PLANKTON  ANALYSIS
TRAINING MANUAL
FEDERAL WATER POLLUTION CONTROL ADMINISTRATION
U.S. DEPARTMENT OF THE INTERIOR

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        PLANKTON ANALYSIS
This course is offered for professional personnel in
the fields of water pollution control,  limnology, and
also water supply.  Primary emphasis is given to
practice in the identification and enumeration of or-
ganisms which may be observed in the microscopic
examination of water.  Problems of significance and
control are also considered.
    U.S. DEPARTMENT OF THE INTERIOR
  Federal Water Pollution Control Administration
          TRAINING PROGRAM
                    May 1970

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                          FOREWORD
These manuals are prepared for reference use of students enrolled
in scheduled training courses of the Federal Water Pollution Control
Administration.
     Due.  to the. limited pn.odu.at4.on and availability
     at the. ma.nua.tA,  it it  not appiopiiate. to  c-Lte.
     the.m at tic.hnic.al teje'ienceA in  bibliogiaphie.4
     •hi othe.fi fioimt  oj publication.
     Re.6e.ie.nc.e.t to  pn.odvic.ti>  and manu6ac.tuie.it  it
     illuttiation  onty; tuck ie.$e.ie.nc.e.t  do not imply
     product e.ndon.te.me.nt by  the. fe.de.iai  Wate.1  Pollution
     Control tidmini&tiation  on. the. U.S.  Ve.paitme.nt
     0(5  Ake. lnte.iioi.
The reference outlines in this manual have been selected and developed
with a goal of providing the student with a fund of the best available
current information pertinent to the subject matter of the course. Indi-
vidual instructors may provide  additional material to cover special
aspects of their own presentations.

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

Constructive suggestions for  improvement in the coverage, content, and
format of the manual are solicited and will be given full consideration.
                                  H. M. Freeman
                                  Chief, Direct Training Branch
                                  Division of Manpower and Training
                                  Federal Water Pollution Control
                                    A dministration

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                       TRAINING   PROGRAM
The Federal Water Pollution Control Administration of the U. S. Department
of the Interior conducts programs of research,  technical assistance, enforce-
ment, and technical training for water pollution control.

Training is  available at five installations of the Administration.  These are:
the National Training Center located at the Robert A. Taft Sanitary Engineering
Center in Cincinnati, Ohio; the Robert S. Kerr Water Research Center, Ada,
Oklahoma; the Southeast Water Laboratory, Athens.  Georgia; the Pacific
Northwest Water Laboratory, Corvallis,  Oregon, and the Hudson-Delaware
Basins Office, Edison,  New Jersey.

The objectives of the Training Program are to provide specialized training in
the field of water pollution control which will lead to rapid application of new
research findings through updating of skills of technical and professional
personnel, and to train new employees  recruited from other professional or
technical areas in the special skills required.  Increasing attention is being
given to development of special courses providing an overview of the nature.
causes, prevention, and control of water pollution.  These courses are being
designed for nontechnical audiences, including administrators at the policy and
decision-making levels,  representatives of public action groups, and others not
requiring the  depth of detail of the more specialized courses.

Scientists, engineers, and recognized authorities from other FWPCA programs,
from other government agencies, universities,  and industry supplement the
training staff by serving as guest lecturers.   Most training is conducted in the
form of short-term  courses of one or two weeks' duration. Subject matter
includes selected practical features of plant operation and design, and water
quality evaluation in field and laboratory.  Specialized aspects and recent
developments of sanitary engineering, chemistry, aquatic biology, microbiology,
and field and laboratory techniques not  generally available elsewhere, are
included.

The primary role and responsibility of  the States in the training of wastewater
treatment plant operators are recognized. Technical support of operator-
training programs of the States is available through technical consultations in
the planning and development of operator-training courses.  Guest appearances
of instructors from the Federal Water Pollution Control Administration, and the
loan of instructional materials such as  lesson plans  and  visual training aids,  may
be obtained  through special arrangement.  These training aids,  including
training manuals,  may be reproduced freely  by the states for their own  training
programs.  Special categories of training for personnel  engaged in treatment
plant operations may be developed and made  available to the States for  their own
further production and presentation.

An annual Bulletin of Courses is prepared and distributed by the Water Pollution
Control Training Program.  The Bulletin includes descriptions  of courses,
schedules, application blanks, and other appropriate information.  Organizations
and interested individuals not on the mailing  list should request a copy from one
of the training centers mentioned above.

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                      D£PAKTMENT  OF THE
                              MANPOWER AND TRAINING DIVISION
                                 DIRECT TRAINING BRANCH
                                   H. M. Freeman, Chief



                  TRAINING  ACTIVITIES OF THE ADMINISTRATION
SOUTHEAST WATER LABORATORY
Athens, Georgia

R. Roth, Sanitary Engineer,  Chief
W. R. Davis, Chemist
ROBERT S. KERR WATER RESEARCH CENTER
Ada, Oklahoma

(Mrs.)M.  E. Smith, Sanitary Engineer,  Chief
J. E. Matthews,  Aquatic Biologist
ROBERT A. TAFT SANITARY ENGINEERING CENTER
Cincinnati, Ohio

H. L. Jeter,  Microbiologist, Director
(Miss) A.  E.  Donahue, Chemist
C  R. Feldmann, Chemist
P. F. Hallbach. Chemist
H. W. Jackson, Chief Biologist
F. J.  Ludzack, Chemist
R. Russomanno, Microbiologist
R. M. Sinclair, Aquatic Biologist
C. E  Sponagle. Sanitary Engineer
PACIFIC NORTHWEST WATER LABORATORY
Corvallis, Oregon

L. J. Nielson, Sanitary Engineer,  Chief
D. S. May, Microbiologist
J. Wooley, Aquatic Biologist
HUDSON-DELAWARE BASINS OFFICE
Edison, New Jersey

F. P. Nixon, Deputy Regional Training Officer
R. B. Fagan, Aquatic Biologist
10.69

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            PLANKTON  ANALYSIS  (141)

                        2  weeks

  CINCINNATI, OHIO             May 11-22,  1970

                                  October 5-16,1970

         This course is offered for professional personnel
  concerned  with  the  evaluation  of  natural  and polluted
  waters   by  means  of  plankton  examination.  Limited
  attention is also  devoted to the examination  and  inter-
  pretation of  the  fauna of  activated sludge  and waste
  stabilization  ponds.

         Instruction  enables the student to carry out  basic
  laboratory procedures in the identification and counting of
  both phytoplankton and zooplankton. He  will be capable
  of applying  laxonomic proceduresto plankton and recognize
  the  major types he  is likely to encounter.  He  will be
  able to  calibrate a microscope and to carry counting and
  group  identification  to  the  point  of obtaining  results
  which   are  qualitatively   and   quantitatively  reliable.

        Attention is gixen  to  the  significance  of  various
  types of counts.  Forms  frequently   found  in  water and
  wastewater  treatment  plants  and polluted  environments
  are  emphasized.  Techniques  for plankton control are
  presented.  Time  is  provided for discussion  of  local
  problems, both  in class and with  specialists at  the train-
  ing facility.

  Representative course topics  usually  include

        Water quality problems of biological origin
        Identification of planktonic animals and plants
               (a series of  lectures and laboratories  com-
               prising  approximately   half of the course)
       Microscope calibration
        Plankton analysis
               Sampling and preparation
               Techniques of counting
               (enumeration, methods selection
       Plant operation problems
               Plankton in stabilization ponds
               Activated sludge fauna
               Toxic algae
               Other biological treatment problems
        Plankton control
               Plant control
              Control  in surface waters

       Although microscopes  arc available for class  use,
more  effective training results when  it is given on  the
same instrument that will be used  in the home laboratory.
The microscope should have magnifications up  to approx-
imately  400X,  oil  immersion  is  optional. The student
consequently is urged to hand-carry his own microscope
to the course.

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                                    CONTENTS



 Title or Description                                                Outline Number

 CHAPTER I    INTRODUCTION

    Water Resources and Needs                                             1

    Limnology and Ecology of Plankton                                      2

    Optics and the Microscope                                              3

    The Aquatic Environment                                               4


 CHAPTER H   IDENTIFICATION OF PLANKTON AND
                ASSOCIATED ORGANISMS

    Structure and Function of Cells                                          1

    Aquatic Organisms of  Significance in Plankton Surveys                    2

    Types of Algae                                                         3

    Blue-Green Algae                                                      4

    Green and Other Pigmented Flagellates                                  5

    Filamentous Green Algae                                               6

    Coccoid Green Algae                                                   7

    Diatoms                                                               8

    Filamentous Bacteria                                                   9

    Protozoa,  Nematodes,  and Rotifers                                     10

    Free-Living Amoebae  and Nematodes                                   11

    Animal Plankton                                                      12

    Laboratory Exercises

    General Laboratory Instructions                                        15

    Types of Algae                                                        16

    Identification of Diatoms                                               17

    Identification of Animal Plankton                                        18




141.5.70

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                                      Contents
Title or Description                                                  Outline Number




CHAPTER HI    TECHNIQUES OF PLANKTON METHODOLOGY




   Techniques of Plankton Sampling Programs                               1




   Preparation and Enumeration of Plankton in the Laboratory                 2




   Calibration and Use of Plankton Counting Equipment                        3




   Preparation of Permanent Diatom Mounts                                 4




   Determination of Odors                                                  5




   Collection and Interpretation of Biological Lake Data                       6




   Determination of Plankton Productivity                                   8




   Methods of  Measuring Standing Crops of Plankton                          9




   Aerial Reconnaissance in Pollution  Surveillance                          10




   Laboratory Exercises




   Proportional Counting of Plankton                                      11




   Calibration of Plankton Counting Equipment                             12




   Fundamentals of Quantitative Counting                                  13




   Class Problem in Plankton Analysis                                    14






CHAPTER IV    INTERPRETATION AND SIGNIFICANCE OF PLANKTON




   Algae and Actinomycetes in Water Supplies                                1




   Algae as Indicators of Pollution                                          2




   Public Health Significance of Toxic Algae                                  3




   Odor Production by Algae and Other Organisms                            4




   Organic Enrichment and Dissolved Oxygen Relationships in Water           5




   Plankton in  Oligotrophic Lakes                                            6




   The Effects of Pollution on Lakes                                         7

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                                     Contents
Title or Description                                                  Outline Number

CHAPTER V   PLANKTON CONTROL

   Control of Plankton in Surface Waters                                      3

   Control of Interference Organisms in Water Supplies                        4

   Nutrients: The Basis of Productivity                                      5


CHAPTER VI    RELATED STUDIES

   The Problem of Synthetic Organic Wastes                                  2

   Beneficial Aspects of Algae                                 •              3

   Behavior of Radionuclides in Food Chains - Freshwater Studies              4

   FWPCA Responsibilities for Water Quality Standards                        5

   Marine and Estuarine Plankton                                            6

   Attached Growths (Periphyton or Aufwuchs)                                7

   Artificial and Related Substances ~ References                             8


CHAPTER VII    IDENTIFICATION KEYS

   Key to Selected Groups of Freshwater Animals                             1

   Key to Algae of Importance in Water Pollution                              2


APPENDIX

   Foreword

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

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                                                    Intervale
                                                        Reservoir
CHAPTER I

INTRODUCTION

Water Resources and Needs
Limnology and Ecology of Plankton
Optics and the Microscope
The Aquatic  Environment
1
2
3
4

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                             WATER RESOURCES AND NEEDS
I  WATER RESOURCES

A  The source of all freshwater is the
   hydrologic cycle, shown in Figure 1.
           CIRCULATION


              EVAPORATION
   \      \   -=	-^     I WATER TABLE

    \      IMPOUNDMENT

   GROUNDWATER          GROUNDWATER
            THE HYDROLOGIC CYCLE
                Figure 1

   1 Precipitation of water as rain, snow,
     hail,  sleet or dew.

   2 Percolation of water through soil to an
     aquifer to form  groundwater.

   3 Runoff of water  which forms lakes,
     streams and rivers.

   4 Evaporation of surface water or trans-
     piration of water from green plants to
     the atmosphere.

   5 Atmospheric recirculation of the water
     vapor.

B  The world's supply of water is contained
   within the hydrologic cycle as:

   1 Oceanic water

   2 Water vapor in the atmosphere

   3 Ice and snow in  glaciers and snowpack

   4 Runoff water in  lakes and streams

   5 Groundwater
 C Withdrawals for use are mostly from those
   waters in the runoff and groundwater
   phases, although some oceanic waters are
   being utilized.

 D Precipitation--which serves to recharge
   groundwaters and surface supplies--is at
   a relatively fixed annual rate.

   1  Average precipitation in the U.S. is
      30 inches  per year or 3, 900 billion
      gallons per day.

   2  Evapo-transpiration losses total
      approximately 21 inches per year or
      approximately 2, 740 billion gallons per
      day.

   3  The  available water totals approximately
      9 inches per year or 1, 160 billion
      gallons per day.
II   THE DISTRIBUTION OF U.S. WATER
    RESOURCES

 Although the water supply in the hydrologic
 cycle is fixed in amount,  it is not distributed
 evenly.  A wide disparity of water distribution
 exists both in time and space.   Distribution
 of the annual average precipitation is shown
 in Figure 2.

 A  Distribution of Precipitation

    1  Dependent upon:

      a Atmospheric conditions such as
        temperature and  winds

      b The geography of the region

      c The general climate of the area

    2  U. S. areas of high annual precipitation

      a The Pacific slope varies from  10 inches
        to greater than 100 inches annually.
 W.RE.28d.4.70
                                 I 1-1

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Water Resources and Needs
                                  Distribution of Precipitation
                                        (Average Annual)
                                             Inciies

                                          Figure 2
     b  The gulf states precipitation varies
        from 20 to 60 inches annually.

     c  Precipitation in the midwest and
        Great Lakes area ranges from 25 to
        50 inches  per year.

     d  Precipitation along the Atlantic Coast
        averages between 35 to  50 inches
        per year.

   3 Areas of low annual precipitation

     a  The Rocky Mountain area precipitation
        ranges between 10 and 20 inches per
        year.

     b  Much of the southwest has less than
        10 inches  of precipitation annually.

   4 Distribution  of precipitation with time

     a  The rainy or wet season varies from
        summer to winter, or in some areas
        there is relatively little change
        throughout the year.
     b  Local storms of high intensity may
        reach as much as 30 inches in 24
        hours.

B  Distribution of Runoff

   1  Dependent upon:

     a  Precipitation in the region

     b  Infiltration - which is controlled by
        the geologic formations and the time
        lapse between rains.

     c  Season of the year controls evaporation,
        and snow melt.

     d  Topography controls the time available
        to percolate through the soil.

     e  Vegetation type and density affects
        interception and evapotranspiration.

   2  Areas of high annual runoff

     a  Sections of the Pacific  slope have
        greater than 80 inches  annually.

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                                                              Water Resources and Needs
     b  The eastern 1/3 of the U.S.  averages
        greater than 20 inches of runoff
        annually.

   3 Much of the western U.S.has less than
     1 inch of runoff annually.

     a  Southwest

     b  Rocky Mountain states

     c  Rocky Mountain plateau

   4 Time distribution of runoff

     a  Overflow--runoff during and immediately
        following precipitation.

     b  Base flow--sustained or fair weather
        runoff composed of delayed sub-
        surface and groundwater runoff.
        See Figure 3 for runoff cycle.

C  Distribution of Groundwater

   1 Groundwater volume is affected by the
     same factors as runoff.

   2 Geologic formations and soils control
     percolation and storage of groundwater.
    3 Topography controls time available
      for percolation.

    4 Evapo-transpiration varies with the
      season, as does precipitation and
      ground saturation.
Ill  WATER USE

 A  Present Water Use in the U. S.

    1 Water available for use

      a  Nine  inches or 1, 160 billion gallons
         per day are not lost through evapo-
         transpiration,  and is therefore
         theoretically available.

      b  Water use in the U.S.  at the present
         time  is approximately  390 billion
         gallons per day or 3 inches of our
         total  supply.

      c  Twenty-one inches are lost through
         evapo-transpiration.
                              storage i  Surface detention = sheet of water
            lnfiltratipn!J?r"*8^S&»4^La»J£— Overland flow

                                                             Surface runoff
                 Perchea""wate'r  table   ^1 mpervfou's
                                     ^_ SS^K. —
                        Water table  -===

                          Ground-water flow ^—"^Stream  channel
                                THE RUNOFF  CYCLE
                                 (Davis  &  DeWiest)
                                       Figure 3

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Water Resources and Needs
   2  The way in which water is used

      Water uses can be grouped into two
      classes.  Those uses which are in situ
      such as recreation,  fishing,  and wildlife
      and those uses requiring withdrawal
      from the stream. These withdrawals are:

      a Agricultural uses take 46% of our
        supply or  180 billion gallons/day;
        only 40% of this water is returned  to
        the streams.

      b Industrial uses take another 46% of our
        supply.  2%  of the water used by
        industry is consumed.

      c Municipal uses total approximately
        25 billion  gallons  daily or 8% of the total.

   3  Source of water used in U. S.

      a National averages show 80% or 312
        billion gallons per day to be from
        surface  sources, while 20% is taken
        from the ground.

      b The ratio of surface water to ground-
        water varies and is dependent on the
        quantity and quality available in each
        locality, as well as the cost.

   4  Seasonal uses of water

      a Irrigation  waters are used during the
        growing season only.

      b Some water using industries such as
        the canning industry are seasonal.

      c The majority of industries needs
        water throughout the year.

      d Municipal  use is higher  in the summer.

B  Demand for water is increasing

   1  The predicted demand of water in  1980
      is approximately 600 billion gallons of
      water per day,  or 220, 000 billion per
      year.
     2  This is mainly due to expansion of
       industry and irrigated agriculture.

     3  Much of the demand for  water will be
       in areas such as the southwest, that
       are already short on water.

  C  Methods for the Development  of U. S.
     Water Resources for Future Needs

     1  Utilization of our present sources of
       water,  surface and groundwater,  must
       be increased.  This would mean
       increased storage, both on  the surface
       and in underground reservoirs.

     2  Desalinization of ocean waters and
       brackish waters  holds some promise
       for regions where transportation will
       not be expensive.

     3  Reduction of evapo-transpiration losses
       will greatly increase our total available
       supply.

     4  Weather modification methods could
       possibly give us  precipitation in the
       right place  at the right time.

     5  Greater reuse of our present supply is
       both through multiple use and better
       waste treatment  methods.
IV  SUMMARY

 The total amount of water available appears
 to be fixed.  In view of the increasing
 demands and the currently inefficient
 utilization of the supply, the demand may
 very shortly exceed the supply.  Better
 management of the resource and more
 engineering research are urgently needed.
 I 1-4

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                                                             Water resources and Needs
                       Table 1.  AVAILABILITY OF GROUND WATER
Areas
A
B
C
D
£
F-l
F-2
G
Atlantic and Gulf Coastal Plain area
Southern Great Plains area
Appalachian Mountain and Piedmont area
Rocky Mountains, northern Great Plains,
and northern Pacific Coast area
Unglaciated central plateaus and lowlands
Basin and range
Columbia Plateau
Glaciated area of the East and Midwest
U. S. Total (rounded)
Water Use
(excluding water power)
Use in mgd and Percent
of total from Ground
Water Sources
Total
mgd
32,000
21.000
8,000
28, 000
26,000
41, 000
24, 000
57, 000
240, 000
Ground
water (%)
25
45
50
12
10
42
7
10
20
A CKNOWLEDGEMENT:

Certain portions of this outline contains
training material from prior outlines by
Peter F. Atkins, F.P. Nixon.
REFERENCES

1  Ackerman,  Edward A., Lof, George O.G.,
      Technology in American Water
      Development.  The Johns Hopkins
      Press.   Baltimore.  1959.

2  Senate Select Committee on  National Water
      Resources:  Water Resources Activities
      in the United States:  Committee Print
      No.  3.   U.S. Gov. Printing Office.
      January 1960.

3  Senate Select Committee on  National Water
      Resources:  Water Resources Activities
      in the United States: Committee Print
      No. 24.  U.S. Gov. Printing Office.
      January 1960.
4  Linsley, RayK..  Kohler. Max A.,
      Paulttus, Joseph H.  Hydrology for
      Engineers.   McGraw-Hill Book Co.,
      Inc., New York.  1958.

5  Chow, Ven Te.   Handbook of Applied
      Hydrology.   McGraw-Hill Book Co.,
      Inc., New York.  1964.

6  Davis,  Stanley N. and DeWiest, Roger,
      J.M.   Hydrogeology.  John Wiley
      and Sons, Inc.,  New York.  1966.

7  American  Chemical Society.   Cleaning
      Our Environment the Chemical Basis
      for Action.   ACS.  Washington, DC
      20036.  249pp.  (2.75)  1969.

This outline was prepared by Edward D.
Schroeder, Former Engineer, FWPCA
Training Activities, SEC and revised by
L. J. Nielson, Chief Technical Training,
Pacific Northwest Water Laboratory,
Corvallis, Oregon.
                                                                              I  1-5

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                        LIMNOLOGY AND ECOLOGY OF PLANKTON
I     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.


 II    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 m 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.
             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.

         Density stratification affects
         aquatic life and water uses.

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

         b   Ice cover and annual spring
             and fall overturns are due to
             successive seasonal changes
             in the relative densities of
             the epilimmon and the hypo-
 BI. MIC.eco.4b.4.70
                                                                                      I  2-1

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

     c   The sudden exchange of
         water masses having differ-
         ent chemical characteris-
         tics may have catastrophic
         effects on certain biota.

     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.

     b   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).

Shore development,  depth, inflow  -
outflow pattern,  and topographic
features  affect the behavior of the
water.

Water movements that may affect
organisms include waves,  currents,
tides, seiches, and floods.
                                                          1    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.

                                                         2    Currents

                                                              a   Currents are arhythmic
                                                                  water  movements which have
                                                                  had major study  only in ocean-
                                                                  ography.   They  primarily are
                                                                  concerned with the translo-
                                                                  cation of water masses.  They
                                                                  may be generated internally
                                                                  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
                                                                  tnan mere laminar flow.

                                                     F   Surface Tension and the Surface  Film

                                                         1   The surface film is the habitat
                                                             of the "neuston", a group of
                                                             particular importance in water
                                                             supplies.

                                                         2   The biological effects of small
                                                             amounts of detergents and simi-
                                                             lar agents are yet to be evaluated.
                                                Ill  DISSOLVED SUBSTANCES

                                                    A   Carbon dioxide is released by plants
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                                                    Limnology and Ecology of Plankton
    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 fun-
    damental nutrients for plant life.

    1    Occur in great dilution, con-
         centrated by plants.

    2    The distribution of nitrogen com-
         pounds is generally correlated
         with the oxygen curve.

D   Iron, manganese, sulphur, and
    silicon are other minerals impor-
    tant to aquatic life which exhibit
    biological stratification.

E   Many other minerals are present
    but their biological distribution in
    waters is less well known.

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

A   Nutritional Classification of Or-
     ganisms

     1    Holophytic or independent or-
         ganisms,  like green plants, pro-
         duce their own basic food ele-
         ments from the physical  environ-
         ment.

     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 Hormomc Relationships

        1   Some organisms such as certain
            blue green algae and some ar-
            mored flagellates 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

    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.

        1    Those that pass through a plankton
             net (No. 25 silk bolting cloth or
             equivalent) or sand filter are
             known as nannoplankton (which
             usually greatly exceed the net
             plankton in 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, 2  fall,  3 summer,
                 4 winter

    B  Benthic organisms (benthos) are those
        living on or near the bottom,  frequently
        attached.  Typically  benthic organisms
        such as certain filamentous algae,  may
        on occasion be broken or washed free,
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Limnology and Ecology of Plankton
          and mingled with the typical plankton.

      C   The emergent vascular shore plants
          are often a very influential commun-
          ity; on death and decay,  they add
          nutrients to the water.

      D   The shallow water or littoral com-
          munity is one of the most varied and
          productive  areas - a fluctuating water
          level will discourage this community.

      E   The deep water and sludge or ooze
          communities may contribute tastes,
          odors, and undesirable chemical
          characteristics.
VI    THE EVOLUTION OF WATERS

      A   The history of a body of water de -
          termines 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 or 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 the
              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 in-
              crease 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.)

 Geological factors which significantly
 affect the nature of 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 pre-
     cipitation.

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

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.
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                                                  Limnology and Ecology of Plankton
2   Maturing 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 irreg-
        ulars 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 pre-
        cipitated in the deeper
        portions of the lake  in part
        through the action of plants.

    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)
VI  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 productivity is a "poor"
        water biologically, and also a rela-
        tively "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 of the factors
        which influence the productivity of
        waters  are as follows:

    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,
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Limnology and Ecology of Plankton
              determines the overall produc-
              tivity.

      C   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 stim-
              ulate 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 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 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
             deliberately increase or 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
             drawdo'vn is the rule.

             The level at which water is re-
             moved from a reservoir is also
             important.   The hypolimnion may
             may be anaerobic while the epi-
             limnion is  aerobic.

             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,  or provide inade-
             quate dilution for toxic waste.
VIII CLASSIFICATION OF LAKES AND RESER-
    VOIRS

    A   The productivity of lakes and impound-
        ments is such a conspicuous feature
        that it is often used as a convenient
        menas 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 - 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 into
I 2-6

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                                FACTORS AFFECTING  PRODUCTIVITY
                                          Geographic Location
           Human
           Influence
           Sewage
         Agriculture
           Mining
         Primary
         Nutritive
         Materials
                                                       Latitude
                                                             Longitude
                                                                     Altitude
Geological
Formation
                     Topography
          Composition
          of Substrate
                        Shape of Basin
                                                                       Wind

                                                           Precipitation // I Insolation
                          Area     Bottom
                                Conformation
Drainage
 Area
        Nature at
         Bottom
         Depo
               S
  Inflow of
Allochthonous
 "Materials
    . Trans-
    parency
               -Light
              Penetration
 leat Penetration
and Stratification-
 i  Penetra  Develop
^- and       Littoral
Utilization    Region
  Seasonal Cycle
Circulat. Stagnation
  Growing Season
                                       Trophic Nature of a Lake
to
I
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Limnology and Ecology of Plankton
IX
          two types, storage and run of the
          river.

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

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

          According to location,  lakes and
          reservoirs may be classified as
          polar,  temperate,  or tropical.
          Differences  in climatic and geo-
          graphic conditions result in dif-
          ferences in their biology.
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
   C  The artificial introduction of nutrients (sewage pollution or


      C   The artificial introduction of
          nutrients (sewage pollution or fer-
          tilizer) thus tneds to eliminate ex-
          isting limiting mmimums for some
          species  and create intolerable maxi-
          mums for other species.

          1   Known limiting mmimums may
              sometimes be deliberately
              maintained.

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

          3   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).

    4   Eutrophic ation leads to treatment
        troubles.

D   Control of eutrophication may be
    accomplished 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.
                                                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 mea-
                                                    sures to achieve a desired result.
                                            REFERENCES

                                            1    Chamberlin,  Thomas C.,  and Salisburg,
                                                  Rollin P.,  Geology Vol.  1,  "Geological
                                                  Processes and Their Results", pp i-xix,
                                                  and 1-654, Henry Holt and Company,
                                                  New York, 1904.
I 2-8

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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 1). 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. 18.6.68
   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.

                                  I 3-1

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 Optics and the Microscope
    Object
                                   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 m-ist 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:

           -   +   -      1
           P       q       f

      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
I  3-2

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                                                                    Optic3 and the Microscope
D  Refraction of Light

   Turning now to lenses r.ithrr than  nmtors
   we find that the mo.sl impoi Unit i h.ir.n ler-
   istic is refraction.  Ki-Ti-.n linn ivli-is to
   the change* ol direi lion .incl/oi  veloi ily ol
   light as it passes from one  in •diuin ID
   another.   The ratio of tin- \,ehn it\  in .in
   (or more correi tlv in i\ v.u  mini) lo tin-
   velocity in the medium is i ailed the
   refractive  index.  Som;' t.>pieal v.ilues ol
   refractive  index measureil with mono-
   chromatic  light (sodium D line) .ire listed
   in Table  1.

   Refraction causes an olijet t imnvrsed in
   a medium of higher refractive  index than
   air to appear closer to the surfai e than it
   actually is (Figure 5). This cffoc t may
   into I'oi us and the new mu.rometer reading
   is taken.  Km.illy, the microscope is re-
   rot used until the surface of the liquid appears
   in sha i p foi us   The micrometer reading
   is Liken again and, with this information,
   the refrac live index m?y be calculated from
   the simplified equalion
                      .      actual depth
                   index = - "- -
                           apparent depth
T.ihle  I   KKKRACT1VK INDICES OK COMMON
MATKRIA1.S MKASURKD  WITH SODIUM  LIGHT
Vac uum
Air
CO.,
Water
1.0000000
1 0002') 18
1. 0004498
1. VISO
Crown glass
Rock salt
Diamond
Lead sulfide
1.48 to
1.5443
2.417
3. 912
1. 61



                                     Air
Actuol i
depth "
Apparent 1
depth \
\
Medium
	 	 image
i
.' Dhiarl
   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
                "9     L.
                —* , where
                nl
                                                                               
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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 arc 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   65631
Carbon disulfide
Crown glass
Flint glass
Water
1.
1.
1.
1.
6523
5240
6391
3372
1.6276
1.5172
1. 6270
1. 3330
1.
1.
1.
1.
6182
5145
6221
3312
    The dispersion of a material can be defined
    quantitatively as:
                     n (yellow)  - 1
    v = dispersion
                     n (blue) - n (red)
        n (593mji) - 1
        n (486mn) -
    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.
I 3-4

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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 arc- not located on the optical
axis of the lens and results in the formation
of an mclistiml image.  The simplest
remedy for astigmatism is to place the
ohjei I (lose to the axif, of the lens system.

Interfere™ e Phenomena

Intcrfcrem c and diffraction are two phe-
nomena whieh arc  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
different cs 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.
                                                                                    I  3-5

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    jtics and the Microscope
                                    otb
   Figure 9b.  Rays 1 and 2 are now 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  twipe 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 + —


     where \ 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).
I 3-6
                 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
               Cover  slip- •-
                Specimen
 x-100%
/ Mirror
               Figure 11
MICROSCOPICAL METHOD OF VIEWING
          INTERFERENCE IMAGES
 a  Examination is 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:

        ns= nm(l
                                        d =
                                                             2.44 fX
                                     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
                                                           1.22 f X
                                        resolving power =  ——	.
     where ns
            X
            t
J Diffraction
refractive index of the
specimen
refractive index of the
surrounding medium
phase shift of the two
beams, degrees
wavelength of the light
thickness of  the specimen.
   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 its 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:
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
                                                                                       I  3-7

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Optics and the  Microscope
                                                                           Eye
     I mog»
                      Mognifitr
                Figure 12

     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:

         M= f  +1

      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
   2 OX.

   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
         Victual
                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.
I  3-8

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Table 3. NOMINAL CHARACTERISTICS OK 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. K
4.4
3.9
1.4
0.7
0.4
0.35
0.21
Maximum
useful
magnif.
SOX
90X
250X
500X
660X
850X
1250X
Eyepiece
for max.
useful magnif.
30X
20X
25X
25X
15X
20X
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 ins ides 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 projpction 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.

                                   I  3-9

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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  30X oculars
   have slightly poorer imagery than medium
   powers and have a very low eyepomt.  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
30X
50X
100X
200X
400X
900X
Eyepiece
15X
45X
75X
150X
300X
600X
1350X

20X
60X
100X
200X
40 OX
800X
1800X

25X
75X
125X
250X
500X
1000X
2250X
MUMa
(1000 NA)
SOX
100X
250X
50 OX
660X
1250X
            aMUM = maximum useful magnification
 I 3-10

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                                                                    Optic3 and the Microscope
      objertivr and shclo 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 centerable 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-west 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
                                                                                      I  3-11

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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.

      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. micromampulation.
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  Kohler.  Also useful for intense illumi-
     nation, Kohler 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
 I 3-12

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                                                                Optics and the Microscope
                        Table 5.  COMPARISON OF CRITICAL.
                      KOHLER AND JfCOR 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
m 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 subs:age condenser is very
slightly defocused.

Critical Illumination

With 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 lamj, 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.

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.
  n
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
                                                                                     I  3-13

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Optics and the Microscope
                Critical
Kohler
  Focal .plane
Poor man's
  Objective
  Preparation

  Substaqe
    condenser

  Substage  —j
      iris
  Lamp iris —

  Lamp
    condenser

  Light  source
I 3-14

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                                                              Opties and the Microscope
   substagc condenser opening.  II il
   docs not,  move the lamp aw.ix I nun
   the wall to enlarge the I'll.inicnl inuj;r,
   refocus.

d  Turn the lamp JIN! .inn  il .it Ihe mu 111-
   scope mirroi t»o a* to m.iml.i in the
   same 18 inches (01 atljusled l.imp
   distance).

e  Place a specimen  on  the mil i cisi ope
   stage and foeiib .sluirplx. with a Id-mm
   (10X) objective. Open fully the
   aperture diaphragm  in the substage
   condenser.  If the  light is  too 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 bulb base  and
   slide it up or down.  If the base is
   fixed, tilt the lamp housing in a
   vertical arc with the field diaphragm
      .is tin- i enter of movement (again
      enile.ivormg to Keep the lamp dia-
      plitM^ni in tht  t entered position).
      II you have- mistered this step,  you
      ll.ivr ac (umplished the most d iff H Ult
      |)()ilioii   (Heller mil lost Ope lamps
      h.ivc .id iiislmcnls lo move the bulb
      independently ul the la mo housing to
      simplify this step.)

   I   ljul the  specimen in plate, replace
      the eyepiei e and the desired ob|ec-
      tjvc ,md  relot us.

   k  Open or i lose 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.
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Optics and the Microscope
         It should be possible to direi t it in
         the general direction of the substage
         mirror, very close thereto 01 in
         place thereof.

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

      c  Remove the top lens ol the londenser
         and, by racking the i ondenser up or,
         more often, down, bring into foe us
         (in the  same plane as  the spei iirn>n)
         a finger, pencil or other objei t plat ed
         in the same general region at>  the
         ground  glass diffuser  on the-  lamp.
         The glass  surface  itself ian then be
         focused in the plane of the spei invn.

      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.
I  3-16
H  Kesolvmg Power

   The resolving power of the microscope is
   its .iluhly U> distinguish separate details
   of i  losely spai ed microscopic structures.
   The Iheoreln al limit of resolving two
   (list rele points,  a distant e X apart, is


         x   TS.T.

     where \  - wavelength  of light used  to
                 illuminate the  specimen
          N.A. = numerical aperture of the
                 objective

   Substituting a wavelength of 4, 500
   Angstroms and a numerical aperture  of
   1. i, about the best that can be done with
   visible light,  we find that  two points about
   2, OOOA (or 0. 2 micron) apart can be seen
   as two separate points.  Further increase
   in resolving  power can be  achieved  for the
   light mic roscope by using light or shorter
   wavelength.   Ultraviolet light near  2,  000
   Angstroms lowers the limit to about 0. 1
   micron, the  lower limit for the light
   microscope.

   The  numerical aperture of an objective  is
   usually engraved on the  objective and  is
   related to  the angular aperture,  AA
   (Figure 15),  by the formula:
        N.A.  = n sin
AA
 2
     where n = the lowest index in the space
               between the object and the
               objective.
                          Angular aperture
     Object
                                                                  Figure 15

                                                          ANGULAR APERTURE OF
                                                          MICROSCOPE OBJECTIVE

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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.
                              /
             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 apochromatic  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.
                                                                                   I 3-17

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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.

   Photomic-rographic 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 1/10-second exposure
                and a magnification of 100X.
                If the magnification is now
  I  3-18

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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    .   ,200.2
     lold magnification '     '   M00;

   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.

2  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

   /\  i^™"^^™^»^^^r"~ *
      new N.A.
   say, 1/50 second.
= 1/40 or,
   It is seen that more light reaches the
   photographic film with higher numeri-
   cal apertures at the same magnification.

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

   Example:   A good picture was obtained
              with Eastman Tn-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
                                                                                  I  3-19

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 Optics and the Microscope
    on the number and size of the crystals in
    the field or, alternatively,  on the area of
    the field covered by birefringcnt 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 reading
   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 = 40X 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 10-5mch).
    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
I 3-20

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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. X E. 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
                                                                                   I 3-21

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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 lOOti (0. 1 mm) apart; one
      or two of these are usually subdivided
      into 10n (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 with 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 micrpns p^er
      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 s.tage  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. 8^!
                   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. 8n,  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 ^emd is by means
       of a table.
 I 3-22

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                                                            Optics and the Microscope

Objective
32-mm
16- mm
4-mm

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

emd
= 44
= 38
= 30


1 emd
40.
15.
3.
9 LI
OLJL
33^
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).
                    h-P H
            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)t page 26.

   The averages^ with respect  to number,
   dj, surface,  d3, and weight or volume,
   £4,  are calculated as follows for the
   data in Table 9.
                                                                                I  3-23

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Optics and the Microscope
                Table 8.  PARTICLE SIZE TALLY FOR A SAMPLE OF STARCH GRAINS
         ins
            (emd*)
              Number of particles
                                                  Total
                             rwj  rt-w  n-*4  rt-*j  rt-u  r-su
                      rt-4j  rtsj  MHJ  rr-w  r*-u  rt-*j
                      «-4j  M-W  I-MJ  rt-ij  r*-*j  111
rt-*j
                                                       rt-ia
                                                tt-u  r-*-*j   r»-*j
                                          t-t-w
                      rt-u  rt-w  rt-*-i  rtHj  11-4.1  r-*oa
                      rt-*j
                      1 1
                                         rt-u
                                                   16

                                                   98




                                                  110
                                                  107
                                                                         71
                            ri-*j
                      1 1
                                                   45



                                                   II

                                                    3

                                                  470
             •emd • exeniece mlcrometer-dixiaiona
           d1 = Znd/Zn = 1758/470

              = 3. 74 emd X 2. 82* = 10. 5(i

           d3 = Znd3/ 2nd2 = 37440/7662

              = 4. 89 emdx 2.82 = 13. Sp.

           d4 = Snd4/Znd3 =  199194/37440

              = 5.32 emdX 2. 82 = 15.0^

           *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 = 15
                                                     nd4 X  100
                                   d = 1
                                                   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,  O,  is known, the  surface
                                average d3, is used.

                                IfD=l.l,  Sm =  6/daD =  6/13. 8(1. 1)

                                               =  0. 395m2/g.
I 3-24

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                                                                   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
428
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
ln~a~sarm)le 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
                   p.        1-ji slice, ji3
    nylon

 fiberglass
18. 5
13.2
268

117
 The percent by volume is,  then:
                      262X268
                               _
                (262X268K(168X117)

              = 78. 1% by volume.

 Still we must take into account the density of
 each in order to calculate the  weight percent.
                                                                                       I  3-25

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Optics and the Microscope
   If the densities are 1. 6 for nylon and 2. 2
   for glass then the percent by weight is
               	262 X 2C8 X 1. C	
      % nylon = (262 x 2fig x i.G)+(if,8X 117 X 2.2)

              = 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,  d4, 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 nd4/6X Dq
                X 100
                 238 X ird4/6X Dq + 467 X TT d|/6 X 0. 80 X Dg
                                                           X 100
   where Dq and Dg are the densities of quartz
   and gypsum_respe£tively, 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 its 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, 59.  (1960).

                 3 Chamot, Emile Monnin,  and Mason,
                      Clyde Walter.  Handbook of Chemical
                      Microscopy,  Vol.  1, third  ed.  John
                      Wiley and Sons, New York  (1959).
I 3-26

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                               THE AQUATIC ENVIRONMENT

                          Part 1: The Nature and Behavior of Water
I  INTRODUCTION

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

A Upon the hydrosphere are based a number
   of sciences which represent different
   approaches.  Hydrology is the general
   science of water itself with its various
   special fields such as 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.

    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.
                                           TABLE 1
                                UNIQUE PROPERTIES OF WATER
                  Property
          Significance
         Highest heat capacity (specific heat) of any
         solid or liquid (except NH.)
Stabilizes temperatures of organisms and
geographical regions
         Highest latent heat of fusion (except
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.21b.2.70
                                     I 4-1

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The Aquatic Environment
B  Physical Factors of Significance

   1 Water substance

     Water is not simply "HgO" but in
     reality is a mixture of some 33
     different substances involving three
     isotopes each of hydrogen and oxygen
     (ordinary hydrogen H  , deuterium H ,
     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)
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)
                            SUBSTANCE  OF WATER
                                        Figure 1
I 4-2

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                                                                The Aquatic Environment
                TABLE 2

EFFECTS OF TEMPERATURE ON DENSITY
        OF PURE WATER AND ICE*
Temperature (°C)
Density
                      Water
        Ice **
-10
- 8
- 6
- 4
- 2
0
2
4
6
8
10
.99815
.99869
.99912
.99945
.99970
.99987 	
.99997
1.00000
.99997
.99988
0.00973
.9397
.9360
.9020
.9277
.9229
.9168





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

** Regular ice is known as  "ice I".  Four or
   more other "forms" of ice are known to
   exist (ice II, ice m, etc.), having densities
   at 1 atm. pressure ranging from 1.1595
   to 1. 67.  These are of extremely restricted
   occurrence and may be ignored in most
   routine operations.

          This ensures that ice usually
          forms on top of a body of water
          and tends to insulate the remain-
          ing water mass from further loss
          of heat.  Did ice sink,  there
          could be little or no carryover of
          aquatic life from season to season
          in the higher latitudes.  Frazil or
          needle ice forms colloidally at a
          few thousandths of a degree
          below DO C.  It is adhesive and
          may build up on submerged objects
          as "anchor ice", but it is still
          typical ice.
 1)   Seasonal increase in solar
     radiation annually warms
     surface waters in summer
     while other factors result in
     winter cooling.  The density
     differences resulting estab-
     lish two classic layers: the
     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 (and probably other
     qualities too) 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
    Oo c upward.

5)  It should also be emphasized
    that when epilimnion and
    hypolimnion achieve the same
    temperature, stratification no
    longer exists, and the entire
    body of water behaves
    hydrologically as a unit,  and
    tends to assume uniform
    chemical and physical
    characteristics.  Such periods
    are called overturns and
                                                                                 I 4-3

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The Aquatic Environment
              usually result in considerable
              water quality changes of
              physical, chemical, and
              biological significance.

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

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

          8)   The relative densities of the
              various isotopes of water also
              influence its molecular com-
              position. For example, the
              lighter Olg tends to go off
              first in the  process of
              evaporation, leading to the
              relative enrichment of air by
              Oig and the enrichment of
              water by O17 and Olg. This
              can lead to  a measurably
              higher Ojo  content in warmer
              climates. Also, the temper-
              ature of water in past geologic
              ages can be closely estimated
              from the ratio of O]^ in the
              carbonate of mollusc shells.

       b  Dissolved and/or suspended solids
          may also affect the  density of
          natural waters.

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

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

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

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

                         4)  Seasonal stratification involves
                             the annual establishment of
                             the epilimnion, hypolimnion,
                             and thermocline as described
                             above.  The spring and fall
                             overturns of such waters
                             materially affect biological
                             productivity.
   5)  Density stratification is not
       limited to two-layered systems;
       three,  four, or even more
       layers may be encountered in
       larger bodies of water.

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
tropical waters.
I 4-4

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                                                             The Aquatic Environment
                   TABLE 4

VISCOSITY OF WATER (In millipoises at 1 atm)
Temp, o C
-10
- 5
0
5
10
30
100
Dissolved solids in g/L
0
26.0
O1 A
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
	
        Surface tension has been reported
        from 34 to 160 atm.   This has
        biological as well as physical sig-
        nificance.  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.

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

        a  For a given body of water,
            immediate sources of energy
            include in addition to solar
            irradiation: terrestrial heat.
            transformation of kinetic energy
            (wave and current action) to heat,
            chemical and  biochemical
            reactions, convection from  the
            atmosphere, and condensation of
            water vapor.
   b  The proportion of light reflected
      depends on the angle of incidence,
      the temperature,  color, and other
      qualities of the water.  In general,
      as the depth increases arithmet-
      ically, the light tends to decrease
      geometrically. Blues, greens,
      and yellows tend to penetrate most
      deeply while ultra violet, violets,
      and orange-reds are most quickly
      absorbed.  On the order of 90%
      of the total illumination which
      penetrates the surface film is
      absorbed in the first 10 meters of
      even the clearest water, thus
      tending to warm the upper layers.

5  Water movements

   a  Waves or rhythmic movement

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

      Standing waves or seiches occur
      in lakes, estuaries,  and other
      enclosed bodies of water,  but are
      seldom large enough to be
      observed.  An "internal wave or
      seich" is an oscillation in a
      submersed mass of water such
      as a hypolimnion.  accompanied
      by compensating oscillation in the
      overlying water such that no
      significant change in surface level
      is detected. Shifts in submerged
      water masses  of this type can have
      severe effects on the biota and
      also on human water uses where
      withdrawals are confined to a given
      depth. Descriptions and analyses
      of many other  types and sub-types
      of waves and wave-like movements
      may be found in the literature.
                                                                                   I 4-5

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The Aquatic Environment
     b  Tides

        Tides are the longest waves known
        in the ocean, and are evident along
        the coast by the rhythmic rise and
        fall of the water.  While part and
        parcel of the same phenomenon, it
        is often convenient to refer to the
        rise and fall of the water level as
        "tide",  and to the accompanying
        currents as "tidal currents".

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

     c  Currents (except tidal cur rents )are
        arhythmic water movements which
        have had major study only in
        oceanography.  They primarily are
        concerned with  the translocation of
        water masses.   They may be
        generated internally by virtue of
        density  changes, or externally by
        wind or terrestrial topography.
        Turbulence phenomena or eddy-
        currents are largely responsible for
        lateral mixing in a current.  These
        are of far more importance in the
        economy of a body of water than
        mere laminar flow.

     d  Coriolis force is a result of inter-
        action between the rotation of the
        earth, and the movement of masses
        or bodies on the earth.  The net
        result is a slight tendency for moving
        objects to veer  to the right in the
        northern hemisphere, and to the
        left in the southern hemisphere.
        While the result in fresh waters is
        usually negligible, it may be con-
        siderable in marine waters. For
        example,  other factors permitting,
        there is a tendency in 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.

   6  The pH of pure water has been deterr
      mined between 5.7 and 7.01 by various
      workers.   The latter value is most
      widely accepted at the present time.
      Natural waters of course vary widely
      according to circumstances.

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

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

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

3  Hutcheson, George E.  A Treatise on
     Limnology.  John Wiley Company.
     1957.
I 4-6

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                             THE AQUATIC ENVIRONMENT

                    Part 2:  The Aquatic Environment as an Ecosystem
 I  INTRODUCTION

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

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

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

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

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

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

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

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

   5   Respiration of plants and animals
       releases carbon dioxide to the
       atmosphere in influential quantities.

   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.
ID  ECOLOGY IS THE STUDY OF THE
    INTERRELATIONSHIPS BETWEEN
    ORGANISMS, AND BETWEEN
    ORGANISMS 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 pacts (Odum,  1959).

    1  From a functional standpoint, an
       ecosystem has two component parts.
       (Figures 1. 2,3)

       a  Autotrophic (self-nourishing)
          organisms are able to fix light
          energy and manufacture food from
          simple inorganic substances.

       b  Heterotrophic (other-nourishing)
          organisms utilize, rearrange,
          and decompose the complex
          materials synthesized by the
          autotrophs.

    2  From a structural standpoint, it is
       convenient to recognize four con-
       stituents as comprising an ecosystem.

       a  Abiotic substances are basic or
          essential mineral elements and
          compounds.

       b  Autotrophic (holophytic)  organisms
          are the producers, largely the
          green plants.

       c  Heterotrophic (or holozoic)
          organisms are chiefly animals
          that ingest or consume other
          organisms or particulate organic
          matter.
BI.21b.2.70
                                I 4-7

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i
oo
                                  ESSENTIAL COMPONENTS
                        NON-ESSENTIAL
                         COMPONENTS
                 (SEWAGEJ
                                         PRIMARY
                                        DECOMPOSERS
                                                           HERBIVORES
                                                           CARNIVORES
                                                          SCAVENGERS
                                         SECONDARY
                                        DECOMPOSERS
                                       TRANSFORMERS
         f  MINERALIZED
          (  EFFLUENT )(oETRITUS
                                     SUBSTRATE



                                     PRODUCERS
                                                                                           CONSUMERS
                                                                                           TERMINAL
                r NON-LIVING
                COMPONENTS
  LIVING
COMPONENTS
               PRINCIPAL STEPS  AND COMPONENTS  IN THE TRICKLING FILTER ECOSYSTEM
                                          FROM. COOKE. ECOLOGY. 40(2) 1959               BI. ECO pi. 1 7 59
CD

a

I
H"
O


!

§
3
(D

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                                                                        Aquatic Environment
        Rg«r« 2.  Diagram of the pond ecosystem. Basic units are as follows: I, abiotic substances-basic inorganic and
       organic compounds;  IIA, producers-rooted vegetation; IIB, producers-phytoplankton; IIMA, primary consumers
       (herbivores)-bottom forms; IIMB, primary consumers (herbivores )-zooplankton; III-2, secondary consumers (car-
       wvorei); III-3. tertiary consumers (secondary carnivores); IV, decomposers-bacteria and fungi of decay.
      d   Decomposers are heterotrophic
          organisms, chiefly bacteria and
          fungi that return or reduce the
          complex compounds of dead
          protoplasm to their original
          mineral condition.

B  Functioning of the Ecosystem

   1  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 inter-
      connected.

   2  A  food web is the interlocking pattern
      of food chains in an ecosystem.
      (Figure 1)   In complex natural com-
      munities, organisms whose  food is
      obtained by the same number of steps
      are said to belong to the same trophic
      (feeding) level.

   3  Trophic  levels

      a   First -  Green plants (producers)
         (Figure 4)fix biochemical energy and
          synthesize basic organic substances.
   b   Second  - Plant eating animals
       (herbivores) depend on the
       producer organisms  for food.

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

   d   Fourth  - Secondary carnivores
       feed on  primary carnivores.

   e   Last - Ultimate carnivores are
       the last or ultimate level of
       consumers.

4  Total  assimilation

   The amount of energy which flows
   through a trophic level is distributed
   between the production of biomass
   and the demands of respiration in a
   ratio of approximately 1:10.

5  Trophic structure of the  ecosystem

   The interaction  of the food chain
   phenomena  (with energy loss at  each
   transfer) results in various com-
   munities having definite trophic
                                                                                         I  4-9

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   The Aquatic Environment
                     ^/T\ Light'.':;•;.	
                    - j"   •* ^^ *" i'" %   '.^—j»- w->-^-^   T L •  i "i^'ai  '' ~  i tm r '  \^L^p^r^^*«^^7 -^^^
                 Nutrient
                 supply
                   Bacterial
                    action
Death and decay
 \
                                                                              Predatory
                                                                               animals
                                                                     )TE ROT HO Ills

                                                                     CAiOilVORKS)

              Figure 3.  A MARINE ECOSYSTEM (After Clark, 1954 and Patten,  1966)
I  4-10

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                                                                The Aquatic Environment

1






\r<
(a)
Decomposers 11 Carnivores (Secondar
[| Carnivores (Primary
1 | Herbivores
Producers |
(b)
A
1 1
1 I
(c)
,, — &O — -
f
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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).   196.6.
     Ecology 40(2):273-291.  1959.

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

4  Hedgpeth, J.W.  Aspects of the Estuarine
     Ecosystem.  Amer. Fish. Soc., Spec.
     Publ. No. 3.  1966.
 I  4-12

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                             THE AQUATIC ENVIRONMENT

                          Part 3.  The Freshwater Environment
 I   INTRODUCTION

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

 Certain atypical inland waters such as saline
 or alkaline lakes,  springs, etc., are not
 treated,  as the main objective 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 QUA LITY ASA
    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 general
    stages of development which may be
    called: birth, youth, maturity,  and old
    age.

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

    2   Youthful streams; when the 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 approached
      geologic base level.  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.
      (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.
 BI.21b.2.70
                                                                                   I  4-13

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 The Aquatic Environment
   6  The natural vegetative cover of the
      land is of course responsible to many
      of the above factors and is also
      severely subject to the whims of
      civilization.  This is one of the major
      factors determining runoff versus
      soil absorption,  etc.

D  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
         ,ane developed through erosion of
         the shore 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  Rooted aquatic  plants 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.

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

          Filling by the accumulation of the
          remains of vegetable materials
          growing in the lake itself.
          (Often two or three processes may
          act concurrently)
III  PRODUCTIVITY IN FRESH WATERS

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

 B  Factors Affecting Stream Productivity
    (See Table 1)

                 TABLE 1
    EFFECT OF SUBSTRATE ON STREAM
                PRODUCTIVITY*
    (The productivity of sand bottoms is
    taken as 1)
Bottom Material
Sand
Marl
Fine Gravel
Gravel and silt
Coarse gravel
Moss on fine gravel
Fissidens (moss) on coarse gravel
Ranunculus (water buttercup)
Watercress
Anacharis (water weed)
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.
I 4-14

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                                                               The Aquatic Environment
   1  Youthful streams, especially on rock
      or sand substrates are low in essential
      nutrients.  Temperatures in moun-
     1 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

   1   The size, shape, and depth of the lake
      basin.  Shallow water is more pro-
      ductive than deeper water since more
      light will reach the bottom to stimulate
      rooted plant growth.  Asa 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 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.

                    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

     3  Turbidity reduces productivity as
        light penetration is  reduced.

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

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

   )  Factors Affecting the Productivity of
     Reservoirs

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

     2  The level at which water is removed
        from a reservoir  is important to the
        productivity of the stream below.
                                                                                I  4-15

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  The Aquatic Environment
        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.

        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.
VII  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  Resei voirs 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.
   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.
VIII  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. it-xix,  and 1-654.  Henry Holt
         and Company.   New York.  1904.

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

  3  Hutcheson, George E.  A Treatise on
         Limnology Vol. I  Geography,
         Physics and Chemistry. 1957.
         Vol II.  Introduction to Lake
         Biology and the Limnoplankton.
         1115 pp.    1967.  John Wiley Co.
  4   Ruttner, Franz.
         Limnology.
         Press,   pp.
 Fundamentals of
University of Toronto
1-242.  1953.
      Tarzwell,  Clarence M.  Experimental
         Evidence on the Value of Trout 1937
         Stream Improvement in Michigan.
         American Fisheries Society Trans.
         66:177-187.  1936.
  I 4-16

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                                                            The Aquatic Environment
U. S. Dept. of Health. Education,  and
    Welfare. Public Health Service.
    Algae and Metropolitan Wastes.
    Transactions of a seminar held
    April 27-29,  I960 at the Robert A.
    Taft Sanitary Engineering Center.
    Cincinnati,  Ohio. No.  SEC TR W61-3.
7  Ward and Whipple.   Fresh Water
       Biology.  (Introduction).   John
       Wiley Company.   1918.

This outline was prepared by H. W. Jackson,
Chief Biologist,  National Training Center.
FWPCA. Cincinnati, OH 45226.
                                                                              I  4-17

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                                 THE AQUATIC ENVIRONMENT

        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
   within.  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 rel-
   atively large surface area of the earth
   is covered with water,  roughly 70 percent
   of the  earth's rainfall is on the seas.
   (Figure 1)
             Flgura 1. IDE WATBt C1CU

   Since roughly one third of the earth's
   rain which falls on the land is again
   recycled through the  stratosphere
   (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 con-
   siderable burden of dissolved and
   suspended solids picked up from the  land.
   This is the substance of geological
   erosion.(Table 1)
                   TABLE i
    PERCENTAGE COMPOSITION OF THE MAJOR IONS
         OF TWO STREAMS AND SEA WATER

  (Data from Clark, F.W., 1924. "The Composition of River
  and Lake Waters of the United States",  U. S. Geol. Surv.,
  Prof. Paper No. 135. Harvey, H.W., 1957, "The Chemistry
  and Fertility of Sea Waters", Cambridge University Press,
  Cambridge)
Ion
Na
K
Ca
Mg
Cl
so4
C03
Delaware River
at
Lambertville, N.J.
6.70
1.46
17.49
4.81
4.23
17 49
32.95
Rio Grande
at
Laredo, Texas
14.78
.85
13.73
3.03
21.65
30.10
11.55
Sea Water
30.4
1. 1
1.16
3.7
55.2
7.7
»-Hco3 0.35
 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).
II   FRESHWATER,  ESTUARINE, AND
    MARINE ENVIRONMENTS

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

 A   Physical and Chemical Factors
    (Figure 2)

    1   Rivers

    2   Estuary and coastal waters

    3   Oceans
BI.21b.2.70
                                  I 4-19

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

     1  A complex of physical and chemical
        factors determine the biotic composi-
        tion of an environment.  In general,
        the number of species in a 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 oceanic
                               regions together are often classified in
                               relation to light penetration and water
                               depth.
                                  Neritic - Relatively shallow-water
                                  zone which extends from the high-
                                  tide mark to the edge of the
                                  continental shelf. (Figure 3)
  I 4-20

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                                                         The Aquatic Environment
                                        Pelagiat
                                                                         -J600
            Primary subdivisions of the marine habitat.
                                  Figure 3.
a  Stability of physical factors is
   intermediate between estuarine
   and oceanic environments.

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

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

Oceanic - The region of the ocean
beyond the continental shelf.  Divided
into three parts, all relatively
poorly populated compared to the
neritic zone.
   Euphotic zone - Waters into which
   sunlight penetrates (often to the
   bottom in the neritic zone). The
   zone of basic productivity.  Often
   extends to 600 feet below the
   surface.
   1)  Physical factors fluctuate
       less than in the neritic zone.

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

b  Bathyal zone - From the bottom
   of the euphotic zone to about
   6, 000 feet.

   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 not as abundant
       as in the bathyal zone.
                                                                             I  4-21

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  The Aquatic Environment
IE  SEA WATER AND THE BODY FLUIDS

 A Sea water is a most suitable environment
    for living cells, because it contains all
    of the chemical elements essential to the
    growth and maintenance of plants and
    animals.  The ratio and often the con-
    centration of the major salts of sea water
    are strikingly similar in the cytoplasma
    and body fluids of marine organisms.
    This similarity is also evident, although
    modified somewhat in the body fluids  of
    both fresh water and terrestrial animals.
    For example,  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 externally
       (hypotonic). In order to prevent
       dehydration, water is ingested and salts
       are excreted through special cells in
       the giUs.
IV  FACTORS AFFECTING THE DISTRI-
    BUTION OF MARINE ORGANISMS

 A Salinity - The concentration of salts is
    not the same everywhere in the sea; in
    the open ocean salinity is much less
    variable than in the ever changing
    estuary or coastal water.  Organisms
    have different tolerances to salinity
    which limit their distribution.  The
    distributions may be in large water
    masses, such as the Gulf Stream,
    Sargasso Sea, etc.,  or in bays and
    estuaries.

    1   In general,  animals in the estuarine
        environment are able to withstand
        large and rapid changes in salinity
        and temperature.  These animals are
        classified as:

        a  Euryhaline ("eury" meaning wide) •
          wide tolerance to salinity changes.
                EURYHALINE
  Fresh Water
  Stenohaline
Marine
Stenohaline
                   Salinity
       ca. 35
  Figure 4. Salinity Tolerance of Organisms
       b  Eurythermal - wide tolerance to
          temperature changes.
   I  4-22

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                                                       The Aquatic Environment
SNAILS

Littorina neritoides
L. rudis
L. obtusata
L. littorea

BARNACLES

Chthamalus stellatus
Balanus balanoides
B. perforatus
%&?%&*£*• ^° •* «4%T>  •
                                                  l^mm»Q
                                                  \ \f\il J 1 -I HI i V»v> IM\\O^I.C
                                Figure 5
       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)
                                                                         I  4-23

-------
The Aquatic Environment
   2  In general, animals in river and
     oceanic environments cannot withstand
     large and rapid changes in salinity and
     temperature.  These animals are
     classified as:

     a  Stenohaline ("steno" meaning narrow) •
        narrow tolerance to salinity changes.

     b  Stenothernal - narrow tolerance to
        temperature changes.

   3  Among euryhaline animals, those living
     in lowered salinities often have a
     smaller maximum size than those 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/".

   4  Usually the larvae of marine organisms
     are more sensitive to changes in salinity
     than are the adults.  This character-
     istic  limits both the distribution and
     size of populations.

B  Tides

   Tidal fluctuation is a phenomenon unique
   to the seas (with minor exceptions).  It is
   a twice daily rise and fall in the sea level
   caused by the complicated interaction of
   many factors including sun, moon, and the
   daily rotation of the earth.  Tidal heights
   vary from day to day and place to place.
   and are  often accentuated by local
   meteorological conditions.  The rise and
   fall may range from a few inches or less
   to fifty feet or more.
 V  FACTORS AFFECTING THE
    PRODUCTIVITY OF THE MARINE
    ENVIRONMENT

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


ACKNOWLEDGEMENT:

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


REFERENCES

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

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

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

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

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

6  Sverdrup, Johnson,  and Fleming.
       The Oceans.  Prentice-Hall. Inc..
       New York.  1087 pp.    1942.

This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
FWPCA.  Cincinnati. OH  45226.
 I  4-24

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CHAPTER II




IDENTIFICATION OF PLANKTON AND ASSOCIATED ORGANISMS





Structure and Function of Cells                                     1




Aquatic Organisms of Significance in Plankton Surveys                2




Types of Algae                                                    3




Blue-Green Algae                                                 4




Green and Other Pigmented Flagellates                              5




Filamentous Green Algae                                           6




Coccoid Green Algae                                               7




Diatoms                                                          8




Filamentous Bacteria                                              9




Protozoa, Nematodes, and Rotifers                                10




Free-Living Amoebae and Nematodes                              11




Animal Plankton                                                  12

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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.
E   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.

b  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.

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

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

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

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

g  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.

h  "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.

i  "Mitochondria" are small mem-
   branous structures containing
   enzymes that oxidize food to produce
   energy transfer compounds (ATP).
 BI.CEL. la.3.70
                                                                                   H  1-1

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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
   ceUs.

   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
        (ONA) 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
     photosynthetic 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
      ceU.

   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.
 H  1-2

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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.
IH  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 cource 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.
                                                                                    II 1-3

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            AQUATIC ORGANISMS OF SIGNIFICANCE IN PLANKTON SURVEYS
I  INTRODUCTION

A Any organism encountered in a survey is
   of significance.  Out problem is thus not
   to determine which are of significance but
   rather to decide,  "what is the significance
   of each?"

B The first step in interpretation is
   recognition.

C Recognition implies identification and an
   understanding of general relationships.
   The following outline will thus review the
   general relationships of living (as  con-
   trasted to fossil)  organisms, and briefly
   describe the various types.
H  THE GENERAL RELATIONSHIPS OF
   LIVING ORGANISMS

 A Living organisms have been long grouped
   into two kingdoms:  Plant and Animal.
   Modern developments however,  have made
   this simple pattern technically untenable.
   It has become evident that there are as
   great and fundamental differences between
   certain other groups and these (two), as
   there are between traditional "plant" and
   "animal. " The accompanying chart con-
   sequently shows the fungi as  a third
   "kingdom."

 B The three groups are essentially defined
   as follows on the basis of their nutritional
   mechanisms:

   1  Plante: photosynthetic, synthetizing
      their own organic substance from
      inorganic minerals.  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.
       Ecologically known as REDUCERS.

    Each of these groups includes simple,
    single-celled representatives,  per-
    sisting at lower levels on the evolutionary
    stems of the higher organisms (as well
    as the higher and more complex types).

    1  These groups span the gaps between
       the higher kingdoms with a multitude
       of transitional form.  They are
       collectively called the PROTISTA.

    2  Within the Protista, two principle
       sub-groups can be  defined on the basis
       of relative complexity of structure.

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

       b  The single-celled algae and protozoa
          are best referred to as the higher
          protista.

    Distributed throughout these groups will
    be found the traditional "phyla" of classic
    biology.
Ill  PLANTS

 A The vascular plants are usually larger
    and possess roots, stems and leaves.

    1  Some types emerge above the surface.

    2  Submersed types typically do not
       extend to the surface.
 BI.AQ. 10d.4.70
                                                                                  II  2-1

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Aquatic Organisms of Significance in Plankton Surveys
                                 CONSUMERS
         PRO DUCERS
         REDUCERS
                                     NUTRIENT
                                     MINERALS
   3 Floating types may be rooted or
     free-floating.

B Algae generally smaller, more delicate,
   less complex in structure, possess
   chlorophyll like other green plants.
   For convenience the following artificial
   grouping is used in sanitary science:

   1 "Blue-green algae" are typically small
     and lack an organized nucleus,  pigments
     are dissolved in cell sap.  Structure
     very simple.

   2 "Pigmented flagellates" possess nuclei,
     chloroplasts, flagellae and a red eye
     spot.  This is an artificial group con-
     taining several remotely related
     organisms, may be green, red, brown,
     etc.
"Diatoms" have "pillbox" structure of
SiOp - may move.  Extremely common.
Many minute in size, but colonial forms
may produce hair-like filaments.
Golden brown in color.

"Non-motile green algae" lack
locomotor structure or ability in
mature condition.  Another artificial
group.

a  Unicellular representatives may be
   extremely small.

b  Multicellular forms may produce
   great floating mats of material.
II 2-2

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                                      Aquatic Organisms of Significance in Plankton Surveys
IV  FUNGI

  Lack chlorophyll and consequently most are
  dependent on other organisms.  They secrete
  extracellular enzymes and reduce complex
  organic material to simple compounds which
  they can absorb directly through the cell wall.

  A Schizomycetes or bacteria are typically
    very small and do not have an organized
    nucleus.

    1  Autotrophic bacteria utilize basic food
       materials from inorganic substrates.
       They may be photo-synthetic or
       chemosynthetic.

    2  Heterotrophic bacteria are most common.
       They require organic material on which
       to feed.

  B "True fungi" usually exhibit hyphae as the
    basis of structure.
 V  ANIMALS

 A Lack chlorophyll and consequently feed on
    or consume other organisms.  Typically
    ingest and digest their food.

 B The Animal Phyla

    1  PROTOZOA are single celled organisms;
       many resembling algae but lacking
       chlorophyll (cf: illustration in "Oxygen"
       lecture).

    2  PORIFERA are the sponges; both
       marine and freshwater representatives.

    3  CNIDARIA (=COELENTERATA) include
       corals,  marine and freshwater jelly
       fishes, marine and freshwater hydroids.

    4  PLATYHELMINTHES are the flat worms
       such as tape worms,  flukes and Planaria.
       NEMATHELMINTHES are the round
       worms and include both free-living
       forms and many dangerous parasites.

       ROTIFERS are multicellular micro-
       scopic predators.
 7  BRYOZOA are small colonial sessile
    forms,  marine or freshwater.

 8  MOLLUSCA include snails and slugs,
    clams,  mussels and oysters, squids
    and octopi.

 9  BRACMOPODS are bivalved marine
    organisms usually observed as fossils.

10  ANNELIDS are the segmented worms
    such as earthworms, sludge worms
    and many marine species.

11  ECHINODERMS include starfish, sea
    urchins and brittle stars.  They are
    exclusively marine.

12  CTENOPHORES, or comb jellies, are
    delicate jelly-like marine organisms.

13  ARTHROPODA, the largest of all
    animal phyla.  They have jointed
    appendages and a chitinous exoskelton.

    a  CRUSTACEA are divided into a
      cephalothorax and abdomen, and
      have many pairs of appendages,
      including two pairs of antennae.

      1) CLADOCERA include Daphnia
         a common freshwater micro-
         crustacean; swim by means of
         branched antennae.

      2) ANOSTRACA  (=PHYLLOPODS)
         are the fairy shrimps, given to
         eruptive appearances in tem-
         porary pools.

      3) COPEPODS are marine and
         freshwater microcrustacea--
         swim by means of unbranched
         antennae.

      4) OSTRA CODS are the microscopic
         "clams with legs. " Generally
         substrate oriented.

      5) ISOPODS are  dorsoventrally
         compressed; called sowbugs.
         Terrestrial and aquatic, marine
         and freshwater.
                                                                                H 2-3

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Aquatic Organisms of Significance.in Plankton Surveys
        6) AMPfflPODA - known as scuds.
           laterally compressed.  Marine
           and freshwater.

        7) DECAPODA - crabs, shrimp,
           crayfish, lobsters, etc.  Marine
           and freshwater.

        INSECTA - body divided into head,
        thorax and abdomen; 3 pairs of legs;
        adults typically with 2 pairs of wings
        and one pair of antennae.  No
        common marine species.  Nine of the
        twenty-odd orders include species
        with freshwater-inhabiting stages in
        their life history as follows:

        1) DIPTERA  - two-winged flies

        2) COLEOPTERA - beetles

        3) EPHEMEROPTERA - may flies

        4) TRICHOPTERA - caddis flies

        5) PLECOPTERA - stone flies

        6) ODONATA - dragon flies and
           damsel flies

        7) NEUROPTERA - alder flies,
           Dobson flies and  fish flies

        8) HEMIPTERA - true bugs, sucking
           insects such as water striders,
           electric light bugs and water
           boatman

        9) LEPIDOPTERA - butterflies
           and moths, includes a few fresh-
           water moths

        ARACHINIDA - body divided into
        cephalothorax and abdomen; 4 pairs
        of legs - spiders, scorpions,  ticks
        and mites. Few aquatic represent-
        atives except for the freshwater
        mites and tardigrades.
  C CHORDATA

    1  PROCHORDATES - primitive marine
       forms such as acorn worms,  sea
       squirts and lancelets

    2  VERTEBRATES - all animals which
       have a backbone

       a PISCES or fishes;  including such
         forms as sharks and rays,
         lampreys, and higher fishes; both
         marine and freshwater

       b AMPHIBIA - frogs, toads, and
         salamanders - marine species rare

       c REPTILA -  snails, lizards and
         turtles

       d MAMMALS - whales and other
         warm-blooded vertebrates with hair

       e AVES - birds  - warm-blooded
         vertebrates with feathers
VI  THE CLASSIFICATION OF ORGANISMS

 A INTRODUCTION

    There are few major groups of orga-
    nisms that are either exclusively
    terrestrial or generally aquatic.  The
    following remarks apply to both, however,
    primary attention will be directed to
    aquatic types.

 B One of the first questions usually posed
    about an organism seen for the first time
    is:  "what is it? " usually meaning,
    "what is its name ? "  The naming or
    classification of biological organisms is
    a science in  itself (taxonomy).  Some of
    the principles involved need to be under-
    stood by anyone working with organisms
    however.
H  2-4

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                          Aquatic Organisms of Significance in Plankton Surveys
RELATIONSHIPS  BETWEEN  FREE  LIVING AQUATIC  ORGANISMS

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

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

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

Coelenterates
Basidiomycetes

Fungi Imperfecti
Ascomycetes

Higher Phycomycetes
          DEVELOPMENT OF MULTICELLULAR OR COENOCYTIC STRUCTURE


                H  I  G" H  E R      P  R 0  T   I  S  T  A

                                 Protozoa

  Unicellular Green Algae

  Diatoms

  Pigmented Flagellates
Amoeboid


Flagellated,
 (non-pigmented)
Cilliated


Suctoria
Lower


  Phycomycetes


  (Chytridiales, et. al )
                      DEVELOPMENT OF A NUCLEAR MEMBRANE
                 LOWER      PROTISTA
                                (or    M o n -e r a )
  Blue Green Algae



          Phototropic Racteria



                 Chemotro|iic Hacteria
                                    Actinomycetes


                               Spuochaetes
                     Sapt ophytic
                     Bacterial
                     Types
                                                                             II 2-5

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 Aquatic Organisms of Significance in Plankton Surveys
      Names are the "key number", code
      designation", or "file references"
      which we must have to find information
      about an unknown organism.

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

      Common names are rarely available for
      most invertebrates and algae.
      Exceptions to this are common among
      the molluscs, many of which have
      common names  which are fairly
      standard for the same species through-
      out its range. This may be due to their
      status as a commercial harvest or to
      the activities of devoted groups of
      amateur collectors.  Certain scientific
      societies have also assigned "official"
      common names  to particular species;
      for example,  aquatic weeds - American
      Weed Society; fish - American Fisheries
      Society; amphibians (salamanders and
      frogs) - American Society  of Ichthyolo-
      gists and Herpetologists.

      The system of biological nomenclature
      is  regulated by international congresses.

      a  It is based on a system  of groups
         and super groups, of which the
         foundation (which actually exists in
         nature) is the species.

      b  The taxa (categories) employed are
         as follows:

         The species is the foundation
         (plural:  species)

         Similar species are grouped into
         genera (singular:  genus).

         Similar genera are grouped into
         families.
   Similar families are grouped into
   orders.

   Similar orders are grouped into
   classes.

   Similar classes are grouped into
   phyla (phylum).

   Similar phyla are grouped into
   kingdoms.

Other categories  such as sub-species,
variety,  strain, division, tribe, etc.
are employed in special circumstances.

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

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

   Homo sapiens - (=sentiens) modern
   man

   Homo heidelbergensis - heidelberg
   man

   Homo neanderthalis - neanderthal
   man

   Oncorhynchus gorbuscha - pink
   salmon

   Oncorhynchus kisutch - coho salmon

   Oncorhynchus tshawytscha -
   Chinook salmon

b  Common names do not exist for
   most of the smaller and less familiar
   organisms. For example,  if we
   wish to refer to members of the
   genus Gomphonema (a diatom) we
   must simply use the generic name,
   and:
II  2-6

-------
                                 Aquatic Organisms of Significance in Plankton Surveys
   Gomphonema olivaceum

   Gomphonema parvulum

   Gomphonema abbreviatum

   three distinct species which have
   different significances to algologists
   interpreting water quality.

A complete list of the various categories
to which an organism belongs is known
as its "classification".  For example,
the classification of a type of diatom
Gomphonema olivaceum is:
Kingdom
Phylum
Class
Order
Family
Genus
Species
Plantae
Chrysophyta
Bacillariophyceae
Pennales
Gomphonema ceae
Gomphonema
olivaceum
        Additional accuracy is gained by
        citing the name of the authority who
        first described a species (and the
        date) immediately following the
        species name.  Authors are also
        often cited for genera or other
        groups.

        It should be  emphasized that since
        most categories above the species
        level are essentially human con-
        cepts,  there is often divergence of
        opinion in regard to how certain
        organisms should be grouped.
        Changes result as knowledge grows.

        The most appropriate or correct
        names too are subject to change.
        The species itself, however,  as
        an entity in nature,  is relatively
        timeless and so does not change
        to man's eye.
                                           REFERENCE

                                           Whittaker,  R.H.
                                              of Organisms.
                                              1969.
                 New Concepts of Kingdoms
                  Science 163:150-160.
a  These seven basic levels of
   organization are often not enough
   for the complete designation of one
   species among thousands; however,
   and so additional echelons of terms
   are provided by grouping the various
   categories into "super... " groups
   and sub-dividing them into "sub... "
   groups as:

   Superorder, Order, Suborder, etc..
   Still other category names such as
   "tribe", "division",  "variety",
   "race", "section", etc.,  are used
   on occasion.
This outline was prepared by H. W.  Jackson,
Chief Biologist,  National Training Center.
Revised 1970 by R.  M. Sinclair,  Aquatic
Biologist, National Training Center, FWPCA,
Cincinnati, OH 45226.
                                                                            H 2-7

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

 A A Igae - Simple plants with an autotrophic
   mode of nutrition.  They contain chloro-
   phyll, but lack the highly differentiated
   reproductive and body structures of higher
   plants.  The algae range in size from less
   than one micron, to the giant kelp more
   than 300 ft. in length.

 B Algae are carried about by water currents,
   wind, aquatic animals, water fowl, and
   insects, and are found everywhere.  They
   grow actively at temperatures ranging
   from -2°C to 6QOC.

 C The algae play a more important role in
   aquatic habitats than on land. They serve
   as the basic energy source for other
   aquatic organisms, providing food for
   zoomicrobes, higher invertebrates and
   fish.

 D There are two basic types of freshwater
   algal communities:

   1  The phytoplankton - free floating algae

   2  Phycoperiphyton - attached or bottom
      forms
II  Algae are classified by their pigments,
 food pigments, food reserves,  morphology,
 and method of reporduction.  Recent classifi-
 cation schemes recognize eight major groups
 (phyla).

 A Blue-green (Cyanophyta)

 B Green (Chlorophyta)

 C Yellow-brown (Chrysophyta)

 D Euglenoid (Euglenophyta)

 E Dinoflagellates (Pyrrophyta)

 F Red (Rhodophyta)
 G Brown (Phaeophyta)

 H Chloromonads (Chloromonadophyta)

 The most numerous freshwater algae are the
 yellow browns, greens, and blue-greens.


Ill  BLUE-GREEN ALGAE

 This group contains many organisms that
 form nuisance blooms in eutrophic waters
 in later summer.  They are considered to
 be the most primitive algae,  and are
 similar to the bacteria in many respects.
 They lack many of the basic cell structures
 found in other algae and terrestrial plants,
 and have no sexual reproduction or  motile
 forms.  Many have the ability to fix nitrogen.

 A The chlorophyll-bearing membranes are
    not organized to form distinct bodies
    (chloroplasts).

 B The cells contain chlorophyll a,  but are
    blue-green because of the presence of the
    blue pigment,  phycocyanin (Table 1).

 C The nucleus is not enclosed by a
    membrane.

 D Reproduction is by cell division, colony
    fragmentation, and spore formation:

    Spores of three types are observed.

    1  Akinetes - enlarged,  (often) thick-
       walled resting cells (see Anabaena).

    2  Heterocysts - appear "empty",  but
       have been observed to germinate
       (see Anabaena).

    3  Endospores - formed by repeated
       division of protoplast in a cell
       (Chamaeosiphon).
  BI.MIC.cla.22.3.70
                                  II 3-1

-------
 "IVpes  of Algae
                                                       TABLE   1
                                      The distribution of pigmrnts in the algal groups. • = major
                             pigmenl(s) of (he group;  3 = a pigment comprising less than half the
                             total pigment content;  O = present in small amounts. The data given by
                             earlier authors varies considerably and the present table is compiled from
                             tables in Fogg (1953), Strain (1958), Goodwin (1960), Egle (1960) and
                                 Haxo and O'Heocha (1960) with additions from their discussions.
X. Alfil
^S^ tmw
\.
Fitments ^N^^
^\
"chlorCh
Chlorophyll!
Chlorophyll <
Chlorophyll 1
Chlorophyll i
Cntnu
a'Carotene
p^Carotenc
r^Caroune
•-Carotene
Fuwaccne
Zeaunthin
Vulounlhin
Flavoxaxilhm

Fucounthin

Neofucoximhm (A and B)
Dutounthin
Diadinoxanlhin
Neodiadinonnlhin
Dmoxanlhin
Neodinoxanlhin
Pendimn
Mrmuthint-Echmenone]
Myraunlhophtll
OKillounlhui
Ailuaalhin
Unlnovm nanlhophyU
HUnUM
R-ph»coer)thrm
R'phyooc) an in
C-phi coert ihf in

Alloph) cocyanin








T

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8


















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I



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o1
o






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1
4
4

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o
0

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£
e

o

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•


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o
o
Q















a
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±0
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REMARKS



•Bui not In all fenerft

Kol in higher planu
•In Ktriiflt uriuam

•Tlraunlhm'


Occun il • chroma-
protein







Alu^^huHr..^

MMU and PMWI/AM



Alia in Cryptomonadi
Also in Cryptoinoiuai.
but tbwrpuon priu
net Ihe lame
•In PprpA/ra n*i«Afm
                          I. 5-4 times Ihe content of/9-nrolene and lutein, but famtles ofFuna almost cntiivl> ^-carotene
                          B. aomelimes in gnalcr irnouDl than P carotene
                          S In large amounts possibly in Ihe c>loplism
                          4 Dominant in some speciei 1 he carolenoid content! of Euitta* iff. are hifher than other ali
-------
                                                                           Types of Algae
 E For convenience, the phylum is divided
    into two groups:

    1  Coccoid - cells variously shaped,
       solitary or in masses,  non-filamentous

    2  Filamentous - filaments solitary or in
       masses,  branched or unbranched,
       sheathed or unsheathed; specialized
       cells may be present

 F Important Genera:

    Anacystis (Microcystis), Gleotrichia.
    Anabaena, Aphanizomenon,  Schizothrix,  and
    Oscillatoria.
IV  GREEN ALGAE

 This group possesses intracellular structures
 (such as chloroplasts) and chlorophyll a and b
 in the ratio 2:1, similar to higher plants.

 A The cells are grass green because of the
    predominance of the chlorophylls over the
    carotenes and xanthophylls.

 B Sexual reproduction and motile cells are
    common.

 C These algae are commonly divided into
    three groups:

    1  Coccoid - non-motile, solitary or
       colonial.  Actinastrum.  Ankistrodesmus,
       Coelastrium. Golenkinia.  Oocystis,
       Pediastrum,  Scenedesmus.

    2  Filamentous  - attached or planktonic,
       branched or unbranched, Ulothrix,
       Mougeotia. Spirogyra, Stigeoclonium,
       Cladophora.

    3  Flagellated -2-4 flagella, solitary or
       colonial

 D Important Genera:  Chlamydomonas.
    Pandorina, Eudorina.  Volvox.  Carte ria
V  YELLOW-BROWN

 These algae are yellowish green to golden
 brown because of a predominance of carotenes
 and xanthophylls.  This group is usually the
 dominant form in the plankton and periphyton.
 The phylum contains coccoid, filamentous,
 and flagellate forms of varying importance.

 A  Coccoid - includes the diatoms (see below)

 B  Filamentous - important genera include
    Tribonema,  Vane her ia

 C  Flagellates - flagella single or paired,
    equal or unequal in length; ceUs solitary
    or colonial; some cells are armored.
    Important genera:  Chromulina,
    Chrysococcus. Kephrion. Synura,
    Dinobryon.

 D  Diatoms

    This group generally contributes 70-80%
    of the  phytoplankton and periphyton.
    The cell walls contain silica and possess
    distinctive markings.  The chloroplasts
    contain chlorophyll a and £ in nearly
    equal amounts, but appear yellow brown
    because of the predominance of /3-carotene
    and fucoxanthin.  The diatoms are
    generally divided into two major groups:

    1  Centric

      The cells are circular in cross-section;
      drum-shaped, cylindrical, or spindle-
      shaped in side view.  Important genera
      include:   Cyclotella, Stephanodiscus,
      Melosira, Rhizosolenia.

    2  Pennate

      The cells are usually rectangular in
      cross-section and boat-shaped,  with
      transverse rows of linear markings.
      Important genera include:  Fragilaria,
      Synedra.  Asterionella. Navicula,
      Nitzschia. Gomphonema.
                                                                                   II 3-3

-------
   Types of Algae
  VI  EUGLENOPHYTA

   A  This group contains chlorophyll ji and b
      and consists primarily of motile forms
      that have a single flagellum.

   B  The common genera are Euglena. Phacus.
      and Trachelomonas.
X  CHLOROMONDOPHYTA

 This is a small group of miscellaneous,
 little-understood, protozoa-like forms.
 There are no common genera in this phylum.
 REFERENCES
 VII   PYRROPHYTA

   A  These algae are greenish tan to golden
      brown, and contain chlorophyll a and £.
      The dominant forms (Dinoflagellates) are
      unicellular, motile, and are encircled by
      a transverse or spiral groove.  One of the
      two flagella lies in the groove and encircles
      the cell; the other extends backward from
      the cell.  Some forms lack a  cell wall,
      while others have thick walls which appear
      to be composed of many plates.

   B  The common genera include Gymnodinium,
      Glenodinium.  Gonyaulax. Peridinium.
      and Ceratium.
VIE  RHODOPHYTA

   A This phylum is largely marine, and contains
     chlorophyll a, /3-carotene,  phycoerythrin,
     and phycocyanin.  The common freshwater
     species are filamentous, attached forms,
     and are not found in the plankton.

   B Important freshwater genera include the
     filamentous forms, Batrachospernum,
     Audouinella and Lemanea.
     PHAEOPHYTA

   The brown algae are almost entirely marine,
   and  have no important freshwater forms.
   They contain chlorophyll a and c and /3-carotene,
   but are brown because of the predominance
   of fucoxanthin and other xanthophylls.  This
   group is usually macroscopic, and contains
   the large "sea weeds" or kelp.
 1  Davis,  C. C.  The marine and freshwater
      plankton.  Mich. State Univ. Press,
      East Lansing.  1955.

 2  Fritsch,  F. E.  The structure and
      reproduction of the algae.  Vol. I.
      Cambridge University Press.   1956.

 3  Fritsch,  F.E.  The structure and
      reproduction of the algae.  Vol. n.
      Cambridge University Press.   1965.

 4  Palmer,  C.M.  Algae and water supplies,
      USDHEW, PHS, DWSPC, Cincinnati,
      1962.

 5  Prescott, G.W.   How to know the fresh-
      water  algae.   Win. C. Brown Company,
      Dubuque. (revision in press)   1954.

 6  Prescott, G.W.  Algae of the Western
      Great  Lakes area.  2nd ed.  Wm. C.
      Brown, Dubuque.   1962.

 7  Prescott, G.W.   The algae: A Review.
      Houghton Mifflin Co., Boston.   1968.

 8  Round,  F.E.  The biology of the algae.
      Edward Arnold, Ltd.,  London.  1965.

 9  Smith,  G.M.  The freshwater algae of
      the United States.  2nd ed.  McGraw-
      Hill  Book Co.,  New York.  1950

10  Tiffany, L.H. and Britton, M.E.
      The  Algae of Illinois.   University
      Chicago Press, Chicago (University
      Microfilms, Ann Arbor, Xerox).  1952.

 This outline  was prepared by Dr. C.I.  Weber,
 Chief, Biological Methods Section, Analytical
 Quality Control Laboratory,  FWPCA,
 1014 Broadway, Cincinnati, OH 45202.
  II 3-4

-------
                                                           Types of Algae
             GREEN ALGAE, NON-FILAMENTOUS
                                                  Dictyosphaerium
Polyedriopsi9
                                                                           II  3-5

-------
Types of Algae
                                      GREEN  ALGAE, FILAMENTOUS
                                                                               Spirogyra
II  3-6

-------
                                                                          Types of Algae
                                BLUE-GREEN ALGAE
Coccochlor IB
(Gloeothece)
   Anacystis
   (Chroococcus)
                       I.yngbya
                                   Aphanizomenon
                                       Gomphosphae ria
                                                                Schizothrix
                                                                             Calothrix
Agmenellum
(Merismophedia)
                                                                                     n  3-7

-------
Types of Algae
                                      FLAGELLATE ALGAE
                          Chlorogoniu
                                                                 Pandorina
II 3-8

-------
                                                                    Types of Algae
                                 DIATOMS
Navicula
(Girdle View)
                                                            Cyclotella
                                                            (Valve View)
                                                            Cyclotella
                                                            (Girdle View)
                                                              die View)
                     Nitzschia
                                                                               n  3-9

-------
                                  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 (phyocyanm-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-
 water 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 do
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-
         tems.  The gelatinous sheath is com-
         posed of pectic substances,  cellulose
         and related compounds.
 BI. MIC. cla. ICa. 8. 69
                                                                                     II  4-1

-------
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.
VIII  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, is divided
        into two regions.  The peripheral
        pigmented portion called chroma-
        toplasm, and an inner centroplasm,
        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-
        tenstic blue-green color from its
        dissolved photosynthetic pigments.
IX  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.
XII 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.
 II  4-2

-------
                                                                          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 Tolypothnx.

    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.

B   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.

C   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.
                                                                                     II 4-3

-------
Blue-Green Algat;
                       SOME   BLUE-GREEN   ALGAE
        1.  Notvfitamentp.tfs ( cbccoid)..Blue-Green Algae:
                     :>i-'i;i;l'.''. •- •":-. /'/xT
                                      II' >-| .   iti'.
-------
                                                                    Blue-Green Algae
    5   Often imparts grassy or nastur-
        tium-like odors to water.

D   Oscillator la 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
        hormongoma only.

    5   Live species exhibit "oscillatona"
        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
             tnchome.
REFERENCES

1  Bartsch, A.  F.  (ed.)  Environmental
      Requirements of Blue-Green Algae.
      FWPCA.  Pacific Northwest Water
      Laboratory. Corvallis. Oregon.
      Ill pp.  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.
         Vegetative cells, heterocysts,
         and even the akinetes are broader
         than long.
                                                                               II 4-5

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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.
 H  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
    encountered.

 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.  sangulnea has red pigment.

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

   7  IS.  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.3.70
                                 II  5-1

-------
   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  Chlamydotnonas 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
      ceU.

   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).
 II 5-2

-------
                                                   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. morum 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.

   3  E. 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 (Oinophyceae)
 (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".
                                                                               H 5-3

-------
  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  P. 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
 Baciliariophyceae),  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  £. 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 biflagellate form growing
   in radially arranged, naked colonies
   (Figure 11).

   a  Flagella equal in length

   b  Cells pyriform or egg shaped

   c  S. 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  IJ. 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.
 II  5-4

-------
                                              Green and Other Figmented Flagellates
(fig 1-13 from Lackey and Callaway)
    Euglena
                  Phacus
                                  Lepocinclis
                                                   Trachelomonas
                                                            GREEN   EUGLENOIDS
        ^

     Chlamydomonas
                                                                   Pandorina
                      Chlorogonium
                                                           GREEN   PHYTOMONADS
io  p





 Chromulina
                  11
                           Synura
                                      Dinobryon
                                                        YELLOW   CHRYSOMONADS
 14
     Massartia
                             15
                                  Peridinium
                                                               Ceratium
                                                 YELLOW-BROWN   DINOFLAGELLATES
                                                                      E 5-5

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  Green and Other Pigmented Flagellates
                                          FLAGELLATES
                                          (MASTIGOPHORA)
                           PLANT FLAGELLATES
                            (PHYTOMASTIGINA)
                       CHRYSOMONADINA

                             CRYPTOMONADINA

                                   PHYTOMONADINA
 ANIMAL FLAGELLATES
   (Z
      OOMASTIGIN
RHIZOMASTIGINA
     PROTOMONADINA
                                          EUGLENOIDINA
           POLYMASTIGINA
                      Figure 17 Phylogenetic Family Tree of the Flagellates
                                   (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,
  Chief Biologist, National Training Center,
  FWPCA,  Cincinnati, OH 45226.
      5-6

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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
   stone worts.
 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).
H  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 ceU( "axial").

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

1  Including a few yellow-brown and red algae.
IE  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 Zoo spores (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
  *Planktonic or occasionally planktonic
 BI.MIC.cla. 14b.3.70
                                  II  6-1

-------
 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
    H.S 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.

 O 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.
   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  De smidiaceae

   Desmidium.  Hyalotheca

I  Tribonemataceae

   Tribonema.  Bumilleria

J  Characeae

   Chara. Nitella. Tolypella
 E 6-2

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                                                          Filamentous Green Algae
          13. GREENS,  FILAMENTOUS
   MAMWULDU
     It*
II  6-3

-------
   Filamentous Green Algae
VIE  IDENTIFICATION

   A Branching and attenuation are of primary
     importance.

   B P last ids:  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.
   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.
    II  6-4

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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.
 BI.MIC. cla.9c. 3.70
                                                                                 II  7-1

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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.
B  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  A nkistrodesmus cells are usually long
      and slender,  tapering to sharp point at
      both ends.  They may be straight,
      curved, or twisted into loose aggregations.
      A nkistrodesmus 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 S. quadricauda are common
      planktonic 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.
II  7-2

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                                                                  Coccoid Green Algae
D
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 maybe 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.

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.  Oocvstis
   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.  Dimorphococ cus
   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.
                                                                                 II  7-3

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  Coccoid Green Algae
       Micrasterias is relatively common,
       ornate.

       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
       spined.

       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.

       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.
in  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,
Chief Biologist,  National Training Center,
FWPCA, Cincinnati, OH 45226.
  II 7-4

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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
      are in filaments or other shapes of
      colonies.
        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.
    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 cylmdnc ("centric") one view
    of which is circular.

    1  Pennate diatoms may be symmetrical,
       transversely unsymetrical,  or longitudi-
       nally unsymmetrical.
Ill   EXAMPLES OF COMMON DIATOMS:
  A  Pennate,  symmetrical:

          Navicula
          Pinnulana
          Synedra
          Nitzschia
          Diatoma
          Fragilana
          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.
  B Pennate,  unsymmetrical:

          Gomphonema
          Surirella
          Cymbella
          Achnanthes
          Astenonella
          Meridiem
                                                  C  Centric:
 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.
          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. MIC.cla. lOa. 8.69
                                     II  8-1

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  Diatoms
    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  Naviculmeae.  True raphe group with
        raphe in center of valve.

        a  Representative genera:

           Navicula
           Pinnulana
           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
          Stephanodisc us
          Melosira
          Rhizosolema
          Biddulpnia


 B Pennals Group

    1  Fragilanneae.  The  false raphe group.

       Representative genera:

          Tabe liana
          Meridion
          Diatoma
     4  Sunrellmeae.  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.
  II  8-2

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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.
                                                                                II 8-3

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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.  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 I^S is present.
 Beggiatoa is distinguished by its ability to
 deposit sulfur within its cells; the sulfur
 deposits appear as large refractile globules.
 When I^S is no longer present in the environ-
 ment,  the 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 HgS  and using this energy to fix
      into all material.  It can also use certain
  organic materials if they are present  along
  with the H2S.

  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.
  The only major nuisance effect of Beggiatoa
  known has been overgrowth on trickling
  filters receiving waste waters rich in I^S.
  The normal microflora of the filter was
  suppressed and the filter failed to give good
  treatment.  Removal of the I^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 HgS 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 re-
 produced by fission or by means of special
 conidia.   Their filamentous habit and method
 of sporulation is  reminiscent of fungi.  How-
 ever, their size, chemical composition, and
 other characteristics are more  similar to
 bacteria.  These organisms  may be con-
 sidered 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
 BA.8. 1.67
                                    H  9-1

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  Filamentous Bacteria
  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 Actmomycetes
  Related to Water Supplies. "  But the actmo-
  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 clorination will kill the
  organisms in the treatment plant or distribu-
  tion system, the odors often are present
  before the  water enters the plant.   Use of
  permanganate 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 rams or to prevent possible
  development of the actmomycetes in water
  rich in decaying organic matter is still needed.
IV  FILAMENTOUS IRON BACTERIA

 The filamentous iron bacteria of the
 Sphaerotilus-Lepthothrix 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 organ-
 ism is easily recognized by its special
 morphology.   Dr.  Wolfe of the University of
 Illinois has published photomicrographs of
 the organism.

 Organisms of the Sphaerotilus-Leptothnx
 group have been extensively studied by many
 investigators (Dondero e_t_. _al. , Oondero,
 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.  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 pro-
cessing 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 mter-
mittant 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 oxi-
dizing ferrous iron to ferric iron and uses
only CO2 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 chlormation
(up to 100 ppm of sodium hypochlorite for 48
hours) followed by  flushing will often remove
the masses of growth and periodic treatment
  II  9-2

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                                                                     Filamentous Bacteria
will prevent the nuisance effects of the ex-
tensive masses of Galhonella.
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  Kowalhk,  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. et_._al.  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 Enumera-
      tion of Actinomycetes  Related to Water
      Supplies.  Robert A. Taft Sanitary
      Engineering Center Techn. Report
      W-62-10.  1962.

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 Galhonella
      ferruginea.  Jour. Bacteriol.   74:344
      349.  1957.
 9  Wolfe,  R. S.  Observations and Studies
      of Crenothnx 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 Organ-
      ism Sp_haerotilu£ natans.  Quart. Jour.
      Fla. Acad. Sci., 21(4):335-340.  1958.

13  Dondero, N.C.,  Philips,  R.A., and
      Henkelkian, H.  Isolation and Preser-
      vation 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.  Inves-
      tigations on the Sphaerotilus -Leptothrix
      Group.  Antonie van Leewenhoek,  29:
      121-153.  1963.

16  Amberg, H. R. and Cormack,  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.

 This outline was prepared by R. F. Lewis,
 Bacteriologist,  Microbiological Activities,
 Research and Development,  Cincinnati Water
 Research Laboratory, FWPCA.
                                                                                   II 9-3

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                                          FUNGI
I    INTRODUCTION

A    Description

     Fungi are heterotrophicachylorophyllous
     plant-like organisms which possess true
     nuclei with nuclear membranes and nu-
     cleolL.  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   i
     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 freshwaters 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 microaerophihc 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.6.4.69
                                  II 9-5

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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 truefungalhyphae.
         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) and both seem
   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
  II 9-6

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                                  PLATE I
                      "SEWAGE FUNGUS" COMMUNITY
                       (Attached "filamentous" growths)
                                                                           Fungi
   Zoogloea
                                         Sphaerotilus natans
                              Beggiatoa alba
                                                        BACTERIA
Fusarium aqueductum
                                                    Leptomitus lacteus
                              Geotrichum candidum
                                                          FUNGI
//   Epistylis   8
                                                                       10
                                                               Opercularia

                                                                 PROTOZOA

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Fungi
                                                            PLATE  II
                                                 REPRESENTATIVE  FUNGI
       Figure       •*•
       Ftuarium aquacductuum
       (Radlmacher and
       Rabcnhorst) Saccardo
         Microconidia (A) produced
       from phialides u in  Ctphalo-
       iporium,  remaining  in ali:
       balls. Macroconidia (B), with
       one  to  several   cross walls,
       produced from collared phial-
       ides. Drawn from culture.


             Figure  3
              Gcotrichiun candidum
              Link ex Persoon
                Mycelium -with  short  cells
              and arthrospores.  Young hy-
              pha (A);  and mature arthro-
              spores (B).  Drawn from cul-
              ture.
    Figure  O
    Achlya americana Humphrey
      Ooogonium with  three oo-
    spores  (A);  young zoospor-
    angium  with delimited  zoo-
    spores (B) ; and zoosporangia
    (C)  with released  zoosporcs
    that  remain encysted in clus-
    ters  at the month of the dis-
    charge tube.  Drawn from cul-
    ture.
Leptomitiu lacleia (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.
            Mycelium with hypbal 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.
                                                                                                                                B.
                                                                                   FIGURE   /    Haplosporidivm costale.  A—mature spore;
                                                                                     B—early  plnsmodimn.
     Figures 1  through  5 from Cooke; Figures 6 and 7  from  Galtsoff.

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                                                                                    Fungi
       sugars and grows most luxuriantly
       in the  presence of organic nitro-
       genous wastes.

    3  Ecological Roles

       Although the "sewage fungi" on
       occasion attain visually notice-
       able concentrations,  the less
       obvious populations of deutero-
       mycetes may be more important
       in the  ecology of  the  aquatic
       habitat.   Investigations of the
       past decade indicate that nu-
       merous 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.
basis of the morphology of the sexual and
zoosporic stage s.  In practical schematics,
however,  numerous fungi do not demon-
strate these stages.  Classification must
therefore be based on the sum total of the
morphological and/or physiological char-
acteristics.  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 pre-
sented on the following pages.
IV  Classification

In recent classification schemes, classes of
fungi are distinguished primarily on the
This outline was prepared by Dr. Donald G.
Ahearn,  Professor of Biology,   Georgia
State College,  Atlanta. Georgia.  30303
                                                                                    II 9-9

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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 comdia-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
              mycehal and very extensive. The outstanding  characteristics of the thallus is a tendency
              to be nonseptate and, in most groups, multinuchate, 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
              Dermocvstidium may be provisionally grouped with  the chytrids    Species of this genus
              cause serious epidemics of oysters and marine and  fresh water fish.

      51       Zoospores anteriorly uniflagellate,  formed inside or outside the sporangium           , .class
                                                                                  . Hyphochytridiomycetes

                   'Lliesc fungi are aquatic (fresh water or marine) chytrid-hke  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-
              stiating cellulose content  Little information is available on the  biology of this class and
              at present it is  limited to less than 20 species.

      6 (41)   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 cocnocytic,  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 thalh 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 parasitical  Members of the family Saprolegniaceac are the common

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                                                                                                       Fungi
         'water molds'  and are among the most ubiquitous fungi in nature   The order Lagemdiales
         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 cndobiotic   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  lacleus may
         produce rather extensive fouling floes 01 slimes in organically enriched waters

6'        Flagella of unequal size, both uhiplash                       class   Plasmodiophoromycetcs

             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  zygospore                   class      7.\ tiomycetes

             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 aie
         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-hke   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)

71        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 iji 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 (21)    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) Deuteromycetes

             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 comdium 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 l

       1        Reproduction by means of conidia, oidia, or by budding	2
       I1        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 (21)    Conidia borne in acervuh  	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
       21        No ballistospores	   Cryptococcaceae

       3        Comdiophores,  if present,  not united into sporodochia or synnemata	    4
       3'        Sporodochia present	      	          . Tuberculanaceae
       3"       Synnemata present                                                             Stilbellaceae

       4 (3)     Conidia and conidiophores or oidia hyaline  or brightly colored    	Monihaceae
       4'        Conidia and/or  conidiophores, containing dark melanin pigment	Dematiaceae

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                                                                                  Fungi
SELECTED1 REFERENCES

Ahearn, D.G., Roth, F.J.  Jr., Meyers,  S.P.
    Ecology and Charact erization of Yeasts
    from Aquatic Regions of South Florida.
    Marine Biology 1:291-308.  1968

Alexopoulos,  J. C.  Introductory Mycology.
    2nd ed.  John Wiley and.Soms,  New York,
    613 pp.  1962

Barron,  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 Knapp, E.  Yeasts
    in Polluted Water and Sewage.
    Mycologia 52:210-230.  1960

Emerson, Ralph and Weston, W.H.
    Aqualinderella fermentans  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.
      1187pp.  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. Bactenol. 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
                                                                                II  9-13

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                        PROTOZOA,  NEMATODES, AND ROTIFERS
I  GENERAL CONSIDERATIONS

A  Microbial 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.

 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 nutriment
   and zoomicrobes adapted to water are
   those that feed on algae.

 E More species and lower densities of
   microbes in  clean  water and fewer species
   and higher densities in polluted water.

 F Pollution-tolerance or nontole ranee 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
    Rotifer a.
 II  BACTERIA

 A No ideal method for studying distribution
    and ecology of bacteria in freshwater.

 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 spp.,  Aerogenes
    spp.,  and nitrogen-fixation bacteria are
    primarily soil dwellers and may be washed
    into the water by runoffs.

 D  E. coli,  streptococci,  and_Cl.  perfringens
    are true indicators of fecal pollution.
HI  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)  - Sub-
          class Phytomastigina dealt with  '
          under algae;  only subclass Zoo-
          mastigina 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) Polymastigma  - with 3 to 8 flagella,
             mostly parasitic in elementary tract
             of animals and man

          4) Hypermastigina - all inhabitants
             of elementary tract of insects
 W.BA.45b. 11.67
                                                                                  n 10-1

-------
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)  Proteomyxa - with radiating
               pseudopodia; without test or
               shell

            b)  Mycetozoa - forming plasmodium;
               resembling fungi in sporagium
               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 u); 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; c/i ;stome
      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; suctona sucking
     through tenacles; bacteriaand small
 II 10-2

-------
                                                          Protozoa, Nemaiodes,  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 encystation.
    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,
         Chromdonda,  Monhysterida,  Enoplida,
         and Trichosyrmgida no papillae on
         male trail, caudal glands absent.
      Orders encountered in water and sewage
      treatment - Free-living forms inhabit at-
      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, caudal
      glands present, mostly bacteria-feeders,
      common genera:  Rhabiditis, Diplogaster,
      Diplogasteroides, Monochoides, Pelodera,
      Panagrellus,  and Turbatrix.

      Dorylaimida  - Relatively large nematodes;
      stylet in mouth, feeding on other nematodes
      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 Tn-
   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 elementary 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.
                                                                                    II 10-3

-------
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, Rhabditis 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 femals,
      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
      Aschelminthes (various forms of worms)
      or as a separate phylum (Rotifera); com-
      monly called wheel animalcules, on
      account of circular movement of cilia
      around head (corona); corona contracted
      when crawling or swimming and expanded
      when attached to catch food.

•   2   Of the 3 classes, 2 (Seisomdeaand
      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, Philodmedae
      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,

 H  10-4
      Keratella,  Monostyla. Trichocerca,
      Asplanchna, Polyarthra. Synchaeta.
      Microcodon; common genera under the
      order Flosculariaceae: Flosculana,
      and Atrochus.  Common genera under
      order Melicertida:  Limnias and
      Conochilus.

   5  Unfortunately orders and families of
      rotifers partly based on character of
      corona 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 femals 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 Philodma,  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.

-------
                                                            Protozoa. Nenv.todes, and Rotifers
 VI  SANITARY SIGNIFICANCE

  A Pollution tolerant and pollution non-
     tolerant species - hard to differentiate -
     requiring specialist training in protozoa,
     nematodes.  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
     numbers of these zoomicrobes; natural
     waters receiving such effluents showing
     significant increase in all 3 categories.

  D Possible Pathogen Carriers

     1  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  free-
        living amoebae parasitizing humans.

     2  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.

     3  Nematodes concentrated from sewage
        effluent in Cincinnati area showing
        live E. coli  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  Sample Bottles - One-gallon glass or
      plastic bottles with metal or plastic
      screw caps, thoroughly washed and
      rinsed three times with distilled water.

   2  Capillary Pipettes and Rubber Bulbs -
      Long (9 in.) Pasteur capillary pipettes
      and rubber bulbs of 2 ml capacity.

   3  Filtration Unit - Any filter holder
      assembly used in bacteriological
      examination.'^'  The funnel should be
      at least 650 ml and the filter flask at
      least 2 liter capacity.

   4  Filter Membranes -  Millipore 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 dechlormating 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 trans-
   ported 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 be
     filtered through a single membrane
     within 15 minutes unless the water has
     turbidity.  At least one gallon of sample
     should be used in a single examination.
     Immediately after the last of the water
     has disappeared through the membrane,
     the suction line is disconnected and the
     membrane placed on the wall of a clean
     50 to 100 ml beaker and flushed re-
     peatedly with about 2 ml of sterile
                                                                                      II  10-5

-------
Protozoa, Nematodes, and Rotifers
      distilled water with the aid of a capillary
      pipette and a rubber bulb.  The concen-
      trate is then pipetted into a clean
      Sedgewick-Rafter Counting Cell and is
      ready for examination.

   2  In concentration of raw water samples
      having visible turbidity, two to four
      membranes may be required per sample,
      with filtration through each membrane
      being  limited to not more than 20
      minutes.  Samples ranging from 500 ml
      to 2 liters may be filtered, depending on
      whether turbidity is high or low.  After
      filtration the membranes are placed  on
      the  walls of separate beakers and
      washed as above.  To prevent the parti-
      culates 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.
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
VIII
         sluggishly motile nematodes may be
         confused with root fibers,  plant fila-
         ments of various types,  elongated
         ciliates such as Homalozoon 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. (10>

      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, W
         Goodey's Soil  and Freshwater Nema-
                 or other books on nematology.
USE OF ZOOMICROBES AS
POLLUTION INDEX
   A 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.
 H 10-6

-------
                                                         Protozoa, Nematodes, and Rotifers
    Can use them on a quantitative basis -
    nematodes, rotifers, 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.
    Most practical method involves the
    equation:  (A + B) /A = Z. P. I., where
    A = number of pigmented protozoa,
    B = other  zoomicrobes,  in a unit volume
    of sample, and Z.P.I.  = zoological  pol-
    lution 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,  or sewage during
    recovery.
K  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 SEWAGE TREATMENT PROCESS -
 TRICKLING FILTERS
 PROTOZOA
    Sarcodina - Amoebae
       Amoeba proteus, A radios a
       Hartmannella Spp
       Arcella Vulgaris
       Noegleria gruberi
       Actmophrys Sol
FLAGELLATA
   Bodo caudatus
   Pleuromonas jaculans
   Oikomonas termo
   Cercomonas longicauda
   Peranema trichophorium

   Swimming type
      Ciliophora:
        Colpidium colpoda
        Colpoda cuculus
        Glaucoma pyriforinis
        Paramecium candatum; P bursaria
   Stalked type
      Opercularia spp. (short stalk
                       dichotomous)
      Vorticella Spp.  (stalk single and
                     contractile)
      Epistylis plicatilis (like opercularia
                        more colonial)
      Carchesium Spp. (like vorticella but
                       colonial)
   Crawling type
      Euplotes patella
      Stylonychia mylitus
      Urostyla Spp.
      Oxytricha Spp.
NEMATODA
   Diplogaster Spp.       Rhabditolaimus Sp.
   Monochoides Spp.      Monhystera Sp.
   Diplogasteroides Spp.  Trilobus Sp.
   Rhabditis Spp.
   Pelodera Spp.
   Aphelenchoides Sp.
   Dorylamus Sp.
   Cylmdrocorpus Sp.
   Cephalobus Sp.
                                                                                 II 10-7

-------
 Protozoa.  Nematodes, and Rotifers
 ROTATORIA

   Diglena

   Monstyla

   Polyarthra

   Philodina

   Keratella
   Brachionus

 OLIGOCHAETA (bristle worms)

   Aelosoma hemprichi

   Aulophorus limosa

   Tubifex tubifex

   Lumbricillus lineatus


 INSECT LARVAE

   Chironomus

   Psychoda Spp. (trickling filter fly)


 ARTHROPODA

   Lessertia Sp.

   Porrhomma Sp.

   Achoratus subuiaticus (collembola)

   Folsomia Sp. (collembola)

   Tomocerus Sp.  (collembola)
 3  Chang, S. L   Proposed Method for Examina-
       tion of Water for Free-Living Nematodes.
       J. A.W. W.A.  52:695-698.  i960.

 4  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

 5  Chang, S. L.  Growth of Small Free-Living
       Amoebae  in Bacterial and Bacteria-Free
       Cultures.   Can.  J. Microbial.
       6:397-405.  1960

 6  Chang, S. L. and Kabler,  P.W  Free-Living
       Nematodes in Aerobic Treatment Plant
       Effluents.  J. W. P. C.F.  34:1256-1261
       1963.

 7  Chitwood,  B G. and Chitwood, M.B.  An
       Introduction to Nematology  SectionT:
       Anatomy   1st ed. Monumental Printing
       Co.  Baltimore.  1950.  pp 8-9.

 8  Cobb, N. A.  Contributions to the Science
       of Nematology VII.  Williams and
       Wilkins Co.  Baltimore.   1918

 9  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)

10  Edmondson,  W. T , et al.   Ward-Whipple's
       Fresh Water Biology.  2nd ed.  John
                         fork
   Wiley It Sons, New
   pp 368-401.
1959
11
Goodey, T.  Soil and Freshwater Nematodes
   (A Monograph) 1st ed.  ivietnuen and Co.
   Ltd.  London   1951.
REFERENCES

1  American Public Health Association,
      American Water Works Association and
      Water Pollution Control Federation.
      Standard Methods for the E xamination
      of Water ana wastewater7 J..un ea.
      New York.
 This outline was prepared by S.  L  Chang, Chief,
 Etiology, Disease Studies & Quality Control
 Laboratories, Water Supply & Sea Resources
 Program,  National Center for Urban & Industrial
 Health.
2  Chang, S. L. , et al.  Survey of Free-
      Living Nematodes and Amoebas in
      Municipal Supplies.  J. A. W. W. A. 52:
      613-618.
II  10-8

-------
                                        Protozoa, Nematodes,  and Rotifers
Effluent
                Insects
             Oligochaetes &
             insect larvae
              Nematodes
              & rotifers
           4-4
          Nonpigmented
            protozoa        *
            I    I  I  fit
         Heterotrophic
           bacteria
              Fungi
              Algae
        Autotrophic bacteria
       Pathogenic organisms"
Suspended organic matter

         (by hydrolysis)
                                 Dissolved organic matter
   (respiration,
    deamination,
    decarboxylation,  etc.)
                                                              Raw Sewage
     Inorganic C, P, N,
              S comp.

 (NH3>  NO". C0°, P)

   (Nitrification, sulfur
    & iron bacteria)
      Food Chain in Aerobic Sewage Treatment Processes
                                                                 II  10-9

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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 and
             natural,  fresh waters - hence,
             frequently encountered in ex-
             amination of raw water.

        2    Cysts not infrequently found in
             municipal supplies  - not patho-
             gen carriers.

        3    Cysts not to be confused with
             those of  Endamoeba histolytica
             in water-borne epidemics.

        4    Cysts useful in evaluation water
             treatment efficiency in remov-
             ing or destroying cysts of path-
             ogenic amoeba.

    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:  Gen-
             era Naegleria, Didascalus. and
             Schizopyrenus - first two being
             flagellate amoebae.

             Family Hartmannellidae:
             Genera Hartmenella (Acantha-
             moeba)

        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 with
             nucleus

        2   Morphology of cysts - Single

 BI.AQ. 14a.4.70
            or double wall with or without
            pores

    D   Cultural Characteristics of Small,
        Free-Living Amoebae

        1   How to cultivate these amoebae

        2   Growth characteristics on plate
            cultures

            3   Complex growth requirements
                for most of these amoebae

    E   Resistance of Amoebic Cysts to
        Physical and Chemical Agents

    F   Practical Use of Small, Free-Living
        Amoebae as Testing Organisms H.
        rhysodes. however,  can grow in or-
        dinary broth.

        1   Culture-induced cysts for study-
            ing removal efficiency of floc-
            culation and sand filtration and
            cysticidal efficiency of water
            disinfectants.
II   FREE-LIVING NEMATODES

    A   Classification of Those  Commonly
        Found in Water Supplies

        1   Phasmidia:  Genera Rhabditis,
            Diplogaster.  Diplogasteroides.
            Cheilobus, Panagrolaimus
         2   Aphasmidia: Genera Monhystera,
            Aphelenchus, Turbatrix (vinegar
            eel).  Dorylaimus, and Rhabdol-
            aimus

    B   Morphological Features

         1   Phassids

         2   Aphasmids

    C   Life Cycle

         1   Methods of mating

         2   Stages of development

                                  II  11-1

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Amoebae and Nematodes in Water Supplies
         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 of 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 nematode-carriers.

    G   Control

         1    Chlorination of sewage effluent

         2    Floeculation and sedimentation
             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 Small Free -
      Living Amoebae in Various Bacterial
      and in Bacteria-Free Cultures".  Can.
      Jour. Microbiol. 6:397-405, 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., etal., "Occurrence of a
      Nematode Worm in a City Water Supply".
      J.A.W.W.A., £1:671-676,  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
      & Hygiene, £: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., 52:695-698,
      1960.

7   Chang, S. L., "Viruses,  Amoebas,
      and Nematodes and Public Water
      Supplies".  J.A.W.W.A., 53:288-296,
      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 Shih L.  Chang,
 M.D., Chief, Etiology, Disease Studies &
 Quality Control Laboratories, Water Supply
 & Sea Resources Program,  NCUIH, SEC.
II  11-2

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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 (euplankton)
      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,
   heptcropods, 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.
 BI.AQ.20c. 4. 70
   (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:

      Codonella fluviatile

      Codonella cratera

      Tintinnidium (usually with organic  matter)

      Tintinnopsis

                                  II  12-1

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  Animal Plankton
III  PHYLUM ROTIFERA

  A Some forma such as Anuraea cochlearis
    and Asplanchna pridonta tend to be present
    at all dmes of the year.  Others such as
    Notholca striata, N. longispina and Poly-
    artnra piatyptera are reported to be essen-
    tiany winter torms.

  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,
   Acroperus, Cerlodapnnia, Bytho-
   trephes,  and the carnivorous
   Leptodora 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
   found by the National Water Quality
   Sampling Network activities.  Eucy-
   clops, Paracyclops, Diaptomus,
   Canth'ocamptus,
   J-.imnocalanus~are other forms
   reported to be planktonic.

   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.
  II  12-2

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                                                                          Animal Plankton
B  Class Insecta

   1  Only a single species of insect can be
      ranked as a true plankton, this is the
      midge fly Chaborus (approx. 8 spp,
      formerly Corethra).

   2  The larva of this insect has hydrostatic
      organs that enable it to remain perman-
      ently suspended in the water.

   3  It is usually found in the depths during
      the daytime, but comes 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 Hydra may become
      detached and float about hanging 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 readily 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
      species 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, and penetrate the human
      skin directly on contact.
D  Phylum Nemathelminth.es

   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 out 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 consid-
      erable numbers 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
                                                                                   II  12-3

-------
Animal Plankton
        weed beds or near shore.  Nekto-
        planktonic forms include Pontoporela
        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 planktotrophic
   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.
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.   221 pp.  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.
II  12-4

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                                                                     Animal Plankton
8  Pennak,  R.W.   Freshwater Invertebrates     10 Welch,  P.S.   Limnology, McGraw-Hill
     of 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.   Chief Biologist,  National Training Center,
     1942.                                     FWPCA,  Cincinnati, OH 45226.
                                                                             II  12-5

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 Animal Plankton
       3/4
       Phylum PROTOZOA

Free Living Representatives
     I. Flagellated Protozoa, Class Mastigophora
    Anthophysis
Pollution to Her ant
    Pollution tollerant
           19/1
                                                    Colony of Poteriodendron
                                                    Pollution tollerant,  35/1
     II. Ameboid Protozoa, Class  Saroodina
   Dimastigamoetoa
 Pollution tollerant
       10-50/i
     Huelearia.reported
    to be intollerant of
    pollution, 45/i-
     III. Ciliated Protozoa, Class Ciliophora
      Colpoda
Pollution tollerant
     20-120 jai
      Holophrya,reported
     to be intollerant of
     pollution, 35/i
        Difflueia
    Pollution tollerant
                    60-500/4
 Bpistylis. pollution
tollerant. Colonies often
maerosoopio.
                                                         H.W.Jaokson

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                                                             Animal Plankton
                       PLANKTONIC PROTOZOA
                       Peranema trichophorum
                Top
               Side
 Chaos
Arcella
vulgaris
Actinosphaerium
                                »

                               &
   , wrcii.cJii

   111
    **5fe'
Vorticella
                           Codonella
                           cratera
                                Tintinnidium

                                fluviatile

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Animal Plankton
                            PLANKTONIC  ROTIFERS
                   Various Forms of Keratella cochlearis
           Synchaeta
           pectinata
Polygarthra
vulgaris
Brachionus
quadridentata
                                Rotaria sp

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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
                                                                        II 12-9

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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 la turn, class Cestoda.  A.  adult as in human
                  intestine;  B.  procercoid larva in copepod; C.  plerocercoid
                  larva in flesh of pickerel (X-ray view).
                                                              H.W. Jackson
II  12-10

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                LABORATORY EXERCISES
General Laboratory Instructions                    15




Types of Algae                                    16




Identification of Diatoms                           17




Identification of Animal Plankton                   18

-------
                        GENERAL LABORATORY INSTRUCTIONS
I  GROUND RULES

A Students will be assigned to specific
   benches or seats.

B Certain items of equipment such as
   microscopes or other instruments
   will be assigned for the use of specific
   students or groups.

C Certain reference books and other items
   will be assigned to a "Central Stores"
   table from which they may be borrowed,
   and to which they should be returned
   when not in use.  The person at whose
   desk they are located at the end of the
   period is responsible for returning them
   to Central Stores.
II   FLASH CARDS

 A  A major objective of certain laboratory
    sessions is to become familiar with a
    given group or groups of organisms.
    This means associating names with
    organisms and/or parts.  One of the
    most effective devices to accomplish
    this objective has been found to be the
    individually prepared "flash card. "

    This consists of a card (3X5.  for ex-
    ample) with a sketch of an organism on
    one side, and its classification written
    on the other side.  For later study, a
    pack of such cards is arranged with all
    sketches up, for  example.  Leaf through
    the pack.  Your sketch will remind you
    of the organism you observed in labora-
    tory or elsewhere. Think its name.  If
    you are in doubt, turn the card over to
    check yourself.  When you have learned
    to recognize all the sketches in the pack
    in hand, turn it over and go over the
    names, recalling in your  mind the
    organisms (sketch) each refers to.

 B  A few sample flash cards will be fur-
    nished.  You are herby instructed to
    prepare additional cards for yourself
    of as many different organisms as
 BI. MET. lab. la. 4. 69
     possible in every laboratory or field
     session where it is appropriate. The
     instructors will be glad to check your
     identification or other items on request
     but cannot take it upon themselves to
     call in or check all cards.  Make fre-
     quent references to the  "Finder. " A
     supply of blank cards will be maintained
     at Central Stores.

 C  Identification represents an expenditure
     of time and effort.  If this achievement
     Is retained on a 3X5 card, then repeti-
     tious time in identification may be saved
     later. Partial identification or even no
     identification can be completed later,
     as one's competence and resource aids
     are enlarged.  Code letters or numbers
     can be used on these unknowns and when
     identification becomes more complete,
     data may be more meaningful.  Date
     collected, location, and selected known
     environmental parameters can enhance
     the usefulness of such a set,  if these
     are added to each card   (see Figure 1).
Ill    LABORATORY SESSIONS

 The laboratory sessions will not all be con-
 ducted in exactly the same way, as several
 different instructors will be involved. Where
 no special procedure is indicated at the end
 of the lecture outline covering a given group,
 the instructor will give verbal instructions
 on the spot.
IV    PRINCIPA L OBJECTIVES OF THE FIRST
      LABORATORY SESSION WILL BE:

 A   Assignment of Places and Equipment

 B   Use of the Microscope

 C   Use of a Biological Key
 These laboratory instructions were prepared
 by H. W.  Jackson, Chief Biologist, and
 Ralph M.  Sinclair, Aquatic Biologist.
 National Training Center, FWPCA,
 Cincinnati, OH 45226.
                                   II  15-1

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                          General Laboratory Instructions
Figure  1

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    Cyanophyta
                                                  fr
    Hormogonales

    Lyngbya	

    Species of this genus have been found to occur
    in surface waters, attached to substrates and
    in some cases associated with pollution.   The
    filament of cells is enclosed by a sheath.  Cells
    range from 4 to 20 microns in diameter and 4 to
    10 microns in length.
P.  Arthropoda
C.  Ins e eta
O.  Ephemeroptera  (Mayflies)

Mayflies are common inhabitants of both lakes and
streams.   They are gill breathers which are usually
found crawling about on rocks.  Most genera can be
distinguished from the stoneflies by the presence of
3 caudal filaments.
P.  Rotatoria (Rotifers)
C.  Monogononta
F.  Brachionidae
G.  Keratella
The genus Keratella represents one of the forms of
rotifers which are often found in the plankton.
                             suoporujsui

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                       LABORATORY EXERCISES ON TYPES OF ALGAE
 I   OBJECTIVES
 A  To learn the terminology and techniques
    for identifying the major groups of algae.
 B  To learn to recognize the more common
    genera on sight.
B  A representative sample of the algal group
   under consideration will be distributed.
   Make a wet mount and focus at lOOx on a
   portion of the algal specimen.

C  Using the algae key in your manual follow
   through the identification with  the in-
   structor, making notes and drawings of
   important characteristics.
II  MATERIALS
 A  Compound microscopes equipped with
    10.20,40, and lOOx objectives and
    mechanical stages.
 B  Microscope lamps, glass slides,  cover
    glasses,  pipettes, algal samples, iden-
    tification keys, and supplemental books.
Ill  PROCEDURE
D  A discussion will accompany and follow
   the identification pointing out the most
   important characters and how they were
   used in the classification.
   This same procedure will be followed
   with several other algal specimens
   which illustrate the major morphological
   forms within a certain group.
F Unknown samples will be distributed for
   identification by the class.
 A A short preview of the characteristics of
    the specific group will be presented.
   A discussion of the classification and
   importance of the specimen will follow
   each identification.
                                                    This outline was prepared by M.E.
                                                    Bender,  Former Biologist,  FWPCA
                                                    Training Activities,  SEC.
  BI. MIC. cla. lab. 11.4.70
                                                                                     II 16-1

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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. "
       What is the predominant color of the
       diatom?  How many plastids ?  In
       diatoms,  the  identification is based al-
       most entirely on the characteristics of
       the cell wall.

       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 image an end
       view or cross(transverse) section view
       would be like.

       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.
                                                                                II  17-1

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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 Bntton,  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.
 II 17-2

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                   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).
    From time to time, however,  animals
    appear (holozoic or nonchlorophyll bearing
    forms), and the ability to recognize 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
        (Ib) 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
  BL MIC. cla. lab. 5b. 10. 66
                                   II 18-1

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Laboratory:  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 (labeled "III C")
   as to phylum and class, and then genus
   and species if possible (do not spend
   undue time on the species without
   assistance).

   1  Make a sketch of at least one organism
     of each phylum observed as an example
     of a type.
     Review the collection of "Explano-
     mounts. " Sketch and study any with
     which you are not familiar.
     Work each one through the keys as  if
     you did not know its name.
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,
 Chief Biologist,  National Training Center,
 FWPCA, Cincinnati, OH 45226.
 II  18-2

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CHAPTER El




TECHNIQUES OF PLANKTON METHODOLOGY






Techniques of Plankton Sampling Programs




Preparation and Enumeration of Plankton in the Laboratory




Calibration and Use of Plankton Counting Equipment




Preparation of Permanent Diatom Mounts




Determination of Odors




Collection and Interpretation of Biological Lake Data




Determination of Plankton Productivity




Methods of Measuring Standing Crops of Plankton




Aerial Reconnaissance in Pollution Surveillance
 1




 2




 3




 4




 5




 6




 8




 9




10

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                       TECHNIQUES OF PLANKTON SAMPLING PROGRAMS
I  INTRODUCTION

A  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.

B  A well-planned study or analysis of the
   growth pattern ot 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 trouble maker approaches, the
      frequency of analysis may be increased.

 C  Detection of a bloom in its early stages
   will facilitate more economical  control.
II  FIELD ASPECTS OF THE ANALYSIS
   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
      if best.
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.
 BI. MIC. enu.9g. 3.70
                                                                                    III  1-1

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  Techniques of Plankton Sampling Programs
          A standardized vertical haul however,
          can be useful for routine comparisons.
Ill   RECORDS

  Field conditions greatly affect the plankton,
  and a record thereof should be carefully
  identified with the collection.

  A  Time of day, turbidity of the water, and
     relative cloudiness affect the amount of
     light, which affects the vertical distribu-
     tion of many forms.

  B  Water temperature affects growth rate and
     behavior.

  C  Wind causes water drift,  and wave action
     breaks up colonial forms  and disperses
     accumulations.

  D  Other conditions may be usefully recorded
     if circumstances permit.
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% formaline is time-
    tested and widely used.  Formaline 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 sometimes useful.
 E  A special thimerosal preservative was
    developed by the FWPCA Water Pollution
    Surveillance System which has proved very

    satisfactory and is described in reference
    No.  2.
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.
 REFERENCES

 1  Hutcheson, George E.  A Treatise on
      Limnology.  John Wiley and Co. New
      York.  1957.

2  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.
 3  Lackey,  J. B.  The Manipulation and Count-
      ing of River Plankton and Changes in
      Some Organisms Due to Formalin Pre-
      servation. Public Health Reports, 53:
      2080-93.  1938.
   Ill 1-2

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                                                 Techniques of Plankton Sampling Programs
4  Olson,  Theodore A. and Burgess,
      Frederick J.   Pollution and Marine
      Ecology.   Interscience Publishers.
      364pp.   1967.

5  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.

6  Weber, C. I.   Methods of Collection and
      Analysis of Plankton and Periphyton
      Sda Samples in the Water Pollution
      Surveillance System.   App. and
      Devel. Rep. (AQC Lab., 1014 Broadway,
      Cincinnati, OH 45202)  19 pp.  1966.
7  Welch, P.S.   Limnological Methods.
     The Blakiston Co., Phila.  Toronto.
     1948.

8  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,
Chief Biologist,  National Training Center,
FWPCA, Cincinnati, OH 45226.
                                                                                  in 1-3

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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 establishment
   of a permanent or semi-permanent program.

B  Concentration or sedimentation of preserved
   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 THIMEROSAL
   PRESERVATIVE

A  The Water Pollution Surveillance System
   of the FWPCA has developed a modified
   thimerosal preservative.  (Williams, 1962;
   Weber. 1967) Sufficient stock to make an
   approximately 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 Thimerosal Preservative
    1  Thimerosal is available from many
      chemical laboratory supply houses;
      one should  specify the water soluble
      sodium salt.

    2  Thimerosal stock: dissolve approximately
      1 gram of sodium borate (borax) and
      approximately 1 gram of thimerosal in
      1 liter of distilled water.
       The amount of sodium borate and
       thimerosal may be varied slightly to
       adjust to different waters, climates,
       and organic contents.
     3  Prepare a saturated aqueous Lugol's
       solution as follows:

       a Add 60 grams of potassium iodide (KI)
         and 40 grams of iodine to 1 liter of
         distilled water.
       Prepare the preservative solution by
       adding approximately 1.0 ml of the
       Lugol's solution to 1 liter of
       thimerosal 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.
   approx.
   approx
   approx.
                       10X(16 mm)
                       20X(8 mm)
                       40X(4 mm)
                       95X(1 8 mm)(optional)
 BI.MIC.enu.l5e.6.68
                                    III  2-1

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 Preparation and Enumeration of Plankton
        A 4OX 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

        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
        expensive for routine plant use.

      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 hemacytometer,
        the Lackey drop method, or by use of
        an inverted microscope.

      Previous to starting serious analytical
      work, the microscope should be
      calibrated as  described elsewhere
      Dimensions of the S-R cell should also
      be checked, especially the depth

      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.
      The organisms in a predetermined
      number of microscope fields or other
      known area are then observed, and by
      means of a suitable series of multiplier
      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,
      introduce 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 identified,
        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
 III 2-2

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                                               Preparation and Enumeration of Plankton
     in the center of the Whipple 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
     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
      extension of the separate field count
      A considerable increase in accuracy
      has recently been shown to accrue by
      emptying and refilling the S-R cell,
      after each group of fields are counted
      and making up to 5 additional such
      counts.  This may not be practical with
      high counts.

   7  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 count-
      ed instead of separate isolated fields.
      Marking the bottom of the cell by evenly
      spaced cross lines as explained elsewhere
      greatly facilitates counting.
      a When the count obtained is multiplied
        by the ratio of the width of the strip
        counted to the width of the cell, the
        product is the estimated 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
   8  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.

D Differential or qualitativej'counts" are
   essentially lists of the kinds of organisms
   found.

E Proportional or relative counts of special
   groups are often very useful.  For example,
   diatoms.  It is best to always count a stan-
   dard number 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.

                                 Ill 2-3

-------
 Preparation and Enumeration of Plankton
      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

      A method for  chlorophyll extraction
      involves filtration, drying for 24 hours,
      extraction with methyl alcohol, and
      evaluation in a colorimeter or acetone
      extracts, using a spectrophotometer
      This is widely used in "productivity"
      studies.

      The membrane (molecular)  filter has
      a great potential,  but a generally accept-
      able technique has yet to  be perfected.
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.
      a  Bacteriological techniques for
         cohform 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 predilection 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
   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
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  25_(4):739-744. 1943.

3  Goldberg, E D , Baker, M ,  and Fox,  D. L.
      Microfiltration m Oceanographic
      Research Sears Foundation. Jour.
      Mar  Res  jj_- 194-204  1952

4  Ingram, W. M. , and Palmer,  C M.
      Simplified Procedures for Collecting,
      Examining,  and Recording Plankton in
      Water.  Jour. AWWA.   44(7).617-624.
      1952.

5  Jackson,  H. W.  Biological  Examination
      (of plankton) Part III in Simplified Pro-
      cedures for Water Examination.  AWWA
      Manual M 12. Am. Water Works Assoc.
      N.Y. 1964

6  Lund, J W G. , and Tailing, J.F.
      Botanical Limnological Methods  with
      Special References to the Algae.
      Botanical Review.  23_(8&9)- 489-583.
      October,  1957
  III 2-4

-------
                                                  Preparation and Enumeration of Plankton
7 Weber, C.I.  The Preservation of Plankton
     Grab Samples. Water Pollution
     Surveillance System,  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)
  Wohlschlag, 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.
Chief Biologist,  National Training Center,
FWPCA, Cincinnati, OH 45226.
                                                                                    Ill 2-5

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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 Whippie
   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 W hippie
   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. 4. 70
                                      in 3-1

-------
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  20X 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.
E   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 mmj 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.
                                    q
    The volume of such a strip in mm  is:

       Vj =  50 X width of field X depth

          =  SOXwX 1

          =  50 w

    In the  example given below on the plate
    entitled Calibration Data, at a magnification
    of approximately 200X 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  (mm )

 B Calculation of Multiplier F actor

    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 tinres the volume of the strip
    examined (V}) would  be contained in 1 ml or
     1000 mm 3.    Thus  in the example given
    above:
                                    3
       ,-,         volume of cell in mm
              volume examined in mm
             1000     1000
          =  	  =  2775
= 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
 III 3-2

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                                          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 1()OX, 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.).
                                                                                     Ill 3-3

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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) Whipple 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.
 Ill 3-4

-------
                                          Calibration and Use of Plankton Counting Equipment
   strip counted.  Thus for two strips in the
   example cited above:

     V=  100W  =  100 X 0.55 = 55 mm3

           1000  _  1000 _ 1n  9
     F2=  -V             18'2
   It will however be noted that F  =  -j- .


   Likewise a factor F_ for three strips
               F,
   would equal -*- 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 mnv* 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:

                =  36.36  or approx. 36
      (as above)

   If two strips are counted:
          andF2 =

                     = 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 18 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 Seal*
           [•.2 mm
                                       Enlargement of Micrometer Scale
                                                                                       III 3-5

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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 car efully unscrewed,  held in the left
            hand,  and the Whipple disc,  held in the right hand.   (Photo by
                                         Don Moran).
HI 3-6

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                                             Calibration and Use of Plankton Counting Equipment
                                CALIBRATION OK WHIPPLE SQUARE

                              as seen with 10X Ocular and 43X Objective
                               (approximately 430X total magnification)
                 Whipple Square as
                 seen through ocular
                  ("Whipple field")
                                                      "Small •quarei" subtend
                                                      one fifth of large squares:
                                                      .0052 mm or 5. 2w
    "Large square" subtends
    one lenth of entire Whipple
    Square: . 026 nun or 26n
                                                             Apparent lines of sight
                                                             subtend . 26 mm or 260p
                                                             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 43 OX (10X ocular and 43 X
objective).
                                                                                           Ill 3-7

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Calibration and Use of Plankton Counting Equipment
MICROSCOPE CALIBRATION DATA


                Microscope No
                                                                     79
Approximate
Mortification
Tube
Length, or
Interpupillary
Setting
Linear dimensions of Whipple
squaies in millimeters*
Whole
Large
Small
Factor for
Conversion
to count/ ml
100X. obtained with (2 S-R Stripe)
Objective
Serial No.
mil, 421/10$
and Ocular
Serial No
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                                    (2 S-R Strips)
Objective
Serial No.
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and Ocular
Serial No.


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               400X, obtained with
                                  (Nannoplankton)
                                  (cell-20 fields )'
Objective
Serial No.
IM/Ai/A&x}
and Ocular
Serial No.
/i? 9^7*4 (fox)


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                *1 mm = 1000 microns
   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
BI.AQ.pl. 8b. 7.66

HI 3-8

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                                     Calibration and Use of Plankton Counting Equipment
                          MICROSCOPE CALIBRATION DATA
                              i

                                             Microscope No.
Approximate
Magnification
Tube
Length, or
Interpupillary
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


























200X, obtained with (2 S-R Strips)
Objective
Serial No.
and Ocular
Serial No.
400X, obtaine
Objective
Serial No.
and Ocular
Serial No.


























(Nannoplankton)
d with (cell- 20 fields )

























     *lmm = 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.
                                                                             in 3-9

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Calibration and Use of Plankton Counting Equipment
                     S-R COVER
                       GLASS	1
                         WATER IN   1MM
                          S-R CELL
                                                 I—TMir ICKICCC C_
                                                   THICKNESS S-R SLIDE
                                         Figure 7

      A cube of water as seen through a Whipple square at IOOX 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.
Ill 3-10

-------
                                         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:
                              3
     _      volume cell in mm
           volume examined in mm*

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.

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=  side2 X depth X no. of fields

        =  1.13 X 1.13 X 1 X10 = 12.8 mm3

     The multiplier factor is obtained as
     above (Section IV A):
                              3
  volume cell in mm1"

volume examined in mm

      = (approx.) 78
      F4 =
      (If one field were counted, the factor
      would be 781, for 100 fields it would
      be 7.8.)
   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= .54mm3
                                     and  F   =
                 = (approx. ) 1850
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.
      13th Edition. Am. Public Health Assoc.
      New York. 1970.

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.
                                                                                    Ill 3-11

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Calibration and Use of Plankton Counting Equipment
                         Area
                       Uncounted
          _ Blrtpt.
            Counted
                                         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.
                                         o
                                          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,
Chief Biologist,  National Training Center,
FWQA,  Cincinnati, OH  45226.
 m 3-12

-------
                      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 F

      b  7000 F

   5  Disposable pipettes

   6  3X6X1/4 inch steel plate
UI  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 distiDed 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.l
                                                                                     III 4-1

-------
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  180° F 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 700° F
     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 F.
   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.
Ill 4-2

-------
                               DETERMINATION OF ODORS
I  INTRODUCTION

Odor shall be determined substantially as
prescribed by the llth edition of "Standard
Methods for the Examination of Water and
Sewage", subject to certain stipulations and
modifications made necessary by the Inter-
national Joint Commission.

The procedure and technique to be followed
are described below.
II   REAGENTS AND APPARATUS
 A  Odor-free water1 - prepared by passing
    tap water through activated carbon at a
    slow rate of speed.  Activated carbon
    can be placed at the bottom of a 20-liter
    glass bottle.  The bottle can be connected
    to the tap by rubber tubing leading to glass
    tubing above the water.  The  outlet from
    the bottom of the bottle should be glass
    tubing.   A trap made of inch glass tubing
    filled with activated carbon is placed at
    the end of the outlet.
    500 ml glass-stoppered Erlenmeyer
    flasks, each flask with a  number.  Glass-
    ware must be thoroughly  cleaned and
    rinsed several times with odor-free
    water before each use.
 C  Chemical Thermometer (0-100°C)
    10 ml Mohr pipettes,  25 ml graduated
    cylinders, 50 ml graduated cylinders,
    200 ml graduated cylinders, 500 ml
    graduated cylinders.  Other pipettes
    and cylinders as needed.
    One liter glass-stoppered bottles to hold
    samples of water being examined. Other
    glass bottles and flasks as needed.
                                                Ill   PRECAUTIONS
Certain conditions are required to
obtain consistent results.  Considerable
practice with the test is desirable to
develop consistent sensitivity to the
sense of smell.

1  In view of the perishability of the
   odor test, these determinations
   should be made immediately after
   collection.

2  The prepared odor-free water should
   be truly free of all detectable odor.

3  All glassware must be free  of odor.
   This is accomplished by thorough
   cleansing followed by several rinses
   with odor-free water.

4  All dilutions should be compared with
   an odorless standard.  This aids the
   observer in deciding whether air odor
   is present or not.

5  All dilutions when examined for odor
   should be of a uniform temperature,
   deviation not to exceed 1°C.

6  A sudden change in the character of
   the odor during the testing procedure
   should be considered as a warning
   that there may be interference from
   outside odors or that the diluting
   water may not be odor-free. The
   character of odor should always be
   recorded for future consideration.
To eliminate psychological influences,
the samples should be coded and in-
termixed so as not to suggest to the
observer what odor concentration is
being observed.

1  Bottles should be colored or covered
   with odor-free  material or the observer
   blindfolded to eliminate auto suggestion
 WS. TO. lab. la. 1.66
                                                                                   III 5-1

-------
 Determination of Odors
       since many samples may possess
       color or turbidity.

       Test should be conducted in a room
       free of outside odors.  The observer
       should be cautioned to refrain from
       smoking or eating for an appreciable
       time before taking test.  Odors should
       be washed from the hands prior to
       taking test.

       The test should not be prolonged to a
       point where the sense of smell becomes
       fatigued.
IV  PROCEDURE

 A To obtain the approximate range of odor
    value take 50 ml, 14 ml and 5 ml of sample
    and make each sample up to 200 ml with
    odor-free water. Compare the odor  of
    these three with 200 ml of odor-free
    water.

    1  Cold odor:  Bring dilutions to temper-
       ature of 24 -  25°C.

    2  Shake each flask uniformly before
       smelling for odor.   Observer should
       characterize  type of odor.

    3  Note which flasks contain odor and
       which do not.  According to results
       obtained, prepare intermediate di-
       lutions,  in each case using sufficient
       odor-free water to make a total
       volume of 200 ml.
      Include a flask with 200 ml of odor-
      free water with each series,  as a
      blank for comparison.
B  Arrange flasks so that their identity is
   unknown and bring to desired temperature.

   1 Observe for odor and make chart with
     a "plus" or "zero" for each dilution.

   2 The results are  reported in "threshold
     odor numbers".   The threshold odor
     number is calculated from the amount
     of sample in the most diluted portion
     which gives perceptible odor.  The
     volume of the dilution (200 ml) divided by
     the  volume of the sample in the dilution
     equals the threshold  odor number.  For
     example,  if 5 ml diluted to 200 ml is
     the  most dilute portion giving perceptible
     odor:
        200
                40, the threshold odor is
                numbered 40.
  The threshold odor number shall not be
  confused with the "threshold odor con-
  centration".  The threshold odor concentra-
  tion is the  smallest amount of odor-producing
  material in mg/1 required to give perceptible
  odor.  If the threshold odor concentration is
  known,  that value multiplied by the threshold
  odor number will give the concentration of
  the odor-producing material in the sample.
                                                  This outline was prepared by E. L.
                                                  Robinson,  Research Aquatic Biologist,
                                                  Fish Toxicology Laboratory, 3411 Church
                                                  Street, Newtown, OH  45244.
 Ill 5-2

-------
                                                Determination of Odors
     ROBERT A. TAFT SANITARY ENGINEERING CENTER
                      AQUATIC BIOLOGY
           ALGAL THRESHOLD ODOR EXPERIMENT
Amount of Culture

Age of Culture	
No. Cells per ml_
Mixed. Unialgal, Pure
ml    Exp. No.
days   Temp. Tested at_

       Culture Medium_

       Date	

  Recorde r	
Observer No.
Observer
Flask
No.










Culture
No.










Dilution
No.










Threshold Odor No.
Description of Odor
1

R












2

R












3

R












4

R












5

R












6

R












7

R








i



          +  =  Odor Detected

   Remarks
   Estimated Composite* T. O.  No.
                                          O  =  No  Odor Detected
 *Geometric average of  T.O. No. of individual observers     E.L. R.  1956
                                                                  III 5-3

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           COLLECTION AND INTERPRETATION OF BIOLOGICAL LAKE DATA
 I   INTRODUCTION

     A   The innumerable interrelationships
         of an organism with its environment
         are most complicated within the
         environs of a lake.  The impact of
         these interrelationships upon a
         lake sampling program and upon the
         data collected must be recognized.

     B   Lake Sampling and Data Collection
         Entails:

         1   A definition of the problem.

         2   A determination of the types of
            samples necessary to point to
            a solution.

         3   A delineation of sampling sites.

         4   A judgment on the number of
            samples necessary.

         5   A decision on the proper time
            and periodicity of sample collec-
            tions.

         6   Some knowledge or understanding of
            the science of limnology.

     C   In any lake sampling program the
         primary interest  is in the relation-
         ship of the aquatic organism with
         its environment,  and to fully
        appreciate this, one must have as
        a prerequisite some knowledge of
        the  science of limnology.
 II  LIMNOLOGY - The study of inland waters

    A   Definitions of common terms:

        1  Epihmnion - the turbulent super-
           ficial layer of a lake lying above
           the thermocline which does not
           have a permanent  thermal strati-
           fication.
 2.  Thermoclme - the layer of water
     in a lake between the epilimnion
     and hypolimnion in which the
     temperature exhibits the greatest
     difference in a vertical direction.

 3   Hypolimnion - the deep layer of a
     lake lying below the thermocline
     and removed from surface in-
     fluence.

 4   Littoral - shorward region of a
     body of water; waters edge to the
     lake ward limit of rooted aquatic
     vegetation.

 5   Sublittoral - from lakeward limit
     of rooted aquatic vegetation to
     the level of the upper limit of the
     hypolimnion.

 6   Profundal - all of the lake floor
     bounding the hypolimnion below
     the light controlled limit of
     plant growth.

 7   Limnetic or Pelagic - region of
     free water in which green plants
     are  present only as phytoplankton.

 8   Benthic - the region of the shore
     and the bottom of waters.

 9   Trophogenic layer - the  superficial
     layer of a lake in which organic
     production from mineral sub-
     stances takes place on the basis
     of light energy.

10   Tropholytic layer - the deep layer
     of the lake where organic dissimilation
     predominates because of light de-
     ficiency.

11   Oligotrophic - waters with a small
     supply of nutrients and, hence, a
     small organic production.
W. RE. Ik. 3. 10.66
                              III 6-1

-------
 Collection and Interpretation of Biological Lake Data
         12  Eutrophic - waters with a good
             supply of nutrients and, hence,  a
             rich organic production.

         13  Dystrophic - bog-type lakes con-
             taining yellow-brown water and
             much humus.

        14,  Liebig's  law of the minimum  -
             the yield of a plant or animal
             is determined by the quantity
             of that particular necessary
             substance which is present in
             minimal amounts as determined
             by the demands of the organism.

        15.  Seiches - oscillations of the
             water level--standing waves.

        16.  Standing crop - the total amount
             of biological growth present in
             the water on a selected date.
 IH  FACTORS WHICH INFLUENCE SAMPLING
     AND DATA COLLECTION

     A   Physical Features

         !„  Temperature

            a   Lakes are warmed in spring
                principally 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,
               biological activity,  and time
               of day

               (1)  Effective length of daylight
                   dimemshes with the depth
                   of the lake.
     6   Wind velocity

     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 sun-
            light

        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 quantity of
           aquatic  life.  Biological
           production is determined
           primarily by the nutrients  in
           solution in the water, and an
           increase in basic fertility will
           increase biological activity.

        b   Basic suppliers  of nutrients
           include  tributary streams,
           precipitation from the atmos-
           phere, and interchange with
           lake bottom sediments.

C   Biological Communities

    1   The littoral community composed
        of rooted vegetation, attached
        algae,  fish, and a host of inver-
        tebrates

    2   The limnetic community composed
        of fish and plankton
III 6-2

-------
                                      Collection and Interpretation of Biological Lake Data
       3   The benthic community composed
           of midge larvae, sludgeworms,
           fingernail clams, and other bottom
           dwelling organisms
IV THEORY AND PRACTICE OF LAKE
   SAMPLING

    A   Purpose of Program

        1   To point toward a logical and
            satisfactory solution to the
            specific problem.

        2  To  correlate the physical,
            chemical, and bib logical pheno-
            mena.

        3   To reach an understanding of the
            interrelationships of the biota
           with the environment.

    B   Types of samples

        1  Chemical

           a   Grab

           b   Composite

           c   Other

        2.  Biological

           a   Plankton

           b,   Benthic organisms

           c.   Fish

           d   Littoral vegetation

           e,  Bottom ooze

           f   Core samples of lake bottom

   C   Possible sample locations

       1   Lake inlets and outlets

       2   Random vertical samples in
           limnetic region away from  littoral
           influence.
         3   Samples collected at intervals
             on line transecting lake basin.

         4.  Individual  "grab"or composited
             samples from similar regions
             and /or depths of lake.

         5   Selected sites to define a specific
             problem.

      D   Number and periodicity of collections

          1  The use of statistics is a valuable
             and necessary tool both in analyzing
             the data and in determining the
             number of samples necessary.

          2  The question,  "How many samples
             must I take?", is one of the more
             difficult ones facing the investiga-
             tor.  In reality, the availability
             of funds, personnel,  and time are
             often the determining factors in a
             sampling program.
  REFERENCES:

  1  Birge, E.A.,  and Juday, C.,  The Inland
       Lakes  of Wisconsin.  The Plankton. I.,
       Its Quantity and Chemical Composition.
       Wis. Geol. Nat. Hist. Sur.  Bulletin 64,
       Science Series 1.  No. 13,  222 pp.  1922.

  2  Coker, Robert E., Streams,  Lakes,
       Ponds.  The University of North Carolina
       Press, Chapel Hill,  327 pp. 1954.

  3  Hutchmson,  G. E., A Treatise on Limnology.
       John Wiley and Sons,  Inc., 1,015 pp.
       1957.

4  Mackenthun, K. M., Ingram, W. M., and
      Ralph Forges.  Limnological Aspects  of
      Recreational Lakes, DHEW,  PHS Publi-
      cation No. 1167, 1964.
5   Needham,  J. A., and Doyd, J. T., The
      Life of Inland Waters.  Comstock
      Publishing Company, Ithaca, New York,
      1937.

6   Reid, George K. Ecology of Inland Waters
      and Estuaries.  Remhold Publishing
      Corporation, New York, 375 pp. 1961.
                                                                                Ill 6-3

-------
Collection and Interpretation of Biological Lake Data
Ruttner,  F.,  Fundamentals of Limnology.
  University  of Toronto Press., 242 pp.
  1953.

Standard Methods for the Examination
  of Water and Wastewater.  American
  Public Health Association,  llth Ed.,
  626 pp., 1960.
                                               10  Welch, P. S.,  Limnology. McGraw Hill
                                                     Book Company, Inc., 471 pp., 1935.

                                               11  Welch, P. S.,  Limnological Methods.
                                                     The Blakiston Company,  Philadelphia,
                                                     381pp.,  1948.
   Symons,  J. M., Weibel. S.R.,  and Robeck,
      G.G.  Influence of Impoundment on Water
      Quality. DHEW, PHS Publication No.
      9999 - WP - 18.  1964.
                                            This outline was prepared by K. M.
                                            Mackenthun, Biologist, formerly with
                                            Technical Advisory and Investigations
                                            Activities,  FWPCA, SEC.
Ill 6-4

-------
                     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 Og.  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
                   -  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
 BI. ECO. pro. la. 4.70
                                   HI 8-1

-------
 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 On - Final "dark" bottle O0 =
       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 0%, resulting in
    erroneously high respiration and low net
    photosynthesis values.

 B  The lower limit of sensitivity of the  Winkler
    Method is 0. 02 mg O% /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 £.  Steemann
 Nielsen (1952).  The method is simple and
 very sensitive.

 A Carbon-14 labelled sodium bicarbonate
    (4 - lOuc /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 jipore 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
  HI  8-2

-------
                                                     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  Steemann Nielsen, E.  The Use of Radio-
      active Carbon (C-14) for Measuring
      Organic Production in  the Sea.   J. Con.
      Internal.  Explor. Mer. 18:117-140.   1952.

9  Strickland,  J. D. H.  Measuring the
      Production of Marine Phytoplankton.
      BuU. Fish. Res. Bd.  Can. No.  122:
      1-172.   1960.
10  Verdum.  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.  Intern at.
      Rev. Hydrobiol. 49:1-61.   1964.

13  Yentsch,  Charles S.   The Measurement
      of Cnloroplastic 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,
 1014 Broadway,  Cincinnati,  OH 45202.
                                                                               HI  8-3

-------
                 AERIAL RECONNAISSANCE IN POLLUTION SURVEILLANCE
 I  INTRODUCTION

 A Definition

   The word "reconnaissance" is derived from
   the word "reconnoiter" which means to
   conduct a preliminary examination or survey.
   Its earlier applications to engineering and
   military requirements has been expanded
   to include photomapping and interpretations
   of natural resources.  Aerial reconnaissance
   can be defined as "airborne examination or
   survey procedures performed by heavier-
   than-air craft,  lighter-than-air craft,  or
   earth orbiting satellites. "

 B Types of Aerial Reconnaissance

   1  Visual

      Examination of the flight path by a human
      observer with no provision for permanent
      recording for later study.  This form
      of aerial observation has limited use but
      in many cases can complement  the other
      forms.

   2  Image forming sensor

      Image recording of the covered flight
      path where maximum advantage can be
      made of image interpretation  techniques.
      Image forming sensors includes such
      instruments as cameras, infrared
      scanners, and radar.

   3  Nommage forming sensors

      Nommage forming sensors include such
      devices as oscilloscopes, strip charts,
      and dial indicators which directly indicate
      parameter differentials as received by
      the sensing elements from the target  scan.

   4  Combination  image forming and nonimage
      sensors

      It is advantageous at times to  combine
      both of these techniques in order to
      rapidly interpret flight path imagery.
     This advantage is shown in Figure 1
     where a heated effluent discharge is
     traced to a canal by infrared imagery
     and indicated by the "lighter" plume
     which parallels the shoreline.  The
     oscilloscope phototracing indicates the
     temperature differential of the  discharge
     point as compared to the offshore bay
     area.

C  Detected images can be identified by one
   or more of the following criteria.

   1 Size of image

   2 Shape of image

   3 Tonal qualities of image

   4 Image profile shadowing

   5 Location of image

   6 Texture patterns  of grouped images

   7 Spatial  relationship of image to
     surrounding bodies

D  Selection of Remote Sensing Method

   1 A knowledge of the electromagnetic
     spectrum (Figure 2) is important in
     order to select the most advantageous
     method of remote sensing. It will be
     noted that there are transmissibility
     and sensitivity limits as well as existing
     film limits with respect to equipment
     wave length patterns.

   2 Remote sensing methods can  be
     categorized into two areas.

     a  Source active:  Utilization of an
       instrument which is capable of
       emitting a source of energy which
       is transmitted to the target area and
       emanations are received whose
       characteristics are dependent upon
       the nature of the specific target.
WP.SUR.fm.6.8.69
                                   HI 10-1

-------
Aerial Reconnaissance in Pollution Surveillance
        An example of such a device is the
        Radar set (Figure 3) or special
        Infrared Emitters both of which are
        capable of being utilized in total
        darkness.

     b  Source passive: Utilization of
        instrumentation which is only capable
        of receiving target area emanations.
        Such remote sensing instruments,
        therefore,  do not transmit artificial
        energy sources but depend upon the
        sun as a source of energy which is
        selectively received and individually
        reflected by each object in the target
        area.  Aerial photography and most
        of the remote sensing is by this
        method and the included regions of
        the  electromagnetic spectrum are the
        upper portions  of the UV band to the
        near infrared region.

     Selection of desired wavelength is
     dependent upon the optimum emission
     capability of the instrument which has
     the shortest wavelength possible to
     sharply differentiate small objects while
     being long enough to preclude excessive
      energy "scattering" and yet not long
      enough so that photographic recording
      is impossible.
II   TECHNIQUES

 A  Radar(RAdio Detection And Ranging)

    Radar is particularly useful for discerning
    certain types of vegetation which may not
    appear in the best visual quality in
    Panchromatic Color Film (sensitive to the
    entire visible spectrum) and also has all-
    weather and around-the-clock capabilities.
    Radar in the longer wavelength ranges is
    capable of penetrating dense vegetative
    cover and  this advantage has been found
    to be useful in detecting drainage networks
    and geological features.

 B  Conventional Photography

    Conventional photography covers the entire
    range of the visible spectrum  and employs
    both black-and-white film and color film.
    Some of the special films are  capable of
    photographing a portion of the infrared band
    (the "near" infrared region) and this
    capability  is useful in special  situations.

-------
                          Aerial Reconnaissance in Pollution Surveillance
            ELECTROMAGNETIC SPECTRUM
                                                 WAVELENGTH
                               3m             300km

             I  I  I  I  I  I  I   I  I  I  I   I  I  I  I
AMM/
RAY
1 1

1 1
20
10
k uv
X-RAY
1

1
18
10
1 1

1 1
1
S
1
L
E
IR
1 1

r i i
16 14
10 10
RADAR UHF
(MICROWAVE)
1 1 1 1 1

1 1 1 1 1
12 10 8
10 10 10
RADIO
1

1


1

1
6
10
LF
1 1

1 1
AUDIO
1

1
4
10
AC
1 1
FREQ
1 1
2
10

1
UE
1

i
VISIBLE LIGHT (LIMITS OF HUMAN VISION)



A.






                                                         (cps)
THE PHOTOGRAPHIC
WAVE ANGSTROM UNITS
LENGTH
§ § §
°. o o
0. m rx

INFRA
RED
SPECTRUM
(1 Angstrom Unit1
o
0
o
•0
III II
| (Red Orange Yellow





'0 1 Millimicrons = 10 ^
§ §
° °.
i 7 i ii
Green BlueVioletl



Centimeters)
o
0
o
1"

ULTRA
VIOLET


§
0
cJ



TO 11,000 A

TO 12,000 A
  PHOTOTUBE SENSITIVITY

  SPECIAL FILMS	
                             h
               ORDINARY FILM-

         ORTHO FILM	

PANCHROMATIC FILM	
                                      I  TRANSMISSION LIMIT
                                      h—GLASS LENSES
                    FIGURE 2

-------
Aerial Reconnaissance in Pollution Surveillance
                                                                               Target
                                               FIGURE 3
   1 Direct photomapping and panoramic scan

     Figure 4 illustrated the manner in which
     direct photomapping differs from the
     panoramic scanning technique and it can
     be seen that continual attached mapping
     scans can be made of all or portions of
     the flight path to be analyzed.

   2 Multiband photography

     Multiband photography is,  as its name
     implies, the synchronized photography
     of up to nine separate photographs of the
     same target area each of which is
     photographed in a  different wavelength.
     A distinct advantage of this method is
     that, during photo analysis, distinct
     anomalies may be evident between
     objects appearing  in the different bands
  and further investigation may be
  warranted. An example of this is shown
  in Figure 5 where it was observed that
  an  anomaly in vegetative growth was
  apparent in the Infrared wavelength
  while not being apparent in the other
  visual spectral ranges.  Further
  investigations proved that  gravel washings
  in slug intervals had affected normal
  growth of the streamside vegetation.

3 Infrared photography

  Infrared photography has been found
  useful in depicting certain hydrological
  features.  Within the photographic
  ranges it is not a heat-measuring tool
  and therefore cannot detect thermal
  anomalies  within bodies of water but
  instead sharply delineates  shoreline and
  tributary features and has  haze
  penetrating qualities.  The blues appear
  much darker by infrared photography
  and the reds,  greens,  and yellows will
  show up much lighter when compared to
  a panchromatic color photograph.

-------
                                       Aerial Reconnaissance in Pollution Surveillance
Direct
Photomapping
                                       FIGURE 4
                                                                 impaired or
                                                                ' dead vegetation
                                                       normal
                                                        vegetation
                                              Infrared   Photograph
                                     FIGURE 5

-------
Aerial Reconnaissance in Pollution Surveillance
   4 Gamma Ray Spectrometer

     This device functions in the very short
     wavelengths and has been found to have
     excellent detecting capabilities for
     radioactivity.  Such a device would serve
     as an excellent means of searching for
     radioactive waste spills which have
     occurred either accidentally or
     intentionally.

C  Infrared Radiometry

   Infrared imagery is different than infrared
   photography since the tone of the imagery
   is directly related to the infrared radiation
   emitted from the target area.  This type
   of scanner converts the normally
   unphotographic IR band to a thermal
   imagery.  An optical-mechanical device
   is necessary since the normal thermal
   energy emitted by the camera itself will
   influence radiations received from the
   target area and this elimination of detector
   internal radiation is accomplished by
   miniturization and application of extremely
   low temperatures.  One example of the
   usefulness of infrared imagery is shown in
   Figure 6 where the dark areas along the
   shoreline were found to be caused by cold
   water infiltration by a previously unknown
   source.  Such a finding could be indicative,
   for  example, of a new untapped freshwater
   source or a polluting influence from a
   remote  source.  This cold water infiltration
   was not evident by normal visual spectrum
   photography and the IR photograph would
   only indicate a more distinct shoreline.

D  Automatic Analysis

   In the analysis of large numbers of prints
   use can be made of photoelectric scanners
   wherein automatic recording of degree of
   brightness can be recorded on a tape
   printout.  An example of this procedure is
   shown in Figure 7 where each symbol has
   a significance relating to  "tone signature. "
   The "D" zones are the darker areas while
   the  other symbols relate to lighter zones.
   In the center the clear area is a river
   tributary.  Future predictions are that
   automatic encoding can be channeled to
   computers wherein decisions can be made,
   for  instance, in water management policies.
 E Stereoscopic Analysis

    The principles and methods developed
    during World War II for the stereoscopic
    analysis of aerial photographs has been a
    continuing and expanding science. Its
    principles are based upon the fact that the
    human eyes are normally spaced about
    65 millimeters apart and are capable of
    compositing, via the optic nerve, the two
    separate and distinct images viewed by
    each orbit.  Aerial stereoscopic analysis
    depends upon the placing of two  photographs
    precise distances apart with precise
    overlapping as required by normal human
    visual acuity.  With the aid of special
    stereoscopic viewing lenses the viewer is
    able,  in most cases,  to composite both
    of the photographs in the same manner as
    with normal vision and thus a sense of
    depth can be imparted materially enhancing
    the analyzing of the terrain.
Ill  APPLICATIONS

 Aerial reconnaissance in pollution sur-
 veillance can be accomplished in a variety
 of ways and the method chosen usually is
 dictated by costs involved and what is
 desired with regard to immediate or
 potential information.

 A Visual Observations

    The simplest and least expensive method
    is to visually observe the study area and
    this can be useful in determining sampling
    sites,  locating previously undetermined
    tributaries, pinpointing visual gross
    pollutant effluents, etc.  To augment
    these observations it is usually better
    to have a  permanent recording of the
    survey area in the form of photographic
    or imagery recordings.

 B Photographic Techniques

    Photographic evidence, either color,
    black-and-white, or special film,  can be
    recorded  in various attitudes or positions
    of the aircraft  in order to make best use
    of interpretation techniques such as
    stereoscopic analysis.
 Ill  10-6

-------
                         Aerial Reconnaissance in Pollution Surveillance
                       FIGURE 6
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                                         D D D n D n :
             "\v r>    ,,,  -•-"-"- r  D  D X XX  D D D D D D
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              ( cw/ C  tt*£fe •::-   Cp r,D XCC  DD  DD D D
                (  ( )  )  "-::- -" CD  D  D ODD C C DDD   DDL
                       (  -::-::- •;:-::- DDDD   ^ 'JDD D D D D L
                                          ,0
                      v-
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DDDD
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     J
D DDDD'  (
DDDD DDDD   (
D D  D DDiO  ())  )
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  DDDE  X XCCC C  (  )
    D X XXX X  (())((
     **»*c c c  ** 0)  )  )  0)
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        •--"-  C    X  "-  CC ( )   ) ))   )

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               X » D (  )   ( ^ (  ) )
X D D D D D   DDD  D  D  D D D
  X D D D D CCDDD D D D D D
               , DDtD  D  DDDL
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       DDDDD  OCX  DDD  D D D DC
                            D L
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                                             (
                                              3D D
                                               DD D D  D L
                        FIGURE 7

-------
Aerial Reconnaissance in Pollution Surveillance
   1  Current patterns and flow velocity can
      be ascertained by a method developed
      by the U.S. Coast and Geodetic Survey
      whereby powdered aluminum is surface
      distributed to a wide  area and subsequent
      photographic patterns can be analyzed.
      Flowing bodies  can either appear as
      "depressions" or "elevations" depending
      upon direction of flow with relation to
      the photographing aircraft and from these
      "parallax" anomalies the velocity can
      be determined.

   2  Photographic IR has found applications
      in the areas of detecting sharp
      delineations of shorelines and tributaries
      especially when haze is prevalent.

   3  The work of Strandberg  concludes that
      "In aerial photographs, oxygen-deficient
      water frequently appears black,  or at
      least darker in tone and this appearance
      may be caused by the incomplete reduction
      of wastes by anaerobic bacteria. " Thus
      his black-and-white photographs of
      waterbodies with low values of dissolved
      oxygen (DO) following a source of waste
      discharge are characterized by a plume
      discharge followed downstream by dark
      water tones.

      Further values ascribed to photography
      in this manual include color photographs
      dealing with fish kills where the extent
      of damage can be better ascessed and
      the use of "false" color infrared
      photography where algal masses can be
      differentiated and, again, the low DO
      concentration is manifested by a color
      anomaly.

C Imagery Techniques

   1  Utilization of the "mid" and "far"
      infrared wavelengths in the form of
      "imagery" has been found useful in
      analyzing for thermal pollution and its
      concomitant  circulation and diffusion
      patterns for which "thermographs" can
      be described for large water bodies.2
      It is also possible to establish naturally
      occurring patterns of seasonal and
      diurnal thermal ranges for surface waters.
       It has been ascertained, in principle,
       that it is possible to identify pollutants
       by study of spectral emissivity
       characteristics for particular  wave-
       lengths and this can be of value, for
       instance,  when one is searching for a
       particular pollutant and the emissivity
       characteristics for the waterbody can
       be compared with known patterns for
       the specific pollutant. 2

       Utilization of radar and orbiting
       satellite platforms can produce imagery
       of great usefulness.  The radar technique
       can precisely delineate shoreline
       characteristics without vegetative
       interferences and thus  can thoroughly
       image drainage basins.  Satellite
       imagery and photography can obtain the
       same information but on a much broader
       scale and with great rapidity.   Recent
       lines of thought have postulated that this
       orbiting scanner can be fitted with
       computing devices to render decisions
       upon analysis of its constant data
       collection. 3 Such decisions can be of
       immeasurable aid to the water con-
       servationist who may require long
       range predictions based upon analysis
       of whole river basins.
IV  SUMMARY

  Development of aerial reconnaissance in
  pollution surveillance has proceeded in the
  areas of technique and interpretation.  With
  regard to the techniques available most of
  the earlier problems have been overcome to
  a degree where available material is amenable
  to analysis.  Such early problems as pitch
  and yaw of the  aircraft,  cloud cover, imagery
  instrumentation, etc.,  has been overcome
  to a large degree by available engineering
  skills and the need for improvement due to
  military needs. In the area of interpretation,
  however,  much has yet to be accomplished
  to develop this tool as an extension of
  laboratory analysis which it will augment
  rather than replace.
 Ill  10-8

-------
                                           Aerial Reconnaissance in Pollution Surveillance
REFERENCES

1 Strandberg, C.H.  "Aerial Discovery
     Manual. "  John Wiley &. Sons,  Inc.,
     New York.

2 Van Lopik, J. R.,  Ramble, G.S. and
     Pressman, A.E.  "Pollution Surveillance
     by Noncontact Infrared Techniques."
     Jour. Water Poll. Control Fed., 40, 3,
     425, March 1968.
3 Colwell, R. N.  "Remote Sensing of
     Natural Resources. "  Scientific
     American. January  1968.
This outline was prepared by R. Russomanno,
Microbiologist, National Training Center,
FWPCA, Cincinnati, OH 45226.
                                                                                 ffl 10-9

-------
                   LABORATORY EXERCISES
Proportional Counting of Plankton                                11




Calibration of Plankton Counting Equipment                       12




Fundamentals of Quantitative Counting                            13




Class Problem in Plankton Analysis                              14

-------
                  LABORATORY:  PROPORTIONAL COUNTING OF PLANKTON
  I  OBJECTIVE

  To learn and practice the techniques of
  proportional counting of mixed plankton
  samples.
 H  MATERIALS

 A Several plankton samples, each containing
    a number of plankton forms.

 B Class slides, cover slips, and dropping
    pipets.
HI  PROCEDURES

 A Make an ordinary wet mount of the
    sample provided.

 B Scan the slide. Identify and list 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 then enumerate
          the other forms.
       b  Move the slide at random and
          repeat the process. Do this for
          5 or 10 fields.

       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.
 BI.MIC. enu. lab. 6a. 8.69
                                in 11-1

-------
            LABORATORY:  CALIBRATION OF PLANKTON COUNTING EQUIPMENT
 I  OBJECTIVES
 A To Become Familiar with Microscope
    Calibration Procedures

 B To Calibrate the Particular Equipment
    Assigned to you
 II  MATERIALS


 A Whipple, Plankton Counting Reticule

 B Compound Microscope as Assigned

 C Stage Micrometer


III  PROCEDURE
 A Adjust the interpupillary distance to the
    position most comfortable for your eyes.
    and record the setting on the "Microscope
    Calibration Data" sheet.

 B Install a Whipple plankton counting reticule
    in the right eyepiece.

 C Obtain a stage micrometer and focus on the
    scale at 100X magnification.
D  Record the exact dimensions of the entire
   field in the column marked "Whole" on the
   plate "Microscope Calibration Data. "

E  Do the same with the 200X and 400X
   magnifications.

F  Return the stage micrometer to the supply
   table.

G  Values for the "Large" and "Small"
   columns may now be calculated
   arithmetically. There are ten large
   squares across the whole field, and 5
   small squares across the large square
   which is subdivided,  in the center of the
   field.

H  Calculate  the conversion factors to counts
   per ml according to the formulae in the
   lecture entitled "Calibration and Use of
   Plankton Counting Equipment. "
 This outline was prepared by H. W. Jackson,
 Chief Biologist,  National Training Center,
 FWPCA, Cincinnati, OH 45226.
 BI. MET. mic. lab. la. 5. 70
                                                                                     III 12-1

-------
 Laboratory:  Calibration of Plankton Counting Equipment
                            MICROSCOPE CALIBRATION DATA
                                               Microscope No.
Approximate
Magnification
Tube
Length, or
Interpupillary
Setting
Linear dimensions of Whipple
squares in millimeters*
Whole
Large
Small
Factor for
Conversion
to count/ ml
100X. obtained with
Objective
Serial No.
and Ocular
Serial No
200X. obtaine
Objective
Serial No.
and Ocular
Serial No.






(2 S-R Strios)









•










d with (2 s-R Strios)

























.-_„ . (Nannoplankton)
400X, obtained with (cell- 20 fields )
Objective
Serial No.
and Ocular
Serial No.



























      *lmm = 1000 microns
                                                          BI.AQ.pl 8 10. 60.
Ill 12-2

-------
                             Laboratory:  Calibration of Plankton Counting Equipment
                     MICROSCOPE CALIBRATION DATA
                                       Microscope No
Approximate
Magnification
Tube
Length, or
Interpupillary
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


























200X, obtainei
Objective
Serial No.

and Ocular
Serial No.


400X, obtaine
Objective
Serial No.

and Ocular
Serial No.


J with





d with
















-
























(2 S-R Strips)





(Nannoplankton)
(cell- 20 fields )





*lmm = 1000 microns
                                                    BI. AQ. pi 8 10. 60.
                                                                           Ill 12-3

-------
               LABORATORY:  FUNDAMENTALS OF QUANTITATIVE COUNTING
 I  OBJECTIVE

 To learn and practice the basic techniques of
 quantitative plankton counting
 II  MATERIALS
 A Plankton Samples Containing a Variety of
    Plankton Forms

 B 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
    follows:
as
       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.
        C 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 correction 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.
  BI. MIC. enu. lab. 7.6. 68
                                                                                  III  13-1

-------
 Laboratory:   Fundamentals of Quantitative Counting
                                               riANKTON COUNT IECOID
              Body of witcr
                                                  Dau Coltoeud
                                                                        . DIM Amljrnid.
                                   Dopth_

TOTAL COUNT
Difft rvntlal
Count |














Notes. Talleyi
















Total
















Vol, Area,
or Typo of Count
















FIELD CONDITIONS

(feather toda}
Previous Wont her
Turbidity Moth*
Method of Collo
i T MadlBff
•tlan
Total Vol Collaeted
Preservative


Fllaawntoti
Other Plan
Surface 8c
Dead Fl«h
. Alu.




Other "hyilc
al or CIWBlcal Dat



HiRniflcatlon
















LABORATORY
Method of Prc
Dopartun frc
Significance
Tret taw nt Rt
Kultl factor
















ANALYSIS

•1

















per
11 tor

















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O .-.
CHAPTER IV

INTERPRETATION AND SIGNIFICANCE OF PLANKTON


Algae and Actinomycetes in Water Supplies

Algae as Indicators of Pollution

Public Health Significance of Toxic Algae

Odor Production by Algae and Other Organisms

Organic Enrichment and Dissolved Oxygen Relationships
in Water

Plankton in Oligotrophic Lakes

The  Effects of Pollution on Lakes
                   1

                   2

                   3

                   4

                   5


                   6

                   7

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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.
  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.


  At Oklahoma City,  prominent earthy odors
  have appeared frequently. The organisms
  blamed for this trouble are the mold-like
  actinomycetes.

  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.

  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.

  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.  Oscillatona 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.3.70
                                                                                      IV 1-1

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    	Algae and Actinomycetot in Water Supplies
III
       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.
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 actmomycete 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, bryozoans, fresh water
  IV  1-2

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                                             Algae and Actinomycetes in Water Supplies
    sponges, water mites and larval stages of
    various insects.

    Plant forms include algae,  actinomycetes
    and other bacteria,  molds and larger
    aquatic green plants.
 V  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.
                                                                                       IV 1-3

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   Algae and Actinomycetes in Water Supplies
      3  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 Hartley. 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.
    IV  1-4

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ALGAE  IMPORTANT  IN  WATER  SUPPLIES
          TASTE AND ODOR ALGAE

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                PLATE I

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FILTER  CLOGGING  ALGAE
            CHROOCOCCUS
        PLATE  2

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POLLUTED WATER ALGAE
       PLATE  3

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CLEAN  WATER  ALGAE
                                 CLAOOPHOBA
      PLATE 4

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                      SURFACE  WATER  ALGAE
SCENEOfSMUS
                            PLATE  5

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ALGAE GROWING ON  RESERVOIR WALLS
             PLATE 6

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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.
 BLIND. 10a.8. i
                                                                                IV  2-1

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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:  Oscillator la,  Euglena,  Navicula,
     Chlorella,  Chlamydomonas, Nitzschia,
     Stigeoclonium,  Phormidium, Scenedesmus,
     Ankistrodesmus.  Phacus.

  B Species: Euglena viridis, Nitzschia
     palea, Oscillatoria chlorina,
     Oscillator la 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   Brmley, 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 A Igae
         to Pollution.   Folia Limnological
         Scandinavia.   No. 5.   1950.

   4   Fjerdingstad,  E.   Taxonomy and Saprobic
         Valency of Benthic Phytomicro-
         Orgamsms.   Intern.  Rev. Ges.
         Hydrobiol.   50:475-604.   1965.
 5  Hawkes, H.A.   The Biological Assess-
      ment of Pollution in Birmingham
      Streams.  The Institute of Sewage
      Purification, Journal and Proceeding's.
      177-186.   1956.

 6   Kolkwitz, R.   Oekologie der Saprobien.
      Schriftenreiche des Vereins fu"r
      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
      Frisbhwasser - 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 an'd
      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.
   IV  2-2

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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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                    PUBLIC HEALTH SIGNIFICANCE OF TOXIC ALGAE
  I  INTRODUCTION

     A  Increasing interest is being shown
         in toxic algae as a result of a recent
         increase in the number of reported
         cases of animal poisoning, and sus-
         pected cases of direct human poison-
         ing thereby.  Indirect poisoning of
         man from eating poisoned shellfish
         has been well demonstrated, and the
         poisoning of fish (for human con-
         sumption) by algae is suspected.

     B   Two general groups of algae are of
         outstanding importance:

         1,   The blue green algae (Cyanophy-
             ceae).

         2    Certain armored flagellates
             (Dmophyceae).

         3   Although both groups occur in
            both fresh and salt waters, the
            blue green algae alone  are known
            to be toxic in fresh water.  In
            marine waters, the armored
            flagellates predominate but
            other groups are also involved.

     C.   The biochemical nature of algae
         toxins is not well understood. They
         seem in general to be substances of
         low molecular weight.  They are
         capable of producing death  or illness
         in  mammals and fish even when  the
         algae cells themselves have been
         excluded.
 II  TOXIC FRESH WATER ALGAE

    A.  Blue green algae associated with
        toxic outbreaks include the following
        (not all blue  green algae are toxic):

                TABLE I

           Toxic Fresh-Water Algae

        Anabaena
        Anabaena circmahs
        Anabaena flos-aquae
        Anabaena lemmermanni
B
 Anacystis (Microcystis)
 Anacystis cyanea (Microcystis aerugmosa)
 Anacystis cyanea (Microcystis flos-aquae)
 Anacystis cyanea (Microcystis toxica)
 Aphamzomenon flos-aquae
 Gloeotrichia echinulata
 Gloeotrichia pisum
 Gomphosphaena lacustris
   (Coelosphaerium kuetzmgianum)
 Lyngbya contorta
 Nodularia spurnigena
 Rivularia fluitans

 Instances of animal poisoning from
 algae polluted waters are numerous.

 1   Toxicity to domestic animals
    has been reported from around the
    world.

    a   First published  account in
        1878 reported sheep, horses,
        dogs, and pigs dying in South
        Australia after drinking water
        containing a heavy concentra-
        tion of Nodularia spumigena.

    b  Arthur 1886 reported cattle
       in Minnesota dying within  one-
       half hour after drinking algae
       polluted water.

    c   Hatch 1959 has reported stock
       killed and made  ill by drinking
       algae polluted water from  barn-
       yard watering tanks.

    d   Symptoms generally included
       prostration and convulsions,
       followed by death.

2   Chickens, turkeys, ducks and
   geese have also been killed.

3  Dying and decomposing  masses
   of Aphanizomenon flos-aquae have
   killed a variety of fish under ex-
   perimental conditions.

L  Most  of the outbreaks of algae
   poisoning have occurred during
   periods of continuous hot weather
   when the water had a high organic
   content, and when the algae were
BI. MIC.txa.3b.4.70
                                                                                  IV  3-1

-------
Public Health Significance of Toxic Alpae
           concentrated at one end of a lake
           or pond by a gentle wind.

       5   Only a single species of alga has
           been involved in each instance.

       Until recently,  no cases of human
       illness or death have been reliably
       traced to drinking water containing
       toxic algae.

       1   During droughts of '30s widespread
           gastroenteritis in several eastern
           cities was ascribed by some in-
           vestigators to  huge growths of
           algae in rivers used as water
           supplies, even though water was
           run through water treatment
           processes including heavy
           chlonnation.

       2   Recent reports from Saskatchawan
           indicate that heavy blue green algae
           growths there  have seriously m-
           terferred with the recreational
           use of water at certain  resort
           areas.

       3   The toxic material from certain
           algae may survive  the laboratory
           equivalent of water treatment using
           alum coagulation, filtration and
           chlonnation.   It may survive ac-
           tivated carbon treatment  in amounts
           ordinarily used in water treatment
           plants.
Ill TOXIC MARINE ALGAE

   A  Toxic marine algae are found pre-
       dominately among the armored flag-
       ellates.  The greens, blue greens,
       aiid others also contribute toxic
       forms.  The following have been
       listed as significant:

               TABLE  II

           Toxic Marine Algae

       Armored flagellates (Dinoflagellates)

       Cochlodimum catenatum
       Exuviaella baltica
    Gonyaulax catenella
    Gonyaulax polyedra
    Gonyaulax tamarensis
    Gymnodmium brevis
    Gymnodinium veneficum
    Gymnodmium splendens
    Gymnodinium mitimoto
    Pyrodmium phoneus

             Flagellate

    Hornelha marina
    Prymnesium parvum

             Brown

    Egregia laevigata
    Hesperophycus harveyanus
    JViacrocystis pyrifera
    Pelvetia fastigiata

             Red

    Gelidium cartilagmeum var. robustum

             Green

    Caulerpa serrulata

             Blue Green

    Lyngbya aestuarn
    Lyngbya majuscula
    Trichodesmium erythraeum

B   Blooms of planktomic marine algae
    may be of various colors, often
    called  '!Redtide"or  "Red water".

    1   May or may not be toxic.

    2   Toxicity may be due to a true toxic
        fraction or to secondary conditions
        such as oxygen deficiency, hydro-
        gen sulfide liberation on decom-
        position, or associated bacterial
        pollution.

C   Occurrence of Red Tides

    1   Trichodesmium - widespread and
        recurrent patches in the Philippines,
        East IndiAn Archipelogo, and along
        the coast of South America.
 IV 3-2

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                                                 Public Health Significance of Toxic Algae
D
2   Tnchodesmium and dinoflageHates  -
    Red Sea and north-west Indian
    Ocean.

3   Chloromonadmeae (Hornellia
    marina) occurs off the south-
    east and south-west Indian Coast.

4   Chrysophysean flagellate
    (Prymnesium paryum) occurs in
    Denmark and Israeli ponds.

5   Dinoflagellates blooms associated
    with untoward conditions have
    been universally recorded from
    all over the world.

    a  Southern California - Gonyaulax
       polyedra and G. catenella.

    b  Washington State - Gymnodmium
       splendens.

    c  Florida and Gulf of Mexico -
       Gymnodmium brevis, and
       Gonyaulax catenella.

    d  Bay of Fundy - Gonyaulax ta-
       marensis.

    e  Belgium -  Pyrodmium phoneus.

    f  Portugal - Gonyaulax polyedra.

    g  Angola, West Africa - Exuyiaella
       baltica.

    h  Japan - Gymnodmium miki-
       motoi and Cochlodinium
       catenatum.

    i  Sidney Australia - Gonyaulax
       polyedra.

Toxic Dinoflagellates may be present
and cause damage even though not
always present in sufficient numbers
to cause visible red tides.

1    Gonyaulax catenella occurs from
    Southern California to Alaska.
    It is the primary source of the
    poison in shellfish on the West
    Coast.
        a  Organism found in stomachs
           of toxic mussels in consid-
           erable numbers.

        b  Yearly maximun of Gonyaulax
           occurs during and preceding
           the poison in mussles.

        c  Mussles kept in the laboratory
           in clean aerated sea water
           lose their toxicity,  whereas
           if Gonyaulax is present they
           increase in  toxicity.

        d  An acid extract of collected
           organisms contains the poison.

        e  Organisms have been grown
           in pure culture and shown to
           contain the poison.

    2   Gonyaulax Tamerensis is believed
        to be the cause of poisonous shell-
        fish in the Bay of Fundy.

    3   Pyrodinium phoneus is believed to
        be the cause of poisonous shell-
        fish in Belgium.

    4   Gymnodmium yeneficum toxin has
        been  obtained salt-free by dialysis
        and concentrated by evaporation
        under reduced pressure.  Isolated
        from an area near Plymouth,
        England.

    5-   Gymnodmium brevis - Bacterial
        free cultures with concentrations
        from  2. 3 to 4.8  million organisms
        per liter were toxic to three
        species of  fish.

E   Physiological Effects

    1   Gonyaulax  catenella toxin causes
        paraesthesia,  loss of strength in
        mussels of the extremities and
        neck, and death  by respiratory
        failure.  Toxin apparently does
        not harm shellfish,  but shellfish
        may concentrate and store the
        poison in their tissue.  Subsequent
        ingestion of the shellfish by man
        or other animals may cause death.
                                                                              IV  3-3

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Public Health Significance of Toxic Algae
       2,  Gymnodmium veneficium toxin
           may cause massive fish kills.
           Small fish, namely gobies, die
           within 10 minutes in toxic
           cultures.  Action seems to be
           on the nervous system and prob-
           ably is due to respiratory failure.
           Injection of the toxin into the
           dosal lymph  sac  of a frog has an
           immediate paralyzing effect.

       Ecological factors leading to bloom
       are not well understood. It appears
       that high concentrations of nutrients,
       particularly nitrate and phosphate
       must be available.  Temperatures
       must be suitable and there must be
       enough phytoplankton present as an
       inoculum to take advantage of  these
       conditions.

       1   Oceanographic factors

           a.   Water temperature - Gonyaulax
              tamerensis has been shown
              to be most prevalent in the
              Bay of Fundy when the  water
              temperature is between 10
              and 14°C, with an optimum
              of 13.9°C.

          b   Salinity - optimum unknown but
              believed to be a factor.

          c   Nutrients - phosphates, and
              Vitamin B12 have been shown
              to be essential to dinoflagellate
              metabolism.

          d   Turbulence of water.

               1)  Upwellings  of ocean currents
                  along the  California Coast,
                  occasionally and unpredic-
                  tably  bring a supply of
                  critical nutrients to the
                  s urface from abyssal depths.

               2) Tidal turbulence as in  the
                 Bay of Fundy may expose
                 geologic deposits of nut-
                 rients.

               3) Heavy rains on land have
            washed nutrient materials
            into the sea and caused
            blooms in Florida.

 2  Small amounts of sewage pollution
    also may favor growth by contri-
    buting nutrient material.

 3  Associated phenomena

    a   Extraordinary wide and rapid
        variations in the dissolved
        oxygen content of the water
        have been observed during
        and  immediately following
        heavy concentrations of
        Gonyaulax.

    b   Biochemical oxygen demand
        when Gonyaulax are present
        is far in excess of what
        might be explained  by sewage
        or other organic  pollutants.

    c   Dinoflagellate blooms are often
        succeeded by blooms of or-
        ganisms  which prey on them.
        In the Bay of Fundy the ciliate
        protozoa, Fevella,  ebrenbergii
        was  found feeding upon Gony-
        aulax.

    d   Other organisms such as
        diatoms may compete with
        Gonyaulax for  food or light
        and check their growth.

Some of the  complexities of dino-
flagellate ecology are suggested by
the epidemiology of fish poisoning
suffered by  people throughout
tropical regions.

1   It has long been known that some
    species of coral are crammed with
    zooxanthelae symbiotic  algae whose
    role in the corals  economy is still
    obscure.

    a   The dinoflagellate Gymnodmium
       adriaticum has recently been
       isolated from a jellyfish, some
       sea animones and corals,
       G. adriaticum  is not toxic but
IV  3-4

-------
                                          Public Health Significance of Toxic Algae
     other local species may be.

 The parasitic dinoflagellate
 Odinum which lives on fish gills
 or skin should also not be over-
 looked.

 Coral animals and shellfish which
 feed upon free swimming dino-
 flagellates  could also pass their
 poison up the food chain to fish
 and prediators which feed on fish.

 Lyngbya and a few other algae also
 have been suggested as a cause
 of fish poisoning.

 It has  been  suggested  that there
 are as many as eight different
 kinds of fish poisoning, but only
 two or three of these have been
 studied sufficiently to indicate their
 similarity or dissimilarity.

 a   Puffer fish poisoning - very
    similar  to shellfish poison but
    not identical.  Symplons most
    frequently develop in 10 to 45
    minutes and include paresthesia,
    hypersahvation, sub-normal
    temperature, decreased blood
    pressure and a weak pulse.
    The paresthesia gradually
    develops into severe numbness
    and finally terminates in ex-
    tensive muscular paralysis.
    If death  occurs,  it is generally
    within 4  to 24 hours and results
    from a progressive ascending
    paralysis involving the res-
    piratory muscles.

b   Ciguatera poisoning - barracuda,
    snappers and various reef fish
    are most commonly incriminated.
    The poison is fat soluble and on
    this basis appears  to be distinct
   from shellfish poison or puffer
   fish poison.  Symptoms develop
   in from 1 to 6 hours and include
   paresthesia, headache, fever,
   profuse sweating, rapid weak
   pulse, prostration, muscle
   pains, and joint aches.  Initial
   symptoms sometimes may
                consist of nausea, vomiting
                and diarrhea,  and in severe
                intoxications,  paradoxical
                sensory disturbances may
                be present in which hot ob-
                jects may feel cold.  Death
                may occur within a few
                minutes but generally re-
                quires  several days.  Mus-
                cular twitchings,  tremors
                and convulsions are followed
                by death due to respiratory
                failure.

                Gymnothorax poisoning -
                frequently caused by co^sum -
                ing moray eels.  As with
                ciguatera and puffer poisoning,
                the initial  symptoms  include
                tingling and numbness about
                the lips, tongue,  hands and
                feet, sometimes followed by
                nausea, vomiting, a metallic
                taste, diarrhea and abdominal
                pain as  in ciguatera.  The
                characteristic signs, however,
                appear  to be profuse perspira-
                tion, excessive mucus produc-
                tion, rapid respiration, high
               fever, purposeless movements
               and violent convulsions.  In
               severe  intoxication,  death does
               not occur until after 14 to 25
               days.
REFERENCES:

1   Ingram, W. M., and Prescott,  G. W.,
      "Toxic Fresh-water Algae, " American
      Midland Naturalist,  5^:75, 1954.

2   Palmer,  C. M., "Algae in Water Supplies, "
      U. S. Dept. of Health, Education, and
      Welfare, Public Health Service,
      Pub. No. 657,  1959.

3   Hatch,  Ray D., Personal Communication,
      Dept. of Veterinary Medicine, Univer-
      sity of Illinois, Urbana, Illinois, 1959.

4   Dillenberg, H. O., and Dehnel,  M. K.,
      Toxic Waterbloom in Saskatchewan,
      1959, Can.  Med.  Assoc.  J. 83_:1151,
      19GO.
                                                                         IV  3-5

-------
 Public Health Significance of Toxic Algae
 5  Bishop,  C.T., Anet, E.F.L. J., and
      Gorham, P.R., Isolation and Iden-
      tification of the Fast-Death Factor
      in Microcystis aeruginosa NRC-1.
      Can.  J.  Biochem. Physiol.  37:453.
      1959.

 6  McFarren,  E.F.. Schafer,  M.L.,
      Campbell,  J. E.,  and Lewis, K.H.,
      "Public Health Significance of
      Paralytic Shellfish Poison, " Advances
      in Food Research JJ):135, Academic
      Press, Inc., New York,  1960.

 7  Hutner,  S. H., and McLaughlin,
      "Poisonous Tides. "  Scientific
      American,  199:92.  1958.

 8  Ballantine,  D., Abot,  B.C., "Toxic
      Marine Flagellates:  Their Occurrence
      and Physiological Effects on Animals, "
      J.Gen. Microbiol. .16:274, 1957.

 9  Ray,  S.M., and Wilson, W.B., "The
      Effects of Unialgal and Bacteria-Free
      Cultures of Gvmnodinium brevis on
      Fish and Notes on Related Studies
      with Bacteria. " Special  Scientific
      Report - Fisheries No. 211.  United
      States Department of Interior, Fish
      and Wildlife Service, 1957.

10  Habekast, R.C., Fraser, I. M., Halsted,
      B.W., "Toxicology - Observations on
      Toxic Marine Algae, "  J. Washington
      Academy Sci.  45_:101, 1955.

11  Banner,  A.H.,  A Dermatitis-Producing
      Alga in Hawaii.   Hawaii Medical
      Journal .19:35,  1959.
12  McFarren,  E.F., Report on Collaborative
      Studies of the Bioassay for Paralytic
      Shellfish Poison,  J.  Assoc. Offic. Agri.
      Chemists 42:263, 1959.

13  McFarren,  E.F., and Bartsch, A.F.,
      Application of the Paralytic Shellfish
      Poison Assay to Poisonous Fishes,
      J. Assoc. Offic. Agri. Chemists
      43:548, 1960.

14  Ulitzur, S. , Purification and Separation
      of the Toxins Produced by the Phyto-
      flagellate Frymnesium parvum.   Verh.
      Internat. Verein. Limnol.  17:771-777.
      1969.

15  Jackson,  Daniel F. , Algae, Man,  and the
      Environment.  Syracuse Univ.  Press.
      554 pp.   1968.
 This outline was prepared by H.W. Jackson,
 Chief Biologist, National Training Center,
 FWPCA,  Cincinnati,  OH 45226.   Portions
 of this outline were taken from previous
 training outlines by W. M. Ingram and
 E.  F. McFarren.
 IV 3-6

-------
             ORGANIC ENRICHMENT AND'DISSOLVED OXYGEN RELATIONSHIPS
                                         IN WATER
 I  INTRODUCTION

 A Oxygen normally composes approximately
   20% of the atmosphere, but from only 0. 5
   to 1% of unpolluted water by volume
   (9 - 14  milligrams per liter, or parts per
   million).

 B Physically,  oxygen is a colorless gas,
   responsive to the laws of solubility,
   temperature,  etc.

 C The amount of oxygen present can be
   readily determined by  various methods,
   both chemical and physical.

 D Biologically,  oxygen is one of the key
   elements  to all life,  all living organisms
   contain it along with hydrogen and carbon.
   It is the physiological  "complement" of
   carbon  dioxide.

 E When properly interpreted, oxygen con-
   centration can serve as an excellent index
   to water quality.

   1  It is widely present.

   2  It is intimately involved in many
      processes, chemical, physical,  and
      biological.
H   BIOLOGICAL OR PHYSIOLOGICAL
    RELATIONSHIPS (Figure 1)

 A  Photosynthesis is the biological starting
    point for synthesizing living substance out
    of non-living carbon dioxide, water, and
    other materials. In the process, radiant
    energy is adsorbed from the sun (hence it
    is an endothermic reaction) and free
    molecular oxygen is released to the environ-
    ment as long as sufficient light is available,
    i. e., during daylight only.

    1  The conventional empirical formula for
      photosynthesis is a very great sim-
      plification, as many as twenty steps
may actually be involved (like
respiration, controlled by systems
of enzymes).
6 CO_ + 6 H,0 + 673 Cal
     £•      &
    (Energy sunlight)
         chlorophyll
         (enzyme system)
C6H12°6+6°2
 (Simple sugar)
The end product is a basic organic
material known as a simple  sugar
(carbohydrate,  monosaccharide,  of
which "glucose" is an example,
see Figures 1, A) which serves as a
starting point for the elaboration  of
higher organic substances, and which
may also be oxidized or "respired" by
the cell to release the stored chemical
energy.

Synthesis of organic materials (living
protoplasm) from simple sugars and
other substances in bacteria, algae,
and man.

a  Glucose and other simple sugar
   molecules can be combined by
   living organisms such as algae into
   other carbohydrates such as starch.

b  After modification into fatty acids
   and glycerol, fats can be made.

c  Combination with nitrogen and
   other minerals taken in from the
   surrounding water produces amino
   acids.  These can then be combined
   into proteins, the most complicated
   of aU life substances.  Plants  are
   the only organisms that can synthesize
   amino acids completely "from scratch. "
 BI. ECO. 23.5. 70
                           IV  5-1

-------
Organic Enrichment and Dissolved Oxygen Relationships in Water
                                           ENERGY CYCLES

                                            H  W  Jackson


                                    A . THE GENERAL FOOD CYCLE
mineral* in the protoplasm
of algae and other plant*
carbohydrates
fate
protein*
S
k r1
mineral* in
microcruitacea.
iniect larvae,
other animal*
                         a
                             ABS
                   A
                        PHOTO1,
                      CHEMICA
                      FIXATION
Mineral* in
forage fi*h
i

mineral* in
predator*
                                                                  ANIMAL
                                                                 EXCRETION
coz
H20

other
mineral*
P
K
N0j

ANIMAL AND
   PLANT
RESPIRATION
                                               BACTERIAI
                                              lESPIRATIOr
                                         mineral* in
                                         bacterial
                                         protoplasm
                                                                                   a
                         B.   SYNTHESIS AND DEGRADATION OF PROTOPLASM


                    THE ENVIRONMENT
                  3
                  5
               Iengymea of photosynthesU (chlorophyll)
              ^   enayrne* ol respiration


Carbohydrate*: poly*accharlde*_ dlsaccharides** monosaccharide*

Fat*:  fat* and llpid*s= fatty acid* and glycerol ap=i-J    |

Protein.: protein.* feStaSEHr

             \      i       S
             urn and other mineral -
              containing excreta
                                                                  combined with N
                                                                  and other
                                                                  mineral*
                                                                 BI.ECO. 2. 11.55
                                               Figure 1
 IV  5-2

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                        Organic Enrichment and Dissolved Oxygen Relationships in Water
                              TS£ S&eOK-SXYfiEN CYCLE m

                                CARBON DIOXIEtt ,
      RESPIRATION
              PHOTOSYNTHESIS
             E1EMY for L
             PROCESSES
ORGANIC MATERIAL DESTROYED
     (OXYGEN CONSUMED)
     ORGANIC MATERIAL, CONSTRUCTED
           (OXYGEN RELEASE!?
                           ecoto«H,o«
                                                                        BI.EC0.2a.3.58
                                        Figure 2
 4  All of these synthetic or growth
   processes are endothermic and require
   energy to consummate.  This energy
   is obtained by respiration.

 Respiration.   Life processes such as those
 mentioned previously (II A 3, also physical
 movement) are energized by the controlled
 oxidation or "respiration ' of single sugars.
 This is a cellular or physiological process
 and  continues constantly in all living things,
 independently of  light,  as long as life
 persists.

 1  Types of respiration.  Respiration is
   an energy releasing or exothermic
   biochemical process and is to be
   distinguished  from the external bodily
act of "breathing", which is a process
involving a lung, gill, or other organ,
whereby molecular oxygen is brought
inside the body of the organism and
carbon dioxide is  released.  Breathing
is transportation; respiration is
burning or biochemical utilization.

a  Aerobic respiration.  The most
   common form of respiration is
   illustrated by the oxidation of
   glucose,  using free dissolved
   oxygen from the environment.
   This is also the most efficient
   type  since virtually all of the
   chemical energy contained in the
   glucose molecule is released
   (673  Cal.) and  the residue is com-
   pletely mineralized or stabilized to
   carbon dioxide and water.

-------
Organic Enrichment and Dissolved Oxygen Relationships in Water
        Although actually taking place in
        more than twenty steps involving
        as many different enzymes, the
        overall process can be empirically
        represented by the  following
        formula:
C6H12°6
                   6°
        (a simple sugar such as glucose)
        6 CO2 + 6 H2O + 673 Cal
        (enzymes)
               (energy)
     b  Anaerobic respiration.   If free
        dissolved oxygen (DO) is not available
        from the environment, certain types
        of bacteria, fungi, and other orga-
        nisms known as "facultative aerobes"
        can switch to another, less efficient
        type of respiration and still obtain
        energy by breaking down simple
        sugars.  This is known as anaerobic
        respiration.  Some types of micro-
        organisms, the true anaerobes, can
        thrive only under anaerobic conditions.

        The anaerobic respiration of glucose
        involves splitting the molecule with-
        out the use of outside oxygen so as
        to release some of the contained
        energy, but not all.  The remainder
        is still available in the end products.
        A common empirical relationship is
        as follows:
        3 C3H12°6-
                 2 CH3(COOH)2
        (glucose)  (enzymes)  (lactic acid)
        + 2 CH_CH0OH + 2 CH, COOH
              J   Ct           O
        (ethyl alcohol)

        +  59  1/2 Cal
            (energy)
                (acetic acid)
c  Anaerobic respiration of proteins
   and their derivations by micro-
   organisms (known as putrefaction)
   often leads to the production of foul
   smelling end products.  Aerobic
   respiration of proteins (known as
   decay) seldom produces offensive
   odors.  In addition to carbohydrates,
   proteins, fats,  and various other
   natural and unnatural hydrocarbons,
   and other substances can also be
   broken down or respired to rel-
   atively stable end products by
   microorganisms.

The adaptability of life is remarkable.
By the same mechanisms of genetics
and natural selection which have
resulted in the evolution of present
day life on earth, microorganisms
have evolved which are capable of
obtaining energy from, or in other
words,  "respiring" a great variety
of new synthetic materials,  the products
of modern industry.  This is the basis
for the biological treatment of wastes.

a  The types of biological mechanisms
   involved, in general, are known
   and to a degree, understood.

b  Many species or kinds of orga-
   nisms may be involved in the
   stabilization of a single type of
   waste.

Significance of respiration in sanitary
engineering.  The process of respi-
ration uses or "demands" oxygen from
the environment. Any material, such
as sewage, an organic industrial waste,
motor oil, etc., which will support
life of any kind will thus exert (through
the organisms living on it) a "BOD".

a  Respiration has two end  "products":
   energy, by which the organisms
   live; and degraded, mineralized,
   or stabilized material such as
   digested sewage sludge which
   retains little or no energy available
   to organisms.  That is, they have
   no biochemical oxygen demand or
   BOD remaining.
 IV  5-4

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                         Organic Enrichment and Dissolved Oxygen Relationships in Water
          Since it is independent of light, it
          goes on night and day.
IE  PHYSICAL PHENOMENA

 A Solubility of oxygen in pure water varies
    with temperature and pressure according
    to well known laws and values.

    1  We expect to find less oxygen in warmer
       waters and more in colder.  This has
       both  seasonal and geographical
       implications.

    2  Dissolved substances such as salts tend
       to reduce the solubility of oxygen at a
       given temperature (See Figure 3 -
       Oxygen Solubility at Selected Salinities).
           T   B    9   IO  M   12   13  14
                 DISSOLVED OXYGEN pern

          OXYGEN SOLU9ILITY AT SELECTED SALINITIES
                Figure  3
       The concentration of oxygen often is
       expressed as percent of saturation.

       a  Organisms,  however, are affected
          by the real quantities present, and
          actually demand the most oxygen at
          the highest temperature, when the
          solubility is  lowest.

       b  It is therefore more significant,
          where organisms and stream con-
          ditions are concerned,  to express
          concentration as milligrams per
          liter  (or parts per million).
  B The movement or distribution of oxygen
    throughout a water mass is not entirely
    dependent on molecular diffusion, but
    also involves various types of gross
    water movements. Reaeration (or aeration)
    is the transfer of oxygen from the air into
    the water mass.
IV  CHEMICAL FACTORS

 A Chemical toxicity may delay the exertion
    of biological oxygen demands, or the
    release of oxygen by biological mechanisms.

 B Chemical substances may themselves
    demand oxygen from the water.

    1  If oxygen is available, the chemical
       oxygen demands tend to be satisfied
       relatively quickly and hence are likely
       to be a more local problem.

    2  They are independent of any biological
       effect.

    3  They are in addition to biological
       action.
 V  OXYGEN AND EUTROPfflCATION

 A  Diurnal interactions of photosynthesis
    and respiration.  (Figure 6)

    1  Life, growth,  and hence the need for
       oxygen for respiration continue
       twenty-four hours a day, as long as
       life persists.

    2  Since photosynthesis in nature is
       activated by radiant energy from the
       sun, it is  operative only during the
       daytime.  It releases approximately
       twenty times as much oxygen as is
       consumed in cellular respiration,
       however,  so there is a great excess
       left over which diffuses out into the
       surrounding water.  Here it is available
       to biochemical oxidative demands of
       other organisms with the result that
       the  ecological system becomes "aerobic. "
                                                                                    IV 5-5

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 Organic Enrichment and Dissolved Oxygen Relationships in Water
   3   DO's affected by photosynthesis thus
      tend to be highest in the daytime
      (particularly mid-afternoon) and lowest
      at night (3  - 4A.M.).

B  Organic materials from domestic sewage
   or similar wastes are readily available
   to microorganisms.  Assimilation of food,
   and growth and multiplication of the pop-
   ulation can thus begin relatively quickly,
   thus establishing a demand for oxygen for
   respiration.

   1   It takes a significant period of time  for
      a population of bacteria, fungi,  and
      other microorganisms such as protozoa
      to become  established,  after the
      initiation of growth of the few original
      organisms, even under the best of
      conditions.

   2   Dissolved oxygen does not thus disappear
      instantly from the water upon the
      admixture  of sewage, but rather begins
      to diminish slowly.  As the population of
      oxygen consuming organisms builds up,
      the deficit  in the water  increases.  As
      this occurs,  any equilibrium which may
      have existed with the atmosphere is
      destroyed, and the rate of aeration
      through the surface film increases.

      a As the population of  microorganisms
        grows still larger, its rate of increase
        begins to increase (logarithmic
        growth).  Very soon a size of pop-
        ulation is achieved where the demand
        for oxygen exceeds the amount
        which can be supplied through the
        surface film.  The concentration of
        free DO now begins to drop sharply
        and may go to  zero.

      b If sufficient food for the micro-
        organisms remains, anaerobic
        conditions will now prevail.
        Oxygen, of course, continues to
        enter at a maximum' rate through
        the surface film, but is immediately
        used up.  The overall rate of
        oxidation of the waste or food
        material however, is now very low
        (although the total amount may be
large), as surface aeration can
supply but a small portion of the
oxygen needed.

If there is no replenishment of the
food supply by repollution,  the big
population of fungi and bacteria
eventually uses up most of the
available substrate or food,  and
begins to starve.  Growth now
begins to slow down,  and the pop-
ulation eventually drops back to
its original low level.

Meanwhile time  has passed.  An
abundance of fundamental plant
nutrients such as nitrate, phosphate,
potash, etc., have been released
to the water as a result of micro -
bial respiration. Algae begin to
grow on this substrate, and increase
rapidly to tremendous numbers.

Algal photosynthesis thus suddenly
begins to release great quantities
of oxygen in the  daytime.  At first
this is exhausted each night by
respiration of the algae and other
microorganisms, but soon persists
around the clock until the algae in
turn have exhausted their food
supply and return to numbers
normal for an unpolluted stream.

With the above mentioned advent
of free DO from the algae,  the more
efficient aerobic type of respiration
can again be employed by the
microorganisms.  This hastens
the oxidation or  stabilization process,
and leaves behind but a minimum
residue of well mineralized material
to accumulate on the bottom.

It should be mentioned that turbidity
from suspended  microorganisms
and other organic solids frequently
inhibit the establishment of an algal
population until the biochemical
oxidation or stabilization process
has been well started. Turbidity
inhibits algal growth primarily by
shading and suppression of photo-
synthesis.
 IV 5-6

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                        Organic Enrichment and Dissolved Oxygen Relationships in Water
When organic material is dumped into a
body of water faster than it can be oxidized
by the mechanism described above,  it
tends to accumulate on the bottom in a
partially digested condition known as
sludge.
1  Great quantities of nutrient energy are
   still present, as well as a huge popu-
   lation of microorganisms.  There is a
   very great need for oxygen which, if
   available, is avidly taken up by the
   organisms in the surface layers.

   a  Aerobic conditions in the overlying
      waters thus  tend to hasten the
      stabilization of sludge banks by
      providing oxygen for aerobic type
      respiration.

   b  Anaerobic waters, on the other hand,
      tend to preserve the  sludge by
      restricting all organisms to anaer-
      obic types of respiration. It has
      been reported that the speed of
      decomposition begins to be restricted
      at concentrations below 1 ppm.
                    Figure 4
         Non-Pigmented, Non-Oxygen-Producing,
                Protozoan Flagellates
      2 Anaerobic conditions are present
        throughout the sludge mass below the
        surface.  Stabilization is still
        proceeding,  but at a much slower
        rate than if oxygen were present.

   D  In the deeper reservoirs, lakes,  rivers,
      and estuaries thermal stratification may
      develop in the summer.

      1 The epilimnion or upper layer, being
        in contact with the atmosphere,
        receives  a continual replenishment of
        oxygen by aeration processes.

      2 If turbidity is not excessive, photo-
        synthesis by algae (Figure 5) will
        also provide oxygen.

      3 The hypolimnion or lower layer, being
        separated from the atmosphere and
        frequently from the light, tends to
        acquire an excess of carbon dioxide
        and a deficiency of oxygen due to the
        respiratory activities of micro-
        organisms (such as protozoa,  fungi,
        and bacteria).  (Figure 4)
        This effect is heightened if the bottom
        material  is high in organic content
        as  has been shown below.

         Figure 5
Pigmented, Oxygen-Producing,  Algal
          Flagellates
                                                                                     7

-------
 Organic Enrichment and Dissolved Oxygen Relationships in Water
VI  PROBLEMS AND BENEFITS RESULTING
    FROM ALGAE PRODUCTION
    (Figures 4,  5, 6)

 A Problems to man may result when the total
    "primary production" by algae leads to an
    increase in the total organic content of the
    water that interferes with a desired use.
                                                Death of large algal populations may
                                                lead to obnoxious odors through
                                                bacterial decomposition.  Oxygen
                                                deficits may result at any time of day
                                                in this process.  Deposition of masses
                                                of organic sediment or sludge  in
                                                estuaries and back waters may be
                                                considerable.
       This may consist of a high algal
       population that produces a water with
       high turbidity, taste and odor,  or other
       undesirable effect.  High respiratory
       needs may lead to nocturnal oxygen
       deficit.

       Certain algae may cause tastes and
       odors, clog filters; or otherwise inter-
       fere with potable water processing.
                                          B  The primary production of algae can also
                                             serve as a supply of food to consumer
                                             organisms (animals), resulting in
                                             increased production at  several (trophic)
                                             levels:   (Figure 7)

                                                zobmicrobes,  microinvertebrates,
                                                macroinvertebrates,  fishes.
            9.0
                	      (OCTOBER 2, 1957)
  e.o
   2

   i

   0


I i.o
•

4 o.s


  0.0
                                                                (OCTOBER 3, I9S7)
                                                       SOUR RADIATION, MAM CALORIES/
                                                            SO,. CM./MIMUTE
                                                     RESPIRATORY USE AND 0.0. PRODUCTION
                                                            AT THREE DEPTHS
                                                   O.S FT.
                                                                     MEAN
                                                                     RESPIRATION
                      DISSOLVED OXYGEN RELATIONS IN OHIO RIVER  (REACH II, BROMLEY)
                                         OCT. 3,  1067

                                          Figure  6
  IV  5-8

-------
         Organic Enrichment and Dissolved Oxygen Relationships in Water
Mean Surface  Water Productivity Rates of the  Great lakes
       and Other  Freshwater Lakes and Ocean Areas.
Lake or Ocean
and Location
Lake Superior
Lake Hvron
Lake Michigan
Lake Erie
Grand Traverse Bay,
N.E. Lake Michigan
Douglas Lake, Mich.
Bras d'Or Lake,
Nova Scotia, Canada
Brooks Lake, Alaska
Maknck Lake, Alaska
Torne Trask, Sweden
Ransaren, Sweden
Lake Erken, Sweden
Lake Esrom, Denmark
Lake Purest, Denmark
N.E. Atlantic -
near Denmark
North' Atlantic, same
lat. as Great Lakes
Southeast Pacific
Southwest Pacific
North Pacific,
Sub-Arctic Region
Productivity Rate
in mg.C/ m^ /day
16.62
23.04
37.62
175.20
0.34
0.23
23.5
3.2
8.8
6.4
8.8
221.0
1600
2100
21
7.5
3.3
7.2
6.4
Investigator
Parkos (1967-68)
Parkos (1968)
Parkos (19'67)
Parkos (1968)
Saunders e± al. (1962)
Saunders et_ al,. (1962)
Geen and Hargrave (1966)
Goldnan (1960)
Goldman (1960)
Rodhe (1958b)
Rodhe (1958b)
Rodhe (1958b)
Jonasson and
Mathiesen (1959)
Jonasson and
Mathiesen (1959)
Steemann-Nielsen (1960)
Steemann-Nielsen (1958b)
Holmes (1961)
Angot (1961)
Koblentz-Mishke (1961)
                         Figure 7

-------
  Organic Enrichment and Dissolved Oxygen Relationships in Water
   1  Earlier notation cited the release of
      oxygen during utilization of CO9 during
      algal photosynthesis.  This encourages
      fungal or bacterial breakdown of
      pollutants.

   2  Photosynthesis occurs in the presence
      of adequate light and favorable conditions.
      In darkness, the cells continue to
      respire and may consume more oxygen
      than they produced because photo-
      synthesis increases the organic load.

   3  Photosynthesis tends to occur at the
      surface where light intensity is greatest
      (Figure 6). Poor vertical mixing
      would result in stratification of water
      supersaturated with oxygen over oxygen
      deficient water.  Depending upon con-
      ditions, a significant fraction of the
      oxygen could be lost to the atmosphere.

ACKNOWLEDGEMENTS:

This outline contains certain material sub-
mitted by F. J. Ludzack and M. E.  Bender.
REFERENCES

1  Anonymous.  Aquatic Life Water Quality
      Criteria.  Aquatic Life Advisory
      Committee on the Ohio River Valley
      Water Sanitation Commission.   Second
      Progress Report.  Sewage and Ind.
      Wastes.   28(5):678-690.   1956.

2  Anonymous.  Oxygen Relationships in
      Streams.  Proc. of Sem. at R.  A.
      Taft Sanitary Engineering Center.
      October 30 - November 1,  1957.

3  Bartsch,  A.F.  Algae in Relation to
      Oxidation Processes in Natural Waters.
      Special Publ. No. 2.   Ecology of Algae.
      Pymatuning Lab. of Field Biology.
      U.  of Pittsburgh.  Pittsburgh, Pa.
      pp. 56-71.
 4  Carpenter, J.H., Pritchard, D.W., and
       Whaley, R.C.  Observations of
       Eutrophication and Nutrient Cycles in
       Some Coastal Plain Estuaries, in:
       Eutrophication:  Causes,  Consequences,
       Correctives;  pp. 210-221.  Proc. of
       Symposium.  June  11-15, 1967.
       Publ. Nat.  A cad. Sci. Washington,
       DC.   1969.

 5  Custer, S.W.  and Krutchkoff, R.G.
       Stochastic Model for BOD and DO
       in Estuaries.   ASCE.   Jour. San.
       Eng. Div.  Vol.  95.   pp. 865-886.
       1969.

 6  Ketchum, B.H.  Eutrophication of
       Estuaries,  in Eutrophication:
       Causes, Consequences,  Correctives;
       pp.  197-209.   Proc. of Symposium.
       June 11-15, 1967.   Publ.  Nat. A cad.
       Sci.   Washington,  DC.   1969.

 7  Olsen,  Theodore A.   Some Observations
       on the Interrelationship of Sunlight,
       Aquatic Plant Life,  and Fishes.
       62nd Annual Meeting of the Am. Fish
       Soc.  Baltimore.   1932.

 8  Richards, F.A., and Corwin, N.   Some
       Oceanographic Applications of Recent
       Determinations of the Solubility of
       Oxygen in Sea Water.   Limnology and
       Oceanography.  l(4):263-267.
       October 1956.

 9  Ruttner,  Franz.   (Translated by D. G.
       Frey and F. E. J. Fry.)  Fundamentals
       of Limnology.  Univ. of Toronto Press.
       pp. 1-242.  1951.

10  Trues dale, G. A., Downing, Al,  and
       Lowden, G. F.  The Solubility of
       Oxygen in Pure Water and  Sea Water.
       J. Appl. Chem.   5(2):53-62.   1955.

 This outline was prepared by H.W. Jackson,
 Chief Biologist, National Training Center,
 FWPCA,  Cincinnati, OH  45226.
   IV 5-10

-------
                            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  PHYSICA L A ND CHEMICA L CHA RA CTER-
    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 15° C

 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
III  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, A sterionella,
     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+Eugleniaceac
                     Desmidiaceae

        if2.5, eutrophic

     c  Diatom quotient

   Centrales _ if 0-0.2, oligotrophic
   Pennales  "if 0.2-3.0,  eutrophic
  BI.ECO.mic.2. 10.66
                                                                                     IV  6-1

-------
Plankton in Oligotrophic 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 lunar is
                     Tetraeadon spp.
                     Pediastrum spp.
                     Fragilaria crotonensis
                     Attheya zachariasii
                     Melosira granulata
                           Oligotrophic    Asterionella formosa
      Eutrophic
      Pronounced
        Eutrophy
Aphanizomenon spp.
Anabaena flos-aquae
Anabaena circinalis

Microcystis aeruginosa
Microcystis viridis
                                                Mesotrophic
Melosira islandica*
Tabellaria fenestrata
Tabellaria flocculosa
Dinobryon divergens
Fragilaria capucina
Stephanodiscus niagarae
Staurastrum spp.
Melosira granulata

Fragilaria crotonensis
Ceratium hirundinella
Pediastrum boryanum
Pediastrum duplex
Coelosphaerium
  naegelianum
                                                              Anabaena spp.
                                                              Aphanizomenon flos-aquae
                                                              Microcystis aeruginosa
                                                Eutrophic
                                          Microcystis flos-aquae
 IV  6-2

-------
                                                       Plankton ii  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.
                                                                             IV 6-3

-------
en
 i
                      Dominant
                      Phytoplankton
                      Dominant
                      Zooplankton
                                         Raw son.
                                         Great Slave Lake
USPHS,
Lake Superior
Milliard,
Karluk Lake
WPSS,
Lake Tahoe
Melosira islandica
Asterionella formosa
Dmobryon divergens
Ceratium hirundmella
Pediastrum boryanum
Tabellaria fenestrata
Cyclotella meneghiniana
Fragilana crotonensis
Fragilaria capucina
Synedia ulna
Eunotia lunar is
Keratella cochlea r is
Kellicottia longispina
Diaptomus tenuicaudatus
Limnocalanus macrurus
Senecella calanoides
Daphnia longispina
Bosmina obtusirostns
Melosira islandica
Tabellaria fenestrata
Cyclotella kutzmgiana
Melosira granulata
Melosira ambigua
Astenonella formosa
Synedra nana
Scenedesmus spp
Ankistrodesmus spp.
Dictyosphaerium spp.
Keratella cochlearis
Kellicottia longispina

Asterionella formosa
Tabellaria flocculosa
Fragilaria crotonensis
Cyclotella bodanica
Cymbella turgida
Dictyosphaerium spp.
Sphaerocystis spp
Staurastrum spp.
Not reported
Fragilaria crotonensis
Synedra nana
Fragilaria construens
Fragilaria pinnata
Nitzschia acicularis
Asterionella formosa
Kellicottia longispina
Daphnia spp.
Diaptomus tyrelli
Epischura nevadensis

O

B'
O
F.
•8
rt-
n
o
"S-
                                                                                                                                                                         5T
                                                                                                                                                                         fl>
                                                                                                                                                                         CD

-------
                                                         Plankton in OLgotrophic 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 Billiard, 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.  The net plankton of
     Great Slave Lake.   J.  Fish.  Res.  Bd.
     Can.  13:53-127.
 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. Bot. 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 Telling, 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,
 FWPCA, 1014 Broadway,  Cincinnati,  OH
 45202.
                                                                                   IV  6-5

-------
                        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

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  HISTORICAL REVIEW

 The cultural eutrophication of a number of
 lakes in Europe and America has been well
 documented.

 A Zurichsee,  Switzerland
WP.LK. lc.4.70
                                                                                   IV  7-1

-------
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 hantzschii
      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 (40n)
      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 Asterionella appeared, followed
               by Synedra

             3  About 200 years ago, Asterionella
               again became abundant

             4  Asterionella 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

              Chlorides
              Dissolved organics
   Date
       1888
       1916

       1888
       1914
        Value

       1.3mg/l
       4.9 mg/1

       9.0 mg/1
      20.0 mg/1
              Secchi Disk
before 1910
1905 - 1910
1914 - 1928
              Dissolved  oxygen, at     1910 - 1930
                 100 M, mid-summer    1930 - 1942
  Max.

  16. 8M
  10. OM
  10. OM

Minimum
   it
    Min.

    3.1M
    2.1M
    1.4M
100% saturation
  9% saturation
  IV  7-2

-------
                                                             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 Bosmina 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.  Raw son 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
                                                                                    IV 7-3

-------
The Effects .of Pollution on Lakes
            TABLE 2  PARAMETERS COMMONLY USED TO DESCRIBE CONDITIONS
                                                              Oligotrophic Condition

                                                                  > 10 meters

                                                                  < lug/1
                                                                  < 200 ng/1
                                                              near 100% saturation

                                                                  < 1 mg/m
                                                                  < 0.1 mg/1
                                                                  < 500/ml
1  Transparency

2  Phosphorus
3  NO, - Nitrogen
     0
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                 <1
        number of species of Desmids

   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.
 IV  7-4

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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 = 8mg/l. P< 5Mg/l. 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
                      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.
 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
                                    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.
                                                                                      IV 7-5

-------
 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.  661 pp.  1969.
      (Nat. A cad. 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,
 FWPCA,  1014  Broadway, Cincinnati, OH.
IV  7-6

-------
                                                  Copper Sulfate
                                                    Applicator
CHAPTER V


PLANKTON CONTROL


Control of Plankton in Surface Waters

Control of Interference Organisms in Water Supplies

Nutrients: The Basis of Productivity
3

4

5

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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 CuSO4 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. con. lOb. 4.70
                                                                                     V 3-1

-------
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 N2)

      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+) and others prefer
        it in the form of NOs".  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 maxmum 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
        001 mg/l  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.
 V  3-2

-------
                          ATMOSPHERE
      STORM
      WATER
     (SURFACE
     RUN-OFF)
  WASTE
  WATER
(DOM. SEW.
IND. WASTE
                GROUND
                 WATER
                (SPRINGS)
    LAKE
    OR

    RESERVOIR
FIG. I   SOURCES OF FERTILIZING
      MATERIALS OF CONCERN
      IN  SURFACE WATERS
I
cc
                                                                 o
                                                                 o

                                                                 £
                                                                 o
                                                                 p
                                                                 3
                                                                 O
                                                                 n
                                                                 o
                                                                 P
                                                                 o
                                                                 "1
                                                                 tn

-------
CO



£>•
                           ATMOSPHERE
                                           HCO~ + OH
                                                                      o
                                                                      o
                                                                      o
                                                                      !U
                                                                      O
                                                                      C/3
                                                                      c


                                                                      V
                                                                      n
                                                                      ro


                                                                      £
                                                                      p

                                                                      (D
      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. (CorneU 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.
                                                                          V  3-5

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             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.

    2  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.
         Bartch 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, quinonesj 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. 4. 70
                                  V  4-1

-------
 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.
  V 4-2

-------
                                      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
      d  By reducing the amount of fertilizing
        nutrients entering the reservoir

      e  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.
This outline was prepared by C. M. Palmer,
Former Aquatic Biologist,  Biological Treat-
ment Research Activities,  Cincinnati Water
Research Laboratory,  FWPCA, SEC.
                                                                              V 4-3

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                          NUTRIENTS: THE BASIS OF PRODUCTIVITY
 I   INTRODUCTION

 A  Nutrients of importance include macro-
    nutrients:  those needed m large quantities,
    and micronutnents:  those needed in small
    amounts.

 B  These nutrients are important because
    they promote biological responses which
    may interfere with some desired use of
    the water by man.

 C  Other factors (e. g.  temperature, light)
    affect the use of these nutrients and should
    be considered in an evaluation of the effects
    of nutrients upon the ecosystem.
II  Algae, bacteria, fungi and aquatic plants
 are the forms of life which nutrients affect
 most directly.

 A  Algae are of Several Types

    1  Phytoplankton are small algae suspended
       in the water and form the basis of pro-
       ductivity in the  aquatic environment.

    2  Benthic algae are those forms anchored
       to substrates of rock and bottom
       materials.

 B  Aquatic plants are of several types.  In
    general they may be referred to as rooted
    or floating forms.

 C  Heterotrophic bacteria are fungi which
    respond to organic nutrients introduced
    into water.  Autotrophic bacteria may re-
    spond and grow due to inorganic nutrient
    sources.
Ill  BIOLOGICAL LAWS

 A Liebig's "law" of the minimum:  the essen-
    tial material available in amounts most
    closely approaching the critical minimum
    needed will tend to be the limiting.

 B Shelford's "law" of tolerance:  survival
    of an organism can be controlled by the
    quantitative or qualitative deficiency or
    excess with respect to any one of several
    factors which may approach the limits of
    tolerance for that organism.

 C Q10  "law": with a temperature increase
    of 10 degrees centigrade metabolic pro-
    cesses (rates) are approximately doubled.
IV  The process of photosynthesis is the fixa-
 tion of the sun's energy with the production
 of organic matter by plants.

 A The general reaction is given below:
       CO,
  B Chlorophyll contains basicaUy C, O, H,
    N and Mg,  and in general makes up about
    5% of the dry weight of algal cells.
 V  MEASUREMENT OF PHOTOSYNTHESIS

 A Oxygen production can be used as a
    measure of photosynthesis because for
    each mole of CO2 reduced to organic
    carbon one mole of free oxygen is liberated.

    1  The value of O2/CO2 has been found
       experimentally to be 1. 25 rather than
       1.0.

 B CO2 Assimilation

    1  The CO2 taken up by algae does not all
       originate from the dissolved gas.  Some
       algae can use bicarbonate directly as
       a source of carbon.
 W.RE.ntr.2c.4.70
                                    V  5-1

-------
  Nutrients:  The Basis of Productivity
     2  Hence measurement of CO2 uptake from
        water is a complicated problem which
        must consider pH,  HCO^,  and COg"
        concentrations.

  C  Fixation of Carbon-14

     1  The use of C14 as a tracer of C12 in
        plant metabolism and productivity
        estimation has been widely used since
        the early nineteen fifties.

     2  In this method a known amount of C14
        is added to the water and after a period
        of time the proportion of C*-4 in the
        plant cells to C14 added is found.  The
        amount of carbon assimilated is then
        estimated from the following equation.
         activity of
       phytoplankton
        activity of
              added
(K)  =
total carbon
assimilated
total carbon
 available
     3  Where K is a constant relating to the
       slower uptake of C14.

     4  The total carbon available is determined
       chemically.

  D  Uptake of Mineral Nutrients

     1  The measurement of depletion of
       nutrients in solution has been tried
       but found unreliable.

  E  Chlorophyll

     1  The quantity of chlorophyll present has
       been found to bear some relation to
       productivity but not a  reliable one.
VI  Nutrients of significance in the growth and
 production of algae and plants are discussed
 below.

 A Carbon

    1  Sources

       a  Gaseous CO2

       b  HCOl
      c  CO-

      d  Other carbon compounds

   2  Effects of the removal of carbon upon
      the water

      a  Lowered pH

      b  Deposition of CaCO,

   3  The quantity of carbon available is
      great and it usually is not a limiting
      factor.

B Nitrogen

   1  Nitrogen can be taken up by most algae
      as either ammonium salts or as nitrates.
      Nitrites can also be used but a high con-
      centration is usually inhibitory.  Some
      blue green algae can fix atmospheric
      nitrogen.  Certain algae varieties
      require supplementary amines, growth
      factors, etc.

   2  The quantity of  nitrogen in waters has
      definitely been shown to limit algal
      populations.

C Phosphorus

   1   Phosphate seems to be the only inorganic
      source of this nutrient.

   2   Limiting concentrations of P have been
      found to range from . 05 ppm at a mini-
      mum and an inhibitory affect if P con-
      centrations exceed 20 ppm.

   3   Optimum concentrations have been found
      to range from .018 ppm to 15 ppm.

   4   Storage of inorganic phosphate by algae
      has been demonstrated.  The extent of
      this storage may reach 80% of the total
      phosphorus in algal cells.

D Silicon

   1   Nutrient ratios in the algal cells of
      some areas have been found to be Si 23:
      N 16: PI. It can be seen from this
      ratio that silicon is an important ele-
      ment in algal growth.
 V  5-2

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                                                    Nutrients: The Basis of Productivity
   2  Silicon is especially important in the      VII
      population growth of diatoms and may
      be the limiting growth factor in these
      populations.                              A

E  Inorganic micronutrients - Many elements
   are needed in very small quantities by
   algal cells.  Some of these have a known
   function in algal metabolism; others do
   not.

   1  Mg is a cation of major importance in
      the chlorophyll molecule.

   2  Co is known to be necessary for vitamin
      B12-

   3  Mn is necessary  for several enzyme
      systems.

   4  Mo, V, Zn,  and Cu are necessary  but
      these functions are not as well known.

F  Organic Micronutrients

   1  Of 179 algal strains investigated about
      40% required vitamin supplementation
      for optimum growth.   Principal growth
      factors that were not synthesized in
      sufficient quantity are given as follows
      along with the percentage of the vitamin     B
      deficient strains  showing marked pro-
      ductivity gain after supplementation:
             addition increased growth on
             of the strains.
      b  Thiamin addition increased growth
        on 53% of the strains.

      c  Biotm addition increased growth on
        10% of the strains.

      Algae  can use and may require many
      organic compounds depending upon
      environmental conditions and  the ability
      of the  organism to synthesize required
      building blocks from mineral  forms of
      C.N. & P.  This is  an area for con-
      tinued investigation  with many unappre-
      ciated or vaguely understood ecological
      factors.
PROBLEMS AND BENEFITS RESULTING
FROM ALGAE PRODUCTION

Problems to man may result when the
total "primary production" by algae leads
to an increase in the total organic content
of the water that interferes with a desired
use.

1  This may consist of a high algal popu-
   lation that produces a water with high
   turbidity, taste and odor, or other
   undesirable effect. High respiratory
   needs may lead to nocturnal oxygen
   deficit.

2  Certain algae may  cause tastes and
   odors,  clog filters; or otherwise inter-
   fere with potable water processing.

3  Death of large algal populations may
   lead to tastes and/or odors through
   bacterial decomposition.  Oxygen
   deficits may result at any time of day
   in this process.  Deposition of masses
   of organic sediment or sludge may be
   considerable.

4  Other problems might be cited.

The primary production of algae can also
serve as a supply of food to consumer
organisms (animals), resulting in increased
production at several (trophic) levels:

   zodmicrobes. microinvertebrates,
   macroinvertebrates,  fishes.
    Earlier notation cited the release of
    oxygen during utilization of CO2 during
    algal photosynthesis.  This encourages
    fungal or bacterial breakdown of
    pollutants.

    Photosynthesis occurs in the presence
    of adequate light and favorable condi-
    tions.  In darkness,  the cells continue
    to respire and may consume  more
    oxygen than they produced because
    photosynthesis increases the organic
    load.
                                                                                   V  5-3

-------
  Nutrients: The Basis of Productivity
        Photosynthesis tends to occur at the
        surface where light intensity is greatest.
        Poor vertical mixing would result in
        stratification of water supersaturated
        with oxygen over oxygen deficient water.
        Depending upon conditions, a significant
        fraction of the oxygen could be lost to
        the atmosphere.

        Increased productivity may result in
        temporary reduction of the free dissolved
        nutrient level in the water but harvesting
        at some level is essential to prevent
        later recycle.
VIE   CYCLE OF NUTRIENTS

   A  Once nutrients enter a body of water they
      are cycled through a food chain.

   B  Factors affecting this food chain (e.g.
      toxicity, removal) will affect the con-
      centration and distribution of the nutrients.
2  Lewin, Ralph A.  Physiology and
     Biochemistry of Algae.  Academic
     Press.  1962.

3  Odum,  Eugene P.   Fundamentals of
     Ecology.   W.B.  Saunders Co.  1959.

4  Odum,  H. T.   Primary Production in
     Flowing Waters.   Limnology and
     Oceanography.   1(2):102-117.
     April 1956.

5  Ryther, John H.  The Measurement of
     Primary Production.  Limnology and
     Oceanography.   l(2):72-84. April 1956.

6  Verduin,  Jacob.  Primary Production in
     Lakes.  Limnology and Oceanography.
     1(2):85-91.  April 1956.
   ACKNOWLEDGEMENT:

   This outline contains certain material
   submitted by F. J.  Ludzack and H.W. Jackson.
   REFERENCES

   1  Golterman, H.L. and Clymo, R.S.
        Chemical Environment in the Aquatic
        Habitat (Proc. of an IBP - symposium,
        Amsterdam and Nieuwersluis Oct. 1966).
        322pp.  (N.V. Noord-Hollandsche
        Uitgevers Maatschappij,  Amsterdam.
        8.95).
This outline was prepared by Michael E.
Bender,  Biologist, Formerly with Training
Activities, Ohio Basin Region, SEC.
    V  5-4

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CHAPTER VI

RELATED STUDIES


The Problem of Synthetic Organic Wastes

Beneficial Aspects of Algae

Behavior of Radionuclides in Food Chains -
Freshwater Studies

FWPCA Responsibilities for Water Quality Standards

Marine and Estuarine Plankton

Attached Growths (Periphyton or Aufwuchs)

Artificial and Related Substances - References
2

3

4


5

6

7

8

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                     THE PROBLEM OF SYNTHETIC ORGANIC WASTES
I  Sources of organic chemicals in water
are varied and of differing complexity. * ^
A  Natural pollutants,  such as algae, actino-
   mycetes, etc. contribute to organic
   pollution.

   1  Tastes and odors associated with these
      materials are probably  not merely a
      result of decomposition, but are closely
      associated with materials produced
      during the life cycle of the organisms
      and plants.

   2  Discharge of nutrients in the form of
      phosphorus and nitrogen compounds
      from domestic or other wastes fre-
      quently stimulate the  production of
      natural pollutants.
B  Industrial  wastes, due to the rate of
   population increase and industrial ex-
   pansion,  have made the problem of
   effective water treatment an acute one in
   many places.

   1  The production of synthetic organic
      chemicals has risen steadily over the
      past years,  representing many new
      and complex products - and of im-
      portance to us - new and complex wastes.

   2  The ideal method  of handling industrial
      waste is  at its source.

      a  However, what is often considered
         good  treatment, still results in
         materials present in sufficient
         quantities to affect the taste and odor
         of water.

      b  Many problems are caused by  slug
         discharges, often accidental.
C  Domestic wastes in various stages of
   treatment.
 D  Miscellaneous sources also contribute to
    the problem.

    1  Wastes from private and commercial
      boats.
    2  Chemicals applied to the land may be
      washed into streams.

    3  Chemicals applied directly to water.

      a  Evaporation control

      b  Killing off rough fish

      c  Aquatic plant control
II   Concentrations of organic chemicals
 in water, even in comparatively minor
 quantities may cause difficulties
 A  Wastes may contain from a few mg/1 to
    several hundred mg/1 of organic
    contaminants.
 B Surface waters may contain from a few
   jjig/1 of organics to several mg/1.

   1  Some of the chemicals isolated from
      water,  along with the concentrations
      which can be detected by odor,  are:'^'

                             Concentration
    Substance
                            Detectable*,
Formalde hyde
Picolmes
Phenohcs
Xylenes
Refinery hydrocarbons
Petrochemical waste
Phenyl ether
Chlorinated phenohcs
50,
500
250
300
25
15
13
1
000
- 1,000
- 4, 000
- 1, 000
- 50
- 100

- 100
 ^Concentrations were determined by taking
 the median of 4-12 observations.
CH.OTS. 40a. 4.70
                                                                                     VI  2-1

-------
 The Problem of Synthetic Organic Wastes
III  The damaging effects of organics in water
 are becoming more apparent.

 A Taste and odor in water is usually the first
    noticed effect from organics.  This  is a
    serious public relations and economic
    problem; it also may be a health problem.

 B Organic contaminants may interfere with
    coagulation, damage ion exchangers, and
    create chlorine  and  carbon demand.

 C In the stream they may have adverse
    effects  on aquatic forms that support higher
    aquatic life, cause off-flavors in fish flesh,
    or have direct toxic  effects on fish.
IV  The methods of study employed in the
 collection and identification  of organic
 chemicals in water involve physical and
 chemical methods and instrumental analysis.
    The comparatively small amounts of
    organic materials may be concentrated
    by adsorption on activated carbon.

    1  This carbon is then extracted with
       appropriate organic solvents, the
       solvent extract is taken to dry ness,  the
       weighed extract is subjected to solubility
       group separation, and these individual
       groups  may then be analyzed by various
       methods.

    2  Employing the above method on Ohio
       River water,  the following results were
       obtained:*3^
Chemical Group
Water solubles
Ether and water
insolubles
Neutral
Amine
Weak acid
Strong acid
Amphoteric
Loss
%of
Total
20
22
14
4
8
6
10
16
TOC
in (ig/1
860
110
3
575
645
365
5, 000+
--
Relative Odor
Contribution
23
200
4,670
7
12
16
--
B  Chemical separation and analyses may be
   accomplished by means of column chromato-
   graphy, formation by derivatives,  gas
   chromatography,  infrared and ultraviolet
   spectroscopy,  x-ray diffraction,  etc.

   1  Specific organic chemicals recovered
      from river  and drinking waters by
      these methods include: synthetic
      detergents (ABS), phenylether, phenol,
      DDT, aidrin, o-nitrochlorobenzene,
      a-conedendrin, and xylene.

V Some of the types of problems that may
be attributed to organic wastes,  and more
specifically to problems of taste and odor,
may be represented by the following examples:
  VI 2-2

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                                                 The Problem of Synthetic _ Organic Wastes
A  By applying the previously mentioned
   carbon adsorption method, the odor po-
   tential of organic pollutants and the
   dilution necessary to reduce this odor
   potential to a barely perceptible level has
   been determined:'^'
Industry Source
Brewery
Chemical
Corn Refining
Meat Packing
Metal Fabrication
Paint
Pharmaceutical
Refinery
Soap
Cone. Required for
Detectable Odor ng/1
CHC13
Sol. Org.
770
28
1,000
1,200
890
390
290
84
900
Total
Org.
1,400
32
3,600
3,600
1,600
1,000
340
510
1,800
Dilution Factor
CHC13
Sol. Org.
14
11,000
1.4
92
2.8
69
10
780
640
Total
Org.
86
14, 000
2. 1
140
4.8
98
32
760
350
   Of all the organic pollutants that can affect
   the taste and odor of drinking water,
   phenol has been the most extensively
   studied.

   1  The potential sources of this chemical,
      both natural and  synthetic, have  been
      discussed. (5)

   2  The course of chlorination of phenol,
      a common method of treatment in the
      water plant, has been shown to proceed
      by  a process which starts with the pure
      compound (in itself relatively tasteless)
      and proceeds through strong-tasting
      intermediates to tasteless end
      products. (6)
   1970 the petrochemical production on a
   tonnage basis may be equal to 41% of all
   chemicals.

   1  The three principal groups of petro-
      chemicals are the paraffins, the
      naphthenes, and the aromatics.  From
      these, over 200 basic products  are
      manufactured, having thousands of
      subordinate uses.

   2  Correspondingly, more than 100 identi-
      fiable compounds have been found in
      waste streams from petrochemical
      processes.


BIBLIOGRAPHY
C  The effects of petrochemical wastes^
   on water quality are becoming increasingly
   important, it has been predicted that by
1  Middleton,  F. M.  Taste and Odor Sources
      and Methods of Measurement.  Taste
      and Odor Control Journal. 26:1.  1960.
                                                                                 VI 2-3

-------
The Problem of Synthetic Organic Wastes
 2  Middleton, F.M., Rosen, A.A.,  and
      Burttschell, R. H.  Taste and Odor
      Research Tools for Water Utilities.
      Jour. A.W. W.A.  50:21.  1958.

 3  Anonymous.  Objectionable Organic Con-
      taminants in Water.  San. Eng. Center
      Activ.Rep. No. 25,  1955.

 4  Sproul, O. J., and Ryckman, D. W.   The
      Significance of Trace Organics in Water
      Pollution.  PCF  33; 1188.   1961.

 5  Hoak,  R. D.  The Causes of Tastes and
      Odors in Drinking Water.  Water  and
      Sewage Works.  ^04:243.  1957.
6  Burttschell,  R. H., Rosen, A. A.,
     Middleton, F.M.,  and Ettinger, M. B.
     Chlorine Derivatives of Phenol Caus-
     ing Taste and Odor.  Jour. A. W. W. A.
     5T205.  1959.

7  Gloyna,  E.F., and Malina, Jr.,  J. F.
     Petrochemical Wastes Effects on
     Water. Indus. Water & Wastes, 7_:
     #5.  134.  Sept.-Oct.    1962.

8  Baker, R. A.  Problems of Tastes and
     Odors. WPCF. 33:1099.  1961.

 This outline was prepared by R. L.  Booth,
 Chief, Analyses Unit, Analytical Quality
 Control Laboratory, 1014 Broadway,
 Cincinnati, OH  45202.
 VI 2-4

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                      BEHAVIOR OF RADIONUCLIDES IN FOOD CHAINS
                                  FRESH WATER STUDIES
INTRODUCTION

Fresh water lakes and streams,  in their
traditional roll of receiving and transporting
all manner of materials,  seem destined to
continue this function in the atomic age and
have radionuclides.  In many cases the water
may serve as an effective medium through
which the nuclides can become incorporated
into living organisms.  Ultimately some of the
radioactive materials may be deposited in fish
or crops which are eaten  by man and thus
provide an additional source of radiation
exposure.

This discussion is primarily directed toward
the mechanisms through which aquatic forms
may accumulate radioactive materials from
their environment and the manner in which we
may expect certain nuclides to become
distributed in biological systems of fresh
water lakes and streams.  In a broad sense,
the subject of the behavior of radionuclides in
fresh water food chains might also include
irrigated crops, and animals which drink from
streams and ponds.  Such problems,  are how-
ever, more closely allied with terrestial
studies and in this case consideration will be
given only to those forms  which actually live
in the water.

SOURCES

Fresh  water streams may receive radioactive
materials from a great  variety of sources.
Naturally occurring radioisotopes removed
from the atmosphere and  eroded from radio-
active  ores  have been carried in both surface
and underground waters since early in the „„„
history^ the earth.  Typically,  U238.  Ra   ,
and Th   are present in  measurable amounts,
but other isotopes such  as K   and C    are
also  present.  The background levels are  in
the range of 10 ...to 10    |ic/ml for most
surface waters    but values in excess  of
10  uc/ml have  been reported for certain hot
springs of high radium content.   In most
streams the natural concentration of the heavy
metals is so low and their uptake by aquatic
forms  is so small that they contribute but
little to the  radioactive  content of the organisms.
Most of the natural radioactivity in the biota
originates from K   which, in fish,  amounts
to about 3 X10'6 jic/g.

With the creation of a demand for large amounts
of uranium as a basic source of nuclear energy
the uranium ore refining industry has expanded
rapidly during the past decade.  As pointed
out by  Tsivoglou,  et al.  '  , the concentrations
of radioelements of the uranium-radium
family are substantially increased in streams
which receive the effluent directly from
uranium mills or seepage from ponds used to
retain  their tailings. The  concentration of
radium in the biota of the streams is likewise
increased.

Liquid wastes from the fabrication of refined
uranium into fuel for reactors can be another
source of naturally radioactive isotopes to
streams.   In comparison with releases at the
mills and with the problems which accompany
irradiation of the fuel, however,  the  contribu-
tion from the fabrication plants will usually
be quite insignificant.

The large quantities of artifically radioactive
nuclides produced in reactors form a new type
of disposal problem, the handling of which
requires a technology considerably more com-
plex than that associated with the natural
isotopes.   Dependent upon  individual  design,
the reactors themselves  may or may not
contribute significant quantities  of radio-
nuclides to fresh water streams.  The mega-
curie quantities of fission products which
accumulate in the  reactor fuel do not, under
ordinary circumstances, escape from the fuel
while itis in the reactor.  As pointed out by
Terrill  , "Present practice involves extensive
control measures through design and operation
to confine the fission products within the
reactor core and its immediate environs. "
Failure of the cladding of fuel elements does,
of course,  provide the opportunity for entry
of fission products into the primary coolant
stream or moderator.  Under normal circum-
stances,  the principal source of radioactive
materials in the fluids which pass through the
reactor core is the activation of materials
which are exposed to the neutron flux. These
activation products may originate  from materials
dissolved in the coolant or they may corrode from
11. l.C. (6.62)
                                   VI  4-1

-------
 Behavior of Radionuelides' in Food Chains - Fresh Water Studies
 the surfaces of the fuel elements,  cooling
 system, or structural materials.  In the Han-
 ford-type reactor, the coolant is filtered
 Columbia River water which is returned to
 the river after  a single pass through the re-
 actor.  Figure  1 shows the isotopes which
 predominate in effluent from the Hanford re-
 actors four hours after irradiation.  Many
 of these have such short half-lives that they
 have decayed to very low levels by the end
 of the first day. Minor amounts of fission
 products are also found in the  Hanford reac-
 tor effluent not  only as a consequence of the
 occasional failure of a fuel element, but also
 because of natural amounts of fissionable
 uranium in the  Columbia River water.   The
 release of radioactive effluent to the Colum-
 bia River is accompanied by extensive monit-
 oring, both before and after discharge, in
 order to provide assurance that the radiation
 exposures to people who subsequently use
 the river or eat fish or irrigated crops are
 well within permissible limits.

                  Figure 1
-
Mn =

}
	 1
Cu«<


—
«,*<
Q COMPOSITION AT 4 HOURS
READ SCALE AT LEFT
rrrq COMPOSITION AT 24 HOURS
I'.:'.':* READ SCALE AT RIGHT
nRan^ra:
C,5I Np233 A,7« 5iJI 2n«> Q,Ti
      Sr92
                                 a a
RADIO-ELEMENT CONTENT OF REACTOR
EFFLUENT 4 HOURS AND 24 HOURS AFTER
IRRADIATION.  (Radioelements marked * ore
are routinely measured).

"Radioactive Waste Management Operations
 at the Hanford Works", included in Con-
 gressional Hearings on Industrial Radio-
 active Waste Disposal (1959).
Thd kinds and amounts of radionuclides dis-
charged to the Columbia River with the Han-
ford reactor effluent should not be considered
as characteristic of other types of reactors.
The concentrations of  various activation
products which build up in the primary cir-
culating fluid will be dependent upon the in-
tended chemical composition of the fluid,
impurities present,  the corrosion of cladding
on fuel elements, piping and vessels,  film
formation, neutron energy and flux, radioactive
half-life,  and the effectiveness of ion-exchange
"clean-up" loops where these are used. Sig-
nificant amounts of activation products of
common elements ranging from tritium to
neptunium can conceivably be present.  In
most cases there probably will not be  inten-
tional release of radioactive materials accumu-
lated in the re-circulated fluids directly to fresh
water streams.  However, minor leakage from
the complex  piping and fuel handling  systems
into the plant effluents appears almost inevit-
able.

Excluding nuclear detonations, the largest
potential source of radionuclides to the environ-
ment is the fission products which accumulate
in reactor fuels.  At the present time, the
bulk of the high-level waste which results from
the processing of the fuel elements, is confined
in some artificial or natural container. Even
so, large  quantities of water with low or inter-
mediate concentrations of radionuclides result
from the later stages of most separation pro-
cesses and these must usually be disposed of
at,  or very near, the processing plant. In
many cases,  disposal of such wastes will be
to the ground or to pits on the surface in order
to take advantage of the ion-exchange  capacity
of the soil and the time lags which allow for
decay of the isotopes with short and intermedi-
ate half-lives.  At the Hanford plant the cumu-
lative volume of intermediate-level waste dis-
charged to the ground through 1958 amounted
to nearly 4 billion gallons, containing nearly
2 million curies of beta-emitters.  The topo-
graphy, soil  structure, and geology at Han-
ford favor retention and none of this material
has been detected in the Columbia River^4^
Conditions for surface disposal are less favor-
able at other existing major installations,
and wastes containing fission products dis-
charged to pits at both Oak Ridge<5) and Chalk
River*6' have seeped through the soil  and
appeared in streams and lakes which  receive
the drainage.


Radioactive wastes may also enter fresh water
streams via the drains from industrial, medical.
VI  4-2

-------
                               Behavior of Radionuclides in Food Chains - F^-esh Water Studies
  or research facilities which use isotopes.
  Although the amounts involved are compara-
  tively small,  such sources should not be
  entirely disregarded since the Oak Ridge
  National Laboratory is now shipping on the
  order of 150, 000 curies of isotopes a year
  to domestic users.

  On the basis of high yield from nuclear fission,
  significantly long half-life,  and biological
  importance,  the fission products which one
  would expect to find in greatest abundance
  in the biota of fresh water streams and lakes
  are:  Sr89, Sr9<>, Y91,  Zr95+ Nb95,  Ru106,
  1*31, cs137, Ba140. Ce144, and one or more
  rare earths.   In addition to these, neutron
  activation products which appear to be of
  importance include:  P32, Sc46, Cr51,  Mn54,
  Fe55,  Fe59.  Co", Co60 and Zn".  In the
  immediate proximity to reactors, .very short-
 lived isotopes such as Na  , Cu^4, and As7**
 may be detected.
 As a result of the use of nuclear energy, the
 associated mining and refinement of fission-
 able materials, and the widespread use of
 radioisotopes, the radioactivity in a few sur-
 face waters of the world has now been in-
 creased above the original background level.
 As the use of atomic energy and its byproducts
 becomes more extensive, the numbers of
 rivers and lakes which receive radionuclides
 will increase, and the organisms which in-
 habit these waters will also acquire the radio-
 active materials.  Some appreciation of the
 mechanisms involved in the uptake of radio-
 nuclides by aquatic organisms is necessary
 to anticipate potential sources of exposure
 to man,  and,  if necessary,  to effectively
 control or reduce the amounts accumulated.
UPTAKE BY LIVING ORGANISMS

Radioactive materials present in the water
may be taken up by aquatic organisms via
three different mechanisms:   1.  adsorption
onto exposed surface areas,  2.  absorption
through membranes exposed  to the surround-
ing water, possibly with the aid of active
transport mechanisms, and 3. engulfment
of food or inert particles which contain the
nuclides.  The relative importance of these
mechanisms is different for various kinds of
organisms and even similar species.  It also
 fluctuates widely between different elements
 and different environments.

 Adsorption, which is a physical process,
 occurs very rapidly.  It is of greatest signifi-
 cance among those organisms which nave
 large surface to volume ratios such  as sponge
 and diatoms. A clear distinction between
 adsorption and biological absorption is not
 easy to make however, since the  physical
 process of adsorption may be a necessary
 primary step in the movement of  ions into
 the cell.  Films of bacteria and other micro-
 organisms  may also fix radioactive materials
 on the surfaces of organisms to give an ad-
 sorption-like effect.

 The process of absorption is obviously of
 prime importance to the plants since this is
 the means by which nutrient materials are
 removed from solution and metabolized.
 Many non-essential elements will also enter
 the plant cells because of passive diffusion
 through the membranes or because the or-
 ganism is handling them in a manner similar
 to the essential elements.   The uptake of radio-
 active materials by the plants is,  of  paramount
 importance to the higher forms since the
 plants form the food base.

 Direct absorption of some radioisotopes from
 the water also occurs in aquatic animals, in-
 cluding the  fish,  with the gill membranes
 probably playing the important role.  Such
 direct uptake by fresh water fish has been
 demonstrated for  Sr90(7) (8) (9), Ba140 +
 Lal40 (7),  Ca45 (10) (11) (12),  P32 (13) Na2*
 (7), and Csl37 (14).  However,  the dominant
 means by which many radioactive  materials
 are accumulated by aquatic animals is through
 ingestion, since this is the manner in which
 they obtain the bulk of their nutrient materials.
 Ingestion does not insure that the isotope will
 be assimilated through the gut.  Some mater-
 ials may pass through the gut with virtually
 no uptake,  and seldom,  if ever, will  all of
 the ingested radioactive material be metaboli-
 zed.  For example,  Watson*15* found that after
 24 hours,  rainbow trout retained about 60 per
cent of the radiophosphorus which they ingested.
Schiffman*16) found that only about 7  per cent
of Sr90 incorporated into natural food organ-
isms was retained by trout after one  day.
                                                                                  VI  4-3

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 Behavior of Radionuclides in Food Chains - Fresh Water Studies
 CONCENTRATION FACTORS

 The uptake of radionuclides by organisms is
 obviously not a phenomenon which is peculiar
 to the radioactive forms of the elements.
 Rather,  the radioisotope acts only as a tracer
 to demonstrate the difference between the
 abundance of the element in the water and in
 the organism.  The so-called "concentration
 factor"
            of organism)
      ((AC /ml of water  )

 for any radionuclide cannot exceed the ratio
 which exists between the concentrations of
 the element in the organism and in the water.
 On this basis it should be possible to predict
 the ultimate concentration factors for various
 radionuclides from conventional quantitative
 chemical measurements,  provided the nuclide
 has become distributed throughout the  system
 and is uniformly mixed with its stable  counter-
 part. In practice, however,  it may  be quite
 difficult to measure the concentration of some
 elements because of the minute quantities
 involved.

 Estimation of concentration factors for fresh
 water organisms involves, at the present
 time, a considerable  amount of speculation.
 While the concentration of some elements in
 a variety of marine organisms is available; (17)
 and the  chemical composition of sea water is
 relatively stable, there is very little infor-
 mation readily available on the chemical com-
 position of fresh water organisms.   Chemistry
 of various lakes and streams differs so great-
 ly from one region to another that no general-
 izations can be made.  The picture is clouded
 further  by the fact that the concentration of
 many elements in aquatic  forms is partially
 dependent upon their abundance in the surround'
 ing water.  Although it is  virtually impossible
 to write down probable concentration factors
 which will apply to more than one locality,
 an indication of the differences in the orders
 of magnitude of the concentration for a few
 of the common elements in some organisms
 and in water is shown in Table 1.

It is evident then, that radioisotopes of the
 different elements will be concentrated to
various  amounts by the different organisms.
 The concentration 'of phosphorus in Columbia
River water is about 0. 01 ppm, while in the
fish it amount* to about 6, 000 ppm.  These
values would lead to an estimated concentra-
tion factor of about 600, 000 for the fish.
Since radiophosphorus is one of the isotopes
present in the effluent which is discharged in-
to the Columbia River from the Hanford reac-
tors, the actual concentration factor for this
nuclide  can be measured.   In contrast to the
theoretical value of 600, 000,  the maximum
observed value is only about 100, 000.  The
observed value is less because the biological
processes required to deposit and exchange
the phosphorus in the body of the fish are
long in comparison to the radioactive half-
life of P32,  which  is about 14 days.  Conse-
quently, the radioisotope is not uniformly
mixed with all of the stable phosphorus in-
volved in the system. Such a reduction would
not be expected for long-lived isotopes.

                  Table 1

Concentration (.ug/g wet weight or ml) of
some elements in fresh water organisms and
in some major rivers of the United States



ailloan
SoiJam
Fboqbm,
bleha
Ira.
ttratlB

U(M
(Smrocrm)
1.900
1.900
290
1.900
4. SCO
2
o^o. V
IhMet Urw
(Cidtfli fly)
20
TOO
WO
300
300
0.2
fl*
(nm»i)
10
1000
iooo
3000
1
OO
ViurJ/

In
1
1
40.001
2
<0.01
CUJH,

HUEL
29
200
1.9
200
6J
l.»
l_l Values are only estimates of orders of
   magnitude obtained by spectrophotometric
   analysis at the Hanford Laboratories.  They
   are recorded here to illustrate differences
   which can exist and are not intended for
   use in precision work.

2j Abstracted from Moyle, J. B., "Relation-
   ships between the chemistry of Minnesota
   Surface Waters and Wildlife Management".
   Journal of Wildlife Management 00: 303-320
   1956, and Clarke. F. W.. "The Composi-
   tion of the River and Lake Waters of the
   United States".   U. S. Geological Survey,
   Prof. Paper 135, 1924.  Strontium values
   from Alexander, Nusbaum, and MacDonald,
   "Strontium and Calcium in Municipal Water
   Supplies", A.W.W.A.. Journal 46(7): 643-654.
   (1954).
VI 4-4

-------
                                  Behavior of Radionuclides in Food Chains - Fresh Water Studies
  From measured amounts of radionuclides in
  the effluent from Hanford reactors and in or-
  ganisms living in the Columbia River im-
  mediately below the reactors, concentration
  factors have been calculated for the more a-
  bundnat isotopes.  These are listed in Table
  2 for the season when maximum values occur.
                                   7fi    Afi
  The  concentration factors for As  , Sc   ,
  Cr51,  and Cu6^ are quite low in fish in com-
  parison to those in algae and insect larvae.
  This illustrates that food chains, which  on
  the one hand provide the mechanism for trans-
  ferring radioactive  materials from one or-
  ganism to  another,  can also serve,, in some
  cases, to reduce the concentration of radio-
  nuclides in large animals.  Two different
  processes  can be involved.   Where possible
  the organisms will eliminate non-essential
  elements,  and thus there will be a repeated
  selection against such nuclides along the food
  chain; and  short-lived isotopes will decay to
  low levels  as they pass through the chain.

                    Table 2*
       Observed Concentration Factors
     For Significant Isotopes Found In Columbia River Organisms
Isotope
P32
Zn85
cs,37(d)
Sr80
Na24
As76
So46
Cr51
Cu64
AUae(a>
100.000-1.000,000
100. 000
1,000-5.000
10,000
100
10. 000
100, 000
100-1,000
10, 000
Insect'1"
Larvae
100, 000
10,000
1,000
100
100
1,000
1,000
100-1,000
1,000
Fish
100. 000
1,000-10,000
5,000-10,000
1, 000
100-1,000
100
10
10
10
  Many of these values are not yet well defined a'nd are subject to revision.

                               Richardsonium ap.
    Sligeoclonliim ap.   Hydropayche ap
    Data for Cs  obtained from a pond environment rather than the Columbia
    River and species are different from those listed above.

  (Values are based partly on data reported by J. D. Davis, R. W. Perkins,
  R.F. Palmer, W.C. Hanson, andJ.F. Cline (18).and R. C. Pendleton and
  W.C. Hanson (19).)
•This table reproduced from "The Need for Biological Monitoring of Radioactive
Waste Streams, " by R. F. Footer presented at the Nuclear Engineering and
Science Conference April 6-9,  1959, Cleveland, Ohio.
 The isotopes which are most likely to have
 high concentration factors in fish and other
 large aquatic animals  are those which are
 readily assimilated, which are retained for
 relatively long periods of time in various
 body structures, and which have long radio-
 active half-lives.  These are characteristics
 which result in relatively high body burdens
 for humans as well,  and which influence  a
 low  maximum permissible concentration for
 the isotope, in drinking water or food.
DIFFERENCES BETWEEN SPECIES

Differences in the concentration factors for
various isotopes among different species leads,
to different levels of radioactivity in the or-
ganisms.   Columbia River organisms provide
a good example of this as shown in Figure 2.
The short-lived isotopes contribute significant-
ly to the activity density (quantity of radio-
active materials per unit mass of substance)
of plankton, algae, and sponge.  But in the
higher animal forms, about 95 per cent of the
activity originates from P".

                   Figure 2

      Radioactivity in Different Columbia
             River Organisms

   RELATIVE CONCENTRATION OF RADIOACTIVE MATERIALS
 0         Z5        SO        75       100
                                                                                SESSILE ALGAE
                                                                                STIGEOCLONIUM
                                                                                SPONGE
                                                                                SPONGILLA
                                                                             CAODIS LARVAE
                                                                             HYOROPSYCHE
             MAY FLY NYMPHS
             PARALEPTOPHLEBIA
             SNAIL FLESH
             STAGNICOLA
                  SUCKERS
                  CATOSTOMUS
            SHINERS   I
            RICHARDSONIUS
                                                                   i
                                                             CRAYFISH
                                                             ASTACUS
                                                         A
                                                         P  WATER
           Davis and Foster, "Bioaccumulation of
           Radioisotopes through Aquatic Food
           Chains", Ecology 39: 530-535 U05B)
                                                                                         VI--4-5

-------
 Behavior of Radionuclides in Food Chains - Fresh Water Studies
                                 32
Because of the dominance of the P  , it is of
interest to consider this isotope separately
from the others.  Figure 3 shows the relative
concentrations of P   in most of the species
included in Figure 2.   The different concen-
trations result from a combination of several
factors: the chemical composition of the
various species is different, the moisture
and organic content varies,, and there are
different lag times along the food  chain which
permit  radioactive decay.

               Figure 3


        RELATIVE  CONCENTRATION OF P32 IN
             COLUMBIA RIVER  ORGANISMS
 As mentioned before,  the reduction in the
 concentration of a radioisotope of an essen-
 tial element along the food chain may occur with
 relatively short-lived isotopes because the
 ratio of radioactive to non-radioactive atoms
 of the element does not remain constant
 throughout the entire biological system.  This
 ratio may properly be called the specific
 activity since it is  the concentration of the
 Isotope per .gram of stable element - in this
 case - jie P^2 per.gram of P^l. The Colum-
 bia River below the Hanford reactors pro-
 vides an unusual opportunity for the  study
 of the specific activity of P82 in various or-
 ganisms of the food chain, since a near
 steady state is maintained^  A "fresh" supply
 of the isotope is added to the river water at
 a more or less constant rate with the reactor
 effluent.  Since the concentration of stable
 phosphorus in the river water is also rela-
 tively constant, the specific activity of P32,
 remains about the  same in the water through-
 out much of the year. If there were a near
 instantaneous and complete exchange of phos-
 phorus between the river water and all aquatic
                                                   organisms,  then the specific activity of the
                                                   organisms would be identical with that of the
                                                   water.  Figure 4 shows that this is not the
                                                   case.  For plankton and sessile algae the
                                                   specific activity is  virtually the same as in
                                                   the water:  This indicates a very rapid exchange
                                                   of phosphorus between these forms and the
                                                   water.  The specific activity is progressively
                                                   less  in the insect larvae,  snails, fish, and
                                                   crayfish (scavengers), indicating that re-
                                                   placement of phosphorus in these animals goes
                                                   on at a relatively slow rate, and thus the
                                                   phosphorus  "ages"  in the organism.

                                                                     Figure 4
                                                    SPECIFIC ACTIVITY OF COLUMBIA RIVER ORGANISMS
                                                   i   CRAYFISH
                                                      MOIC: THOC WLUCS WOK ESTOMTtD HUt HATtMAL COLLKHD AT I
                                                         TIMES MK> AM SUSJCCT TO REVISION
                                                  If one compares the specific activity of caddis
                                                  fly larvae with that of their food," which is
                                                  predominantly plankton for the species rep-
                                                  resented here,  it can be shown that the aver-
                                                  age phosphorus atom remains in the caddis
                                                  larvae for about 9 days.
                                                                                32
                                                  The low specific activities of P   in the lar-
                                                  ger animals results not only from slow phos-
                                                  phorus exchange within the animals, but also
                                                  from the reduced specific activity of its food.
                                                  The effect is thus cumulative along the food
                                                  chain.  For isotopes which are shorter lived
                                                  than P32, the reduction in specific activity
                                                  along the food chain may be even more spec-
                                                  tacular; for isotopes with relatively long half-
                                                  lives the reduction may not be apparent.
                                                  It must be remembered, however,  that a re-
                                                  duction in specific activity along the food chain
                                                  results from a combination of two factors:
                                                  one is a comparatively short half-life, and
                                                  the other is a relatively long retention of the
                                                  element in the organisms.
yi 4-6

-------
                              Behavior of Radionuclides in Food Chains - Fresh Water Studies
EXCHANGE BETWEEN ORGANISMS

Although we talk in terms of food chains with
the implication that radionuclides and other
materials are transferred sequentially from
small food species to larger predatory forms,
such simple, straight-forward systems are
not strictly characteristic of aquatic  com-
munities.  Such communities might better
be thought  of as complex chemical exchange
systems in which there is a kind of equilibrium
established between the concentration of the
nuclide in the water and in the various bio-
logical forms,  silt, and other exposed sur-
faces.   Several experiments have been carried
out in both the laboratory and the field with
the use of P^  in order to measure the rates
of exchange between various components of
the systems (20-31).  Figure 5 is a highly
schematic  representation of the kind  of phos-
phorus exchange which operates in a  fresh
water community.  The size of the arrows is
intended to give some conception of the quan-
tity of phosphorus (or radiophosphorus in this
case) moving in a given direction in some
short unit of time.  A large fraction of the
available isotope  moves rapidly back and forth
between the water and the photosynthetic
plants.  Animals  which eat the plants obtain
an appreciable part of their radiophosphorus
from the plants, but they  also carry on at
least some exchange directly with the water.
A similar arrangement exists at the higher
trophic levels. Each of the groups of organ-
isms contributes  some isotope to the sedi-
ments,  either through death or via fecal
pellets, with bacteria playing an important
role in decomposition.  Some of the isotope
remains with the  sediments and, in time, is
buried deeper. However, much of it is re-
leased back into the water and recycled
through the biota.
                  Figure 5
In many fresh water communities the great-
est mass of material will be made up of the
photosynthetic plants - the primary producers
which constitute the first trophic level.  The
mass of successive trophic levels will usually
be smaller and smaller, so that relatively
few carnivorus fish are supported at the top
of the biomass pyramid.  However, in some
open-water  communities where the primary
trophic level is made up almost exclusively of
phytoplankton,  with a rapid "turnover", a
part of the pyramid may be inverted, (32).
Of the total  inventory of radiophosphorus and
most other radionuclides,  one would expect
to find the greatest fraction held in those or-
ganisms which make up the greatest portion
of the biomass. In fresh water systems this
will usually be the green plants,  which also
have rapid exchange rates and an affinity for
a large variety of nuclides. Nevertheless,
only a small fraction may be held in the large
                                                                                      VI  4-7

-------
 Behavior of Radionuclides in Food Chains - Fresh Water Studies
 fish, this may well be the part of greatest
 interest to the radiation protection specialists.

 It should be evident then,  that radioisotopes
 of biologically essential elements,  when in-
 troduced into streams or lakes, are not apt to
 remain exclusively in the water.  Rather,
 they will rapidly become distributed between
 the  water,  the biomass, and the sediments:
 In many cases the portion which is removed
 from the water will exceed that which remains.
 If it is desired to reduce the specific activity
 of a contaminating radionuclide by adding
 carrier to the water,  consideration must be
 given to the probability that there is a greater
 reservoir of essential elements in the solids
 (including the biota) than is  dissolved in fresh
 water.
 SEASONAL VARIATIONS

 Where continued or repetitive release of
 radioactive waste occurs, a true equilibrium
 in distribution of radionuclides between water
 and organisms will seldom be attained.  Even
 under stable conditions of flow and effluent
 discharge,  seasonal fluctuations in the composi-
 tion and metabolism of the community will keep
 the system in a dynamic state. New blooms
 of plankton or vascular plants will create a
 demand which will draw material from the
 water and from other parts of the biomass.
 Conversely,  the dying-off of large masses of
 vegetation may  release significant quantities
 of radionuclides back into the  water where they
 will be available to other components of the
 community.

 Most aquatic animals are cold blooded, and
 thus their metabolic rates change with varia-
 tions  in temperature and so, with the seasons.
 Concentrations of those radionuclides affected
 by metabolic rates in aquatic organisms will
 also fluctuate with the seasons.  During the
 cold-water period food intake is drastically
 reduced.  If inges.tion  is the principal mode
of uptake of a radionuclide, the supply to the
animal will likewise be reduced, and the
quantity of nuclide  in the animal will diminish
as a result of radioactive decay and biological
elimination.  The  reverse is true when tem-
peratures are high.
Figure 6 shows a smoothed curve for the
activity density of plankton and small fish of
the Columbia River over the period of a year.
Fluctuations in the plankton (predominantly
diatoms), are quite similar to those in the
water since there is a rapid exchange of nu-
clides between the  water and the plant cells
by direct absorption and adsorption; tempera-
ture has little effect in this case.

A pronounced decrease in  the activity density
of the plankton occurs during the late spring,
because the flow of the Columbia River increases
greatly at this  time to provide additional dilu-
tion of the effluent  from the reactors.  Fluc-
tuations in the  activity density of the fish are
on the other hand,  more closely  related to
temperature.   As indicated above, the marked
increase in activity density of the fish during
the time of high temperature reflects an increase
in metabolic rate all along the food chain.
Not only are the fish eating more food, but each
food organism  has  become more radioactive,
and the effective retention time for radioac-
tive decay within each trophic  level has be-
come less.  The effect is thus cumulative along
the food chain.

                  Figure  6


 SEASONAL VARIATIONS  IN THE CONCENTRATION  OF
   P3Z IN COLUMBIA  RIVER PLANKTON AND  FISH
                          AUG SEP OCT NOV DEC
Because of the rapid exchange of some nuclides
between the water and algal cells,  the activity
density of these organisms will rapidly reflect
changes in the concentration of the radionuclides
in the water.  However, the uptake and elimi-
nation  of most nuclides in the larger forms,
such as the fish, is  a comparatively slow
  VI  4-8

-------
                                Behavior of Radionuclides in Food Chairs - Fresh Water Studies
  process.  Their activity density will then
  fluctuate in a rather sluggish manner in com-
  parison to changes in the water or in their
  food.  Figure 7 shows that trout fed a ration
  of P32 each day required about 2 weeks to
  reach an equilibrium level.  The activity
  density of the fish may not therefore, provide
  a good indication of the conditions in the
  water at the time fish are  collected.  What
  may be more  significant however,  is that
  the fish samples may provide a reasonably
  good index of  the average conditions which
  have existed in the water over a period of
  hours  or even weeks, dependent on the  par-
  ticular nuclide involved.

                   Figure 7

      UPTAKE AND RETENTION OF P32 BY TROUT TISSUES
                        -UN, p-r d«   ENti OF P32 FEEDING

                        ,1,1,1     . I
   0001,
 Watson, George,  and Hackett, U.S.A.E.G.
 Document HW-59500 (1959)
 CONCLUSION

 Radionuclides which enter fresh water streams
 and lakes may enter the food chain and thus
 appear in fish and other aquatic forms eaten
 by man.  The radioactive contamination levels
 which are apt to result in the various organ-
 isms, are affected by a large number of phy-
 sical, chemical,  and biological factors.   For
 radioisotopes of some of the more abundant
 essential elements, we can estimate within
 broad limits what the maximum levels may
 be.  The characteristics of fresh water com-
 munities vary so greatly between different
localitits however,  that the continued release
of substantial quantities of radionuclides must
be carried out with considerable prudence
 and with monitoring of the concentrations of
 radionuclides which actually result in the
 environs at the point of release.
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                                                                                 VI  4-9

-------
 Behavior of Radionuclides in Food Chains - Fresh Water Studies
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VI  4-10

-------
                             Behavior of Radionuclides in Food Chains - Fresh Water Studies
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      Radiophosphorus",  (Hanford Works)
      Biology Research - Annual Report
      1953: 16-23,  U.S.  Atomic Energy
      Commission Document HW-30437,
      Unclassified  1954.

32 Odum, E.P., "Fundamentals of Ecology, "
      W.B. Saunders Company,  pages 1-546,
      1959.
This outline was prepared by R. F. Foster,
Manager, Aquatic Biology, Biology Operation,
Hanford  Laboratories, General Electric
Company, Richland,  Washington.   The report
is based  on work performed under contract
No. AT(45-D-1350 for the U.S. Atomic
Energy Commission.
Additional plates by L. G. Williams, Former
Aquatic Biologist, Aquatic Biology Section,
Basic and Applied Sciences Branch. Division
of Water Supply and Pollution Control.
                                                                                 VI 4-11

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                FWPCA RESPONSIBILITIES FOR WATER QUALITY STANDARDS
I  INTRODUCTION

The Water Quality Act of 1965 provided for
the most forceful program yet devised to
protect and enhance the quality of the  Nation's
waters.  Under this law,  the States had until
June 30, 1967, to adopt-  water  quality criteria
for their interstate and coastal waters and a
plan for implementing and enforcing the
criteria adopted.  Criteria and plans acceptable
to the Secretary of the Interior become the
water quality  standards applicable to the
State's  interstate and coastal waters.

In the event any State standards are unacceptable,
the Secretary of the Interior has the authority
to establish standards, but only after  the
States  and all other  affected  interests have
had a full opportunity to be heard.  Once
adopted, standards are enforceable by the
States  and the Federal Government if  any
discharge of material occurs which reduces
the quality of  the water below the established
standards.

The standards make it possible  for munic-
ipalities,  industries, and other  water users
to know in advance what their responsibilities
are for keeping clean waters  clean and for
restoring polluted waters to  a reasonable
degree of purity.  The standards also give
the Federal Government authority to prevent
pollution before it occurs, instead of instituting
enforcement action after health  and welfare
are proven to be endangered.

The standards are not intended to "lock in"
present uses of water,  nor to exclude uses
not now possible.  Nor are they intended to
be least common denominators of water
quality and use.  Instead,  they are intended
to enhance the quality and productivity of the
Nation's water resources in  an orderly,
programmed manner.

 Standards prepared by the States were sub-
mitted and reviewed by the Administration
 prior to submitting  them to the  Secretary.
 The standards-setting effort is  the Federal
Water Pollution Control Administration's
 top priority program.
When standards are reviewed,  the respective
types of water uses determined to be
desirable are evaluated against the water
quality criteria  prescribed to achieve those
uses.  Close attention is also paid in the
review to plans  prepared for implementing
the standards and to surveillance programs
developed for assessing effectiveness of the
proposed program.

To assist States in  delineating factors to be
considered in reviewing standards, the
Federal Water  Pollution Control Administra-
tion prepared and widely distributed
"Guidelines for  Establishing Water Quality
Standards for Interstate Waters" and
"Necessary Supporting Material and
Implementation  Plan Contents. " To assist
him in evaluating State submissions,  the
Secretary of the Interior appointed a  variety
of experts to the National Technical Advisory
Committee, with the request that they pre-
pare water quality criteria for water uses
in the area of their competence. Sub-
committees were established to develop
criteria in the following areas: fish, other
aquatic life and  wildlife, municipal water
supplies,  industrial water supplies,
agriculture,  and recreation and aesthetics.

A  comprehensive report has been completed
and is available in limited quantities.
Standards submitted for review have
generally been of a high caliber and rep-
resent a substantial Federal-State-local
cooperative effort.  The standards-setting
process has resulted in actions not visible
on the horizon initially.  Specific programs
with a definite schedule have been prepared
for cleaning up rivers, lakes,  streams, and
coastal waters; State laws to permit com-
pliance with the Water Quality Act have been
revised where necessary;  and  information
previously unavailable or in an unassembled
form has been developed and collected in a
single set of documents available to all groups
with an interest in preserving  and protecting
water quality.  Most important, however, is
the spirit aroused in the preparation of these
blueprints  for clean water. Officials  at all
W.Q. STO.4a. 9. 69
                                   VI 5-1

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 FWPCA Responsibilities for Water Quality Standards
levels have been surprised at the interest
expressed by citizen groups and have themselves
become actively involved in assuring the
program's success.

Significant as they are,  standards are basically
a guide for obtaining the longer range objective
of improving and protecting water quality.
Standards run through the clean-up program,
holding together and  guiding efforts in planning,
waste treatment works construction, research,
enforcement, pollution surveillance, training,
and technical assistance.

Standards have  been  partially or wholely
approved for all States, three territories,
and the District of Columbia.  Present
efforts are to effect resolution of unaccept-
able portions of the submitted standards
and to develop acceptable anti-degradation
statements.  Development of additional and
more definitive standards may be  anticipated
as  the program progresses.

Additional information on this program may
be  obtained from the Regional Coordinator of
Water Quality Standards or the Water Quality
Standards Staff, Office of Program Plans and
Development, Federal Water Pollution Control
Administration, Washington, D. C.  20242.

The act of setting standards or  selecting words
or  numbers  to describe permissible water
quality ranges is obviously not an  end to itself.
Surveillance is  required to determine if the
standards are being met,  and enforcement
may be necessary if  cleanup operations
designed  to meet standards are not proceeding
on  schedule.

The mission of  the water quality surveillance
program  is:

A   To monitor water quality and waste facility
    information for compliance or non-
    compliance with water  quality standards
    and to report these findings to those in a
    position, to take action.

B   To provide the specific information required
    to assess pollution problems and to evaluate
    the effects of remedial actions by:

    1  Acting as a support  service to other
      FWPCA programs;

    2  Providing technical  assistance to the
      states;

    3  Cooperating with other Federal agencies
      and states in the collection of pollution-
      related information.
C To supply water quality and related
   pollution information for basin-wide
   water pollution control planning, to
   document pollution control violations for
   enforcement actions, and to fulfill
   responsibilities as assigned to the program.

The highest priorities for all organizational
elements  responsible for this program are
the planning, developing, and coordinating
of the surveillance systems necessary to
determine the compliance or noncompliance
of the water quality standards and
implementation plans.

This will  require that Headquarters provide
technical  guidance to ensure that all data
gathering and evaluation are accomplished
in a uniform and efficient manner.
Particular attention will be given to improving
the systems for the collection of water quality
data and the establishment of an Early Warning
System  which identifies trends in water quality
in advance of the potential violation.

It will be  the responsibility of Headquarters
to identify water quality surveillance program
activities  as they are presently handled in
other programs so that these may be con-
solidated  and that  sufficient funds and
personnel are allocated from these programs
to adequately continue the level of
surveillance.

Regions will plan and design their surveillance
systems on a major basin basis ensuring that
the information systems output is current
and accurate.

Efforts  in information collection,  evaluation,
and dissemination must be highly coordinated
with appropriate Federal, state,  local,  and
private  groups or agencies to avoid duplication
of effort,  to ensure proper allocation of
resources, and to consider  alternative
methods or courses of action.

Each  region will provide support for the
National effort in analytical quality control
in sample handling and analysis.  All essential
data developed in the  region will be put in a
storage and retrieval system that is accessible
to aU of FWPCA.
VI  5-2

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                                         FWPCA Responsibilities for Water Quality Standards
The order of priority for work assignments
in this program will be:

A  Water Quality Standards

B  Enforcement Activities

C  Pollution Control Planning

D  Waste Facilities Inventories

E  Others as Required

The word "surveillance" suggests a possible
observance or inspection of activities.
Surveillance as practiced by the Federal
Water Pollution Control Administration,
however, involves the collecting of discrete
information expressly to define the status of
water pollution control on a point by point
basis so that timely evaluation and remedial
action can be  effected.

Water pollution control in the United States
was launched as a permanent,  national
program in 1956 with the enactment of Public
Law 84-660, the Federal Water PoUution
Control Act.  Some entirely new concepts were
created by the Water Quality Act of 1965 and
the Clean Water Restoration Act of 1966.  In
several respects the recent legislation bears
significantly on the water pollution surveillance
activity in the United States.

The Clean Water Restoration Act of 1966
transferred administration of the Oil Pollution
Act of 1924 from the Secretary of the Army
to  the Secretary of the Interior.  In the process,
the Oil Pollution Act, which was formerly
limited to vessel pollution affecting coastal
navigable waters and the sea within the
territorial jurisdiction of the United States,
was extended to inland navigable waters and
adjacent  shorelines subject to pollution from
boats and vessels.   This new authority
complements and expands upon previous
responsibilities vested in the FWPCA  by
PL 84-660, as amended, for the control of
pollution resulting from  land-based sources,
including oil pollution from shore-based
facilities.
II   SURVEILLANCE ACTIVITIES

 A  Indices

    The fresh waters  of our nation must be put
    to various uses.  Among these, municipal
    water supply has number one priority.
    Asa consequence, statistics are  kept on
    the location, quantity,  and type of water
    treatment provided by every municipality
    in the United States.  Similarly,
    inventories are maintained of sewerage
    systems (including waste treatment
    facilities) delineating the populations
    served,  the type of treatment provided,
    and the additional facilities needed.
    National data are  also collected,  evaluated,
    and published periodically on the  financing
    and construction of sewage collection and
    treatment facilities and on pollution-
    caused fishkills.  At present,  the major
    unknowns, insofar as waste  discharges
    are concerned,  include those associated
    with industrial manufacturing, acid mine
    drainage, thermal pollution,  and  irrigation
    return flows. In appreciation of this, the
    Secretary of the Interior  announced
    that the FWPCA will initiate an industrial
    waste discharge inventory as a continuing
    program.

 B  Water Quality Data

    In the final analysis,  progress in the field
    of water pollution control will be  measured
    by the presence or absence of desired
    levels of quality in the  streams and water-
    ways of the Nation.  Thus, while  the
    aforementioned  statistics are  essential.
    Federal  surveillance resources must be
    aimed primarily at monitoring in-stream
    conditions for immediate relation to the
    established water  quality standards --
    point by  point,  stream by stream, and
    basin by basin on  a day-to-day basis.

   To carry out this mission, the FWPCA's
   Division of Pollution  Surveillance pursues
   the following major stream quality oriented
   activities via a central facility and regional
   laboratories.
                                                                                    VI  5-3

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 FWPCA Responsibilities for Water Quality Standards
    1  Water Quality Compliance System -
      Flexible basin-wide systems of water
      quality monitoring stations, including
      both central laboratory analyses and
      in-stream analyses via automatic
      equipment.

    2  Oil Pollution Act Laboratory Services  -
      A  unit at the FWPCA laboratory in
      Cincinnati specializing in techniques
      required for the detection and analysis
      of petroleum compounds.


    3  Specialized Analytical Services  and
      Instrumentation -  Expert analytical
      services and specialized analytical
      equipment for national consultation
      and services and the development,
      installation, and operation of appropriate
      automatic water quality sensing and
      transmitting instruments.

   4  Analytical Quality  Control - To  assess
      and assure the reliability of laboratory
      and field analytical processes and results,
      including the field  testing of new laboratory
      analytical procedures, and the initiation
      of a complementary program with state
      and interstate water pollution control
      agencies to assure the validity of data
      used in litigation.

   5  Data Operations, Evaluation, and Control -
      To provide and operate a computerized
      system for the timely storage, retrieval,
      processing, and analysis of necessary
      water quality data and related  statistics--
      including interstate water quality standards
      and implementation plans.

C  Surveillance Stations

   Aside from the many  sampling stations
   established for limited duration as  part
   of  special studies and investigations,
   FWPCA has, since 1956,  established
   stations at critical points in major  basins
   of the United States.   Each of the basic
   stations is being critically reviewed as  to
   location, collection frequency, and the
   parametric analyses which will be required
   in determining compliance with specific
   water quality standards.   Over 100 of the
   stations are located at or near international
or interstate boundaries.  Other stations
are located at or near the current or
proposed municipal water intakes of such
major cities as Kansas City,  St.  Louis,
Cincinnati,  Washington, Philadelphia,
Buffalo,  and New York City.  Stipulating
the development of standards aimed at
protecting and  enhancing water quality
at water supply intakes, many stations
obviously must also be located on inter-
state  streams below major municipal and
industrial complexes.

Under the terms of an interagency agree-
ment with the U. S. Geological Survey,
the Survey will operate these critical
long-term monitoring stations.  The
Survey's responsibilities include sample
collection and analysis and entry of the
data in the STORET System.  FWPCA's
responsibilities include continuing review
and evaluation of the  data to determine
adequacy of station location and  parameters,
frequency of sampling, and the detection
of trends or standards violations.

1  Number  - It is currently impossible to
   forecast the eventual requirements for
   either state or Federal pollution oriented
   monitoring.  We do know that the number
   of stations must be substantially
   increased in each  major basin to fulfill
   the Federal responsibility as  well as
   give technical assistance to the states
   and to permit the Secretary to act
   quickly if a  state fails to establish or
   maintain satisfactory interstate water
   quality standards.   As in the case of the
   basic  surveillance stations, all new
   stations including  reactivation of
   stations occupied for short periods
   during previous investigations, will
   be scrutinized to assure that they
   conform to certain basic criteria.

2  Locations required - The locating of the
   Federal water quality surveillance
   stations will be influenced by  the state
   implementation plans.  However, it is
   quite predictable that Federal stations
   will be located at the mouths of rivers
   and tributaries,  the points at which
   streams cross state lines, points above
   and below major municipal and industrial
   complexes,  and  points designated for
   specific water uses, including water-
  based recreation and propagation of
  fish and wildlife.  Some station loca-
  tions may be thought of as  permanent
VI  5-4

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                                     FWPCA Responsibilities for Water Quality Standards
   installations inasmuch as it may be
   impossible to predict the time when
   data will not be needed for that point.
   Conversely, a significant number of
   stations may measure progress toward
   water quality objectives which can be
   met within a predictable,  definite period
   of time.  For example,  a station
   established  to  monitor a point downstream
   from a specific waste problem may well
   be deactivated, or at least curtailed,
   once the problem is  ameliorated.

3  Monitoring criteria - In order to maximize
   the results of vested resources, pollution
   control monitoring systems must be
   designed for both flexibility and specificity
   in regard to sampling locations, analyses
   performed,  and the frequency of analysis.
   Water pollution surveillance systems must
   be directed primarily toward detecting
   changes in the degree of water pollution
   associated with man's activities. These
   changes occur with  relative rapidity as
   compared with changes associated with
   most natural phenomena; for example,
   the changes  occasioned by the passage
   of water over or through the ground.
   A new industrial complex, a new sewage
   treatment plant,  or any  new water
   resources structure can effect rapid
   changes in water quality; hence, the
   basin system and its component  monitoring
   stations must be periodically reviewed
   to ensure flexibility to respond quickly
   to such physical changes.  Each year
   such reviews will undoubtedly result
   in the relocating of a number of  stations,
   curtailing sampling at some, and expanding
   the sampling frequency and diversity of
   analyses at others.   Thus, each basin
   system must be designed to  detect and
   assist in the control of waste discharges,
   to determine the effectiveness of waste
   treatment and control, and to measure
   pollution abatement  progress.

4  Parameters  required - Although the
   adoption of an identical,  inviolate list
   of parameters to be associated with
   each surveillance point would simplify
   the administration of each basin
   surveillance system, such rigidity
   would be neither useful nor desirable.
   Surveillance for water pollution control
   purposes will be oriented to specifically
   stated water quality criteria.  Water
   pollution problems of varying severity
   exist in most rivers and  streams of the
   United  States; yet they are never
   identical.  The  surveillance program
   will be tailored to the very special
   set of circumstances and needs associ-
   ated with the specific  problem at
   individual locations.   In this sense, the
   water quality criteria received from the
   states show a diversity of parameters
   and serve to define those which are
   particularly significant for major
   reaches of interstate waterways.  Thus,
   the parameters requiring analyses may
   range both within a basin and on a
   national basis.  They  may vary from
   readily obtainable onsite  information on
   dissolved oxygen, conductivity, pH, and
   temperature to the sophisticated
   analyses of numerous biological entities,
   a growing number of radionuclides,
   toxic substances such as  arsenic and
   heavy metals, plus a formidable array
   of bacteria, viruses and individual
   organic contaminants  including pesticides
   and petroleum-based materials, all
   requiring detailed laboratory evaluation.

5  Frequency of analysis and automatic
   equipment - The frequency of sampling
   cannot be irrevocably  fixed for an entire
   system of surveillance stations. Indeed,
   it cannot be fixed permanently  for a
   single station.   Periodic adjustments
   of frequencies will be  necessary for a
   variety of reasons.  The lowest sampling
   frequency capable of providing desired
   information is the best frequency.
  Automatic sensors can facilitate the
   collection of large amounts  of data at
   closely spaced time intervals and can
   be used advantageously for a variety of
  purposes.  There is a definite place for
   such equipment,  and there will be more
  need for it as the water quality standards
  are implemented. However, each
   situation will be examined critically to
  determine if instrumentation is really
  needed.  For example, many physical
  and chemical characteristics of surface
  water remain relatively constant,  or
                                                                                 VI 5-5

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  FWPCA Responsibilities for Water Quality Standards
       change slowly.  For such parameters,
       laboratory analyses of periodic grab
       samples often are sufficient.  Conversely,
       a critical water quality parameter,  such
       as dissolved oxygen, which is vital to
       fish and other aquatic life as well as
       being generally indicative of water quality
       conditions, may vary under certain
       conditions from an acceptable to a non-
       acceptable value in a short time.  In
       general,  instrumentation will be applied
       only  to those situations where the
       parameters are subject to quick change,
       are essential to the development and
       verification of mathematical models for
       evaluating alternate pollution control
       programs within a basin, or are required
       to "sound  an alarm" at a critical location.
       The guiding principles in selecting
       automatic instrumentation,  as related to
       water pollution control, are presented
       in a "Program Guide for Automated
       Instrumentation for Water Pollution
       Surveillance."
III  WATER QUALITY MONITORING ACTIVITIES
    OF THE STATES

 Consideration of water quality monitoring
 activities which can best be conducted by the
 states, indicates at least three levels of
 surveillance to be satisfied.  First, on-site
 inspection of municipal and industrial waste
 handling procedures must be  recognized as a
 basic state or local responsibility; this  will
 assure both more efficient operation of  treat-
 ment and control facilities and the highest
 level of return per unit of regulatory effort.
 Second,  monitoring of individual plant effluent
 lines should be an important part of state
 programs.  Third, the states should examine
 the receiving water body to assure attainment
 of desired water quality levels consistent with
 adopted water quality standards.  The latter
 will require the utmost care to assure
 coordination and cooperation among the  states
 and the Federal government in fulfilling their
 mutual responsibilities.  Indeed,  the need for
  coordination to assure wise programming of
  the limited resources of the responsible
  agency is FWPCA's main reason for not
  defining the specific stations  to be included
  in each of its basin monitoring systems.
IV  ANA LYTICA L QUA LITY CONTROL

 A Need

    A principal concern of the FWPCA is the
    reliability of water quality measurements
    used to determine compliance with water
    quality  standards.  When violations occur,
    these data will be used to defend litigation
    proceedings.  In anticipation of such
    circumstances,  new emphasis is being
    given to analytical quality control within
    FWPCA.  The agency and those who  work
    with it must expect court challenges  by
    those who carry the burden of defense.
    Data which cannot be successfully defended
    under spirited and argumentative challenges
    will mean serious loss of state and Federal
    program effectiveness.  Thus, the Federal
    Water Pollution Control Administration is
    taking a strong position  toward upgrading
    the present system for analytical quality
    control and methods validation; before
    using information from other sources the
    Administration will scrutinize the data--as
    it will screen its own--to assure the
    successful presentation  of the facts
    associated with a specific pollution
    situation.
 V  DATA HANDLING

 A  Coordination

    Vital to the FWPCA mission and essential
    to a coordinated and cooperative state/
    Federal pollution control effort is a unified
    or compatible approach to the processing
    and timely reporting of surveillance data.
    This includes both water quality information
    and facilities statistics.   Data of the highest
    scientific accuracy and precision are of
    little value unless they can be applied at
    the right time.
  VI 5-6

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                                       FWPCA Responsibilities for Water Quality Standards
B  STORET

   1 Computer center - In the past, various
     computer systems have been used to
     handle the volume of information which
    . must flow among the responsible pollution
     control agencies.  To assist this operation,
     FWPCA has established the STORET
     system (STOrage and RETrieval) to
     assure  that all collected information
     is available in a timely and concise
     fashion. This system has been converted,
     along with subsystems now being
     developed, for use at the Department of
     the Interior's computer center which
    , utilizes the IBM 360/65.

   2 Extent  of use - All FWPCA water quality
     data and related information are currently
     being placed in the STORET system so
     that they can be rapidly retrieved to
     support all operating arms of the
     Administration throughout the United
     States.  FWPCA invites the use of the
     STORET system by all water pollution
     control agencies having need of such
     data, and FWPCA frequently inserts
     information developed by other Federal
     and state agencies if it has a direct use
     consistent with the pollution control
    • mission.  At the present time, STORET
     contains water quality data and, in many
     instances, flow data for over 6, 000
     stations which have been operating for
     one year or more. At least 3, 500
     station  records include flow data and/or
     quality  data collected by the U. S.
     Geological Survey which are being used
     in enforcement, comprehensive planning,
     and water quality standards development
     activities of FWPCA.  Similarly,  FWPCA
     projects have inserted data from ten
     states,  as collected by state water
     pollution control or water  resource
     agencies. In addition, water quality
     and quantity data for over  100 locations
     have been inserted in STORET to assist
     the Tennessee Valley Authority to
     implement pollution control responsibilities.

   3 Retrieval of data - Since statistical
    , printouts of STORET data  for specific
     locations are available upon request,
     many states are now tapping the STORET
       reservoir of information.  The
       availability of concise printouts,
       including all available data of reputable
       quality at a specific location, is
       particularly valuable in judging whether
       a proposed  criteria will in fact result
       in water quality enhancement.   At
       present, turn-around time for variable
       retrievals from the system,  as offered
       to the various agencies, is dependent
       on the mails. Ultimately, FWPCA is
       planning a national communications
       data handling system which would
       further reduce the time required to
       respond to requests for services.  The
       cost of such a system will be small as
       compared to the cost of developing
       analytical results and will be minor
       when one considers the estimated 20 to
       50 billion dollar cost of the remedial
       construction program.  By connecting
       the central  computer with terminal
       facilities located in FWPCA  regional
       offices,  laboratories, and other major
       program offices,  it will be possible to
       utilize the full power of the STORET
       system.  Such a network will make
       STORET and the developing subsystems
       available to all elements of FWPCA and
       other water pollution control agencies.
VI  SUMMARY

 In this new era of water pollution control in
 the United States, the fight to restore water
 quality in our environment has just begun.
 Accordingly, the necessary operations  are
 still in the formative stages as related  to the
 ultimate needs.   The type of information to
 be gathered must be carefully selected; the
 data must be verified to assure that they meet
 their intended purpose.  The coordinated
 exchange of information among cooperating
 and responsible agencies will play a major
 role in minimizing the cost of surveillance
 activities as well as speeding water pollution
 control toward its ultimate goal--enhancement
 of the quality and value of the Nation's
 water resources.

 This outline was prepared for the Training
 Program by M. L.  Wood, Director, Robert
 S. Kerr Water Research Center, Ada,
 Oklahoma.
                                                                                    VI 5-7

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                           MARINE AND ESTUARINE PLANKTON
 I  INTRODUCTION

 In recent years the rate of increase in the
 human population of the earth and the resulting
 need for more food has intensified our interest
 in, and increased emphasis on, studies of those
 organisms forming the key units or  "links" in
 oceanic food chains,  the plankton.  In addition
 to this interest,  the value of these planktonic
 organisms as "indicators" of the origin of
 water masses, both oceanic  and estuarme,
 has become increasingly evident to students of
 oceanography and estuarme hydrography.

 The common organisms making up the estuarme
 and marine plankton and the  general environ-
 mental factors influencing their occurrence,
 abundance and distribution are, therefore,
 worthy of the attention of all persons interested
 in the biology of these environments.
II  THE ESTUARINE ENVIRONMENT

 A Definition

   An estuary may be variously defined.  For
   biological purposes it might best be con-
   sidered as a region v/here river water
   mixes with, and measurably dilutes sea
   water; or, as those water masses which,
   by virtue of their position, are directly
   subject to the combined action of river and
   tidal currents.

 B General Structure

   1  Physical, chemical and environmental
      characteristics of an estuary are depend-
      ent on the degree of mixing and on the
      currents and circulation patterns set up
      when two dissimilar water masses meet.

   2  Types of estuary:

      a  Two-layered
       b  Partially mixed

       c  Completely mixed

       The mixing and circulation of estuaries
       are controlled by such varied factors as
       wind, rate of fresh-water run-off, tidal
       amplitude and physiographic features
       such as the slope, width and depth of the
       estuarme basin.

       These diverse factors acting simultane-
       ously make each estuary a unique con-
       stantly changing environment.
Ill  THE ESTUARINE PLANKTON

 A Plankton Types

    1  Permanent estuarme plankton is com-
       posed of those organisms remaining for
       an appreciable Jength of time within the
       estuary.

    2  Transient estuarme plankton is made  up
       of those transitory forms from:

       a  Fresh water feeding into  the head of
          the estuary.

       b  Marine waters brought into the
          estuary from the sea.

       The stay of transient forms  in the
       estuary is brief and may be  limited to
       a single tidal cycle.

 B Permanent Estuarme Plankton

    1  Example:  St. John River, New
       Brunswick, Canada

       a  Salt water penetrates 25 miles up-
          stream from the river mouth.
 BI.MAR. mic. 2a.4.70
                                    VI 6-1

-------
 Marine and Estuanne Plankton
         1)  At the upper end of the estuary
            barnacle nauplu and Sagitta elegans
            are found in August.

         2)  Both of these planktomc forms are
            less common near the mouth of the
            estuary.

      b  This is an instance where the length
         of the estuarine system leads to a
         water mass and its plankton becoming
         "stranded" in the system and,  there-
         fore,  more or less permanent.

   2  Example: Miramichi River,  New
      Brunswick, Canada

      a  Smelt larvae occur in samples from
         the middle part of the estuary begin-
         ning in June.

      b  During the ensuing summer (June-
         August) catches in the same area
         indicate growth of the larvae.

      c  All evidence indicates a light-
         controlled diurnal vertical migration
         of the larvae.  In the summer months
         this would keep them in the bottom
         layer of inflowing sea water most  of
         the time.

C  Transient Estuarine  Plankton

   1  Example:  Margaree River, Nova
      Scotia, Canada

      a  This is a very  short river as com-
         pared with the  St. John and Miramichi.

      b  Plankton tows reveal organisms of
         marine or freshwater origin depend-
         ing on state of  the tide.

         1) At high tide  marine cladocerans,
           copepods, ctenophores, polychaete
           larvae and marine diatoms are
           found.

         2) At low tide very few planktomc
           organisms are found and these  are
           all  of fresh  water origin; e.g.,
           Dephnia, Cyclops and freshwater
           diatoms.
          No permanent plankton exists in the
          Margaree estuary, since it is a short,
          shallow system.
IV  THE MARINE ENVIRONMENT

 A When compared with that of the estuary the
    environment of the ocean is found to be
    more stable.

 B Currents

    1  No part of the ocean is completely
       stagnant.  Circulation has evolved  in
       all oceans, determined largely by the
       rotation of the earth, positions of the
       sun and moon, the winds and the con-
       figurations of the ocean basins.

    2  There are also currents caused by
       difference in density, by up well ings
       and by the entry of river waters into
       the sea.

 C Planktomc distribution is largely a function
    of temperature,  nutrients,  salinity, light,
    and current patterns.
V  THE MARINE PLANKTON

 A  Types of Marine Plankton

    1  The phytoplankton includes all the freely
       floating photosynthetic forms.  Individu-
       ally they are microscopic in size and
       are  unicellular or in groups of cells
       loosely bound together.

    2  The zooplankton includes a great array
       of micro- and macroscopic free floating
       animals ranging in size from several
       micra to about 50 mm.   (Some jellyfish
       may reach 1 m. in diameter.)

    3  Marine plankton may be  further divided
       into:

       a  Temporary plankton consisting of
         transitory, floating eggs,  larvae and
         juveniles of bent hi c and nektomc
         organisms, medusoid stages  of
         benthic coelenterates, and the spores
         and gametes of various benthic algae.
VI 6-2

-------
                                                             Marine ana Estuanne Plankton
        1) Temporary plankton is often
           seasonal in occurrence and
           generally is confined to neretic
           or coastal waters.

     b  Permanent plankton consists of
        organisms completely adapted  to
        open ocean existence which remain
        floating or feebly swimming through-
        out their entire life cycle.

B  Organisms of the Phytoplankton

   1  Diatoms (Bacillanophyceae) are first
     in rank as producers of organic material
     (especially in northern waters).

     a  Reproduction

        Reproduction in diatoms is  by binary
        fission.  This potential for  geometric
        increase in population size  contributes
        to "blooms" under  favorable
        conditions.

   2 Dmoflagellates are generally considered
     second to diatoms as primary  producers
     though often they are more important in
     southern waters.

     a  Dmoflagellates are most well known
        for their "blooms" which result in
        catastrophic mortalities.   Red tides
        caused by various species of Gym-
        nodmium in southern areas are a
        good example.

   3 Coccolithophores constitute major com-
     ponents of the plankton in some areas.
     They are of special geological signifi-
     cance because of the accumulation of
     their calcium carbonate coccoliths in
     marine sediments.

C  Factors Affecting Distribution of
   Phytoplankton

   1 Light

     The amount of light energy available to
     photosynthesizers diminishes  with depth.
   2  Vertical circulation

     Adequate supplies of plant nutrients
     (especially nitrates and phosphates)
     must be present if the phytoplankton
     is to grow.

     a  The most abundant  source of such
        nutrients  is bottom sediment which
        is out of reach of the photosynthesizing
        organisms.

     b  Methods of returning nutrients to the
        surface are:

        1) Upwellings (most commonly en-
           countered near shore).

        2) Divergences of major currents
           (more common in areas far from
           shore).

   3  Temperature and  salinity act largely
     as selective agents.

D  Marine Zooplankton

   1  The marine zooplankton contains organ-
     isms in every major group of marine
     animals except the sponges,  bryozoans,
     brachiopoda,  and  mammals.  Copepods
     are  the most abundant representatives.

   2  The greatest significance of  the zoo-
     plankton lies in their role as organic
     links in the biological economy of the
     sea.

   3  Cyclic Representation of a General
     Food Chain in the Sea:
 Sun
             CO,
  \
  PH
PHYTOPLANKTON	) GRAZERS

          \
       Nutrients
                        CARNIVORES I
 Death &'
 Sedimentation
                             I
              vDeath &
               Excretion*	CARNIVORES II
                                                                                     VI 6-3

-------
Marine and Estuarine Plankton
REFERENCES

1  Hurt, W.V. and McAlister,  W. B.  Recent
      Studies on the Hydrography of Oregon
      Estuaries.  Res.  Briefs, Fish.  Comm.
      of Oregon,  Vol. 7, pp. 14-27.  1959.

2  Clemens, W.A.  Pastures of the Sea.
      Occasional Pap.  Calif. A cad. Sci.,
      No. 41, 3 figs.,  8 pp.  1963.

3  Hardy, A. C.  The Open Sea, Its Natural
      History:  The World of Plankton.
      Houghton-Mifflin Co., Boston, 335 pp.
      1956.

 4  Johnson, M. W.  Plankton.  IN J.W.
      Hedgpeth,  ed., Treatise on Marine
      Ecology and Paleoecoplogy, Vol.  1,
      Ecology.  Chap.  16, pp.  443-459, Mem.
      67, Geological Soc. of America.  1957.

 5  Ketchum,  B. H.  The Exchanges of Fresh
      and Salt Waters  in Tidal Estuaries.
      Jour. Marine Res.,  Vol.  10, pp. 18-
      38.  1951.
   Olson, Theodore A. and Burgess,
      Frederick J.   Pollution and Marine
      Ecology.  Interscience Publishers.
      364 pp.   1967.
   Rogers, H.M.  Occurrence and Retention
      of Plankton within the Estuary.  Jour.
      Fisheries Res. Bd.  Canada, Vol. 5,
      pp.  164-171.   1940.
This outline was prepared by G. C. Hughes,
Assistant Professor of Biological Sciences,
University of the Pacific, and Assistant
Professor of Marine Science, Pacific
Marine Station, Dillon Beach, Marin County,
California.
 VI  6-4

-------
                                   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, Lais on, 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                A djective
 various         epiholitic, nereiditic, sessile
 plants          epiphytic
 animals        epizooic
 wood           epidendritic,  epixylonic
 rock           epihthic

[After Srameck-HuseM 1946) and via Sladeckova
 (1962)] Most above listed latin-root adjectives
 are derivatives of nouns such as epihola,
 epiphyton, spizoa,  etc.
BI.MIC.enu. 19a.4. 70
                                                                                   VI  7-1

-------
 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.
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
       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); VI
       or the process of collection adds sufficient
       foreign materials  d. e. detritus,  sub-
       strate, etc.) so that some  commonly         A
       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).
    1 It can prevent the settling of smothering
      materials.

    2 It flushes metabolic wastes away and
      introduces nutrients to the colony.
    THE LENGTH OF TIME THE SUBSTRATE
    IS EXPOSED IS IMPORTANT.

    The growths need time to colonize and
    develop on the recently  introduced
    substrate.

    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.

    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.
   VI 7-2

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                                                     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


K  EXPRESSION OF RESULTS

 A Qualitative

    1 Forms found

    2 Ratios of number per group found

    3 Frequency distribution of varieties
      found

  B Quantitative

    1 A real 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 O?/mg of growth/hour
                                                                                     VI  7-3

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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.  Uber 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.
   Srameck~Husek  (On the Uniform
      Classification of Animal and Plant
      Communities in our Waters).
      Sbornik MAP 20:3:213.   Orig. in
      Czech.  1946.

   Thomas,  N.A.   Method for Slide
      Attachment in Periphyton Studies.
      Manuscript.  1968.

   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+pp. (multilith).  1966.
This outline was prepared by Lowell E. Keup,
Acting Supervisory Biologist, Biological and
Chemical Section,  National Field Investigations
Center, FWPCA, U. S. Dept. of the Interior,
Cincinnati, OH.
VI  7-4

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                 ARTIFICIAL AND RELATED SUBSTRATES - REFERENCES
INTRODUCTION

This reference list in the field of artificial
substrates has been prepared for those not
familiar with the literature and for those
planning to use one or more of these
techniques.  It includes references both to
periphyton and benthos including techniques,
rationale, and practical applications. Since
1967, Dr. C. I. Weber of the Analytical
Quality Control Laboratory has compiled an
annual bibliography for the Midwest
Benthological Society.  These bibliographies
are recommended for additional and current
references. This list is thus complementary
to Weber's annual citations for the periphyton.
Addresses and sources are given where
possible.

Priority has been given to comprehensive
works; those of historical note, especially
where the original description of a sampler
is given, and a sampling of recent papers
to show versatility and application for general
and specific uses.
GENERAL

Cooke, William B.  Colonization of Artificial
   Bare Areas by Microorganisms.  Bot.
   Rev. 22(9):613-638.  Nov. 1956.

Sladeckova, Alena.  Limnological  Investiga-
   tion Methods for the Periphyton C'Aufwuchs")
   Community.  Bot.  Rev. 28(2):286-350.
   1962.

Cummins,  K.  W. An Evaluation of Some
   Techniques for the Collection and Analysis
   of Benthic Samples with Special Emphasis
   on Lotic Waters.  American Midland
   Naturalist, Vol.  67, No. 2.  pp. 477-504.
   1962.
PERIPHYTON - GENERAL

Weber, C. E. (FWPCA,  1014 Broadway
   Cincinnati, OH 45202)  Benthic Macro-
   invertebrates and Periphyton.  Select and
   Current Bibliographies.  Midwest
   Benthological Society.

Sladeckova, Alena.  The Significance of the
   Periphyton in Reservoirs for Theoretical
   and Applied Limnology. Verh.  Internat.
   Verein. Limnol.  16:753-758.  1966
PERIPHYTON - PARAFFIN-COATED
SUBSTRATES

Beers, G.  D.  and Neuhold,  J. M.
   Measurement of Stream Periphyton on
   Paraffin-Coated Substrates.  Limnol.
   andOceanogr.  13(3)-559-562. 1968.

PERIPHYTON - GLASS SLIDES

See general references above.

Thomas, Nelson A. (National  Center for
   Field Investigations, FWPCA, 5555
   Ridge Avenue,  Cincinnati,  OH 45213. )
   Method for Slide Attachment in Periphyton
   Studies.  Manuscript.  (Nonhardening
   "Plasti-tak" used for attaching slides to
   bricks. )

Weber,  Cornelius I.,  and McFarland,
   Ben H.  (FWPCA,  Analytical Quality
   Control, 1014 Broadway, Cincinnati, OH
   45202) Periphyton Biomass-Chlorophyll
   Ratio as an Index of Water Quality.  17th
   Annual Meeting, Midwest Benthological
   Society.  19 pp. Multilithed. 1969.

Weber,  C.  E  and Rauschke,  R  L.
   Use of a Floating Periphyton  Sampler
   for Water  Pollution Surveillance.  Water
   Poll.  Sur. Sept. Applications and Develop.
   Report No. 20.  FWPCA-USDI,  Cincinnati,
   Ohio. September 1966.
PERIPHYTON - DIATOMS

Patrick, R , Hohn, M.  H.,  and Wallace,  J.H.
   A New Method for Determining the Pattern
   of the Diatom Flora.  Notulae Naturae,
   Academy of Natural Sciences of Philadelphia.
   No. 259.  pp.  1-12.  1954.

Hohn,  M. H.  The Use of Diatom Populations
   as a Measure of Water Quality in Selected
   Areas of Galveston and Chocolate Bay,
   Texas.  Publications of the Institute of
   Marine Science of the University of Texas.
   Vol. 6.  pp. 206-212.   1959.

Patrick, R.  Factors Affecting the Distribution
   of Diatoms.  Botanical Review.  Vol.  14.
   No. 8. pp. 473-524.   1948.

Patrick. R.  A Discussion of Natural and
   Abnormal Diatom Communities.   In:
   Jackson, D. F. (ed.),  Algae and Man.
   Plenum Press, New York.  pp. 185-204.
   1964.
BI. BIB. 1.5.70
                                 VI 8-1

-------
Artificial and Related Substrates - References
Hohn, M.   Determining the Pattern of the
   Diatom Flora.  Journal of the Water
   Pollution Control Federation, Vol. 33.
   No. 1.  pp.  48-53.  1961.

Hohn, M.   The Relationship Between Species
   Diversity and Population  Density in  Diatom
   Populations from Silver  Springs, Florida.
   Transactions of the American Micro-
   scopical Society.  Vol. 80. No.  2. pp.
   140-165.  1961.
PERIPHYTON - PLASTIC SLIDES AND
PANELS

Grzenda, Alfred R. and Brehmer, Morris L.
   A Quantitative Method for the Collection
   and Measurement of Stream Periphyton.
   Limnol. Oceanog. 5(2):190-194.
PERIPHYTON - DIATOMS - STYROFOAM

Hellerman,  J.  A Study of the Diatoms of
   Mohonk Lake, New York and Vicinity.
   Unpublished M. S. Thesis. Rutgers  - The
   State University.  1962.

Hohn, Matthew H. (Department of Biology,
   Central Michigan University,  Mt. Pleasant,
   Michigan).  Artificial Substrate for Benthic
   Diatoms—Collection, Analysis, and
   Interpretation, p. 87-97.  1966.  In:
   Organism Substrate Relationships in
   Streams Pymatuning Special Publication.
   No. 4.  $2.50. Pymatuning Laboratory
   of Ecology, University of Pittsburgh.
   Pittsburgh, PA 15213.
PERIPHYTON - TRICKLING FILTERS

Cooke, W. B.  Continuous Sampling of
   Trickling Filter Populations.  Sewage and
   Industrial Wastes.  Vol. 30. No. 1.
   pp.  21-27.   1958.

Cooke, W. B.  Fungi in Polluted Water and
   Sewage.  IV  The Occurrence of Fungi in
   a Trickling Filter-type Sewage Treatment
   Plant.  Proceedings of the 13th Industrial
   Waste Conference, Purdue University,
   Series  No.  96,  Vol. 43.  No. 3.  pp. 26-45.
   1959.

Cooke, W. B.  Trickling Filter Ecology.
   Ecology. Vol. 40. No. 2. pp. 273-291.
   1959.
PERIPHYTON - LABORATORY STUDIES

Ehrlich, Gary G. and Slack, Keith V.
   (Water Res. Div., USGS, Menlo Park,
   California  94025).  Uptake and Assimila-
   tion of Nitrogen Microecological Systems.
   Spec. Tech. Pub. 448. ASTM. p.  11-23.
   1969.
MACROINVERTEBRATES - GENERAL

Macan,  T.  T.  Methods of Sampling the
   Bottom Fauna of Stony Streams.  Mitt.
   Intern. Ver.  Limnol.  No. 8. p. 1-21.
   1958.
MACROINVERTEBRATES - BRUSH BOXES

Scott, D. C.  Biological Balance in Streams.
   Sewage and Industrial Wastes.  Vol.  30.
   No. 9.  pp. 1169-1172.  1958
MACROINVERTEBRATES - ROCK BASKETS

Anderson, J. B. and Mason, William T.,  Jr.
   A Comparison of Benthic Macroinvertebrates
   Collected by Dredge and Basket Sampler.
   Jour.  Water Poll. Cont.  Fed. 40(2):252-259.

Anderson, J. B. and Mason, W. T., Jr.
   The Use of Limestone-Filled Samples for
   Collecting Macroinvertebrates from
   Large Streams.  Water Poll. Surv.  Syst.
   Application and Develop.  Report No.  17,
   FWPCA-USDI, Cincinnati, Ohio.  May  1966.

Henson, E. B.,  Jr.  A Cage Sampler for
   Collecting Aquatic Fauna. Turtox News.
   43:298-299.   1965.

Hilsenhoff, William L.  An Artificial Substrate
   Device for Sampling Benthic Stream
   Invertebrates.  Limnology and Oceanography.
   14(3):465-471.  1969.

Mason, W. T., Anderson,  J. B.,  and
   Morrison, G. E.  (FWPCA,  Analytical
   Quality Control,  1014 Broadway, Cincinnati,
   Ohio)  Limestone-filled,  Artificial Substrate
   Sampler-float unit for Collecting Macro-
   invertebrates in large streams.  Progressive
   Fish Culturist.  29:74.  1967.

Wene, G.,  and Wickliff.  E.  L.  Modification
   of the Stream Bottom and Its Effect on  the
   Insect Fauna.  Can. Entomologist.  72:131-
   135.  1940.
 VI  8-2

-------
                                              Artificial and Related Substrates - References
MA GROIN VERTEBRATES - MULTIPLE
PLATES
                                      colonized "than the living plant"
                                      (invertebrate s).
Arthur, JohnW. and Horning, William B.,
   (Federal Water Pollution Control
   Administration,  Duluth, Minnesota,
   National Water Quality Laboratory.
   The Use of Artificial Substrates in
   Pollution Surveys.   The Amer. Midland
   Natur. Vol. 82.  No. 1.  pp. 83-89.  1969.

Hester, F. E.  and Dendy,  J  S.
   A  Multiple-Plate Sampler for Aquatic
   Macroinvertebrates.  Trans.  Am. Fish.
   Soc.  91 (4):420-421. April 1962.

Brewer,  Jesse W. and Gleason,  Gale R.
   Modification of the Dendy Principle for
   Stream Bottom Sampling.  Midwest
   Benthological Society. 12th Annual Mtg.
   Mimeo.  1964.
Dendy, J. S.
   Animals.
   1963.
 Living Food for Aquatic
Turtox News 41(10):258-259.
MACROINVERTEBRATES - CEMENT
PLATES

Britt,  N. W.  New Methods for Collecting
   Bottom Fauna from Shoals or Rubble
   Bottoms of Lakes and Streams.
   Ecology.  36:524-525.  1955.

MACROINVERTEBRATES - BURIED TRAYS

Moon,  H.  P   Methods and Apparatus Suitable
   for  an Investigation of the Littoral Region
   of Oligotrophic Lakes.  Int.  Rev.  Hydrobiol.
   32:319-333.  1935.
MACROINVERTEBRATES - PLASTIC TAPES
FOR BLACKFLIES

Williams, T. R. and Obeng, L.  A Compari-
   son of Two Methods of Estimating Changes
   in Simulium  Larvae Populations, with a
   Description of a New Method.  Ann. Trop.
   Med. Parasit. 56:358-361.   1962.
MACROINVERTEBRATES - ARTIFICIAL
WEEDS

Freshwater Biological Association (The
   Ferry House.  Far Sawrey, Ambleside.
   Westmoreland,  England).  1969.
   Thirty-seventh Annual Report, p. 36.
   Artificial weeds made of strands of
   polyethylene and polypropylene twine.
    'Artificial Littorella is more heavily
                                    MACROINVERTEBRATES - BRICKS

                                    Elvins, B  J.  Investigation of the Animal
                                      Population in Polluted Streams.  Journ.
                                      Inst. Sewage Purif.  Part 6. p. 569.
MACROINVERTEBRATES - 10 or 20 Stone
Surveys

Chutter,  F. M.  On the Ecology of the Fauna
   of Stones in the Current in a South African
   River Supporting a Very Large Simulium
   (Diptera) Population.  J.  App. Ecol.
   5:531-561.  1968.

Williams. T.  R  and Obeng, L.  A  Comparison
   of Two Methods of Estimating Changes in
   Simulium Larval Populations, with a
   Description of a New Method.  Ann. Trop.
   Med.  Parasit. 56:358-361.   1962.

Reed, Roger J.  1966.  Some Effects of DDT
   on the Ecology of Salmon Streams in
   Southeastern Alaska.  USFWS Special
   Scientific Report No. 542.  15 pp.

Cope, Oliver B.   Effects of DDT Spraying
   for Spruce  Budworm on Fish in the
   Yellowstone River System.  Trans. Amer.
   Fish.  Soc.  90:239-251.

MACROINVERTEBRATES - ECOLOGY

Waters,  T. F. Recolonization of Denuded
   Stream Bottom Areas by Drift.  Trans-
   actions of the American Fisheries Society,
   Vol. 93, No.  3, pp. 311-315.  1964.

Waters,  T. F. Standing Crop and Drift of
   Stream Bottom Organisms.   Ecology.
   42:532-537. 1961.

Driscoll, E. G.  Attached Epifauna-Substrate
   Relations.  Limnol.  Oceanogr. 12(4):633-641.
   1967.
                                   MICROINVERTEBRATES  - SESSILE
                                   PROTOZOA

                                   Spoon, D. M.  and Burbanck, W. D.
                                      A New Method for Collecting Sessile
                                      Ciliates in Plastic Petri Dishes with Tight
                                      Fitting Lids.  J. Protozool.  14(4):735-739.
                                      1967.
                                                                                  VI  8-3

-------
 Artificial and Related Substrates - References
 Burbanck, W. D.  (Emory University,
   Atlanta, Georgia) and Spoon, D. M.
   The Use of Sessile Ciliates  Collected
   in Plastic Petri Dishes for Rapid
   Assessment of Water Pollution. J.
   Protozool. 14(4):739-744.  1967.

 Sickel, James B.  A Survey of the Mussel
   Populations (Unionidae) and Protozoa of
   the Altamaha River with Reference to
   Their Use in Monitoring Environmental
   Changes. MS Thesis. Emory University.
   133 pp.  1969.

 Sladecova,  Alena.  (Inst. Chem.  Tech.,
   Prague, Czechoslovakia) Factors
   Affecting the Occurrence and Stratifica-
   tion of Sessile Protozoans in Artificial
   Reservoirs,  (in Russian.Summ. in English
   and Czech) Technology of Water.
   8(1):483-490.  1964.
 SUBSTRATES - BUOYS AND OTHER
 NAVIGATIONAL AIDS

 Miller, Milton A.  Isopoda and Tanaidacea
   from Buoys in Coastal Waters of the
   Continental U. S., Hawaii, and the
   Bahamas (Crustacea).  Proc. U. S. Nat.
   Mus. 125  (3652):53 pp. 1968.

 Fremling, C. R.  Biology and Possible
   Control of Nuisance Caddisflies of the
   Upper  Mississippi River.  Agricultural
   and Home Economics Experiment Station,
   Iowa State University of Science and
   Technology. Ames, Iowa.  Research
   Bulletin 483, pp.  856-879.  1960.
RECOVERY DEVICES

Ziebell, Charles D.,  McConnell, W  J  ,
   and Baldwin, Howard A.  A Sonic
   Recovery Device for Submerged Equipment.
   Limnol. and Ocean.  13(1):198-200.  1968.

Fox,  Alfred C.  (Univ.  of Georgia.
   Cooperative Fisheries  Unit, Athens, Georgia)
   Personal Communication.  Use of Inexpensive
   Detonator;  "Seal Salute. " Miller Fireworks
                           TVA, Div.  of Health and Safety,
                           Water Quality Branch.   1967.

                        Waters, T  F  Notes on the Chlorophyll
                           Method of Estimating the Photosynthetic
                           Capacity of Stream Periphyton.  Limnol.
                           Oceanogr.  6:486-488.   1961.

                        Wetzel, R. G.  Techniques and Problems
                           of Primary Productivity Measurements
                           in Higher Aquatic Plants and Periphyton.
                           In:  C.  R. Goldman et. "Primary
                           Productivity in Aquatic Environments. "
                           Univ. Calif. Press.  Berkeley.  1966.

                        U.  S. Dept. of the Interior, FWPCA.
                          Keup, Lowell and Stewart, Keith.
                          National Field Investigations Center,
                          4676 Columbia Parkway, Cincinnati,
                          OH 45226.  Effects of Pollution on
                          Biota of the Pigeon River, North
                          Carolina and Tennessee. 35 pp.  1966.
                          Utilization of glass slides attached
                          to bricks for periphyton, chlorophyll
                          biomass ratios to evaluate paper  mill
                          and other industrial effluents.

                        	Keup, Lowell.  National Field
                          Investigations Center. Effects of
                          Pollution on Aquatic Life Resources
                          of the South Platte River Basin in
                          Colorado.  Vol.  1  and Vol.  2.   1967.

                        Vollenweider, Richard A.  et al.  (Eds.)
                          A  Manual on Methods for Measuring
                          Primary Production in Aquatic Environ-
                          ments.  International Biological Programme
                          Handbook.   No. 12.  213 pp. Davis.  1969.
                        NOTE:  Mention of commercial products and
                        manufacturers does not imply endorsement
                        by the Federal Water Pollution Control
                        Administration and the U. S. Department of
                        the Interior.
   and Novelty Company,
   Holland, Ohio 43528.
   shipping.  1969.
501 Gleneary Road,
$4.00 gross and
ARTIFICIAL SUBSTRATES - PRACTICAL
APPLICATIONS IN WATER POLLUTION
CONTROL

Taylor, Mahlon P.  Thermal Effects on the
   Periphyton Community in the Green River.
VI  8-4

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CHAPTER VII





IDENTIFICATION KEYS




Key to Selected Groups of Freshwater Animals




Key to Algae of Importance in Water Pollution
1




2

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                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
                  • of the group. Phylum
PROTOZOA.  If you selected "lb'\  proceed
to the couplet indicated.  Continue this
process until the selected statement is
terminated with the name of a group.

If you wish more information about the
group, consult references.  (See reference
list.)
BI.AQ.21E.8.69
                                                                                 VII  1-1

-------
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
      m a colony with little or no differ-
      ence between the cells, i.e. ,  with-
      out forming tissues; or body com-
      prised of masses  of multmucleate
      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 of 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
 VII  1-2

-------
                                              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 ANNELIDIA (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,  nereid worms.
          Phylum ANNELIDIA (Fig. 9)

14a  Segments with bristles and/or fleshy
     lobes or other extentions.  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,  often slimey, 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 PELECYPODA (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
    apparently present.

20a Body elongated, head broad and flat
20
                                                                               'VII  1-3

-------
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 BRANCHIPODA
          (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.
      Cyclops or cladocera.
          Subclass CLADOCERA (Fig.  12)

24b   Locomotion accomplished by        25
      body legs,  not by antennae.
25a Appendages leaflike, flattened,
    more than ten pairs.
        Subclass BRANCHIPODA
        (See 22 a)

25b Animal less than 3 mm, in length.
    Appendages more or less slender
    and jointed, often used for walking.
    Shells opaque.  Ostracodes.
        (Fig. 11)  Subclass OSTRACODA

26a Body  a series of six or more         27
    similar segments, differing
    mainly in size.

26b Front part of body enlarged into      28
    a somewhat separate body unit
    (cephalothorax) often covered
    with a single piece of shell (cara-
    pace). Back part (abdomen) may be
    relatively small,  even folded
    underneath front part. (Fig.  19b)

27a Body  compressed laterally,  i.e.,
    organism is tall and thin.  Scuds.
    amphipods.
        Subclass AMPHIPODA (Fig. 17)

27b Body  compressed dorsoventrally,
    i. e.,  organism low and broad.
    Flat gills contained in chamber
    beneath tail.  Sowbugs.
        Subclass ISOPODA (Fig. 18)

28a Abdomen extending straight out
    behind, ending in two small pro-
    jections.   Two large masses of
    eggs are  often attached to female.
    Locomotion by means of two enlarged,
    unbranched antennae, the only large
    appendages on the body. Copepods.
        Subclass COPEPODA (Fig. 19)

28b Abdomen extending out  behind ending
    in an expanded "flipper" or swim-
    ming paddle.  Crayfish or  craw fish.
    Eyes on movable stalks.  Size range
    usually from one to six inches.
        Subclass DECAPODA

29a Two pairs of functional wings,         39
    one pair may be more or less har-
    dened as  protection for the other
    pair.  Adult insects which normally
    live on or in the water. (Figs.  25. 28)
 VII 1-4

-------
                                              Key to Selected Groups of freshwater Animals
29b
30a
     No functional wings, though
     pads in which wings are develop-
     ing may be visible. Some may
     resemble adult insects very
     closely, others may differ ex-
     tremely from adults.
30
     External pads or cases in which     35
     wings develop clearly visible.(Figs.
     24.16.27)
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 

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. 24Q

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.  24D)
                                                38
                                                                                   VE 1-5

-------
  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.
           Ofcder COLEOPTBRA
           (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).

 4Ob  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
VII 1-6

-------
                                           Key to Selected Groups of Freshwater Animals^
REFERENCES - Invertebrates
REFERENCES - Fishes
     Eddy,  S. and Hodson, A. C.
          Taxonomic Keys to the Common
         Animals of the North Central States"
         Burgess Pub. Co. ,  Minneapolis,
         pp 1-141.  1955.

     Edmondson, W. T.   (ed). and Ward
         and Whipple's Freshwater Biology.
         John Wiley & Sons,  New York.
         pp 1-1248.  1959.

     Jahn,  T. L.  and Jahn, F.F.  "How
         to Know the Protozoa" Win. C.
         Brown Company, Dubuque, Iowa.
         pp 1-234.  1949.

     Kudo,  R.  "Protozoology" Charles
         C. Thomas, Publisher,  Spring-
         field, Illinois,  pp 1-778.  1950.

     Palmer, E. Lawrence "Fieldbook
         of Natural History" Whittlesey
         House,  McGraw-Hill Book Co.,
         Inc., New York.  1949.

     Pennak, R. W.  "Freshwater Inverte-
         brates of the United States"
         The Ronald Press Co. ,  New York
         pp 1-769.  1953

     Pr.afo, W. W.  'M MMtunA of tfa* Common
         Invertebrate Animals Exclusive of
         Insects"  The Blaikston Co. , Phila.
         pp 1-854.  1951
     American Fisheries Society  A List
         of Common and Scientific Names
         of Fishes from the United States
         and Canada.  Special Publication
         No. 2, Am.  FishSoc., Dr. E.A
         Seaman, Sec. -Treas., Box 483,
         McLean, Va   (Price $1. 00 paper,
         $2. 00 cloth).   1960.

     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.)

     Eddy,  Samuel "How to Know the
         Freshwater Fishes" Wm. C
         Brown Co.,  Dubuque, Iowa.  1957.

     Hubbs, C. L. and Lagler, K.F.
         Fishes of the Great Lakes Region.
         Bull.  Cranbrook Inst  Science,
         Bloomfield Hills, Michigan.  1949.
      Lagler, K.F.
          Biology"
          Dubuque,
 "Freshwater Fishery
Wm. C  Brown Co. ,
Iowa.  1952
      Trautman, M. B.   "The Fishes of Ohio"
          Ohio State University Press,
          Columbus   1957.  (An outstanding
          example of a State study  )
                                                                             VII  1-7

-------
Key to Selected Groups of Freshwater Animals
         1. Spongilla spicules
            Up to .2 mm. long.
3A  Rotifer, Polyarthra
  '             ~
                    3B. Rotifer. Keratella
                        Up to .3 mm.
  4A.  Jointed leg
       Caddis fly
                                 4B. Jointed leg
                                     Crayfish
                                                                 2B. Bryosoal mass.  Up to
                                                                     several feet diam.
                                    3C-  Rotifer, Philpdina
                                       Up to. 4 mm.
                                       2A. Bryozoa, Plumatella. Individuals up
                                           to ?• mm.  Intertwined masses maybe
                                           very extensive.
4C. Jointed leg
     Ostracod
                                                                   5.  Tapeworm head.
                                                                      Taenia.  Up to
                                                                      25 yds. long
       6A. Turbellaria, Mesostoma
           Up to 1 cm.
                                     6B. Turbellaria, Dugesia
                                          Up to 1. 6 cm.
                                                               7. Nematodes.  Free living
                                                                  forms commonly up to
                                                                  1 mm. ,  occasionally
                                                                  more.

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                                      Key to Selected Groups ojJFreshwater Animals
                  8B. Diptera, Mosquito
                      pupa. Up to 5mm.
8A.  Diptera, Mosquito larvae
    Up to 15 mm. long.
                      8C. Diptera,  chironomid
                           larvae.  Up to 2 cm.
                                                           ilE'
pupa. Up to 2. 5 cm.
                                   9D. Diptera, Rattailed maggot
                                       Up to 25 mm. without tube
9A.  Annelid,
    segmented
    worm, up to
    1/2 meter
                                                     10B. Alasmidonta,  end view.
                       10A. Pelecyopod, Alasmidonta
                           Side view, up to 18 cm. long.
9B. Annelid,  leech up to 20 cm.
                          12A. Branchiopod,
                               Daphnia.  Up
                               to 4mm.
  11A. Ostracod,  Cypericus
      Side view, up to 7 mm.
                        HB.  Cypericus, end view.
                                                         12 B.  Branchiopod,
                                                                Bosmina.  Up
                                                                to 2mm.

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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
                Up to 25 mm.
                                         18.  Isopod,  Asellus
                                              Up  to  25 mm.
 20. Collembola, Podura
     Up to 2 mm. long

 vn  1-10
19A. Calanoid copepod,   ,
     Female       19B. Cyclopoid copepodr
     Up to 3 mm.       Female
                       Up to 25 mm.

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                            Key to Selected Groups of Freshwater Animals
              V"n
 21A.
                 21B.
21C,
   21D.           21E.

21. Trichoptera,  larval cases,
   mostly 1-2 cm.
                                   22. Megaloptera, alderfly
                                       Up to 2 cm.
                             E/-'
23A. Beetle larvae,    23B. Beetle larvae, 24A. Odonata;  dragonfly
     Dytisidae,            Hydrbphilidae       nymph up  to 3 or
     Usually about  2 cm.   Usually about       4 cm
         I                 1  cm.

                             ra:
                  24B". Odonata, tail
                       of damselfly
                       nymph
                       (side view)
                   Suborder
                    Zygoptera
                    (24B, D)

            24D. Odonata, damselfly
                 nymph (top view)
                                       24E, Odonatar front view
                                  ///" /      of dragonfly nymph
                                            showing "mask"
                                            partially extended
                                          Suborder
                                          Anisoptera
                                                 , E, C)

                                      24C. Odonata, tail of
                                           dragonfly nymph
                                           (top view)
                                                      VII  1-11

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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.
                                 Plecoptera,
                                 Stonefly nymph
                                 Up to 5cm.
             27 .Epheme ropte ra,
               Mayfly nymph
               Up to 3cm.
28A.  Coleoptera,
     Water scavenger
     beetle. Up to 4 cm.
                                                                28B. Coleoptera.
                                                                     Dytiscid beetle
                                                                     Usually up to 4  cm.
                29A. Diptera, Crane
                    fly. Up to 2i cm.
                                                         Diptera, Mosquito
                                                         Up to 20 mm.
VII   1-12

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 II   KEY TO ALGAE OF IMPORTANCE IN WATER POLLUTION

 1        Plant  a tube, thread,  strand, ribbon, or membrane; frequently visible to the unaided eye   .  2
 1'       Plants of microscopic cells which are isolated or in irregular, spherical,  or microscopic
         clusters, cells not grouped into threads  ...........              ..         .          123

 2(1)    Plant a tube,  strand,  ribbon, thread, or membrane composed of cells. .         .             3
 2'       Plant a branching tube with  continuous protoplasm, not divided into cells                  120

 3(2)    Plant a tube,  strand,  ribbon, thread, or a mat of threads.        .       .........      4
 3'       Plant a membrane of  cells one  cell thick (and 2 or more  cells wide)  .        .   .           116

 4 (3)    Calls in  isolated or clustered threads or ribbons which are only one cell thick or wide.       5
 4J       Cells in  a tube, strand, or thread all (or a part)  of which is more than one cell  thick or
         wide [[[      108

 5 (4)    Heterocysts present .....................................        •         •          6
 5'       Heterocysta absent ........       .....................              .....         23

 6 (5)    Threads gradually narrowed to a point at one end ...................    . .   .   •   7
 6'       Threads same width throughout ..................................       •         12

 7(6)    Threads as radii, in a gelatinous bead or mass .............     .    ..        .  .     8
 7'       Threads not in a gelatinous  bead or mass .............................       •     11

 8(7)    Spore  (akinete) present,  adjacent to the terminal heterocyst (Cloeotrichia) .....           9
 8'       No spore (akinete) present  (Rivularia) .........................        .10

 9 (8)    Gelatinous colony a smooth  bead .......................    Cloeotncrua  echinulatc
 9'       Gelatinous colony irregular .....................   Gloeotrichia natans

 10(8')   Cells near the narrow end as long as wide.     ..........      •   •  •   Rivularia dura
 10'      Cells near the narrow end twice as long as wide  ...      .       • .      Rivularia haematites

 11  (7'1    Cells  adjacent to heterocyst wider than heterocyst .......        ... Calothrix braunii
 11'       Cells  adjacent to heterocyst narrower than heterocyst  .   ...             Calothrix parietina
 12(6')   Branching present
 12"      Branching absent
  13(12)   Branches in pairs  ............      •   ••     Scytonema tolypothricoides
  13'      Branches arising singly. ..   ..      ----       •   ••        •             . Tolypothrix tenuis

  14(12')  Heterocyst terminal only  (Cvclindrospermum) ....................................   15
  14'      Hetrocysts intercalary  (within the filament) ----     ...                   .               16

  15 (14)   Heterocyst round . .  . .       ................    ...     Cylindrospermum muscicola
  15'      Heterocyst elongate ...............             . Cylindrospermum stagnate

  16 (141)  Threads encased in a gelatinous bead or mass .....     .      ..............      . .  .17
  16'      Threads not encased in a definite gelatinous mass ...............................  18

  17 (16)   Heterocysts and vegetative cells rounded .........     ........  Nostoc pruniforme
  17'      Heterocysts and vegetative cells oblong ..............     .           Nostoc carneum

  18 (161)  Heterocysts and vegetative cells shorter than the thread width ...... Nodular la spumigena
  18'      Heterocysts and vegetative cells not shorter than the thread width ................... -19

  19(18')  Heterocysts rounded (Anabaena) ............................................. 20
  19'      Heterocysts chndric. ...................................... Aphanizomenon flos -aquae

  20(19)  Cells elongate, depressed in the middle; heterocysts rare. ...........  Anabaena constricta
  20'      Cells rounded, heterocysts  common ............................................... 21

  21(20')  Heterocysts with lateral extension! ....................... Anabaena  planctonica
  21'      Heterocysts without lateral extensions .............................................. £2

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   22(21')  Threads 4-8|» wide	Anabaena flos-ag^ae
   22'      Threads 8-14M wide	Anabaena circinalla

   23(5')  Branching absent	     24
   23'      Branching (including "false" branching) present	    84

   24 (23)  Cell pigments distributed throughout the protoplasm	25
   24'      Cell pigments limited to plastids	49

   25 (23)  Threads short and formed as an even spiral  	285
   25'      Threads very long and not forming an even spiral	    "

   26(25') Several parallel threads of cells in one common sheath	Microcoleus subtorulosus
   26'      One thread per sheath if present	      	   27

   27(26') Sheath or gelatinous matrix present	        ••   28
   27'      No sheath nor  gelatinous matrix apparent (Oscillatoria)	    35

   28(27)  Sheath distinct; no gelatinous matrix between threads  (Lvngbya)    	       29
   28'      Sheath indistinct or absent, threads interwoven with gelatinous matrix between (Phormidium). . .


   29(28)   Cells  rounded	LynKbYa ocracea
   29'      Cells  short cylindric	           	30

   30 (29')  Threads in part forming  spirals	LYnBbYa  lafierheimii
   30'       Threads straight or bent but not in spirals	      .    • •    •      	       31

   31(30')  Maximum cell length 3 5n  ; sheath thin	         	     Lyngbya dipuett
   31'       Maximum cell length 6 SM ; sheath thick	Lyngbya versicolor

   32(28')  Ends  of some threads with a rounded swollen "cap" cell   ....          ..    •      .   . . 33
   32'      Ends  of all threads  without a "cap" cell	             	           34

   33(32)   End of thread  (with "cap")  abruptly bent .       	    Phormidium uncmatum
   33'      End of thread  (with "cap")  straight	              .  Phormidium autumnale

    34 (32')  Threads 3-5ii  in width        ...    	     . .     ...          Phormidium inundatum
    34'      Threads 5-12M in width.     .        	          	   Phormidium retzu

    35 (271)  Cells very short, generally less than 1/3 the thread diameter	         . .          .36
    35'      Cells generally 1/2 as long to longer than the thread diameter  	                  39

    36(35)   Cross walls constricted	       	      	  Oscillatoria ornata
    36'      Cross walls not constricted   	      ...      	  37

    37(36')  Ends of thread,  if mature,  curved   	              .  .      	     38
    37'      Ends of thread straight   	     	       ...   Oscillatoria limosa

    38 (37)   Threads 10-14|> thick .     ...     	       .   .       .  Oscillatoria curvicepa
    38'      Threads 16-60|i thick  	   Oscillatoria pnnceps

    39(35')  Threads appearing red to  purplish	Oscillatoria rubeacena
    39'      Threads yellow-green to blue-green	    .   .    .      	   40

    40(39')  Threads yellow-green   	      	41
    40'      Threads    blue-green   	    	     	    	  43

    41 (40)   Cells 4-7  times  as long as tne thread diameter	Oscillatoria put rid a
    41'       Cells less than 4 times as  long as the thread diameter              	   42

    42 (41')  Prominent granules ("pseudovacuoles") in center  of each cell        Oscillatoria  lauterbornii
    42'      No prominent granules in  center of cells   	Oscillatoria chlorina

    43(40')  Cells 1/2-2 times  as long  as the thread diameter      	44
    43'      Cells 2-3  times  as long as the thread diameter   	48

    44 (43)   Cell  walls between cells thick and transparent   	Oscillatoria pseudogeminata
    44'      Cell  walls thin,  appearing as a dark line  	     45
vn   2-2

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45(44')  Ends of thread straight  ....       .....     ..        .....  Oaeillatona agardhu
45'      Ends of mature threads curved  .  .     .          ....      .....           . . 46

46 (45')  Prominent granules present especially at both ends of each cell     .     .  Oscillatona tenuis
46'      Cells without prominent granules           ..........        .  .    .           .      47

47 (46')  Cross walls constricted      ...................   Oscillatoria chalybea
47'      Cross walls not constricted       .     .......           .....  Oscillatoria formosa

48(43')  End of  thread long tapering  .........................   Oscillatoria  splendida
48'      End of  thread not tapering       .        .................. Oscillatoria amphibia

49 (24')  Cells separate from one  another and enclosed in a tube  (Cymbella)    ......       251'
49'      Cells attached to one another aa a thread or ribbon       .  .........     .   .    .    50

50 (49')  Cells separating readily into discs or  short cylinders, their circular (ace showing radial
         markings    . .     .........................................   233
50'      Cells either not separating readily,  or if so.  no circular end wall with radial markings       51

51 (50')  Cells in a ribbon,  attached side by side or by their corners    ............    52
51'       Cells in a thread,  attached end to  end   .    .....................       ...... 56

52(51)   Numerous regularly spaced markings  in the cell wall   ..............              53
52'      Numerous markings in the cell walJ  absent  (Scenedesmus)      ......    .......      128

53 (52)  Wall markings of two types,  one coarse,  one fine   .....      ........... 185
53'      Wall markings all fine (Fragilana)      ...............     .       .           54

54(53')  Cells attached at middle  portion only        .         ......... Fragilaria crotonensis
54'      Cells attached along entire length         ......................  55

55(54')  Cell length 2 5-100,,    ......         ..................     FraRilana capucina
55'      Cell length 7 -25,,     ..... '.   .            .......         •   •    Fragilaria construens

56(51')  Plastid in the form of a spiral band  (Spirogyra)   ..           ............      57
56'      Plastid not a spiral band    .....................
                                                                                                  CO
57(56)  One plastid per cell   .      ...      ......       ....          .
57'      Two or more plastids per cell  ......................

58(57)  Threads 18-26, wide   ............................. SpiroHyra communi.
58'      Threads 28-50(i wide   ..........................

59(58')  Threads 28-40p wide       .............                            SpiroByra variajs
59'      Threads 40-50M wide         .   .     .   .           ............  SpirofiYra port.calis

60 (57'»  Threads 30-45p wide: 3-4 plastids per cell ........................ SpiroRYra fluviatiha
60'      Threads 50-80»  wide, 5-8 plastids per cell     .                 .      Spirofiyra maiuscula

61(56')  Plastids two per cell  ..................................         '"
61'      Plastids either one or more than  two  per cell    ..........       •     ...

62(61)  Cells with knobs or granules on the wall   .................        •     .......   °
62'      Cells with a smooth outer wall    .......

63 (62)  Each cell with two central knobs on  the wall ......                   Desrmdium  Rrevillii
63'      Each cell with a ring of granules near one end                             Hyalotheca  mucosa
 64(62')  Cells dense green, each plastid reaching to the wall    . .                     ZyBnema
 64'      Cells light green, plastids not completely filling the cell  .

 65(64')  Width of thread 26-32,,,  maximum cell length 6<>  .      .  .         ...     Zyfinema insifine
 65'      Width of thread 30-36p,  maximum cell length 120p .......  ZyRnema pectmatum

 66 (61')  Plastid a wide ribbon, passing through the cell axis (Mougeotia).  ...      .             67
 66'      Plastid or  plastids close to the cell wall  (parietal)  .....      ...         .             69
                                                                                          VII  2-3

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67(66)  Threads with occasional "knee-joint" bends ............... Mouge^tia Renuflexa
67 '      Threads straight ............................................ 68

68 (67') Threads 19-24M wide, pyrenoids 4-16 per cell ........... Moufjeotia sphaerocarpa
68'      Threads 20-34V wide; pyrenoids 4-10 per cell ............      Mouggotia scalaris

69 (661) Occasional cells with one to several transverse wall lines near one end (Oedogonium)       70
69'      Occasional terminal transverse wall lines  not present ............         73

70 (69)  Thread diameter less than  24ji  ................         •                                7*
70'      Thread diameter 25(i or more       .........         ....

71 (70)  Thread diameter 9-14,. .    .          ...        .    -                Oedopjonium suecicum
71'      Thread diameter 14-23M       .......                         •       .  Oedogonium boscii

72(70)  Dwarf male plants attached to normal thread,  when reproducing   Oedoeonium idioandrosporum
72'      No dwarf male plants produced     ..........                  •      Oedogoniurn ^rande
73 (69 ')  Cells with one plastid which has a smooth surface         ........                      7<|
73"      Cells with several plastids or with one nodular plastid   .....                          78

74(73)   Cells with rounded ends                 .                            .  Stichococcus bacillaris
74'      Cells with flat ends  (Ulothrix)    .............
75(74')  Threads 10M or less in diameter  .........       •    •     ••        •
75'      Threads more than 10|i in diameter     ....             •

76(75)  Threads 5-6, in diameter        .....              -                 . . Ulothj-jx variabihs
76'
Threads 6-10H m diameter                           .     •     •          Ulothrix tenemma
 77(75') Threads 11-17,, in diameter                                      •   •      . Ulothrix aequahs
 77'
         Threads 20-60,, in diameter   .                                              Ulothrix Zonata
 78 (73')  Iodine test for starch positive, one nodular plastid per cell    ....                79
 78'      Iodine test for starch negative,  several plastids per cell                                    80

 79 (78)   Thread when broken, forming "H" shape segments                        Microspora amoena
 79'      Thread when fragrrfented,  separating irregularly  or between cells  (Rhizoclonmm)          100

 80 (781)  Side walls of cells straight,  not bulging   A pattern of fine lines or dots present in the wall
          but often  indistinct  (Melosira)	           ...           .                    81
 80'      Side walls of cells slightly bulging   Pattern of wall markings not present (Tribonema)       83

 81(80)   Spine-like teeth at margin of end walls   .  .     	          .     .                     82
 81'       No spine-like teeth present   .                                    .          .Melosira varians

 82 (81)   Wall with fine granules, arranged obliquely                              .  Melosira crenulata
 82'      Wall with coarse granules, arranged parallel to sides                      Melosira granulata

 83 (80')  Plastids 2-4 per cell                   .                         •          Tribonema minus
 83'      Plastids more than 4 per cell                  .        ..        .    Tribonema bombycinum

 84 (231)  Plastids present;  branching  "true"                .     .           .           • •           85
 84'      Plastids absent, branching "false"             .                       Plectonema tomasmiana

 85(84)   Branches reconnected, forming a net       ...                 .  Hydrodictyon reticulatum
 85'      Branches not forming a distinct net                    .                    ...             .86

 86 (85')  Each cell in a conical sheath open at the  broad  end (Dinobryon)	       .   .87
 86'      No conical sheath around each cell.              .             ....        .90

 87(86)   Branches diverging,  often almost at a right angle      .        ...     Dinobryon divergens
 87'      Branches compace often almost parallel      .          . .          ...      •              88

 88(87')  Narrow end of sheath sharp  pointed 	      .  .    -              •            •   ..89
 88'      Narrow end of sheath blunt pointed ...          .   .                   .  .Dinobryon sertulana


 VII  2-4

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89 (88)     Narrow end drawn out into a stalk               ...          .        Dinobryon stipitatum
89'        Narrow end diverging at the base    .       .                            Dinobryon sociale

90 (86')    Short branches on the main thread in whorls of 4 or more (Nitella)                       91
90'        Branching commonly single or in pairs  .                                               92

91 (90)     Short branches on the main thread rebranched once                      .    Nitella flexilis
91'        Short branches on the main thread rebranched two to four times             Nitella gracilis

92 <90')    Terminal cell eacli with a colorless  spine having an abruptly swollen base (Bulbochaete)  93
92'        No terminal spines with abruptly swollen bases      .         .                          94

93         Vegetative cells  20-48»* long      .           ••                    Bulbochaete mirabilis
93'        Vegetative cells  48-88H long    .      .                               .Bulbochaete insignia

94(92')    Cells red, brown, or violet   ..........                   . . . Audouinella violacea
94'        Cells green    .       .                                                                 95

95 (94')    Threads enclosed in a gelatinous bead or mass                                          96
95"        Threads not surrounded by a gelatinous mass   .                                        99

96 (95)     Abrupt change in width from main thread to branches (Draparnaldia)               •      97
96'        Gradual change in width from mam thread  to branches (Chaetophora)                     98

97 (96)     Branches (from  the main thread) with  a central,  mam axis           Draparnaldia plumosa
97'        Branches diverging and with no  central main axis                  Draparnaldia glomerate

98(96')    End cells long-pointed, with colorless tips     .    .               Chaetophora  attenuata
98'        End cells abruptly pointed, mostly without long colorless tips  .  .    . .Chaetophora elegans

99(95')     Light and dense  dark cells intermingled in the thread          .      Pithophora
 991         Most of the cells essentially alike in density                                            10°

 100(99')   Branches few in number, and short, colorless  .......   Rhizoclonium hieroglyphicum
 100'        Branches numerous and green          ...                            •

 101 (1001)   Terminal attenuation gradual,  involving two or more cells  (Stieeoclonium)              102
 101'        Terminal attenuation absent or abrupt, involving only one cell (Cladophora)             104

 102(101)   Branches frequently in pairs                            ......        •    •  •  •   103
 102'
Branches mostly single	   Stigeoclomum stagnate
 103(102)    Cells in main thread 1-2 times as long as wide	StiReoclomum lubricum
 103'         Cells in main thread 2-3 times as  long as wide   .         	     Stigeoclomum tenue

 104(101')    Branching often appearing forked,  or in threes	Cladophora aegagropila
 104'         Branches distinctly lateral	

 105 (104')   Branches forming acute angle with main thread,  thus forming clusters Cladophora glomerata
 105'         Branches forming wide angles with the main thread  . .           	            •lob

 106(105')   Threads crooked and bent   ...                                         Cladophora frac^
 106'         Threads straight      ....            •              ....

 107(106')   Branches few. seldom rebranching   ....             ••             Cladophora insignia
 107'         Branches numerous, often rebranching.                               Cladophora crispata

 108 (41)     Plant or tube with a tight surface layer of cells and with regularly spaced swellings (nodes)
     1                                                       	     .  .      Lemanea annulata
 108'
             Plant not a tube that has both a tight layer of surface cells and nodes     .                109
  109 (108')   Cells spherical and loosely arranged in a gelatinous matrix          Tetraspora Relatinosa
  109'        Cells not as loosely arranged spheres

  110(109')   Plants branch   	        •      •             •   .        V.1
  110'         Plants not branched    	Schizomens  leibleinu

  111(110)     Clustered branching     	11Z
  111'         B ranches single	115


                                                                                       VII  2-5

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112(111)     Thraada embedded in gelatinous matrix (Batrachosoermum)	113
112'        No gelantinous matrix (Chara)	            "4

113 (112)    Nodal masses of branches touching one another 	Batraehospermiim vagum
113'        Nodal masses of branches separated by a narrow space.   . . Batrachospermum moniliforme

114 (1121)   Short branches with 2 naked cells at the tip	  Chara globularis
114'        Short branches with 3-4 naked cells at the tip	    Chara vulgans

115(111')   Heterocysts present, plastids absent	Stigonema minutum
115'        Heterocysts absent, plastids  present	       . Compnopogon coeruleus

116(3')     Red eye spot and two flagella present for each cell	      	     125
116'        No eye spots nor flagella present  	          •          	II7

117(116')   Round to oval cells, held together by a flat gelatinous matrix  (Agmenellum)  .     .   ..131
117'        Cells not round and not enclosed in a gelatinous matrix	         	   118

118(117')   Cells regularly arranged to an unattached disc. Number of cells 2,  4,  8, 16, 32, 64,  or
           128	          133'
118'        Cells numerous, membrane attached on one surface	      	              119

119(118')    Long hairs extending from upper surface of cells   .          	Chaetopeltis megalocystis
119'        No hairs extending from cell surfaces	Hildenbrandia rivularis

120 (21)    Constriction at the base of every branch	Dichotomosiphon tuberosus
120'       No constrictions present in the tube  (Vaucheria)	           . .           121

121 (1201)   Egg sac attached directly,  without  a stalk,  to the  main vegetative tube  . .Vaucheria sessilis
121'        Egg sac attached to an abrupt,  short, side branch  	    122

122(121')   One egg sac per branch  	         .-        Vaucheria terrestris
122"       Two or more egg sacs per branch	      	    Vaucheria geminata

123(1')      Cells in colonies generally of a definite form or arrangement   	             124
123'        Cells isolated, in  pairs or in loose, irregular aggregates    	                173

124 (123)    Cells with many transverse  rows of markings on the wall	           .185
124'        Cells without transverse rows of markings	         125

125(124')   Cells arranged as a layer one cell thick	I26
125'        Cell cluster more than one cell thick and not a flat plate  .          	     137

126(125)    Red eye  spot and two flagella present for each cell  	   Gonium pectorale
126'        No red eye  spots nor flagella present         .           .      	            .127

127 (1261)   Cells elongate, united side by side in 1 or 2 rows  (Scenedesmus)  .                      128
127'        Cells about as long as wide  ....              ...      .       •               131

128 (127)    Middle cells without spines but with pointed ends   	Scenedesmus dimorphus
128'        Middle cells with  rounded ends     	     	     • • •   1*9

 129(128")  Terminal cells with spines        .  .      	      ...        .130
129'        Terminal cells without spines  . .              .      	    Scenedesmus bnuga

130 (129)    Terminal cells with two spines  each	    Scenedesmus quadricauda
 130'        Terminal cells with three or more spines each                 . . .   Scenedesmus abundans

 131 (117)    Cells in regular rows, immersed in  colorless matrix (Agmenellum ouadriduphcatum)   132
 131'        Cells not immersed in colorless matrix    	      .       •-      ...         .133

 132(131)    Cell diameter 1 3 to 2 2|»  .     .   .      . Agmenellum quadriduplicatum . tenulBaima type
 132'        Cell diameter 3-5|i    .       	Agmenellum quadriduphcatum.   glauca type

 133 (1311)   Cells without spines,  projections,  or incisions . .         	Crucigema quadrata
 133'        Cells with spines, projections, or incisions              •  .   .      	134



 VII  2-6

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134(133')   Cells rounded  	       .     .     	                Micractinium pusillum
134'        Cells angular  (Pediastrum) ....                                         .135

135(134')   Numerous spaces between  cells	   Pediastrum duplex
135'        Cells fitted tightly together	                            .        136

136 (135')   Cell incisions  deep and narrow .                                        Pediastrum tetras
136'        Cell incisions  shallow and  wide	               .    Pediastrum boryanum

137(125')   Cells sharp-pointed at both ends, often arcuate ..          .     .                        138
137'        Cells not sharp-pointed at  both ends; not arcuate  .        .                  	   141

138 (137)   Cells embedded in a gelatinous matrix  ...      .                     Kirchnenella lunar is
138'        Cells not embedded in a gelatinous matrix                                         .     139

139 (1381)   Cells all arcuate;  arranged back to back .                             Selenastrum gracile
139'        Cells straight  or bent in various ways, loosely arranged or twisted together
             	     	(Ankistrodesmus)     .                  140

140(139')   Cells bent             	                 Ankistrodesmus falcatus
140'        Cells straight              .           ...   Ankistrodesmus falcatus var  acicularis

141 (1371)   Flagella present,  eye spots often present  .                                             142
141'        No flag el la nor eye spots present       	                                 .      152

142 (141)    Each cell in  a  conical sheath open at the wide end (Dinobryon)                          86
142'        Individual cells not in conical sheaths            .                                       143

143 (142')   Each cell with 1-2  long straight rods extending   .              Chrysosphaerella longispina
143'        No long  straight rods extending from the cells.                                         144

144 (1431)   Cells touching one another  in a dense colony   ....     . .              	        145
144'        Cells embedded separately in a colorless matrix	             .            149

145 (144)   Cells arranged radially,   facing outward  	               ...        .       146
145'        Cells all facing in  one direction ...               .          ....             .  .  147

146(145)   Plastida brown, eye spot absent       .      	Synura uvella
146'        Plastids green, eye spot present in each cell	  Pandorina morum

147(145')   Each cell with 4 flagella .                      	Spondylomorum quaternarium
147'        Each cell  with 2 flagella (Pyrobotrys)	             .148

148(147')   Eye spot in the wider (anterior) end of the cell ...      ...  .            Pyrobotrys stellata
148'        Eye spot in the narrower (posterior) end of the cell . .       . .          Pyrobotrys gracilia

149(1441)   Plastids brown .        	      	             Uroglenopsis americana
149'        Plastids green  .     .       	       . .          .  .          . .  150

150(149')   Cells 16, 32, or 64 per colony	   Eudorma elegans
150'        Cells more than 100 per colony  	           .  .     	    151

151(150')   Colony spherical, each cell with an eye spot. ..              .        •        Volvox aureus
151'        Colony tubular or irregular: no eye  spots  (Tetraspora)             .                     109

15Z (1411)   Elongate cells, attached together at one end, arranged radially (Actmastrum)           153
152'        Cells not elongate, often spherical	                  •  ••                154

153(152)   Cells cyhndric	      	          .  .Actinastrum gracillimum
153'        Cells distinctly bulging  	  Actinastrum hantzschii

154(152')   Plastids present	        •  •   •      •  ••        J55
154'        Plastids absent, pigment throughout each protoplast   .  .              .             •   168

155 (154)   Colonies,  including the outer matrix, orange to red-brown         .  . Botryococcus braunii
155'        Matrix,  if any, not bright colored,  cell plastids green         	              156
                                                                                      VII   2-7

-------
 156 (155')    Colonies  round to oval	                     j^g
 156'         Colonies  not round, often irregular in form	          157

 157(156')    Straight (flat) walls between adjacent cells   (Phytocoms) .    ..               	   Z78
 157'         Walls between neighboring  cells rounded	     	    15g

 158(157')    Cells arranged as a surface layer in a large gelatinous tube  (Tetraspora)    . .       109
 158'         Colony not a tube,  cells in irregular pattern	     159

 159 (1581)    Large cells more than twice the diameter of the small cells  (Chlorococcum) .         .280'
 159'         Large cells not more  than twice the diameter of the small cells (Palmella)	      281

 160(156)     Cells touching one another, tightly grouped	      .    ...  Coe last rum microporum
 160'         Cells loosely grouped	      	     _ , ,     _       161

 161 (1601)    Colorless threads extend from center of colony to cells        ....         . .           162
 161'         No colorless threads attached to cells in colony	    . .     	      164

 162 (161)     Cells rounded or straight, oval  (Dictyosphaerium)              	         163
 162'         Cells elongate, some  cells  curved	Dimorphococcus lunatus

 163(162)     Cells rounded  	  Dictyosphaerium pulchellum
 163'         Cells straight, oval	   Dictyosphaerium ehrenbergianum

 164(161')     Cells rounded    	165
 164'         Cells oval	Oocystis borgei

 165 (164)    One plastid per cell	166
 165'        Two to four plastids per cell   	          Gloeococcus schroeteri

 166 (165)    Outer matrix divided into layers  (Cloeocystis)     ...      . .     . .        .     ...    167
 166'        Outer matrix homogeneous	        Sphaerocystts schroeteri

 167(166)    Colonies angular  	      .       ..     Gloeocystis planctonica
 167'        Colonies rounded      	           Gloeocystis  gigaa

 168(154')   Cells equidistant from center of  colony   (Gomphoaphaeria)  .  .        .    .   .     ..   169
 168'        Cells irregularly distributed in the colony ......        .                     .  172

 169(168)    Cells with pseudovacuoles            	Gomphospaeria  wichurae
 169"        Cells without pseudovacuoles     ....        .       ...      .               ..         170

 170(169')   Cells 2-4|>in diameter (Comphosphaena lacustris)      .                  .             171
 170'        Cells ovate          	            .       .       Gomphosphaeria aponina

 171(170)    Cells spherical.                         .  Gomphosphaeria lacustris, kuetzmgianum type
 17''         Cells 4-15  in diameter   .  .    .        ...      Gomphosphaeria lacustris, colhnaii type

 172 (1681)    Cells ovid, division plane perpendicular to long axis  (Coccochloris)            .        286
 172'         Cells  rounded, or division plane perpendicular to short axis   (Anacystis)    .   .       286'

173 (1231)    Cells with an abrupt median transverse groove or incision	       .     .     - .174
173'         Cells without an abrupt transverse median groove or incision .     ...          .184

174 (173)    Cells brown, flagclla present (armored flagellates)	             .  .    .        175
174'         Cells green, no flagella (desrmds)    .     	     	        178

175(174)    Cell with 3 or  more long horns	Ceratium hirundinella
175'         Cell without  more than 2 horns    	   176

176 (1751)    Cell wall of very thin smooth plates  	Glenodinium palustre
176'         Cell wall of very thick  rough plates  (Peridimum)	177

177 (1761)    Ends of cell pointed  	  Peridimum wisconsinense
177'         Ends of cell  rounded	   Peridimum cinctum

178 (174')    Margin of  cell with sharp pointed , deeply cut lobes or long spikes	      .  . 179
178'         Lobes, if present,  with rounded ends  	182
 VII  2-8

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179 (178)    Median incision narrow,  linear  	Micrasteriaa truncata
179'        Median incision wide. "V" or "U" shaped (Staurastrum)	      180

180 (179)    Margin of cell with long spikes	   Staurastrum paradoxum
ISO1        Margin of cell without long spikes   	    181

181 (180")    Ends of lobes with short spines 	 Staurastrum polymorphum
181'         Ends of lobes without spines 	 Staurastrum punctulatum

182 (1781)    Length of cell  about double the width   	,	Euastrum oblongum
182'        Length of cell  one to one  and one-half times the width   (Coamarium)  ...    .     ...    183

183 (1821)    Median incision narrow linear	   Coamarium botrytia
183'        Median incision wide, "U" shaped  	  Cosmarium portianum

184(173')    Cells triangular  	   Tetraedron muticum
184'        Cells not triangular	      .     185

185 (124)    Cells with one end distinctly different from the other	       ...    186
185'        Cells with both ends essentially alike	225

186 (185)    Numerous transverse (not spiral) regularly spaced wall markings present (diatoms)      187
186'        No transverse regularly  spaced wall markings	   193

187 (186)    Cells curved (bent) in girdle view	Rhoicosphenia curvata
187'        Cells not curved in girdle view	      188

188 (187')    Cells with both fine  and coarse transverse lines	Meridion circulare
188'        Cells with transverse lines all alike in thickness	   189

189 (1881)    Cells essentially linear to rectangular; one terminal swelling larger than the  other	
            	(Asterionella)	     190
189'        Cells wedge-shaped, margins sometimes wavy (Gomphonema)  	191

190 (189)    Larger terminal swelling 1-1/2 to 2 times wider than the other	   Asterionella formosa
190'        Larger terminal swelling less than  1-1/2 times wider than the other. .Asterionella Rracilhma

191 (1891)    Narrow end enlarged in valve  view	   Gomphonema geminatum
191'         Narrow end not enlarged  in valve view	  192

192 (1911)    Tip of broad end about  as wide as tip of narrow end in  valve view  .  Gomphonema parvulum
192'        Tip of broad end much  wider than tip of .narrow end in valve view. Comphonerna olivaceum

193 (1861)    Spine present at each end of cell  	  Schroederia aetigera
193'        No spine on both ends of cell   	      194

194 (1931)    Pigments in one or more plastids	195
194'        No plastid; pigments throughout the  protoplast	Entophysalis lemamae

195 (194)    Cells in a conical sheath  (Dinobryon)	 86
195'        Cells not  in a conical sheath  	      .196

196 (195')    Cell covered with scales and long spines	MaHomenas caudata
196'         Cells not  covered with  scales and long spines	        197

197(196')    Protoplasts separated by a space from a  rigid sheath (lorica)	              198
197'         No loose sheath around the cells 	   202
                                                                                                .199

199 (198')    Lorica opaque; yellow to reddish or brown	    Trachelomonas crebea
199'        Lorica transparent; colorless to brownish  (Chryaococcusl .   ..      .       ..      .  .200

200(199')'  Outer membrane (lorica) oval	Chrysococcus ovahs
200'        Outer membrane (lorica) rounded	       .          201
                                                                                       vn   2-9

-------
 201(200')   Lorlca thickened around opening  	Chry.ococcus rutescens
                                                                                               maior
201"        Lorica not thickened around opening	Chrysococcus
 202 (197 ')   Front end flattened diagonally .............................................    203
 203'        Front end not flattened diagonally ............................ ..........  ....    206

 203(202)    Plaatids bright blue -green  (Chroomonas) ...............................  204
 203'        Plastids brown, red, olive-green, or yellowish ............................  ..    205

 204 (203)    Cell pointed at one end ....................................... Chroomonas nordstetu
 204'        Cell not pointed at one end ................................   Chroomonas setoniensis

 205(203')   Gullet present, furrow absent  ..... ........................   Cryptomonae erosa
 205'        Furrow present, gullet absent  ................................. Rhodomonaa
 206(202')  Plastids yellow-brown ......................................... Chromulina roaanoffi
 206'        Plastids not yellow-brown; generally green •. ..........................      207

 207(206')  One plastid per cell ..................................................  208
 207'        Two to  several plastids per cell ................................     '   '   211

 208(207)   Cells tapering at each end ...................... Chlorogonlum euchlorum
 208"        Cells rounded to  oval ................................................ ~TT. - 209

 209 (208')  Two flagella per  cell (Chlamydomonas) ...................    210
 209'        Four  flagella per cell  ............................ " . ."  Cater'ia multifil'is

 210(209)   Pyrenoid angular, eye spot in front third of cell ........ Chlamydomonas reinhardi
 210        Pyrenoid circular, eye spot in middle third of cell  ........... . Chlarnvdomon"as eloboaa

 211(207.)   Two plastids per cell  ......................... Cryptoglena pjgra
 211'        Several plastids per cell   ................................            — -- ^212

 212 (2111)   Cell compressed (flattened)  (Phacus)     ....          .                        213
 212'        Cell not compressed   .........................               '         '      ,..

 213(212)    Posterior spine short, bent ..................        Phacus pleuronectee
 213         Posterior spine long, straight ...........        .    .     .    .    Phacus longicluda"

 214 (212)    Cell margin rigid ...................................            215
 2141        Cell margin flexible (Euglena)     ...       ....                                217

 215(214)    Cell margin with  spiral ridges     ............................... Phacus pyrum
 ^15         Cell margin without ridges,  but may have spiral lines (Lepocinclis) . . .  .        .   . 216

 216 (215')   Posterior end with an abrupt,  spine-like tip ..............        .    Lepocinclis ovum
 216'        Posterior end rounded .....................           Lepocinclis
217(214')   Green plastids hidden by a red pigment in the cell  .         .       .    . Euglena sanguinea
217'        No red pigment except for the eye spot  .......      .           .           .  ~~ — - 218

218(217')   Plastids at least 1/4 the length of the cell .............      ..........  219
218'        Plastids discoid or at least shorter than 1/4 the length of the cell     .                   220

219 (218)    Plastids two per cell .............................          ...  Euelena agll,8
219'        Plastids several per cell, often extending radiately from the center. .       Euglena viridis

220(218')   Posterior end extending as an abrupt colorless spine ..............            221
220'        Posterior end rounded or at least with no colorless spine ..........           222

221(220)    Spiral markings  very prominent and granular ..................... Euglena spirogyra
221'        Spiral markings  fairly prominent,  not granular  .....................  Euglena oxyuris

222 (220')   Small, length 35-55,    ...........................  Euglena gracilis
222'        Medium to large, length 65, or more  .............     .      ......       - 223

223(222')   Medium in size;  length 65-200,  ..........................                224
223-        Large in size, length 250-290M ................ ".'.'.'.  '.'. .  '.".'..'. .Eugiena e'hren'bergii


vn   2-10

-------
224 (223)   Plastids with irregular edge, flagellum 2 times as long as cell          Euglena polymorpha
224'       Plastids with smooth edge, flagellum about 1/2 the length of the cell   .  .     Euglena deses

225 (1851)  Cells distinctly bent (arcuate); with a spine or nan owing to a point at both ends         226
225'       Cells not arcuate   	„  	            -••                          23°

226 (225)   Vacuole with particles showing Brownian movement at each end of cell   Cells  not in
           clusters     (Closterium)      	                                              2-7
226'       No terminal vacuoles.  Cells may be in clusters  or colonies         .                   228

2Z7 (226)   Cell wide, width 30-70|i  .                    .        .            . Closterium momhferum
227'       Cell long and narrow, width up to 5M                                  Closterium aciculare

228 (2261)  Cell with a narrow abrupt spine  at each blunt end                   Ophiocytium capitatum
2Z81       No blunt ended cells with abrupt terminal spines               .                       •  229

229 (228')  Sharp pointed ends as separate colorless spines                          .               193
2Z91       Sharp pointed ends as part of the green protoplast                                       137

230 (225)   One long spine at each end of cell ...     	                              .  .     231
230'       No long terminal spines   .  .                                                      •    232

231 (230)   Cell gradually narrowed to the spine    .                    •                 •         -137
231'       Cell abruptly narrowed to the  spine                                   Rhizosolema gracihs

232        A regular pattern of fine lines or dots in the wall (diatoms)                            233
232'       No regular pattern of fine lines  or dots in the wall    .            .                    276

233 (50,   Cells  circular in one  (valve) view, short rectangular or square in other (girdle) view   . 234
     232)
233'       Cells  not circular in one view.	                                              -*°

234 (233)   Valve surface with  an inner and outer (marginal) pattern of striae  (Cyclotella)         235
234'        Valve surface with  one  continuous pattern of striae (Stephanodiscus) .                 238

235(234)   Cells  small. 4-10,»  in diameter                                       Cyclotella filomcrata
235'       Cells  medium to large, 10-80 in diameter   .                      .            .        236

236 (2351)  Outer half of valve with two types of lines, one long, one short                         23?
236'       Outer half of valve with radial lines all alike                      Cyclotella meneghiniana

237 (236)  Outer valve zone constituting more than 1/2 the diameter               Cyclotella bodanica
237'       Outer valve zone constituting more than 1/2 the diameter.                Cyclotella compta

                                                                                                 21<)
238(234')  Cell 4-25|i  in diameter.             .                                      _,
238'       Cell25-65M in diameter         .   ..                             Stephanodiscus mafiarae

239(238)  Cell with two transverse bands, in girdle view                    Steohanodiseus binderanus
239'       Cell without two transverse bands, in girdle view   ..             Stephanodiscus hantzschii

240(233')   Cells  flat,  oval  (Cocconeis)	              	           *
240'        Cells  neither flat nor oval

241(240)    Wall markings (striae)  18-20 in 10M	        •     Cocconeis pediculus
241'        Wall markings (striae)  23-25 in 10M.                             •    Cocconeis placentula

                                                                                                 243
242(240')   Cell aigmoid in  one view	              •          •
242'        Cell not sigmoid in either round or point ended (valve) or square ended (girdle) surface
            view.

243(242)   Cell sigmoid in valve surface view     	        . CYro3iBma attenuatum
243'        Cell sigmoid in square ended  (girdle) surface view   . .        ...    Nitzschia aciculans

244 (242')  Cell longitudinally unsymmetrical in at least one view     .      	  24j>
244'        Cell longitudinally  symmetrical   	           	

245(244)   Cell wall with both fine and coarse transverse lines (striae  and  costae)            .     246
245'        Cell wall with fine transverse lines (striae)  only            . -  •           •            247


                                                                                      VII   2-11

-------
246 (245)
246'

247 (245)
247'

248 (247)
248'

249 (248')
249'

250(247')
250'

251 (250)
251'

252 (2511)
    (246)
252'

253 (252')
253'

254 (244')
254'

255 (254)
255'

Z56 (Z551)
256'

2S7 (254)

257'
258 (257)
258'

259 (257')
259'

260 (Z591)
260'

2ol (260)
261'

262 (261)
262'

i
-------
267 (2661)  Striae distinctly composed of clots (punctae)
267'       Striae essentially as continuous lines

268 (2671)  Central clear area on valve face  rectangular
268'       Central clear area on valve face  oval

269 (268')  Cell length 29-40)1.  ends slightly capitate
269'       Cell length 30-l20(i, ends not capitate

270 (260')  Knob at one end larger than at the other  (Asterionella)
270'       Terminal knobs if present  equal in size  (Synedra)
                                                                                  Navicula lann-olata
                                                                     Navicula graciloidcs
                                                                  Navicula eryptoccphala
                                                                         Navicula racliosa

                                                                                       189
                                                                                      271
271 (2701)
271'

272 (2711)
272'

273 (272')
273'

274
274'

275 (274')

275'
276 (232')
276'

277 (276)
277'

278 (277')
278'

279 (278')

279'

280 (2791)
Z80'

281 (159')
281'

282 (281')
282'
Clear space (pseudonodule) in central area
No pseudonoriule in central area

Sides parallel in valve view, each end with an enlarged nodule
Sides converging to the ends in  valve view

Valve linear to lanceolate-linear,  8-12 striae per 10p
Valve narrowly  linear-lanceolate,  12-18  striae per 10(1

Valve 5-6|»  wide                    ....
Valve 2-4M  wide     ...      . .  .
      Synrdra pulchclla
                    272
          Synedra ulna
                   .274
                    275
Cells up to 65 times as long as wide,  central area absent to small oval
                                                               Synedra acus var  radians
Cells 90-120 times as long as wide, central area rectangular
                         .  .                              Synedra acus var  augustissima
Green to brow.i pigment in one or mure plastids
No plastids,  blue and green pigments throughout protoplast

Cells long and narrow or flat
Cells rounded

Straight,  flat wall between adjacent cells in colonies
Rounded wall between adjacent cells in colonies
                    277
                    284

                    233
                    278

  fhytoconis botryoides
                    279
Cell either with 2 opposite wall knobs or colony of 2-4 cells surrounded by distinct mem-
brane or both     ....                                                  l64
Cell witnout  2 wall knobs, colony not of 2-4 cells surrounded by distinct? membrane     280
Cells essentially similar in size within the colony
Cells of very different sizes within the colony  .  .

Cells embedded  in an extensive gelatinous matrix
Cells with little or no gelatinous matrix around them  (Chlorella)

Cells rounded .
Cells ellipsoidal to ovoid  .
                    281
Chlorococcum Hurmcola

      Palmclla murosa
                    282

                    283
   Chlorella rllipsoidoa
 283  (282)
 283'

 284  (276')
 284'

 285  (25)
 285'
Cell 5-10^1 in diameter, pyrenoid indistinct
Cell 3-5u in diameter, pyrenoid distinct

Cell a spiral rod .       .  .
Cell not a spiral rod   	
Thread septate (with crosswalls)
Thread non-septate (without crosswalls)
      Chlorella vulj-aris
  Chlorella pyrpnoidosa

                    285
                    286

    Arthrospira jenncri
   Spirulma nordstrdtii
 286 (172)    Cells dividing in a plane at right angles to the long axis
     (284')
 286' (1721)   Cells sperical or dividing in a plane parallel to the long axis  (Anacystis)

 287 (286')   Cell containing pseudovacuoles
 287'        Cell not containing pseudovacuoles.
                                                                   Coccochluria stagnina
                                                                                      287

                                                                        Anacystis cyanea
                                                                                      288
                                                                                    VII   2-13

-------
288(287')  Cell 2-6B in diameter; aheath often colored	Anacyatis montana
288'       Cell 6-50n in diameter; sheath  colorless	289

289 (2881)  Cell 6-12n in diameter; cells in colonies are mostly spherical .   .  .  . Anacystis thermalis
289'       Cell 12-50(1 in diameter;  cells in colonies are often angular. .           Anacyatis dimidiata
  VII  2-14

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APPENDIX


CLASSIFICATION-FINDER FOR NAMES OF
AQUATIC ORGANISMS IN WATER SUPPLIES
AND POLLUTED WATERS

-------
                         FOREWORD
The following work is more easily defined in terms of what it is not,
than what it is; it is not a "key" in the usual biological sense of the
word, nor is it a glossary of a dictionary.  It is rather a device for
determining what general kind of organism or group is designated by
some unrecognized name, be it common or scientific.

If one has access to one or more of the references cited,  he can find
the same name, and learn much more about it; but not everyone has
all of these books,  and the information is often couched in highly
technical terms.

The nonbiologist would be well advised to read Part I before attemp-
ting to use the  Finder.  The experienced biologist on the other hand
may proceed directly to the index and quickly be referred to the larger
group to which his unknown organism belongs.

No professional systematist will find himself completely at home.  In
an effort to present a relatively simple concept of relationships
couched in standard terms for all groups or  organisms,  some violence
was done to certain highly sophisticated systems of classification.  It
is hoped, however, that the layman will find accuracy sufficient for his
needs,  and the specialist will be referred to technical literature where
he can  satisfy his needs for greater detail.

While every effort has been made to ensure accuracy, it is inevitable
that errors have crept in.  Please call them to our attention.

Grateful appreciation is extended to Michael E. Bender and Charles L.
Brown, Jr., both former Biologists with the Water Pollution Training
Activities,  for their valuable contributions and encouragement.
                                         H.W. Jackson
                                         Chief Biologist
                                         National Training Center

-------
PART I.  The System of Biological Classification




PART II.  Outline of Biological Classification

-------
        PART I
The System  of
Classification

-------
                           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

The scientific name of an or-
ganism is 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. 10.66

-------
                         RELATIONSHIPS BE1WEEN LIVING ORGANISMS
    PLANTS 5

 ORGANIC MATERIAL
PRODUCED, USUALLY
 BY  PHOTOSYNTHESIS
                      ENERGY FLOWS FROM LEFT  TO RIGHT, GENERAL EVOLUTIONARY
                               SEQUENCE IS UPWARD
                       ANIMALS 81

                ORGANIC MATERIAL INGESTED OR
                        CONSUMED
                   DIGESTED INTERNALLY
 ENERGY STORED
                                         ENERGY RELEASED
                               FUNGI  250

                           ORGANIC MATERIAL
                               REDUCED
                           BY EXTRACELLULAR
                           DIGESTION AND IN-
                           TRACELLULAR META-
                           BOLISM TO MINERAL
                              CONDITION
                                                          ENERGY RELEASED
 FLOWERING PLANTS
 AND GYMNOSPERMS 76
 CLUB MOSSES, FERNS 76


 LIVERWORTS,  MOSSES 73





 ALGAE  12
       ARACHNIDS 167

       INSECTS 154

       CRUSTACEANS 129

       SEGMENTED WORMS 121

       MOLLUSCS 172

       MOSS ANIMALS 120,181

       WHEEL ANIMALS 116

       ROUNDWORMS 113

       FLATWORMS 108
MAMMALS 2 3

BIRDS 242

REPTILES 241

AMPHIBIANS 240

FISHES 195

PROCHORDATES 191

STARFISH GROUPS 185
BASIDIOMYCETES 266



ASCOMYCETES 265


HIGHER PHYCOMYCETES
                261
                                     JELLYFISH - CORAL GROUP 103
                            SPONGES 99
                DEVELOPMENTOFMULTICELLULARORCOENOCYTICORGANISMS
 DIATOMS  38
 PIGMENTED FLAGELLATES
               HIGHER PROTISTA
                        PROTOZOA  82
    AMOEBOID PROTOZOA 86         CILLIATED PROTOZOA  92
       FLAGELLATED PROTOZOA 85       SPOROZOA 98
12 I   (COLORLESS  FLAGELLATES  85      SUCTORIA 97  	
                            LOWER PHYCOMYCETES
                                           261
                            DEVELOPMENT OF A NUCLEAR MEMBRANE
 BLUE GREEN ALGAE 7
        PHOTOTROPIC
        BACTERIA  252
 CHEMOTROPIC BACTERIA
                  252
                             LOWER PROTISTA (ORMONERA)
     NOTE:  NUMERALS REFER TO PARAGRAPHS IN PARTS 2 AND 3.

     W. B. COOKE AND H.  W.  JACKSON, AFTER WHITTAKER
                                                           ACTINOMYCETES 253

                                                           SPIROCHAETES 255

                                                           MYXOBACTERIA 254

                                                           PARASITIC
                                                           BACTERIA   251
                                                           AND VIRUSES

                                                           SAPROBIC BACTERIA 251
     BI.ECO.pl.2b.4.66

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                                                         Classification - Finder
         genus  Anabaena  (an alga),
         we  must  simply  use the
         generic  name, and:

         Anabaena^ planctonica,

         Anabaena gonstricta,  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  crassii 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.
     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 Animalia

        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.

II  THE GENERAL  RELATIONSHIPS OF
    LIVING  ORGANISMS
                               :
 A  Living organisms (as contrasted to
    fossil  types)  have long been group-
    ed into two kingdomfi:  Plant King-
    doms and Animal Kingdoms.  Modern
    developments however  have made this

-------
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.

-------
       PART II
       Outline
of  Biological
Classification

-------
                                                         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 Hyxophyceae                8

     Order Chroococcales             9

     Order Hormogonales             10

      Suborder Hetercystineae       11

 C  PHYLUM CHLOROPHYTA - green      12
    algae

    CLASS Chlorophyceae             13

     Order Volvocales               14

     Order Ultrichales              15

     Order Chaetophorales           16

     Order Chlorococcales           17

     Order Siphonales               18

     Order Zygnematales             19

     Order Tetrasporales            20

     Order Ulvales                  21

     Order Schizogonales            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

    Order Heterococcales

   CLASS Chrysophyceae -
   yellow-green algae

    Order Chrysomondales

    Order Rhizochrysidales

    Order Chrysosphaerales

    Order Chrysocapsales

    Order Chrysotrichales
30

31

32


33

34

35

36

37
     Order Rhizochloridales
29
   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              43

    Order Desmomonadales          44

   CLASS Dinophyceae -            45
   dinoflage Hates

    Order Gymnodiniales           46

    Order Peridiniales            47

    Order Dinocapsales            48

    Order Chloromonadales         49

   CLASS Cryptophyceae            50

G  PHYLUM CHLOROMONADOPHYTA       51

H  PHYLUM RHODOPHYTA - red algae  52

   CLASS Rhodophyceae             53

    Order Bangiales               54

    Order Nemalionales            55

    Order Gelidiales              56

-------
  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 Laminarlales             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 phytomastlgina        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 Cestoldea - tapeworms   111
         F  PHYLUM NEMERTEA - proboscis    112
            worms
         G  PHYLUM NEMATODA - threadworms,113
            roundworms

-------
                                                      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 Hirudlnea - leeches      124

   CLASS Archiannelida            125

   CLASS Echiuroidea              126

   CLASS Sipunculoidea - peanut   127
   worms

P  PHYLUM ARTHROPOOA - 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 Dlptera - two winged    163
     flies

     Orders including aquatic      164
     adults

     Order Coleoptera - beetles    165

     Order Hemiptera - true bugs   166

    CLASS Arachnoldea - spiders,   167
    scorpions,  mites

     Order Xlphosoura - horse-     168
     shoe or king crabs

     Order Hydracarina - aquatic   169
     mites

     Order Pantopoda (Pycnogonida)-170
     pycnogonids

     Order Tardigrada              171

Q   PHYLUM MOLLUSCA                 172

    CLASS Amphlneura -  chitons      173

    CLASS Gasteropoda -  snails      174

     Order Prosobranchiata          175

     Order Opisthobranchiata        176

     Order Pulmonata  - air breath-  177
     ing  snails

   CLASS  Scaphopoda  - tusk          173
    shells

   CLASS  Bivalvia                   179
   (Pelecypoda)

   CLASS  Cephalopoda - squid,      ISO
   octipus, nautilus

R  PHYLUM BRYOZOA (Ectoprocta)  -   181
   Moss  animals

S  PHYLUM BRACHIOPODA - lamp       182
   shells

T  PHYLUM CHAETOGNATHA - arrow     183
   worms
U  PHYLUM PHORONIDEA - tufted     184
   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

   Subphylum Hemichordata -      192
   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  -
     chlmaeras
202
    CLASS  Osteichthys  (Pisces)  - 203
    bony fishes

     Order Acipenseriformes  -     204
     sturgeons
    Order Polyodontidae  -
    paddle  fishes
205

-------
                                                   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 Cyprinldae - 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

    CLASS Mammalia - whales,
    seals, walrusses

IV  FUNGUS KINGDOM

 A  Bacteria

    Eubacteria

    Actinomycetes

    Myxobacteria

    Spirochaetes

    Other bacterial types

 B  FUNGI

    "Phycomycete" group
242

243


250

251

252

253

254

255

256

260

261

-------
Classification - Finder
   CLASS Chytrldiomycetes         262



   CLASS Oomycetes                263



   CLASS Zygomycetes              264



   CLASS Ascomycetes              265



   CLASS Basidiomycetes           266



   CLASS Fungi Imperfecti         267
10

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