons, New York. 1959. pp 368-401.
 15-8

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                                                 Protozoa, Nematodes, and Rotifers
14 Goodey,  T.  Soil and Freshwater               This outline was prepared by S. L. Chang,
      Nematodes.  (A Monograph)  1st ed.           Chief, Etiology,  Criteria Development
      Methuen and Co.  Ltd.  London.  1951.        Branch,  Water Supply Research Laboratory,
                                                 NERC, USEPA, Cincinnati, Ohio 45268.

                                                 Descriptors:  Protozoa,  Nematodes,  Rotifers
                                                                            15-9

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Protozoa, Nematodes, and Rotifers
                              Insects
                          Oligochaetes &
                          insect larvae
                           Nematodes
                           & rotifers
                         4-4
                       Nonpigmented
                         protozoa        *
                         I   I   t  Iff
                      Heterotrophic
                        bacteria
                           Fungi
                           Algae
                     Autotrophic bacteria
                   Pathogenic organisms
     Suspended organic matter

              (by hydrolysis)
                                                                            Raw Sewage
      Dissolved organic matter
        (respiration,
        de animation,
        decarboxylation, etc.)
         Inorganic C,  P,  N,
                   S comp.
, f _ (NH3, NO',  CO', P)
       (Nitrification, sulfur
        & iron bacteria)
                   Food Chain in Aerobic Sewage Treatment Processes
 15-10

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                                 ACTIVATED SLUDGE
                                        PROTOZOA
Larger animals (worms, snails,  fly larvae,
etc.) dominate trickling filters.  Why are
these always absent from the activated
sludge process?
Why are there numerous micro-species
common to both trickling filters and
activated sludge?
What organisms besides protozoans and
animals are present in activated sludge?
What is the advantage(s) of microscopic
examination of activated sludge ?
One sampling site would be the one of choice
in sampling an activated sludge plant for
microscopic analysis.  Why?  Where?
What organisms predominate in activated
sludge ?
Why are photosynthetic green plants (in
contrast to animals) basically absent from
the activated sludge process in general and
mixeid liquor specifically?
Why are the same identical species of protozoa
found in activated sludge plants all over the
world?
What is the significance of a microscopic
examination of mixed liquor?
Define and characterize:                       10
    Activated Sludge
    Mixed Liquor
    Floes
What is the relation between bacterial/         11
protozoan populations in activated sludge
and the process itself?
At what total magnification were you able to    12
believe the smallest cells observed were in
fact bacteria?
Activated sludge is a dynamic (although        13
man-manipulated) ecosystem.  How does
it differ from a natural ecosystem?
BLIND. 14a. 6.76                                                                          16_j

-------
Activated Sludge Protozoa
What is the greatest problem(s) with a wet      14
mount slide preparation?
How do you overcome these disadvantages?     15
How do you slow down fast moving protozoans   16
on a wet mount?
Why are quantitative counts of protozoa (like    17
number/ml) generally meaningless?
What is the significance of proportional        18
counts?
Scanning a slide (in making a count) should     19
generally be done at	X.  (Total
magnification)
The iris diaphragm on the microscope is      20
used to adjust light intensity (true-false).
Why sample the surface film of the            21
settleometer?
Why is the thinnest film most ideal for a      22
wet mount?
What did you learn from the microscopic      23
examination of the activated sludge?
What is the physical nature of the floes        24
observed?
What filamentous organisms were             25
observed?
Why are "rare" species of no practical        26
significance in microscopic analyses of
activated sludge?
Why are there no protozoan indicator species  27
of process efficiency in activated sludge?
Activated sludge biological communities       28
are temporal in contrast to biological
communities in trickling filters which
are spatial (TRUE/FALSE).
"Seeding" a newly started activated sludge     29
plant with cultures or material from other
plants  is only a wasted effort (TRUE/FALSE).
Justify your answer.
16-2

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                                                               Activated Sludge Protozoa
If a wet mount slide of mixed liquor is
prepared and  placed in a petri dish with a
wet blotter underneath and allowed to sit
for several hours,  what will be the distri-
bution of the protozoa under the cover slip?
30
In a mixed liquor sample nearly all of the
stalked ciliates have "broken off" the stalks
and are free swimming as "telotrochs. "
What does this indicate?
31
What are Monads?  And are they good, bad,    32
or indifferent in activated sludge?
What are hypotrichs or crawling ciliates,
and are they good,  bad or indifferent in
activated sludge?
33
What are swimming ciliates,  and are they      34
good, bad or indifferent in activated sludge?
What are flagellates, and are they good,  bad   35
or indifferent in activated sludge?
What are amoebae, and are they good, bad,    36
or indifferent in activated sludge?
What are the ideal characteristics of a wet
mount  slide preparation?
37
Why does total community give a better        38
indication of process efficiency in activated
sludge?
In observing and identifying protozoa one looks 39
for what characteristics of an individual
organism?
What is the role of bacteria in activated
sludge?
40
What is the role of protozoa in activated
sludge?
41
Microscopic analysis of the mixed liquor
sample can be very quick, simple, and
meaningful (TRUE/FALSE).
42
                                                                                      16-3

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Activated Sludge Protozoa
Protozoan communities present in activated    43
sludge reveal:

   a.  Plant efficiency
   b.  Settleability
   c.  BOD removal
   d.  Solids removal
   e.  Plant loading

(Circle applicable descriptions)
Protozoan communities in activated sludge     44
reveal complete and instantaneous conditions;
average of physical and chemical conditions;
extremes of chemical and physical conditions.
(Draw a line through  phrases not true.)
Rank in increasing plant efficiency the          45
following protozoan group which would pre-
dominate.
   	Rotifers
   	Stalked ciliates
   	Amoebae
         Swimming ciliates
         Crawling ciliates
   	Flag ellate s

(For example,  use number  1-6.  One would be
startup conditions or least efficient,  and six
would be the most efficient.)
Identification is usually done at	X and    46
sometimes requires	X.
Immersion oil should be used sparingly at      47
what two points on a slide?
Which comes first in microscopic examina-    48
tion; scanning at low power to pick out
unknowns or higher  power to identify?
In making proportional count, which total       49
number to count would be better; total of ten
organisms or a total of 100 organisms? Why?
Why not kill the organisms so you can          50
identify and count them on the slide?
What simple chemical solutions are useful      51
to immobilize protozoa if methyl cellulose
or poly vinyl alcohol is not available?
16-4

-------
                                                              Activated Sludge Protozoa
Initially the wet mount slide should be
racked up close to the low power objective:
by your eye on the eyepiece through the
scope; or by glancing at the actual distance
with the naked eye while you rotate the
coarse adjustment knob. (Underline which)
                                   52
What are par-focal objectives on the
microscope?
                                   53
Why should water on the microscope and all   54
its parts be carefully avoided?
If activated sludge is a man manipulated
system,  are there comparable natural
ecosystems?  Example?
                                   55
What is the " community" concept in exami-  56
nation of activated sludge?
What are the applications of direct micro-    57
scopist examination of activated sludge?
What are rotifers, and are they good,  bad
or indifferent in activated sludge?
                                   58
List the five kingdoms of organisms and give  59
a specific example for each.
What techniques are most useful in           60
identifying an unknown organism, and why
is correct identification important?
Scanning and counting is done at	X
magnification.  Identification of most
PROTOZOA usually requires	X
magnification and occassionally	X
magnification.
                                   61
OBJ.
3.5 X
 10 X
TOTAL MAG.   USE
                                            62
(10 X eyepieces)
                                                                                     16-5

-------
                                                    Activated Sludge Protozoa
            hand is constantly operating the   63
            hand is constantly operating the
The microscope is manually operated and
requires skill and understanding on the part
of the operator.  A microscope no matter
how costly is only as good as the micro-
scopist operating it.
List the basic skills required in utilizing the   64
optimum capability of your microscope.
 This outline was prepared by R. M. Sinclair,
 National Training Center, MOTD, OWPO,
 USEPA, Cincinnati, Ohio 45268.
 Descriptors:  Microorganisms, Protozoa,
 Rotifers, Activated Sludge, Biota
                                                                              16-6

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                     FREE-LIVING AMOEBAE AND NEMATODES
I  FREE-LIVING AMOEBAE

   A  Importance of Recognizing Small,
      Free-Living Amoebae in Water
      Supplies

      1  Commonly found in soil, aerobic
        sewage effluent and natural, fresh
        waters - hence,  frequently en-
        countered in examination of raw
        water.

      2  Cysts not infrequsntly found in
        municipal supplies - not pathogen
        carriers.

      3  Flagellate-amoebae NaegJLeria
        involved in 50 some cases of
        meningoencephalitis,  about half
        in the U. S.; associated with
        swimming in small warm lakes.
        Acanthamoeba rhyjsodes parasitizing
        hyman throats and causing (3  cases)
        nonswimming- associated meningo-
        encephalitis .

      4  Cysts not to be confused with those
        of Endamoeba histolytica in water-
        borne epidemics.

   B  Classification of Small, Free-Living
      Amoebae

      1  Recognized  classification based
        on characteristics in mitosis.

      2  Common species fall into the
        following families  and genera:

        Family Schizopyrenidae: Genera
        Naegleria, Didascalus,  and
        Schizopyrenus -  first two being
        flagellate amoebae.

        Family Hartmannellidae:  Genera
        jiartmanella (Acanthamoeba)

      3  How to prepare materials for
        studying mitosis -  Feulgen stain
    C  Morphological Characteristics of
       Small,  Free-Living Amoebae

       1  Morphology of Trophozoites -
         Ectoplasm and endoplasm usually
         distinct; nucleus with large nucleolus.

       2  Morphology of cysts - Single or
         double wall with or without pores

    D  Cultural Characteristics of Small,
       Free-Living Amoebae

       1  How to cultivate these amoebae -
         plates with bacteria; cell cultures,
         axenic culture.

       2  Growth characteristics on plate,
         cell,  and axenic culture

       3  Complex growth requirements
         for most of these amoebae

    E  Resistance of Amoebic Cysts to
       Physical and Chemical Agents
II   FREE- LIVING NEMATODES

    A  Classification of Those Commonly
       Found in Water Supplies

       1  Phasmidia (Secerneutes):
         Genera Rhabditis, Dip_logaster,
         Diplogasteroides, Chsilobus^
         Panagrolaimus

       2  Aphasmidia (Adenophoro): Genera
         ^onjivjtera^ Aphelenclras, Turbatrix_
         (vinegar eel), Dorylaimus, and
         Rhab dolaimus

    B  Morphological Features

       1  Phasmids: papilla on tail of males,
         mouth adapted to feed on bacteria,
         few exceptions.

       2  Aphasmids:  no papilla on male tail;
         glandular cells in male.
BI.AQ. 14b.6.76
                                                                                 17-1

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Amoebae and Nematodes in Water Supplies  _
   C  Life Cycle

      1  Methods of mating

      2  Stages of development

      3  Parthenogenesis

   D  Cultivation

      1  Bacteria-fed cultures

      2  Axenic cultures

   E  Occurrence in Water Supplies

      1  Relationship between their
        appearance in finished water
        and that in raw water.

      2  Frequency of occurrence in
        different types o.? raw water
        and sources.

      3  Survival of human enteric path-
        ogenic bacteria and viruses in
        nematodes.

      4  Protection of human enteric
        pathogenic bacteria and viruses
        in .lematode-carriers.

   F  Control

      1  Chlorination of sewags effluent

      2  Flocculation and sedimentatioa
        of water

      3  Chlorination of water

      4  Other methods of destruction
REFERENCES

Amoebae

1  Singh, B. N., "Nuclear Division in Nine
      Species of Small, Free-Living Amoe-
      bae and its Bearing on the Classifica-
      tion of the Order Amoebida", Philos.
      Trans.  Royal Soc, London,  Series B,
      236:405-461,  1952.
2  Chang, S, L.,  et al.  "Survey of Free-
      Living Nematodes and Amoebas in
      Municipal Supplies".  J. A.W . W. A.
      _52:613-618,  1960.

3  Chang, S. L.,  "Growth of SmaU Free-
      Living Amoebae in Various Bacterial
      and in Bacteria-Free Cultures".  Can,
      Jour.  Microbiol.  j>:397-405, 1960.

Nematodes

1  Goodey, T., "Freshwater Nematodes",
      1st. Edition, Methuen&Co., London,
      1951.

2  Edmondson, W.T.,  Ed., Ward & Whipple's
      "Fresh-Water  Biology" 10th Edition,
      page 397, 1955.

3  Chang, S. L.,  et al., "Occurrence Of a
      Nematode Worm in a City Water Supply".
      J.A.W.W.A.,  Ł1:671-676,  1959.

4  Chang, S. L.,  etal., "Survival, and
      Protection Against Chlorination, of
      Human Enteric Pathogens in Free-
      Living Nematodes Isolated From Water
      Supplies".  Am. Jour. Trop, Medicine
      & Hygiena.  J): 136-142, 1960.

5  Chang, S. L.,  etal., "Survey of Free-
   Living Nematodes and Amoebas in
   Municipal Supplies".   J.A.W.W.A.,
   52^:613-618,  1960.

6  Chang, S. L.,  "Proposed Method for
      Examination of Water for Free- Living
      Nematodes".   J.A.W.W.A., 5_2:695-698,
      1950.

7  Chang, S. L.,  "Viruses,  Amoebas,
      and Nematodes and Public Water
      Supplies".  J.A.W.W.A., 53:283-296,
      1961.

8  Chang, S. L.,  and Kabler, P.W.,  "Free-
      Living Nematodes in Sewage Effluent
      from  Aerobic  Treatment. Plants". To
	be published.	
This outline was prepared by Shin L.  Chang,
M. D., Chief, Etiology,  Criteria Development
Branch,  Water Supply Research Laboratory,
NERC,  EPA, Cincinnati. OH 45238.

Descriptors: Amoebae, Nematodes
 17-2

-------
                  SUGGESTED CLASSIFICATION OF _SMALL AMOEBAE


Subphylum:     Sarcodina Hertwig and Lesser

   Class:       Rhizopoda von Siebold

      Subclass;    Amoebaea Butschli

        Order:    Amoebida Calkins and Ehrenberg

           Superfamily:   Amoebaceae - free-living
                          (Endamoebaceae - parasitic in animals)

               Family:   Schizopyrenidae  - active limax form common; transcient
                            flagellates present or absent; nucleonus-origin of
                            polar masses; polar caps and interzonal bodies present
                            or absent

                  Genus:    Schizopyrenus - no transcient flagellates; single-walled
                             cysts; no polar caps or interzonal bodies in mitosis

                     Species:    S,,  erythaenusa - reddish orange pigment formed in agar
                                cultures with gram-negative baciliary bacteria

                                j3.  ŁU83eIli - no pigment produced in agar cultures

                  Genus:    Didascalus - morphology and cytology similar to Schizopyrenus^
                               but small numbers of transcient flagellates formed at times

                     Species:    D.  thorntoni - only species described by Singh (1952)

                  Genus:    Naegleria Alexeieff - double-walled cysts; transcient
                             flagellates formed readily; polar caps and interzonal
                             bodies present in mitosis

                     Species:    N.  gruberi (Schardinger) - only species established;
                                   Singh (1952) disclaimed the N. soli_he described in 1951

               Family:   Hartmannellidae  - no transcient flagellate formed; motility
                            sluggish; no Mmax form; nucleolu.3 disappearing, probably
                            forming spindle in mitosis; no polar caps or masses, aster
                            and centrosome not known

                  Genus:    Hartmannella -  ectoplasm clear or less granular than
                               endoplasm; single-walled cysts; single vacuole

                     Species:    H.  glebae - clear ectoplasm
                                H.  _agricola_- ectoplasm less granular than endoplasm

                  Genus:    Acanthamoeba - filamentous processes from ecto-  or
                               endoplasm; growing axenically in fluid bacteriological
                               media
                                                                               17-3

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Suggested Classification of Small Amoebae
                     Species:   A.  rhjrsode_3_

                  Genus:    Slnghella - double-walled cysts; ecto- and endoplastn
                             indistinguishable; many vacuDles

                     Species:   Singhella leptpcnemus
17-4

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                                   ANIMAL PLANKTON
 I  INTRODUCTION

 A Planktonic animals or zooplankton are
   found in nearly every major group of
   animals.

   1  Truly planktonic  species (euplanktcn)
      spend all or most of their active life
      cycle suspended in the water.  Three
      groups are predominantly involved in
      fresh water; the protozoa, rotifers,
      and microcrustacea.

   2  Transient planktonic phases  such as
      floating eggs and cysts,  and  larval
      stages occur in many other groups.

 B Many forms are strictly seasonal in
   occurrence.

 C Certain rare forms  occur in great numbers
   at unpredictable intervals.

 D Techniques of  collection, preservation,
   and identification  strongly influence the
   species reported.

 E In oceanographic work, the zooplankton is
   considered  to include many relatively large
   animals such as siphonophores, ctenophores,
   hepteropods., pteropods, arrowworms, and
   euphausid shrimp.

 F The plant-like  or  phytoplankton on the
   other hand are essentially similar in all
   waters,  and are the nutritional  foundation
   for the animal community.
II   PHYLUM PROTOZOA

 A  The three typically free living classes,
    Mastigophora, Rhizopoda, and Ciliophora,
    all have planktonic representatives.  As
    a group however, the majority of the phylum
    is benthic or bottom-loving.  Nearly any
    of the benthic forms may occasionally be
    washed up into the overlying waters and
    thus be collected along with the euplankton.

 B  Class mastigophora,  the nonpigmented
    zooflagellates.

    These have frequently been confused with
    the phytomastigina or plant-like flagellates.
    The distinction is made here  on the basis
    of the presence or absence of chlorophyll
    as suggested by Palmer and Ingram 1955.
   (Note Figure:  Nonpigmented,  Non-Oxygen
   Producing Protozoan Flagellates in the
   outline Oxygen Relationships.)

   1  Commonly encountered genera

      Bodo

      Peranema

   2  Frequently associated with eutrophic
      conditions

C  Class Rhizopoda - amoeboid protozoans

   1  Forms commonly encountered as
      plankton:
      Chaos

      Arcella

      Difflugia

      Euglypha
(Amoeba)

Centropyxis

Heliozoa
   2  Cysts of some types may be encountered
      in water plants or distribution systems;
      rarely in plankton of open lakes or
      reservoirs.

D  Class Ciliophora

   1  Certain "attached" forms  often found
      floating freely with  plankton:

      Vorticella

      Carchesium

   2  Naked, unattached ciliates.  Halteria
      one of commonest in this group'.   Various
      heavily ciliated forms (holotrichs) may
      occur from time to  time such as
      Colpidium, Enchelys, etc.

   3  Ciliates protected by a shell or test
      (testaceous) are most often recorded
      from preserved samples.  Particularly
      common in the experience of the National
      Water Quality Sampling Network are:

      Codpnella fluviatile

      Codonella cratera

      Tintinnidium (usually with organic matter)

      Tintinnopsis
 BI.AQ.20c. 6. 76
                                    18-1

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     Animal Plankton
III  PHYLUM ROTIFERA

 A Some forms such as Anuraea cochlearis
    and Asplanchna pridonta~tehd to be present
    at all times of the year. Others such as
    Notholca striata, N._ longispina and Poly-
    arthra pTafypTera ar e~ reportecTto be essen-
    tially winter lorms.

 B Species in approximate order of descending
    frequency currently recorded by National
    Water Quality Sampling Network are:

    Keratella cochlearis

    Polyarthra vulgaris

    Synchaeta pectinata

    Brachionus quadridentata

    Trichocerca longiseta

    Rotaria sp.

    Filinia longiseta

    Kellicottia longispina

    Pompholyx sp.

 C Benthic species almost without number may
    be collected with the plankton from time to
    time.


IV  PHYLUM ARTHROPODA

 A Class Crustacea

    1  The Class  Crustacea includes the larger
       common freshwater euplankton. They
       are also the greatest planktonic consum-
       ers of basic nutrients in the form of
       phytoplankton,  and  are themselves the
       greatest planktonic contribution to the
       food of fishes.   Most of them  are herb-
       ivorous. Two groups, the cladocera
       and the copepods are most conspicuous.

    2  Cladocera  (Subclass Branchiopoda,
       Order Cladocera) or Water Fleas

       a  Life History

          1)  During most of the year, eggs
             which will develop without fertil-
             ization (parthenogenetic) are
             deposited by the female in a dorsal
             brood chamber.  Here  they hatch
             into minature adults which escape
             and swim  away.
   2) As unfavorable conditions develop,
     males appear, and thick-walled
     sexual eggs are enclosed in egg
     cases called ephippia which can
     often endure freezing and drying.

   3) Sexual reproduction may occur
     at different seasons in different
     species.

   4) Individuals of a great range of
     sizes, and even ephippia,  are
     thus encountered in the plankton,
     but there is no "larval" form.

b  Seasonal variation - Considerable
   variation may occur between winter
   and  summer forms of the same
   species  in some cases.  Similar
   variation also occurs  between arctic
   and  tropical situations.

c  Forms commonly encountered as
   open water plankton include:

   Bosmina longirostris  and others

   Daphnia galeata and others

   Other less  common genera are:

   Diaphanosoma,  Chydorus, Sida,
   TScroperus/ Ceriodaphnla7 Bytho-
   tr ephe s,~ and" theTcafniyor ous
   LepTocfora and Polyphemus.

d  Heavy blooms of Cladocerans may
   build up in  eutrophic waters.

The copepods  (order Copepoda) are the
perennial microcrustacea of open waters,
both fresh and marine.  They are the
most ubiquitous  of animal plankton.

a  Cyclops is  the genus most often
   TcmricJ by the National Water Quality
   Sampling Network activities.  Eucy-
   clops, Paracyclops. Diaptomus,
   CanthQcamptus, "Epischura,~ and
   Limnocalanus are other forms
   reported TcTEe planktonic.

b  Copepods hatch into a minute char-
   acteristic larvae called a nauplius
   which differs considerably from the
   adults.  After five or  six moults, the
   copepodid stage is reached,  and after
   six more moults, the  adult.   These
   larval stages are often encountered
   and  are  difficult to identify.
    18-2

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                                                                       Animal Plankton
B  Class Insecta

   1  Only a few kinds of insect that can be
      ranked as a truly planktonic.  Mainly
      •the phantom midge fly Chaborus.

   2  The larva of this insect has hydrostatic
      organs that enable it to  remain suspended
      in the water,  or make vertical ascents in
      the water  column.

   3  It is usually benthic during the daytime,
      but ascends to the surface at night.


V  OCCASIONAL PLANKTERS

A  While the protozoa, rotifers,  and micro-
   crustacea make up the bulk of the plankton,
   there  are many other groups as mentioned
   above that may also occur.  Locally or
   periodically these may be of major import-
   ance.  Examples  are given below.

B  Phylum Coelenterata

   1  Polyps of  the genus Hy_dra may become
      detached and float aTJouFnanging from
      the surface film or floating detritus.

   2  The freshwater medusa Craspedacusta
      occasionally appears in lakes or reser-
      voirs  in great  numbers.

C  Phylum Platyhelminthes

   1  Minute Turbellaria (relatives of the
      well known Planaria) are sometimes
      taken  with the  plankton in eutrophic
      conditions. They are often confused
      with ciliate protozoa.

   2  Cercaria larvae of Trematodes (flukes)
      parasitic on certain wild animals,
      frequently appear in  great numbers.
      When  trapped in the droplets of water
      on  a swimmer's skin, they attempt to
      bore in.  Man  not being their natural
      host,  they fail. The resultant irritation
      is called "swimmer's itch".  Some can
      be  identified, but  many  unidentifiable
      sspecies may be found.

   3  In many areas of the world, cercaria
      larvae of human parasites such as the
      blood  fluke Schistosoma japonicum may
      live as plankton,~an3~penefrate"fhe~human
      skin directly on contact.
D  Phylum Nemathelminthes

   1  Nematodes (or nemas) or roundworms
      approach the bacteria and the blue-green
      algae in ubiquity.  They are found in
      the soil and in the water, and in the air
      as dust.  In both marine  and fresh waters
      and  from the Arctic to the tropics.

   2  Although the majority are free living,
      some occur as parasites of plants,
      animals, and man, and some of  these
      parasites are among our most serious.

   3  With this distribution,  it is obvious that
      they will occasionally be encountered as
      plankton.  A more complete discussion
      of nematodes and their public health
      implications in water supplies will be
      found elsewhere  (Chang,  S.  L.).

E  Additional crustacean groups sporadically
   met with in the plankton include the following:

   1  Order Anostraca or fairy shrimps
      (formerly included with the two
      following orders in the Euphyllopoda)
      primarily planktonic in nature.

      a Extremely local and sporadic, but
        when present, may be dominating.

      b Artemia, the  brine  shrimp,  can
        tolerate very high salinities.

      c Very widely distributed,  poorly
        understood.

   2  Order  Notostraca, the tadpole shrimps.
      Essentially southern and western in
      distribution.

   3  Order  Conchostraca,  the clam shrimps.
      Widely distributed, sporadic in occur-
      rence.   Many local species.

   4  Subclass Ostracoda,  the  seed shrimps.
      Up to 3 in.  in length.   Essentially
      benthic but certain species of Cypris,
      and Notodromas  may occur in corisTd"-
      erabTe~numb"ers  as plankton at certain
      times of the year.

   5  Certain members of  the large subclass
      Malacostraca are limnetic,  and  thus,
      planktonic to some extent.

      a The scuds, (order Amphipoda) are
        essentially benthic but are sometimes
        collected in plankton samples around
                                                                                      18-3

-------
 Animal Plankton
         weed beds or near shore.  Nekto-
         planktonic forms include Pontoporeia
         and some species of Gammarus.

      b  The mysid, or opossum shrimps are
         represented among the plankton by
         Mysis relicta, which occurs in the
         deeper waters,  large lakes as far
         north as the Arctic Ocean.

F The Class Archnoidea, Order Hydracarina
   (or Acari) the mites.  Frequent in plankton
   tows near shore although Unionicola crass-
   ipes has been reported to be virtually
   planktonic.

G The phylum Mollusca is but scantily
   represented in the freshwater plankton,
   in contrast to the marine situation.
   Glochidia (ciliated) larvae are occasion-
   ally collected, and snails now and then
   glide  out on a quiet surface film and are
   taken in a plankton net.   An exotic
   bivalve Corbicula has a planktonic
   veliger stage.

H Eggs and other reproductive structures
   of many forms including  fish,  insects, and
   rotifers may be found in  plankton samples.
   Special reproductive structures such as
   the statoblasts of bryozoa and sponges,
   and the ephippia  of cladocerans may also
   be included.

I   Adventitious and Accidental Plankters

   Many  shallow water benthic organisms
   may become accidentally and temporarily
   incorporated into the plankton. Many of
   those  in the preceding section might be
   listed here,  in addition to such forms as
   certain free living nematodes, small
   oligochaetes, and tardigrades, Collembola
   and other surface film livers are also
   taken  at times but should not be mistaken
   for plankton. Fragments and molt skins
   from a variety of arthropods are usually
   observed.

   Pollen from terrestrial or aquatic plants
   is often unrecognized, or confused with
   one of the above.  Leaf hairs from
   terrestrial plants are also confusing to
  the uninitiated, they are sometimes
  mistaken for fungi or other organisms
  (and vice versa).

  In flowing waters, normally benthic
  (bottom living) organisms are often found
  drifting freely in the stream.  This
  phenomenon may be constant or periodic.
  When included in plankton collections,
  they must be reported, but recognized
  for what they are.
    Surfuc'' films are especially rich in
    micro ''biological garbage" and these
    enrich the plankton.

REFERENCES

1  Edmondson, W. F., ed.  Ward and
      Whipples's Freshwater Biology,  2nd
      Edition, Wiley & Sons, Inc., New York.
      1959.

2  Hutchinson, G. Evelyn.   A Treatise on
      Limnology.  Vol. 2.   Introduction to
      Lake Biology and the Limnoplankton.
      Wiley.   1115 pp.  1967.

3  Lackey, J. B.   Quality and Quantity of
      Plankton in the South End of Lake
      Michigan in 1952.  JAWWA.
      36:669-74.   1944.

4  McGauhey, P.H.,  Eich,  H.F., Jackson,
      H. W.,  and Henderson, C.  A Study
      of the Stream Pollution Problem in the
      Roanoke, Virginia, Metropolitan
      District.  Virginia Polytech. Inst.,
      Engr. Expt. Sta.

5  Needham,  J. G. and Lloyd, J.T.   The
      Life of Inland Waters.   Ithaca,  New
      York, Comstock Publishing Co., Inc.
      1937.

6  Newell,  G.E.  and Newell, R. C.
      Marine Plankton.  Hutchinson Educ.
      Ltd.   London.   221pp.   1963.

7  Palmer, C.M. and Ingram,  W.M.
      Suggested Classification of Algae and
      Protozoa in Sanitary Science.
      Sew.  & Ind. Wastes.  27:1183-88.
      1955.
  18-4

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                                                                      Animal Plankton
8  Penriak, R.W.   Freshwater Invertebrates      10  Welch, P.S.   Limnology, McGraw-Hill
     d:f the United States. The Ronald Press,           Book Co., Inc., New York.   1935.
     New York.   1953.

9  Sverdrup, H. W., Johnson, M.W., and
     Fleming,  R.H.  The Oceans,  Their
     Physics, Chemistry and General             This outline was prepared by H. W. Jackson,
     Biology.   Prentice-Hall, Inc., New York.    former Chief Biologist, National Training
     1942.                                      Center,  and revised by R. M. Sinclair,
                                                Aquatic  Biologist, National Training Center
                                                MOTD,  OWPO, USEPA, Cincinnati, Ohio
                                                45268.

                                                Descriptor:  Zooplankton
                                                                                18-5

-------
      Animal Plankton
       3/k
       Phylum PROTOZOA

Free Living Representatives
     I. Flagellated Protozoa, Class Hastigophora
    Ahthophysis
Pollution tollerant
    Pollution tollerant
           19/1
                                                    Colony of Poteriodendron
                                                    Pollution tollerant, 35/1
     II. Ameboid Protozoa, Class  Saroodina
   Pimastigamoeta
 Pollution tollerant
       10-50/j
     Nuelearia.reported
    to be intollerant of
    pollution, 45 /*•
     III. Ciliated Protozoa, Class Ciliophora
      Colpoda
Pollution tollerant
     20-120 /i
      Holophrya.reported
     to be intollerant of
     pollution, 35 >»
        Difflugia
    Pollution tollerant
                    60-500/,
 EpistyliB. pollution
tollerant. Colonies often
naorosoopio.
                                                        H.W.Jaokson

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                                                     Animal Plankton
                        PLANKTONIC PROTOZOA
                        Peranema trichophorum
                Top
                Side
 Chaos
Arcella
vulgaris
Actinosphaerium
Vorticella
                           Codonella
                           cratera
                                Tintinnidium
                                fluviatile

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     Animal Plankton
                                     PLANKTONIC ROTIFERS

                            Various Forms of Keratella cochlearis
                     Synchaeta
                     pectinata
Polyarthra
vulgaris
                                         Rotaria sp
Brachionus
quadridentata
18-8

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                                                                Animal Plankton
                       SOME PLANKTONIC CRUSTACEANS
                                CRUSTACEANS
Copepod; Cyclops. Order Copepoda
             2-3 mm
            Water Flea;
             Daphnia
                                                A Nauplius larva of a Copepod
                                                          1-5 mm
Order Cladocera
                                 2-3 mm
                                 OSTRACODE
            Left:  Shell closed           Right: Appendages extended

                                   1-2 mm

-------
  Animal Plankton
                           PLANKTONIC ARTHROPODA
    A mysid shrimp - crustacean
A water mite - arachnid
                              Chaoborus midge larva - Insect
               Aspects in the life cycle of the human tapeworm
               Diphyllobothrium latum, class Cestoda.  A.  adult as inhuman
               intestine; B. procercoid larva in copepod; C.  plerocercoid
               larva in flesh of pickerel (X-ray view).
                                                            H.W. Jackson
10

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                       TECHNIQUES OF PLANKTON SAMPLING PROGRAMS
 I  INTRODUCTION

 A  A plan is necessary.  "If you fail to plan,
    you are planning to fail. "  Overall objec-
    tives, integration with other survey units,
    statistical design.

 B  A planned program of plankton analysis
    should involve periodic sampling at weekly
    intervals or more  often.

    1  Most interference organisms are small,
       and hence have  relatively short-life
       histories.

    2  Populations of such organisms may
       fluctuate  rapidly in response to chang-
       ing water, weather, or seasons.

    3  Seasonal  growth patterns of plankton
       tend to repeat themselves from year to
       year, thus they are relatively predictable.

 C  A well-planned study or analysis of the
    growth pattern of plankton in one year will
    provide a basis for predicting conditions
    the following year.

    1  Since the seasons and the years differ,
       the more records are accumulated, the
       more useful can they become.

    2  As the time for an anticipated bloom of
       some troub.le maker approaches,  the
       frequency of analysis may be increased.

 D  Detection of a bloom in its early stages
    will facilitate more economical control.

II   FIELD ASPECTS OF THE ANALYSTS
    PROGRAM

 A  Two general aspects of sampling are com-
    monly recognized:  quantitative and
    qualitative.

    1  Qualitative examination tells what is
      present.

    2  Quantitative tells how much.
    3  Either approach is useful,  a combination
      is best.

    BI. MIC.enu. 9h. 6.  76
B  Equipment of collecting samples in the
   field is varied.

   1  A half-liter bottle will suffice for sur-
      face samples of phytoplankton if carefully
      taken.  If zooplankton also are of interest,
      2 or more liters should be collected.
     (See below).

   2  Plankton nets concentrate the sample
      in the act of collecting, and capture
      certain larger forms which escape from
      the bottles.  Only the more elaborate
      types are quantitative however,

   3  A kemmerer-type sampler is suggested
      for depth samples.

   4  Other methods such as the Clark-Bumpus
      sampler or the Juday plankton trap may
      be employed for  special purposes.

C  The location of sampling points is
   important

   1  Both shallow and deep samples are
      suggested.

      a  "Shallow" samples should be taken
         at a depth of 6 inches to one foot.

      b  "Deep" samples should be taken at
         such intervals as the depth of the
         reservoir permits.  There should be
         at least one open water sampling
        point.

      c Each major bay or shoal area should
         have at least one sampling point.

      d Additional sampling stations should
        be established on the basis of ex-
        perience and resources.

      e Samples may  be composited if nec-
        essary to give an overall summary of
         conditions.  Such summaries are  not
         advised and should be interpreted
        with care.
                                     19-1

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  Techniques of Plankton Sampling Programs
          A standardized vertical haul however,
          can be useful for routine  comparisons.
Ill  FACTORS WHICH INCLUENCE SAMPLING
    AND DATA COLLECTION

 A Physical Features

    1  Temperature

       a  Lakes are warmed in spring princi-
          pally by the action of wind forcing
          the warmer water down into the
          cooler water against the forces of
          gravity.

       b  Thermal stratification

    2  Turbidity

    3  Color

    4  Water movement

    5  Light penetration

       a  A factor of turbidity, color, biolog-
          ical activity, and time of day

          (1) Effective length of daylight
             diminishes  with the depth of
             the lake.

    6  Wind velocity and direction

    7  Bottom materials

    8  Size, shape,  and  slope  of lake basin

 B Chemical Factors

    1  Alkalinity, pH, and dissolved minerals
       excluding nitrogen and phosphorus

    2  Dissolved oxygen

       a  From photosynthesis in sunlight

       b  From contact  of lake surface with
          the air

       c  Fluctuates seasonally because  of
          temperature and biological activity,
          and diurnally because of biological
          activity.
     3  Nutrients for biological growth -
        especially nitrogen and phosphorus

        a  A given body of water will produce
           a given quanity of aquatic life.
           Biological production is determined
           primarily by the nutrients in solu-
           tion in the water, and an increase
           in basic fertility will increase
           biological activity.

        b  Basic suppliers of nutrients
           include tributary streams,  precip-
           itation from the atmosphere, and
           interchange with lake bottom sedi-
           ments .
IV  FIELD PRESERVATION OF SAMPLES

 Provision should be made for the field
 stabilization of the sample until the laboratory
 examination can be made.  Techniques and
 materials are listed below.  No "ideal" pre-
 servative or technique has yet been developed
 each has its virtues.

 A Refrigeration or icing.  The container
    containing the sample can be cooled, but
    under no circumstances should ice be
    dropped into the sample.

 B Preservation by 3-5% formalin  is time-
    tested and widely used.   Formalin  shrinks
    animal tissue,  fades colors, and makes all
    forms brittle.

 C Ultra-violet sterilization is useful in the
    laboratory to retard decomposition of
    plankton.

 D Lugol's solution is often used.


   E A special merthiolate preservative
     developed by the FWPCA Water Pollution
     Surveillance System which has proved very

     satisfactory and is described in reference
     No. 9.
      19-2

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                                                  Techniques of Plankton Sampling Programs
V   SUMMARY AND CONCLUSIONS

 A  The field sampling program should be
    carefully planned to  evaluate all signifi-
    cant locations in the  reservoir or stream,
    giving due consideration to the capacity
    of the laboratory.

 B  Adequate  records and notes should be
    made of field conditions and associated
    with the laboratory analyses in a permanent
    file,

C  Once a procedure for processing plankton
   is adopted, it should  be  used exclusively
   by all workers at the plant.

D  Such a procedure should enable the water
   plant operator to prevent plankton troubles
   or at least to anticipate them and have
   corrective materials  or equipment
   stockpiled.

 ACKNOWLEDGMENT:

 Portions of this outline were prepared by
 K. M. Mackenthun, Biologist, formerly with
 Technical Advisory and Investigation Activities,
 FWPCA,  SEC, Cincinnati, Ohio.

REFERENCES

1  APHA. Standard Methods for the
       Examination of Water and Wastewater
       14-th Ed, NY,   1976.
2  Hutcheson, George E.  A Treatise on
      Limnology.  John Wiley and Co.  New
      York. 1957.

 3  Jackson,  H. W. Biological Examination
      (of plankton) Part III in Simplified
      Procedures for Water Examination.
      AWWA Manual M12.  Am. Water
      Works Assoc.,  N.Y. 1964.

4  Lackey,  J. B.  The Manipulation and
      Counting of River Plankton and Changes
      in Some Organisms Due to Formalin
      Preservation.  Public Health Reports
     ,53: 2080-93.  1938.

5  Mackenthun, K. M.,  Ingram,  W. M.,
      and Ralph Forges.  Limnological
       Aspects of Recreation Lakes, DHEW,
       PHS Publication No. 1167, 1964.

 6  Olson, Theodore A. and Burgess,  Fred-
       erick J.  Pollution and Marine Ecology.
       Interscience Publishers.   364 pp. 1967.

 7  Palmer, C. M. Algae in Water Supplies.
       U. S, Department of Health, Education,
       and Welfare, Public Health Service
       Publication No. 657,  Superintendent of
       Documents, Washington 25, D. C.

 8  Schwoerbel, J. Methods of Hydrobiology
       (Freshwater Biology).  Pergamon
       Press,   1970,

 9  Websr,  C. I.  Methods of Collection and
       Analysis of Plankton and Periphyton
       Samples in the Water Pollution
       Surveillance System.   App. and Devel.
       Rep. (AQCLab.,  1014 Broadway,
       Cincinnati,  OH 45202) 19 pp  1966.

10  Welch, P. S.   Limnological Methods.
       The Blakiston Co., Phila.  Toronto.
       1948.

11  Williams,  L.  G.  Plankton Population
       Dynamics, in National Water Quality
       Network, Supplement 2, U.S..  PHS
       Pub. No. 663.  1962.
This outline was prepared by H. W. Jackson,
former Chief Biologist, National Training
Center, and revised by R. M. Sinclair,
Aquatic Biologist, National Training Center,
MOTD. OWPO. USEPA, Cincinnati, Ohio 45268.

Descriptor: Plankton
                                                                                     19-3

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        PREPARATION AND  ENUMERATION  OF PLANKTON IN THE LABORATORY
 I    RECEPTION  AND PREPARATION  OF
     SAMPLES

 A   Preliminary sampling and analysis is  an
     essential preliminary to the establish-
     ment of a permanent or semi-permanent
     program,

 B   Concentration or  sedimentation  of  pre-
     served samples may precede analysis.

     1   Batch centrifuge

     2   Continuous centrifuge

     3   Sedimentation

 C   Unpreserved (living) samples should be
     analyzed at once or refrigerated  for
     future analysis.

II    PREPARATION OF MERTHIOLATE
     PRESERVATIVE

 A   The Water Pollution Surveillance  System
     of the FWPCA  has  developed a modified
     merthiolate preservative.   (Williams,
     1967)  Sufficient stock to  make an approx-
     imately  3.5% solution in the bottle when
     filled is placed in the sample bottle in
     the laboratory.   The bottle is then filled
     with water in the field and returned to
     the laboratory for analysis.

 B   Preparation of  Merthiolate Preservative

     1   Merthiolate  is available from many
        chemical  laboratory supply houses;
        one  should specify the water soluble
        sodium salt.

     2   Merthiolate  stock:  dissolve approxi-
        mately 1. 5 gram  of sodium borate
        (borax and approximately 1 gram of
        merthiolate  in 1  liter  of  distilled water.
       The  amount of sodium borate and
       merthiolate may be varied slightly to
       adjust  to different  waters,  climates,
       and organic contents.

    3  Prepare a  saturated aqueous Lugol1 s
       solution as  follows:

       a  Add 60  grams of potassium iodide
          (KI) and 40 grams of iodine crystals
          to 1 liter of distilled water.

    4  Prepare the preservative solution by
       adding approximately  1. 0 ml of the
       Lugol's solution  to  1  liter of merthi-
       olate stock.
Ill  SAMPLE  ANALYSIS
    Microscopic examination is most  frequently
    employed in the  laboratory to  determine
    what plankton organisms are present  and
    how many there  are:

    1   Optical equipment  need not  be elaborate
        but should include.

        a   Compound microscope with the
           following equipment:

           1)   Mechanical stage
           2)   Ocular:  10X,  with Whipple  type
               counting eyepiece or reticule
           3)   Objectives:
                 approx.   10X(16mm)
                 approx.   20X(8 mm)
                 approx.   40X(4 mm)
                 approx.   95X(1.8 mm)(optional)

           A  40X objective with a working
           distance  of 12.8 mm and an erect
           image may be obtained as special
           equipment.   A  water immersion
           objective (in addition to  oil) might
          be  considered  for use with  water
           mounts.

           Binocular  eyepieces  are  optional.
   BI. MIC.enu.15f. 6. 76
                                                                                      20-1

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Preparation and  Enumeration  of Plankton
          Stage micrometer (this may be
          borrowed,  if necessary,  as it is
          usually  used only once,  when the
          equipment  is calibrated)

       b  Inverted microscopes  offer certain
          advantages but are not widely
          available.   The same  is  true of
          some of the newer optical  systems
          such as phase  contrast microscopy.
          These are  often excellent but ex-
          pensive  for routine plant use.

   2   Precision made counting chambers
       are required for quantitative work with
       liquid mounts.

       a  Sedgwick-Rafter cells  (hereafter
          referred to as S-R cell)  are used
          for routine  counts of medium and
          larger forms.

       b  Extremely  small forms or "nanno-
          plankton" may  be  counted by use of
          the nannoplankton (or  Palmer) cell,
          a Fisher- Littman cell, a hemacyto-
          meter,  the  Lackey drop  method,  or
          by use of an inverted  microscope.

   3   Previous to starting serious analytical
       work, the microscope should be cali-
       brated as described elsewhere.  Di-
       mensions of the S-R  cell should also
       be checked, especially the depth.

   4   Automatic particle counters  may be
      useful for coccoid organisms.


B  Quantitative Plankton  Counts

   1   All quantitative counting techniques
      involve the  filling  of a standard cell
      of known dimensions with either
      straight  sample or a  concentrate or
      dilution thereof.

   2  The organisms in  a  predetermined
      number of microscope fields or other
      known area are then observed,  and by
      means of a suitable  series of  multi-
      plier factors,  projected to a number or
      quantity  per ml gallon,  etc.
       Direct  counting of the unconcentrated
       sample eliminates manipulation,  saves
       time,  and reduces error.   If frequency
       of organisms is low,  more area may
       need to be  examined  or  concentration
       of the  sample may be in order.

       Conventional  techniques  employing
       concentration of the  sample provide more
       organisms for observation,  but because
       they involve more manipulations,  intro-
       duce additional errors and take more
       time.
C  Several methods of counting  plankton are
   in general use.

   1  The numerical  or  clump count is
      regarded as the simplest.

      a   Every organism observed  must be
          enumerated.  If it  cannot  be  identi-
          fied,  assign a symbol or number
          and make a sketch  of  it on the back
          of the record sheet.

      b   Filaments, colonies and other
          associations of cells are counted
          as  units,  equal to single isolated
          cells.  Their identity  as indicated
          on  the record sheet is the  key to
          the significance  of  such a count.

   2  Individual cell count.   In this  method,
      every  cell of every colony  or  clump
      of organisms is counted,  as well as
      each individual single-celled organism.

   3  The areal standard unit method offers
      certain technical advantages,  but also
      involves certain inherrent difficulties.

      a   An areal standard unit is  400 square
          microns.  This  is the area of one
          of the smallest subdivided  squares
          in the center of the Shipple eyepiece
          at a  magnification of 100X.

      b  In operation,  the number of areal
         units  of  each species is recorded on
         the record sheet rather  than the
         number of individuals.    Average
         areas of the common species  are
  20-2

-------
                                                 Preparation and  Enumberation of  Plankton
      are  sometimes printed  on record
      sheets for a particular plant  to
      obviate the necessity of estimating
      the area  of each  cell observed
      individually.

   c  The  advantage of the method  lies in
      the cognizance taken  of the relative
      masses of the various species as
      indicated  by the area presented to
      the viewer.   These areas,  however,
      are often  very difficult  to  estimate.

4  The  cubic standard unit  method is a
   logical  extension  of the areal method,
   but has achieved  less acceptance.

5  Separate field  count

   a  In counting separate fields, the
      question always arises  as to  how
      to count organisms touching or
      crossed by the edge of the Whipple
      field.   Some  workers estimate the
      proportion of the  organism lying
      inside the field as compared  to that
      outside.   Only those  which are over
      half  way  inside are counted.

   b  Another system is to select two
      adjacent, sides of the  square for
      reference,  such as the top and left
      boundaries.   Organisms touching
      these lines in any degree, from
      outside or inside,  are then counted,
      while organisms touching the  opposite
      sides are ignored.   It is important
      to adopt some such system and
      adhere  to it consistently.

   c  It is  suggested that if separate
      microscopic fields are  examined,
      a  standard number of ten be  adopted.
      These should be evenly spaced in two
      rows about one-third  of  the distance
      down from the top and one-third of
      the distance up from  the bottom of
      the S-R cell.

6  Multiple area count.   This  is an ex-
   tension  of the separate  field count.
   A  considerable increase  in  accuracy
   has recently  been shown to accrue by
   emptying  and refilling the  S-R cell,
D
       after each group of fields are  counted
       and making up to 5  additional such
       counts.   This may not be practical with
       high counts.

       The strip count.   When a rectangular
       slide such as the  S-R cell is used, a
       strip (or  strips) the  entire length (or
       known portion thereof) of the  cell may
       be counted instead  of separate isolated
       fields.   Marking the bottom  of the cell
       by evenly spaced cross  lines  as  ex-
       plained  elsewhere greatly facilitates
       counting.

       a  When the  count  obtained is  multi-
          plied by the ratio of the width of
          the  strip  counted to the width of
          the  cell,  the product is the esti-
          mated  number  of organisms in  the
          cell, or per ml.

       b  When the  material in the  cell is
          unconcentrated sample water,  this
          count represents the  condition of
          the  water being evaluated without
          further calculation.

       Survey count.  A  survey count is an
       examination of the  entire area of a
       volumetric cell using a  wide field  low
       power microscope.   The objective  is
       to locate  and record the larger forms,
       especially zooplankton such as copepods
       or large rotifers which  may  be present
       in size.   Special large capacity cells
       are often  employed  for this purpose.
       For still  larger marine  forms,  numerous
       special  devices have been created.

       Once a  procedure for concentration
       and/or counting is  adopted by a plant
       or other organization it  should be
       used consistently from then on so
       that results from year to year can be
       compared.
Differential or qualitative "counts" are
essentially lists  of the kinds of organisms
found.
                                                                                    20-3

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Preparation and  Enumeration  of Plankton
E   Proportional  or relative counts of special
    groups are often very useful.   For ex-
    ample, diatoms.   It is best to always
    count  a standard numbers of cells.

F   Plankton  are  sometimes measured by
    means other than microscopic counts.

    1  Settled volume of killed plankton in an
      Imhoff cone  may be observed after  a
      standard length of time.   This will
      evaluate primarily only the larger  forms.

    2  A gravimetric method  employs drying
      at  60°  C for  24 hours  followed  by
      ashing  at 600° C for 30  minutes.   This
      is  particularly useful for chemical
      and radiochemical analysis.

    3  Chemical and physical  evaluation of
      plankton populations employing various
      instrumental techniques are coming to
      be widely used.  Both  biomass  and
      productivity  rates can be measured.
      Such determinations probably achieve
      their greatest utility when coordinated
      with microscopic examination.

   4  The membrane (molecular) filter has
      a great potential,  but a generally
      acceptable  technique  has yet  to  be
      perfected.

         a Bacteriological techniques for
           coliform determination are
           widely accepted.

         b Nematodes and larger organisms
           can readily  be washed off of  the
           membrane after filtration.

         c It is  also being used to measure
           ultraplankton that pass treatment
           plant operations.

         d Membranes  can be cleared and
           organisms deposited thereon
           observed directly,  although
           accessory staining is desirable.
        e  Difficulties include a predilec-
           tion of extremely fine membranes
           to  clog rapidly with silt  or
           increase  in plankton counts,  and
           the difficulty of making  observations
           on individual cells when  the
           organisms are piled on  top of each
           other.   It is sometimes  necessary
           to  dilute  a sample to  obtain  suitable
           distribution.


IV  SUMMARY AND CONCLUSIONS

A  The field sampling program  should be
    carefully  planned to  evaluate all significant
    locations  in the reservoir or stream,
    giving due consideration to the  capacity
    of the laboratory.

B  Adequate  records and notes should  be
    made of field conditions  and  associated
    with the laboratory analyses  in a
    permanent file.

C  Optical  equipment in the laboratory should
    be calibrated.

D  Once  a  procedure for processing plankton
    is adopted,  it should be  used exclusively
    by all workers at the plant.

E  Such a procedure should enable the water
    plant  operator to prevent plankton troubles
    or at least to anticipate  them and have
    corrective materials  or  equipment stockpiled.
REFERENCES

1  Ely Lilly Company.   Merthiolate as a
       Preservative.   Ely Lilly & Co.
       Indianapolis 6,  Indiana.

2  Gardiner,  A. C.   Measurement of
       Phytoplankton Population by the
       Pigment Extraction Method.   Jour.
       Marine Biol.  Assoc.  ^5(4):739-744.  1943.

3  Goldberg,  E. D.,  Baker, M.,  and Fox,  D. L.
       Microfiltration  in  Oceanographic
       Research Sears Foundation.   Jour.
       Mar. Res.   11:194-204.   1952.
 20-4

-------
                                               Preparation and Enumeration of Plankton
Ingram,  W. M., and Palmer, C.  M.
  Simplified Procedures for  Collecting,
  Examining,  and Recording  Plankton
  in Water.   Jour.   AWWA.   44(7):
  617-624.   1952.

Jackson, H. W. Biological Examination
  (of plankton) Part III in Simplified
  Procedures for Water Examination.
  AWWA Manual M 12.   Am. Water
  Works Assoc.  N. Y.  1964.

Lund,  J.  W.  G.,  and Tailing,  J.  F.
  Botanical Limnological Methods  with
  Special References to the Algae.
  Botanical Review.  Ł3(8&9):489-583.
  October,  1957.
7  Weber,  C. I.  The Preservation of
      Plankton  Grab Samples.   Water
      Pollution Surveillance Systems,
      Applications and Development Report
      No.  26, USDI,  FWPCA,  Cincinnati,
      Ohio.    1967

8  Williams,  L.  G. Plankton Population
      Dynamics.   National Water Quality
      Network Supplement  2.   U. S. Public
      Health  Service  Publ. No. 663.  1962

9  Wohlschag,  D. D.,  and Hasler, A.  D.
      Some Quantitative Aspects of  Algal
      Growth in Lake  Mendota.  Ecology.
      32(4):581-593.    1951
                                             'This outline was prepared by H. W. Jackson,
                                              former Chief Biologist,  National Training
                                              Center,  and revised by R. M. Sinclair,
                                              Aquatic Biologist, National Training Center,
                                              MOTD, OWPO, USEPA, Cincinnati, Ohio 45268

                                              Descriptor:  Plankton
                                                                                  20-5

-------
                                   ATTACHED GROWTHS
                                  (Periphyton or Aufwuchs)
I  The community of attached microscopic
plants and animals is frequently investigated
during water quality studies.  The attached
growth community (periphyton) and suspended
growth community (plankton) are the principal
primary producers in waterways--they con-
vert nutrients to organic living materials and
store light originating energy through the
processes of photosynthesis.  In extensive
deep waters, plankton is probably the pre-
dominant primary producer. In shallow lakes,
ponds, and rivers,  periphyton is the predominant
primary producer.   During the past two
decades, investigators of microscopic
organisms have increasingly placed emphasis
on periphytic growths because of inherent
advantages over the  plankton when interpreting
data from surveys on flowing waters:

A  Blum (1956) ". . .  .workers are generally
   agreed that no distinctive association of
   phytoplankton is found in streams, although
   there is some  evidence  of this for individual
   zooplankters (animals) and for a few
   individual algae and bacteria.  Plankton
   organisms are often introduced into the
   current from impoundments,  backwater
   areas or stagnant arms of the stream....
   Rivers whose plankton is not  dominated by
   species from upstream  lakes or ponds are
   likely to exhibit a majority of forms which
   have been derived from the stream bottom
   directly and which are thus merely
   facultative or opportunistic plankters. "

B  "The transitory nature of stream plankton
   makes it nearly impossible to ascertain at
   which point upstream agents producing
   changes in the algal population were
   introduced, and whether the changes
   occurred at the sampling site  or at some
   unknown point upstream.  In contrast,
   bottom algae (periphyton) are true com-
   ponents  of the  stream biota.  Their
   sessile-attached mode of life subjects
   them to the quality of water continuously
   flowing over them.  By  observing the
   longitudinal distribution of bottom algae
   within a stream,  the sources of the agents
   producing the change can be traced
   (back-tracked)" (Keup,  1966).
II   TERMINOLOGY

 A  Two terms are  equally valid and commonly
    in use to describe the attached community
    of organisms.   Periphyton literally means
     around plants  such as the growths over-
    growing pond-weeds; through usage this
    term means  the attached film of growths
    that rely on  substrates as a "place-to-
    grow" within a waterway. The  components
    of this growth assemblage consists of
    plants, animals, bacteria,  etc.  Aufwuchs
    is an equally acceptable term [probably
    originally  proposed by Seligo (1905)] .
    Aufwuchs is  a German noun without
    equivalent english translation; it is
    essentially a collective term  equivalent
    to the above  American (Latin root) term -
    Periphyton.  (For convenience,  only,
    PERIPHYTON,  with its liberal  modern
    meaning will be used in this outline.)

 B  Other terms, some rarely encountered in
    the literature,  that  are essentially
    synonymous  with periphyton or  describe
    important  and dominant components  of the
    periphytic community are:  Nereiden,
    Bewuchs,  Laison,  Belag, Besatz,  attached,
    sessile, sessile-attached, sedentary,
    seeded-on, attached materials,  slimes,
  » slime-growths, and coatings.

    The academic community occasionally
    employs terminology based on the  nature
    of the substrates the periphyton grows on
    (Table 1).

              TABLE  1

      Periphyton Terminology Based
          on Substrate  Occupied
 Substrate                 Adjective
 various         epiholitic, nereiditic,  sessile
 plants          epiphytic
 animals        epizooic
 wood           epidendritic, epixyIonic
 rock           epilithic

[After Srameck-Husek( 1946) and via Sladeckova
 (1962)] Most above listed latin-root adjectives
 are derivatives of nouns  such as epihola,
 epiphyton, spizoa,  etc.
 BI.MIC.enu. 19b.7.76
                                       21-1

-------
 Attached Growths (Periphyton or Aufwuchs)
III  Periphyton, as with all other components
 of the environment,  can be sampled quali-
 tatively (what is present) and quantitatively
 (how much or many  are present).

 A Qualitative sampling can be performed by
    many methods and may extend from direct
    examination of the growths attached to a
    substrate to unique "cuttings" or scrapings.
    It may also be a portion of quantitative
    sampling.

 B Quantitative sampling is difficult because
    it is nearly impossible to remove the
    entire community from a standardized or
    unit area of substrate.

    1  Areas scraped cannot be determined
       precisely enough when the areas are
       amorphous plants, rocks or logs  that
       serve as the principal periphyton
       substrates.

    2  Collection of the entire  community within
       a standard area usually destroys  individual
       specimens thereby making identification
       difficult (careful scraping can provide
       sufficient intact individuals of the species
       present to make qualitative determinations);
       or the process of collection adds  sufficient
       foreign materials (i. e.  detritus,  sub-
       strate, etc. ) so that some commonly
       employed quantitative procedures are
       not applicable.
IV  Artificial substrates are a technique
 designed to overcome the problems of direct
 sampling.   They serve their purpose, but
 cannot be used without discretion.  They are
 objects standardized as to surface  area,
 texture, position, etc. that are placed  in the
 waterway for pre-selected time periods during
 which periphytic growths accumulate.  They
 are usually made of inert materials, glass
 being most common with plastics second in
 frequency.  Over fifty various devices  and
 methods of support or suspension of the
 substrates have been devised (Sladeckova,
 1962) (Weber,  1966) (Thomas, 1968).
 V  ARTIFICIAL SUBSTRATE PLACEMENT

 A Position or Orientation

    1  Horizontal - Includes effects of settled
       materials.

    2  Vertical - Eliminates many effects of
       settled materials.

 B Depth (light) - A substrate placed in lighted
    waters may not reflect conditions in a
    waterway if much  of the natural substrate
    (bottom) does not receive light or receives
    light at reduced intensity.  (Both excessive
    light and a shortage of light can inhibit
    growths and influence the kinds of organisms
    present. )

 C Current is Important

    1  It can prevent the settling of smothering
       materials.

    2  It flushes metabolic wastes away and
       introduces nutrients to the colony.
VI  THE LENGTH OF TIME THE SUBSTRATE
    IS EXPOSED IS IMPORTANT.

 A  The growths need time to colonize and
    develop on the recently introduced
    substrate.

 B  Established growths may intermittently
    break-away from the substrate because
    of current or weight induced stresses, or
    "over-growth" may "choke" the attachment
    layers (nutrient,  light, etc. restrictions)
    which then weaken or die allowing release
    of the mass.

 C  A  minimum of about ten  days is required
    to produce sufficient growths on an
    artificial substrate;  exposures exceeding
    a longer time than 4-6 weeks may produce
    "erratic results" because of sloughing or
    the accumulation of senile growths in
    situations where the substrate is
    artificially protected from predation and
    other environmental stresses.
  21-2

-------
                                                      Attached Growths (Periphyton or Aufwuchs)
 VII   Determining the variety of growths present
   is presently only practical with microscopic
   examination.  (A few micro-chemical pro-
   cedures for differentiation show promise--
   but, are only in the early stages of development. )
VIII   DETERMINING THE QUANTITY OF
      GROWTH(S)

   A  Direct enumeration of the growths while
      attached to the substrate can be used, but
      is restricted to the larger organisms
      because (1) the problem of maintaining
      material in an acceptable  condition under
      the short working distances of the objective
      lenses on compound microscopes, and
      (2) transmitted light is not adequate
      because of either opaque substrates and/or
      the density of the colonial growths.

   B  Most frequently, periphyton is scraped
      from the substrate and then processed
      according to several available procedures,
      the selection being based on the need, and
      use of the  data.

      1  Aliquots of the sample may be counted
        using methods frequently employed in
        plankton analysis.

        a  Number  of organisms

        b  Standardized units

        c  Volumetric units

        d  Others

      2  Gravimetric

        a  Total dry weight of scrapings

        b  Ash-free dry weight (eliminates
           inorganic sediment)

        c  A  comparison of total and ash-free
           dry weights

      3  Volumetric, involving centrifugation of
        the scrapings to determine a packed
        biomass volume.
    4  Nutrient analyses serve as indices of
       the biomass by measuring the quantity
       of nutrient incorporated.

       a Carbon

         1) Total organic carbon

         2) Carbon equivalents (COD)

       b Organic nitrogen

       c Phosphorus - Has limitations
         because cells can store excess
         above immediate needs.

       d Other

    5  Chlorophyll and other bio-pigment
       extractions.

    6  Carbon-14 uptake

    7  Oxygen production,  or respiratory
       oxygen demand


EK  EXPRESSION OF RESULTS

 A Qualitative

    1  Forms found

    2  Ratios of number per group found

    3  Frequency distribution of varieties
       found

    4  Autotrophic  index (Weber)

    5  Pigment diversity index (Odum)
 B Quantitative
    1  Areal basis--quantity per square
       inch, foot,  centimeter,  or meter.
       For example:
       a 16 mgs/sq. inch
       b 16,000 cells/sq. inch

    2  Rate basis.  For example:

       a 2 mgs/day, of biomass accumulation
       b 1 mg O2/mg of growth/hour
                                                                                     21-3

-------
Attached Growths (Periphyton or Aufwuchs)
REFERENCES

1  Blum,  J. L.  The Ecology of River Algae.
      Botanical Review.  22:5:291.   1956.

2  Dumont,  H. J.  A Quantitative Method for
      the Study of Periphyton.   Limnol.
      Oceanogr.   14(2):584-595.

3  Keup, L.E.  Stream Biology for Assessing
      Sewage Treatment Plant Efficiency.
      Water and Sewage Works.  113:11-411.
      1966.

4  Seligo, A.  Tiber den Ursprung der
      Fischnahrung.  Mitt.  d.  Westpr.
      Fisch. -V.  17:4:52.   1905.

5  Sladeckova, A.   Limnological Investigation
      Methods for the Periphyton Community.
      Botanical Review.   28:2:286.  1962.

6  Srameck-Husek.  (On the Uniform
      Classification of Animal and Plant
      Communities in our Waters).
      Sbornik MAP 20:3:213. Orig. in
      Czech.  1946.
 7  Thomas,  N.A.   Method for Slide
       Attachment in Periphyton Studies.
       Manuscript. 1968.

 8  Weber, C.I.  Methods of Collection and
       Analysis of Plankton and Periphyton
       Samples in the Water Pollution
       Surveillance System.   Water Pollution
       Surveillance System Applications and
       Development Report No. 19,  FWPCA,
       Cincinnati.  19-lTpp. (multilith).  1966.

 9  Weber, C.I.  Annual Bibliography
       Midwest Benthological Society.
       Periphyton.  1014 Broadway,
       Cincinnati,  OH 45202.

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

11  Spoon, Donald M.  Microbial Communities
       of the Upper Potomac Estuary: The
      Aufwuchs in.- The Potomac Estuary,
       Biological Resources,  Trends and
       Options.  1CPRB  Tech Pub.  No. 76-2,
       1976.

 This outline was  prepared by Lowell E. Keup,
 Chief, Technical Studies Branch, Division of
 Technical Support, EPA, Washington,  DC 20242.

 Descriptor:  Periphyton
  21-4

-------
                     DETERMINATION OF PLANKTON PRODUCTIVITY
  I  INTRODUCTION

  Primary production is the synthesis of organic
  matter from inorganic raw materials.  The
  energy required for this process may come
  from light (photosynthesis), or from  chemical
  sources (chemosynthesis).  The primary
  synthesis of organic matter in lakes  and
  streams is carried on by planktonic and ben-
  thic algae  and bacteria, and aquatic
  macrophytes.
 II  PHOTOSYNTHESIS

 The photosynthetic process involves the up-
 take of CO2 and the release of O2.  The
 reactions are enzyme catalyzed and are af-
 fected by the following factors:

 A Temperature

 B Light Intensity

 C Light Quality

 D pH

 E Nutrients

 F Trace Elements


III  MEASURING PRODUCTIVITY

 Methods employed to measure plankton pro-
 ductivity are:

 A Standing Crop

 B Oxygen

 C pH

 D Carbon-14
IV  STANDING CROP METHOD

 The productivity of a body of water is indicated,
 in a general way,  by the density of the plankton
 population.  The standing crop of plankton is
 commonly measured by determining one or
 more of the following:

 A Dry  and Ash-free Weight of Seston

 B Cell or Unit Counts

 C Cell Volume

 D Chlorophyll

 E Particulate and Dissolved Carbohydrate

 F Particulate and Dissolved Organic Carbon

 Increases in the standing crop  over a period
 of time may be used to determine  productivity.
 However, this method  provides only a rough
 approximation of the rate of primary
 production.


 V  OXYGEN METHOD

 The use of dissolved oxygen to determine
 short-term rates of primary production was
 introduced by Gaardner and Gran (1927).
 Estimates of the amount of carbon fixed are
 based on the premise that one molecule of
 oxygen  is given  off for each atom of carbon
 assimilated.
C0
H20  -
                       CH20
 A "Light" and "dark" bottles are filled with
    sample and resuspended at various depths
    for 4-24 hours.

 B The concentration of dissolved oxygen is
    determined (using the Winkler Method) at
  RT. KC.O. oro. la. 6. 76
                                                                                      22-1

-------
 Determination of Plankton Productivity
    the beginning and end of the incubation
    period.  The values obtained are as
    follows:
     1  Final "light" bottle O2 - initial O2 =
       net photosynthesis

     2  Initial O^ - Final "dark" bottle O2 =
       respiration

     3  Net photosynthesis  + respiration =
       gross photosynthesis

 This method has some serious disadvantages:

 A  The bottles provide an artificial substrate
     for the proliferation of bacteria which
     use up large amounts  of C>2, resulting in
     erroneously high respiration and low net
     photosynthesis values.

 B  The lower  limit of sensitivity of the Winkler
     Method is 0.02 mg 02/liter.  This is a
     serious handicap when working in oligo-
     tropic lakes and the open sea.
VI  CARBON-14  METHOD

 The use of carbon-14 for the measurement
 of the rate of carbon assimilation by phyto-
 plankton was pioneered by E.  Steemann
 Nielsen (1952).  The method is simple and
 very sensitive.

 A  Carbon-14 labelled sodium bicarbonate
    (4 -  lOjic/liter) is added to "light" and
    "dark" bottles, which are resuspended in
    the water for 4-24 hours.

 B  An aliquot of the  sample is passed through
    a membrane filter (1.2 upore diameter),
    and the filters are treated with acid  to
    remove any inorganic labelled carbon.

 C  The (beta) activity of the filter is deter-
    mined with an end-window Geiger tube,
    or with gas flow or liquid scintillation
    techniques.

 D  The carbon fixed is determined as
    follows:
                carbon _   activity on filter
                 fixed   "  total activity added
    There are several important disadvantages
    in this method.

    A Some of the labelled photosynthesis pro-
       ducts will be broken down immediately by
       respiration, and the liberated carbon-14
       reused in photosynthesis.  Therefore, it
       is generally agreed that the method mea-
       sures only net photosynthesis.

    B It has been found that the algae rapidly ex-
       crete up to 50% of the photosynthate in the
       form of organic acids, carbohydrates,
       and amino acids.  Since these labelled
       materials are not retained by the filter,
       they escape detection.

  VII  pH METHOD

    The uptake of CO2 by the algae during photo-
    syntheses results in an increase in the pH of
    the surrounding medium.  Periodic pH measure-
    ments are made of the body of water being
    studied, and the carbon uptake is determined
    using published nomographs.

    Verduin (1952) used this method in a study
    of the productivity of Lake Erie.   However,
    the method has not gained wide acceptance
    because it can be used only in waters with
    low alkalinity.
    REFERENCES

    1  Allen, M.B. Excretion of Organic Com-
          pounds by Chlamydomonas.  Arch. f.
          Mikrobiol. 24:163-168.  1956.

    2  Curl,  H. Jr.,  and Small,  L. F.  Variations
          in Photosynthetic Assimilation in Natural
          Marine Photoplankton Communities.
          Limnol. Oceanogr.  10(Suppl.):R67-R73.
          1965.

    3  Gaardner, T. ,  and Gran,  H. H.  Investi-
          gations of the Production of the Plankton
          in the Oslo Fjord. Rapp. et Proc. -
          Verb., Con. Internal. Explor.  Mer.
          42:1-48.  1927.
X
available
 HCO:
X
correction for
  isotope
discrimination
   22-2

-------
                                                      Determination of Plankton Productivity
4  Goldman,  C. R.  Molybdenum as a Factor
      Limiting Primary Productivity in
      Castle Lake,  California.  Science 132:
      1016-1017.   1960.

5  Kamen, M. D.  Primary Processes in
      Photosynthesis.  Academic Press,
      New York.  1963.

6  Marshall,  S.M.,  and Orr, A. P.   Carbo-
      hydrate  as a Measure of Photoplankton.
      J. Mar. Biol.  Assoc. U.K. 42:511-519.
      1962.

7  Ryther, J. H.   Photosynthesis in the Ocean
      as a Function of Light Intensity.
      Limnol. Oceanogr.  1:61-70.  1956.

8  S'teemann Nielsen, E.  The Use of  Radio-
      active Carbon (C-14) for Measuring
      Organic  Production in the Sea.  J. Con.
      Internat. Explor. Mer. 18:117-140.   1952.

9  Strickland,  J. D. H.  Measuring the
      Production of Marine Phytoplankton.
      Bull. Fish..Res. Bd. Can.  No.  122:
      1-172.   1960.
10  Verduin, J.  Photosynthesis and Growth
      Rates of Two Diatom Communities in
      Western Lake Erie.  Ecology 33(2):
      163-168.  1952.

11  Vernon, L. P.  Bacterial Phytosynthesis. '
      Ann. Rev. Plant. Physiol. 15:73-100.1962

12  Wetzel, R. G.  A Comparative Study of the
      Primary Productivity of Higher Aquatic
      Plants, Periphyton, and Phytoplankton
      in a Large,  Shallow Lake.  Internat.
      Rev. Hydrobiol. 49:1-61.  1964.

13  Yentsch, Charles S.   The Measurement
      of Chloroplastic Pigments- Thirty
      Years of Progress?  pp.  255-270 in
      Chemical Environment in the Aquatic
      Habitat.  Proc. IBP Symposium.
      Amsterdam.   1967.  (N.V. Noord-
      Hollandsche Uitgevers Maatschappij.
      Amsterdam, Netherlands. 8.95)
 This outline was prepared by C. I. Weber,
 Chief, Biological Methods Branch,
 Analytical Quality Control Laboratory,
 NERC, EPA, Cincinnati, OH 45268.

 Descriptors:  Productivity, Plankton
                                                                                22-3

-------
                              ALGAL GROWTH POTENTIAL TEST
 I  INTRODUCTION

 Dense growths of algae in surface waters are
 aesthetically undesirable, cause problems  in
 water treatment, produce changes in the aquatic
 environment that are harmful to fish and other
 aquatic life,  and are symptomatic of pollution.
 The density of phytoplankton populations is
 directly related to  the concentration of
 nutrients.  This relationship has been well
 documented, and is now embodied in the
 concept of trophic level or trophic status of
 surface waters.  One or more of the following
 parameters are commonly used to describe
 the trophic status:  (a) nutrient concentration -
 principally N and P,  (b) algal count,
 (c) chlorophyll concentration,  (d) primary
 productivity, (e) particulate organic matter,
 (f) oxygen  depletion in the hypolimnion,  and
 (g) phytoplankton species composition or
 indicator species (Rawson 1956; Davis 1964;
 Goldman & Carter  1965; Oglesby &  Edmondson
 1966; Fruh, Steward, Lee & Rohlich 1966).
 II  EUTROPHICATION

 Three general trophic levels now recognized,
 here arranged in ascending order, are:
 oligotrophic (low),  mesotrophic (intermediate),
 and eutrophic (high).  The addition of nutrients
 to surface waters raises the trophic level and
 results in an increase in phytoplankton density
 and changes in the  species composition.
 This process, commonly referred to as
 eutrophicatiori,  is greatly accelerated by the
 discharge of nutrient-laden domestic and
 industrial wastes (Hasler 1947),  Edmondson
 & Anderson 1956).
Ill  MEASUREMENTS OF TROPHIC LEVELS

 Although chemical analyses provide information
 on the concentration of nutrients, their
 availability to the algae can be determined
 only by biological assay. Biological assays
 to determine the potential (algal) productivity
 of surface water were first used in the late
 twenties (Schreiber  1927) and early thirties
 (Strom 1933), but until recently had been
 used only infrequently (Potash 1956;
 Skulberg 1964, 1967; Shelef & Halperin 1970).
 In 1967, the  Joint Industry-Government Task
 Force on Eutrophication took steps to develop
 a standardized algal growth potential (AGP)
 test.   Using  this test,  one can:

 A  Evaluate the effectiveness of waste
    treatment processes in removing elements
    that support or stimulate the growth of
    algae.

 B  Determine at what point along the time
    scale of progressing eutrophication the
    water of a given lake or stream happens
    to lie (trophic status).

 C  Anticipate the effect on algal production
    of introducing extraneous nutrients.

 D  Determine the extent to which nutrient
    levels must be reduced in  a body of water
    to effect an acceptable remedy.
IV  BASIC STEPS OF ALGAL GROWTH
    POTENTIAL TEST

 A A surface (test) water sample is collected
    and the indigenous microorganisms are
    removed by filtration (0. 45 micron
    membrane filter at 15 inches of mercury)
    or ultracentrifugation.

 B The  surface water and standard medium
    (Table 1) are inoculated with 1000  cells/ml
    of Selenastrum capricornutum,  or 50, 000
    cells/ml of Anabaena flos-aquae or
    Microcystis aerugingsa.

 C The  cultures are prepared in triplicate
    and incubated 7-10 days at 24QC, 200 fc
    (blue-greens) or 400 fc (Selenastrum)
    continuous illumination, with shaking at
    100 oscillations/min (culturing may be by
    flask,  chemostat, or in situ technique).

 D Algal growth is measured daily by
    (1) cell counts, (2) determining the
 BI.BIO. alg. lb.6.76
                                        23-1

-------
Algal Growth Potential Test
                                     TABLE 1

               MAY, 1970 VERSION OF PAAP NUTRIENT BASAL MEDIUM


         (This formula consists of 30% of the concentrations of the macroelements
         listed in the February, 1969,  PAAP Booklet. The Na CO  was replaced by
         NaHCO-, and the EDTA was reduced to 333 /ug/1.)
               o

         MACROELEMENTS:  (milligrams per liter)
Compound

NaNOQ
O
K.HPO.
2 4
MgCl2
MgSO • 7H O

CaCl0-2H0O
6t Łi
NaHCO,
O
If the medium is to be
solution from a single
Final Cone.

25.500
1.044

5.700
14.700

4.410
15.000
filtered, add
combination
filtration, K HPO should be added
solutions of individual salts may be
Element
Furnished
N
P
K
Mg
Mg
S
Ca
Na
Element
Cone.
4.200
0.186
0.469
1.456
1.450
1.911
1.202
11.001









the following trace- element -ir on- EDTA
stock solution after
filtration.
last to avoid iron precipitation
made up in 1000 X 's final cone
With no
. Stock
. or less.
MICROELEMENTS: (micrograms per liter)
H3B°3
MnCl2
ZnCl2
CoCl2
CuCl2
Na2MoO4- 2H2O
FeCl3
Na0EDTA- 2H0O
185.5
264.3
32.7
0.780
0.009
7.26
96.0
300.0
B
Mn
Zn
Co
Cu
Mo
Fe

32.5
115.4
15.7
0.354
0.004
2.88
33.05









 23-2

-------
                                                                 Algal Growth Potential Test
  E
chlorophyll content, in vivo fluorescence,
light scattering or optical density
(600 nm) of the culture, (3) measuring
the C-14 uptake, or (4) determining the
dry weight of the algae at the end of the
Incubation period.  Regardless of the
parameter used to measure growth
response, the result should always be
expressed in terms of the final dry weight
of the culture.

The growth response of the alga in the
test water is compared to its growth in
the standard medium.
 V  PHASES OF THE TEST STILL UNDER
    STUDY INCLUDE:

  A Composition of the standard growth
    medium.

  B Effects of ventilation and shaking on the
    growth response of batch cultures.

  C Techniques of measuring growth response.

  D Techniques of removing indigenous
    microorganisms from test surface waters.
VI  For copies of the Provisional Algal Assay
,  Procedure and information on the availability
  of subcultures of the test organism, contact:

       Dr. A. F. Bartsch, Chairman
       JTF Research Program Group
       Director,  Pacific Northwest
       Water Research Laboratory
       Corvallis, Oregon 97330
 REFERENCES

 1	.  Provisional Algal Assay
       Procedure.  Joint Industry-Government
       Task Force on Eutrophication, P.O.
       Box 3011, Grand Central Station,
       N.Y.  10017.  1969.

    Davis, C.C.  Evidence for the eutrophication
       of  Lake Erie from phytoplankton records.
       Limnol. Oceanogr.  9:275.   1964.
 3  Edmondson, W. T. and Anderson, G. C.
       Artificial eutrophication of Lake
       Washington.   Limnol.  Oceanogr.
       l(l):47-53.   1956.

 4  Fruh, E.G.,  Stewart, K.M., Lee,  G. F.,
       and Rohlich,  G.A.  Measurements of
       Eutrophication and Trends.   JWPCF
       38(8):1237-1258.  1966.

 5  Goldman,  C.R.  and Carter, R.C.
       An investigation by rapid C14
       bioassay of factors affecting the
       cultural eutrophication  of Lake Tahoe,
       California. JWPCF 37:1044-1063.
       1965.

 6  Hasler,  A.D.   Eutrophication of lakes by
       domestic drainage.  Ecology 28(4):
       383-395.  1947.

 7  Oglesby,  R. T. and Edmondson, W. T.
       Control of Eutrophication. JWPCF
       38(9):1452-1460.  1966.

 8  Potash,  M.   A  biological  test for
       determining the potential productivity
       of water.  Ecology 37(4):631-639.
       1956.

 9  Rawson, D. S.  Algal indicators of lake
       types.  Limnol. Oceanogr.  1:18-25.
       1956.

10  Schreiber,  W.  Der Reinkultur von
       marinem Phytoplankton und deren
       Bedeutung fur die Erforschung der
       Produktions-fahigkeit des Meerwassers.
       Wissensch.  Meeresunters., N. F.
       16:1-34.   1927.

11  Shelef, G. and Halperin, R. 1970. Wastewater
       nutrients and algae growth potential.
       In: H.I. Shuval, ed., "Developments
       in Water Quality Research",  Proc.
       Jerusalem Internat'l.  Conf.  on Water
       Quality and Poll. Res.,  June, 1969.
       Ann Arbor-Humphrey Science Publ.,
       p. 211-228.
                                                                                    23-3

-------
 Algal Growth Potential Test
12  Skulberg, O.M.  Algal problems related
      to the eutrophication of European water
      supplies,  and a bioassay method to
      assess fertilizing influences of pollution
      on inland waters.  In:  D.F. Jackson,
      ed., "Algae and Man",  Plenum Press,
      N. Y. p. 262-299.  1964.

13  Skulberg, O.M.  Algal cultures as a means
      to assess the fertilizing incluence of
      pollution.  In: Advances in Water
      Pollution Research, Volumn 1,
      Pergamoa Press, Washington, D. C.
      1967.

14  Strom, K.M.  Nutrition of algae.  Experi-
      ments upon; the feasibility of the
      Sehreiber method in fresh waters;
      the relative inportance of iron and
      manganese in the nutritive medium;
      the nutritive substance given off by
      lake bottom muds.  Arch. Hydrobiol.
      25:38-47.   1933.

 ADDITIONAL RECENT REPORTS:

 1	.   Algal Assay Procedure
      Bottle Test.  82 pp. Environmental
      Protection Agency,  National Eutrophica-
      tion Research Program, Corvallis,
      Oregon.  1971.

 2	.  Inter-Laboratory
      Precision Test.   An Eight-Laboratory
      Evaluation of the  Provisional Algal
      Assay Procedure Bottle Test.   70 pp.
      Environmental Protection Agency,
      National Eutrophication Research
      Program, Corvallis, Oregon.   1971.

 3 Berge, G.,  Predicted Effects of
      Fertilizers Upon  the Algae Production
      in Fern Lake. Fisk Dir. Skr. Serv.
      Hav. Unders., 15:339-355.  1969.

 4 Johnson, J. M.,  T.O.  Odlaug, T.A.  Olson,
      andO.R. Ruschmeyer.  The Potential
      Productivity of Freshwater Environ-
      ments as Determined by an Algal
      Bioassay Technique. Water Resources
      Research Center  Bulletin No.  20,
      University of Minnesota, Minneapolis.
      1970.
5  Maloney, T.E., W.E. Miller, and T.
      Shiroyama.  Algal Responses to
      Nutrient Additions in Natural Waters.
      I. Laboratory Assays.  In:  Special
      Symposia 1:134-140.  Amer. Soc.
      Limnol. Oceanogr. 1972.

6  Miller,  W.E.,  andT.E. Maloney.
      Effects of Secondary and Tertiary
      Wastewater Effluents on Algal Growth
      in a Lake-River System.  JWPCF
      43(12)2361-2365.  1971.

7  Murray, S., J. Scherfig,  andP.S.  Dixon.
      Evaluation of Algal Assay Procedures-
      PAAP Batch Test. JWPCF 43(10):
      1991-2003.   1971.

8  Shapiro, J., and R. Riberiro.   Algal
      Growth and Sewage Effluent in the
      Potomac Estuary.  JWPCF  37(7):
      1034-1043.  1965.

9  Toerien, D. F., C.H. Huang, J. Rad:;msky,
      E.A. Pearson, and J. Scherfig.  Final
      Report, Provisional Algal Assay
      Procedures.  211 pp.  Sanitary Engineer-
      ing Research Laboratory Report No.
      71-6, University of California,
      Berkeley.  1971.
This outline has been prepared by Dr. C. I.
Weber,  Chief, Biological Methods Section,
Analytical Quality Control Laboratory,
NERC, EPA, Cincinnati, OH  45268.

Descriptors: Plankton,  Productivity
 23-4

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                    ALGAE AND ACTINOMYCETES IN WATER SUPPLIES
 I  Water treatment always should include
 detection and control of microorganisms.


 A  Two types of microorganisms are involved:

    1  Pathogenic types include such forms as
       the typhoid bacteria, the dysentery
       ameba, and the infectious hepatitis
       virus.

    2  Interference types include taste and
       odor organisms, filter-clogging
       organisms, pipe-infesting organisms,
       and  others.
 B  Water treatment practices are closely
    associated with these organisms.

    1  For pathogens,  practices include
       coliform tests,  use of chlorine,  and
       guarding the water supply against fecal
       pollution.

    2  For interference organisms,  practices
       include plankton enumeration, use of
       copper sulfate and the covering of
       reserviors.

    3  Many of the other treatment practices
       have significant effects on the organisms.
B  At Indianapolis, copepods were present in
   parts of the distribution system in numbers
   sufficient to be visible in the drinking
   water.  The eggs of the copepods were
   found to pass through the filters and to
   hatch in the distribution system.


C  At Oklahoma City, prominent earthy  odors
   have appeared frequently.  The organisms
   blamed for this trouble are the mold-like
   actinomycetes.

D  At Peoria, white wigglers  up to 3/8"  long
   were reported in the tap water,  during
   early March,  1956.  These chironomid
   larvae had hatched in the city's open
   reservoir, requiring that the reservoir be
   drained,  cleaned and treated with a larvicide.

E  At Chicago, diatoms are a very important
   cause of short filter runs.  The one diatom
   Tabellaria is considered to be more
   responsible than any other organism  for
   this trouble.
F  In Ontario, the alga Cladophora often
   grows in large numbers attached to rocks
   on the shoreline of lakes.  When the alga
   is broken loose it collects near the shore-
   line and gives rise to very offensive odors.
 C  This discussion will be limited to the inter-
    ference organisms.
II   EXAMPLES OF PROBLEMS CAUSED BY
    INTERFERENCE ORGANISMS

 A  At Chicago,  the alga Dinobryon reappears
    almost every year,  generally in June and
    July in numbers  sufficient to impart a
    prominent fishy odor to the water.  In
    1951,  it required an estimated $70, 500
    worth of activated carbon to control the odor
    of this organism for a period of two months.
G  In a water supply impoundment in Utah the
   plankton algae frequently cause the pH of
   the water to increase to 8. 3 or higher,
   requiring that the water be treated  with
   acid to obtain the desired pH of 8 or lower.

H  In Texas a water supply from underground
   sources was  stored in a large open settling
   basin.  Oscillatoria and unicellular green
   algae developed in large numbers in the
   stored  water, turning it green and pro-
   ducing  a strong odor.

I  Los Angeles  has more than 25 open reser-
   voirs of various sizes and ranging in
   elevation from almost sea level to over
   BI.MIC. 12c.6.76
                                                                                      24-1

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       Algae and Actinomycetes in Water Supplies
       7,000 feet.  Many tons of copper sulfate
       are used every year in these reservoirs
       for rigid control of plankton, chiefly
       diatoms and occassionally blue-green
       algae.  This treatment is carried out to
       improve  the water quality including the
       reduction of tastes and odors.
Ill  TYPES OF PROBLEMS CAUSED BY
    INTERFERENCE ORGANISMS


 A Tastes and Odors

    1  May be caused by algae, actinomycetes,
       Crustacea, and anaerobic bacteria.

    2  Common algal odors imparted to water
       are ones described as fish, earthy,
       musty, grassy,  cucumber, geranium,
       nasturtium, and septic.

    3  Common actinomycete odor is earthy.

    4  Tastes produced in water by algae
       include sweet and bitter.

    5  Other causative  agents of tastes and
       odors may be industrial wastes, sludge,
       and compounds  dissolved from soil and
       rock, and chemicals used in treatment.


 B Filter Clogging

    1  Both rapid and slow sand filters are
       affected.

    2  Diatoms are the organisms most
       frequently involved but blue-green
       algae,  filamentous green algae and
       other organisms as well as silt may
       cause it.
 C  Other Problems in the Treatment Plant

    1  Algae may cause variation in the pH,
       hardness, color,  and organic content
       of the water.

    2  Amount of plankton organisms often
       influences the rate and effectiveness
       of coagulation.
     3  Chlorine dosage may depend upon
        amount of plankton organisms present.
     4  Growths of algae may reduce the flow
        through influent channels and screens.
     5  Organisms may be responsible for
        increasing the quantity of sludge to be
        disposed of in sedimentation basins.

     6  Microcrustacea "spot" paper in paper
        mill rolls.
 D  Infestation of Distribution Systems

    1  Attached organisms reduce the rate of
       flow in the pipes.

    2  Iron and sulfur bacteria may initiate
       or stimulate corrosion of pipes.

    3  Organisms may appear as visible
       bodies in tap water.

    4  Tastes  and odors may result from
       presence of organisms.

    5  Chlorine residual is difficult to main-
       tain when organic matter is present.

    6  Organisms could theoretically harbor
       and  protect  against chlorine certain
       pathogenic bacteria.


 E  Profuse Growths of Organisms in Raw
    Water Supplies

    1  A limited and balanced growth of
       various organisms  is generally an
       asset.

    2  Extensive surface mats, blooms and
       marginal growths often cause troubles
       along the shoreline  and eventually in
       the treatment plant.

    3  Some fish kills may be caused by
       profuse growths of  algae by reducing the
       DO during the night.

    4  Certain massive growths of blue-green
       algae are  deadly poisonous to animals.

IV  ORGANISMS INVOLVED
 A  Animal forms include  protozoa, rotifers,
    crustaceans, worms,  bryozoaris, fresh water
     24-2

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                                               Algae and Actinomycetes in Water Supplies
  V
     sponges,  water mites and larval stages of
     various insects.

     Plant forms include algae, actinomycetes
     and other bacteria,  molds and larger
     aquatic green plants.
IMPORTANCE OF BIOLOGICAL
PROBLEMS
  A  The increased use of surface water supplies
     increases the problems caused by organ-
     isms.   Biological problems are less
     common with ground water supplies.

  B  Standards of water quality requested by
     domestic and industrial patrons are rising.

  C  Procedures for detection,  control and
     prevention of problems caused by organisms
     are improving and are receiving more
     extensive use.
VI  A number of methods may be used to
  control the interference organisms or their
  products;

  A Addition to water or an algicide or pesticide
    such as copper sulfate, chlorine dioxide
    or copper-chlorine-ammonia.

  B Mechanical cleaning of distribution lines,
    settling basins,  sand filters,  screens,  and
    reservoir walls.

  C Modification  of coagulation, filtration,
    chemical treatment, or location of intake.

  D Use of absorbent,  such as activated
    carbon, for taste and odor substances.

  E Modification  of Reservoir to Reduce the
    Opportunities for Massive Growths of
    Algae

    1  By covering treated water reservoir to
       exclude sunlight

    2  By increasing the depth of the water in
       reservoirs
                                                 3  By eliminating shallow marginal areas

                                                 4  By reducing the amount of fertilizing
                                                    nutrients entering the  reservoir.
VII It is generally more satisfactory to
 anticipate and prevent problems due to these
 organisms than it is to cope with them later.


 A Routine biological tests are essential to
    detect the initial development or presence
    of interference organisms.

    1  Control measures can then be used
       before problem becomes acute.

    2  These tests should be applied to the
       raw treatment plant water supply and
       distribution system.


 B In the Reservoir or Other Raw Water Supply

    1  Routine plankton counts should be made
       of water samples from selected loca-
       tions.  Plankton counter should be
       aware of the particular organisms
       known to be most troublesome.

    2  During the warmer months routine
       surveys of the reservoir,  lake or
       stream should be made to record any
      visible growths of algae and other
      organisms.

    3 Odor tests of water from several
      locations should be made to obtain
      advance notice of potential trouble at
      the treatment plant.


 C In the Treatment Plant

    1  Records of plankton counts and threshold
      odor between each step in treatment
      gives data on effectiveness of each
      procedure.

    2  Coagulation and filtration can be
      adjusted to remove up to 95% or more
      of organisms in water.
                                                                                       24-3

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         Algae- and Actinomycetes in Water Supplies
         Microscopic analysis of samples of
         filter material for organisms can
         supply data useful in modifying sand
         filtration and treatment of finished
         water.
   D  In the Distribution System With Its
      Finished Water

      1  Open reservoirs require constant
        attention especially during summer.

      2  Parts of the system farthest from the
        treatment plant or  adjacent to dead
        ends  require most  frequent sampling
        for organisms and  tastes and odors.
VIII   SUMMARY


   A  Interference organisms cause problems
      in distribution systems,  treatment plants,
      raw water supplies.

   B  Organisms involved include algae, actino-
      mycetes, other bacteria, and minute
      aquatic animals.

   C  Control is by special chemicals, mechanical
      cleaning, adjustment of chemical or
      mechanical treatment and by modification
      of reservoirs, intakes, etc.,  for the raw
      water supply.

   D  Facilities for detection of problems in
      their early stages are required for most
      efficient and satisfactory control.
   REFERENCES

   1   Palmer, C. M.  Algae in Water Supplies.
        An Illustrated Manual on the Identification,
        Significance, and Control of Algae in
        Water Supplies.  U.S. Public Health
        Service Publication No. 657.  1959.
        p. 88.
 2  Palmer, C.M. and Poston,  H.W.
      Algae and Other Interference
      Organisms in Indiana Water Supplies.
      Jour. Amer. Water Works Assn.
      48:1335-1346.   1956.

 3  Palmer, C.M.  Algae and Other Inter-
      ference Organisms in New England
      Water Supplies.   Jour. New England
      Water WorksAssn.  72:27-46.  1958.

 4  Palmer, C.M.  Algae and Other Orga-
      nisms in Waters of the Chesapeake
      Area.   Jour. Amer. Water Works
      Assn.   50:938-950.   1958.

 5  Palmer, C.M.  Algae and Other Inter-
      ference Organisms in the Waters of
      the South Central United  States.  Jour.
      Amer. Water Works Assn.   52:897-
      914.   1960.

 6  Silvey,  J.K. and Roach, A.W.
      Actinomycetes May Cause Tastes
      and Odors in Water Supplies.  Public
      Works 87.   5:103-106,210,212.    1956.

 7  Ingram, W.M. and Bartsch, A.F.
      Operators Identification Guide to
      Animals Associated with  Potable
      Water Supplies.   Jour. Amer. Water
      WorksAssn.  52:1521-1550.  1960.

 8  Otto, N. E. and Bartley,  T.R.   Aquatic
      Pests on Irrigation Systems.
      Identification Guide.   Bur. of
      Reclamation.  USDI.   72 pp.  1965.

 9  Herbst,  Richard P.   Ecological Factors
      and the Distribution of Cladophera
      glomerata in the Great Lakes.
      Amer.  Midi. Nat.   82:90-98.  1969.
This outline was prepared by C.M. Palmer,
formerly Aquatic Biologist, In Charge,
Interference Organism Studies, Microbiology
Activities, Research & Development,
Cincinnati Water Research Laboratory,
FWPCA.
                                                   Descriptors:  Algae,  Water Supplies,
                                                   Actinomycetes
       24-4

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                    Algae and Actinomycetes in Water Supplies
ALGAE   IMPORTANT  IN  WATER  SUPPLIES
            TASTE AND ODOR ALGAE
                  PLATE I

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Algae and Actinomycetes in Water Supplies
                         FILTER  CLOGGING ALGAE
                                      CHROOCOCCUS
                                 PLATE  2

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        Algae and Actinomycetes in Water Supplies
POLLUTED  WATER  ALGAE
        PLATE  3

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Algae and Actinomycetes in Water Supplies
                          CLEAN  WATER ALGAE
                                                               CLAOOPHORA
                                                              .«*•>-
                                 PLATE 4

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       Algae and Actinomycetes in Water Supplies
SURFACE WATER ALGAE
       PLATE 5

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Algae and Actinomycetes in Water Supplies
               ALGAE  GROWING ON RESERVOIR  WALLS
          PHORMIDIUM
                              PLATE 6
  10

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                         ALGAE AS INDICATORS OF POLLUTION
  I  LIMITATIONS

  A Algae are only one of a number of types
    of organisms present which could be
    considered.

  B Forms recognized here as algae  are
    comparatively simple, pigmented, aquatic
    organisms,  including blue-greens,  greens,
    diatoms and pigmented flagellates.

  C Various pollutants react differently on
    algae.  Organic pollutants such as house-
    hold sewage will be dealt with here.

  D No algae are intestinal organisms.  They
    therefore are not indicators of pollution
    in the same way that coliform bacteria
    are.
 B Wastes may have physical effects on
    certain algae.  May cause plasmolysis,
    change in rate of absorption of nutrients,
    etc.

 C Wastes may reduce available light,
    increase the water temperature, and
    cover up the areas for attachment  to
    rocks.

 D Wastes may prevent algal respiration at
    night by reducing the DO of water.

 E Wastes may stimulate  other organisms
    at the expense of certain algae.

 F Products of waste decomposition may
    act as powerful growth stimulants  for
    certain algae.
 II  ALGAE AND ORGANIC POLLUTION

 A Heavy pollution may tend to limit various
    kinds of algae to certain zones in the
    affected area.

 B These zones are distinguished according
    to the degree of change which has
    occurred in the organic wastes. One set
    of names for these zones includes  the
    Polysaprobic, alpha-mesosaprobic, beta-
    mesosaprobic and oligosaprobic.

 C A few "pollution" algae are common in
    the first two zones.  Many algae are
    common in and often limited to one or
    both of the last two  zones.

 D Some workers have listed separately
    those algae indicative of each of the four
    zones.
Ill  REASONS FOR SELECTIVITY OF
    POLLUTANTS TO ALGAE

 A Certain components of wastes are chemi-
    cals toxic to some algae but not to others.
IV  ALGAE AS INDICATORS OF POLLUTION

 A  Selection of list of "pollution" algae
    follows an evaluation of the kinds re-
    ported in published reports by numerous
    workers as  relatively prominent in, or
    representative of, the polysaprobic and
    alpha-mesosaprobic zones in a stream
    polluted with sewage.  It includes also
    other conditions or areas approximating
    these zones.

 B  A total list of more than 1000 kinds of
    algae has been compiled to date.

    1  In order to tabulate the information,
       an arbitrary numerical value is
       allotted to each author's record of
       each pertinent alga.

    2  The algae are then arranged in order
       of decreasing emphasis by the
       authors as a whole.
  BI. IND. lOa. 6. 76
                                                                                  25-1

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   Algae as Indicators of Pollution
  VI   SOME GENERA AND SPECIES OF ALGAE
      HIGH ON THE LIST ARE AS FOLLOWS:

   A  Genera: Oscillatoria, Euglena, Navicula,
      Chlorella, Chlamydomonas,  Nitzschia,
      Stigeoclonium, Phormidium,  Scenedesmus,
      Ankistrodesmus, Phacus.

   B  Species:  Euglena viridis, Nitzschia
      palea, Oscillatoria chlorina,
      Oscillatoria limosa, Oscillatoria  tenuis
      Scenedesmus quadricauda, Stigeoclonium
      tenue, Synedra ulna and Pandorina morum.
 VII   SOME ALGAE REPRESENTATIVE OF
      CLEAN WATER ZONES IN STREAMS:

   Chrysococcus rufescens, Cocconeis
   placentula, Entophysalis lemaniae, and
   Rhodomonas lacustris.
VIII   RELIABILITY IN USE OF INDICATORS
      DEPENDS IN PART UPON ACCURATE
      IDENTIFICATION OF SPECIMENS
   REPRESENTATIVE LITERATURE

   1  Brinley, F.J.   Biological Studies.   Ohio
         River Pollution Survey.  I.
         Biological Zones in a Polluted Stream.
         II.  Plankton Algae as Indicators of
         the Sanitary Condition of a Stream.
         Sewage Works Journal,  14:147-159.
         1942.

   2  Butcher, R.W.   Pollution and
         Repurification as Indicated by the
         Algae.   Fourth International
         Congress for Microbiology (held)  1947.
         Report  of Proceedings.   1949.

   3  Fjerdingstad, E.   The Microflora of the
         River Moelleaa with Special  Reference
         to the Relation of the Benthal Algae
         to Pollution.   Folia Limnological
         Scandinavia.   No.  5.   1950.

   4  Fjerdingstad, E.   Taxonomy and Saprobic
         Valency of Benthic Phytomicro-
         Organisms.   Intern. Rev. Ges.
         Hydrobiol.   50:475-604.  1965.
   25-2
 5  Hawkes, H.A.  The Biological Assess-
       ment of Pollution in Birmingham
       Streams.  The Institute of Sewage
       Purification,  Journal and Proceedings.
       177-186.   1956.

 6  Kolkwitz,  R.  Oekologie der Saprobien.
       Schriftenreiche des Vereins fUr
       Wasser-,  Boden-, und Lufthygiene
       Berlin-Dahlem.   Piscator - Verlage,
       Stuttgart.

 7  Lackey, J. B.  The Significance of
       Plankton in Relation to the Sanitary
       Condition  of Streams.   Symposium
       on Hydrobiology.   University of
       Wisconsin.  311-328.   1941.

 8  Liebmann, H.   Handbuch der
       Frischwasser - und Abwasserbiologie.
       R. Oldenbourg, Munchen.

 9  Palmer, C.M.  Algae as Biological
       Indicators of Pollution.   In
       Biological Problems in Water
       Pollution.  Trans,  of 1956 Seminar.
       Robert A. Taft Sanitary Engineering
       Center.   1957.

10  Palmer, C.M.  The Effect of  Pollution
       on River Algae.   Annal. N.Y. Acad.
       Sci.  108:389-395.   1963.

11  Palmer, C.M.  A Composite Rating of
       Algae Tolerating Organic Pollution.
       Jour. Phycology 5 (l):78-82.  1969.

12  Palmer, C.M.  Algae in Water Supplies
       of the United States. In: Algae and
       Man,Ch  12, Plenum Press.  N.Y.
       pp. 239-261.  1964.

13  Patrick, R.  Factors Effecting the
       Distribution of Diatoms.   Botanical
       Review.  14:  473-524.  1948.

14  Whipple,  G.C., Fair, G.M. and
       Whipple, M. C.   The Microscopy of
       Drinking Water,  4th ed.  J.  Wiley
       and Sons.   New York.   1948.
 This outline was prepared by C.M.  Palmer,
 Aquatic Biologist,  Cincinnati Water Research
 Laboratory, FWPCA.

 Descriptors: Bioindicators,  Algae

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                              Algae as Indicators of Pollution
       POLLUTED  WATER  ALGAE
PHORMIDIUM
                PLATE  3

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Algae as Indicators of Pollution
                        CLEAN   WATER  ALGAE
                                                                CLADOPHORA
                                PLATE 4

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                   ODOR PRODUCTION BY ALGAE AND OTHER ORGANISMS
  I  Most biological odors present in our water
  supplies are derived from algae, actinomycetes,
  and bacteria.

  A The odor produced by algae and actino-
    mycetes is generally the result of
    intracellular metabolic activity while
    the odor caused by bacteria usually
    results from extracellular enzymatic
    activity upon other organisms.

  B The odors produced by actinomycetes are
    usually earthy while those produced by the
    algae are aromatic,  grassy, and fishy.
 H  SOME SPECIES OF ALGAE CAUSING
    ODORS

 A Diatoms

    1  Asterionella (aromatic, fish)

    2  Cyclotella (aromatic)

 B Pigmented Flagellates

    1  Synura (cucumber)

    2  Dinobryon (fishy)

 C Blue-green Algae

    1  Anabaena (grassy, green corn,
       nasturtium)

    2  Aphanizomenon (grassy,  nasturtium)

 D Green Algae

    1  Chlorococcum (grassy)


IE  RESEARCH ON A LGAE ODORS

 A Growing Algae for Odor Research

    1  Obtaining unialgal bacteria-free
       cultures
       a  Plating out on semi-solid medium

       b  Single  cell isolation

       c  Use of antibiotics

       d  Exposure to ultra-violet  light

     2  Determining nutritional requirements

       a  Inorganic salts

       b  Organic growth factors

  B  Methods of extracting odoriferous
     material from algal cultures

     1  Distillation  - steam and vacuum

     2  Solvent extraction

     3  Use of ion exchange resins

     4  Freeze out methods

  C  Some Results of Research

     1  Effect of culture age upon odor
       production

     2  Effect of pH on odor intensity

     3  Comparison of odor intensity in intact
       and broken cells

     4  Groups of chemicals which may be
       responsible  for causing algal odors


IV   RESEARCH ON ACTINOMYCETE ODORS

 A  A number of actinomycetes were isolated
     from water and muds of rivers and lakes.
  BI.MIC.to. lOc.6.76
                                     26-1

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Odor Production by Algae and Other Organisms	
      1  Large numbers were found to
        be present in muds, while there
        were relatively few in the water.

      2  Most, species belonged to tha
        Streptomyces and a few to the
        Micromonospora.

   B  Extraction of Odoriferous Material

      1  Streptomyces griseoluteus was
        used in this work.

        a  Cultured in a defined medium

           (1)  Cultures have threshhold
               odor of 20, 000 to 50, 000

      2  Primary extraction was by
        distilling the culture at  100°C
        at atmospheric pressure.

        a  Distillation of 10% of the
           culture volume resulted
           in 90%  odor removal.

      3  Odor was  further concentrated
        by two methods

        a  Ether extraction of the
           distilling off of the ether
           in vacuo.

           (1)  Re suite d in y ello wish
               brown concentrate
               having a threshold odor
               of approximately 6 billion.

        b  Absorption on activated carbon
           followed by elution of material
           with chloroform

   C  Effect of Activated Carbon  in Re-
      moving the Earthy Odor
      1  The odor is practically elimi-
         nated by 10 ppm carbon.

   D  Effect of Chlorine on Odor

      1  Chlorine does not eliminate
         the odor but does not intensify
         the odor.

   E  Soil perfusion Tests

      1  Conducted to determine the
         extent to which actinomycetes
         impart odors to a water environ-
         ment.
REFERENCES

1  Fogg., G.E.,  "The Metabolism of
      Algae",  John Wiley and Sons, Inc.,
      New York, N.  Y., 1953.

2  Fox, Leo,  ''Microscopic Organisms
      in Drinking Water", Taste arid Odor
      Journal, Vol. 19, No. 10, 1953.

3  Palmer,  C. M., and Tarzwell, C. M.,
      "Algae of Importance in Water
      Supplies",  Public Works Magazine,
      1955.

4  Whipple,  G. C., Fair, G. M.,  and
      Whipale, M. C., "The Microscopy
      of Drinking Water", Fourth Edition,
      John Wiley and Sons, Inc.,  New York,
      N.Y., 1948.
This outline was prepared by T. E. Maloney,
P'ormer Research Biologist, Aquatic Biology
Activities,  Research and Development,
Cincinnati Water Research Laboratory,  FWPCA.

Descriptor:  Odor Producing Algae
 26-2

-------
                            PLANKTON IN OLIGOTROPHIC LAKES
  I  INTRODUCTION

  The term oligotrophic was taken from the
  Greek words oligos --  small and trophein --
  to nourish,  meaning poor in nutrients.
  Lakes with  low nutrient levels have low
  standing crops of plankton.  The term is now
  commonly applied to any water which has a
  low  productivity, regardless of the reason.
 II  PHYSICAL AND CHEMICAL CHARACTER-
    ISTICS OF OLIGOTROPHIC LAKES*

 A Very deep; high volume to surface ratio

 B Thermal stratification common; volume
    of the hypolimnium large compared to the
    volume of the epilimnion

 C Maximum surface temperature  rarely
    greater than 15OC

 D Low concentrations of dissolved minerals
    and organic matter.

    1  Phosphorus, less than 1 microgram
       per liter

    2  NO -Nitrogen, less than 200 micrograms
       per liter

 E Dissolved oxygen near saturation  from
    surface to  bottom

 F Water very transparent, Secchi disk
    readings of 20-40 meters are common

 G Color dark blue, blue-green,  or green
IH  PLANKTON

 A Quantity

    1  Standing crop very low

       a Ash-free weight of plankton, less
         than 0. 1 mg per liter (compared to
         1 mg per liter or more in eutrophic
         lakes).
        b Chlorophyll,  1 mg per M  or less

        c Cells counts,  less than 500 per ml

   2 Zooplankton to phytoplankton volume
     ratio,  19:1.

B  Quality

   1 European biologists have found
     oligotrophic lakes to be dominated by
     Chlorophyta (usually desmids),
     chrysophyta (such as Dinobryon), and
     Diatomaceae (Cyclotella and Tabellaria).
     Eutrophic lakes are dominated by
     Synedea, Fragilaria, Asterionella,
     Melosira, blue-green algae, Ceratium,
     and Pediastrum. Nygaard devised
     several phytoplankton quotients based
     on these relationships

     a  Simple quotient

        Number of species of

    Chlorococcales _ if <1,  oligotrophic
    Desmidiaceae     if > 1, eutrophic
     b  Compound index
    Myxophyceae+Chlorococcales+Centrales+Eugleniaceae
                     Desmidiaceae


        if <1,  oligotrophic

        if 1-2.5,  mesotrophic

        if > 2. 5, eutrophic

     c  Diatom quotient

   Centrales  _ if  0-0.2, oligotrophic
   Pennales  "if  0.2-3.0,  eutrophic
   BI. ECO. mic.2. 6.76
                                                                                    27-1

-------
Plankton in Qligotrophic Lakes
   2 Several lists of trophic indicators have
     been published:
     Two are listed here
               Teiling,
               Swedish Lakes
                                   Rawson,
                                   Canadian Lakes
      Oligotrophic    Tabelleria flocculosa
                     Dactylococcopsis
                        ellipsoideus
      Mesotrophic    Kirchneriella lunaris
                     Tetraeadon spp.
                     Pediastrum spp.
                     Fragilaria crotonensis
                     Attheya zachariasii
                     Melosira granulata
      Eutrophic
      Pronounced
        Eutrophy
Aphanizomenon spp.
Anabaena flos-aquae
Anabaena circinalis

Microcystis aeruginosa
Microcystis viridis
                           Oligotrophic   Asterionella formosa
                                          Melosira islandica*
                                                Mesotrophic
                                          Tabellaria fenestrata
                                          Tabellaria flocculosa
                                          Dinobryon divergens
                                          Fragilaria capucina
                                          Stephanodiscus niagarae
                                          Staurastrum spp.
                                          Melosira  granulata
Fragilaria crotonensis
Ceratium hirundinella
                                                               Pediastrum boryanum
                                                               Pedia strum duplex
Coelosphaerium
  naegelianum
                                                               Anabaena spp.
                                                               A phani zom enon flos-aquae
                                                               Microcystis aeruginosa
                                                Eutrophic
                                          Microcystis flos-aquae

-------
                                                       Plankton in Oligotrophic Lakes
Some discrepancies can be seen in the
ranking of species in the lists.  These
may be the result of true differences in
the composition of the plankton,  or may
be only apparent differences which
resulted from different sampling methods.
Many studies (e.g. those by Milliard,
Olive, and Rawson) have been based on
netted samples, which may be highly
biased because they contain little of the
nannoplankton. Also, it is not uncommon
to characterize populations on the basis
of one or two samples collected  during
the summer months.

The dominant plankton in four
oligotrophic North American  lakes are
listed below.  The Great Slave Lake
and Karluk Lake data are from netted
samples  taken during the summer, and
monthly, respectively.  The Lake
Superior and Lake Tahoe data are from
grab samples taken twice monthly, and
quarterly, respectively.
The dominant diatoms are generally
similar in the four lakes. Asterionella
formosa and Fragilaria crotonensis
are common to all.  There are also
some obvious differences. Melosira
islandica, the dominant diatom in the
Great Slave Lake and Lake Superior,
is absent from Lake Tahoe and Karluk
Lake.  It was not found in Crater Lake
by Sovereign (1958), in the Mountain
lakes of Colorado by Olive (1955) or
Brinley (1950), and does not occur  in
WPSS samples in streams west of the
Great Lakes.  Tabellaria is also
absent from Lake Tahoe.  It was
reported in  Colorado lakes by Olive,
but was not abundant.  Brinley makes
no reference to it, and Sovereign
indicated that it was rare in Crater
Lake samples. It is apparent that the
absence of these two diatoms from
Lake Tahoe is not related to the lake.

Except for the absence of Keratella
cochlearis from Lake Tahoe,  the
rotifer populations are very similar.
Data on other segments of the zoo-
plankton population are insufficient to
permit comparison.
                                                                               27-3

-------
-J
Dominant
Phytoplankton
Raw son.
Great Slave Lake
Melosira islandica
Asterionella formosa
Dinobryon divergens
Ceratium hirundinella
Pediastrum boryanum
Tabellaria fenestrata
Cyclotella meneghiniana
Fragilaria crotonensis
Fragilaria capucina
Synedra ulna
Eunotia lunaris
USPHS,
Lake Superior
Melosira islandica
Tabellaria fenestrata
Cyclotella kutzingiana
Melosira granulata
Melosira ambigua
Asterionella formosa
Synedra nana
Scenedesmus spp.
Ankistrodesmus spp.
Dictyosphaerium spp.

Hilliard,
Karluk Lake
Asterionella formosa
Tabellaria flocculosa
Fragilaria crotonensis
Cyclotella bodanica
Cymbella turgida
Dictyosphaerium spp.
Sphaerocystis spp.
Staurastrum spp.

WPSS,
Lake Tahoe
Fragilaria crotonensis
Synedra nana
Fragilaria construens
Fragilaria pinnata
Nitzschia acicularis
Asterionella formosa

                                                                                                                                                                                 O
                                                                                                                                                                                 }-'•
                                                                                                                                                                                 o
                                                                                                                                                                                 o
                                                                                                                                                                                 M.
                                                                                                                                                                                 o
                       Dominant
                       Zooplankton
Keratella cochlearis
Kellicottia longispina
Diaptomus tenuicaudatus
Limnocalanus macrurus
Senecella calanoides
Daphnia longispina
Bosmina obtusirostris
Keratella cochlearis
Kellicottia longispina
Not reported
Kellicottia longispina
Daphnia spp.
Diaptomus tyrelli
Epischura nevadensis

-------
                                                          Plankton in Oligotrophic Lakes
REFERENCES

1  Brinley, F.J.  1950.  Plankton population
     of certain lakes and streams in the
     Rocky Mountain National Park,
     Colorado.  Ohio J. Sci. 50:243-250.

2  Milliard, O.K., 1959.  Notes on the
     phytoplankton of Karluk Lake, Kodiak
     Island,  Alaska. Canadian Field-
     Naturalist 43:135-143.

3  Jarnefelt,  H.,  1952.   Plankton als
     Indikator der Trophiegruppen der seen.
     Ann. Acad.  Sci. Fennicae A.IV:l-29.

4  Knudson, B.M.,  1955. The distribution of
     Tabellaria in the English  Lake District.
     Proc. Int. Assoc.  Limnol. 12:216-218.

5  Nygaard, G.,  1949.  Hydrobiological studies
     in some ponds and lakes II.  The
     quotient hypothesis and some new or
     little known phytoplankton organisms.
     Klg. Danske Vidensk.  Selsk. Biol.
     Skrifter 7:1-293.

6  Olive, J.R., 1955. Some aspects of
     plankton associations in the high
     mountains lakes of Colorado.  Proc.
     Int. Assoc.  Limnol. 12:425-435.

7  Rawson, D.S., 1953.  The standing crop
     of net plankton  in lakes.  J. Fish.  Res.
     Bd. Can. 10:224-237.
8 Rawson, D. S.,  1956.
     Great Slave Lake.
     Can. 13:53-127.
The net plankton of
J. Fish.  Res. Bd.
                         9  Rawson, D. S.,  1956.  Algal indicators
                              of trophic lake types.  Limnol.
                              Oceanogr.   1:18-25.

                        10  Rodhe,  W., 1948.  Environmental
                              requirements of fresh-water plankton
                              algae.  Symb. Hot. Upsal. 10:1-149.

                        11  Ruttner, F., 1953.  Fundamentals of
                              Limnology,  2nd ed., Univ. Toronto
                              Press,  Toronto.

                        12  Sovereign, H.E., 1958.  The diatoms of
                              Crater  Lake,  Oregon.  Trans. Amer.
                              Microsc.  Soc.  77:96-134.

                        13  Teiling, E., 1955.  Some mesotrophic
                              phytoplankton indicators.   Proc. Int.
                              Assoc.  Limnol. 12:212-215.

                        14  USPHS,  1962.  National Water Quality
                              Network, Annual Compilation of Data,
                              PHS Publ. No. 663.

                        15  Welch,  P.S.,  1952.  Limnology, 2nded.,
                              McGraw Hill Book Co., New York.
This outline was prepared by C.I. Weber,
Chief,  Biological Methods Section,
Analytical Quality Control Laboratory,
NERC,. EPA,. Cincinnati, OH  45268.

Descriptors: Oligotrophy, Plankton
                                                                                    27-5

-------
                   BIOLOGICAL INTEGRITY OF STREAM COMMUNITIES
 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"--substrates
     of oceans, lakes,  streams, etc.
     They may be attached, burrowing, or
     move on the interface.
     1  Bacteria

       A wide variety of decomposers work
       on organic materials,  breaking them
       down to elemental or simple com-
       pounds.

     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, (Riverweeds,
       Pondweeds)
      The largest flora, composed of
      complex and differentiated tissues.
      May be emersed, floating, or sub-
      mersed according to habit.

    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 materi^lc
       or occupy  the interstices between rocks,
       floral or faunal materials.
    5  Meioiauna

       Meiofauna occupy the interstitial zone
       (like between sand grains) in benthic
       and hyporheic habitats.  They are inter-
       mediate in size between the microfauna
       (protozoa and rotifers) and the macro-
       fauna (insects, etc.).  They pass a No. 30
       sieve (0. 5 mm approximately).  In fresh-
       water they include nematodes, copepods,
       tardigrades,  naiad worms, and  some flat
       worms.  They are usually ignored in fresh-
       water studies, since they pass the standard
       sieve and/or sampling devices.

    6 Macrofauna (macroinvertebrates)

       Animals that are retained on a No. 30 mesh
       sieve (0. 5 mm approximately).  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, settles
       darters, and madtoms.

 B The benthos is a  self-contained community,
   though there is interchange with other
   communities. For example:   Plankton
   settles to it, fish prey on  it and lay their
   eggs there,  terrestrial detritus and leaves
   are added to it, and many aquatic insects
   migrate from it to the terrestrial environ-
   ment for their mating cycles.

 c It is an in-situ water quality monitor.   The
   low mobility of the biotic  components 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, reproduction, and re-
   cruitment from the hyporheic zone.

D Between the benthic  zone (substrate/water
   interface) and the underground water table
   is the hyporheic zone. There is considerable
   interchange from one zone to another.

Ill HISTORY OF BENTHIC OBSERVATIONS

A  Ancient literature records the vermin associ-
   ated with fouled waters.
BI.MET.fm.8j. 3.80
                                      28-1

-------
  Biological Integrity of Stream Communities
B  500 -year- old fishing literature refers
    to animal forms that are fish food and
    used as bait.

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's 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
      a  bacteria
      b  fungi
    2  Producers (plants)
   28-2
    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

    8 Sensitivity to perturbation

F  "For subtle chemical changes,
   unequivocal data, and observations
   suited to some statistical evaluation will
   be needed. This requirement favors the
   macrofauna as a parameter.  Macro-
   invertebrates are easier to  sample
   reproductively than other organisms,
   numerical estimates are possible and
   taxonomy needed for synoptic investi-
   gations is within the  reach of a non-
   specialist. '' (Wuhrmann)
G  "It is self-evident that for a multitude of
   non-identifiable though biologically active
   changes of chemical conditions in rivers,
   small organisms with high physiological
   differentiation are most responsive.
   Thus the small macroinvertebrates
   (e. g. insects) are doubtlessly the most
   sensitive organisms for demonstrating

-------
                                             Biological Integrity of Stream Communities
V
   unspecified changes of water
   chemistry called 'pollution' .
   Progress in knowledge on useful
   autecological properties of
   organisms or of transfer of such
   knowledge into bioassay  practice
   has been very small in the past.
   Thus, the bioassay concept
   (relation of organisms in a
   stream to water quality) in
   water chemistry has brought not
   much more than visual demon-
   stration of a few overall chemical
   effects.   Our capability to derive
   chemical conditions from biological
   observations is, therefore,  almost
   on the same level as fifty years ago.
   In the author's opinion it is  idle to
   expect much more in the future because
   of the limitations inherent to natural bio-
   a,ssay systems (relation  of organisms
   In a stream to water quality). "  (Wuhrmann)
REACTIONS OF THE BENTHIC MACRO-
INVERTEBRATE COMMUNITY TO
PERTURBATION
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 generalized order of macro-
      invertebrate disappearance on a
      sensitivity scale below pollution
      sources is shown in Figure 2.
Water
Quality
Deteriorating
            Stoneflies
            Mayflies
            Caddisflies
            Amphipods
             Isopods
             Midges
             Oligochaetes
Vater
Duality
improving
      As water quality improves, these
      reappear in the same order.

B  The Number of Survivors Increase

    1 Competition and predation are reduced
      between different species.

    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
    and mayfly nymphs, hellgrammites,
    and caddisfly larvae represent a grouping
    (sensitive  or intolerant) that is  generally
    quite  sensitive to environmental
    changes.   Blackfly larvae,  scuds,  sow-
    bugs,  snails, fingernail clams, dragon-
   fly and damselfly naiads, and most
    kinds of midge larvae are facultative
   (or intermediate)  in  tolerance.
    Sludge-worms, some kinds of midge
   larvae (bloodworms), and some leeches
                                                                                  28-3

-------
   Biological Integrity of Stream Communities
                  DIRECTION OF FLOW
    X
    ui
    03
    U
    I-
    4
    Ul
    
        B.
      /-•*
W    i  t
E    '   '
<
(0
ui
                               W
                              D.
w
Ul
K
(0
S
                               I

                             Pi:
                    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 animals; 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 for they are
   essentially air-breathers.

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 respiratory 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 deluge 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.
28-4

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                                               Biological Integrity of Stream Communities
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 re-
       duced from 100 to 1 per  square meter.
       Qualitative data would indicate  the
       presence of both species, but might not
       necessarily delineate the change in pre-
       dominance 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, Ekman, and  Ponar
       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, unless hydraulically
       operated.
                             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.

                             Kick samples of one minute duration will
                             usually yield around 1, 000  macroinvert-
                             ebrates per square meter (10. 5 X a one
                             minute kick=  organisms/m2).

                             Manipulated substrates (often referred to
                             as "artificial substrates") are
                             placed in a stream and left for a specific
                             time period.  Benthic macroinvertebrates
                             readily colonize these forming a manipu-
                             lated community.  Substrates may be con-
                             structed of natural materials or synthetic;
                             may be placed in a natural  situation or
                             unnatural; and may or may not resemble
                             the normal stream community.   The
                             point being that a great number of  envi-
                             ronmental variables are  standardized and
                             thus upstream and downstream  stations
                             may be legitimately compared in terms of
                             water quality of the moving water column.
                             They naturally do not evaluate what may
                             or may not be happening to  the substrate
                             beneath said monitor. The latter could
                             easily be the more important.
    A
    B
    C

    D
    E
    F
    G
    H
              REPRESENTATIVE BOTTOM-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.
(Plecoptera)
(Ephemeroptera)
Stonefly nymph
Mayfly nymph
Hellgrammite or
 Dobsonfly larvae (Megaloptera)
Caddisfly larvae  (Trichoptera)
Black fly larvae  (Simulildae)
Scud             (Amphipoda)
Aquatic sowbug   (Isopoda)
Snail            (Gastropoda)
I
J
K
L

M
N
O
P
Fingernail clam   (Sphaeriidae)
Dams elf ly naiad   (Zygoptera)
Dragonfly naiad   (Anisoptera)
Bloodworm or midge
                        fly larvae
                        Leech
                        Sludgeworm
                        Sewage fly larvae
                        Rat-tailed maggot
                                    KEY  TO FIGURE 2
                  (Chironomidae)
                  (Hirudinea) •
                  (Tubificidae)
                  (Psychodidae)
                  (Tubifera-Eristalis)
                                                                                    28-5

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                 B      X^ C

                 SENSITIVE
1—\
',. _. J  c
             F           G

              INTERMEDIATE
H
  M

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                                       Biological Integrity of Stream Communities
 Invertebrates which are part of the
 benthos, but under certain conditions
 become carried downstream in
 appreciable numbers,  are known as
 Drift.

 Groups which have members forming
 a conspicuous part of the  drift
 include the insect orders  Ephemeroptera,
 Trichoptera, Plecoptera and the
 crustacean order Amphipoda.

 Drift net studies are widely used and
 have a proven validity in stream
 water quality studies.

 The collected sample is screened with
 a standard sieve to concentrate  the
 organisms; these are separated from
 substrate and debris, and the number
 of each kind of organism determined.
 Data are then adjusted to number per
 unit area, usually to number of  bottom
 organisms 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.

 Studies have shown that a significant
 number  and variety of macroinverte-
brates inhabit the hyporheic zone in streams.
 As much as 80% of the macroinverte-
 brates may be below 5 cm  in this
hyporheic zone.   Most samples and
 sampling techniques do not penetrate
 the substrate below the 5 cm   depth.
 All quantitative studies must take this
 and other substrate factors into  account
 when absolute figures are  presented on
 standing crop and numbers per square
 meter,  etc.
                                               Flora
      Direct quantitative sampling of natu-
       rally growing bottom algae is difficult.
       It is basically one of collecting algae
       from a standard or 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.

       Manipulated 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 quantity 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.
VII ANALYSES OF MICROFLORA

    A  Enumeration

       1  The quantity of algae on manipulated
          substrates can be measured in several
          ways.   Microscopic counts of algal
          cells and dry weight of a algal mater-
          ial are long established methods.

       2  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
          igniting the sample to burn off the
          algal materials, leaving inert inorganic
          materials that are again weighed.
          The difference between initial dry weight
          and weight after ignition is attributed to
          algae.

       3  Any organic sediments, however,
          that settle on the  substrate along
          with the algae are processed also.
                                                                             28-7

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 Biological Integrity of Stream Communities
    Thus,  if organic wastes are present
    appreciable errors may enter into
    this method.

B   Chlorophyll Analysis

    1  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.

    2  The algae are scrubbed from the
       placed 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.

    3  Because the cholorophyll extracts fade
       with time, colorimetry should  be used
        for permanent records. For routine
        records, simple colorimeters will
        suffice.  At very high cholorophyll
        densities,  interference with colori-
        metry occurs,  which must be corrected
        through  serial dilution  of the sample
        or with a nomograph.
C   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 sur-
    face 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 particulate  organic matter is present
    in high concentrations, large populations
    of filamentous bacteria, stalked protozoa,
    and other nonchlorophyll bearing micro-
    organisms develop and the percentage
    of chlorophyll is then reduced.  If the
    biomass-chlorophyll a  relationship
    is expressed as a ratio (the autotro-
    phic index),  values greater than 100
    may result from organic pollution
    (Weber and McFarland, 1969; Weber,
    1973).
     Autotrophic Index
Ash-free Wgt (mg/m )
Chlorophyll a (mg/m2)
VIII  MACROINVERTEBRATE ANALYSES

  A  Taxonomic

     The taxonomic level to which animals are
     identified depends on the needs, experience,
     and available resources.  However, the
     taxonomic level to which identifications are
     carried in each major group should be
     constant throughout a given study.

  B  Biomass

     Macroinvertebrate biomass (weight of
     organisms per unit area) is a useful
     quantitative  estimation of standing crop.

  C  Reporting Units

     Data from quantitative samples may be used
     to obtain:

     1  Total  standing crop of individuals, or
       biomass,  or both per unit area or unit
       volume or sample unit, and

     2  Numbers  of biomass,  or both, of individual
       taxa per unit area or unit volume or sample
       unit.

     3  Data from devices  sampling a unit area
       of bottom will be reported in grams dry
       weight or ashrfree dry weight per square
       meter (gm/m ), or numbers of indi-
       viduals per square meter, or both.

     4  Data from multiplate samplers will be
       reported in terms of the total surface
       area of the plates in grams dry weight
       or ash-free dry weight or numbers of
       individuals per square meter, or both.

     5  Data from rock-filled basket samplers
       will be reported as grams dry weight
       or numbers of individuals per sampler,
       or both.
  28-8

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                                                 Biological Integrity of Stream Communities
IX  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.
    Tne 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 that
    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
    than  rivers.  The number of kinds
    present in a lake may be less than that
    found in a stream because of a more
    uniform 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, or stream  pool cannot be
   directly compared with that of a flowing
   stream riffle.

D  Extrapolation

   How can bottom-dwelling  macrofauna data
   be extrapolated to other environmental
   components?  It must be borne in mind
   that a component of the total environment
   is being sampled. If the sampled com-
   ponent 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 would be  expected to have a
   wide variety  of desirable  bottom fishes;
   when pollution reduces the number of bottom
   organisms,  a comparable reduction  would
   be expected in the number of fishes.  More-
   over, it would be logical to conclude that
   any  factor that eliminates all bottom organ-
   isms would eliminate most other aquatic
   forms of life. A clean stream with a wide
   variety of desirable  bottom organisms
   would be expected to permit a variety of
   recreational,  municipal and industrial  uses.

E  Expression of Data

   1  Standing crop and taxonomic composition

      Standing crop and numbers of taxa (types
      or kinds) in a community are highly
      sensitive to environmental perturbations
      resulting from the introduction of contam-
      inants.  These parameters, particularly
      standing crop,  may vary considerably in
      unpolluted habitats, where they may range
      from the typically high standing crop of
      littoral zones of glacial lakes to the
      sparse fauna of torrential soft-water
      streams.  Thus,  it is important that
      comparisons are made only between truly
      comparable environments.
                                                                                  28-9

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Biological Integrity of Stream Communities
2  Diversity

   Diversity indices are an additional tool
   for measuring the quality of the environ-
   ment and the effect of perturbation on
   the structure of a community of macro-
   invertebrates.   Their use is based on
   the generally observed phenomenon that
   relatively undisturbed environments
   support communities having large
   numbers of species with no individual
   species present in overwhelming
   abundance.  If the species in such a
   community are ranked on the basis of
   their numerical abundance, there will
   be relatively few species with large
   numbers of individuals and large
   numbers of species represented by only
   a few individuals.  Perturbation tends
   to reduce diversity by making the
   environment unsuitable for some species
   or by giving other species a competitive
   advantage.

3  Indicator-organism scheme ("rat-tailed
   maggot studies")

      a For this technique, the individual
        taxa are classified on the basis of
        their tolerance or intolerance to
        various levels of putrescible wastes.
        Taxa are classified according to
        their presence or absence of
        different environments as  deter-
        mined by field studies.  Some
        reduce data based on the presence
        or absence of indicator organisms
        to a simple numerical form for ease
        in presentation.
       i;
     b  Biologists  are engaging  in fruit-
        less exercise if they intend to make
        any decisions about indicator
        organisms, by operating at the
        generic level of macroinvertebrate
        identifications." (Resh and Unzicker)

4 Reference station methods

  Comparative or control station methods
  compare the qualitative characteristics
  of the fauna in clean water habitats with
  those of fauna in habitats subject to stress.
  Stations are compared on the basis of
  richness of species.
     If adequate background data are avail-
     able to an experienced investigator,
     these techniques can prove quite useful—
     particularly for the purpose of demon-
     strating the effects of gross to moderate
     organic contamination on the macro-
     invertebrate community.  To detect
     more subtle changes in the macroinver-
     tebrate community, collect quantitative
     data on numbers or biomass of organisms.
     Data on the presence of tolerant and
     intolerant taxa and richness of species
     may be effectively summarized for evalu-
     ation and presentation by means of line
     graphs, bar graphs, pie diagrams,
     histograms,  or pictoral diagrams.

 X   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.

XI AREAS IN WHICH BENTHIC STUDIES
   CAN BEST BE APPLIED

 A Damage Assessment or Stream Health

   If a stream is suffering from abuse the
   biota will so indicate.  A biologist can
   determine damages by looking at the
   "critter" assemblage in a matter  of
   minutes.  Usually, if damages are not
   found, it will not be necessary to  alert
   the remainder of the agency's staff,
   28-10

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                                                 Biological Integrity of Stream Communities
   pack all the equipment,  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 chemi-
   cal,  physical,  and engineering data can
   be used to predict the direction of future
   changes and to estimate the amount of
   pollutants that need to be removed from
   the waterways to make them productive
   and useful once more.

D The benthic macroinvertebrates are an
   easily used index to stream health that
   citizens may use in stream improve-
   ment programs.  "Adopt-a-stream"
   efforts have successfully used simple
   macroinvertebrate indices.

E The potential for restoring biological
   integrity in our flowing  streams using
   macroinvertebrates has barely been
   touched.

REFERENCES
1  Freed, J. Randall and Slimak,  Michael \\ .
    Biological Indices in Water Quality
    Assessment.  (In The Freshwater
    Potomac Aquatic Communities and
    Environmental Stresses). Interstate
    Commission on the Potomac  River
    Basin.  1978.

2  Hellawell,  J. M. ,  Biological Surveillance
    of Rivers. Water Research Center.
    Stevenage and Medenham.  333 pp.
    1978.

3  Hynes, H. B. N.  The Ecology of Running
    Waters.  Univ.  Toronto Press.  1970
 4  Mackenthun,  K. M.  The Practice of
    Water Pollution Biology.  FWQA.
    281 pp.   1969.

5  Weber, Cornelius I.,  Biological Field
    and Laboratory Methods for Measuring
    the Quality of Surface Waters and
    Effluents. U.S. Environmental Pro-
    tection Agency, NERC, Cincinnati,
    OH .   Environmental Monitoring Series
    670/4.73.001  July 1973

6  Keup,  L.  E. and Stewart, R.  K.  Effects
    of Pollution on Biota of the Pigeon River,
   North Carolina and Tennessee.  U.  S.  EPA,
   National  Field Investigations Center.  35 pp.
    1966.  (Reprinted 1973, National Training
   Center)

 7  v/uhrmann, K., Some Problems  and
    Perspectives in Applied Limnology
    Mitt. Internat.  Verein Limol. 20:324-402.
    1974.

,'i  Armitag, P. D.,  Machale, AngeluM.,  and
   Crisp,  Diane  C.  A Survey of Stream
   Invertebrates in the Cow Green Basin
   (Upper Teesdale) Before Inundation.
   Freshwater  Biol.  4:369-398. 1974.

9  Resh, Vincent H. andUnzicker,  John D.
   Water Quality Monitoring and Aquatic
   Organisms: the JWPCF 47:9-19.  1975.
 This outline was prepared by Lowell Keup,
 Chief, Technical Studies Branch,
 Division of Technical Support,  EPA,
 Washington, D. C.  20460, and revised by
 R. M. Sinclair, National  Training and
 Operational Technology Center, OWPO,
 US EPA,  Cincinnati, Ohio 45268.

 Descriptors: Aquatic Life,  Benthos, Water
 Quality,  Degradation, Environmental Effects,
 Trophic  Level, Biological Communities,
 Ecological Distributions
                                                                                        28-11

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                                       ECOLOGY PRIMER
                        (from Aldo Leopold's A SAND COUNTY ALMANAC)
 I   Ecology is a belated attempt to convert
     our collective knowledge of biotic materials
     into a collective wisdom of biotic manage-
     ment.

 II   The outstanding scientific discovery of
     the twentieth century is not television or
     radio, but rather the complexity of the
     land organism.

Ill   One of the penalties of an ecological edu-
     cation is that one lives alone in a world
     of wounds.  Much of the damage inflicted
     on land is quite invisible to laymen.  An
     ecologist must either harden his shell
     and make believe that the consequences
     of science are none of his business, or
     he must be the doctor who sees the marks
     of death in a community that believes
     itself well and does not want to be told
     otherwise.

 IV   Elcosystems  have been sketched out as
     pyramids, cycles,  and energy circuits.
     The concept  of land as an energy circuit
     conveys three basic ideas:

  A  That land is  not merely soil.

  B  That the native plants and animals kept
     the energy circuit open; others may or
     may not.

  C  That man-made changes are of a different
     order than evolutionary changes,  and have
     effects more comprehensive than is intend-
     ed or foreseen (See figures 1-4).

  D  To keep every cog and wheel is the first
     precaution of intelligent tinkering.

 V   The process of altering the pyramid for
     human occupation releases stored energy,
     and this often gives rise, during the
     pioneering period,  to a deceptive exuber-
     ance of plant and animal life,  both wild
     and tame.  These releases of biotic
     capital tend to becloud or postpone the
     penalties of violence.
 VI   A thing is right when it tends to preserve
      the integrity, stability, and beauty of the
      biotic community.  It is wrong when it
      tends otherwise.

 VII   Every farm is a textbook on animal
      ecology;  every stream is a textbook
      on aquatic ecology; conservation is
      the translation of the book.

VIII   There are three spiritual dangers in not
      owning a farm

   A  One is the danger of supposing that break-
      fast comes from the grocery.

   B  Two is that heat comes from the furnace.

   C  And three is that gas comes from  the
      pump.

 IX   Tn  general, the trend of the  evidence
      indicates that in land,  just as  in the
      fishes body, the symptoms may lie in
      one organ and the cause in another.  The
      practices we now call conservation are,
      to  a large extent  local alleviations of
      biotic pain.  They are necessary,  but
      they must not be confused with cures.

  X   An Atom at large in the biota is too free
      to  know freedom; an atom back in  the sea
      has forgotten it.  For every atom  lost to
      the sea,  the prairie pulls another  out of
      the decaying rocks.  The only certain
      truth is that its creatures must suck
      hard, live fast, and die often, lest its
      losses exceed its gains.

   REFERENCES

   1  Leopold, Luna B.  (ed.) Round River.
          Oxford University Press.  1953.

   2  Leopold, Aldo.  A Sand County Almanac.
          Oxford University Press.  1966.

   3  United States Environmental Protection
          Agency.  The Integrity  of Water.
          Washington,  B.C.  1977.
   BI..ECO. 26b. 9.79
                                         29-1

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Ecology Primer (from Aldo Leopold's A Sand County Almanac)
REFERENCES  (Continued)

4  United States Geological Survey.
      "Topographic Maps"
      Reston, Virginia.  1978.
This outline was prepared by R. M.  Sinclair
National Training and Operational Technology
Center, OWPO,  USEPA, Cincinnati, Ohio
45268.

Descriptor:  Ecology

                  Mapped In 1949

                 Figure 1.
                                                                  Revised In 1956
                  Figure 2.
                                                                               ^.if?^
                                                                               i-.l.t»3taji'i.-iV:i
                 Photorevised in 1969
                  Figure 3.
               Photorevised in 1973
                 Figure 4.
29-2

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                            GLOBAL ENVIRONMENTAL QUALITY
I  FROM LOCAL TO REGIONAL TO GLOBAL
   PROBLEMS

A  Environmental problems do not stop at
   national frontiers, or ideological barriers.
   P'ollution in the atmosphere and oceans
   taints all nations,  even those benignly
   favored by geography, climate, or natural
   resources.

   1  The smokestacks of one country  often
      pollute the air and water of another.

   2  Toxic effluents poured into an  inter-
      national river can kill fish in a
      neighboring nation and ultimately
      pollute international seas.

B  In Antarctica,  thousands of miles from
   .pollution sources, penguins and fish
   contain DDT in their fat.  Recent layers
   of snow and ice on the white continent
   contain measurable amounts of lead.
   The increase can be correlated with the
   earliest days of  lead smelting and com-
   bustion of leaded gasolines.  PCB's are
   universally distributed.

C  International cooperation, therefore, is
   necessary on many environmental fronts.

   1  Sudden accidents that chaotically
      damage the environment - such as oil
      spills from a tanker at sea - require
      international  cooperation both  for
      prevention and for cleanup.

   2  Environmental effects cannot be
      effectively treated by unilateral action.

   3  The ocean can no longer be considered
      a dump.

D  "One of the penalties of an ecological
   education is that one lives alone in a
   world of wounds.  Much of the damage
   inflicted on land is quite invisible to
   laymen.  An ecologist must either harden
   his shell and make believe that the conse-
   quences of science are none of his
II
business, or he must be the doctor who
sees the marks of death in a community that
believes itself well and does not want to
be told otherwise. "  Aldo Leopold

CHANGES IN ECOSYSTEMS ARE
OCCURRING CONTINUOUSLY
 A Myriad interactions take place at every
   moment of the day as plants and animals
   respond to variations in their surroundings
   and to each other.  Evolution has produced
   for each species,  including man, a genetic
   composition that limits how far that
   species can go in adjusting to sudden
   changes in its surroundings.  But within
   these limits the several thousand species
   in an ecosystem, or for that matter,  the
   millions in the biosphere,  continuously
   adjust to outside stimuli.  Since inter-
   actions are so numerous, they form long
   chains of reactions.

 B Small changes in one part of an ecosystem
   are likely to be felt and compensated for
   eventually throughout the system.
   Dramatic examples of change can be  seen
   where man has altered the course of
   nature.  It is vividly evident in his well-
   intentioned but poorly thought out tampering
   with river,  lake, and other ecosystems.

   1  The Aswan High Dam

   2  The St. Lawrence Seaway

   3  Lake Kariba

   4  The Great  Lakes

   5  Valley of Mexico

   6  California earthquake (Scientific
      American 3981, p. 333)

   7  Everglades and the Miami, Florida
      Jetport

   8  Copper hill, Tennessee (Copper Basin)

   9  (You may add others)
  BI. ECO. hum. 2f. 7. 79
                                                                                 30-1

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   'Global Environmental Quality
 C Ecosystem Stability

   1  The stability of a particular ecosystem
      depends on its diversity.  The more
      interdependencies in an ecosystem, the
      greater the chances that it will be able
      to compensate for changes imposed
      upon it.

   2  A cornfield or lawn has little natural
      stability.   If they are not  constantly
      and carefully cultivated, they will not
      remain cornfields or lawns  but will
      soon bs overgrown with a wide variety
      of hardier plants constituting a more
      stable ecosystem.

   3  The  chemical elements that make up
      living systems also depend on complex,
      diverse sources to prevent cyclic
      shortages or oversupply.

   4  Similar diversity is essential for the
      continued functioning of the cycle by
      which atmospheric nitrogen is made
      available to allow life to exist.  This
      cycle depends on a wide variety of
      organisms, including soil bacteria and
      fungi,  which are often destroyed by
      pesticides in the soil.

   5  A numerical expression of diversity
      is often used in defining stream water
      quality.

D  Biological Pollution

   Contamination of living native biotas by
   introduction of exotic life forms has been
   called biological pollution by Lachner
   et  al. Some of these introductions are
   compared to contamination as severe as
   a dangerous chemical release.  They
   also threaten to replace known  wildlife
   resources with species of little or un-
   known value.

1  Tropical areas have especially been
   vulnerable.  Florida is referred to as
   "a  biological  cesspool of introduced life. "
    2  Invertebrates

       a  Asian Clams have a pelagic veliger
          larvae,  thus, a variety of hydro
          installations are vulnerable to sub-
          sequent pipe clogging by the adult
          clams.

       b  Melanian snails are intermediate
          hosts for various trematodes
          parasitic on man.

    3  Vertebrates

       a  At least 25 exotic species of fish
          have been established in North
          America.

       b  Birds,  including starlings and
          cattle egrets.

       c  Mammals,  including nutria.

    4  Aquatic plants

       Over twenty common exotic species
       are growing wild in the United States.
       The problem of waterway clogging has
       been especially severe in parts of the
       Southeast.

    5  Pathogens and Pests

       Introduction of insect pests and tree
       pathogens have had severe economic
       effects.
Ill   LAWS OF ECOLOGY

 A Four principles have been enunciated by
    Dr.  Barry Commoner.

    1  Everything is connected to everything
       else.

    2  Everything must go somewhere.

    3  Nature knows best.

    4  There is no such thing as a free lunch.

 B These  may be summarized by the principle,
    "you can't do just one thing.
30-2

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                                                               Global Environmental Quality
IV THE THREE PRINCIPLES OF
   ENVIRONMENTAL CONTROL (Wolman)

A  You can't escape.

B  You have to organize.

C  You have to pay.

V  LEOPOLD'S PRINCIPLE OF BIOTIC
   CAPITAL

   "The releases of  biotic capital tend to
   becloud or  postpone the penalties of
   violence".  Can you apply this to other
   parts of this outline?

VI POLLUTION COMES IN MANY PACKAGES

A  The sources of air, water, and land
   pollution are interrelated and often
   interchangeable.

   1   A single source may pollute the air
      with smoke and chemicals, the land
      with solid wastes,  and a river or lake
      with chemical  and other wastes.

   2   Control of air  pollution may produce
      more solid wastes,  which then pollute
      the  land or water.

   3   Control of wastewater  effluent may
      convert it into solid wastes,  which
      must be disposed of on land, or by
      combustion to  the air.

   4  Some pollutants - chemicals, radiation,
      pesticides - appear in all media.

B  "Disposal" is as  important and as costly
   as purification.

VII PERSISTENT CHEMICALS IN THE
   ENVIRONMENT

Increasingly complex manufacturing processes,
coupled with rising industrialization, create
greater amounts of exotic wastes potentially
toxic to humans and aquatic  life.

They may also be teratogenic (toxicants
responsible for changes  in the embryo with
resulting birth defects, ex., thalidomide),
mutagenic (insults which produce mutations,
ex., radiation), or carcinogenic (insults
which induce cancer, ex., benzopyrenes) in
effect.  Most carcinogens are also muta-
genic.   For all of these there are no thresh-
hold levels as in toxicity.  Fortunately there
are simple rapid tests for mutagenicity using
bacteria.   Tests with animals are not always
conclusive.

A  Metals - current levels of cadmium, lead,
   and other substances  are  a growing concern
   for they affect not only fish and wildlife but
   ultimately man himself.   Mercury pollution,
   for example,  has become  a serious  problem,
   yet mercury has been present on earth since
   time immemorial.

B  Pesticides

   1  A pesticide and its metabolites may
     move through an ecosystem in many
     ways.  Hard (pesticides which are
     persistent, having a long half-life in
     the environment includes the organo-
     chlorines, ex., DDT) pesticides
     ingested or otherwise borne by the
     target species will stay in the
     environment,  possibly to be recycled
     or concentrated further through the
     natural action of food chains if the
     species is eaten.   Most of the volume
     of pesticides do not reach their target
     at all.

   2  Biological magnification

     Initially, low levels of  persistent
     pesticides  in air,  soil,  and water
     may be concentrated at every step
     up the food chain.   Minute aquatic
     organisms and scavengers, which
     screen water and bottom mud having
     pesticide levels of a few parts per
     billion, can accumulate levels
     measured in parts per  million -
     a thousandfold increase.  The sediments
     including fecal deposits are continuously
     recycled by the bottom  animals.

     a  Oysters, for instance, will con-
        centrate DDT 70, 000 times higher
        in their tissues than it's concentration
        in surrounding water.  They can
        also partially cleanse themselves
        in water free of DDT.
                                                                                 30-3

-------
 Global Environmental Quality
      b  Fish feeding on lower organisms
         build up concentrations in their
         visceral fat which may reach several
         thousand parts per million and levels
         in their edible flesh of hundreds of
         parts per million.

      c  Larger animals, such as fish-
         eating gulls and other birds,  can
         further concentrate the chemicals.
         A survey on organochlorine residues
         in aquatic birds in the Cnadian
         prairie provinces showed that
         California and ring-billed gulls were
         among the most contaminated.
         Since gulls breed in colonies, breed-
         ing population changes can be
         detected and related to levels of
         chemical contamination.  Ecological
         research on colonial birds to monitor
         the effects of chemical pollution on
         the environment is useful.

C  "Polychlorinated biphenyls" (PCB's).
   PCB's are used in plasticizers, asphalt,
   ink, paper, and a host of other products!
   Action has been taken to curtail their
   release to the environment, since their
   effects are similar to hard pesticides.

D  Other compounds which are toxic and
   accumulate in the ecosystem:

   1  Phalate esters - may interfere with
      pesticide analyses

   2  Benzopyrenes

E  Refractory compounds like pentachloro-
   phenal and hexachlorophene are poorly
   removed by both water treatment plants
   and wastewater treatment plants.

F  It is estimated that 80% to 90% of cancers
   are caused by chemicals both in the work-
   ing environment and total environment.
   This is shown by high risk industries and
   living areas.

G  Most of the problems of persistent  and
   dangerous chemicals in the environment
   are "after-the-fact".  The solution
   obviously  is tied to prevention.  This is
   extremely complicated by economics,
  H
ignorance, and decision as to risks
involved.  Some advertising slogans now
have more than an intended meaning.

Wittingly or unwittingly we have all become
a King Mithridates.  And even a fish is no
longer a fish!
VIII ACID RAIN

    Acid rain is also becoming a problem
    in this country.

IX  EXAMPLES OF  SOME EARLY WARNING
    SIGNALS THAT  HAVE BEEN DETECTED
    BUT FORGOTTEN, OR IGNORED.

  A  Magnetic micro-spherules in lake sediments
     now used to detect changes in industriali-
     zation indicate our slowness to recognize
     indicators of environmental change.

  B  Salmonid fish kills in poorly buffered clean
     lakes in Sweden.  Over the past years there
     had been  a successive increase of SO? in the
     air and precipitation. Thus,  air-borne con-
     tamination from industrialized European
     countries had a  great influence on previously
     unpolluted waters and their life.

  C  Minimata, Japan and mercury pollution.

  D  Organochlorine  levels in commercial and
     sport fishing stocks,  ex.,  the lower
     Mississippi River fish kills.

  .X  SUMMARY

  A  Ecosystems of the world are linked
     together through biogeochemical cycles
     which are determined, by patterns of
     transfer and concentrations of substances
     in the biosphere and  surface rocks.

  B  Organisms determine or strongly influence
     chemical and physical characteristics of
     the atmosphere, soil, and waters.

  C  The inability of man  to adequately predict
     or control his effects on the environment
     is indicated by his lack of knowledge con-
     cerning the net effect of atmospheric
     pollution on the  earth's climate.
   30-4

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                                                          Global Environmental Quality
D  Serious potential hazards for man which
   are all globally dispersed,  are radio-
   nuclides, organic chemicals, pesticides,
   and combustion products.

E  Environmental destruction is in lockstep
   with our population growth.

REFERENCES

1  Lachner, Ernest A., Robins,  C. Richard,
   and Courtenay,  Walter R.,  Jr.
   Exotic Fishes and Other Aquatic
   Organisms Introduced into North
   America.  Smithsonian Contrib.  to
   Zool.   59:1-29.  1970

2  Commoner,  Barry.  The Closing Circle,
   Nature, Man, and Technology.  Alfred
   A. Knopf.  326 p.  1971.

3.  Dansereau, Pierre ed.  Challenge for
   Survival.  Land, Air, and Water for
   Man in Megalopolis,  Columbia Univ.
   Press.  235  p.  1970.

4  Wiens, John A. ed. Ecosystem Structure
   and Function.  Oregon State Univ. Press.
   176 p.  1972.

5  Leopold, Aldo.  A Sand County Almanac
   with Essays  on Conservation from
   Round River. Sierra Club/Ballantine
   Books.  295  p.  1970.

6  Whiteside, Thomas.  The Pendulum and
   the Toxic Cloud, The course of Dioxin
   Contamination.  Yale University Press.
   1979.

7  Butler, G. C. (editor) Principles of
   Ecotoxicology..  Scope 12.   Int. Council
   of Sci. Unions.  J. Wiley & Sons.  1978.
This outline was prepared by R. M. Sinclair,
National Training and Operational Technology
Center, MOTD,  OWPO, USEPA, Cincinnati,
Ohio  45268.
Descriptors:
Ecosystems
Environmental Effects,
                                                                                     30-5

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                          THE EFFECTS OF POLLUTION ON LAKES
I  INTRODUCTION

The pollution of lakes inevitably results in a
number of undesirable changes in water
quality which are directly or indirectly
related to  changes in the aquatic  community.

A  Industrial Wastes may contain the following:

   1  Sewage

   2  Dissolved organics--synthetics,  food
      processing wastes, etc.

   3  Dissolved minerals--salts,  metals
      (toxic and nontoxic), pigments,  acids, etc.

   4  Suspended solids--fibers, minerals,
      degradable and non-degradable organics

   5  Petroleum products--oils, greases

   6  Waste heat

B  The Materials in Domestic Wastes which
   affect Water Quality are:

   1  Pathogenic fecal microorganisms

   2  Dissolved nutrients: minerals,  vitamins,
      and other dissolved organic substances

   3  Suspended solids (sludge)--degradable
      and non-degradable organic materials

   4  Toxic materials which line the counters
      of supermarkets, drugstores, and
      discount stores

C  Pollution and Eutrophication

   The discharge of domestic wastes often
   renders the  receiving water unsafe for
   contact water sports and water supplies.
   For example, some beaches on the eastern
   seaboard and in metropolitan regions of
   the Great Lakes  are unfit for swimming
   because of high coliform counts.  Other
   effects of domestic pollution include
   changes in the abundance and composition
   of populations of aquatic organisms.
    1  As the nutrient level increases,  so does
      the rate of primary production.

    2  Shore-line algae and rooted aquatics
      become more abundant.  For example,
      problems  have been experienced with
      Cladophora and Dichotomosiphon along
      the shores of Lakes Ontario,  Erie,
      and Michigan.  These growths interfere
      with swimming, boating, and fishing,
      and cause odors when the organisms
      die and decay.

    3  The standing crop of phytoplankton
      increases, resulting in higher counts
      and greater chlorophyll content.
      Increases in phytoplankton  abundance
      may result in taste and odor problems
      in water supplies, filter clogging,
      high turbidity, changes in water color,
      and oxygen depletion in the hypolimnion.

    4  Populations of fish and larger swimming
      invertebrates increase, based on the
      increase in basic food production.

    5  Changes in dominant species

      a Diatom communities give way to
        blue-greens.  Toxic blue-greens may
        pose a problem.

      b Zooplankton changes include
        replacement of Bosmina coregoni
        by B^ longirostris.

      c Trout and whitefish are replaced by
        perch,  bass, and rough fish.

      d Hypolimnion becomes anaerobic in
        summer; bottom  sludge buildup
        results in loss of fish food organisms,
        accompanied by increase in density
        of sludgeworms (oligochaeta).
II   HISTORICA L REVIEW

 The cultural eutrophication of a number of
 lakes in Europe and America has been well
 documented.

 A  Zurichsee,  Switzerland
WP. LK. Id.3. 80
                                                                                       31-1

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The Effects of Pollution on Lakes
   1  1896 - sudden increase in Tabellaria
      fenestrata

   2  1898 - sudden appearance of Oscillatoria
      rubescens which displaced Fragilaria
      capucina

   3  1905 - Melosira islandica var. helvetica
      appeared

   4  1907 - Stephanodiscus hantzschil
      appeared

   5  1911 - Bosmina longirostris replaced
      B.  coregoni

   6  1920
      1924 - O. rubescens occurred in great
      quantities

   7  1920 - milky-water phenomenon;
      precipitation of CaCCL crystals (40[i)
      due to pH increase resulting from
      photosynthesis

   8  Trout and whitefish replaced by perch,
      bass, and rough fish
          B Hallwilersee,  Switzerland

             1  1897 - Oscillataria rubescens not
                observed up to this time

             2  1898 - O. rubescens bloomed,
                decomposed, formed H S,  killing off
                trout and whitefish

          C Lake Windermere, England (core study)

             1  Little change in diatoms from glacial
                period until recent times

             2  Then A sterionella appeared,  followed
                by Synedra

             3  About 200 years ago, A sterionella
                again became abundant

             4 A sterionella abundance ascribed to
                domestic wastes

          D Finnish Lakes

             Aphanizomenon,  Coelosphaerium,
             Anabaena,  Microcystis, are the most
             common indication of eutrophy.
                TABLE 1  CHANGES IN PHYSIO-CHEMICAL PARAMETERS

                                   Zurichsee, Switzerland

              Parameter                  Date
              Chlorides
              Dissolved organics
       1888
       1916

       1888
       1914
        Value

       1.3mg/l
       4.9 mg/1

       9.0mg/l
      20.0 mg/1
              Secchi Disk
              Dissolved oxygen, at
                100 M, mid-summer
before 1910
1905 - 1910
1914 - 1928
1910 - 1930
1930 - 1942
  Max.

  16. 8M
  10. OM
  10. OM

Minimum
    Min.
    3. 1M
    2.1M
    1.4M
100% saturation
  9% saturation
31-2

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                                                             The Effects of Pollution on Lakes
E  Linsley Pond, Connecticut

   1  Species making modern appearance
      include Asterionella formosa,
      Cyclotella glomerata, Melosira
      italica, Fragilaria crotonensis,
      Synedra ulna

   2  Asterionella formosa and Melosira
      italica were considered by Patrick to
      indicate high dissolved organics

   3  Bosmlna coregoni replaced by B.
      longirostris

F  Lake Monona, Wisconsin

   1  Began receiving treated sewage in 1920,
      developed blue-green algal blooms.

G  Lake Washington,  Washington

   1  1940 - Bosmina longirostris appeared

   2  1955 - Oscillatoria rubescens seen for
      the first time,  and constituted 96% of
      phytoplankton,  July 1

H  Lake Erie

   1  Phytoplankton counts at Cleveland have
      increased steadily from less than
      500  cells/ml in the 1920's to over
      1500 cells/ml in the 1960's

   2  Abundance of burrowing mayflies
      (Hexagenia  spp.)in Western Lake Erie
      decreased from 139/m2 in 1930,  to
      less than  1/m2 in  1961.

I   Lake Michigan

   1  Milky water observed in south end, and
      in limnetic  region in mid-1950's and
      again in 1967.

   2  During the period  1965-1967 the Chicago
      water treatment plant has found it
      necessary to increase the carbon dosage
      from 23 Ibs/mil gal to 43 Ibs/mil gal,
      and the chlorine dosage from 20 Ibs/mil
      gal to 25 Ibs/mil gal.
       Phytoplankton counts in the south end
       now exceed 10, 000/ml during the
       spring bloom.
Ill  FACTORS AFFECTING THE RESPONSE
    OF LAKES TO POLLUTION INCLUDE:

 A Depth-surface area ratio:  A large
    hypolimnion will act as a reservoir to
    keep nutrients from recirculating in the
    trophogenic zone during the summer
    stratification period.  Rawson found an
    inverse relationship between the standing
    crop of plankton, benthos,  and fish,  and
    the mean  depth.

 B Climate:  Low annual water temperatures
    may restrict the response  of the
    phytoplankton to enrichment.

 C Natural color or turbidity:  Dystrophic
    (brown-water) lakes may not develop
    phytoplankton blooms because of the low
    transparency of the water.
IV  TROPHIC LEVEL

  Except in cases where massive algal blooms
  occur,  the trophic status of lakes is often
  difficult to determine.  Core studies are
  used to determine trends in diatom populations
  which  might indicate changes in nutrient
  levels over an extended period of time.
'V  CONTROL OF POLLUTION

 The success of efforts to arrest the
 eutrophication process,  and where desirable,
 reduce the trophic level of a lake, will
 depend on a thorough knowledge of the
 nutrient budget.

 A  Significant quantities of nutrients may
    enter a lake from one or more of the
    following sources:

    1  Rainfall

    2  Ground water
                                                                                   31-3

-------
The Effects of Pollution on Lakes
            TABLE 2  PARAMETERS COMMONLY USED TO DESCRIBE CONDITIONS
          1  Transparency

          2  Phosphorus

          3  NO  - Nitrogen
               O
          4  Minimum annual
              hypolimnetic oxygen concentration

          5  Chlorophyll

          6  Ash-free weight of seston

          7  Phytoplankton count

          8  Phytoplankton quotients

            a  number of species of Chlorococcales
                 number of species of Desmids
              Oligotrophic Condition

                 >  10 meters

                 <  lug/1
                 <  200  ng/1
              near 100% saturation

                           3
                 <  1 mg/m

                 <  0. 1  mg/1
                 <  500/ml
                 <1
            b Myxophycease+Chlorococcales+Centrales+Euglenaceae <1
                                  Desmidaceae

            c Centrales                                              0-0.2
              Pennales

         9  Phytoplankton species present (see outline on
            plankton in oligotrophic lakes).
   3 Watershed runoff

   4 Shoreline domestic and industrial outfalls

   5 Pleasure craft and commercial vessels

   6 Waterfowl

   7 Leaves,  pollen, and other organic
     debris from riparian vegetation

B  The supply of nutrients from "natural"
   sources in  some cases may be greater
   than that from cultural sources, and be
   sufficient to independently cause a rapid
   rate of eutrophication regardless of the
   level of efficiency of treatment of domestic
   and industrial wastes.
C  Many methods have been employed to
   treat the symptoms, reduce the
   eutrophication rate, or completely
   arrest and even reverse the eutrophication
   process.

   1 Use of copper sulfate,  sodium arsenite,
     and organic algicides: It is not
     economically feasible to use algicides
     in large lakes.

   2 Addition of carbon black to reduce
     transparency.  This is likewise
     frequently impractical.

   3 Harvesting algae by foam fractionation
     or chemical precipitation.
 31-4

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                                                           The Effects of Pollution on Lakes
      4 Reducing nutrient supply by (a) removal
        of N and P from effluents,  (b) diversion
        of effluents, and  (c) dilution with
        nutrient-poor water.

   D  Examples of lakes where control has been
      attempted by  reducing the nutrient supply,
      are:

      1 Lake Washington, Seattle

        The natural water supply for this lake
        is nutrient poor
        (Ca = 8 mg/1,  P < 5 Mg/1, TDS=76mg/l).
        Since the turnover time of the water in
        this lake is only three years, it was
        expected that diversion of sewage
        would result in a  rapid improvement of
        water quality.  Diversion began in 1963,
        and improvements were noticeable by
        1965 - including an  increase in
        transparency,  and a reduction in seston,
        chlorophyll, and  epilimnetic phosphorus.


                   TABLE  3

PHOSPHORUS REDUCTION  IN LAKE WASHINGTON
                                 3  Lake Tahoe

                                   This lake is still decidedly oligotrophic.
                                   To maintain its high level of purity,
                                   tertiary treatment facilities were
                                   installed in the major sewage treat-
                                   ment plant, and construction is now
                                   underway to transport all domestic
                                   wastes out of the lake basin.
                               REFERENCES
    Year

    1963

    1964

    1965
Maximum phosphorus in
   upper 10 meters
       (Mg/D	
         70

         66

         63
      2  Green Lake, Washington

        The lake has a long history of heavy
        blooms of blue-green algae.  Beginning
        in 1959,  low-nutrient city water was
        added to  the lake, reducing the  con-
        centration of phosphorus by 70% in the
        inflowing water.  By 1966,  the lake had
        been flushed three times. Evidence of
        improvement in water quality was noted
        in 1965,  when Aphanizomenon was
        replaced by Gleotrichia.
                                                                    Eds.
1  Ayers, J. C. and Chandler,  D. C.
      Studies on the environment and
      eutrophication of Lake Michigan.
      Special Report No. 30.  Great Lakes
      Research Division, Institute of
      Science and Technology,  University
      of Michigan,  Ann Arbor.  1967.

2  Brezonik, P.L., Morgan, W.H.,
      Shannon,  E.E., and Putnam, H.D.
      Eutrophication factors in North
      Central Florida Lakes.  University
      of Florida Water Res. Center.
      Pub. #5,  101 pp.   1969.

3  Carr, J.F., Hiltunen,  J. K.  Changes
      in the bottom fauna of Western Lake
      Erie from 1930 to 1961.   Limnol.
      Oceanogr.  10(4):551-569.   1965.

4  Frey, David G.  Remains of animals
      in Quaternary lake and bog sediments
      and their interpretation.
      Schweizerbartsche Verlagsbuchhandlung.
      Stuttgart.   1964.

5  Edmondson, W.T., and Anderson, G.C.
      Artificial eutrophication of Lake
      Washington.  Limnol. Oceanogr.
      l(l):47-53.    1956.

6  Fruh, E.G.   The overall picture of
      eutrophication.   Paper presented
      at the Texas Water and Sewage
      Works Association's Eutrophication
      Seminar, College Station, Texas.
      March 9, 1966.

7  Fruh, E.G., Stewart, K.M., Lee,  G.F.,
      and Rohlich, G.A.   Measurements
      of eutrophication and trends.
      J.W.P.C.F.  38(8):1237-1258.  1966.
                                                                                    31-5

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 The Effects of Pollution on Lakes
 8  Hasler,  A.D.   Eutrophication of lakes
      by domestic drainage.  Ecology
      28(4):383-395.  1947.

 9  Hasler,  A.D.   Cultural Eutrophication
      is Reversible.  Bioscience 19(5):
      425-443.   1969.

10  Herbst,  Richard P.  Ecological Factors
      and the Distribution of Cladophora
      glomerata in the Great Lakes.
      Amer. Midi. Nat. 82(l):90-98.   1969.

11  National Academy of  Sciences.
      Eutrophication:  Causes,  Consequences,
      Correction.   661pp.   1969.
      (Nat. Acad.  Sci. ,2101 Constitution
      Avenue,  Washington, DC 20418, 13.50).
12  Neel,  Joe Kendall.   Reservoir
       Eutrophication and Dystrophication
       following Impoundment.   Reservoir
       Fisheries Res.  Symp.  322-332.

13  Oglesby,  R.T. and Edmondson, W.T.
       Control of Eutrophication.
       J.W.P.C.F. 38(9):1452-1460.   1966.

14  Stewart,  K.M. and Rohlich,  G.A.
       Eutrophication - A Review.
       Publication No. 34,  State Water
       Resources Control Board, The
       Resources Agency, State  of California.
       1967.

 This outline was prepared by C. I. Weber,
 Chief, Biological Methods Section,
 Analytical Quality Control Laboratory,
 NERC, EPA,.  Cincinnati, OH 45268.

 Descriptor: Eutrophication
 31-6

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                               APPLICATION OF BIOLOGICAL DATA
 I   ECOLOGICAL DATA HAS TRADITIONALLY
    BEEN DIVIDED INTO TWO GENERAL
    CLASSES:

 A  Qualitative - dealing with the taxonomic
    composition of communities

 B  Quantitative - dealing with the  population
    density or rates of processes occurring
    in the communities

    Each kind of data has been useful in its own
    way.
II  QUALITATIVE DATA

 A Certain species have been identified as:

   1  Clean water (sensitive) or oligotrophic

   2  Facultative,  or tolerant

   3  Preferring polluted regions
      (see:  Fjerdinstad 1964, 1965; Gaufin
      & Tarzwell 1956; Palmer 1963,  1969;
      Rawson 1956; Teiling 1955)

 B Using our knowledge  about ecological
   requirements the biologist may compare
   the species present

   1  At different stations in the same river
      (Gaufin 1958) or lake (Holland 1968)

   2  In different rivers or lakes (Robertson
      and Powers 1967)

   or changes in the species in a river or/lake
   over a period of several years.  (Carr
   & Hiltunen 1965; Edmondson & Anderson
   1956; Fruh, Stewart,  Lee & Rohlich 1966;
   Hasler 1947).

 C Until comparatively recent times taxonomic
   data were not subject to statistical treat-
   ment.
Ill  QUANTITATIVE DATA; Typical
    Parameters of this type include:
                                 2
 A Counts - algae /ml; benthos/m ;
    fish/net/day
                 3
 B Volume - mm  algae/liter

 C Weight - dry wgt; ash-free wgt.

 D Chemical content - chlorophyll;
    carbohydrate; ATP; DNA; etc.

 E Calories (or caloric equivalents)

 F Processes -  productivity; respiration
IV  Historically, the chief use of statistics
 in treating biological data has been in the
 collection and analysis of samples for these
 parameters.  Recently,  many methods have
 been devised to convert taxonomic data into
 numerical form to permit:

 A Better communication between the
    biologists and other scientific disciplines

 B Statistical treatment of taxonomic data

 C In the field of pollution biology these
    methods include:

    1  Numerical ratings of organisms on the
       basis of their pollution tolerance

       (saprobic valency:  Zelinka & Sladecek
       1964)

       (pollution index: Palmer  1969)

    2  Use of quotients or ratios of species in
       different taxonomic groups (Nygaard
       1949)
  BI.EN. 3a. 6.76
                                                                                     32-1

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Application of Biological Data
   3  Simple indices of community diversity:

      a  Organisms are placed in taxonomic
         groups which behave similarly under
         the same ecological conditions.  The
         number of species in these groups
         found at "healthy" stations is com-
         pared to that 'found at "experimental"
         stations.   (Patrick 1950)

      b  A truncated log normal curve is
         plotted on the basis of the number
         of individuals per diatom species.
         (Patrick, Hohn, & Wallace 1954)
                                          j  Information theory:

                                             The basic equation used for
                                             information theory applications was
                                             developed by Margalef (1957).
                                                                N!
                                                 _
                                                 N   g2  N  !  N'. . . . N  !
                                             where I - information/individual;
                                             N , N  . . .N  are the number of
                                             individuals in species a,  b,  ...
                                             s,  and N is their sum.
      c  Sequential comparison index.
         (Cairns, Albough, Busey & Chanay
         1968).  In this technique, similar
         organisms encountered sequentially
         are grouped into "runs".
      SCI =
                       runs
             total organisms examined
        Ratio of carotenoids to chlorophyll
        in phytoplankton populations:


        °D430/°D665(Margalef 1968)
               OD67()(Tanaka,  et al 1961)
      e  The number of diatom species present
        at a station is considered indicative
        of water quality or pollution level.
        (Williams 1964)
        number of species (S)
        number of individuals (N)
        _ number of species (S)    _
        square root of number of individuals (N/ N)
                                             This equation has also been used
                                             with:

                                             1)  The fatty acid content of algae
                                                (Mclntire, Tinsley, and Lowry
                                                1969)

                                             2)  Algal productivity (Dickman 1968)

                                             3)  Benthic biomass (Wilhm 1968)
         S - 1
                (Menhlnick 1964)
        d =
En. (n. - 1) (Simpson 1949)

N (N -  1)
        where n. = number of individuals
                   belonging to the i-th species,
                   and
               N = total number of individuals
REFERENCES

1  Cairns,  J., Jr., Albough,  D.W.,
      Busey, F, and Chaney, M.D.
      The sequential comparison index -
      a simplified method for non-biologists
      to estimate relative differences in
      biological diversity in stream pollution
      studies.  J. Water Poll. Contr. Fed.
      40(9):1607-1613.   1968.

2  Carr,  J. F. and Hiltunen, J.K.   Changes
      in the bottom fauna of Western Lake
      Erie  from 1930 to 1961.   Limnol.
      Oceanogr. 10(4):551-569.   1965.

3  Dickman,  M.  Some indices of diversity.
      Ecology 49(6): 1191-1193.  1968.
  32-2

-------
                                                             Application of Biological Data
 4  Edmondson, W.T. and Anderson, G. C.
       Artificial Eutrophication of Lake
       Washington.   Limnol.  Oceanogr.
       l(l):47-53.  1956.

 5  Fjerdingstad,  E.  Pollution of Streams
       estimated by benthal phytomicro-
       organisms.  I.  A saprobic system
       based on communities of organisms
       and ecological factors.  Internat'l
       Rev. Ges. Hydrobiol. 49(1):63-131.1964.

 6  Fjerdingstad,  E.   Taxonomy and saprobic
       valency  of benthic phytomicro-
       organisms.  Hydrobiol. 50 (4):475-604.
       1965.

 7  Fruh, E.G.,  Stewart,  K.M.,  Lee,  G.F.
       and Rohlich, G.A.   Measurements of
       eutrophication  and trends.   J. Water
       Poll. Contr. Fed. 38(8):1237-1258.
       1966.

 8  Gaufin, A.R.   Effects of Pollution on a
       midwestern stream.   Ohio J. Sci.
       58(4):197-208.    1958.

 9  Gaufin, A.R.  and Tarzwell, C.M.   Aquatic
       macroinvertebrate communities as
       indicators  of organic pollution in Lytle
       Creek.  Sew.  Ind. Wastes.  28(7):906-
       924.  1956.

10  Hasler, A.D.  Eutrophication of lakes by
       domestic drainage.   Ecology 28(4):383-
       395. 1947.

11  Holland, R.E. Correlation of Melosira
       species with trophic conditions in Lake
       Michigan.   Limnol.   Oceanogr.
       13(3):555-557.    1968.

12  Margalef,  R.   Information theory in
       ecology.   Gen. Syst.   3:36-71.  1957.

13  Margalef,  R.   Perspectives in ecological
       theory.  Univ. Chicago Press.   1968.

14  Mclntire, C.D.,  Tinsley,  I.J.  and
       Lowry, R.R.   Fatty acids in lotic
       periphyton: another measure of
       community  structure.  J. Phycol.
       5:26-32.   1969.
15  Menhinick, E.F.   A comparison of some
       species - individuals diversity indices
       applied to samples of field insects.
       Ecology 45:859.  1964.
16  Nygaard, G.   Hydrobiological studies in
       some ponds and lakes.   II.  The
       quotient hypothesis and some new or
       little-known phytoplankton organisms.
       Klg. Danske Vidensk.  Selsk. Biol.
       Skrifter 7:1-293.  1949.

17  Patten, B.C.  Species diversity in net
       plankton of Raritan Bay.   J.  Mar.
       Res.  20:57-75.   1962.

18  Palmer,  C.M.  The effect of pollution on
       river algae.   Ann. New York Acad.
       Sci.   108:389-395.   1963.

19  Palmer,  C.M.   A composite rating of
       algae tolerating organic pollution.
       J. Phycol. 5(l):78-82.   1969.

20  Patrick,  R.,  Hohn, M.H. and Wallace,
       J.H.   A new method for determining
       the pattern of the diatom flora.   Not.
       Natl. Acad. Sci.,  No. 259.
       Philadelphia.    1954.

21  Rawson,  D. S.  Algal indicators of trophic
       lake types. Limnol. Oceanogr.
       1:18-25.   1956.

22  Robertson, S. and Powers, C.F.
       Comparison of the distribution of
       organic matter in the five  Great Lakes.
       in: J. C. AyersandD.C.  Chandler,
       eds.   Studies on the environment and
       eutrophication of Lake Michigan.
       Spec.  Rpt. No.  30,  Great  Lakes Res.
       Div.,Inst. Sci. &Techn.,  Univ.
       Michigan,  Ann Arbor.   1967.

23  Simpson, E.H.   Measurement of diversity.
       Nature (London) 163:688.   1949.

24  Tanaka, O. H.,  Irie,  S. Izuka,  and Koga,  F
       The fundamental investigation on the
       biological productivity in the Northwest
       of Kyushu.  I. The  investigation of
       plankton.  Rec. Oceanogr.  W. Japan,
       Spec.  Rpt. No.  5, 1-57.   1961.
                                                                                       32-3

-------
 Application of Biological Data
25  Tailing, E.   Some mesotrophic phyto-
       plankton indicators.  Proc. Intern.
       Assoc.  Limnol.    12:212-215.   1955.

26  Wilhm, J. L.   Comparison of some
       diversity indices applied to populations
       of benthic macroinvertebrates in a
       stream receiving organic wastes.  J.
       Water Poll. Contr. Fed.  39(10):1673-1683.
       1967.

27  Wilhm, J. L.   Use of biomass units in
       Shannon's formula.  Ecology 49:153-156.
       1968.
28  Williams, L. G.   Possible relationships
      between diatom numbers and water
      quality.   Ecology 45(4):810-823.   1964.
29 .Zelinka, M. and Sladecek, V.   Hydro-
      biology for water management.
      State Publ. House for Technical
      Literature, Prague.   122 p.   1964.
 This outline was prepared by C.I.  Weber,
 Chief,  Biological Methods Section, Analytical
 Quality Control Laboratory, NERC,  EPA,
 Cincinnati, Ohio, 45268
                                                  Descriptors:  Analytical Techniques, Indicators
  32-4

-------
            SIGNIFICANCE OF "LIMITING FACTORS" TO POPULATION VARIATION
 I  INTRODUCTION

 A All aquatic organisms do not react uniformly
   to the various chemical,  physical and
   biological features in their environment.
   Through normal evolutionary processes
   various organisms have become adapted
   to certain combinations of environmental
   conditions.  The successful development
   and maintenance of a population or community
   depend upon  harmonious ecological balance
   between environmental conditions and
   tolerance of  the organisms to variations
   in one or more of these conditions.

 B A factor whose presence  or absence  exerts
   some restraining influence upon a population
   through incompatibility with species
   requirements or tolerance  is said to be a
   limiting factor. The principle of limiting
   factors is one of the major aspects of the
   environmental control of  aquatic organisms
   (Figure  1).
II  PRINCIPLE OF LIMITING FACTORS

 This principle rests essentially upon two basic
 concepts.  One of these relates organisms to
 the environmental supply of materials essential
 for their growth and development.  The second
 pertains to the tolerance which organisms
 exhibit toward environmental conditions.
                /UNLIMITED GROWTH
                '              DECREASE IN
POPULATION GROW
/
	 LTWTfATIOMNS
EQUILIBRIUM WITH
/X" xs ENVIRONMENT
/ ^^ INCREASE IN
'/ ^^ "TlMTtXriONS
,/ * POPULATION DECLINE
                  TIME
 L                                        J
 Figure 1.  The relationships of limiting factors
           to population growth and development.
   Liebig's Law of the Minimum enunciates
   the first basic concept.  In order for an
   organism to inhabit a particular environ-
   ment, specified levels of the materials
   necessary for growth and development
   (nutrients,  respiratory gases,  etc.) must
   be present.  If one of these materials is
   absent from the environment or present
   in minimal quantities, a given  species
   will only survive in limited numbers, if
   at all (Figure 2).
   Copper, for example,                  -j
   is essential in trace amounts for
   many species.
                    OPTIMUM
                                                          LOW —
                                                                   CRITICAL RANGE
                                                                MAGNITUDE OF FACTOR —
                                  "men
Figure 2.  Relationships of environmental
           factors and the abundance of organisms.
     The subsidiary principle of factor
     interaction states that high concentration
     or availability of some substance,  or
     the action of some factor in the environ-
     ment,  may modify utilization of the
     minimum one.  For example:

     a  The uptake of phosphorus by the
        algae Nitzchia closterium is influenced
        by the relative  quantities of nitrate
        and phosphate in the environment;
        however, nitrate utilization appears
        to be unaffected by the phosphate
        (Reid,  1961).

     b  The assimilation of some algae is
        closely related to temperature.

     c  The rate of oxygen utilization by fish
        may be affected by many other sub-
        stances or factors in the environment.
 BI.ECO. 20a. 7. 79
                                                                                        33-1

-------
Significance of "Limiting Factors" to Population Variation
B
   d Where strontium is abundant,  mollusks
     are able to substitute it,  to a partial
     extent,  for calcium in their shells
     (Odum,  1959).

2  If a material is present in large amounts,
   but only a  small amount is available for
   use by the organism, the amount available
   and not the total amount present deter-
   mines whether or not the particular
   material is limiting (calcium in the form
   of CaCO ).
          O

Shelford pointed out  in his Law of Tolerance
that there are maximum as well as minimum
values of most environmental factors which
can be tolerated.  Absence or failure of an
organism can be controlled by the deficiency
or excess of  any factor which may approach
the limits of  tolerance for that  organism
(Figure 3).
                                                  
                                                  Ł
                                                  <
Minimum Limi'. of
Toleration
Absent
Decreasing
Abundance
Range of Optimum
of Factors
Greatest Abundance
Maximum Limit of
Toleration
Decreasing
Abundance
Absent
Figure 3.  Shelford's Law of Tolerance.
     Organisms have an ecological minimum
     and maximum for each environmental
     factor with a range in between called
     the critical range which represents the
     range of tolerance (Figure 2).  The
     actual range thru which an organism can
     grow, develop and reproduce normally
     is usually much smaller than its total
     range of tolerance.

     Purely deleterious factors (heavy metals,
     pesticides, etc.) have a maximum
     tolerable value, but no optimum (Figure  4).
                                                        CONCENTRATION

                                                 Figure 4.  Relationship of purely harmful
                                                            factors and the abundance of
                                                            organisms.
                                                 3  Tolerance to environmental factors
                                                    varies widely among aquatic organisms.

                                                    a  A species may exhibit a wide range
                                                      of tolerance toward one factor and a
                                                      narrow range toward another.  Trout,
                                                      for instance, have a wide  range of
                                                      tolerance for salinity and  a narrow
                                                      range for temperature.

                                                    b  All stages in the life history of an
                                                      organism do not necessarily have the
                                                      same ranges of tolerance.  The
                                                      period of reproduction is a critical
                                                      time in the life  cycle of most
                                                      organisms.

                                                    c  The range of tolerance toward one
                                                      factor may be modified by another
                                                      factor.  The toxicity of most sub-
                                                      stances increases as the temperature
                                                      increases.

                                                    d  The range of tolerance toward a given
                                                      factor may vary geographically within
                                                      the same species. Organisms that
                                                      adjust to local conditions are called
                                                      ecotypes.

-------
                                   Significance of "Limiting Factors" to Population Variation
     e  The range of tolerance toward a given
        factor may vary seasonally.   In general
        organisms tend to be more sensitive
        to environmental changes in summer
        than in other seasons.  This is
        primarily due to the higher summer
        temperatures.

     A  wide range of distribution of a species
     is usually the result of a wide range of
     tolerances.  Organisms with a wide
     range of tolerance for all factors are
     likely to be the most widely distributed,
     although their  growth rate may vary
     greatly.  A one-year old carp, for
     instance,  may  vary in size  from less
     than an ounce to more than  a pound
     depending on the habitat.

     To express the relative degree of
     tolerance for a particular environmental
     factor the prefix eury (wide) or steno
     (narrow) is added to a term for that
     feature (Figure 5).
                                                Ill
The law of the minimum as it pertains to
factors affecting metabolism, and the law
of tolerance as it relates to density and
distribution,  can be combined to form a
broad principle of limiting factors.

1  The abundance, distribution, activity
   and growth of a population are deter-
   mined by a combination of factors, any
   one of which may through scarcity or
   overabundance be limiting.

2  The artificial introduction of various
   substances into the environment tends
   to eliminate limiting minimums  for
   some species and create intolerable
   maximums for others.

3  The biological productivity of any body
   of water is the end result of interaction
   of the organisms present with the
   surrounding environment.
VALUE AND USE OF THE PRINCIPLE OF
LIMITING FACTORS
 o
 z
 <
 0
 z
 >
 Ł?
 •<
       STENOTHERMAL -...„,........ STENOTHERMAL
       (OLIGOTHERMAL|EUIIY™EIIMAl (POLYTHERMAl]
                  TEMPERATURE
The organism-environment relationship
is apt to be so complex that not all factors
are of equal importance in a given situation;
some links of the chain guiding the organism
are weaker than others.  Understanding
the broad principle of limiting factors and
the subsidiary principles involved make
the task of ferreting out the weak link in
a given situation much easier and possibly
less  time consuming and expensive.

1  If  an organism has a wide range of
   tolerance for a factor which is
   relatively constant in the environment
   that factor is not likely to be limiting.
   The factor cannot be completely
   eliminated from consideration, however,
   because of factor interaction.
Figure 5.  Comparison of relative limits of
           tolerance of stenothermal and
           eurythermal organisms.
   If an organism is known to have narrow
   limits of tolerance for a factor which is
   also variable in the environment, that
   factor merits careful study since it
   might be limiting.

-------
Significance of "Limiting Factors" to Population Variation
B  Because of the complexity of the aquatic
   environment, it is not always easy to
   isolate the  factor in the environment that
   is limiting  a particular population.
   Premature conclusions may result from
   limited observations of a particular
   situations.  Many important factors may
   be overlooked unless a sufficiently long
   period of time is covered to permit the
   factors to fluctuate within their ranges of
   possible variation.  Much time and money
   may be wasted on control measures without
   the real limiting factor ever being dis-
   covered or the situation being improved.

C  Knowledge  of the principle of limiting
   factors may be used to limit the number
   of parameters that need to be measured or
   observed for a particular study.   Not all
   of the numerous physical,  chemical and
   biological parameters need to be measured
   or observed for each study  undertaken.
   The aims of a pollution survey are not to
   make and observe long lists of possible
   limiting factors but to discover which
   factors are significant, how they bring
   about their effects, the source or sources
   of the problem, and what control measures
   should be taken.
D Specific factors in the aquatic environment
   determine rather precisely what kinds of
   organisms will be present in a particular
   area.  Therefore, organisms present or
   absent can be used to indicate environ-
   mental conditions.  The diversity of
   organisms provides a better indication of
   environmental conditions than does any
   single species.  Strong physio-chemical
   limiting factors tend to reduce the diversity
   within a community;  more tolerant species
   are then able to undergo population growth.
REFERENCES

1  Odum,  Eugene P.  Fundamentals of
     Ecology, W. B. Saunders Company,
     Philadelphia.  (1959)

2  Reid, George K.   Ecology of Inland Waters
     and Estuaries.  Reinhold Publishing
     Corporatiua, New York. (1961)

3   Rosenthal H. and Alderdice,  D. F.
    Sublethal Effects of Environmental
    Stressors, Natural and Pollutional,
    on Marine Fish Eggs and Larvae.
    J.  Fish. Res. Board Can. 33:2047-2065,
    1976.
                                                    Thorp,  James H. and Gibbons, J.
                                                      Whitfield (editors), Energy and
                                                      Environmental Stress in Aquatic
                                                      Systems.  Tech Inform Ctr,
                                                      U. S. Dept of Energy.  1978.
                                                 This outline was prepared by John E.
                                                 Matthews, Aquatic Biologist, Robert S. Kerr
                                                 Water Research Center, Ada, Oklahoma.

                                                 Descriptors:  Population, Limiting Factors
 33-4

-------
                        ALGAE AND CULTURAL EUTROPHICATION
I  INTRODUCTION

This topic covers a wide spectrum of items
often depending upon the individual discussing
the subject and the particular situation or
objectives that he is trying to "prove".
Since the writer is not a biologist,  these
viewpoints are "from the outside-looking in".
Any impression of bias is intentional.

A  Some Definitions are in Order to Clarify
   Terminology:

   1 Eutrophication - a process or action of
     becoming eutrophic,  an enrichment.
     To me,  this is a dynamic progression
     characterized by nutrient enrichment.
     Like many  definitions,  this one is not
     precise; stages of eutrophication are
     classified as olig-, meso-, and  eutrophic
     depending upon increasing degree.  Just
     how a  given body of water may be
     classified is open to  question. It
     depends upon whether you look at quiet.
     or turbulent water, top or bottom
     samples, season of the year, whether
     it is a first impression or seasoned
     judgement. It also depends upon the
     water  use in which you are interested,
     such as  for fishing or waste discharge.
     The transitional stages are the major
     problems - it is  loud and clear to a
     trout fisherman encountering carp and
     scum.

   2 Culture

     Fostering of plant or animal growth;
     cultivation of living material and
     products of such cultivation,  both fit.
     Some degree of control is implied but,
     the control may have limitations as
     well as advantages.  Human cultural
     development has fostered human num-
     bers successfully, but, has promoted
     rapid degradation of  his natural  environ-
     ment.
    3  Nutrients

       A component or element essential to
       sustain life or living organisms.  This
       includes many different materials,
       some in gross quantities - others in
       minor quantities.  Deficiency of any
       one essential item make living
       impossible.  Nutrients needed in large
       quantities include carbon,  hydrogen,
       oxygen, nitrogen,  phosphorus,  sulfur
       and silica.  N and P frequently  are
       loosely considered as  "the "nutrients
       because of certain solubility,  con-
       version and "known" behavior
       characteristics.

    4  Algae

       A group of nonvascular plants,  capable
       of growth on mineralized nutrients with
       the aid of chlorophyll and light energy -
       known as producer organisms,  since
       the food chain is based directly or
       indirectly upon the organic material
       produced by algae.

 B Now that we have "backed into" the title
    words via definitions,  some of the
    ramifications of eutrophication, nutrient
    enrichment,  and cultural behavior  are
    possible.
II   NUTRIENTS INTERRELATIONSHIPS

 A All nutrients are interchangeable in form,
    solubility,  availability, etc.  There are
    no "end" products.  We can isolate, cover,
    convert to  gas liquid or solid, oxidize,
    reduce, complex, dilute,  etc. -  some
    time, some place,  that nutrient may
    recycle as part of cultural behavior.

    1  Water contact is a major factor in
       recycle dynamics just  as water
       represents two-thirds  or more of cell
 BI. ECO. hum. 3. 6.76
                                      34-1

-------
Algae and Cultural Eutrophication
      mass and appears to be the medium in
      which living forms started.  Waste
      disposal interrelationships (Figure 1)
      suggests physical interrelationships of
      soil, air and water.  The wet apex of
      this triangle is the basis for life. It's
      difficult to isolate water from the soil
      or atmosphere - water contact means
      solution of available nutrients.
                WASTi: DISPOSAL
              INTERRELATIONSHIPS
                    ATMOSPHERE
                           X
     WATER
   2  Figure 2 takes us into the biosphere (1)
     via the soluble element cycle.  This
     refers mainly to phosphorus interchange.
     Phosphorus of geological origin may be
     solubilized in water,  used by plants or
     animals and returned to water.  Natural
     movement is toward the ocean.  Less
     phosphorus returns by water transport.
     Phosphorus does not vaporize; hence,
     atmospheric transport occurs mainly
     as windblown dust. Man and geological
     upheaval, partially reverse the flow of
     phopshorus toward the ocean sink.
         SOLUDLE  ELEMENT CYCLE
   ATMOSPHERE
                                                     LITHOSPHERE
                                                                                        HYDROSPHERE
3  The nitrogen cycle starts with ele-
   mental nitrogen in the atmosphere.
   It can be converted to combined form
   by electrical discharge, certain
   bacteria and algae,  some plants and
   by industrial fixation.  Nitrogen gas
   thus may go directly into plant form
   or be fixed before entry. Denitrification
   occurs mainly via saprophytes.
   (Figure 3)  Industrial fixation is a
   relatively new contribution to
   eutrophication.
              NITROGEN  CYCLE

-------
                                                             Algae and Cultural Eutrophication
      Carbon Conversions (Figure 4) show
      most of the carbon in the form of
      geological carbonate (1) but bicarbonate
      and CO  readily are converted to plant
      cell mass and into other life forms.
      Note the relatively small fraction of
      carbon in living mass.
                    CIRCULATION
               IN  BIOSPHERE
B  Nutrient - Growth Relationships

   Nutrient cycles could go on, but,  life
   depends upon a mixture of essential
   nutrients under favorable conditions.
   Too much of any significant item in the
   wrong place may be considered as
   pollution.  Since toxicity is related to
   chemical concentration, time of exposure
   and organism sensitivity, too much
   becomes toxic. If it happens to be too
   much growth,  its  a result of eutrophication.
   'How much' is generally more important
   than the 'what'?  Both natural and manmade
   processes lead to biological conversions,
   to pollution,  to eutrophication and to
   toxicity.  Man is the only animal that can
   concentrate, speed up,  invent, or otherwise
   alter these conversions to make a collossal
   mess.

   1  Life forms have been formulated in
     terms of elemental or nutrient com-
     ponents many times. The simplest is
     C  HgO-N.  A more  complex formula
     is C10oS6°80N20 Ca6C17P2CuF2
     SiMgMn2K2NaS21Zn. This includes
       16 elements.  More than 30 have been
       implicated as essential and they still
       would not "live", unless they were
       correctly assembled.  Asa nutrient
       Mnemonic H.  COPKINS - - Mg(r)-
       CaFe-MoB does fairly well.  It also
       indicates  Iodine-I,  Iron-Fe,
       Molybdenum-Mo, and Boron-B that
       were not included earlier.

       The  Law of Distribution states that
       "Any given habitat tends to favor all
       suitable species - any given species
       tends to be present in all suitable
       habitats. " Selection tends to  favor the
       most suitable species  at a given place
       and time.

       Liebigs Law  of the  Minimum, states
       that  "The essential material available
       in amounts most closely approaching
       the critical minimum will tend to be
       the limiting growth factor. "

       Shelfords law recognizes that there
       will be some  low concentration of any
       nutrient that will not support growth.
       Some higher  concentration will stimulate
       growth.  Each nutrient will have some
       still higher concentration that will be
       bacteriostatic or toxic. This has been
       discussed earlier but was considered in
       a different manner.
Ill  BIO LOGICAL PROGRESSIONS

 The biological "balance" appears to be a very
 transitory condition in cultural behavior.
 Man favors production.  A steady state
 "balance" does not persist very long unless
 energy of the  system is too low to permit
 significant growth.  A progression of species
 where each predominent form thrives  for a
 time, then is  displaced by another temporarily
 favored group is usual.  Yearly events in the
 lawn start with chickweed, then dandelion,
 plantain,  crab grass, ragweed, etc.,  in
 successive predominence.  Occasionally,
 more desirable  grasses may appear on the
 lawn.  Grass  is a  selected unstable  "culture".

-------
Algae and Cultural Eutrophication
A Figure 3 shows a biological progression (2)
   following introduction of wastewater in an
   unnamed stream.  Sewage or slime bacteria
   proliferate rapidly at first followed by
   ciliates, rotifers, etc.
                                                                   THE  BIOTA
                                      SEWAGE BACTERIA
                                          NO. PER ml.

              300 r-
                                                         CILIATES
                                                      NO. PER ml. x 1000    ROTIFERS
                                                                 '    &CRUSTACEANS
                                                                    NO. PER ml.x25,000
              200-
           Q  100

                       2    1

                      24   12
      23456789
           DAYS
12   24   36   48   60   72   84   96  108
           MILES
              5.  Bacteria thrive and finally become prey of the ciliates, which in turn are food for the rotifers and crustaceans.
B  Figure 4 shows another progression of
   bottom dwelling larva.  Here the sequence
   of organisms changes after^wastewater
   introduction from aquatic insects to sludge
   worms, midges, sow bugs and then to
   re-establishment of insects.
                                                                         THE BIOTA
                                                               SLUDGE WORMS
                                                  I   I
                                                  14   12
                                                              I   1
                                                                            I   I   7
                               >   4
                               DAYS
                       It   14   31   41  II   11   14
                               MILES
I
III
                                          Figure 6. The population curve of Figure 7 is composed of a series of maxi—
                                          for individual species, each multiplying and dying off as stream conditions vary.

-------
                                                             Algae and Cultural Eutrophication
  C  Another progression after waste
     introduction changes the biota from
     an algal culture to sewage moulds with
     later return to algal predominence.
                                                "CTOQIO*WFT"CT'"   THE BIOTA
                                24  12
      DAYS
12  24  36  48
     MILES
                                                        60  72 84  96  108
                        9 Figure  /Shortly after sewage discharge, the moulds attain maximum growth.
                        These are associated with sludge deposition shown in the tower curve. The sludge is
                        decomposed gradually; as conditions clear up, algae gain a  foothold and multiply.
    Figures 5,  6,  and 7 are shown separately
    only because one visual would be unreadable
    with all possible progressions on it.  There
    are progressions for fungi, protista,  insect
    larvae, worms,  fish, algae, etc.  Each
    species will perform as it may perform.
    It it cannot compete successfully, it will
    be replaced by those that can compete
    under prevailing conditions at the time.
    Conditions  shift rapidly with rapid growth.
IV  The interactions of bacteria or fungi and
 algae  (Figure 8)  are particularly
 significant to eutrophication.

 A The bacteria or the saprophytic group
    among them tend to work on preformed
    organic materials  - pre-existing organics
    from dead or less  favored organisms.
    Algal cells produce the organics from
    light energy chlorophyll and mineralized
    nutrients. This is a happy  combination
    for both:  The algae release the oxygen
    for use by the bacteria while the bacteria
    release the CO  needed by the algae.
             Since the algae also acquire CO_ from
             the atmosphere, from wastewater and
             from geological sources, it always ends
             up with more enrichment of nutrients in
             the water - more enrichment means  more
             growth and growing organisms eventually
             clump and deposit.   The nature of growth
             shifts from free growth to rooted forms,
             starting in the shallows.  Another
             progression occurs  (Figures 9 and 10).

             It is this relationship that favors profuse
             nuisance growth of algae below significant
             waste discharges.  There is a tremendous
             pool of carbon dioxide available in
             geological formations and in the air.
             Transfer to the water is significant and
             encourages algal productivity and eventual
             eutrophication of any body of water,  but,
             this does not occur  as rapidly as when the
             water body is super saturated with CO_
             from bacterial decay of wastewater
             discharges or benthic deposits from  them.

-------
AIgae and Cultural Eutrophication
                            NATION
         DEAD
         ALGAE
       SEWAGE
         DEAD
       BACTERIA
                                   PROFUNDAL
                            PHOTOSYNTHESIS

-------
                                                            CHARAPHYTES
                                                                       LADOPHORA
   Nitrogen and phosphorus are essential
   for growth.  They also are prominently
   considered in eutrophication control.
   Algal cell mass is about 50% carbon,
   15% nitrogen and approximately 1%
   phosphorus not considering luxury uptake
   in excess of immediate use.  Phosphorus
   is considered as the most controllable
   limiting nutrient.  It's control is com-
   plicated by the feedback of P from benthic
   sediments  and surface wash.  Phosphorus
   removal means solids removal.  Good
   clarification is essential to obtain good
   removal of P.  This also means improved
   removal of other nutrients- a major
   advantage of the P removal route.  Both
   N & P are  easily converted from one form
   to another; most forms are water soluble.
V  SUMMARY

 Control of eutrophication is not entirely
 possible.  Lakes must eventually fill with
 benthic sediments, surface wash and
 vegetation. Natural processes eventually
 cause filling.  Increased nutrient discharges
 from added activities grossly increase filling
 rate.
A  We produce more nutrients per capita per
   day in the United States than in other
   nations and much more today than 100
   years ago.  More people in population
   centers accentuate the problem.

B  Technology is available to remove most
   of the nutrients from the water carriage
   system.

   1  This technology will not be used unless
      water  is recognized to be in short
      supply.

   2  It  will not be used unless we place a
      realistic commodity value on the water
      and are willing to pay for cleanup for
      reuse  purposes.

C  Removal  must be followed by isolation of
   acceptable gases to the atmosphere
   acceptable solids into the soil for reuse
   or  storage.  Water contact cannot be
   prevented, but it must be limited or the
   enrichment of the water body is hastened.

-------
 Algae and Cultural Eutrophication
REFERENCES                                   	
                                                This outline was prepared by F. J. Ludzack,
1  A collection of articles on the Biosphere        Chemist,  National Training Center,
      Sci. Am. 223:(No.  3). pp. 44-208.          MOTD,  OWPO, USEPA,  Cincinnati,  Ohio
      September 1970.                           45268.

2  Bartch,  A.E. and Ingram, W.M.              Descriptors: Algae, Eutrophication
      Stream life and the Pollution Environ-
      ment.  Public Works 90:(No. 7) 104-
      110.   July 1959.
 34-8

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                         CONTROL OF PLANKTON IN SURFACE WATERS
 I   PHILOSOPHICAL CONSIDERATIONS

 A  Plankton growths are as natural to aquatic
    areas as green plants are to land areas
    and respond to the same stimuli.
 B  Man is currently harnessing plankton forms
    to accomplish useful work.

    1  For generation of oxygen

      a  Stabilization of waste waters in
         oxidation ponds

      b  Oxygen recovery from CO2 in space
         travel

    2  For augmentation of food supply

      a  Fish ponds

      b  Nitrogen fixation in rice growing

      c  Harvesting of algae for direct use
         as food
    A. growing knowledge of the nutrient re-
    quirements of plankton organisms will
    lead to a more enlightened approach to
    ways and means of controlling their growth
    when desirable.
II   CLASSICAL METHODS OF CONTROL

 A  Chemical

    1  Inorganic

      a  Copper sulfate is used most exten-
         sively.  It is most effective in pre-
         ventive rather than curative treat-
         ment.  It  has long lasting effects in
         soft  waters but is  short-lived in hard
         waters due to precipitation of the
         Cu++ as a basic carbonate.  The pre-
         cipitated material accumulates in
          bottom muds and is toxic to certain
          benthal forms,  some of which serve
          as important fish food.

          Dosages are normally based on the
          alkalinity of the water.  When alka-
          linity is < 40 mg/1, the recommended
          dosage is 0. 3 mg/1 of CuSO^ 5H2O
          in total volume  of water.  When
          alkalinity is > 40 mg/1, recommended
          dosage is 2. 0 mg/1 in surface foot
          of water.

       b  Chlorine is preferable to copper
          sulfate in the control of certain
          forms of algae.   However,  it is
          difficult to apply in most instances
          and is very short-lived due to photo
          catalytic decomposition of HC1O —
          HC1 + O.

       Organic - Numerous organic  compounds
       have been evaluated, especially in re-
       lation to control of blue-green algae.
       "Phygon",  2, 3-dichloronaphthoquinone,
       has been  field  tested but is too specific
       in its action for general application.
Ill  ECOLOGICAL CONTROL

 A  Theory - Ecological control is based upon
    the principle of preventing or restricting
    growth by limiting one or more of the
    essential requirements.  This is an ap-
    plication of Liebig's Law of the Minimum.
    The logical avenues of control are as
    follows:

    1  Elimination of light

    2  Limiting  nutrient materials

 B  Light - Many cities have solved the prob-
    lem of plankton growths by the use of
    covered reservoirs, underground and
    elevated.  Concurrently, they have solved
    contamination problems  created by birds
    and atmospheric fallout. In open reservoirs,
 BI. MIC. coh. lOb. 6.76
                                                                                       35-1

-------
Control of Plankton in Surface Waters
   some  success has been obtained by
   limiting light through the use of a film of
   activated carbon.
C. Nutrients - Since phytoplankton (algae)
   serve as the base of the food chain, know-
   ledge concerning their nutrient require-
   ments is required for ecological control,
   when limitation of light is impractical.
   The nutrient requirements of phytoplankton
   are as follows:

   1  Nature of - The major nutrients are:

      a Carbon dioxide

      b Nitrogen - ammonia and nitrates
        (also
      c  Phosphorus - phosphates.

      Minor nutrients are:

      d  Sulfur - sulfates

      e  Potassium

      f  Trace inorganics - magnesium, iron,
        etc.

      g  Trace organics -vitamins,  amino
        acids

   2  Sources of - See Fig.  1

      a  Atmosphere

      b  Groundwater - springs

      c  Storm water  or surface runoff

      d  Waste waters - domestic sewage and
        industrial wastes.

   3  Significance of each major nutrient

      a  Carbon dioxide - See Fig.  2
            Usually present in great abundance.
        Rapidly replenished from atmosphere
        and bacterial decomposition of organ-
        ic matter.  No reasonable  possibility
        of human control.   Nature, however,
        does provide some  control through
         elevated pH levels if carbon dioxide
         becomes depleted rapidly.

         Nitrogen - Like land plants, certain
         algal forms prefer nitrogen in the
         form of NH3(NH4"f") and others prefer
         it in the form of NC>3~.  Both forms
         often become depleted during the
         growing season and reach maximum
         concentrations during the winter
         season.  A level of 0. 30 mg/1 of
         inorganic nitrogen at the time of the
         spring turnover is considered to be
         the maximum permissible level .

         All natural surface waters are
         saturated with nitrogen gas.  This
         serves as a source of nitrogen for
         bacteria and algae capable of fixing
         it.

         Phosphorus - A key element in all
         plant and animal nutrition.  The
         critical level is considered to be
         0.01 mg/1 at the time of the spring
         turnover .     Phosphorus is needed
         to sustain nitrogen fixing forms .
D  Practice Of

   1  Exclusion of  light - Practice well
      established in distribution system
      reservoirs but impractical on large
      storage reservoirs.

   2  Nutrient  limitation

      a  Control of surface run-off quality

        1) Agricultural
        2) Other

      b  Diversion of sewage plant effluents

        1) Madison,  Wisconsin
        2) Detroit Lakes,  Minnesota
        3) Pending - State College, Pa.


      c  Tertiary treatment of sewage

        1) Nitrogen removal -  Because of
           the several forms is very difficult.
 35-2

-------
                      ATMOSPHERE
                        STORM
                        WATER
                  WASTE
                  WATER
 GROUND
 WATER
(SPRINGS)
                       (SURFACE  A (DOM. SEW.
                        RUN-OFF )/^IND. WASTE
LAKE
OR

RESERVOIR
FIG. I   SOURCES  OF FERTILIZING
      MATERIALS  OF CONCERN
      IN SURFACE  WATERS
                                                              o
                                                              JO
                                                              o
                                                              I—'
                                                              o
                                            p
                                            FT
                                            O
                                                              o
                                                              fD
                                                              P_
                                                              iS"
                                                              I'-i

-------
                                                                 n
                                                                 o
                     ATMOSPHERE
                                                                 &
                                                                 o
ORGANIC
 CARBON
                                    ^ HCO-
                                                                 o
                                                                 3
                                                                 3

                                                                 C
                                                                 o
                                                                 (D
                                                                 P
                                                                 0)
                                                                 1
                                                                 en
FIG. 2  CARBON  DIOXIDE - BICARBONATE - CARBONATE - HYDROXIDE
      RELATIONSHIPS IN NATURAL WATERS

-------
                                                Control of Plankton in Surface Waters
     Also, may be unsuccessful in
     control unless phosphorus is con-
     trolled, too, because of nitrogen
     fixing forms.

   2) Phosphorus removal - Phosphorus
     can be effectively removed by
     coagulation methods employing
     lime, alum or ferric salts.   It
     is expensive and no one has
     proven  its value beyond laboratory
     experiments.

d  By Biological Engineering

   Laboratory studies have shown that
   effluents essentially free  of plant
   fertilizing elements can be produced
   by biological treatment of wastes
   with proper ratios of C to N and P.
   3  Experiences

      a Madison

      b Detroit Lakes

      c State College

      d Lake Winnisquam, N. H.

E  Practical Aspects

   1  Diversion

   2  Nutrient  control

REFERENCE

Mullican,  Hugh F. (Cornell Univ.)
   Management of Aquatic Vascular Plants
   and Algae,  pp. 464-482.  (in Eutro-
   phication;  Causes, Consequences, and
   Correction.  Nat. Acad. Sci.)  1969.

This outline was prepared by C. N. Sawyer,
Director of Research, Metcalf & Eddy
Engineers, Boston, Massachusetts.

Descriptors:  Algicides,  Eutrophication
                                                                            35-5

-------
             CONTROL OF INTERFERENCE ORGANISMS IN WATER SUPPLIES
 I  NECESSITY FOR DATA

 A Information on the number, kinds, and
   effects of interference organisms in a
   particular water supply is essential for
   determining adequate control measures.

 B Collection of the biological data should be
   on a regular routine basis.

 C Interpretation of data requires  information
   on relationship of number and kinds of
   organisms to the effects produced.

 D It is generally more  satisfactory to an-
   ticipate and prevent problems due to these
   organisms than it is  to cope with them later.
II   CONTROL IN RAW WATER SUPPLY

 A  Use of algicides

    1  Application of an algicide is to prevent
      or  destroy excessive growths of algae
      which occur as blooms,  mats or a high
      concentration of plankton.

    21  Algicide may be applied to control even
      low concentrations of certain algae such
      as  Synura.

    3  Copper sulfate is the only algicide in
      common use at present.

      a  Application may be by dusting,
          spraying or dissolving from  a porous
          container over all or part of the water
          surface, or by continuous feeding
         of the algicide at the  intake of the
          reservoir or pre-treatment basin.

      b  Effective dosage depends upon the
         Alkalinity and pH and temperature
         of the water and the amount and
         kinds of algae to be controlled.
         Bartsh states that the following
         arbitrary dosages have been found
         to be generally effective and safe:

         M.O. alkalinity >  50  p.p. m.   =
          2 p. p. m.  in the surface foot of
         water only (5.4 pounds per acre).
      M. O.  alkalinity < 50 p. p. m.  = 0. 3
      p. p. m. in total volume of water
      (0. 9 pound per acre foot).

   c  Application of copper sulfate should
      be limited to the minimum effective
      dosage because of its corrosive
      properties,  and its toxicity to fish and
      other aquatic animals.

4  Other algicides

   a  Promising types include  inorganic
      salts,  organic salts, rosin amines,
      antibiotics,  quinones, substituted
      hydrocarbons, quaternary ammonium
      compounds,  amide derivatives and
      phenols.  Cuprichloramine which is
      a combination of copper, chlorine and
      ammonia, and also chlorine dioxide
      have shown promise  as general algi-
      cides.

   b  For domestic water supplies they will
      have to be not only economically fea-
      sible but  nontoxic to  animal life and
      to green plants other than algae.

   c  Due to higher costs they  will prob-
      ably be used only when adequate plank-
      ton and algal records are kept, which
      would permit early localized treat-
      ment.

   d  Algicides selectively toxic to the
      particular algae of greatest signifi-
      cance  would be useful.

5  Mechanical removal or  spreading out
   to permit rapid drying may  be the sim-
   plest way of handling massive growths
   which are detached and  washed ashore.

6  Turbidity due to silt keeps down the
   plankton population.  In shallow reservoirs,
   fish which stir up the bottom mud will
   aid in keeping turbidity  due  to silt high.

7  Provisions for keeping the amounts of
   nutrients to  a minimum may be em-
   phasized more in the future.

8  For new reservoirs, clearing the site
  BI. MIC. con. 6b. 6.76
                                                                                       36-1

-------
 Control of Interference Organisms in Water Supplies
       of vegetation and organic debris before
       filling will reduce  the algal nutrients.
       Steep rather than gentle slopes will
       reduce the areas which allow marginal
       growths to occur.
Ill  CONTROL IN TREATMENT PLANT

 A Coagulation and sedimentation

    1  When well regulated they often will re-
       move 90 per cent or more of the plank-
       ton.

    2  With low plankton counts,  a coagulant
       aid may be required.

    3  Frequent removal of sludge from the
       basins,  especially during the warm
       seasons may help to reduce tastes and
       odors originating from decomposing
       organic sediment.

 B Sand filtration

    1  Both slow and rapid sand filters tend to
       reduce the plankton count of the effluent by
       90 per cent or more, when well regu-
       lated.

    2  For rapid filters, accumulated plankton
       can be removed or reduced by surface
       scraping and by back washing.

 C Micro-straining

    1  This involves the passing of the water
       through  a finely woven fabric of stain-
       less  steel. All but  the smaller plankton
       organisms tend to be removed from the
       water.  It is being used in some  treat-
       ment plants in England and elsewhere.

 D Activated carbon

    1  The slightly soluble, organic, taste
       and odor compounds tend to be readily
       adsorbed by the activated carbon.  It
       is probably most often applied prior to
       coagulation, but may be used prior to
       filtration or in the raw water.

 E Chlorination
     1  Treatment with chlorine is practiced
       primarily to destroy pathogenic organ-
       isms.  The dosages commonly used are
       toxic also to many algae and to some of
       the other groups of aquatic organisms.
       However,  dead as well as living organ-
       isms are often capable of causing tastes
       and odors and of clogging filters.

     The  depth and position of the intake for
     entrance of raw water into the treatment
     plant may determine the kinds and amount
     of plankton which will be drawn into the
     plant. Plankton algae generally are more
     concentrated near the surface of the water
     in lakes and reservoirs.
IV  CONTROL IN DISTRIBUTION SYSTEM

 A Maintenance of a chlorine residual con-
    trols the chlorine sensitive organisms.

 B Other pesticides  such as cuprichloramine
    have been used in attempts to control the
    resistant organisms such as worms,
    nematodes and copepod eggs.

 C Flushing of infested portions of the system,
    especially  dead ends may be practiced.

 D Covering of treated water reservoirs to
    prevent the entrance of light will stop  the
    growth of algae.

 E Organisms associated with pipe corrosion
    are probably the  most active when the water
    itself is corrosive.

 F Mechanical cleaning of the distribution
    system may be an effective but expensive
    method of reducing infestations of attached
    organisms.
 V  SUMMARY

 A Adequate control is dependent upon ade-
    quate procedures for detecting and record-
    ing of organisms.

 B Control may involve the following:

    1  Use of an algicide or pesticide.
   36-2

-------
                                      Control of Interference Organisms in Water Supplies
2  Mechanical cleaning of distribution
   lines, settling basins,  and filters,
   screens, intake channels and reservoir
   margins.

3  Modification of coagulation,  filtration,
   chemical treatment or location of raw
   water intake.

4  Use of adsorbent, such as activated
   carbon,  for taste and odor substances.

5  Modification of reservoir  to reduce the
   opportunities for  massive growths.

   a  By covering treated water reservoirs

   b  By increasing  the depth of the water

   c  By eliminating shallow marginal
      areas
         By reducing the amount of fertilizing
         nutrients entering the reservoir

         By encouraging a balanced develop-
         ment of the aquatic organisms
REFERENCE

Mackenthun,  Kenneth M.   The Practice of
   Water Pollution Biology.   FWPCA.
   U.S. Dept. of Interior,  Washington, DC.
   1969.

Smalls, I.C. and Greaves, G.F., A Survey
   of Animals in Distribution Systems.
   Water Treat. Exam.  17 (3): 150-186.
   1968.

Houghton, G. U. ,  Observations on the  Asellus
   Problem in South Essex.  Water Treat.
   Exam.  17 (2):  127-133.  1968.

Bellinger,  E. C., A Key to the Identification of
   the  More Common Algae Found in Water
   Undertakings in Britain. Water Treat. Exam.
   18 (2):  106-127.  1969.  (see also 23 (1)
   76-131.  1974)

Bays,  L. R. , Pesticide Pollution and the
   Effects on the  Biota of Chew Valley Lake.
   Water Treat. Exam. 18 (4): 295-326.  1969.

Blogoslawski, Walter J. and Rice,  Rip G..
Aquatic Applications of Ozone.  International
Ozone  Institute. Syracuse Univ.  1975.
                                              This outline was prepared by C. M.  Palmer,
                                              former Aquatic Biologist,  Biological Treat-
                                              ment Research Activities, Cincinnati Water
                                              Research Laboratory, FWPCA, SEC.

                                              Descriptor: Nuisance Organisms
                                                                                 36-3

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                    THE BIOLOGY OF PIPES, CONDUITS, AND CANALS
 I   INTRODUCTION

    Water moving in man made structures of
    metal and concrete offers another niche
    for over 100 types of aquatic organisms
    from protozoa,  to five centimeter clams.

    Unfortunately these structures are
    planned,  constructed, and managed by
    people who are  unaware of the biolog-
    ical potential for  mischief and this be-
    comes translated into often severe
    economic liabilities.

II   CRUSTACEA

 A  Copepods

    Often appear in finished water and clear-
    wells, because  the minute eggs pass
    rapid sand filters.

 B  Isopods

    Are often present in  numbers up to  100
    per meter of conduit.  Their presence
    may go undetected unless special
    sampling procedures are used,  such as
    foam plugs sent hydraulically through
    a. line with a sieving  device at the other
    end to capture the dislodged organisms.

    In England, where the problem appears tc
    be more acute,  it is  associated with sur-
    face water sources and rapid sand filters.
    Apparently only a chance introduction of
    an individual is needed, then populations
    propagate within  the mains and smaller
    lines.

Ill  MOLLUSCS

    Bivalves (clams and  mussels) are often
    more spectacular when invading a water
    distribution system because of their shell
    size and greater biomass.

A  Life History

    The problem here relates to the  "D"
    stage larvae (see  Fig.  1) or veliger
   (around 200 microns in size) which is
   free living and thereby passes screens
   and obstructions along with the water
   mass.  The adults live in or  on the
   substrate and during breeding periods
   billions of these tiny veligers are
   discharged into the water currents.
   All hydro installations thereby are
   very  vulnerable to an infestation of
   clams.

B  Veliger to Byssiger

   Notice  in figure one how the larval
   mollusc changes from a swimming
   and/or floating plankter to a  byssus
   attached benthic form.  This life
   history (shared by none  of our native
   bivalves) graphically demonstrates
   how the numerous veligers in the
   water are drawn in with the intake
   water and dispersed through  the system.
   The transitional stage, the pediveliger,
   can opt for another substrate if the
   first is unsuitable, for it still retains the
   velum  for free movement while having
   a ciliated notched  foot for climbing and
   exploring the substrate.  When the velum
   is  finally lost as development proceeds
   the byssiger can still explore about by
   foot while retaining elastic anchored life
   lines.  A study of this figure explains
   the severe mechanical problem in stopping
   water by heavy shelled adults.

C  Pest Species

   The worst are usually exotic, that is
   imported, and exotics are nearly always
   bad whether it be plants or animals.

   1  Dreissena polymorpha the Zebra Clain.

      Not yet in North America but has
      created many problems in Europe where
      it took over a century to spread.

   2  Corbicula manilensis the Asian Clam.
      or Good Luck Clam. (Figures  2 and 3).

      Initially it became a problem in the
      40's in California, and now it is found
BI. MIC. con. 13. 3.80
                                                                                   37-1

-------
VELIGER
                         PEDIVELIGER     BYSSIGER
                                                BENTHIC
                           Figure 1 D-Stage

                   Bivalve Larval Stages (app. 200 microns)
              Figure 2

    Corbicula or Exotic Asian Clam
Approximately 6 cm maximum size. Notice
serrated "teeth".
                                                Figure 3

                                       Corbicula or Exotic Asian Clam
                                  Approximately 6 cm maximum size. Notice
                                  heavy corrugated outer shell.
                                                            37-2

-------
                                             The Biology of Pipes. Conduits,  and Canals
 D
   in most North American drainage
   systems.  Literature on this species
   is voluminous. It has been a severe
   pest in TVA steam plants and numerous
   water diversion schemes.  Most
   probably it was introduced through
   immigrants from Canton, China.  In
   its native range it is of value as a
   commercial food  species.

3  Limnoperna fortune!

   A serious pest in Hong Kong water
   supplies.

4  Modiolus  striatulus

   A serious pest in Calcutta water
   supplies.

As Morton points out these species are
problems in water supplies and distribu-
tion  systems because they are/or have a
    1  Opportunist
    2  Grow fast
    3  Long breeding season
    4  High reproductive potential
    5  Free swimming larval phase (the
      veliger).
    6  Quick to colonize all available
      substrates.

 E  Treatment and prevention has largely
    (and successfully) been limited to
    chlorine.

IV  Pipe Ecology

    A pipe  is simply a place to  live.

 A  Habitat

    Even under great velocities a pipe's inner
    surface offers a place of attachment.
    Rough surfaces afford micro-habitats.

 B  Niche

    Even though potable water may be very
    low in nutrients and particulate organic
    matter, the constant velocity makes it
    a rich food  supply.  Predators may be
    completely  absent.
C  It may be dark inside but a link to the
   outside photosynthetic world of food
   plants and decomposing bacteria
   (bacteria are food to a variety of
   pipe dwelling animals) is in constant
   motion.

V  In conclusion it is no surprise
   to biologists to discover a variety and
   abundance of organisms (micro and
   macro in size) well adapted to living in
   just about any pumped water supply,
   even when lines are subject to
   chlorine.

REFERENCES

1  Morton, Brian.  The  Colonization of
   Hong Kong's Raw Water Supply System
   by Limnoperna fortunei.  (Dunker  1857)
   (Bivalvia; Mytilacea)  from China.
   Malacol. Rev. 8:91-105. 1975.

2  Clarke,  K. B.  The Infestation of Water-
   works by Dreissena polymorpha.  a
   freshwater mussel. J. Inst. Water Engrs.
   6:370-379.   1952.

3  Sinclair, Ralph M.  Annotated Bibliography
   of The Exotic Bivalve Corbicula in North
   America.   Sterkiana 43:11-18.   1971.
                                                  Mattice, J. S.
                                                  Newsletter  .
                 and Tilly, L. J.  Corbicula
                 Envir.  Sci. Div., Oak Ridge
                                                 National Laboratory, Oak Ridge, TN 37830.
                                                 Issued irregularly,  names added to mailing
                                                 list by request to Dr.  Mattice at address
                                                 above.  Contains summary s of ongoing
                                                 research on control of Corbicula.

                                                 Smalls, I.C.  and Greaves, G. F. A Survey
                                                 of Animals in Distribution Systems. Water
                                                 Tr. and Exam.  17:150-186.   1968
                                                 (Thirty six water supply systems were
                                                 examined taking flush samples from the
                                                 distribution system.  Over 100 types of
                                                 animals were taken. )
                                               This outline was prepared by R. M. Sinclair,
                                               National Training Center, MOTD,  OWPO,
                                               USEPA, Cincinnati, Ohio 45268.

                                               Descriptors: Molluscs,  Isopods, Fouling,
                                               Nuisance Organisms,  Pipelines
                                                                                      37-3

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                 SAN FRANCISCO EXPERIENCE WITH NUISANCE ORGANISMS
 I   INTRODUCTION

 In order to have a clear picture of the
 problems in the San Francisco Water Supply
 system by nuisance organisms an understand-
 ing of the basic system should be helpful.

 The largest quantity of water is produced in
 the Hetch Hetchy watershed, located in
 Yosemite National Park.  This water is
 transmitted some 150 miles by tunnel and
 pipe to the San Francisco Bay Area,  where
 the water not sold enroute is discharged
 into Crystal Springs Reservoir.  From
 Crystal Springs Reservoir, water either
 flows by gravity to the lower elevations
 of the San Francisco Peninsula and the  City
 proper, or  is pumped to nearby San Andreas
 Reservoir.   This latter reservoir supplies
 the higher areas.  The capacity of Hetch
 Hetchy reservoir is 117 billion gallons,
 Crystal Springs 22. 5 billion gallons and
 San Andreas 6 billion gallons.  All water
 flowing from the local reservoirs is chlor-
 inated and fluoridated along with the custom-
 ary copper  sulphate treatment for algae
 control of surface waters.

 Two other parts of the system should be
 mentioned,  although these waters have not
 been involved particularly with nuisance
 organisms.   On the East Side of San Fran-
 cisco Bay the San Francisco Water Depart-
 ment owns and operates two large reservoirs,
 San Antonio and Calaveras.  The water from
 these reservoirs passes through the 80  m. g. d.
dual media,  Sunol Valley Water Filtration
 Plant capable of complete treatment.

 All watersheds tributary to Crystal Springs
 and San Andreas Reservoirs are owned  by
 the Water Department and only seven water-
 shed keepers live on the 25, 000 acres.  The
 other reservoirs are quite remote and human
 activity is small compared to'the area
 involved.

 The distribution and transmission system
within  San Francisco consists of eighteen
 pressure zones; ten large covered reservoirs
 with capacities ranging from 2. 5 to 176. 7
 million gallons; 1, 163  miles of pipe and the
 usual pumping stations, valves, etc.  The
 goal of this presentation is to describe the
 problems caused by nuisance organisms in
 the San Francisco  system and briefly relate
 what was done about them, if anything.

 The following described outbreaks of nuisance
 organisms occurred during the period 1934
 to the present.

II  AQUATIC ACTINOMYCETES

 During Spring of 1956 there appeared in the
 distribution system increasing incidents of
 earthy taste and odor  complaints,  many by
 our staff.  Even the management was of the
 opinion that the water was becoming poorer
 and poorer;  so  a graduate student in micro-
 biology was  hired for  the summer to investi-
 gate the possibility of actinomycetes being the
 source.  Positive results were obtained from
 many samples of water but a definite pattern
 could not be established that would implicate
 any part of the  system.

 Before finite data could be established the
 need to raise our chlorine residuals to com-
 bat persistant coliforms became our over-
 whelming problem, and in the course of events
 chlorine taste complaints took the place of
 earthy and woody complaints.  No doubt the
 chlorine residuals of lmg/1  and better
 distribution  maintenance with regards to
 water quality were responsible for the control
 of these organisms.

 Those having complaints of this nature are
 referred to the many articles in the Journal
 by Professor J. K. G.  Silvey and his co-
 workers. (1)

III  BRYOZOAN

 This outbreak occurred during the Spring
 of 1956.
 BI. MET. con. 6. 6.76
                                    38-1

-------
 San Francisco Experience With Nuisance Organisms
 A routine complaint was turned over to the
 Department Laboratory for inspection.  A
 customer had complained that little black
 specks were caught in her nylons after
 washing. Inspection showed that they were
 coming from the tap and flushing of nearby
 fire hydrant produced clusters of the black
 organisms  up to the size of a quarter.

 The black specks were identified as
 statoblasts  of Bryozoan, and Whipples
 description of a Brooklyn Water Depart-
 ment outbreak in  1897 really shook the staff
 up.  His description states,  "in  a number
 of instances this material stopped  up the
 taps, and even large pipes were choked". (2)
 Great black masses of these statoblasts
 could be imagined blocking our pipes and
 valves.

 The area for several blocks around was
 flushed until no further statoblasts were
 observed.  In spite of a close inspection
 of other parts of the system, including
 distribution and impounding reservoirs no
 additional organisms were found.  In fact
 these Bryozoans have never been encountered
 again.

 In the laboratory, attempts were made to
 germinate the statoblasts to no avail and
 it was assumed that the organisms were
 dead.  Several months later a test tube of
 statoblasts  in water which had laid on a
 table formed colonies.

 Although the San Francisco Water  Supply has
 experienced only the one outbreak, Bryozoans
 are a common nuisance in water systems.
 Prokopovich and Hebert (3) described a
 problem in  California's Delta-Mendota
 Canal and Whipple also described outbreaks
 in Hartford, Connecticut and Boston,
 Massachusetts.

IV CHLOROPHYTA: BULBOCHAETA AND
   SPIROGRYRA

 Although plankton net catches from the large
 Hetch Hetchy  reservoir contain  high counts
 of Crustacea the reservoir has never
 required copper sulphate treatment.
During November 1964 a number of complaints
from the Department wholesale customers
were received, stating water meters were
clogging with a growth.   At the time both
Hetch Hetchy and Calaveras Reservoirs
were supplying the Bay Division Lines and
Department personnel jumped to the con-
clusion that the growth must be originating
in Calaveras Reservoir which was not
filtered at this time and routinely needs
copper sulphate treatment.  Accordingly,
Calaveras was shut down and all customers
were supplied from Hetch Hetchy, but meters
continued to clog.  Further investigations were
made and the trouble traced to the source
which was a short section of the Tuolumne
River where the Hetch Hetchy water ran in
the river a distance of twelve  miles before
entering the tunnel and pipe system.

As this section of the system was in the
Yosemite National Park,  permission to
treat parts of the river with copper sulphate
was  requested of the Department of Interior,
and tests were run at the nearby Moccasin
Fish Hatchery.  These tests showed treat-
ment could be effective without killing trout.

Prior to the conclusion of the  above tests a
telegram was  received from Washington
which read "Technicians  this Service and
Bureau of Sport Fisheries  and Wildlife
consider knowledge about use  of the chem-
ical  inadequate at this time to insure pre-
servation of ecological conditions based on
potential threat to the river's  native aquatic
organisms and possible fish kill.  We
cannot grant approval for this program. "

As the purpose of the treatment was to kill
some of the  native aquatic  organisms,  the
project to copper sulphate  the river was
abandoned without argument,  but two small
regulating reservoirs were treated.

Luckily the organisms disappeared through-
out the system and have not reoccurred.
Since the above incident,  this  section of  river
has been by-passed by a  tunnel and the water
from Hetch Hetchy Reservoir  enters the pipe
and tunnel system directly.
 38-2

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                                             San Francisco Experience With Nuisance Organisms
V HYDROIDES

Within the boundries of the City and County
of San Francisco lies the 2-f billion gallon
Lagana de la Merced or as the Water
Department calls it,  Lake Merced.

Lake Merced,  formerly connected with the
Pacific Ocean  by a Channel which was
closed sometime between  1869 and 1894 is
now a freshwater lake and was used for
domestic water purposes from  1895 to 1932
at which time it was placed on a standby
basis.  The lake has characteristic fresh-
water fauna, except for five species of
definitely marine affinities.  The organism
which could give trouble in the system if
this source were ever used again is
Cordylophora lacustris  (Allman)
(Coelenterata Hydroidea). (4)

Hydroides were first noticed by the author
when the City's boating  concessionaire
complained of  growths  covering the bottoms
of his row boats.  This was solved by using
a non-fouling paint, but one cannot help
speculating on the problems which would be
encountered if an earthquake severed the
normal water supply lines to the city,  in
which case Lake Merced would be the sole
source until repairs were made.

An experimental microstrainer was installed
to treat water  from Crystal Springs Reservoir
in conjunction  with coliform investigations in
1963 and after a number of months of operation
hyclroides established themsleves in the tanks.
Although this organism  did not cause opera-
tional difficulties,  it was unsightly and the
tanks had to be manually cleaned.  Strangely,
these organisms have never become
established in  the distribution system even
though there are distribution tanks and
reservoirs in the system with approximately
the same light intensity. It can only be
assumed that the chlorine residual is keep-
ing  the hydroides from becoming a problem.

VI MYRIOPHYLLUM OR EURASIAN
   WATERMILFOIL

Over a number of years this  fern like plant
growth has been a problem in a 13. 5 million
gallon open distribution reservoir within the
City of San Francisco.   Every two to three
years the growth would be so abundant that
the weed would require harvesting by
dragging large rakes across the reservoir.
Of course, the solution was to  clean.line
and roof the reservoir which was  done in
1960.

Myriophyllum has now thoroughly established
itself in San Andreas Reservoir and because
of its size, 550 acres,  it is impossible to
control it by dragging.  The only effective
control has been to lower the reservoir and
let the banks dry out killing the surface
growth but,  of course,  not the  roots and the
following year if the reservoir water level
remains relatively constant the cycle is
repeated.

With the construction of an 80 m. g. d.  water
treatment plant employing coagulation,  sedi-
mentation and dual media filtration, problems
are anticipated when the stems fragment
break off and either settle in the basins or
mat the filters thus shortening the filter runs.
This will at least transfer the problem from
the water consumer to ourselves.  At the
present many  consumers find the  small fern
like leaves in  their tap  water and  become
most unhappy  when told they are not
receiving filtered water and there is nothing
that can be done to resolve the problem;
although if the problem seems  severe and
localized, the mains in the area are flushed.

This summer  some experiments are to be
performed utilizing a blanket of air bubbles
rising around  the intake structure in an
attempt to keep the floating debris from
being sucked into the intake adits.

San Francisco is not alone with this problem
as the Watermilfoil has established itself in
reservoirs throughout the country.  The
TV A project has fought the problem for
over ten years. If you  are confronted with
this weed and  your management asks where
it came from,  Smith of the Vector Control
Branch TVA states, "We suspect  that the
original TVA milfoil infestation in Peny
River embayment of Watts Bar Reservoir
started either intentionally or otherwise
from a misplaced fish bowl plant.  From
such a modest beginning, it thrived but
remained unnoticed until its amazing spread
began to interfere with  fishing  and boating.
                                                                                  38-3

-------
 San Francisco Experience With Nuisance Organisms
 Within a very short time (probably 3 to 5
 years) it embraced about  1, 500 acres." (5)

VII PLANKTON

 This group of organisms gives trouble in almost
 any unfiltered surface supply by being observed
 by the consumer as a white swimming speck
 in a glass of water; by dying off in the dis-
 tribution piping and adding to the organic load
 or, as has been found in San Francisco,
 protecting coliform bacteria from the action
 of chlorine.  The author presented a paper
 before this Association in 1966 detailing this
 problem wherein Cyclops.  Daphnia, etc. were
 so protecting the coliform bacteria that 5 tubes
 positive could be obtained from samples which
 had chlorine contact times of 2 hours at a free
 residual of 1.4 mg/1. (6)

 To the present time this problem continues
 and laboratory tests confirm the original
 hypothesis.  Since the publication of the
 original paper, discussions with the operators
 of water systems similar  to San Francisco's
 also support this thinking.

Vlll SLIME ORGANISMS

 Hetch Hetchy water began to flow into the San
 Francisco Water System in October 1934,
 carrying a flow of  45  m. g. d.  Part of this
 flow,  16 m.g. d., was diverted into a 36-inch
 wrought iron pipe that had been laid in 1887.

 Three weeks after this diversion the carrying
 capacity started to drop off at the rate of 0. 2
 m. g. d.  After another three week period the
 flow decreased from 16. 2 to 13. 6 m. g. d. a
 capacity loss of 16 per cent.  Tests failed to
 indicate any air pockets or other obstructions
 along  the line and in March  1935 the line was
 opened for inspection. A  slimy, gelatinous
 growth was found,  light brown in color
 covering the entire inner surface of the pipe
 from  1/8 to 1/4 inch thick.

 Microscopic examination showed this growth
 to contain the iron bacteria Crenothrix
 and the sulphur bacteria   Beggiatoa.

 Experiments were performed utilizing 4-inch
 pipes,  varying flows and chemical dosages.
 Hydrated lime to raise the pH  of the water to
 10; and copper sulphate treatment did not
 produce beneficial results.  Treating the
 water with l-Ł lb. of ammonia and 6 Ib. of
 chlorine per m. g. was effective in controlling
 the growth.

 A large chlorination plant was then built and
 growths have been controlled since by this
 treatment with the only change being the use
 of chlorine alone.  (7, 8)  This use of chlorine
 alone has  been effective probably due to the
 higher flows of 100 - 275 m.g. d. now  required
 by consumption.  Generally only a slight
 taste or odor of chlorine is  encountered.

 At least once since the initial start of  treat-
 ment an outbreak of slime has occurred and this
 was caused  by lowering the  chlorine dosage
 below the  required level for 100% control.

 A visual inspection was made by shutting
 down the tunnel system.  This inspection
 showed strings of mucoid slime hanging
 from the tunnel ceiling.  Heavy chlorination
 cleaned the  tunnel and maintenance of  . 5 to
 . 75 mg/1  chlorine residual  keeps the tunnel
 system clean of these slime organisms.

IX  FRESHWATER SPONGES

 During July 1961 water being withdrawn
 from San Andreas Reservoir through Outlet
 No.  3 had a very objectionable  odor.   The
 outlet system was drained and visual
 inspection showed large freshwater sponges
 growing on the walls of the 72-inch bitumastic
 lined steel pipe upstream from the point of
 chlorine injection.  None of these growths
 could be found downstream from the point
 of chlorination but numerous growths could
 be found all the way upstream to the adit
 shut-off valve.  No growths were ever found
 on the intake screens.

 Microscopic examination showed this growth
 to be a freshwater sponge.   Because growths
 could not be found below the point of chlorine
 injection the solution obviously was to move
 the point of  chlorine injection as far upstream
 as possible, namely 1276 feet.   Crews brushed
 the walls of the pipes free of growths,  the
 line was flushed and placed  back in service.
 Funds were  budgeted for a new chlorine
 station and prior to its being placed into
 service the  pipeline was again inspected, and
 found to have sponge growths,  this required
 38-4

-------
                                               San Francisco Experience With Nuisance Organisms
 another cleaning and the new chlorine
 station was then placed in service.  No
 problems have been encountered since.

 Strangely, there are two other outlets  from
 this same reservoir and neither of these two
 have ever been found to have a sponge  problem
 although all points of chlorine injection
 originally were about the same.   Possibly
 the different velocities in the three lines
 could be a factor, but one line has a
 greater velocity and the other a lower
 velocity.

 The author was interested to learn of a
 similar outbreak which was observed during
 1966 in the St. Louis County Water District.
 Those with a particular interest in fresh-
 water sponges and the  resultant problems
 are  particularly referred to the article in
 the Journal by King,  Ray and Tuepker. (9)
 This article goes into scientific description
 of the freshwater sponge Trochospongilla
 Leidyi  and their methods of control with
 chlorine, and there is no need to repeat
 them here.

X  SUMMARY

 As San Francisco's experience with the
 common blue-green and green algae is typical,
 this  paper has not included any discussion of
 these algae.  Although  the local impounding
 reservoirs do have growths which are easily
 controlled through the normal bluestone
 treatment, in a few instances the more
 exotic organisms just vanished before control
 methods were instituted.

 Operators needing assistance in identification
 or additional references are referred to the
 work of Ingram and Bartsch '10) as excellent
 source material.

 Almost all the nuisance organisms encountered
 in the San Francisco Water Supply System have
 been susceptible to control by chlorination but
 the resulting complaints of excessive chlorine
 taste must be  accepted. These complaints
 become less numerous  as water users  become
 accustomed to the chlorinous taste.   The
 ultimate goal, of course, is filtration and
 judicious use of post chlorination.
REFERENCES

1  Silvey,  J. K. G. and Roach,  A. W.
      Laboratory Culture and Odor  Producing
      Aquatic Actinomycetes.  Journal AWWA,
      51:20 (January 1959).

2  Microscopy of Drinking Water, by G. C.
      Whipple.  Revised by G.  M.  Fair and
      M. C. Whipple (4th ed.,  1927).

3  Prokopovich, N. P. and Herbert, D. J.
      Sedimentation in the Delta Mendota
      Canal.  Journal AWWA,  57:375
      (March 1965).

4  Miller,  R. C.  The Relict Fauna of Lake
      Merced, San Francisco.   Sears
      Foundation:  Journal of Marine Research.
      17:375 (November 1958)

5  Smith, Gordon  E.  Eurasian Watermilfoil
      (Myriophyllum spicatum) in the
      Tennessee Valley, Paper presented  at
      the meeting  of Southern Weed  Con-
      vention.  Chattanooga, Tennessee.
      (January 1962).

6  Tracy,  H.W. et. al. Coliform Persistance
      in  Highly Chlorinated Waters.  Journal
      AWWA 58:1151 (September 1966).

7  Arnold,  G. E. Crenothrix Chokes Conduits.
      Engineering News - Record (May 28,
      1936).

8  Arnold,  G. E. Tesla Portal  Chloramination
      Station.  Water Works and Sewage
      (April  1938).

9  King,  D. L. et.  al. Freshwater Sponges
      in  Raw-water Transmission Lines.
      Journal AWWA 61:473 (September 1969).

10 Ingram, W. M.  and Bartsch, A.F.
      Operator's Identification  Guide to
      Animals Associated with  Potable Water
      Supplies.  Journal AWWA 52:1521
      (December 1960).
This outline was prepared by Harry W. Tracy,
Manager Purification Division, San Francisco
Water Department.

Descriptor: Nuisance Organisms
                                                                                 38-5

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                      LABORATORY:  IDENTIFICATION OF DIATOMS
 I   OBJECTIVES

 A  To become familiar with important
    structural features of diatoms.

 B  To learn to recognize some common
    forms at sight.

 C  To learn to identify less common forms
    using technical keys.

II   PROCEDURE

 A  Transfer a drop of the water sample con-
    taining diatoms to a microscope slide.
    Cover with cover glass and observe under
    low power  (10X) of microscope.

    1   Do all of the diatom cells appear to
       have the same shape? Do some have
       square  ends and some rounded ends?
       Touch the cover glass with  your pencil
       several times as you observe through
       the microscope and note the relationship
       of the two types of ends to one another.

    2   Find a place where a round-ended and a
       square-ended cell are close together
       and observe these under oil power
       (90X).  The round-ended view is that of
       the top  or bottom of the diatom  cell and
       is called the "valve" view.  The square
       ended view is that of the side of the cell
       and is called the "girdle" view.

    3   In the valve view note the cross lines
       in the wall.  In this diatom  there are
       many fine lines and a smaller number
       of coarse lines.   The former are present
       in all diatoms and are called "striae" or
       "striations. "  The latter are present
       only in  some genera of diatoms and are
       called "septa, " or in other  genera,
       "costae. " Which of the two types of
      lines are continuous from side to side?
       The  space left in the center by the in-
      terrupted lines is known as  a "false
       (pseudo) raphe."
    4  What is the predominant color of the
       diatom?  How many plastids?  In
       diatoms, the identification is based
       almost entirely on the characteristics
       of the cell wall.

    5  Make an outline drawing,  at least 3
       inches long, of a valve view and a girdle
       view of the diatom. Show the markings
       in the upper third of each.  Label the
       striae, septa, and false-raphe.   Make
       a drawing of what you imagine an end
       view or cross (tiansverse) section view
       would be like.

    6  Using the key, identify your specimen,
       listing the alternatives selected.

 B  Use the key to identify other  unknowns as
    far as possible, listing the alternatives
    selected in the key.  Make a  sketch  of
    Navicula and  Cyclotella if you identify these
    forms.

Ill  IMPORTANT TERMS

 Capitate - having a known-like end.

 Costae - coarse transverse ribs in wall.

 False raphe  - (see pseudoraphe)

 Frustule - the wall of the diatom.

 Girdle view - the side view,  in which the
 diatom appears to have  square or blunt ends.

 Nodule - a lump-like swelling in the center or
 ends of the valve.

 Pseudoraphe - a  clear space extending the
 length of the diatom and  bordered on both
 sides by striae.

 Punctae - the dots which comprise the
 striae.
BI.MIC.cla.lab. 9. 6.76
                                                                                 39-1

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 Laboratory: Identification of Diatoms
 Raphe - a longitudinal line (cleft) bordered on
 both sides of striae.

 Septa - a self-like partition in the diatom,
 appearing often as a coarse line.

 Striae - fine transverse lines especially
 evident in the valve view.

 Valve view  - the top or bottom view, in which
 the diatom has rounded ends, or is circular
 in outline.
REFERENCES

1  Boyer,  C. S. The Diatomaceae of
      Philadelphia and Vicinity.  J. B. Lippin-
      cott  Co.  Philadelphia.  1916. p 143.

2  Boyer,  C.S.  Synopsis of North America
      Diatomaceae,  Parts I (1927) and II
      (1928).  Proceedings of the Academy
      of Natural Sciences; Philadelphia.

3  Elmore, C.J.  The Diatoms of Nebraska.
      University of Nebraska Studies,  21:22-
      215.   1921.

4  Hohn, M. H.  A Study of the Distribution
      of Diatoms in Western New York State.
      Cornell University Agricultural Experi-
      mental Station. Memoir 308, pp 1-39.
      1951.

5  Pascher, A.  Bacillariophyta  (Diatomeae).
      Heft  10 in Die  Susswasser-Flora
      Mitteleuropas,  Jena.  1930. p 466.

6  Patrick, R.   A Taxonomic and Ecological
      Study of Some  Diatoms from the
      Pocono Plateau and Adjucant Regions,
      Farlowia.  2:143-221.   1945.
 7  Smith, G. M.  Class Bacillariophyceae.
      Freshwater Algae of the United States,
      McGraw-Hill Book Co.  New York.
      pp 440-510  2nd Ed.   1950.

 8  Tiffany,  L. H., and Britton, M. E.  Class
      Bacillariophyceae. The Algae of
      Illinois, University of Chicago Press.
      pp 214-296.  1952.

 9  Ward, H. B.,  and  Whipple, G. C.  Class I,
      Bacillariaceae  (Diatoms).  Freshwater
      Biology, J.  Wiley & Sons.  New York.
      pp 171-189.  1948.

10  Weber, C.I.  A Guide to the Common
      Diatoms at Water Pollution Surveillance
      System  Stations.  USDI. FWPCA,
      Cincinnati, OH.  1966.

11  Whipple, G. C., Fair, G. M., and Whipple,
      M. C.  Diatomaceae. Microscopy of
      Drinking Water.  Chapter 21, 4th ed.
      J. Wiley and Sons, New York.  1948.
 This outline was prepared by L. G. Williams,
 Aquatic Biologist, Formerly with Research
 and Development,  Cincinnati Water Research
 Laboratory,  FWPCA,  SEC.

 Descriptors:  Diatoms, Analytical Techniques
39-2

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                      PREPARATION OF PERMANENT DIATOM MOUNTS
 I   The identification of many diatoms to
 genus and all diatoms to species requires
 that the cells be free of organic contents.
 This is necessary because the taxonomy  of
 the diatoms is based on the structure of the
 frustule (shells) of the organisms and many
 features are masked by the presence of
 organic materials which may remain inside.
 It is also necessary that at least 1000X
 magnification (oil immersion) be used to
 detect the structural features used in
 identification.  No simple procedure for  the
 accurate routine counting of diatoms has yet
 been developed.
II  MATERIALS NECESSARY

 A Sample Concentration

   1  Centrifuge (such as Universal DU)

   2  100 ml centrifuge tubes

   3  Membrane filter apparatus

   4  Vacuum

 B Slide Preparation

   1  Slides. 1X3 inch,  frosted-end

   2  Cover glasses, circular #1, 18mm,
      0. 13 -  . 16 mm thick

   3  Resinous mounting medium (such as
      Harleco microscope mounting medium)

   4  Hot plates

      a  1800 p

      b  7000 F

   5  Disposable pipettes

   6  3X6X1/4 inch steel plate
E   PROCEDURE

 A  The volume of sample needed will vary
    according to the density of diatoms and
    silt, and  only with experience can the
    correct sample size be determined.  In
    most cases,  100 ml will be sufficient.

    1  Spin 100 ml at 1000 G for 20 minutes.

    2  Withdraw the supernatant liquid with
      an aspirator, being careful not to
      disturb the concentrate at the bottom
      of the centrifuge tube.  (Draw off all
      but 2-3 ml.)

    3  Transfer the concentrate to a labelled
      10 ml disposable vial.  Label the vial
      with a  magic marker,  diamond pencil,
      or "time" label.

    4  If the sample has been preserved with
      formalin,  or contains more than
      1.0 gram per liter dissolved solids,
      it will  be necessary to wash the
      concentrate with distilled water.  In
      this case,  transfer the entire concen-
      trate to a  15 ml centrifuge tube.
      Dilute  to 15 ml with distilled water,
      making certain that the sample is well
      mixed.  Spin for 10 minutes at full
      speed in a clinical centrifuge.   With-
      draw the supernatant liquid, and refill
      with distilled water. Spin again for  10
      minutes.  Withdraw the supernatant
      liquid as before, return the concentrate
      to the rinsed vial in 2-3 ml of distilled
      water and proceed with the mounting.

    5  If more than 200 ml of sample must be
      centrifuged to obtain sufficient material
      to prepare a diatom slide, concentrate
      the  diatoms by filtering the sample
      through a 1.2 micron pore diameter
      membrane filter. Transfer the filter
      to a 15 ml centrifuge tube, and dissolve
      with 90% acetone.  Centrifuge  10
      minutes (full speed) and decant with an
      aspirator.  Refill with 90% acetone,
 BI. MIC. enu. lab. 5b. 6. 76
                                    40-i

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Preparation of Permanent Diatom Mounts
     agitate, and spin again for 10 minutes.
     Repeat until three fresh acetone washes
     have been used.  Replace the acetone
     with 2-3 ml of distilled water and
     transfer to a labelled vial as described
     in #4.

B  If the loss of minute forms in supernatant
   is suspected, spin 100 ml at 1000 gs in
   a batch centrifuge for as long as may be
   necessary, then proceed as below.

C  Mounting

   1 Heat the hot plates to the prescribed
     temperatures.

   2 Place one cover glass on the steel plate
     for each sample.

   3 Place the steel plate on the  18QOF hot
     plate.

   4 Transfer a drop of sample to a cover
     glass.

   5 Allow the water to evaporate (caution:
     do not allow it to boil.)

   6 Continue to add more sample until a
     thin layer of material is  noticeable on
     the dry cover  glass, or until all of the
     concentrate has been used.  This step
     is especially critical, and can be
     learned only by trial and error.

   7 Transfer the steel plate to the 700op
     hot plate for 20-30  minutes.  (The
     plate should be hot  enough to incinerate
     paper.)

   8 While the material  is on  the high
     temperature hot plate, label the
     microscope slides (use a #2 pencil
     or a fine point drawing pen); place
     them on the low temperature hot plate,
     which now has been reset to approxi-
     mately 2750 p.
   9  Place a drop of mounting resin on the
      microscope slides and allow the solvent
      to evaporate.

  10  When the incineration of the material
      on the cover glasses  is complete,
      transfer the cover glasses, while  still
      hot, to the mounting medium.

  11  Allow the resin to penetrate the
      frustules (1-2 minutes).

  12  Remove the slide, place it on a cool
      desk top, and press the cover glass
      lightly with a pencil eraser for a few
      seconds.  The medium will harden in
      5-10 seconds.

  13  Scrape off the excess resin with a
      razor blade.

D The preparation is now  ready for exam-
   ination under an  oil immersion objective.
ACKNOWLEDGEMENT:

Certain portions of this outline contains
training material from a prior outline by
M.E. Bender.
This outline was prepared by Dr. C.I.
Weber, Chief,  Biological Methods Section,
Analytical Quality Control Laboratory,
1014 Broadway, Cincinnati,  OH 45202.
Descriptors-  Diatoms,  Analytical
 Techniques
  40-2

-------
                    LABORATORY:  IDENTIFICATION OF ANIMAL PLANKTON
 I  INTRODUCTION
 A The great majority of organisms commonly
    encountered in plankton analysis work are
    plants or at least plant-like (holophytic).
    Animals, however, (holozoic or nonchloro-
    phyll bearing forms) are an important part
    of the community, and the ability to recog-
    nize them may be quite important.

 B Many animals are soft bodied and so  are
    best observed in the living condition,  as
    they shrink and become otherwise distorted
    on preservation. There are consequently
    many which will not be available in a
    suitable form for the following exercise.
    Only such forms will be  dealt with as can
    readily be  obtained alive, or which retain
    essential characteristics on preservation.
        Examine your specimen carefully,
        then read the first couplet of
        statements in the key (la and Ib).

        Since the specimen is large enough
        to see,  it obviously could not be  the
        object of statement la.   Therefore
        due to the nature of the key (as
        explained in  the second paragraph of
        the introduction) the second alternative
        (lb) must apply.  This alternative
        instructs us  to proceed to couplet 2.

        From here on, follow from couplet
        to couplet, considering each couplet
        by itself, until a final selection leads
        to a name.  If this name or couplet
        is, followed  by another couplet
        number, this means that the group
        named is further subdivided.
 II  OBJECTIVES
  A To Study the nature and use of a key for
    identifying organisms

  B To Introduce the Beginners to the Use of
    the Microscope

  C To Learn to Recognize Basic Animal Types

  D To Identify Animal Plankton Species as
    Available,  and to Become Familiar with
    the Literature
III  PROCEDURE
 A The Use of the Biological Key

    1  Obtain a  "Basic Invertebrate Collection"
       from the instructor.

    2  Select a specimen designated by the
       instructor, and turn to the "Key to
       Selected  Larger Groups of Aquatic
       Animals. "
      Identify the other specimens in the
     Basic Invertebrate Collection in  the
      same way.

      Carry the identification further,  to
      genus and species if possible,  in one
      or more of  the more detailed keys
      listed at the end of the  "Key to Selected
      Larger Groups of Aquatic Animals. "
B  The Use of the Microscope

   1   Obtain preliminary information from
      the instructor as to how to set up and
      operate the instrument.

   2   Place a prepared slide of a printed letter
      on the stage and observe it successively
      under low (100X) and high (45X) powers.
      When the letter is right  side up to you,
      how does it appear through the microscope?

   3   Place a prepared slide of a micro-
      crustacean on the stage  and identify it
      using the "Key to Selected Larger Groups
      of Aquatic  Animals. "  Continue your
 BI.MIC.cla.lab.5c.6.76
                                                                                      41-1

-------
Lab oratory: Identification of Animal Plankton
      identification as far as possible using
      Eddy and Hodson's "Taxonomic Keys. "

      Prepare a "wet mount" under the
      direction of the instructor and identify
      the organism.  Confirm your identifica-
      tion in one or more of the technical
      reference books available.
   Identify each of the specimens in the
   reference collection as to phylum and
   class, and then genus and species if
   possible (do not spend undue time on the
   species without assistance).
   Make a flash card sketch of at least
   one organism of each phylum observed
   as an example of a type.
D  Examine the living material provided.
   Sketch and identify animal forms
   encountered as far as time permits.
   Can you draw any conclusions as to the
   types of animal life found in the various
   habitats indicated?
                                                This outline was prepared by H.W.  Jackson,
                                                former Chief Biologist, National Training
                                                Center,  and revised by R. M. Sinclair,
                                                Aquatic  Biologist,  National Training Center,
                                                MOTD,  OWPO, USEPA,  Cincinnati,  Ohio
                                                45268
                                                Descriptors:
                                                Zooplankton
             Analytical Techniques,
41-2

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               LABORATORY: PROPORTIONAL COUNTING OF PLANKTON
 I  OBJECTIVE

 To learn and practice the techniques of
 proportional counting of mixed plankton
 samples.
 II  MATERIALS

 A  Several plankton samples,  each con-
    taining a number of plankton forms.

 B  Class slides, cover slips,  and dropping
    pipets.
Ill  PROCEDURES

 A  Make an ordinary wet mount of the
    sample provided.

 B  Scan the slide. Identify and lists all
    types of plankton present.

 C  Proportional Counting (use clump count)

    1  Field count

       a  Count and tally all individuals of
          each type present in a field.  The
          best way to do this is to list the
          most common types separately
          and  record the counts and the
          enumerate the other forms.
       b  Move the slide at random and
          repeat the process.  Do this for
          5 or 10 fields, or for one or
          two strips.

       c  Tally the results and compute
          the percent of each type.

    2  Five hundred count

       a  Moving the slide at random count
          and tally all the types of plankton
          as before until a total of 500 cells
          or clumps have been counted.

       b  Tally the results and compute the
          percentage of each type as before.
IV  RESULTS

 A  Record your results for both methods
    on the board.

 B  Discuss the two methods and the use of
    the proportional count results.
 This outline was prepared by M. E. Bender,
 Biologist, formerly with FWPCA Training
 Activities, SEC.

 Descriptors:  Plankton, Analytical
 Techniques
  BL MIC. enu.lab. 6a. 6. 76
                                       42-1

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               CALIBRATION AND USE OF PLANKTON COUNTING EQUIPMENT
I  INTRODUCTION

A  With the exception of factory-set
   instruments, no two microscopes can
   be counted upon to provide exactly the
   same magnification with any given com-
   bination of oculars and objectives.  For
   accurate quantitative studies, it is there-
   fore necessary to standardize or "calibrate"
   each instrument against a known standard
   scale. One scale frequently used is a
   microscope slide on which two millimeters
   are  subdivided into  tenths, and two addi-
   tional tenths are subdivided into hundredths.
   Figure 3.

B  In order to provide an accurate measuring
   device in the microscope,  a Whipple
   Plankton Counting Square or reticule
   (Figure 2a)  is installed in one ocular
   (there are many different types of reticules).
   This square is theoretically of such a size
   that with a 10X objective, a 10X ocular,
   and a tube length of 160 mm, the image
   of the square covers a square area on the
   slide one mm  on a slide.  Since this
   objective is rarely attained however, most
   .microscopes must be standardized or
   "calibrated" as described below in order
   to ascertain the actual size of the Whipple
   Square as seen through the microscope
   (hereinafter referred to as the "Whipple
   field").  This  process is schematically
   represented in Figures 5 and 7. If the
   Whipple eyepiece is to be used at more
   than one magnification,  it must be recali-
   brated for each.  A basic type of monocular
   microscope is shown in Figure 1.

C  Microscopes with two eyepieces (binocular)
   are a convenience but not essential.  Like
   modern cars they are not only great
   "performers, " but also  complicated to
   service or,  in this instance,  calibrate.
   On some instruments, changing the inter-
   pupillary distance  also changes the  tube
   length, on others it does not.   The  "zoom"
   feature on certain scopes is also essentially
   a system for changing the tube length.

   The resultant is that in addition to calibra-
   tion at each combination  of eyepiece and
   objective,  any other  factor which may
   affect magnification must also be considered.
   In some instances this may mean setting up
   a table of calibrations at a series of micro-
   scope settings.
   Another procedure is to select a value
   for each of the variables involved (inter-
   pupillary distance, zoom,  etc.) and
   calibrate the scope at that combination.
   Then each time the scope is to be used for
   quantitative work, re-set each variable to
   the value selected.  A separate multipli-
   cation factor must be calculated for each
   adjustment which changes the magnification
   of the instrument.

   Since the Whipple Square can be used to
   measure both linear dimensions and
   square areas,  both should be recorded on
   an appropriate form.  A suggested format
   is shown in Figure 6.

   (Data written in are used as an illustration
   and are not intended to apply to any
   particular microscope.  An unused form
   is included as Figure 6-A. )
II   THE CALIBRATION PROCEDURE

 A  Installing the Whipple Square or Reticule

    To install the reticule in the ocular
    (usually the right one on a binocular
    microscope), carefully unscrew the upper
    lens mounting and place the reticule on
    the circular  diaphragm or shelf which  will
    be found approximately half way down inside
    (Figure 4).   Replace the lens mounting and
    observe the markings on the reticule.  If
    they are not  in sharp focus,  remove and
    turn the reticule over.

    On reticules with the markings etched  on
    one side of a glass disc, the etched sur-
    face can usually be recognized by shining
    the disc at the proper angle in a light.
    The markings will usually be in the best
    focus with the etched surface down.  If the
    markings are sandwiched between two  glass
    discs cemented together, both sides are
    alike, and the focus may not be quite as
    sharp.

 B  Observation  of the Stage Micrometer

    Replace the ocular in the microscope and
    observe the stage micrometer as is illus-
    trated schematically in Figure 5:  Calibration
    of the Whipple Square.  On a suitably ruled
    form such as the one illustrated, Figure 6,
    Calibration Data, record the actual distance
    in millimeters subtended by the image  of
 BI, MET.mic. Id. 6. 76
                                                                                      43-1

-------
 Calibration and Use of Plankton Counting Equipment^
    the entire Whipple field and also by each
    of its subdivisions.  This should be
    determined for each significant settling of
    the interpupillary distance for a binocular
    microscope, and also for each combination
    of lenses employed.  Since oculars and
    objectives  marked with identical magnifi-
    cation,  and since microscope frames too
    may differ, the serial or other identifying
    number of  those actually calibrated should
    be recorded.  It is  thus apparent that the
    determinations recorded will only be valid
    when used  with the  lenses listed and on that
    particular  microscope.

  C Use of the  2OX Objective

    Due to the  short working distance beneath
    a 46X (4mm) objective, it is impossible
    to focus to the bottom of the  Sedgewick-
    Rafter plankton counting cell with this lens.
    A  10X (16mm) lens on the other hand
    "wastes" space between the front of the
    lens and the coverglass, even when focused
    on the bottom of the cell. In order to make
    the most efficient use possible of this cell
    then, an objective of intermediate focal
    length is desirable.  A lens with a focal
    length of approximately 8 mm, having a
    magnification of 20 or 2IX will meet these
    requirements. Such lenses are available
    from American manufacturers and are
    recommended for this type of work.
in  CHECKING THE CELL

 The internal dimensions of a Sedgewick-Rafter
 plankton counting cell should be 50 mm long
 by 20 mm wide by 1 mm deep (Figure 8).

 The actual horizontal dimensions of each new
 cell should be checked with calipers, and the
 depth of the cell checked at several points
 around the edge using the vertical focusing
 scale engraved on the fine adjustment knob of
 most microscopes.  One complete  rotation of
 the knob usually raises or lowers the objective
 1 mm  or 100 microns (and each single mark
 equals 1 micron).  Thus,  approximately ten
 turns of the fine adjustment knob should raise
 the focus from the bottom of the cell to the
 underside of a coverglass resting on the rim.
 Make these measurements on an empty cell.
 The use of a No.  1  or 1-1/2, 24 X 60 mm
 coverglass is recommended rather than the
 heavy  coverglass that comes with the S-R
 cell, as the thinner glass will somewhat con-
 form to any irregularities of the cell rim
 (hence, also making a tighter seal  and reduc-
 ing evaporation when in actual use).  Do not
 attempt to focus on the upper surface of the
 rim of an empty cell for the above depth
 measurements, as the coverglass is supported
 by the highest points of the rim only, which
 are very difficult to identify.  Use the average
 of all depth measurements as the  "true" depth
 of the cell.  To simplify calculations below,  it
 will be assumed that we are dealing with a
 cell with an average depth of exactly 1. 0 mm.
IV  PROCEDURE FOR STRIP COUNTS USING
    THE SEDGEWICK-RAFTER CELL

 A Principles

    Since the total area of the cell is 1000 mm2,
    the total volume is 1000 mm3 or 1 ml.  A
    "strip" the length of the cell thus constitutes
    a volume (V.) 50 mm long,  1 mm deep, and
    the width of the Whipple field.
                                    o
    The volume of such a strip in mm  is:

       V1 = 50  X width of field X depth

          = 50  XwX 1

          = 50  w

    In the example given below on the plate
    entitled Calibration Data, at a magnification
    of approximately 20OX with an interpupillary
    setting of  "60", the width of the Whipple
    field is recorded as approximately 0. 55 mm
    (or 550 microns).  In this case, the  volume
    of the strip  is:

       Vj = 50 w = 50 X 0. 55 = 27. 5  (mm3)

 B Calculation  of Multiplier Factor

    In order to convert plankton counts per
    strip to counts per ml, it is simply
    necessary to multiply the count  obtained
    by a factor (F..) which represents the
    number of times the  volume of the strip
    examined (Vi) would be contained in 1 ml or
    1000 mm3.     Thus in the example  given
    above:
                                    3
       F  -     volume of cell in mm
        1     volume examined in mnv*
             1000
              V
1000
27.5
= 36.36
          =  approx. 36
    If more than one strip is to be counted,
    the factor for two, three, etc.,  strips
    could be calculated separately using the
    same relationships outlined above,  changing
    only the measurement for the length of
  43-2

-------
                                          Calibration and Use of Plankton Counting Equipment
                       Figure 1.  THE COMPOUND MICROSCOPE
A) coarse adjustment; B) fine adjustment;
C) arm or pillar; D) mechanical stage which
holds slides and is movable in two directions
by means of the two knobs; E) pivot or joint.
This should not be used or "broken" while
counting plankton; F) eyepiece (or ocular cf:
figure 4); G) draw tube.  This will be found
on monocular microscopes only (those having
only one eyepiece).  Adjustment of this tube
is very helpful in calibrating the microscope
for quantitative counting (Sec. 5. 5. 2. 2. ).
H) body tube.   In some makes of microscopes
this can be replaced with a body tube having
two eyepieces, thus making the  'scope into
a "binocular. " I) revolving nosepiece on
which the objectives are mounted; J) through
M are objectives, any one of which can be
turned toward the object being studied.  In
this case J is a 40X,  K is a 100X, L is a 20X,
and M is a 10X objective.  The product of
the magnification power of the objective being
used times the magnification power  of the
eyepiece gives the total magnification of the
microscope.   Different makes of microscopes
employ objectives of  slightly different powers,
but all are approximately equivalent.  N) stage
of the microscope; O) Sedgwick-Rafter cell in
place for observation; P) substage condenser;
Q) mirror; R) base or stand; note:  for
information on the optical system, consult
reference 3.   (Photo  by Don Moran.).

-------
 Calibration and Use of Plankton Counting Equipment
                                          Figure 2
 Types of eyepiece micrometer discs or
 reticules (reticules,  graticules, etc.).
 When dimensions are mentioned in the
 following description, they refer to the
 markings on the reticule discs and not to
 the measurements  subtended on the micro-
 scope slide.  The latter must be determined
 by calibration procedures  such as those
 described elsewhere, (a) Wnipple plankton
 counting eyepiece.  The fine rulings in the
 subdivided square are sometimes extended to
the margin of the large square to facilitate
the estimation of sizes of organisms in
different parts of the field,  (b) Quadrant
ruling with 8. 0 mm circle, for counting
bacteria in milk smears for example,   (c)
Linear scale 5. 0 mm divided into tenths.
For measurement of linear dimensions.
(d) Porton reticule for estimating the size
of particles.  The sizes  of the  series of discs
is based on the square root of two so that the
areas of successive discs double as they
progress in size.
43-4

-------
                                          Calibration and Use of Plankton Counting Equipment
   strip counted.  Thus for two strips in the
   example cited above:

      V2=   100W = 100 XO. 55  = 55 mm3
         _   1000  _ 1000  _
         '   -  ---     -
                    55-
                         _    „
                         - 18'2
   It will however be noted that F  =  -=i .


   Likewise a factor F  for three strips

               Fl
   would equal -5- or approximately 12, etc.


C  An Empirical "Step-Off" Method

   A simpler but more empirical procedure
   for determining the factor is to consider
   that if a strip 20 mm wide were to be
   counted the length of the cell,  that the
   entire 1000 mm3 would be included since
   the cell is 20 mm wide and 1 mm deep.

   This 20 mm strip width can be equated to
   1000 mm3.  If a strip (or the total of 2 or
   more strips) is less than 20 mm in width,
   the quotient  of 20  divided by this width will
   be a multiplier  factor for converting from
   count per strip(s) to count per ml.
V
    Thus in the example cited above where at
    an approximate magnification of 200X and
    with an interpupillary setting of 60, the
    width of the Whipple field is . 55 mm.  Then:
                                                             on
   Fl=
                                                       (as above)
                        or approx. 36
    If two strips are counted:
       .55
       .55
      1.10
                         = 18.2 = approx.  18,etc.
    This same value could be obtained without
    the use of a stage micrometer by carefully
    moving the cell sidewise across the field
    of vision by the use of a mechanical stage.
    Count the number of Whipple fields in the
    width of the cell.  There should be approxi-
    mately 36 in the instance cited  above.
SEPARATE FIELD COUNT USING THE
SEDGEWICK-RAFTER CELL
 A  Circumstances of Use

    The use of concentrated samples, local
    established programs,  or other circumstances
                              Figure 3.  STAGE MICROMETER

     The type illustrated has two millimeters divided into tenths, plus two additional
     tenths subdivided into hundredths.
                                 Micrometer Scale,
                                     Enlargement of Micrometer Scale
                                                                                     43-5

-------
Calibration and Use of Plankton Counting Equipment
     Figure 4.  Method of Mounting the Whipple Disc in an Ocular.  Note the upper
        lens of the ocular which has been carefully unscrewed, held in the left
           hand, and the Whipple disc, held in the right hand.  (Photo by
                                        Don Moran).

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                                             Calibration and Use of Plankton Counting Equipment
                                CALIBRATION OF WHIPPLE SQUARE
                              as seen with 10X Ocular and 43X Objective
                                (approximately 430X total magnification)
                  Whipple Square as
                  seen through ocular
                   ("Whipple field")
                                                      "Small squares" subtend
                                                      one fifth of large squares-
                                                      . 0052 mm or 5. 2n
    "Large square" subtends
    one tenth of entire Whipple
    Square: .026 mm or 26|i
                                                             Apparent lines of sight
                                                             subtend . 26 mm or 260>i
                                                             on stage micrometer
                                                             scale
                   PORTION OF MAGNIFIED IMAGE OF STAGE MICROMETER SCALE —
                                             Figure 5

                           CALIBRATION OF THE WHIPPLE SQUARE
The apparent relationship of the Whipple
Square is shown as it is viewed through a
microscope while looking at a stage
micrometer with a magnification of
approximately 43OX (10X ocular and 43X
objective).
                                                                                           43-7

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Calibration and Use of Plankton Counting Equipment
                                   MICROSCOPE CALIBRATION DATA


                                                    Microscope No.
Apr"oxirnate
Mollification
Tube
Length, or
"nterpupillary
Setting
Linear dimensions of Whipple
squares in millimeters*
Whole
Large
Small
Factor for
Conversion
to count/ ml
                100X.  obtained with
                                                        (2 S-R Strips)
Objective
Serial No.
and Ocular
Serial No
/3Qt,74L6hX\


SO
(,0
10


1. /3o
l.llŁ
1. 100


0.113
D.lll
o. no


0.03 at*
6.0J.3.Z
0.03.22


Ł9
9.0
9J

l^^i-^yl1-"0.?1-^^
Objective
Serial No.
                and Ocular
                Serial No.
                400X, obtained with
                                                                        (2 S-R Stripe)

Ł~0
(*Q
70


O.StoO
0. S5'0
O.SVs


O.O5L,
0. GSS
o. $sy


0, 0 //A
0.0110
0.0109


/7.1
/f.A
/J>.3

                                                      (Nannoplankton)
                                                      (cell-20 fields )'
Objective
Serial No.
and Ocular
Serial No.
/39Ł>7yt.('ot)


^0
(,0
'7/9


0.3.6,7
0.U2
0.3LO


&GU?
/).6263
n.OAUO


,00-53
.0053
. 0052


/734.
lift,.


                *1 mm - 1000 irij.•.!•!
   Microscope calibration data.  The form
   shown is suggested for the recording of
   data pertaining to a particular microscope.
   Headings could be modified to suit local
                                    situations.  For example,  "Interpupillary
                                    Setting" could be replaced by "Tube Length"
                                    or the "2S-R  Strips  could be replaced by
                                    "per field" or "per 10 fields. "
                                            Figure 6
 43-8

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                                	Calibration and Use of Plankton Counting Equipment
                            MICROSCOPE CALIBRATION DATA
                                               Microscope No.
Approximate
IVI 3. unification

Tube
Length, or
Inte rpupillary
Setting
Linear dimensions of Whipple
squares in millimeters*
Whole 1 Large 1 Small
Factor for
Conversion
to count/ ml
100X. obtaine
Objective
Serial No.

and Ocular
Serial No.


J with























<2 S-H Strips)





      200X, obtained with
  (2 S-R Strips)
Objective
Serial No.
and Ocular
Serial No.


























      400X, obtained with
(Nannoplankton)
(cell-20 fields )
Objective
Serial No.
and Ocular
Serial No.


























      *lrnm = 1000 microns
                                                            BI. AQ.pl. 8. 10. 60.
                                      Figure 6-A

                          MICROSCOPE CALIBRATION DATA

Suggested work sheet for the calibration of a microscope.  Details will need to be adapted
to the particular instrument and situation.
                                                                                 43-9

-------
Calibrn I OMU nt I
                                 ^^             _______
                    S-R COVER
                     GLASS —
                       WATER IN   1MM
                        S-R CELL
                                                  WHIPPLE SQUARE
                                                        PLANKTERS
                                                 THICKNESS S-R SLIDE
                                        Figure 7

    A cube of water as seen through a Whipple square at 100X magnification in
    a Sedgewick-Rafter cell.  The figure is drawn as if the microscope were
    focused on the bottom of the cell, making visible only those organisms lying
    on the bottom of the cell.  The little "bug" (copepod) halfway up,  and the
    algae filament at the top would be out of focus.   The focus must be moved up
    and down in order to study (or count) the entire cube.
43-10

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                                          Calibration and Use of Plankton Counting Equipment
   may make it necessary to employ the more
   conventional technique of counting one or
   more  separate Whipple fields instead of
   the strip count method. The basic relation
   ships  outlined above still hold,  namely:
             volume cell in mm
f~3
           volume examined in mfh~

B  Principles Involved

   The volume  examined in this case will
   consist of one or more squares the dimen-
   sions of the  Whipple field in area and 1 mm
   in depth (Figure 7).  Common practice
   for routine work is to examine 10 fields,
   but exceptionally high or low counts or
   other circumstances may indicate that
   some other number of fields  should be
   employed.  In this case a "per field"
   factor may be determined to be subsequently
   divided by the number of fields examined
   as with the strip count.  The following
   description however is based on an assumed
   count of 10 fields.
                                         NANNOPLANKTON COUNTING

                                      For counting nannoplankton using the high
                                      dry power (10X ocular and 43X objective)
                                      and the "nannoplankton counting cell"
                                      (Figure 9) which is 0. 4 mm deep, a minimum
                                      of 20 separate Whipple fields is  suggested.
                                      The same general relationships presented
                                      above (Section IV) can be used to obtain a
                                      multiplier or  factor (F5) to convert counts
                                      per 20 fields to counts per ml.

                                      To take another example from Figure 4, at
                                      an approximate magnification of  400X and an
                                      interpupillary setting of 70 (see also Figure 3)
                                      we observe that one side of the Whipple field
                                      measures 0. 260 mm.  The volume of the
                                      fields examined is thus obtained  as follows:

                                      V5 =  side2 X  depth X no.  of fields

                                         =  0. 26 X 0.26 X 0.4 X 20 = .54 mm3
                                      and  F
                                  =  (approx. ) 1850
C  Calculation of Multiplier Factor

   As stated above, the total volume
   represented in the fields examined con-
   sists of the total area of the Whipple fields
   multiplied by the depth.

     V4 =   (side of Whipple field)2 X depth

     (1 mm) X no. of fields counted)

   For example,  let us assume an approxi-
   mate magnification of 100X (see Figures
   6 and 7 and an interpupillary setting of
   "50".  The observed length of  one side
   of the Whipple field in this case is 1. 13
   mm. The calculation of V. is thus:

     V4=   side  X depth X no.  of fields

        =   1.13 X 1.13 X 1 X 10 = 12.8 mm3

     The  multiplier factor is obtained as
     above  (Section IV A):
                              3
     F4 =
	volume cell in mm"
volume examined in mm"
        =  ~  = (approx.) 78

     (If one field were counted, the factor
     would be 781, for 100 fields it would
     be 7.8. )
                 It should be noted that the volume of the
                 nannoplankton cell, . 1 ml, is of no significance
                 in this particular calculation.
                 REFERENCES

                 1  American Public Health Association,  et. al.
                      Standard Methods for the Examination
                      of Water, Sewage, and Industrial Wastes.
                      14th Edition. Am. Public Health Assoc.
                      New York.  1976.

                 2  Jackson,  H.W. and Williams,  L. G.
                      The Calibration and Use of Certain
                      Plankton Counting Equipment.  Trans.
                      Am.  Mic. Soc.   LXXXI(1);96-103. 1962.

                 3  Ingram, W. M. and Palmer,  C. M.
                      Simplified Procedures for Collecting,
                      Examining,  and Recording Plankton in
                      Water. Jour. Am. Water Works.
                      Assoc. 44(7): 617-724.   1952.

                 4  Palmer,  C. M.  Algae in Water Supplies.
                      U. S.  D. H.  E. W.  Public Health Service
                      Pub.  No.  657.  1959.

                 5  Palmer,  C. M. and Maloney, T. E. A
                      New Counting Slide for  Nannoplankton.
                      American Soc. Limnol.  and Oceanog.
                      Special Publications No. 21.  pp. 1-6.
                      1954.
                                                                                      43-11

-------
Calibration and Use of Plankton Counting Equipment
                         Area
                        Uncounted
                                         Figure 8

   Sedgewick-Rafter counting cell showing bottom scored across for ease in counting
   strips.  The "strips" as shown in the illustration simply represent the area counted,
   and are not marked on the slide.  The conventional dimensions are 50 X 20 X 1 mm, but
   these should be checked for accurate work.
                                          Figure 9

   Nannoplankton cell.  Dimensions of the circular part of the cell are 17.9 mm diameter
   X 0. 4 mm depth.  When covered with a coverglass, the volume contained is 0. 1 ml.
   The channels for the introduction of sample and the release of air are 2 mm wide and
   approximately 5 mm long.  This slide is designed to be used with the 4 mm or  43X
   (high dry) objective.
6  Welch, Paul S.  Limnological Methods.
      Blakiston Company.  Phila.  Toronto.
      1948.

7  Whipple, G. C., Fair, G. M., and
      Whipple, M. C.  The Microscopy of
     Drinking Water.  John Wiley and Sons.
     New York.   1948.
This outline was prepared by H. W. Jackson,
former Chief Biologist, National Training
Center, and revised by R. M. Sinclair,
Aquatic Biologist,  National Training Center
MOTD, OWPO. USEPA, Cincinnati, Ohio 45268

Descriptors:  Plankton, Microscopy
 43-12

-------
               LABORATORY:  FUNDAMENTALS OF QUANTITATIVE COUNTING
 I  OBJECTIVE

 To learn and practice the basic techniques of
 quantitative plankton counting
 II  MATERIALS
    Plankton Samples Containing a Variety of
    Plankton Forms

    S-R Cells and Coverglasses, Large Bore
    1 ml Pipettes,  Whipple Discs,  Plankton
    Record Form
III  PROCEDURE
    Fill the S-R cell with sample number 1 as
    follow s:
       Place the coverglass diagonally across
       the S-R cell.  This leaves the other two
       corners uncovered; one for putting in
       the sample fluid, the other to allow
       air to be driven out as it is replaced
       by the incoming aliquot.  Shake the
       sample to disperse the plankton.  Before
       settling occurs in the sample draw about
       1-1/4 ml of the fluid into the pipette
       and quickly fill the S-R cell by delivering
       the aliquot into one of the open corners
       of the chamber.
   Starting from one end of the S-R cell and
   preceding to the opposite (this is called a
   strip count, begin counting (clump counts)
   the plankton forms.  The length of the
   cell may be traversed in several ways.

   1  Count all the forms in the Whipple
      square or in a portion of the square,
      record the count and move the slide so
      that the square covers the adjoining
      area.

   2  Move  the slide very slowly counting
      and recording the various forms as
      they pass the leading  edge of the
      Whipple disc.
                                                 IV  RESULTS
A  Using the conversion factor obtained in
   the previous laboratory compute the
   number of plankton organisms per ml.

B  Record the results on the board.

C  Discussion of Results

D  Refill the slide with a fresh aliquot and
   recount the sample.  Compare results
   with the first count.

E  Count the other samples of mixed plankton
   as assigned, following the same procedure.
    Using lOOx focus on the sample.  After
    focus has been obtained switch to 200x.
    Scan the slide and list the plankton forms
    present.
 This outline was prepared by M. E. Bender,
 Former Biologist,  FWPCA,  Water Pollution
 Training Activities, SEC.

 Descriptors:  Plankton, Analytical Techniques
    BI. MIC. enu. lab. 7. 6. 76
                                                                                    44-1

-------
Laboratory:  Fundamentals of Quantitative Counting
               idy of Water: .
                                                 PLANKTON COUNT RECORD
                                                     Date Collected:.
                                                                             , Date Analyzed.


                                                                             .  Analyst	

TOTAL COUNT:
Organisms,
Differential
Count 1














Notes, Talleys
















Total
















FIELD CONDITIONS
Tlno nf Hay ,, ,
Vol, Area.
or Type of Count

















Air Tenor Vater Tenp*
Voather tndav •
Previous Weather •
Turbidity Met ho
lr T. reading-


Preservative •

Filanentou
Other Plan
Surface Sc
Dead Fish
Odor of Wa
Other Physic
s Alffaer,
tB •



al nr Chemical Dat
a




















LABORATORY
Method of Pr<
Departure fr
Significance
Treatment Re


















ANALYSIS
Count
per
ml.

















per
liter

















Group
Totals























PLANT OR OTHER DATA, FOR B
Treatment fo, J?Ł,tSJ
A * r
Other Cnevic
X:
.1.

Taate and Odor:
Filter Runs:
Othgr



                                            SUGGESTED BASIC FORM FOR PLANKTON RECORDS
                                                                                                              HWJ
                                                                                                              4/68
                 .UIC.enu.pl.2.4.68

-------
                 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 "ib", 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
list.)
         . 2j.b.6.76
                                                                                      45-1

-------
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,  accordian-
      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-
    eton may be external or internal.

5b  Body soft and/or wormlike.
    Skin may range from soft to
    parchment-like.
6a  Three or more pairs of well
    formed jointed legs present.
        Phylum ARTHROPODA (Fig. 4)

6b  Legs  or appendages, if present,
    limited to pairs of bumps or hooks.
    Lobes or tenacles,  if present,
    soft and fleshy, not jointed.

7a  Body  strongly depressed or
    flattened in cross section.

7b  Body  oval, round,  or shaped like
    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
    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
15
19
10
11
  45-2

-------
                                              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.        40
    (Vertebrates)

15b Body covered with an external         16
    skeleton or shell.
         (Figs.  10,  13,  17, 18,  24,
         25, 28)

16a External skeleton jointed, shell       19
    covers legs and other appendages,
    often leathery in nature.
         Phylum ARTHROPODA

16b External shell entire, not jointed,     17
    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
                                                                                     45-3

-------
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. 
-------
                                               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
                                                                                     45-5

-------
 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.
41a  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
 45-6

-------
                                             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.
      398pp.   1966.
G.P. Putnam's Sons.
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. 151pp.   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.
                                                                                     45-7

-------
  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
         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
45-8
                    ' aria, Meaostoma
                   L i,m.
                                       6B.  Turbellaria, Dugesia
                                           Up to 1. 6 cm.
                              7.  Nematodes. Free living
                                 forms commonly up to
                                 1mm., occasionally
                                 more.

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                                     Key to Selected Groups of Freshwater Animals
                  8B. Diptera, Mosquito
                      pupa. Up to 5mm.
8A.  Diptura, Mosquito larvae
    Up to 15 mm. long.
                      8C. Diptera,  chironomid  8E'
                           larvae.  Up to 2 cm.
                                  9D. Diptera, Rattailed maggot
                                      Up to 25mm. 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.
                   12A. Branchiopod,
                         Daphnia.  Up
                         to 4mm.
 11A. Ostracod, Cypericus
     Side view, up to 7 mm.
                        11B. Cypericus, end view.
                                                              12 B.  Branchiopod,
                                                                     Bosmina.  Up
                                                                     to 2mm.
                                                                   45-9

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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, Eubranchipus
    Up to 5 cm.
            17."Amphipod,  Pontoporeia
                   to 25 mm.
                                         18. Isopod, Asellus
                                             Up to 25 mm.
20. Collembola,  Podura
    Up to 2 mm.  long
    45-10
19A. Calanoid copepod,
     Female       19B. Cyclopoid cope pod-
     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, larval cases,
       mostly 1-2 cm.
                   22. Megaloptera,--alderfly
                       Up to  2  cm.
23A. Beetle larvae,   23B. Beetle  larvae,  24A.  Odonata, dragonfly
     Dytisidae,            Hydrophilidae        nymph up to 3 or
     Usually about 2 cm.   Usually about        4 cm
                            1  cm.
                   24E. Odonata, tail
                        of damsel fly     ,,. ^ _._   ^J
                        nymph            >=^ 24E,  Odonata, front view
                        (side view)    ///"• I      of dragonfly nymph
                    Suborder           \5&       showing "mask"
                     Zygoptera         ^^        partially extended
                     (24B, D)       |—^__,  Suborder
                                    T"f  Anisoptera
             24D.  Odonata, damselfly V	—V       (24A, E, C)
                  nymph (top view)    V—•—y
                                       \f]j24C. Odonata, tail of
                                                dragonfly nymph
                                                 (top view)
                                                               45-11

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Key to Selected Groups of Freshwater Animals
            25A. Hemiptera,
                 Water Boatman
                About 1 cm.
                                      25B. Hemiptera,
                                            Water  Scorpion
                                            About 4 cm.
                             26. Plecoptera,
                                Stonefly nymph
                                Up to 5cm.
            27.Ephemeroptera,
              Mayfly nymph
              Up to 3cm.
28A.  Coleoptera.
     Water scavenger
     beetle.  Up to 4 cm.
                                                              28B. Coleoptera,
                                                                   Dytiscid beetle
                                                                   Usually up to 4
                                                                                 cm,
    45-12
               29A. Diptera, Crane
                   fly. Up to 2i cm.
                                                       Diptera, Mosquito
                                                       Up to 20 mm.

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                                     KEY TO FRESH WATER ALGAE
                     COMMON IN WATER  SUPPLIES AND IN  POLLUTED WATER
    2a.
  Beginning with "1a" and  "1b," choose one of the two
contrasting statements and follow this procedure with the
"a" and "b" statements of the number given at the end of
the chosen statement. Continue until the;.name of the alga
is given instead of another  key number.  (Where recent
changes in names of algae have been made, the new name
is given followed by the  old name in brackets.)
  la.    Plastid (separate color body) absent; com-
        plete protoplast  pigmented;  generally blue-
        green; iodine starch test*  negative 	
        	(blue-green  algae)     4
  1b.    Plastid or plastids present;  parts of  proto-
        plast free of some  or all pigments;  gener-
        ally  green, brown,  red,   etc.,  but  not
        blue-green;  iodine  starch test*  positive or
        negative  	     2
        Cell wall permanently rigid  (never showing
        evidence of collapse), and with  regular pat-
        tern of fine markings (striations, etc.); plas-
       tids brown-to-green; iodine  starch  test*
        negative; flagella absent; wall of two  essen-
        tially similar halves, one  placed over  the
        other as a cover	(diatoms)
        Cell wall  is present, capable  of sagging,
        wrinkling, bulging, or rigidity, depending on
        existing turgor pressure of cell, protoplast;
        regular pattern of fine markings oh wall gen-
       erally  absent;  plastids green, red, brown,
       etc.; iodine starch test* positive  or negative;
       flagella present or absent; cell wall continu-
       ous and generally not of two parts	
       Cell or colony motile; flagella present (often
       not readily visible);   anterior and posterior
       ends of cell different from one  another in
       contents and often in shape	
        	(flagellate  algae)
        Non-motile; true flagella  absent;  ends of
       cells often not differentiated	
        	(green algae and associated forms)
    2b.
    3a.
    3b.
                     Blue-Green Algae
    4a.   Cells in filaments  (or  much  elongated to
         form a thread)	
                                                      75
                                                     198
                                                    262
   'Add one drop Lugol's (iodine) solution, diluted 1:1 with  distilled
    water. In about 1 min, if positive, starch is stained blue.
  4b.    Cells not in (or as) filaments	    61
  5a.    Heterocysts present 	     6
  5b.    Heterocysts absent	    25
  6a.    Heterocyst located at one end of filament..     7
  6b.    Heterocyst at various locations-in filament. .    15
  7a.    Filaments radially arranged in a gelatinous
        bead 	     8
  7b.    Filaments isolated or irregularly  grouped...    11
  8a.   Akinetes present  	(C/oeotr/chia)     9
  8b.    Akinetes absent	(Rivularia)    10
  9a.    Gelatinous colony a smooth bead	
        	G/oeotr/cn/a ech/nu/ata
  9b.    Gelatinous colony irregular	
        	Gloeotrichia  natans
10a.    Cells near the narrow end as long as wide..
        	Rivularia dura
10b.    Cells near the narrow end twice as long as
       wide	Rivularia haematites
11a.    Filament gradually narrowed to one end....
        	(Calothrix)    12
11b.    Filament not gradually narrowed to one end    13
12a.   Cells adjacent to heterocyst wider than het-
       erocyst	Calothrix braunii
12b.    Cells adjacent to heterocyst narrower than
       heterocyst 	Calothrix parietina
13a.   Heterocysts at both ends; filaments bent. . .
        	/Anafaaenops/s
13b.    Heterocysts at one  end;  filaments  straight
        	(Cylindrospermum)    14
14a.   Heterocysts round	
        	Cylindrospermum muscicola
14b.   Heterocysts elongate 	
        	Cylindrospermum stagnate
15a.   Filaments unbranched or with true branches    16
15b.    Filament with occasional false branches....    24
16a.   True  branching  present;  filament  all   or
       partly multiseriate ... .Stigonema minutum
16b.   Branching absent; filaments uniseriate	    17
17a.   Cross-walls much closer together than width
       of filament	Nodularia spumigena
17b.   Cross-walls at least as far apart  as width of
       filament  	    18
18a.   Filaments normally in tight parallel  clusters;
       heterocysts and spores cylindric to long oval
       	Aphanizomenon flos-aquae
BLMIC.cla. 23.4. 80
                                                                                                           46-1

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Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                                       Key
 18b.   Filaments not in tight parallel clusters; het-
        erocysts and spores often round to oval....   19
 19a.   Filaments in a common  gelatinous  mass ..
        	  (Nostoc)   20
 19b.   Filaments not in a common gelatinous mass
        	  (Anabaena)   21
 20a.   Heterocysts and vegetative cells rounded..
        	Nostoc pruniforme
 20b,   Heterocysts and vegetative cells oblong —
        	 Nostoc carneum
 21a.   Cells elongate, depressed in the middle; het-
        erocysts rare	Anabaena constricta
 21b.   Cells rounded; heterocysts common	   22
 22a.   Heterocysts with lateral extensions	
        	Anabaena p/anctom'ca
 22b.   Heterocysts without lateral extensions	   23
 23a.   Threads 4-8 microns wide	
        	Anabaena flos-aquae
 23b.   Threads 8-14 microns wide	
        	Anabaena drdnalis
 24a,   False branches in pairs	
        	  Scytonema to/ypothr/coides
 24b.   False branches single.. .  . To/ypothnx  tenuis
 25a.   Filament or elongated cell attached at one
        end  and  with  one  or  more  round  cells
        (spores) at the other end	
        	Entophysalis lemaniae
 25b.   Filament generally not attached at one end;
        no terminal spores present	    26
 26a.    Filament with regular spiral form throughout   27
 26b.   Filament not spiral or with spiral limited  to
        a portion  of filament	    30
 27a.    Filament septate	Arthrospira jenneri
 27b.   Filaments not septate	(Spirulina)   28
 28a.   Thread 0.9 micron or  less in diameter	
        	Spirulina subtillissima
 28b.    Thread  1.2 microns or more in diameter...    29
 29a.   Thread 1.2-1.7 microns in diameter	
        	Spirulina  major
 29b.    Thread  2.0 microns in diameter	
        	Spirulina  nordstedtii
 30a.   Filament aggregates forming conical tufts..
        	Symp/oca muscorum
 30b.    Filament aggregates  not forming  conical'
       tufts  	    31
 31a.    Filament very  narrow, only 0.5-2.0 microns
       wide	Schizothrix caldcola
 31 b.    Filament 3.0-95.0 microns wide	     32
32a.   Filaments loosely aggregated or not  in clus-
       ters  	     33
32b.   Filaments tightly  aggregated and surrounded
       by a common gelatinous secretion that may
       be invisible  	     56
33a.   Filament surrounded  by  a wall-like sheath
       that frequently extends beyond  the ends of
       the filament of cells; filament  generally
       without  movement	     34
 33b.    Filament  not  surrounded  by  a wall-like
        sheath; filament may show movement	   41
 34a.    False branching present	
        	P/ectonema tomas/n/ana
 34b.    False branches absent 	   35
 35a.    Cells separated from one another by a space
        	  /ohannesbapt/st/a
 35b.    Cells in contact with adjacent cells	
        	 (Lyngbya)   36
 36a.    Threads in part forming spirals	
        	Lyngbya  lagerheimii
 36b.    Straight or bent but not in spirals	   37
 37a.    Threads colored yellowish to brown	   38
 37b.    Sheaths colorless  	   39
 38a.    Cells rounded	Lyngbya ocnracea
 38b.    Cells short discs	Lyngbya aestuarii
 39a.    Cells constricted at the joints	
        	Lyngbya putealis
 39b.    Cells not  constricted at the joints	   40
 40a.    Sheath very thick	Lyngbya versicolor
 40b.    Sheath very thin  	Lyngbya digueti
 41 a.    All filaments short, with  less than 20 cells;
        one or both ends of filament sharp pointed
        	 Raphidiopsis
 41 b.    Filaments long with  more  than 20  cells;
        filaments  commonly without sharp-pointed
       ends	(Osdllatoria)   42
 42a.   Cells very short,  generally  less than one-
       third  the thread diameter	    43
 42b.    Cells generally one-half  as  long to  longer
       than thread  diameter	    46
 43a.   Cross walls constricted	
        	Osdllatoria  ornata
 43b.    Cross walls not constricted	    44
 44a.    Ends of mature threads curved	    45
 44b.    Ends  of mature threads straight	
        	Osdllatoria  limosa
 45a.   Threads 10-14 microns thick	
        	Osdllatoria curv/ceps
 45b.   Threads 16-60 microns thick	
        	Osdllatoria princeps
 46a.   Threads appearing red to purplish	
        	Osdllatoria  rubescens
 46b.   Threads yellow-green  to blue-green	    47
 47a.   Threads blue-green 	    48
 47b.   Threads yellow-green 	    53
 48a.   Cells 1A-2  times as long as thread diameter.    49
48b.   Cells 2-3 times as  long as thread diameter.    55
 49a.   Cell  walls  between  cells thick  and  trans-
       parent 	Osdllatoria pseudogeminata
 49b.    Cell walls  thin, appearing as  a dark line....    50
 50a.   Ends of thread straight 	
       	 Osdllatoria agardhii
 50b.   Ends of mature threads curved	    51
51a.   Prominent  granules  present especially at
       both ends of each cell	
       	Osdllatoria  tenuis
 46-2

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                Key to Fresh Water Algae  Common in Water Supplies and in Polluted Water
                                      ALGAE AND WATER  POLLUTION
51 b.   Cells without prominent granules	    52
52a.   Cross walls  constricted	
       	Oscillatoria chalybea
52b.   Cross walls  not constricted	
       	Oscillatoria tormosa
53a.   Cells 4-7 times as long as thread diameter..
       	  Osc///ator/a  putr/da
53b.   Cells less than 4 times as long as the thread
       diameter  	    54
54a.   Prominent granules (pseudovacuoles) in cen-
       ter of each cell .... Oscillatoria lauterbornii
54b.   No prominent granules  in center of cells  ..
       	 Oscillatoria chlorina
55a.   End  of thread long tapering ....;	
       	  Oscillatoria splendida
Ł>5b.   End  of thread not tapering	
       	Oscillatoria amphibia
56a.   Filaments arranged in a tight, essentially par-
       allel  bundle	Microcoleus subterulosus
56b.   Filaments arranged in irregular fashion, often
       forming a mat	(Phormidium)    57
;>7a.   Ends of some threads with  a rounded swol-
       len "cap" cell  	    58
57b.   Ends of all threads without a "cap" cell.  . .    60
58a.   Cells quadrate	Phormidium autumnale
58b.   Cells much shorter than broad	    59
!>9a.   Ends of some  threads with round  cap and
       abruptly bent	Phormidium  uncinatum
59b.   Ends of some threads with conical cap and
       straight	Phormidium  subfuscum
60a.   Threads"3-5  microns in width	
       	Phormidium  inundatum
60b.   Threads 5-12 microns in width 	
       	Phormidium retzii
61 a.   Cells in a regular pattern of parallel  rows,
       forming a plate	
       	(Agmene//um quadriduplicatum)    62
61 b.   Cells not regularly arranged to form a plate    63
62a.   Cell diameter 1.3-2.2 microns	Agmen-
       e//um  quadriduplicatum,  tenuissima  type
62b.   Cell  diameter 3-5 microns  	
       Agmenellum quadriduplicatum, glauca type
63a.   Cells regularly arranged  near surface of a
       spherical gelatinous bead  	    64
63b.   Gelatinous bead,  if present, not spherical..    68
64a.   Cells ovate  to heart-shaped;  connected  to
       center of bead by colorless stalks	
       	(Gomphosphaer/a)    65
64b.   Cells round; without gelatinous stalks	
       (Gomphosphaer/a  [Coelosphaerium  type])    65
65a.   Cells with pseudovacuoles  	
       	  Gomphosphaer/a w/'churae
65b.   Cells without pseudovacuoles	    66
66a.   Cells 2-4 microns diameter	
       	(Gomphosphaer/a lacustris)    67
66b.   Cells 4-15 microns diameter	
       	  Gomphosphaer/'a aponina
67a.   Cells  spherical   	Gom-
       phosphaer/a  lacustris,  kuetzingianum  type
67b.   Cells  ovate 	
       .... Gomphosphaer/a lacustris, collinsii type
68a.   Cells attached	Dermocarpa
68b.   Cells  unattached	    69
69a.   Cells cylindric-oval  .. Coccoch/or/s stagn/na
69b.   Cells  spherical 	    70
70a.   Two or more distinct layers of gelatinous
       sheath around each cell or cell cluster	
       	  (Anacystis [G/oeocapsap   72
70b.   Gelatinous sheath around cells not distinctly
       layered  	    71
71a.   Cells  isolated or in  colonies of 2-32  cells..
       	  (Anacystis  [Chroococcus],)   72
71b.   Cells  in colonies of many cells 	
       	  (Anacystis [Microcystis])   72
72a.   Cell containing pseudovacuoles 	
       	Anacystis  cyanea
72b.   Cell not containing pseudovacuoles	    73
73a.   Cell  2-6  microns  diameter; sheath  often
       colored 	Anacystis montana
73b.   Cell 6-50 microns diameter; sheath colorless   74
74a.   Cell 6-12 microns diameter; cells  in colonies
       are mostly spherical . . . .Anacystis thermalls
74b.   Cell 12-50 microns  diameter; cells in colo-
       nies are often irregular.  Anacystis dimidiata

                      Diatoms
75a.   Transverse wall markings not in one  or two
       longitudinal rows; front (valve) view gener-
       ally circular in outline; markings, if present,
       radial  in arrangement; cells may form a fila-
       ment  	(centric diatoms)    76
75b.   Front  (valve) view  elongate,  not circular;
       transverse wall markings in one or two longi-
       tudinal rows; cells if grouped, not forming
       a filament but a ribbon, star, etc	
       	(pennate diatoms)   107
76a.   Cells  pillow shaped in girdle view  with  a
       blunt  process at each corner	
       	  Biddulphia  laevis
76b.   Cells  without blunt processes 	    77
77a.   Cells  very long, cylindrical  in girdle view,
       with a long spine at each end	
       .'	  (Rhizosolenia)    78
77b.   Cells a disc or short cylinder in girdle view
       with no long spine  at each end of side....    79
78a.   Setae  shorter than cell length	
       	 Rhizosolenia  eriensis
78b.   Setae  longer than cell length	
       	Rhizosolenia gracilis
79a.   Cells  in persistent filaments with  valve faces
       in contact; therefore, cells commonly seen
       in side (girdle) view 	(Melosira)   80
79b.   Cells  isolated  or  in fragile filaments, often
       seen in front  (valve) view	    88
                                                                                                          46-3

-------
Key to Fresh Water Algae Common in Water  Supplies and in Polluted Water
                                                       Key
 80a.   Distinct pores on valve mantle (shoulder)..
        	 Me/os/ra binderana
 80b.   No distinct pores on valve mantle (shoulder)    81
 81a.   No visible ornamentation. .Me/os/ra var/ans
 81 b.   Ornamentation visible	    82
 82a.   Terminal cells with long  spines	    83
 82b.   Terminal cells without long spines	    84
 83a.   Diameter 5-21 microns. .Me/os/ra granulata
 83b.   Diameter 3-5 microns	
        	Me/os/ra granulata var. angust/ss/ma
 84a.   Sulcus (groove) angular at base	
        	Melosira ambigua
 84b.   Sulcus (groove) not angular at base	    85
 85a.   Semi-cells shorter  than wide	
        	Me/os/ra distans var. a/p/gena
 85b.   Semi-cells about as long as wide	    86
 86a.   With  robust short  spines.. .Me/os/ra ita//ca
 86b.   With  fine teeth 	    87
 87a.   Sulcus distinctly acute-angled	
        	Me/os/ra crenu/ata
 87b.   Sulcus not distinctly acute-angled	
        	Me/os/ra islandica
 88a.   Radial markings (striations), in valve  view,
        extending from center to  margin; short mar-
        ginal spines sometimes present in valve view    89
 88b.   Area of prominent markings, in valve  view,
        limited to about outer half of circle;  mar-
        ginal spines generally absent ... (Cydotella)    90
 89a.   Radiate hyaline areas on  valve  view	
        	(Stephanodiscus)    99
 89b.   No radial hyaline areas on valve view	
        	 (Coscinodiscus)  106
 90a.   Cells with marginal spines	
        	  Cydotella  pseudostelligera
 90b.   Cells without marginal spines	    91
 91a.   Central area with  3-4  round,  raised  spots
        	  Cydotella ocellata
 91 b.   Central area without such ocelli	
 92a.   Central area with star-shaped  lines around
        a  central dot  	Cyc/ote//a stelligera
 92b.   Central area otherwise	
 93a.   Cells small; 4-10 microns diameter	
 93b.   Cells larger; 10-80  microns diameter	    95
 94a.   Cells in chains; single ocellus in central area
        	  Cydotella atomus
 94b.   Cells all isolated; no ocellus in central area
        	Cydotella glomerata
 95a.   Central area clear. .Cydotella meneghiniana
 95b.   Central area with markings  	    96
 96a.   Circular shadow  line  passes  through the
        costae	 Cydotella striata
 96b.   No circular shadow line	    97
 97a.   Central area with punctae or short lines....
        	Cydotella kutzingiana
 97b.   Central area with fine radial striae	    98
 98a.   A puncta at inner end of several shortened
        marginal costae	Cydotella bodanica
92
93
94
 98b.    No puncta at inner end of several marginal
         costae	Cydotella comta
 99a.    Cell diameter 4-30 microns 	  100
 99b.    Cell diameter 30-80 microns 	  104
100a.    Prominent  rib-like  structures  over outer
         third of cell	Stephanodiscus dubius
100b.    No prominent  rib-like structures	  101
101a.    Spine at end of each striation	
         	 Stephanodiscus tenuis
101b.    Spines not  as above	  102
102a.    Spines alternating with striae	
         	Stephanodiscus hantzschii
102b.    Spines not  as above	  103
103a.    Girdle view with two transverse bands... .
         	Stephanodiscus binderanus
103b.    Girdle view without transverse bands	
         	Stephanodiscus astraea var. minutula
104a.    Outer punctae  of striae 12 in 10 microns...
         	  Stephanodiscus  astraea
104b.    Outer punctae  of  striae 16 in 10  microns..  105
105a.    Cell diameter 30-60 microns	
         	 Stephanodiscus niagarae
105b.    Cell diameter 72-80 microns	
         .... Stepnanod/'scus  niagarae var. magnifies
106a.    Surface  slightly undulate;   markings poly-
         gonal 	Coscinodiscus rothii
106b.    Surface  flat; markings  angular  with  central
         dots	Cosc/nod/scus denarius
107a.    Cell longitudinally symmetrical in valve view  108
107b.    Cell longitudinally unsymmetrical (two sides
         unequal in shape), at least in valve view...   188
108a.    Raphe at or near the edge of the valve....   109
108b.    Raphe or   pseudoraphe median  or  sub-
         median  	   124
109a.    Cells lying side by side in colonies	
         	 Badllaria paradoxa
109b.    Cells isolated or in  twos	   110
110a.    Valve face  transversely undulate	
         	 Cymatopleura so/ea
110b.    Valve face not transversely undulate	   111
111a.    Marginal,  keeled raphe areas  lie  opposite
         one another on the two valves	
         	 Hantzschia amphioxys
111 b.    Marginal,  keeled raphe areas lie diagonal to
         one another on the  two valves. .(Nitzschia)  112
112a.   Valve long-pointed, spine-like 	
         	 Nitzschia acicularis
112b.   Valve not  long-pointed and  spine-like  ....   113
113a.   Valve axis sigmoid  	   114
113b.    Valve axis  not  sigmoid  	   116
114a.    Cell 20-40 microns long . .Nitzschia parvula
114b.    Cell more than  40 microns long	   115
115a.   Cell 50-70 microns long .. .Nitzschia sigma
115b.    Cell 160-500 microns  long	
         	 Nitzschia sigmoidea
116a.    Carinal dots extended far across the valve
         	Nitzschia denticula
  46-4

-------
                     Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                        ALCAE AND  WATER  POLLUTION
 116b.    Carinal dots  not extended  far  across the
         valve	  117
 117a.    Cells in star-shaped colonies	
         	  N/tzsch/a holsatica
 117b.    Cells not in star-shaped colonies	  118
 118a.    Keel  only  slightly excentric  	
         	  Nitzschia dissipata
 118b.    Keel  distinctly excentric 	  119
 119a.    Cell distinctly  pulled in at the middle	
         	Nitzschia linearis
 119b.    Cell not distinctly pulled in at the middle. .  120
 120a.    Cell with longitudinal fold	  121
 120b.    Cell without longitudinal fold	  122
 121a.    Cell 6-9 microns broad. .Nitzschia hungarica
 121b.    Cell 16-35 microns broad	
         	  Nitzschia tryblionella
 122a.    Striae 15-17 in 10 microns 	
         	Nitzschia amphibia
 122.b.    Striae more than 25 in  10 microns	  123
 123a.    Striae 28-30 in 10  microns;  cells elliptical-
         lanceolate  	Nitzschia fonticula
 123b.    Striae 35-40 in 10 microns; cells linear-lan-
         ceolate  	 Nitzschia palea
 124a.    Cell transversely  symmetrical in  valve view  125
 124b.    Cell transversely  unsymmetrical  (two ends
         unequal in shape or size), at least in valve
         view  	  175
 125a.    Cell  round-oval  in  valve  view;  not more
         than twice as long as wide	(Coccone/s)  126
 125b.    Cell elongate,  more than  twice  as long as
         wide	  127
 126a.    Wall  markings (striae)  18-20  in 10 microns
         	Coccone/s pediculus
I26b.    Wall  markings (striae)  23-25  in 10 microns
         	  Coccone/s placentula
127a.   Cell flat; girdle face wide, valve face narrow
         	(Tabellaria)  128
127b.    Girdle and valve faces  about  equal in width  129
128a.   Girdle face less than one-fourth as wide as
         long  	  Jabellaria fenestrata
128b.    Girdle face more than one-half as wide as
         long  	  Tabellaria flocculosa
129a.    Cell with several markings (septa) extending
         without interruption across the valve face;
         no marginal line of pores present (Diatoma)  130
129b.    Cross-markings  (striations or  costae)  on
         valve  surface,  either interrupted by  longi-
        tudinal space (pseudoraphe), line  (raphe), or
         line of pores (carinal dots)	  132
13Da.    Cells  2-4 microns wide. Diatoma  elongatum
130b.    Cells  4-13  microns wide	  131
131a.    Cells  4-8 microns wide . .. .Diatoma anceps
131b.    Cells  10-13 microns wide. .D/atoma vulgare
132a.    Cells  attached side by side to form a ribbon
        of several-to-many cells	(Fragilaria)  133
132b.    Cells  isolated or in pairs	  138
133a.

133b.
134a.
134b.
135a.
135b.
136a.

136b.

137a.

137b.

138a.

138b.

139a.
139b.
140a.
140b.
14la.

141b.

142a.

142b.

143a.

143b.

144a.
144b.
145a.

145b.

146a.

146b.

147a.


147b.

148a.

148b.

149a.

149b.
 Cells attached at middle portion only	
 	  Fragilaria crotonensis
 Cells attached  along entire length	   134
 Central area clear	Fragilaria capucina
 Central area not clear; has striations	   135
 Striae coarse 	   136
 Striae fine  	   137
Valves much inflated at center  	
 	 Fragilaria leptostauron
 Valves not  inflated at  center  	
 	 Fragilaria  pinnata
Striae very short; cells  3-5  microns  wide..
 	  Fragilaria brevistriata
 Striae long; cells 5-12  microns wide	
 	  Fragilaria construens
Cell narrow, linear, often narrowed  to both
ends; true raphe absent	(Synedra)   139
 Cell  commonly  "boat"  shaped  in valve
view; true raphe present  	   147
Cell width 1-2  microns  	Synedra nana
Cell width  2-10 microns  	   140
Cell width 2-5  microns  	   141
Cell width 5-10 microns 	   144
Central clear area on one side of valve only
 	Synedra vaucheriae
 No clear area on one side of center of valve
only  	   142
Central clear  area  present;  striae  almost
continuous  	   143
Central clear area absent; striae short	
 	Synedra tabulata
Cell length  about  500   microns  	
 	 . Synedra acus  var. angust/ss/ma
Cell length 40-200 microns	
 	 Synedra  acus var.  radians
Valves linear 	  Synedra capitata
Valves lanceolate to linear-lanceolate	   145
Valves narrow lanceolate; striae 12-14 in 10
microns  	  Synedra acus
Valves linear-lanceolate; striae not 12-14 in
 10 microns 	   146
Large  clear refractive   central area;  ends
generally capitate	Synedra  pulchella
Large clear non-refractive central area; ends
non-capitate	Synedra ulna
Cell longitudinally  unsymmetrical  in girdle
view; sometimes with  attachment  stalk . .  .
 	  (Achnanthes;   148
Cell symmetrical in girdle as  well as valve
view; generally not attached	   150
Valves constricted  toward  poles 	
 	 Achnanthes  microcephala
Valves gradually tapering toward  the poles
Striations  pronounced  	
	 Achnanthes lanceolata
Striations  fine ... Achnanthes minutissima
                                           149
                                                                                                            46-5

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Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                                       Key
 150a.   Area without striations extending as a trans-
         verse belt around middle of cell 	
         	  Staurone/s phoenicenteron
 150b.   No continuous clear belt around middle of
         cell  	   151
 151a.   Cell with coarse transverse markings (costae),
         which appear as  solid lines even under high
         magnification 	 (Pinnularia)   152
 151b.   Cell  with fine transverse markings (striae),
         which appear as lines  of dots under high
         magnification  	   153
 152a.   Cell 5-6 microns broad  	
         	 Pinnularia subcapitata
 152b.   Cell 34-50 microns  broad	,	
         	 Pinnularia nobilis
 153a.   Girdle view  hour-glass  shaped; valves with
         median longitudinal keel (extension)	
         	 Amphiprora alata
 153b.   Girdle view  not hour-glass  shaped; valves
         without  median  longitudinal keel  	   154
 154a.   Longitudinal  black spaces extending across
         striations  	   155
 154b.   No longitudinal  black  spaces extending
         across striations  	   157
 155a.   Longitudinal  black spaces wavy	
         	  /4nomoeone/'s ex/'//s
 155b.   Longitudinal black spaces straight	   156
 156a.   Longitudinal  black spaces near margin ....
         	  Ca/one/s
 156b.   Longitudinal black spaces near raphe: cen-
         tral nodules  with pair  of extensions along
         each side of raphe  	Diploneis smithii
 157a.   Raphe and valve sigmoid 	   158
 157b.   Raphe and valve not sigmoid  	   160
 158a.   Valve striae  forming transverse and longi-
         tudinal  rows 	  (Cyrosigma)   159
 158b.   Valve striae forming transverse and  oblique
         rows 	 Pleurosigma delicatulum
 159a.   Cell length 150-240 microns 	
         	  Cyrosigma attenuatum
 159b.   Cell length 80-120  microns  	
         	  Gyros/gma  kutzingii
 160a.   Pair  of  longitudinal extensions of central
         nodule along sides of raphe	
         	Frustulia ovu/gar/s
 160b.   Central nodule without longitudinal exten-
         sions 	(Navicula)   161
 161a.   Striae irregularly shortened  in  central area
         	 Navicula mutica
 161 b.   Striae not irregularly shortened in central
         area  	   162
 162a.   Broad clear  lanceolate  area over much  of
         valve	 Navicula confervacea
 162b.   No broad clear area over much of valve  ..   163
 163a.   Central area long, rectangular	
         	Navicula accomoda
 163b.   Central area not long and rectangular ....   164
164a.   Margin of valve undulate 	
        	  Navilcula contenta
164b.   Margin of valve not undulate 	   165
165a.   Short septum  at apices of valve  	
        	 Navicula incomposita
165b.   No short septum at  apices of valve  	   166
166a.   Central area large, irregularly rectangular ..
        	  Navicula  exigua var. cap/fata
166b.   Central area not irregularly  rectangular ...   167
167a.   Central area  strongly widened transversely  168
167b.   Central area round, rhombic, lanceolate, or
        small  	   169
168a.   Valve  length  less than 25 microns  	
        	  Navicula  canalis
168b.   Valve length more than 25 microns  	
        	  Navicula graciloides
169a.   Valve ends distinctly narrowed 	   170
169b.   Valve ends truncate, rounded, or acute ...   172
170a.   Valve  broadly lanceolate; 5-7 microns ...   171
170b.   Valve  narrowly  lanceolate;  width 4-5 mi-
        crons 	  Navicula notha
171a.   Central area large, rounded; ends not capi-
        tate 	 Navicula viridula
171b.   Central area medium sized, irregular;  ends
        capitate	  Navicula cryptocephala
172a.   Terminal striae more strongly marked than
        elsewhere	Navicula hungarica
172b.   Terminal striae not  more strongly marked
        than elsewhere 	   173
173a.   Valves almost linear  	
      .  	  Navicula tripunctata
173b.   Valves lanceolate  	   174
174a.   Central area large; valve lanceolate  	
        	Navicula lanceolata
174b.   Central area  small;  valve  linear-lanceolate
        	  Navicula radiosa
175a.   Cells attached together at one end only to
        form radiating colony	(Asterionella)  176
175b.   Cells  not forming  a  loose radiating  colony  177
176a.   Larger terminal swelling 1Vz  to 2  times
        wider than the other . .Asterionella formosa
176b.   Larger terminal swelling less than 11/z times
        wider than  the other	
        	  Asterionella gracillima
177a.   Cells  in fan-shaped  colonies 	
        	  Meridion circulars
177b.   Cells  isolated  or in  pairs 	   178
178a.   Prominent  wall markings  in  addition  to
        striations present just below lateral margins
        on valve	 (Surirella)  179
178b.   Wall markings  along sides of valve  limited
        to striations 	   183
179a.   Cell width  40-160 microns	   180
179b.   Cell width  8-30 microns	   181
180a.   Cell transversely symmetrical	
        	Surirella striatula
180b.   Cell transversely unsymmetrical	
        	  Surirella splendida
 46-6

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                       Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                       ALCAE AND  WATER POLLUTION
181a.    Cell  linear, symmetrical  	
        	 Surirella  angustata
181b.    Cell  wider at one  end 	  182
182a,    Longitudinal folds  marginal  	
        	Surirella brightwellii
182b.    Longitudinal folds extend to the center ...
        	  Surirella ovata
183a.    Cell  elongate; sides of  valve almost parallel
        except for terminal  knobs ... (Asterionella)  176
183b.    Sides of valve converging toward one  end  184
184a.    Cells bent in girdle view  	
        	  Rhoicosphenia curvata
184b.    Cells straight in girdle  view	  185
185a.    Longitudinal line crossing striae near both
        sides of valve	Gomphoneis
185b.    No  longitudinal line  crossing  striae near
        both sides of valve	(Gomp/ionema)  186
186a.    Narrow end enlarged in valve view	
        	Gomphonema geminatum
186b.    Narrow end not enlarged in valve view . ..  187
187a.    Tip of broad end  about  as  wide as tip of
        narrow end in valve view	
        	  Gomphonema  parvulum
187b.    Tip of broad end  much  wider than tip of
        narrow end in valve view	
        	  Comphonema olivaceum
188a.    Valve with transverse septa or costae	  189
188b.    Valve with no transverse septa  or costae ..  191
189a.    Central portion of  raphe "V" shaped	
        	   (Epithemia)  190
189b.    Central portion of raphe straight	
        	  Rhopalodia gibba
190ai.    Cells 8-15 microns wide;  constricted below
        the recurved  capitate poles  	
        	Epithemia sorex
190b.    Cells 15-18 microns wide;  only slightly con-
        stricted below the recurved somewhat capi-
        tate poles	Epithemia turgida
191 a.    Convex margin of valve  undulate at least
        near the  ends  	  (Eunotia)  192
191b.    Convex margin  of valve not  undulate	  193
192a.    Valve arcuate	  Eunotia lunaris
192b.    Valve linear, only slightly  curved	
        	  Eunotia  pectinalis
193a.    Raphe  located  almost through center of
        valve  	 (Cymbella)  194
193b.    Raphe excentric; near concave edge of valve
        	  Amphora ovalis
194a.    Cell  only  slightly unsymmetrical 	
        	  Cymbella cesati
194b.    Cell  distinctly unsymmetrical 	  195
195a.    Central area with a puncta  	
        	  Cymbella  tumida
195b.    Central area without puncta 	  196
196a.    Striations  distinctly cross-lined  	
        	 Cymbe//a prostrata
196b.    Striations  not distinctly cross-lined 	  197
197a.   Striae 7-9 in 10 microns; cells 30-100 mi-
        crons long	Cymbella turgida
197b.   Striae 12-18 in  10  microns; cells 10-40 mi-
        crons long	 Cymbella ventricosa

                    Flagellate Algae
198a.   Cell  in a loose, rigid, conical sac {lorica);
        isolated or  in a branching colony	
        	 (Dinobryon)   199
198b.   Case or sac, if present, not conical; colony,
        if present, not  branching 	   202
199a.   Branches diverging, often almost at a right
        angle	Dinobryon divergens
199b.   Branches compact, often almost  parallel  ..   200
200a.   Narrow end of lorica  sharp pointed 	   201
200b.   Narrow end of  lorica blunt pointed	
        	  Dinobryon sertularia
201 a.   Narrow end drawn out into a stalk	
        	   Dinobryon  stipitatum
201 b.   Narrow end diverging at the base 	
        	  Dinobryon sociale
202a.   Cells isolated  or in pairs 	   203
202b.   Cells in a colony of four or more cells ...   252
203a.   Prominent  transverse  groove encircles the
        cell  	   209
203b.   Cell  without transverse  groove  	   204
204a.   Plastid golden-brown  	   205
204b.   Plastid green, yellow-green,  red,  or  blue-
        green  	   215
205a.   Anterior end of cell rounded; one flagellum
        	 (Chromulina)   206
205b.   Anterior end of cell oblique; two flagella  ..   207
206a.   Plastid in anterior half of cell; posterior por-
        tion of cell attenuate	
        	  Chromulina rosanoffii
206b.   Plastid almost full  length of cell; Posterior
        portion of cell  wide, rounded 	
        	  Chromulina vagans
207a.   Flagella extending from  gullet;  flagella al-
        most equal in length	(Cryptomonas.)   208
207b.   No gullet; flagella very unequal in length  . .
        	  Oc/iromonas
208a.   Cell  narrowed  to posterior end  	
        	 Cryptomonas cylindrica
208b.   Cell  not narrowed to  posterior end 	
        	  Cryptomonas  erosa
209a.   Cell  with prominent projections,  rigid, one
        forward and two or three on posterior end
        	   Ceratium hirundinella
209b.   Cell  without several rigid, polar projections   210
21 Oa.   Portions above  and below transverse groove
        about equal 	   211
210b.   Front portion distinctly larger than posterior
        portion 	   214
211 a.   Cells naked, with no cell wall Cymnodinium
211b.   Cells with  cell wall composed  of several
        plates  	   212
                                                                                                           46-7

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Key to Fresh Water Algae  Common in Water Supplies and in Polluted Water
                                                       Key
 212a.   Cell wall thin but composed of plates	
         	 Glenodinium palustre
 212b.   Cell wall  thick with clearly evident plates
         	(Peridinium)   213
 213a.   Ends of cells pointed 	
         	  Peridinium wisconsinense
 213b.   Ends of cell rounded ..Peridinium cinctum
 214a.   Transverse furrow extends about half  way
         around cell 	  Hemidinium
 214b.   Transverse furrow extends all  way around
         cell 	  Massartia vorticella
 215a.   Cell with  long bristles extending from  sur-
         face plates	Mallomonas caudata
 215b.   Cells without bristles and surface plates  ..   216
 216a.   Plastids blue-green	Cyanomonas
 216b.   Plastids green, yellow-green, or red 	   217
 217a.   Cell naked, not covered  by wall or lorica  or
         rigid membrane	 Dunaliella
 217b.   Cell covered by wall or loose rigid covering
         or rigid membrane 	   218
 218a.   Space  between  protoplast  and wall with
         radial strands of protoplasm  Haematococcus
 218b.   No radial  strands  of  cytoplasm between
         protoplast and wall or lorica	   219
 219a.   Cell protoplast enclosed  in loose rigid cov-
         ering  (lorica)  	   220
 219b.   Cell with  membrane or  wall but no loose
         rigid covering  	   225
 220a.   Cell with  four flagella  	  Pedinopera
 220b.   Cell with  one or two flagella  	   221
 221 a.   Lorica flattened; cell with two flagella	
         	  Phacotus  lenticularis
 221 b.   Lorica not flattened; cell with one flagellum   222
 222a.   Lorica often opaque, generally  dark brown
         to red; plastid green Trache/omonas crebea
 222b.   Lorica often transparent, colorless to light
         brown;  plastid light  brown  (Chrysococcus)   223
 223a.   Outer membrane  (lorica) oval  	
         	  Chrysococcus ova//s
 223b.   Outer membrane (lorica) rounded 	   224
 224a.   Lorica thickened  around  opening 	
         	  Chrysococcus rubescens
 224b.   Lorica not thickened around opening ....
         	  Chrysococcus major
 225a.   Plastids brown to red to olive- or blue-green   226
 225b.   Plastids  grass green 	   229
 226a.   Plastid blue-green to blue  .. (Chroomonas)   227
 226b.   Plastids  brown to red to olive-green	
         	  Rhodomonas lacustris
 227a.   Cell not pointed at one  end 	
         	  Chroomonas  setoniensis
 227b.   Cell pointed at posterior end  	   228
 228a.   Plastid one per cell .. . Chroomonas caudata
 228b.   Plastids two per cell Chroomonas nordstetii
 229a.   Cell with colorless, rectangular wing	
         	  Pteromonas angulosa
 229b.   No wing extending from cell  	   230
230a.   Cells  with  two chloroplasts, one  on each
        side 	  Cryptoglena n/'gra
230b.   Cells with more than two chloroplasts	   231
231a.   Cells  flattened; margin rigid 	(Phacus)   232
231 b.   Cell not flattened;  margin rigid or flexible   233
232a.   Posterior spine short,  bent	
        	  Phacus p/euronectes
232b.   Posterior spine long, straight	
        	  Phacus longicauda
233a.   Pyrenoid present in the  single  plastid; no
        paramylon;  margin  not  flexible;   two  or
        more  flagella per cell  	   234
233b.   Pyrenoid absent; paramylon present; several
        plastids per cell; margin flexible  or  rigid;
        one flagellum  per cell  	   240
234a.   Cells  long  fusiform (tapering at each end)
        	  (Chlorogonium)   397
234b.   Cells  not fusiform,  generally almost spher-
        ical 	,   235
235a.   Plastids numerous Vacuo/ar/a novo-munda
235b.   Plastids few, commonly one	   236
236a.   Two flagella per cell  	   237
236b.   Four flagella per cell  ... .Carter/a  multifilis
237a.   Cell with sheath of different shape from pro-
        toplast 	Sphaerellopsis
237b.   Cell not as above  	(Ch/amydomonas)   238
238a.   Distinct clear area across middle of  cell ....
        	Ch/amydomonas pertusa
238b. •  No distinct clear area across  middle of cell   239
239a.   Pyrenoid angular;  eyspot in  front third  of
       . cell	Ch/amydomonas  reinhardi
239b.   Pyrenoid circular; eyespot in middle third of
        cell	 Ch/amydomonas g/obosa
240a.   Cell flexible in form; paramylon a capsule or
        disc; cell elongate  	(Euglena)   241
240b.   Cell rigid in form; paramylon ring-shaped;
        cell almost  spherical	   250
241a.   Green plastids hidden  by a red pigment  ...
        	  Euglena sanguinea
241 b.   No red  pigment except for the  eyespot  . . .   242
242a.   Plastids at least one-fourth the length of the
        cell 	   243
242b.   Plastids discoid or at least shorter than one-
        fourth the length of the cell	   244
243a.   Plastids two per cell	Euglena agilis
243b.   Plastids several  per cell, often extending
        radiately from the center . .Euglena viridis
244a.   Posterior end extending as a colorless spine   245
244b.   Posterior end  rounded or at least with no
        colorless spine	   247
245a.   Posterior end gradually narrowed to a spine
        	 Euglena acus
245b.   Posterior end with an  abrupt spine	   246
246a.   Spiral  markings very prominent and granular
        	  Euglena  spirogyra
246b.   Spiral markings fairly prominent, not granu-
        lar  	Euglena oxyuris
  46-8

-------
                        Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                       ALCAE AND WATER POLLUTION
247a.    Small; length 35-55 microns Euglena gracilis
247b.    Medium to large; length 65 microns or more  248
248a.    Medium in size; length  65-200 microns	  249
248b.    Large in size; length 250-290 microns	
        	  Euglena  ehrenbergii
249a.    Plastids with irregular edge; flagellum  two
        times as long as cell ..  Euglena polymorpha
249b.    Plastids with smooth edge;  flagellum about
        one-half as long as the cell ... Euglena deses
250a.    Cell almost spherical or with abrupt poste-
        rior tip;  paramylon ring-shaped  	
        	  (Lepocinclis)  251
250b.    Posterior end of cell gradually pointed; cell
        margin with  spiral ridges . .. .Phacus pyrum
25>1a.    Posterior end with  an abrupt, spine-like tip
        	  Lepocinclis ovum
251 b.    Posterior end rounded  ...Lepocinclis texta
252a.    Plastids brown		  253
2S2b.    Plastids  green  	  254
253a.    Cells in contact with one another	
        	Synura uve//a
253b.    Cells separated from one another by a space
        	Uroglenopsis americana
254a.    Colony flat; one cell thick Gonium pectorale
254b.    Colony rounded; more than one cell thick  .  255
2.55a.    A long straight rod extending from each cell
        	  Chrysosphaerella longispina
255b.    No long straight rod extending from each
        cell  	  256
256a.    Cells in contact with one another	  257
256b.    Cells  separated  from  one another  by  a
        space 	  260
257a.    Cells radially arranged  . . Pandorina morum
257b.    Cells  all facing one direction 	  258
258a.    Cells each with two flagella . .(Pyrobotrys)  259
258b.    Cells  each with four flagella  	
        	  Spondylomorum quaternarium
259a.    Eyespot in the  wider (anterior) end  of  the
        cell	Pyrobotrys stellata
259b.   Eyespot in the  narrower  (posterior) end of
        the cell  	 Pyrobotrys gracilis
260a.    Cells more than 400 per colony 	
        	 Volvox aureus
260b.   Cells less than 150 per colony	  261
261 a.    Cells of two distinct sizes in colony	
        	  Pleodorina
261 b.   Cells all of one size  in colony	
        	 Eudorina  elegans

            Green Algae and Associated Forms
262a.    Cells joined together to form a net	
        	  Hydrodictyon reticulatum
262b.   Cells not forming  a net  	  263
263a.    Cells attached side by  side to form a plate
        or ribbon one  cell thick and one (or  two)
        cells wide; Number of  cells commonly 2, 4,
        or 8  	 (Sceneries™ us.)  264
263b.   Cells not attached side by side 	  268    279b.
264a.

264b.
265a.

265b.

266a.
266b.

267a.

267b.

268a.

268b.

269a.

269b.

270a.

270b.
271a.

271 b.
272a.

272b.

273a.

273b.

274a.

274b.

275a.

275b.

276a.

276b.
277a.

277b.

278a.
278b.
279a.
Middle  cells   without   spines  but  with
pointed ends	   265
Middle cells with rounded ends	   266
All cells in colony erect  	
	  Scenedesmus  obliquus
Median cells erect, terminal cells lunate  ...
	  Scenedesmus  dimorphus
Terminal cells with  spines 	   267
Terminal cells without spines	
	 Scenedesmus bijuga
Terminal cells  with two spines each 	
	  Scenedesmus quadricauda
Terminal cells with  three or more spines
each 	  Scenedesmus abundans
Cells isolated or in  nonfilamentous or non-
tubular thalli 	   269
Cells in filaments or other tubular or thread-
like thalli 	   335
Cells isolated and narrowest  at the center
due to incomplete  fissure 	(desmids)   270
Cells isolated or in clusters but without cen-
tral fissure 	   276
Each half of cell with three spine-like  or
pointed knobular extensions . .(Staurastrum)   271
Cell margin with no such extensions  	   273
Margin of cell with  long  spikes 	
	Staurastrum paradoxum
Margin of cell without long spikes  	   272
Ends of lobes with short  spines	
	 Staurastrum  polymorphum
Ends of lobes without spines 	
	Staurastrum punctulatum
Semi-cells with no  median  incision or  de-
pression 	 (Cosmarium)   274
Semi-cells  with a median incision or  de-
pression 	   275
Median incision narrow linear 	
	 Cosmarium botrytis
Median  incision wide, "U" shaped  	
	 Cosmarium portianum
Margin with  rounded lobes  	
	  Euastrum  oblongum
Margin with sharp-pointed  teeth  	
	 Micrasterias truncata
Lunate or  otherwise  bent cells in a wide
gelatinous  matrix  	(Kirchneriella)   277
Cells  otherwise  	   278
Cells sharply pointed 	
	 Kirchneriella lunaris
Cells bluntly pointed 	
 	  Kirchneriella subsolitaria
Cells elongate  	   279
Cells round to oval to angular	   297
Cells quadrately arranged in fours 	
	  Tetradesmus
Cells not quadrately arranged  	   280
                                                                                                         46-9

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 Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                                       Key
280a.   Cells radiating from a central point 	
        	(Act/'nastrum;   281
280b.   Cells isolated or in irregular clusters 	   282
281a.   Cells cylindric ..../\ct/nastrum  gradllimum
281 b.   Cells distinctly bulging  	
        	  /\ct/nastrum hantzschii
282a.   One or both cell ends  gradually narrowed
        to an acute spine-like point	
        	  Ourococcus bicaudatus
282b.   Cells  either  with  true  spines  or without
        spine-like points  	   283
283a.   Cells with terminal  spines  	   284
283b.   Cells without terminal spines  	   285
284a.   Cell ends blunt  .. . Ophiocytium capitatum
284b.   Cell ends tapering .. . .Schroederia setigera
285a.   Cells with colorless attachment  area at one
        end 	  Characium
285b.   No attachment area at one end of cell... .   286
286a.   Plastids two  per cell;  unpigmented  area
        across  center of  cell 	 (Closterium)   287
286b.   Cell  with plastid that continues longitudi-
        nally across  the center of the  cell 	   290
287a.   Cell small; length up to 177 microns	
        	  Closterium acutum
287b.   Cell larger;  length more than 240 microns   288
288a.   Cell long and narrow;  width  up to 5  mi-
        crons  	 Closterium  aciculare
288b.   Cell wide; minimum width 19 microns. .. .   289
289a.   Inner margin of cell straight	
        	 Closterium acerosum
289b.   Inner margin of  cell tumid and curved  ...
        	  Closterium moniliferum
290a.   Two, four, or many  cells surrounded by
        homogenous envelope  	
        	 Elakatothrix getatinosa
290b.   No gelatinous envelope  	   291
291a.   Cell five to ten times as long as broad  ....   292
291 b.   Cell two to four  times as long as broad .. .   294
292a.   Pyrenoid absent  or one per cell 	
        	  (Ankistrodesmus)   293
292b.   Pyrenoids several per cell	
        	 Closteriopsis  brevicula
293a.   Cells bent	Ankistrodesmus falcatus
293b.   Cells straight	
        .... Ankistrodesmus falcatus var. acicularis
294a.   Plastid  pale yellow-green; reddish  oil drop-
        lets present	 Pleurogaster
294b.   Plastid grass-green;  storage food  is starch   295
295a.   Cells semi-circular; cell  ends pointed  but
        with no terminal spines	fSe/enastrum)   296
295b.   Cells  arcuate  but less  than  semi-circular;
        cell ends pointed and  each with  a  short
        spine   	Closteridium lunula
296a.   Cells with rounded  ends	
        	Selenastrum capricornutum
296b.   Cells with pointed  ends 	
        	  Selenastrum gracile
297a.   Plastids green 	   298
297b.   Plastids golden-brown;  cells with pseudo-
        podia  	   306
298a.   Numerous gelatinous  setae extending from
        surface of colony Chaetopeltis megalocystis
298b.   No gelatinous setae extending from surface
        of colony  	   299
299a.   Cells arranged in a flat regular colony	   300
299b.   Cells not in  a tight, flat, regular colony  ...   307
300a.   Marginal cells with one or  two or  more
        spines or spine-like extensions	   301
300b.   Marginal cells otherwise  	   304
301 a.   Colonies limited to  four  cells with  true
        spines  	  Tetrastrum
301 b.   Colonies generally of  eight or more cells, if
        limited  to four  cells, without  true  spines
        	  (Pediastrum)   302
302a.   Numerous spaces between cells 	
        	 Pediastrum duplex
302b.   Cells fitted tightly together 	   303
303a.   Cell incisions deep and  narrow	
        	 Pediastrum  tetras
303b.   Cell incisions deep and wide 	
        	  Pediastrum boryanum
304a.   All cells in contact with  neighboring cells..   305
304b.   At least some cells lie  free  from one  an-
        other 	 Dispora
305a.   Quadrangular space in center of each group
        of four cells	Crucigenia quadrata
305b.   No quadrangular space  in  center of each
        group of four cells .... Pras/'o/a nevadense
306a.   Cells isolated  	 C/irysamoeba
306b.   Cells in colonies	 Chrysidiastrum
307a.   Plastid  distinctly  central	Apiococcus
307b.   Plastid parietal  	   308
308a.   Cells angular  	   309
308b.   Cells round  to oval 	   311
309a.   Two or more  spines at each  angle	
        	 Polyedriopsis spinulosa
309b.   Spines none  or  less than two at each angle
        	  (Tetraedron)   310
31 Oa.   Corners produced into processes 	
        	Tetraedron limneticum
310b.   Corners not  produced into processes  ....
        	   Tetraedron muticum
311a.   Cells with long  sharp spines 	   312
311b.   Long sharp spines absent	   315
312a.   Cells round   	   313
312b.   Cells oval  	   314
313a.   Cells isolated  	Colenkinia  radiata
313b.   Cells in colonies ... Micractinium pusillum
314a.   Each cell end with one spine	
        	Diacanthos belenophoris
314b.   Each cell end with more than one spine ...
        	  Chodatella quadriseta
315a.   Each cell with a sheath  which is not con-
        fluent with sheaths of adjacent cells	
        	 (Cloeocystis)   316
315b.   Cells or sheaths otherwise 	   317
  46-10

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                    Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                       ALGAE AND  WATER POLLUTION
316a.    Colonies angular .. .Gloeocystis planctonica
316b.    Colonies rounded	  Gloeocystis gigas
317a.    Each cell group with two shapes of cells ..
        	 Dimorphococcus lunatus
317b.    All  cells of essentially the same shape ...   318
318a.    Colony of definite, regular form, round to
        oval  	   319
318b.    Colony, if  present,  not a definite oval or
        sphere;  or cells may  be isolated 	   325
319a.    Colony a tight sphere of cells 	   320
319b.    Colony a loose sphere of cells enclosed by
        a membrane 	   321
320a.    Sphere solid, slightly irregular; no connect-
        ing processes between cells  	
        	  Planktosphaeria gelatinosa
320b.    Sphere  hollow,  regular;  short  connecting
        processes between cells	
        	  Coelastrum microporum
321a.    Cells  round 	   323
321b.    Cells oval	(Oocystis)  322
322a.    Cells with polar nodules .Oocystis lacustris
322b.    Cells without polar nodules Oocystis  borgei
323a.    Cells  connected  to  center  of  colony by
       • branching stalk	(Dictyosphaerium)  324
323b.    No stalk connecting the cells	
        	  Sphaerocystis schroeteri
324a.    Cells  rounded D/ctyospnaerium  pulchellum
324b.    Cells  straight, oval 	
        	  Dictyosphaerium ehrenbergianum
325a.    Oval cells enclosed in a somewhat spherical,
        often  orange-colored matrix	
        	  Bofryococcus braunii
325b.    Cells  round,  isolated or in colorless  matrix  326
326a.    Adjoining cells with  straight, flat walls be-
        tween their protoplasts  	   327
326b.    Adjoining cells with rounded walls between
        their protoplasts 	  328
327a.    Cells  embedded  in a common gelatinous
        matrix	Palmella mucosa
327b.   No matrix or sheath  outside  of cell walls
        	  Phytoconis botryoides
328a.    Cells  loosely arranged in a large gelatinous
        matrix	Tetraspora gelatinosa
328b.   Cells isolated or tightly grouped in a small
        colony  	  329
329a.    Cells  located inside of protozoa 	
        	  Zoochlorella
329b.   Cells not inside of protozoa 	  330
330a.    Cells  with two or more plastids 	
        	  Palmellococcus
330b.   Each cell with a single plastid 	  331
331a.    Plastid filling two-thirds or less of the cell..  332
331 b.   Plastid filling three-fourths or more of the
        cell 	 Chlorococcum humicola
332a.    Cell diameter 2 microns or  less; reproduc-
        tion by  cell division	  Nannochloris
332b.   Cell diameter 2.5 microns or more; repro-
        duction by internal spores	(Chlorella)   333
333a.   Cells rounded 	   334
333b.   Cells ellipsoidal  to ovoid  	
        	 Chlorella ellipsoidea
334a.   Cell 5-10  microns  in  diameter; pyrenoid
        indistinct  	 Chlorella  vulgaris
334b.   Cell 3-5  microns in  diameter; pyrenoid
        distinct 	  Chlorella  pyrenoidosa
335a.   Cells attached end to end in an unbranched
        filament  	   336
335b.   Thallus branched  or more  than one  cell
        wide  	   362
336a.   Plastids in  form  of one or more marginal,
        spiral ribbons; spirals may be incomplete..   337
336b.   Plastids not in form of spiral  ribbons	   342
337a.   Spiral turn of plastic  incomplete Sirogonium
337b.   Plastid forming one or more  spiral turns .. .
        	 (Spirogyra)   338
338a.   One plastid per cell	   339
338b.   Two or more  plastids per cell  	   341
339a.   Threads 18-26 microns wide	
        	  Spirogyra communis
339b.   Threads 28-50 microns wide 	   340
340a.   Threads 28-40 microns  wide	
        	  Spirogyra varians
340b.   Threads 40-50 microns wide 	
        	Sp;>ogyra porticalis
341a.   Threads 30-45 microns wide;  3-4  plastids
        per cell  	 Spirogyra fluviatilis
341 b.   Threads  50-80 microns wide;  5-8  plastids
        per cell	Spirogyra majuscula
342a.   Filaments  when  breaking, separating
        through  middle of  cells  	   343
342b.   Filaments, when breaking, separating irregu-
        larly or at ends of cells 	   346
343a.   Starch  test positive; cell margin  straight; one
        plastid, granular  	(Microspora)   344
343b.   Starch test negative;  cell margin   slightly
        bulging; several  plastids	(Tribonema)   345
344a.   Cells 22-33 microns  broad 	
        	 Microspora  amoena
344b.   Cells 11-20 microns  broad 	
        	 Microspora wittrockii
345a.   Plastids two to four  per cell 	
        	  Tribonema minus
345b.   Plastids more than four per cell 	
        	 Tribonema bombycinum
346a.   Filaments short;  generally 2-3 cells long ...
        	 St/chococcus  bacillaris
346b.   Filaments longer than 2-3 cells		   347
347a.   Marginal indentations between cells	   348
347b.   No marginal  indentations between  cells . .   349
348a.   Cells  much shorter than broad  	
        	  Desmidium  grevillii
348b.   Cells almost  as  long as broad  	
        	  Hyalotheca mucosa
349a.   Plastids two  per cell 	(Zygnema)   350
                                                                                                            46-11

-------
Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                                                       Key
 349b.   Plastid one per cell  (sometimes appearing
         numerous)  	   352   365b.
 350a.   Cell dense  green, each plastid  reaching to        366a.
         the wall	Zygnema sterile        366b.
 350b.   Cells  light  green;  plastids  not  completely        367a.
         filling the cell  	   351
 351 a.   Width of thread 26-32 microns; maximum        367b.
         cell length 60 microns ... Zygnema insigne        368a.
 351 b.   Width of thread 30-36 microns; maximum
         cell length 120 microns	
         	 Zygnema  pectinatum        368b.
 352a.   Some cells  with  walls having  transverse
         wrinkles near one end; plastid an irregular        369a.
         net 	   (Oedogonium)  353
 352b.   No apical  wrinkles  in wall;   plastid  not        369b.
         porous  	   356
 353a.   Thread diameter less than 25 microns  ....   354   370a.
 353b.   Thread diameter 25  microns or more  ....   355
 354a.   Thread diameter 9-14 microns 	
         	  Oedogonium suedcum        370b.
 354b.   Thread diameter 14-23 microns	         371a.
         	 Oedogonium boscii
 355a.   Dwarf male plants attached to normal thread        371 b.
         when reproducing 	         372a.
         	  Oedogonium idioandrosporum        372b.
 355b.   No dwarf male plants produced 	         373a.
         	 Oedogonium grande
 356a.   Plastid a flat or twisted axial ribbon  	         373b.
         	  (Mougeotia)  357   374a.
 356b.   Plastid an arcuate marginal band (Ulothrix)  359
 357a.   Threads with occasional "knee-joint" bends        374b.
         	 Mougeot/'a genurVexa
 357b.   Threads straight 	   358
 358a.   Threads 19-24 microns wide; pyrenoids 4-16        375a.
         per cell	Mougeotia sphaerocarpa        375b.
 358b.   Threads 20-34 microns wide; pyrenoids 4-10        376a.
         per cell  	 Mougeot/'a sca/ar/s
 359a.   Threads 10  microns or less  in diameter ...   360   376b.
 359b.   Threads more  than 10 microns  in diameter  361
 360a.   Threads 5-6 microns in diameter  	         377a.
         	  Ulothrix variabilis
 360b.   Threads 6-10 microns in diameter	         377b.
         	 Ulothrlx tenerrima
 361a.   Threads 11-17  microns in diameter	        378a.
         	  Ulothrix aequalis
 361 b.   Threads 20-60 microns in diameter	        378b.
         	  Ulothrix zonata
 362a.   Thallus a tangled  tubular mass of filaments        379a.
         	 Thorea ramosissima
 362b.   Thallus  otherwise  	  363   379(3.
 363a.   Thallus a gelatinous tube in which cells are
         embedded  	  Hydrurus        380a.
 363b.   Thallus  otherwise  	  364   380b.
 364a.   Thallus a flat plate of cells  	
         	   Hildenbrandia rivularis        381 a
 364b.   Thallus otherwise 	  365
 365a.   Thallus a tubular layer of cells	
 	 Enteromorpha intestinalis
 Thallus otherwise 	  366
 Thallus a long tube without cross-walls ...  367
 Thallus otherwise   	  370
 Tube with constrictions especially at base of
 branches — D/chotomos/'phon tuberosus
 Tube with  no constrictions ...  .(Vaucheria)  368
 Egg  sac attached directly,  without  a  stalk,
 to the main  vegetative  tube 	
 	  Vaucheria sessilis
 Egg  sac attached to an abrupt, short, side
 branch 	  369
 One egg sac per branch 	
 	Vaucheria terrestris
 Two or more egg sacs per branch  	
 	  Vaucheria geminata
 Thallus a  leathery  strand  with regularly
 spaced swellings and  a continuous mem-
 brane  of cells 	  396
 Thallus otherwise 	  371
 Filament unbranched  	
 	Schizomeris leibleinii
 Filament branched   	  372
 Branches in whorls (clusters) 	  373
 Branches single  or  in pairs 	  378
 Thallus embedded in gelatinous matrix ...
 	  (Batrachospermum)  374
 Thallus not embedded in gelatinous matrix  375
 Nodal  masses of branches touching  one an-
 other  	 Batrachospermum vagum
' Nodal masses of branches separated from
 one another by  a  narrow space 	
 	 Batrachospermum  moniliforme
 Main filament one cell thick	(Nitella)  376
 Main filament three cells thick  ...  .(Chara)  377
 Short  branches  on the  main   thread  re-
 branched once  	 Nitella flexilis
 Short  branches  on the  main   thread  re-
 branched two to four times Nitella gracilis
 Short  branches  with  2 naked  cells at the
 tip 	  Chara globularis
 Short  branches with 3-4 naked cells at the
 tip	 Chara vu/gar/s
 Most of filament surrounded by a  layer  of
 cells  	  Compsopogon coeruleus
 Filament not surrounded by a layer of cells
 	  379
 End cell  of branches with a  rounded  or
 blunt-pointed tip  	  380
 End cell of branches with a sharp-pointed
 tip 	  387
 Plastid green; starch test positive 	  381
 Plastids red; starch test negative	
 	  Audouinella  violacea
 Some  cells   dense,  swollen,  dark  green
 (spores); others  light green, cylindric	
 	  Pithophora oedogonia
 46-12

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Key to Fresh Water Algae Common in Water Supplies and in Polluted Water
                ALGAE AND  WATER POLLUTION
                           382
                           383
381 b.   All cells essentially alike, being light to me-
        dium green, cylindric	(Cladophora)
382a.   Branches arising from below apices of cells
           Cladophora profunda var. nordstedt/ana
382b.   Branches arising from apices of cells	
383a.   Branches often appearing forked or in threes
        	  Cladophora  aegagropila
383b.   Branches distinctly lateral  	   384
384a.   Branches forming  acute angles with main
        thread, thus forming clusters  	
        	 Cladophora glomerata
384b.   Branches forming wide  angles with the main
        thread  	   385
385a.   Threads crooked and bent	
        	  Cladophora Iracta
385b.   Threads straight 	   386
386a.   Branches few, seldom  rebranching  	
        	  Cladophora insignis
386b.   Branching  numerous, often rebranching ...
        	  Cladophora crispata
387a.   Filaments embedded in gelatinous  matrix .
387b.   Filaments  not embedded  in gelatinous ...
        matrix  	
388a.   Cells of main filament much  wider than
        even the basal cells of the  branches	
        	  (Draparnaldia)
388b.   No abrupt change in  width of cells from
        main filament to branches .. (Chaetophora)
389a.   Branches (from the main thread) with  a cen-
        tral main axis	Draparnaldia  plumosa
3fl9b.   Branches  diverging and  with no central
        main axis	Draparnaldia glomerata
                            388
                            391
                            389
                            390
390a.    End cells long-pointed  with  colorless tips
        	 Chaetophora attenuata
390b.   End cells abruptly pointed, mostly without
        long colorless tips ... Chaetophora e/egans
391 a.    Branches very short,  with no  cross-walls
        	 Rhizodonium hieroglyphicum
391 b.   Branches long, with cross-walls  	  392
392a.    Branches ending  in an abrupt spine having
        a bulbous base  	  (Bulbochaete)  393
392b.   Branches gradually reduced in width, end-
        ing in a long pointed cell, with  or without
        color  	  (Stigeodonium)  394
393a.   Vegetative cells 20-48 microns long	
        	  Bulbochaete mirabilis
393b.   Vegetative cells 48-88 microns long 	
        	   Bulbochaete  insignis
394a.   Branches frequently in  pairs  	  395
394b.   Branches mostly  single  	
        	 Stigeodonium stagnatile
395a.   Cells  in main thread  1-2 times  as  long as
        wide	Stigeodonium lubricum
395b.   Cells  in main thread  2-3 times  as  long as
        wide  	  Stigeodonium tenue
396a.   Nodes covered by a ring of antheridial tis-
        sue 	 Lemanea annulata
396b.   Nodes covered  by wart-like  outgrowths of
        antheridial  tissue  	 Sacheria
397a.   Pyrenoids two per cell 	
        	  Chlorogonium elongatum
397b.   Pyrenoids several per cell 	
        	  Chlorogonium  euchlorum
                                     This outline was prepared by C. M. Palmer
                                     former Aquatic Biologist,  (from Algae
                                     and Water Pollution ,  MERL,  ORD,
                                     U.S.  EPA, Cincinnati, Ohio  45268.)

                                     Descriptors:  Algae, Plankton,
                                     Identification Keys
                                                                                 46-13

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             A KEY FOR  THE INITIAL SEPARATION OF  SOME COMMON
                                PLANKTON ORGANISMS
1.  No chlorophyll present,  unless  through ingestion	8
1.  At  least some chlorophyll present	2

    2.  Pigments not in plastids	Cyanophyta
    2.  Pigments in one or  more plastids	3

3.  Cell wall of over-lapping halves and distinctly sculptured	Bacillariophyta
3.  Cell wall not of over-lapping halves,  or if  so,  then not  sculptured	4

    4.  Pyrenoids present; color usually  bright green	Chlorophyta
    4.  Pyrenoids absent;  color green, yellow-green or yellow-brown	  .  5

5.  Bright green,  motile,  usually with one anterior flagellum	Euglenophyta
5.  Yellowish to brownish,  motile  or not	6

    6.  With a distinct lateral  groove,  motile	Pyrrophyta
    6.  Without  a lateral groove	7

7.  Seldom motile;  unicellular,  colonial or filamentous	Xanthophyta
7.  Motile, unicellular or  colonial	Chrysophyta

    8.  Unicellular; naked or enclosed in a smooth or  sculptured shell	   9
    8.  Multicellular; body usually  with a  distinct  exoskeleton	   11

9.  Amoeboid; sometimes  with  shell,  no cilia or flagella	Ameoboid Protozoa
9.  Actively motile;  never with shell;  cilia or flagella  obvious	10

    10.  Body more or less covered  by short cilia;
         movement "darting"	Ciliate Protozoa
    10.  Body with one or more flexible whip-like falgella;
          movement  "continous"	Flagellate  Protozoa

11.  Shell bivalved  (clam-like)	12
11.  Shell not  composed of two halves	   13

    12.  With  distinct  head anterior to valves	Cladocera
    12.  No head anterior to valves	Ostracoda

13,  Usually microscopic;  body extended into  a  tail or foot with  one
     or more toes	Rotifera
13,,  Usually macroscopic if mature	14

    14.  Appendages bilateral; head not prominent	Copepoda
    14.  Appendages unilateral; head  prominent	Phyllopoda
 BI.AQ. 33. 6.16                                                                      47-1

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                                      CHLOROPHYTA

                   A Key to Some of the Common Filamentous  Genera
1.  Filaments unbranched	2
1.  Filaments branched (sometimes parenchymatous)	12

    2.   Chloroplast  single, parietal band	3
    2.   Chloroplast  one  or more,  if parietal not  a  band	   5

3.  Chloroplast encircling more  than  half the cell (Napkin-ring  like)	Ulothrix
3.  Chloroplast encircling less than half the cell	4

    4.   Filaments of indefinite length; cells with  square ends	Hormidium
    4.   Filaments usually short,  of 3-8 cells, with ends  round	Stichococcus

5.  Cell .wall of H pieces; pyrenoids lacking	Microspora
5.  Cell wall not of H pieces	6

    6.   Some  cells with apical caps	Oedogonium
    6.   Cells  without apical caps	7

7.  Chloroplast (s)  parietal	8
7.  Chloroplast (s)  axial	   9

    8.   Chloroplast  one  or more,  spiral bands	Spirogyra
    8.   Chloroplast  several,  longitudinal bands	Sirogonium

9.  Cell walls without a median  constriction	10
9.  Cell walls with a median constriction	11

    10.  Chloroplast  stellate	Zygnema
    10.  Chloroplast  an  axial band	Mougeotia

11.  Filaments cylindrical	Hyalotheca
11.  Filaments triangular, twisted	Desmidium

    12.  Coenocytic  dichotomously branched,  with constrictions	Dichotomosiphon
    12.  Filaments with regular  cross walls	13

13.  Parenchymatous,  discoid, epiphytic	Coleochaete
13.  Not parenchymatous	14

    14.  Main axis cells  much broader than branch cells	Draparnaldia
    14.  Main axis and branch cells approximately the  same breadth	16

15.  Main  axis and lateral branches attenuated into  long multicellualr hairs	16
15.  Axis  and branches not attenuated  into long multicellular hairs	17

    16.  Sparsely or loosely  branched	Stigeoclonium
    16.  Densely and compactly  branched	Chaetophora
 47-2

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17.   Cells bearing swollen or bulbous-based setae	   18
17.   Cells without setae	19

     18.   Swollen-based setae on dorsal surface of cells; prostrate
         epiphytes; little or  not al all  branched	Aphanochaete
     18.   Bulbous-based setae terminal on branches; not
          prostrate epiphytes	Bulbochaete

19.   With terminal and/or intercalary  akinites	Pithophora
19.   Without distinctive akinites	20

     20.   Cells of erect filaments becoming  shorter and broader toward
          filament apex; usually  growing on back of turtles; branching only
          from base	Basicladia
    20.   Thallus not as above	 .   21

21.  Filaments irregularly branched; branches short  1 -  or few celled .  .  . Rhizoclonium
21.  Filaments repeatedly branched; branches narrowed toward tips	Cladophora
                                                                                     47-3

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                                      CHLOROPHYTA

                  A Key to Some Common Non-Filamentous  Genera
1.  Unicellular	".....    2
1.  Colonial	27

    2.   Motile in  vegetative state,  flagella 2-4	3
    2.   Non-motile in vegetative state	5

3.  Cells with  2 flagella	   4
3.  Cells with  4 flagella	Carteria

    4.   Cell enclosed by bicovex shell	Phacotus
    4.   Cell not enclosed by a shell	Chlamy domonas

5.  Cells with  median constriction  (often slight), or  of chloroplast only	6
5.  Cells without a median constriction	'.  .  15

    6.   Cells  lunate	Closterium
    6.   Cells  not  lunate  in any  degree	7

7.  Cells cylindrical,  noticeably  longer than  broad	8
7.  Cells almost never cylindrical; flattened  or  triangular  in apical view	9

    8.   Length 2-3 times the  breadth,  contriction nearly lacking	Cylindrocystis
    8.   Length much greater than breadth, nodulose  at constriction	Pleurotaenium

9.  Cells triangular in end view	Staurastrum
9.  Cells not triangular in end view	10

    10.   Semicells with lateral incisions,  appearing  lobed	11
    10.   Semicells without lateral incisions	12

11.  Lateral incisions few, shallow,  lobes rounded	Euastrum
11.  Lateral incisions many,  deep,  lobes  angular	Micrasterias

    12.   Semicells with radiating arms	Staurastrum
    12.   Semicells without arms; spines,  granules,  or teeth may be present	13

13.  Semicells without  spines	Cosmarium
13.  Semicells with  spines	14

    14.   Spines few,  usually  at  the  apical  corners	Arthrode smus
    14.   Spines numerous, scattered	Xanthidium

15.  Cells elongate,  sometimes  needle-like	16
15.  Cells spherical, ovid, angular; not needle-like	18

    16.   Cells with terminal  setae	 Schroederia
    16.   Cells without terminal  setae	    17
   47-4

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17.   Cells acicular, without a row of  pyrenoids	Ankistrodesmus
17.   Cells acicular, very  long, with a  row  of  10-12  pyrenoids	Closteropsis

     18.   Cells without  spines  or  processes	.	19
     18.   Cells with spines or processes	23

19.   Cells embedded in a  gelatinous matrix	20
19.   Cells without  a gelatinous matrix	21

     20.   Gelatin obvious,  lamellate; chloroplast cup-shaped	Gloeocystis
     20.   Gelatin sometimes obvious, chloroplast,  star-like	.Asterococcus

21.   Cells spherical	22
21.   Cells angular	Tetraedron

     22.   Cell wall  smooth	Chlorella
    22.   Cell wall  sculptured	Trochiscia

23.   Cells angular	 24
23.   Cells spherical or oval,  with spines	23

    24.   Angles with furcated  processes	Tetraedron
    24.   Angles with spines	Polyedriopsis

25.   Cells spherical, spines delicate	Golenkinia
25.   Cells oval, spines evident	26

    26.   Spines localized at ends of cell	Lagerheimii
    26.   Spines distributed  over cell	Franceia

27.   Motile,  each  cell with 2  equal-length flagella	28
27.   Non-motile invegetative state	33

    28.   Colony a "flat" plate	29
    28.   Colony spherical or ovid	30

29.   Colony  "horse-shoe" shaped	Platydorina
29.   Colony  quadriangular or circular	Gonium

    30.   Cells 8-16,  crowded-pyriform	Pandorina
    30.   Cells  more than  16,  not  crowded, spherical or nearly  so	31

31.  Cells more than 300  in number	Volvox
31.  Cells less than 300 in number	~~,  T~32

    32.   Cells  (16) - 32 in number	Eudorina
    32.   Cells  64-128  - (256)  in number	Pleodorina

33.  Cells of colony lying  in one plane	34
33.  Cells not in a conspicuous single plane	37

    34.   Colony circular (rarely cruciate)	Pediastrum
    34.   Colony not circular	~ '. ',  ",  735
                                                                                     47-5

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35.   Colony a flat strip; cells  side by  side	Scenedesmus
35.   Colony quadriangular	36

     36.   Colony usually large,  cells  in 4's,  no spines	Crucigenia
     36.   Colony of 4 cells,  each with  1 or  2 marginal spines	Tetrastrum

37.   Cells  acicular (needle-like)	Ankistrodesmus
37.   Cells  not acicular	38

     38.   Colony without a gelatinous  envelope	39
     38.   Colony with a  more or  less conspicuous gelatinous  envelope	47

39.   2-8 oval enclosed by a  distinct  sheath	Oocystis
39.   Cells  not enclosed by a sheath	40

     40.   Cells  with  long spines or setae	41
     40.   Cells  without  spines  or setae	42

41.   Colony pyramidate,  cell spherical, long  spines	Errerella
41.   Colony quadrate, or  a tetrahedron, long setae	Micractinium

     42.   Cells  linear,  radiating from a common center	Actinastrum
     42.   Cells  no linear	43

43.   Cells  strongly lunate, often "back-to-back"	Selena strum
43.   Cells  not lunate	".  . ~ .  .  . 44

     44.   Cells  arranged in a hallow sphere	45
     44.   Cells  not arranged in a hallow sphere	46

45.   Cells  spherical, sometimes joined by processes	Coelastrum
45.   Cells  not spherical,  outer angles  extended  into  stout,  blunt
      teeth or spines	Sorastrum

     46.   Cells  uniform, spherical, in groups of  4 -  8	Westella
     46.   Cells  not uniform,  ellipsoid (oblong) or reniform	Dimorphococcus

47.   Cells  curved to  strongly lunate	48
47.   Cells  not curved or lunate	49

     48.   Cells  lunate,  loosely arranged in colony	Kirchneriella
     48.   Cells  curved or  reniform,  colony compact,  distinct	Nephrocytium

49.   Cells  connected  by branching central  strands	Dictyosphaerium
49.   Cells  not connected by  stands	50

     50.   Cells  cylindrical or fusiform	51
     50.   Cells  spherical or  slightly ovoid	52

51.   Cells  in parallel "bundles" of 2  -  8	Quadrigula
51.   Cells  longitudinally arranged,  not  grouped laterally	Elakatothrix

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    52.  Cells ellipsoid  or  ovoid,  envelope lamellated	Gloeocystis
    52.  Cells spherical, or nearly so	53

53.  Chloroplast axial,  star-shaped	Asterococcus
53.  Chloroplast parietal, not  star-shaped	~'.  '.  '.  . 54

    54.  Cells enclosed  by  lamellated sheaths	Gloeocystis
    54.  Cells in homogenous  envelopes	Sphaerocystis
                                                                                      47-7

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                                    EUGLENOPHYTA
1.  Vegetative cells sessile	Colacium
1.  Vegetative cells motile	2
    2.   Cells with  chlorophyll	3
    2.   Cells without chlorophyll	8
3.  Protoplast within a lorica or test	Trachelomonas
3.  Protoplast naked, no lorica	4
    4,   Body strongly metabolic	Euglena
    4.   Body rigid	5
5.  With two laminate,  longitudinal chloroplasts	Chryptoglena
5.  With numerous  chloroplasts	  .  6
    6.   Body conspicuously flattened, sometimes twisted	Phacus
    6.   Body not compressed,  radially  symmetrical	6
7.  Body broadly ellipsoid to ovoid	Lepocinclis
7.  Body elongate,  narrow	Euglena
    8.   Cells with one flagellum	Astasia
    8.   Cells with two flagella	Peranema
47-8

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        KEY  TO  SOME COMMON DIATOM  GENERA (BACTLLARIOPHYTA) OF
                                        MICHIGAN
Diatoms in  Valve View

1.   Valves  without a dividing line or  cleft; markings of valve radiate about a central
    point,   (centric diatom genera)	 2

    2.   Frustules  usually in filaments or zig-zag chains; rectangular in girdle
         view; valve round,  oblong,  triangular or  elongate	   3

         3.   Frustules  cylindrical; markings prominent on girdle;  sulcus
             present; seldom seen in valve view	Melosira

         3.   Frustule not  cylindrical	4

             4.   Valve ovate to oblong;  two horns or processes present on valve face,
                 scatter small spines often present	Biddulphia

             4.   Valve not ovate to  oblong,  horns or processes absent	5

                 5.   Valve face appearing as two; three  sided pieces  giving the
                     appearance  of  six processes	Hydrosira

                 5.   Valve face three to several times longer than broads;  margins
                     undulate:   "costae" evident	Terpsinoe

    2.   Frustules  usually solitary (sometimes forming short chains)	6

         6.   Frustules  usually  elongated; many intercalary bands,  frustules
             cylindrical	7

             7.   Each valve with a  single long spine	Rhizosolenia

             7.   Each valve with two  long spines	Attheya

         6.   Valves discoid; cylindrical; or spherical; somtimes with spines or
             processes 	  8

             8.   Ornamentation of valves in two concentric parts of unlike
                 pattern	Cyclotella

             8.   Ornamentation of valves radiate;  continuous  from center to
                 margin of valves	9

                 9.   Ornamentation  of valves distinctly radiate;  rows  of punctae
                     single at  center  becoming multiseriate  at  the  margin; margin
                     of valve  with recurved spines	Stephanodiscus

                 9.   Ornamentation  of valves not distinctly radiate or becoming
                     multiseriate at margin	10
                                                                                     47-9

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             10.   Isolated large punctum evident at  the  margin spines
                  not evident	 Thalassiosira

             10.   Isolated punctum not evident at the margin, short
                  spines present	Cascinodiscus

1.   Valve with a  dividing line  or  cleft;  marking of wall bilaterally
    disposed to an axial or excentric line (pennate diatom genera)	11

    11.  Both  valves with  a pseudoraphe	12

         12.   Valves asymetrical to one axis	13

               13.   Valve asmetrical to longitudinal axis,  striae present .  . Ceratoneis

               13.   Valve asymetrical to transverse axis	14

                    14.  Valves clavate	15

                    14.  Valves not  clavate; striae  present;  valves with unequal
                         capitate  ends;  often forming  star like colonies .  .  Asterionella

                         15.   "Costae"  present, frustules may be joined face
                               to face to form fan like filaments	Meridion

                         15.   "Costae:  present,  frustules  single (appears
                               as  asymetrical Fragilaria)	Opephora

         12.   Valves symetrical to both axes	16

               16.   Frustules septate or "costate";  often appearing in
                    zig-zag chains	17

                    17.  Septae present; usually many  partially  septate
                         intercalary  bands;  valves triundulate	Tabellaria

                    17.  Septae absent;  prominant "costae" on valve  .  .  .  Diatoma

               16.   Frustules occuring free or  attached in filaments;
                    sometimes forming  fascicles	18

                    18.   Frustules typically forming long filaments; usually
                         not more than  5 or 6 times longer  than broads;
                         often  appearing costate	Fragilaria

                    18.   Frustules usually  solitary or forming fascicles;
                         usually many times longer  than broad	Synedra
                         (note: the above two  genera are actually seperated
                         only  on the  basis of  growth habit)

    11.  At least one valve with a pseudoraphe	19
 47-10

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19.   One valve with a true raphe the other valve with a pseudoraphe	20

      20.   Valve asymetrical to the transverse axis; partial terminal septae;
           bent about the transverse axis	Rhoicosphenia

      20.   Valves symetrical to both axes	21

           21.   Valve elliptical; valves  with  a marginal and/or  submarginal
                hyaline  ring; often loculiferous; bent about the longitudinal
                axis	Cocconeis

           21.   Valves usually lanceolate or  linera  lanceolate; bent
                around the transverse axis	Achnanthes
                (Those  with a  sigmoid  raphe  are sometimes put in
                Achnanthidium  or  Eucocconeis)

19.   Both valves  with a raphe	22

      22.   Raphe median or nearly  so,  never completely marginal;
           not  in a  canal	..   23

           23.   Valves sigmoid in  outline	24

                24.   Raphe sigmoid; punctae in two series; one transverse
                     and one longitudinal row forming a 90° angle .... Gyrosigma

                24.   Raphe sigmoid; punctae in three series forming
                     angles of other than 90°	Pleurosigma

           23.   Valve not sigmoid  in outline	25

                25.   Valves symetrical to both axes	26

                     26.   Frustules with septate intercalary bands	27

                     27.   Intercalary bands with marginal loculi, punctae
                           distinct	Mastagloia

                     27.   Intercalary bands with two  large faramen  along
                           apical axis,  punctae indistinct	Diatomella

                     26.   Frustules without septate intercalary  bands	28

                           28.   Valve face with a sigmoid  saggital keel;
                               "hourglass" shape outline in girdle view .  .  . Amphiprora

                           28.  Valve face  without a sigmoid saggital keel	29

29.   Valve  with undulate or zig-zag irregular logitudinal lines or
      blank  spaces	 Anomoeneis

29.   Valve  without  undulate or zig-zag  irregular logitudinal lines or
      blank  spaces	30
                                                                                      47-11

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  30.   Valve with thickened,  non-punctate central area; (stauros)
       pseudoseptae sometimes  present,  longitudinal lines and  blank
       spaces lacking	Stauroneis

  30.   Valve with or without stauros, septal absent	31

       31.   Valve with longitudinal lines or blank spaces	32

            32.  Proximal ends of raphe usually  curved in  opposite
                 directions;  "defaut regularier" toward valve apices;
                 longitudinal blank spaces	Neidium

            32.  Proximal ends of raphe straight; valves with fine
                 striae that appear as  costae; longitudinal line
                 near margin	Caloneis

       31.   Valves without longitudinal lines or blank spaces	33

            33.  Valves  with siliceous ribs along each side of the  raphe	   34

                 34.   Raphe bisects siliceous  ribs  on valve; central
                       area small and  orbicular; striae and punctae
                       very distinct	'	Diploneis

                 34.   Raphe short;  less  than  1/2  length of valve; central
                      area long and narrow; terminal nodules  evident,
                      elongate	35

                      35.   Raphe short; 1/4 or less  the length of the  valve;
                            striae not evident	 Amphipleura

                      35.   Raphe longer; usually  about 1/3  the length of
                           the  valve; striae usually fine but evident .  . . Frustulia

            33.  Valve without  siliceous ribs	36

                 36.   Valves with smooth transverse costae; raphe
                       often ribbon like;	Pinnularia

                 36.   Valves with transverse striae	37

                       37.   Raphe  sigmoid	Scoliopleura

                       37.   Raphe  straight	38

  38.   Raphe in straight  and raised keel	Tropodoneis

  38.   Raphe straight and not in a keel	39

       39.   Striae daubly punctate,  central area long  and  narrow  .  .  . Brebessonia

       39.   Striae single to lineate,  central area  variable .... Navicula
47-12

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25.  Valves symetical to one axis only . '	40

     40.   Valves symetrical to the longitudinal axis	41

           41.   Punctae  in one  series; longitudinal line absent	Gomphonema

           41.   Punctae  in two  series; longitudinal line present	Gomphoneis

     40.   Valves symetrical to transverse  axis	42

           42.   Raphe short; vestigial terminal;  with evident terminal nodules,
                central nodule  lacking	43

                43.   Colonial;  forming tree like  colonies; valves usually with
                      evident spines	Desmogonium

                43.   Usually not in colonies or if  colonial,  forming only
                      short chains or  stellate  clumps	44

                      44.   Cells shaped like the  femur of a chicken  . .  .  . Actinella

                      44.   Valves various shaped; lunate to  nearly  straight;
                           valve often with  undulate dorsal  and/or  ventral
                           margin; raphe  prominant in girdle view	45

                           45.   Dorsal margin convex,  ventral margin slightly
                                concave, both margins sinvate-dentate  .  .  . Amphicampa

                           45.   Dorsal margin convex,  ventral margin straight
                                to  concave,  one, both  or neither margin wavy,
                                pseudoraphe often present on  ventral margin . .  . Eunotia

           42.   Raphe not vestigial; usually  as nearly as long as the valve;
                valves usually  cymbiform	46

                46.   Valves  convex;  central nodule  usually  lies very close to the
                      ventral margin; both raphe visible  in  girdle view ....  Amphora

                46.   Valves  flat or nearly  so; raphe a smooth curve with the same
                      curvature  as  the axial field; raphe  not visible in  girdle
                      view	Cymbella

22.  Raphe marginal and in  a  canal	47

     47.   Valves with a single  canal that is usually marginal but  may appear
           to be  somewhat central	48

           48.   Valves symetrical  to  both axes	49

                49.   Transverse internal  septae  that appear as "costae"; canal
                      nearly median	Denticula

                49.   Transverse "costae" lacking;  carnial dots present  . .  . Nitzschia
                                                                                    47-13

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       48.   Valves asymetrical to longitudinal axis	50

             50.   "Costae" quite evident	51

                  51.   Axial field forming an acute angle at the  central nodule;
                       raphe along the  ventral margin of valve	Ephithemia

                  51.   Axial field forming a less acute angle at the  central
                       nodule;  raphe  along the dorsal margin of  the valve  .  .  Rhopalodia

             50.   "Costae" not evident;  carnial dots along the  raphe	52

                  52.   Raphe  of one  valve diagonally  opposite  the raphe on
                       the other valve	Nitzschia
                  52.   Raphe of one valve directly opposite the raphe on the
                       other valve	Hantzschia

   47.  Valves  with a  canal next to both  lateral margins	53

        53.  Valve face  transversely undulate; band  of  short  costae  along each
             lateral margin appearing like a  row of beads	Cymatopleura

        53.  Valve face  not transversely  undulate	54

             54.   Valve shaped like a saddle	Campylodiscus

             54.   Valve face flat; either  isopolar  or heteropolar;  sometimes
                   the  frustule  is slightly spiral in shape	Surirella
47-14

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                                        CHRYSOPHYTA
                       A Key to Some  More  or Less  Common Genera
1.  Filamentous,  branched	Phaeothamnion
1.  Not  filamentous	   2
    2.   Unicellular	   3
    2.   Colonial	7
3.  Protoplast enclosed  by a lorica	4
3.  Protoplast not enclosed  by  a lorica	6
    4.   Epiphytic or epizooic	5
    4.   Motile; cells with siliceous  scales  many  of  which have long,
         siliceous spines	Mallomonas
5.  Epiphytic; lorica flask-shaped	Lagynion
5.  Epiphytic or epizooic; lorica cylindrical	.Epipyxis(= Hyalobryon)
    6.   Motile; protoplast naked	Ochromonas (and assoc.)
    6.   Non-motile;  protoplast  with  long  delicate,  pseudopodia	Rhizochrysis
7.  Sessile; each cell in a long,  cylindrical lorica	       Epipyxis (Hyalobryon)
7.  Motile	.8
    8.   Each cell within a companulate,  basally pointed lorica	Dinobryon
    8.   Lorica absent	9
9.  Colony  bracelet-shaped	Cyclonexis
9.  Colony  spherical	10
   10.   Colony bristling with long siliceous rods	Chrysosphaerella
   1.0.   Colony without siliceous  rods  from each  cell	11
11.  Colonies not enclosed by  a gelatinous  sheath	Synura
11.  Colonies enclosed by a distinct gelatinous sheath	12
   12.   Shorter flagellum  more  than 1/2  length of longer flagellum	Uroglenopsis
   12.   Shorter flagellum  less than  1/2  length of longer flagellum	Uroglena
                                                                                       47-15

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                                       CYANOPHYTA
                        A Key to  Some of the Common Genera
 1.  Cells not in trichomes; unicellular or  colonial	2
 1.  Cells in trichomes	10

     2.   Colony with some regular arrangement of cells	3
     2.   Colony amorphous, no definite form	6

 3.  Colony  a flat plate	 4
 3.  Colony  spherical, cells peripheral	   5

     4.   Cells  regularly arranged	Merismopedia
     4.   Cells  irregularly arranged	.Holopedium

 5.  Colony  with  a central branching  system	Gomphosphaeria
 5.  Colony  without  a  central branching system	Coelosphaerium

     6.   Colony many celled	.7
     6.   Colony mostly few celled	9

 7.  Cells elongate	Aphanothece
 7.  Cells spherical	8

     8.   Cells  close together	Microcystis
     8.   Cells  more than  2-3 diameters apart	Aphanocapsa

 9.  Cells usually hemispherical,   with or without definite gelatinous sheaths . .  Chroococcus
 9.  Cells spherical or ovaod,  gelatinous sheaths very  distinct	Gloeocapsa

     10.  Trichomes without  sheath (not filamentous)	11
     10.  Trichomes in  a  sheath  (filamentous)	20

11.  Heterocysts  absent	12
11.  Heterocysts  present	14

     12.  Trichomes straight .  .	Oscillatoria
     12.  Trichomes regulary spiraled	13

13.  Cross  walls  distinct	Arthrospira
13.  Cross  walls  lacking	Spirulina

     14.  Heterocysts  terminal	15
     14.  Heterocysts  intercalary	18

15.  Trichomes cylindrical	Cylindrospremum
15.  Trichomes attenuate	16

     16.  Trichomes solitary	Calothrix
     16.  Trichomes in masses	  '. '.\  T?
   47-16

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17.  Trichomes without akinetes	Rivularia
17.  Trichomes with akinetes	Gloeotrichia

     18.   Trichomes  straight, parallel in bundles	Aphanizomenon
     18.   Trichomes  solitary, or if in masses,  not  parallel	19

19.  Trichomes solitary,  or if  numerous,  then not in a firm  gelatinous
     matrix	Anabaena
19.  Trichomes entangled in a firm  gelatinous matrix	Nostoc

     20.   Many parallel trichomes in a sheath	Microcoleus
     20.   A single  trichome or row of trichomes in  a sheath	21

21.  Filaments not  branched	22
21.  Filaments branched	23

     22.   Sheath  obvious firm	Lyngbya
     22.   Sheath  indistinct, delicate	Phormidium

23.  Filaments with false branching	  .24
23.  Filaments with true branching	26

     24.   Without heterocysts	Plectonema
     24.   With heterocysts	25

25.  False branches arising single	Tolypothrix
25.  False branches in pairs	Scytonema

     26.   Trichomes  always uniseriate	Haplosiphon
     26.   Trichomes  wholly or  in part  nultiseriate	 Stigonema
                                                                                     47-17

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                                      XANTHOPHYCEAE

                              A Key to  Some  Common Genera
 1.  Filamentous	2
 1.  Not filamentous	'	4

     2.  Filaments branched,  siphonaceaous	Vaucheria
     2.  Filaments not branched	.'	3

 3.  Cell wall of stout H pieces,  cells sometimes barrel-shaped	Tribonema
 3.  Cell wall of delicate H pieces,  cells short and cylindrical	Bumilleria

     4.  Cells embedded in a gelatinous matrix	5
     4.  Cells not embedded in a gelatinous matrix	7

 5.  Colonial gelatinous envelope  dichotomously  branched	Mischococcus
 5.  Colonial gelatinous envelope  not branched	.  .  .6

     6.  Colonial envelope,  tough  "heavy" cartilaginous	Botryococcus
     6.  Colonial envelope watery; colonies small, free  floating	Chlorobotrys

 7.  Cells epiphytic	8
 7.  Cells not epiphytic	11

     8.  Stipe long,  seta-like	Stipitococcus
     8.  Stipe short;  or cell-sessile	Peroniella

 9.  Cell vase-like,  apex flattened	Stipitococcus
 9.  Cell oval or spherical	Peroniella

     10.  Cells  cylindrical,  ends broadly rounded	Ophiocytium
     10.  Cells  not cylindrical	Characiopsis

11.  Cells cylindrical,  straight or contorted	Ophiocytium
11.  Cells globose, with rhyzoids,  on soil	Botrydium
   47-18

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Acicular:  needle-like in shape.  (Ankistrodesmus)

Aerial:  algal habitat on moist soil, rocks, trees,  etc.; involving  a thin film of water;
         sometimes only somewhat  aerial.

Akinete:  A  type  of spore  formed by the  transformation of  a  vegetative cell into a thick-
          walled resting cell,  containing a concentration of food material.  (Pithophora)

Amoeboid:   like an amoeba; creeping by extensions of highly  plastic protoplasm
             (pseudopodia).   (Chrysamoeba)

Amorphous:   without  definite  shape; without regular form.

Anastomose:  to separate and come together again; a meshwork.

Antapical:   the posterior or rear pole  or region of an  organism,   or of a colony of cells.

Anterior:  the forward  end; toward the  top.

Antheridiurn:  a single  cell or a series of cells in which male  gametes are produced; a
               multicellular,  globular male organ in the Characeae,  more properly called
               a globule (a  complicated  and specialized  branch in which antheridial cells
               are produced.

Antherozoid:  male sex cell;  sperm.

Apex:   the summit, the terminus,  end of a projection,  of an  incision, or of a  filament
        of cells.

Apical:  Forward tip.

Aplanospore:  non-motile,  thick-walled  spore formed  many within an unspecialized
               vegetative cell; a small  resting  spore.

Arbuscular:   branched or growing  like a tree or bush.

Armored:  See theca.

Attenuate:  narrowing to a point or becoming reduced in diameter.  (Gloeotrichia)

Autospores:   spore-like bodies  cut  out of the contents of a  cell which  are small replicas
              of the parent cell and which only  enlarge  to become  mature plants.   (Coelastrum)

Axial:   along a median  line bisecting an object  either transversely or  longitudinally
        (especially the later,  e.g.  an axial chloroplast).

Bacilliform:   rod-shaped.

Bilobed:  with two lobes or extensions.
                                                                                    47-19

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Bipapillate:  with two  small protrusions; nipples.

Biscuit-shaped:  a thickened pad; Pillow-shaped.

Bivalve (wall):  wall of  cell which is in two sections, one usually slightly larger
                 than the others.

Blepharoplast:  a granular  body in a  swimming organism from  which a flagellum arises.

Bristle:  a stiff hair;  a  needle-like spine.   (Mallomonas)

Capitate:   with an enlargement  or a  head at one end.  (as in some species of Oscillatoria)

Carotene:  Orange-yellow plant pigment of which there are  four kinds  in  algae; a
            hydrocarbon,  C, H.

Chlorophyll:   a green  pigment of which there are five kinds in  the algae  chlorophyll-a
              occurring  in all of the algal  divisions.

Chloroplast:   a body of  various shapes  within the cell containing the pigments of which
              chlorophyll is the predominating  one.

Chromatophore:  body within  a cell contining  the pigments of which some other tan
                 chlorophyll is the predominating one; may be  red, yellow, yellow-green
                 or  brown.

Chrysolaminarin:  See leucosin.

Coenobium;  a  colony  with  2^ number of cells.  (Scenedesmus)

Coenocytic:  with multinucleate  cells, or cell-like  units; a multinucleate,  non-cellular
             plant.   (Vaucheria)

Collar:  A thickened ring or neck surrounding, the  opening in a shell or lorica through
         which  a flagellum projects from the inclosed organism.  (Trachelomonas)

Colony:  a group or  closely associated  cluster of cells,  adjoined or  merely inclosed by a
         a common investing mucilage or sheath; cells  not  arranged in a linear  series to
         form a filament;  either aggregate or  coenobium.

Constricted:   cut in  or incised, usually form two opposite points on  a  cell so that an
              isthmus  is formed between two parts  or cell halves; indented as at the
              joints  between cells of a  filament.  (Cosmarium)

Contractile vacuole:   a small vacuole (cavity) which is bounded by a membrane that
                      pulsates,  expanding and contracting.

Crenulate:  wavy with  small scallops; with small crenations.

Crescent:  an arc of a circle; a curved figure  tapering to horn-like points from  a wider,
           cylindrical  midregion.

Cross  wall:  a  cross partition.
  47-20

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Cup-shaped:  a more or less complete  plate (as a chloroplast)  which lies  just within
              the cell wall, open  at one side to form a cup.

Cylindrical:   a figure, round in cross section,  elongate with parallel lateral margins
              when seen from the  side,  the ends square or truncate.   (Hyalotheca)

Daughter cells:  cells  produced directly from the division  of a  primary or parent cell;
                 cells  produced from  the same  mother cell.

Dichotomous:  dividing or  branched by  repeated forkings,  usually into  two equal portions
               or segments.

Disc; Disc-shaped:   a  flat (usually circular) figure: a circular plate.

Distal:   the  forwar or anterior end or  region as opposed to the basal  end.

Ellipsoid:   an ellipse,  a plane figure  with curved margins,  the poles more sharply
           rounded than the lateral  margins of an elongate  figure.

Epiphyte:  living upon  a  plant, sometimes living internally  also.

Euplankton:  true or openwater plankton (floating) organisms.

Eye-spot:   a granular  or complex of  granules (red or brown) sensitive to  light and
            related to responses to light by swimming organisms of spores.  (Pandorina)

False Branch:  a branch formed by lateral growth of one or both ends of  a broken
                filament; a branch not formed by  lateral division of  cells in an unbroken
                filament,   (Tolypothrix)

Filament:   a thread  of cells;  one  or more rows of cells;  in the blue-green algae the
           thread of cells together with a sheath  that may be present, the  thread of
           cells alone  referred to as  a trichome.

Flagellum (flagella):   a relatively coarse,  whip-like organ of  locomotion,  arising from
                      a special granule, the blepharoplast,  within a  cell.   (Euglena)

Fucoxanthin:  a  brown pigment predominant in the Phaeophyta and Chrysophyta.  (Synura)

Fusiform:  a figure broadest  in the midregion and gradually tapering to  both poles  which
            may be acute  or  bluntly rounded; shaped  like a  spindle.   (Closterium)

Gamete:  a sex  cell; cells which  unite to produce a fertilized egg or zygospore.

Gas vacuole:   See  pseudovacuole.

Gelatin  (gelatinous):  a mucilage-like  substance.

Glycogen:  a  starch-like  storage product questionably  identified in food granules of  the
           Cyanopjyta.   (Chroococcus)

Gregarious:   an association;  groupings of individuals not necessarily joined together but
             closely  associated.
                                                                                    47-21

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Gullet:   a canal leading from the  opening of  flagellated  cells into  the  reservoir in the
         anterior  end.   (Euglena)

Gypsum:   granules of  calcium sulphate  which occur in the vacuoles of some
           desmids.  (Closterium)

Haematochrome:   a red or orange pigment,  especially in some Chlorophyta and
                   Euglenophyta, which masks the green chlorophyll.

Heterocyst:  an enlarged cell in some of the filamentous  blue-green  algae,  usually
             empty and different in shape from the vegetative  cells,   (Anabeana)

Hold-fast  cell:  a basal cell of a filament or thallus, differentiated to form an
                an attaching organ.   (Oedogonium)

H-pieces:   wall of overlapping H-shaped structures.   (Tribonema)

Intercalary:  arranged in the same series,  as spores or  heterocysts  which  occur in
              series with the vegetative cells rather than  being terminal  or  lateral.
              (heterocyst  of Anabeana)

Laminate:  plate-like;  layered.

Lateral  groove:   a groove in Dinoflagellates  encircling the cell.   (Ceratium)

Leucosin:   a  whitish food  reserve  characteristic of many of the Chrysophytam especially
            the Heterokontae; gives a  metallic lustre  to  cell contents.   (Dinobryon)

Lorica:  a shell-like structure of varying shapes which  houses an organism,  has an
         opening  through  which organs of locomotion are  extended.    (Dinobryon)

Lunate:  crescent-shaped; as of the new moon in shape.   (Selenastrum)

Median construction:   See constricted.

Metabolic:  plastic, changing shape in motion as in many Euglena.

Micron:   a unit of microscopical measurement; one 1/1000 of a millimeter,  determined
          by using a micrometer in the  eyepiece of the  microscope which has  been
          calibrated with a standard stage micrometer; expressed  the  symbol.

Moniliform:  arranged like a string of beads; beadlike;  lemon-shaped.  (Anabeana)

Mother  Cell:  the  cell which by mitosis  or by internal cleavage gives rise to
              other cells (usually  spores).

Multinucleate:  with many  nuclei.

Multiseriate:  cells arranged in more than one  row; a filament two or more cells in
              diameter.   (Stigeonema)

Motile:   motion caused by cilia or flagella.   (Volvox)

Oblong:  a  curved figure,  elongate with the  ends broadly rounded  but  more sharply  curved
         than the  lateral margins.


 47-22

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Obovate:  an  ovate  figure, broader at the anterior end than at the posterior.

Oogonium:  a female  sex organ, usually an enlarged cell; an  egg case.

Oval;  an elongate,  curved figure with convex  margins and with  ends  broadly and
       symmetrically curved but more sharply so than the lateral margin.

Ovoid:  shaped  like an egg; a  curved figure  broader at one end than at the other.

Paramylum:  a solid,  starch-like storage product in the Euglenophyta.  (Euglena)

Parietal:   along the wall; arranged at the circumference; marginal as opposed to
           central or  azial in location.

Pellicle:  a thin membrane.   (Euglena)

Peridinin:   a brown pigment characteristic  of the Dinoflagellata.   (Ceratium)

Periplast:   bounding membrane  of  cells in Euglenoids and Chrysophytes.

Phycocyanin:  a blue  pigment found in the Cyanophyta, and in some Rhodophyta.

Phycoerythrin:  a red pigment found in  the  Rhodophta, and in some Cyanophyta.

Plcinkton:  organisms  srifting in the water,  or if swimming,  not able  to move
           against  currents.

Plastid:   a  body or organelle of the cell, either  containing pigments or in some
          instances  colorless.

Plate:  sections, polygonal in shape,  composing  the cell wall  of  some Dinoflagellata
        (the thecate or armored  dinoflagellates).

Posterior:  toward  the rear; the end opposite the forward (anterior) end of a  cell
            or of an organism.

Protoplast:   the living part  of the  cell;  the  cell membrane and its contents usually
             enclosed  by a cell  wall of dead material.

Pseudocilia:  meaning false cilia; flagella-like  structures not  used for locomotion as
              in Apiocystis and  Tetraspora.

Pseudoparenchymatious:  a false cushion; a  pillow like mound of cells (usually attached)
                         which  actually is a compact  series of short, often branched
                         filaments.  (Coleochaete)

Pseudovacuole:   meaning a false vacuole; a  pocket in  the cytoplasm of many  blue-green
                 algae which  contains  gas or mucilage; is light refractive.  (Microcystis)

Pyrenoid:  a protein body around which  starch or paramylum  collects in a cell,  usually
           buried in a chloroplast  but sometimes free within  the  cytoplasm.   (Oedogonium)

Pylform:  pear-shaped.  (Pandorina)
                                                                                     47-23

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Reniform:  kidney-shaped; bean-shaped.   (Dimorphocacus)

Replicate:  infolded; folded back as in the cross walls of some  species of Spirogyra;  not
            a plane or straight  wall.

Reticulate:  netted,  arranged to form a  network;  with openings.

Scale:  siliceous  or inorganic material covering the  cell.  (Mallomonas)

Semicell:  a cell-half, as in the Placoderm  desmids in which the cell has two parts
           that are mirror images of one another, the two parts often connected by a
           narrow isthmus.  (Staurastrum)

Septum;   a cross-partition, cross wall or a  membrane complete  or incomplete through
          the  short diameter of  a cell, sometimes parallel with the long axis.

Setae:  a hair, usually arising  from within a cell wall; or a hair-like extension
        formed by tapering of a filament of cells  to  a fine point.

Sheath;  a covering, usually of  mucilage, soft or  firm; the  covering of a colony
         of cells,  or an envelope about one or more filaments of cells.

Siphonous:  a  tube; a thallus without cross partitions.  (Vaucheria)

Solitary:  unicellular;  solitary.   (Chlamydomonas)

Spine:  a sharply-pointed projection from the cell wall.  (Mallomonas)

Sproangium:   a cell (sometimes an unspecialized vegetative  cell) which gives rise to
              spores; the case which  forms abcut  the zygospores in the Zygnematales.

Star-shaped:   See  stellate

Stellate:   with radiating projections  from a  common  center; star-like.   (Zygnema)

Stigma:  see eyespot.

Suture:  a groove between plates, as  in  some Dinoflagellata; a cleft-like crack or line
         in some  zygospores of the  Zygnemataceae.   (Ceratium)

Thallus:   a plant  body which is  not  differentiated  into root,  stem and  leal organs; a
          frond; the  algal plant.

Theca; Thecate:   a firm outer wall; a shell, sometimes  with plates as in the
                  Dinoflagellata.   (Peridinium)

Test:  a shell  or covering external to the cell itself.  See Lorica.

Transverse furrow  (groove):  a  groove extending around  the cell as in the
                              Dinoflagellata.  (Ceratium)

Trichrome:  in blue-greens, a  series of cells joined end to end.   (Oscillatoria)
 47-24

-------
True Branch:  a branch formed by means  of lateral division of cells in  a main filament.
               Includes all branched algae except those blue- green algae with false
               branching.

Tychoplankton:  the plankton of waters near shore;  organisms floating and entangled
                among weeds and in algal  mats, not in the open water of a lake
                or  stream.

Undulate:  regularly wavy.

Unicellular:   See  solitary.

Vegetative:   referring to  a non- reproductive  stage,  activity,  or cell  as  opposed to
             activities and stages  involved  in reproduction, especially sexual reproduction.
Xanthophyll:  a yellow pigment of several kinds associated with chlorphyll,

Zoospore:   an animal- like spore equipped with flagella and usually with an eye- spot.
                                                 This key was prepared by Dr. Matthew H. Hohn,
                                                 Professor of Biology, Central Michigan
                                                 University, Mt. Pleasant, Michigan.
                                                 Descriptors: Plankton, Identification Keys
                                                                                     47-25

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                           CLASSIFICATION - FINDER
                                     for
                         NAMES OF AQUATIC  ORGANISMS
                                     in
                     WATER SUPPLIES AND POLLUTED  WATERS

                     Part I.   The System of Classification
I  INTRODUCTION

A  Every type of living creature
   has a favorite place to live.
   There are few major groups that
   are either exclusively terres-
   trial or aquatic.  The following
   remarks will therefore apply in
   large measure to both, but pri-
   mary attention will be directed
   to aquatic types.

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

   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 subdivi-
      sions.  There are over a
      million and a half items (or
      species)  included in the
      system of biological nomen-
      clature (very few libraries
      have a million books).

   3  The system of biological no-
      menclature 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 sub-
         ject to change and revision
         as man's knowledge and
         understanding increase.
   b  The basic categories em-
      ployed are as follows:

      (1)  Similar species are
           grouped into genera
           (genus)

      (2)  Similar genera are
           grouped into families

      (3)  Similar familes are
           grouped into orders

      (4)  Similar orders are
           grouped into classes

      (5)  Similar classes are
           grouped into phyla
           (phylum)

      (6)  Similar phyla are
           grouped into kingdoms

4  The scientific name of an or-
   gan isin~Ts~~ITs~genus" name plus
   its species name.   This is ana-
   logous to our system of sur-
   names (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
BI.AQ. 24.6.76
                             48-i

-------
                        RELATIONSHIPS BL1WEEN LIVING ORGANISMS
                     ENERGY FLOWS FROM LEFT TO RIGHT, GENERAL EVOLUTIONARY
                              SEQUENCE IS UPWARD
PLANTS 5
ORGANIC MATERIAL
PRODUCED, USUALLY
BY PHOTOSYNTHESIS
ENERGY STORED
FLOWERING PLANTS
AND GYHNOSPERHS 76
CLUB MOSSES, FERNS 76
LIVERWORTS, MOSSES 73

ALGAE 12
M_MM_aMI_^^^^_^^^_^M_MMHMMI^^_^B_MMi_^^^_Hi_M
ANIMALS 81
ORGANIC MATERIAL INGESTED OR
CONSUMED
DIGESTED INTERNALLY
ENERGY RELEASED
243
ARACHNIDS 167 MAMMALS
INSECTS 154 BIRDS 242
CRUSTACEANS 129 REPTILES 241
SEGMENTED WORMS 121 AMPHIBIANS 240
MOLLUSCS 172 FISHES 195
MOSS ANIMALS 120,181 PROCHORDATES 191
WHEEL ANIMALS 116 STARFISH GROUPS 185
ROUNDWORMS 113
FLATWORMS 108
JELLYFISH - CORAL GROUP 103
SPONGES 99
^— — — — — ^— ^— — 	 — 	
FUNGI 250
ORGANIC MATERIAL
REDUCED
BY EXTRACELLULAR
DIGESTION AND IN-
TRACELLULAR META-
BOLISM TO MINERAL
CONDITION
ENERGY RELEASED
BASIDIOMYCETES 266
ASCOMYCETES 265
HIGHER PHYCOMYCETES
261

*>
\

: DEVELOPMENT OF MULTICEUU^AROR COENOCYTICORGANISMS
DIATOMS  38
PIGMENTED FLAGELLATES
                   12
BLUE GREEN ALGAE 7
       PHOTOTROPIC
       BACTERIA  252
CHEMOTROPIC BACTERIA
                252
           HIGHER PROTISTA
                    PROTOZOA  82
AMOEBOID PROTOZOA 86         CILLIATED PROTOZOA  92
  FLAGELLATED PROTOZOA  85      SPOROZOA 98
  (COLORLESS FLAGELLATES 85     SUCTORIA 97
                          DEVELOPMENT OF A NUCLEAR MEMBRANE
                           LOWER PROTISTA (ORMONERA)
LOWER PHYCOMYCETES
              261
    NOTE:  NUMERALS REFER TO PARAGRAPHS IN PARTS 2 AND 3.

    W. B. COOKE AND H. W.  JACKSON, AFTER WHITTAKER


    BI.ECO.pl.2b.4.66
                                                     ACTINOMYCETES  253

                                                     SPIROCHAETES 255

                                                     MYXOBACTERIA 254

                                                     PARASITIC
                                                     BACTERIA  251
                                                     AND VIRUSES

                                                     SAPROBIC BACTERIA 251
 4S-2

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                                                           Classification - Finder
          genus Anabaena (an alga),
          we  must simply use the
          generic name,  and:

          Anabaena planctonica,

          Anabaena constricta,  and

          Anabaena flos-aquae

          are three distinct species
          which have  different  signi-
          ficances to water treatment
          plant operations.

       A  complete list of the various
       categories to  which an organism
       belongs  is known  as its  "classi-
       fication".   For example,  the
       classification of a type  of
       frog spittle,  a common fila-
       mentous  alga,  and a crayfish
       or crawdad are shown side by
       side below.  Their scientific
       names  are  Spirogyra crassa and
       Cambarus sciotensis.

       a   Examples of the classifica-
          tion  of an  animal  and  a
          plant:
  (Frog Spittle)

Plantae
Chlorophyta
Chlorophyceae
Zygnematales
Zygnemataceae
Spirogyra
crassa
Kingdom
Phylum
Class
Order
Family
Genus
Species
 (Crayfish)

Animalia
Arthropoda
Crustacea
Decapoda
Palaemonidae
Cambarus
sciotensis
         These seven basic  levels  of
         organization  are often  not
         enough  for the  complete de-
         signation of  one species
         among thousands; however,
         and so  additional  echelons
         of terms are  provided by
         grouping the  various cate-
         gories  into "super..."
         groups  and subdividing  them
         into "sub..." groups as:
         Superorder, Order,  Suborder,
         etc.  Still other  category
         names such as "tribe",  "di-
         vision", "variety", "race",
         "section", etc. are used on
         occasion.
                           II
   c  Additional accuracy is gained
      by citing the name of the
      authority who first described
      a species (and the date) im-
      mediately following the spe-
      cies name.  Authors are also
      often cited for genera or
      other groups.

   d  A more complete classification
      of the above crayfish is as
      follows:

      Kingdom A.nimalia

      Phylum Arthropoda

      Class Crustacea

      Subclass Malacostraca

      Order Decapoda

      Section Nephropsidea

      Family Astacidae

      Subfamily Cambarinae

      Genus Cambarus

      Species sciotensis Rhoades
      1944

   e  It should be emphasized that
      since all categories above
      the species level are essen-
      tially human concepts,- there
      is often divergence of opin-
      ion in regard to how certain
      organisms should be grouped.
      Changes result as  knowledge
       grows.
   f  The most appropriate or cor-
      rect name for a given species
      is also sometimes disputed,
      and so species names too are
      changed.  The species itself,
      as an entity in nature, how-
      ever, is relatively timeless
      and so does not change to
      man's eye.

THE GENERAL RELATIONSHIPS OF
LIVING ORGANISMS           •;

Living organisms (as contrasted  to
fossil types) have long been group-
ed into two kingdoms:  Plant King-
doms and Animal Kingdoms.  Modern
developments however have made this
                                                                           48-3

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Classification - Finder
    simple  pattern technically  unten-
    able.   It  has  become  evident that
    there  are  as great and fundamental
    differences between certain other
    groups  and these  (two),  as  there
    are  between the traditional "plant"
    and  "animal".   The accompanying
    chart  consequently shows the
    Fungi  as a third  kingdom.

 B  The three  groups  are  essentially
    defined as follows on the  basis
    of their  nutritional  mechanisms:

    1  Plantae:   photosynthetic;
       synthesizing their own  organic
       substance  from inorganic min-
       erals.  Ecologically known as
       PRODUCERS.

    2  Animalia:   ingest  and digest
       solid  particles of organic  food
       material.  Ecologically known
       as  CONSUMERS.

    3  Fungi:  extracellular digestion
        (enzymes secreted  externally).
       Food material  then taken in
       through cell membrane where it
       is  metabolized  and reduced  to
       the mineral condition.  Ecolo-
       gically known  as REDUCERS.

 C  Each of these groups  includes
    simple, single celled representa-
    tives, persisting at  lower levels
    on the evolutionary stems of the
    higher organisms.

    1  These groups span  the gaps be-
       tween the  higher kingdoms with
       a multitude of transitional
       forms.  They are collectively
       called PROTISTA.,

    2  Within the protista, two prin-
       ciple sub-groups can be defined
       on the basis of relative com-
       plexity of structure:

       a  The bacteria and blue algae,
          lacking a nuclear membrane,
          may be considered as the
          lower protista or MONERA.

       b  The single celled algae and
          protozoa having a nuclear
          membrane, are best referred
          to simply as the higher
          protista.
 48-4

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                                                         Classification - Finder
                        Part II.   Biological Classification
 I  INTRODUCTION

 A  What is it?

 B  Policies

 C  Procedures

II  PLANT KINGDOM

 A  "Algae" defined
 1

 2

 3

 4

 5

 6
 B  PHYLUM CYANOPHYTA - blue-green   7
    algae

    CLASS Myxophyceae                8

     Order Chroococcales             9

     Order Hormogonales             10

      Suborder Hetercystineae       11

 C  PHYLUM CHLOROPHYTA - green      12
    algae

    CLASS Chlorophyceae             13

     Order Volvocales               14

     Order Ulotrichales               15

     Order Chaetophorales           16

     Order Chlorococcales           17

     Order Siphonales               18

     Order Zygnematales             19

     Order Tetrasporales            20

     Order Ulvales                  21

     Order Schizogoniales             22

     Order Oedogoniales             23

     Order Cladophorales            24

    CLASS Charophyceae              25

     Order Charales                 26

 D  PHYLUM CHRYSOPHYTA - yellow-    27
    green algae or yellow-brown
    algae
    CLASS Xanthophyceae             28
    Order Heterocapsales          30

    Order Heterococcales          31

   CLASS Chrysophyceae -          32
   yellow-green algae

    Order Chrysomondales          33

    Order Rhizochrysidales        34

    Order Chrysosphaerales        35

    Order Chrysocapsales          36

    Order Chrysotrichales         37

   CLASS Bacillariophyceae -      38
   Diatoms

    Order Pennales - pennate      39
    diatoms

    Order Centrales - centric     40
    diatoms

E  PHYLUM EUGLENOPHYTA - eugle-   41
   noid algae

F  PHYLUM PYRRHOPHYTA - yellow    42
   brown algae
            CLASS Desmokontae

             Order Desmomonadales

            CLASS Dinophyceae -
            dinoflage Hates

             Order Gymnodiniales

             Order Peridiniales

             Order Dinocapsales

             Order Chloromonadales

            CLASS Cryptophyceae
                                  43

                                  44

                                  45


                                  46

                                  47

                                  48

                                  49

                                  50
     Order Rhizochloridales
29
G  PHYLUM CHLOROMONADOPHYTA       51

H  PHYLUM RHODOPHYTA - red algae  52

   CLASS Rhodophyceae             53

    Order Bangiales               54

    Order Nemalionales            55

    Order Gelidiales              56
                                                                              4S-5

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  Classification - Finder
      Order Cryptonemiales

      Order Gigartinales

      Order Rhodymeniales

      Order Ceramiales
57

58

59

60
  I  PHYLUM PHAEOPHYTA - brown algae 61

     CLASS Phaeophyceae              62

      Order Ectocarpales             63

      Order Sphacelariales           64

      Order Tilopteridales           65

      Order Chordiales               66

      Order Desmarestiales           67

      Order Punctariales             68

      Order Dictyosiphonales         69

      Order Laminariales             70

      Order Fucales                  71

      Order Dictyotales              72

  J  PHYLUM BRYOPHYTA                73

     CLASS Hepaticae - liverworts    74

     CLASS Musci - mosses            75

  K  VASCULAR PLANT GROUP            76

     Emergent vegetation             77

     Rooted plants - floating leaves 78

     Submerged vegetation            79

     Free floating plants            80

III  ANIMAL KINGDOM                  81

  A  PHYLUM PROTOZOA - protozoa      82

     CLASS Mastigophora              83

      Subclass phytomastigina        84

      Subclass zoomastigina          85

     CLASS Sarcodina - amoeboid      86
     protozoa
Order Amoebina

Order Foraminifera

Order Radiolaria

Order Heliozoa
87

88

89

90
             Order Mycetozoa (Myxomycetes)  91

            CLASS Ciliophora - ciliates    92

             Order Holotricha              93

             Order Spirotricha             94

             Order Peritricha              95

             Order Chonotricha             96

            CLASS Suctoria - suctoria      97

            CLASS Sporozoa                 98

         B  PHYLUM PORIFERA - sponges      99

            CLASS Calcispongea            100

            CLASS Hyalospongea            101

            CLASS Demospongea             102

         C  PHYLUM COELENTERATA           103

            CLASS Hydrozoa - hydroids     104

            CLASS Scyphozoa - jellyfish   105

            CLASS Actinozoa (Anthozoa)  -  106
            corals

         D  PHYLUM CTENOPHORA - comb      107
            jellies

         E  PHYLUM PLATYHELMINTHES -      108
            flatworms

            CLASS Turbellaria - turbella- 109
            rians

            CLASS Trematoda - fluke       110

            CLASS Cestoidea - tapeworms   111

         F  PHYLUM NEMERTEA - proboscis    112
            worms

         G  PHYLUM NEMATODA - threadworms,113
            roundworms
   48-6

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                                                      Classification - Finder
H  PHYLUM NEMATOMORPHA -
   Horsehair worms
114
I  PHYLUM ACANTHOCEPHALA - thorny 115
   headed worms

J  PHYLUM ROTIFERA - rotifer,     116
   wheel animalcules

K  PHYLUM GASTROTRICHA - gastro-  117
   trichs
L  PHYLUM KINORHYNCHIA

M  PHYLUM PRIAPULIDA

N  PHYLUM ENDOPROCTA
118

119

120
0  PHYLUM ANNELIDA - segmented    121
   worms

   CLASS Polychaeta - polychaet   122
   worms

   CLASS Oligochaeta - bristle    123
   worms

   CLASS Hirudinea - leeches      124

   CLASS Archiannelida            125

   CLASS Echiuroidea              126

   CLASS Sipunculoidea - peanut   127
   worms

P  PHYLUM ARTHROPODA -jointed    128
   legged animals

   CLASS Crustacea - crustaceans  129

    Subclass Branchiopoda         130

     Order Anostraca - fairy      131
     shrimps

     Order Notostraca - tadpole   132
     shrimps

     Order Conchostraca - clam    133
     shrimps

     Order Cladocera - water fleas!34

    Subclass Ostracoda - seed     135
    shrimps, ostracodes

    Subclass Copepoda - copepods  136

    Subclass Branchiura - fish    137
    lice
Subclass Cirripedia -
barnacles

Subclass Malacostraca

 Order Leptostraca
138


139

140
               Order Hoplocardia           141
               (Stomatopoda)  - mantis shrimps
 Order Syncarida

 Order Peracarida

  Suborder Mysidacea

  Suborder Cumacea

  Suborder Tanaidacea
142

143

144

145

146
                Suborder Isopoda - sowbugs,147
                 pillbugs

                Suborder Amphipoda - scuds 148

               Order Eucarida              149

                Suborder Euphausiacea -    150
                krill

                Suborder Decapoda - shrimp,151
                lobster, crab

                Macrurous group (4 tribes) 152
                shrimps, prawns, lobsters,
                crayfish

                Brachyurous group          153
                (2 tribes)  - crabs and hermit
                crabs

             CLASS Insecta  - the insects   154

              Orders represented by imma-  155
              ture stages only.

              Order Plecoptera - stone-    156
              flies
              Order Ephemeroptera -
              mayflies
                             157
              Order Odonata - dragon and   158
              damselflies

              Order Megaloptera - alder-   159
              flies, dobsonflies, fishflies

              Order Neuroptera - spongilla-160
              flies

-------
Classification - Finder
    Order Trichoptera - caddis-    161
    flies

    Order Lepidoptera - aquatic   162
    caterpillars

    Order Diptera - two winged    163
    flies

    Orders including aquatic      164
    adults

    Order Coleoptera - beetles    165

    Order Hemiptera - true bugs   166

   CLASS Arachnoidea - spiders,   167
   scorpions, mites

    Order Xiphosoura - horse-     168
    shoe or king crabs

    Order Hydracarina - aquatic   169
    mites

    Order Pantopoda (Pycnogonida)-170
    pycnogonids

    Order Tardigrada              171

Q  PHYLUM MOLLUSCA                172

   CLASS Amphineura - chitons     173

   CLASS Gasteropoda - snails     174

    Order Prosobranchiata         175

    Order Opisthobranchiata       176

    Order Pulmonata - air breath- 177
    ing snails
         U  PHYLUM PHORONIDEA  -  tufted     184
            worms
   CLASS Scaphopoda - tusk
   shells

   CLASS Bivalvia
   (Pelecypoda)
178
179
   CLASS Cephalopoda - squid,     180
   octipus, nautilus

R  PHYLUM BRYOZOA (Ectoprocta) -  181
   Moss animals
S  PHYLUM BRACHIOPODA - lamp
   shells
182
T  PHYLUM CHAETOGNATHA - arrow    183
   worms
         V  PHYLUM ECHINODERMATA  -
            echinoderms
                              185
            CLASS Asteroidea  - starfishes  186

            CLASS Ophiuroidea - brittle    187
            stars

            CLASS Echinoidea - sea urchins   188

            CLASS Holothuroidea -  sea      189
            cucumbers

            CLASS Crinoidea - sea  lilies   190

         W  PHYLUM CHORDATA - chordates    191

                                           192
Subphylum Hemichordata -
Acorn worms

Subphylum Urochordata -
tunicates, sea squirts
                                           193
             Subphylum Cephalochordata  -    194
             lancelets

             Subphylum Vertebrata           195
             (Craniata)  - vertebrates

             CLASS Agnatha  - jawless       196
             fishes

              Order  Myxiniformes  -         197
              hagfishes

              Order  Petromyzontiformes -   198
              lampreys
              CLASS  Chrondrichthys  -
              cartilage  fishes
                              199
  Order Squaliformes - sharks 200

  Order Rajiformes - skates,  201
  rays

  Order Chimaeriformes -      202
  chimaeras

 CLASS Osteichthys (Pisces) - 203
 bony fishes

  Order Acipenseriformes -    204
  sturgeons
               Order  Polyodontidae  -
               paddle fishes
                              205
  48-8

-------
                                                    Classification - Finder
Order Semionoteformes - gars  206

Order Amiiformes - bowfins    207

Order Clupeiformes - soft     208
rayed fishes

 Family Clupeidae - herrings  209

 Family Salmonidae - trouts,  210
 salmon

 Family Esocidae - pikes,     211
 pickerels
               Family Serranidae - sea     228
               basses
Order Myctophiformes -
lizard fishes

Order Cypriniformes -
212
213
 Family Cyprinidae - minnows, 214
 carps

 Family Catostomidae - suckers215

 Family Ictaluridae - fresh-  216
 water catfishes

Order Anguilliformes ~ eel-   217
like fishes
Order Notacanthiformes -
spiny eels
218
Order Beloniformes - needle-  219
fishes, flying fishes

Order Cyprinodontiformes -    220
 killifishes, livebearers

Order Gadiformes - cods and   221
hakes

Order Gasterosteiformes -     222
stickelbacks

Order Lampridiformes - Opahs, 223
ribbon fishes

Order Beryciformes - beard-   224
fishes

Order Percopsiformes - trout  225
and pirate perches
Order Zeiformes - dory
226
Order Perciformes - spiny-    227
rayed fishes
               Family Centrarchidae -
               sunfishes, freshwater
               basses
                                  229
      Family Percidae - perch     230

      Family Sciaenidae - drum    231

      Family Cottidae - sculpins  232

      Family Magilidae - mullets  233

     Order Pleuronectiformes -    234
     flounders

     Order Echeneiformes - remoras235

     Order Gobiesociformes -      236
     clingfishes

     Order Tetraodontiformes  -    237
     spikefishes

     Order Batrachoidiformes -    238
     toadfishes
              Order Lophiiformes -
              goosefishes
                                  239
    CLASS Amphibia - frogs, toads,240
    salamanders

    CLASS Reptilia - turtles,     241
    snakes, lizards

    CLASS Aves - birds            242

    CLASS Mammalia - whales,      243
    seals, walrusses

IV  FUNGUS KINGDOM                250

 A  Bacteria                      251

    Eubacteria                    252

    Actinomycetes                 253

    Myxobacteria                  254

    Spirochaetes                  255

    Other bacterial types         256

 B  FUNGI                         260

    "Phycomycete" group           261
                                                                       48-9

-------
 Classification - Finder
    CLASS Chytridiomycetes

    CLASS Oomycetes

    CLASS Zygomycetes

    CLASS Ascomycetes

    CLASS Basidiomycetes

    CLASS Fungi Imperfecti
262
263

264
265
266

267
This outline was prepared by H. W.
Jackson,  former Chief Biologist,
National Training Center, and revised by
R. M. Sinclair, Aquatic Biologist, National
Training Center,  MOTD, OWPO, USEPA,
Cincinnati, Ohio  45268.

Descriptors: Aquatic Organisms,
Classification
48-10

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