United States
Environmental Protection
Agency
National Training
and Operational
Technology Center
Cincinnati OH 45268
Water
I
I
Benthic Analysis
Training Manual
EPA-430/1-79-001
March 1979
EPA REGION VII IRC
160874
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EPA-430/1-79-001
March 1979
BENTHIC ANALYSES
This course is for technicians and biologists who have responsibility
for collection, identification and interpretation of findings of benthic
communities in surveillance of the aquatic environment. This is an
introductory course in benthic community analysis.
After successfully completing the course, the student will be able
to plan, conduct and evaluate benthic monitoring programs.
The training consists of classroom instruction and activities,
laboratory studies and field experience.
Course coverage emphasizes benthic macroinvertebrate communities
in freshwater, including sampling considerations, taxonomic analysis,
sorting techniques, sampling handling, and data presentation and
interpretation.
U. S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Program Operations
National Training and Operational Technology Center
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DISCLAIMER
Reference to commercial products, trade names or
manufacturers is for purposes of example and
illustration. Such references do not constitute
endorsement by the Office of Water Program Operations,
U. S. Environmental Protection Agency.
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CONTENTS
Title or Description Outline Number
Benthic Communities (Marine) 1
Using Benthic Biota in Water Quality Evaluation 2
Statistics As An Ecological Tool 3
The Interpretation of Biological Data With Reference To Water Quality 4
Application of Biological Data 5
Optics And the Microscope 6
Artificial Substrates 7
Benthic Integrity and Macro Invertebrate Drift 8
Effects of Thermal Pollution On the Benthos 9
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BENTHIC COMMUNITIES (MARINE)
I INTRODUCTION
A The term community in the ecological sense
includes all of the populations of animals
and plants occupying a given area. The
community and the nonliving environment
function together as an ecological system or
ecosystem.
B The community cannot exist without the
cycling of materials and the flow of energy
in the ecosystem.
C The community concept is important in
ecological theory because it emphasizes
the fact that diverse organisms usually
live together in an orderly manner.
D Since organisms in salt water (or in any
other natural habitat) are not arranged in
taxonomic or systematic orders, classi-
fication on an ecological basis as one of the
following is useful:
1 On major niches or position in energy
or food chain
a Producers
b Consumers
c Decomposers
2 On their mode of life
a Benthos
b Periphyton
c Plankton
d Nekton
e Neuston
3 On region or subhabitat (see Figure 1)
a Intertidal
b Subtidal
c Lower Neritic
d Bathyal
e Abyssal
f Hadal
E Among benthic organisms the biotic factors
of the environment are manifested in pre-
dacity, competition for food and space, and
materialisms.
F The zonation of varied and important groups
of plant-like sessile animals on the sea
bottom is often as striking as the zonation
of trees on a mountain and similarly pro-
vides shelter for small organisms.
G Because of their stability benthic organisms
provide a basis for the classification of
zones.
II THE BENTHIC HABITAT
A Distinct faunas in marine environments
depend on the physical characteristics of
the environments, the ecological activities
of the organisms, the geological history of
the region and the biology of the species.
On this basis the following types of assem-
blages and environments can be generalized:
1 Intertidal and rocky shores.
2 Intertidal sand beaches and flates to
10 m.
3 Low salinity lagoons and mangroves.
4 Nearshore, sand and sand-mud, 10-30 m.
5 Intermediate shelf to 65 m.
6 Outer shelf to 130 m.
BI. MAR. eco. 3. 2. 79
1-1
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Benthic Communities
THE MARINE ENVIRONMENT
X
en
o
o
c
6
c.
0>
h-
W
o
03
U.
erres
trial
lanne
Neritic zone
Bathyal zon
Pelagie-neriticl
Infra-littoral Jrr
Abyssal zone
Pelagic-oceanic (Nekton «• Plankton) 1
Hemipelagic Ln Pelagic
Light
Temperature,
Salinity
Movement
' Algae,
(green, red, brown).
a o herbivora.
Variable.
Eurythermal fauna.
Waves »fast currents
(tidal etc.).
Thick shells, of ten broken
Boring and fixed
animals .
Accumulation swift
with diastems, variable.
Ripple marks
Cross-tam i nation
Surface , a o. herbivora.
Jeeper, no herbivora (carnivora ,mud eaters)-
Blind or large eyes, light-organs.
Variable at the surface.
Deeper, constant, low. Stenothermal fauna
In the abyss 1O*
Oceanic
Convect
>ic )
\
ion )
currents, slow
Accumulation
Slow to extremely slow, uninterrupted.
Extensive, uniform deposits
Phytoplankton-
Flora
Calcareous algae,
(Lithothamnium reefs).
Bacteria
precipitating lime.
I
Coccolithophora
„ Diatoms
Fauna
Nekton I
(C«phalopods, Crusttceans, Vertebrates, etc.).
Zooplankton |
^roraminifera (Clobigerina )
Coral-, Bryozoan reefs.
. Shells of benthos.
CaCO,
Radiolarians
(f)
Llthosphere
Terrigenous.-(abrasion, rivers, glaciers, wind ) and Volcanic matter.
j (Transport aqgeous and eolian).
-Coarse >- I |
-* Tine ' >-
• Colloidal •
Evaporation
Limestone, gypsum,
sails
Precipitation,
jartly organic
Limestone, chert,
phosphate, glauconite,
pynte, limonite,
etc.
Manganese nodules.
-a
0)
Shallow water dep
Terrigenous
- Clay -
( Limestone, dolomite.
< Reef rock
«—Ooii.es.
°| Black (organic) mud I
Pelagic deposits
Pteropod
igenna t
Clobigerina 1
Diatom
Red clay
Radiolarian)
Figure 1. THE ENVIRONMENTS OF SEDIMENTATION IN THE OCEAN
1-2
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Benthic Communities
Table 1. SIZE CLASSIFICATION OF SEDIMENTARY PARTICLES
As frequent reference will be made to materials of various grain
sizes, the following table shows the most used size classifications
(grade scales)
Diameters in millimeters
Wentworth
Boulder
Coblilc
Pebble
Granule
Very coarse sand
Coarse sand
Medium sand
Fine sand
Very fine sand
Silt
Oay
Above 256
256-64
64-4
4-2
2-1
1-M
M-W
K-K
>»-«s
M«-hs«
Below M«.
2-1
1-0.5
O.S-0.25
0.25-0. 125
0.125-0.0625
0.0625 0.004
Below 0.004-
Attcrberg
Block
Cobble
Pebble
Coarse sand
Fine sand
Silt
Clay
2000-200
200-20
20-2
2-0.2
0.2-0.02
0.02 0.002
Below 0.002
1 „ ...
Gravel PscPhltl:
Psammitc
(arenaceous)
Petite or lutite
The finest fractions of sediments are frequently called "clay. " But
as this word has a definite mineralogical implication the terms "pelite"
or "lutite" or "lutum" are used by some authors to denote all particles
smaller than the sand fraction, whether consisting of clay minerals,
calcite, or any other mineral. The word "ooze" signifies a fine de-
posit composed principally of the shells and debris of pelagic organisms.
By "mud" the marine geologist means all fine-grained deposits of a
more or less plastic nature in moist condition.
For a more extensive review of this and other problems of sedimen-
tary petrography the reader may consult Krumbein and Pettijohn
(1938), Pettijohn (1949), or other manuals of petrography and also
Twenhofel's books.
FromKuenen, P. H. Marine Geology. 1950.
a clay bottom or
b sand bottom (depth figures are
approximation)
7 Basins and troughs to 1500 m.
8 Upper (inner) Continental Slope.
9 Middle Continental Slope.
10 Lower (outer) Continental Slope.
11 Abyssal basins to 10, 000 m.
12 Hadal basins to 13, 000 m.
B Investigations of marine bottom com-
munities, with relatively few exceptions
have been concerned with describing the
aggregations of species of infaunal in-
vertebrates along the broad areas of
differing sedimentary factors.
1-3
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Benthic Communities
C An investigation of the temporal sequences
and biotic successions in the fouling of
artifacts may be a prime feature in the
detection of pollution.
D In shore the relationship between popu-
lation density and substratum type is
quite striking. This is brought about by
rain wash, stream erosion, aeolian
deposits and glacial action.
E Marine sediments on and in which the
benthos lives are derived from a great
number of different sources
1 Sources
a Skeletons and tests of organisms
b Decomposable organic matter
(1 kg/m^/yr. in coastal waters)
c Precipitates: lime, iron, Mn. , etc.
d Coastal and bottom erosion: waves,
currents, etc.
3
e Rivers (Miss. R. 1 km sand and
silt/yr)
f Glaciers and ice
g Weathering on the sea floor
2 Media of transport
a The atmosphere
1) Meteoric dust (10 to 20 million
particles/day)
2) Volcanos: pumice, ash, etc.
3) Offshore winds; dust, sand
b Sediment slumping and currents
(settling rates)
c Horizontal currents
d Currents and wave turbulence
e Rhythmic accumulations
F The macroscopic fauna of sandy beaches
is composed mostly of burrowers.
G The microscopic fauna of sandy beaches
is highly specialized and depends largely
on the composition of the substratum and
the percent composition of sand grain size.
H In the neritic zone the benthos are con-
sumers and exhibit marked zonation be-
cause of the large number of distinct
sessile forms. They are distinct for each
of the neritic zones.
I The unequal distribution of animal mass
in shallow waters is the result of inter-
twined biological and physical factors in
the bottom, some of which involve aerobic
and anaerobic exchanges with the environ-
ment, but many of which are not understood.
J The burrowing habit is more common in
muddy sand than in sand or solid rock.
K The variety of organisms on the bottom is
greatest where measurable light can reach
to the bottom and where plankton production
is high.
L The eulittoral zone gives rise to many
communities because it is greatly varied
with regard to type of substratum and
also to character of shoreline and degree
of exposure.
M In deeper water, 2000 m or more, gravels,
sands and silts from the land are replaced
by pelagic oozes or red clay. The former
are important as a substratum and because
of their organic matter content.
N On the level bottoms of the sea live 2 eco-
logically distinct groups: the eipfauna com-
prising 3% of all species of animals (more
than 4/5 of all bottom dwellers) and the
infauna making up 11% of known species.
(less than 1/5 of all bottom dwellers).
O Rocky shores from outcrops, boulders,
etc. present characteristic faunas on
their slopes, in crevices and in pools,
communities whose composition is also
dependent on vagueries of the tides.
1-4
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Benthic Communities
P Benthic communities are irregularly
spaced: the same type of level bottom
substrata at similar depths in widely
spread regions support paralleled groups
of invertebrates.
Q The bottom fishes as soles, plaice, halibut,
founders, rays, sculpin, etc. , depend to
a great extent on consistent productivity
of the bottom on which they live for food,
shelter and reproduction.
R Deep sea benthic animals are mainly mud-
dwelling forms variously adapted to this
mode of life. The deepest fauna in the
most widely distributed; the poverty of
individuals being apparently related to
distance from the shore.
S Much of the animal life on the deep sea is
endemic not merely an extension of eury-
bathic forms. This fauna has a relatively
small number of species in proportion to
the number of genera.
T The abyssal and hadal zones are regions
of relatively uniform conditions in terms
of pressure, temperature and light. The
communities in these areas are correspond-
ingly similar while in many respects and
depend on the productivity of the euphotic
zone.
3 Holme, N. A. Methods of Sampling the
Benthos. Adv. Mar. Biol. 2:171-260.
1964.
4 Jones, N. S. Marine Bottom Communities.
Biol. Rev. 25:283-313. 1950.
5 Kanwisher, John . Gas Exchange of
Shallow Marine Sediments. Symposium
on the Environmental Chemistry of
Marine Sediments; Occasional Publ.
1:13-19. 1962.
6 Kuenen, P. H. Marine Geology. New York
City, John Wiley & Sons., Inc. 1952.
7 Marshall, N. 3rd. The Environmental
Chemistry of Marine Sediments. URI
Grad. School of Oceanog. Occ. Publ. 1.
1962.
8 Odum, E. P. , and Odum, H. T. Funda-
mentals of Ecology. Philadelphia,
W. B. Saunders Co. 1959.
9 Southward, A. J. Life on the Sea Shore.
Cambridge, Mass. Harvard University
Press. 1965.
10 Thorson, G. Bottom Communities. Geol.
Soc. Am. Mem. 67(1): 461-534. 1957.
REFERENCES
1 Elton, C. Ecological Communities. J.
Animal Ecology. 5:1-56. 1946.
2 Gorsline, D. S. Ed. Proceedings of the
First National Coastal and Shallow
Water Conference. NSF and ONR,
Tallahassee, Fla. 1962.
This outline was prepared by D. J. Zinn,
Professor of Zoology, Graduate School of
Oceanography, University of Rhode Island,
Kingston, Rhode Island.
Descriptors: Aquatic Life, Benthos,
Benthic fauna, Benthic flora, Biological
communities, Ecological distribution,
Marine biology, and Oceans.
1-5
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USING BENTHIC BIOTA IN WATER QUALITY EVALUATION
I BENTHOS ARE ORGANISMS GROWING
ON OR ASSOCIATED PRINCIPALLY
WITH THE BOTTOM OF WATERWAYS
Benthos is the noun.
Benthonic, benthal and benthic are
adjectives.
II THE BENTHIC COMMUNITY
A Composed of a wide variety of life
forms that are related because they
occupy "common ground"--substrates
of oceans, lakes, streams, etc.
Usually they are attached or have
relatively weak powers of locomotion.
These life forms are:
1 Bacteria
A wide variety of decomposers work
on organic materials, breaking them
down to elemental or simple com-
pounds.
2 Algae
photosynthetic plants having no true
roots, stems, and leaves. The basic
producers of food that nurtures the
animal components of the community.
3 Flowering Aquatic Plants (Pondweeds)
The largest flora, composed of
complex and differentiated tissues.
May be emersed, floating, sub-
mersed according to habit.
4 Microfauna
Animals that pass through a U. S.
Standard Series No. 30 sieve, but
are retained on a No. 100 sieve.
Examples are rotifers and micro-
crustaceans. Some forms have
organs for attachment to substrates,
while others burrow into soft materi"!"
or occupy the interstices between rocks,
floral or faunal materials.
5 Meiofauna
Meiofauna occupy the interstitial zone
(like between sand grains) in benthic
and hyporheic habitats. They are inter-
mediate in size between the microfauna
(protozoa and rotifers) and the macro-
fauna (insects, etc.). They pass a No. 30
sieve (0. 5 mm approximately). In fresh-
water they include nematodes, copepods,
tardigrades, naiad worms, and some flat
worms. They are usually ignored in fresh-
water studies, since they pass the standard
sieve and/or sampling devices.
6 Macrofauna (macroinvertebrates)
Animals that are retained on a No. 30 mesh
sieve (0. 5 mm approximately). This group
includes the insects, worms, molluscs, and
occasionally fish. Fish are not normally
considered as benthos, though there are bottom
dwellers such as sculpins and darters.
B It is a self-contained community, though there
is interchange with other communities. For
example: Plankton settles to it, fish prey on
it and lay their eggs there, terrestrial detritus
leaves are added to it, and many aquatic insects
migrate from it to the terrestrial environment
for their mating cycles.
C it is stationary water quality monitor. The
low mobility of the biotic components requires
that they "live with" the quality changes of the
over-passing waters. Changes imposed in the
long-lived components remain visible for
extended periods, even after the cause has
been eliminated. Only time will allow a cure
for the community by drift, reproduction, and re-
cruitment from the hyporheic zone.
D Between the benthic zone (substrate/water
interface) and the underground water table
is the hyporheic zone. There is considerable
interchange from one zone to another.
Ill HISTORY OF BENTHIC OBSERVATIONS
A Ancient literature records the vermin associ-
ated with fouled waters.
BI. MET. fm. 8i. 8. 78
2-1
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Using Benthic Biota In Water Quality Evaluation
B 500 -year- old fishing literature refers
to animal forms that are fish food and
used as bait.
C The scientific literature associating
biota to water pollution problems is
over 100 years old (Mackenthun and
Ingram, 1964).
D Early this century, applied biological
investigations were initiated.
1 The entrance of state boards of health
into water pollution control activities.
2 Creation of state conservation agencies.
3 Industrialization and urbanization.
4 Growth of limnological programs
at universities.
E A decided increase in benthic studies
occurred in the 1950's and much of
today's activities are strongly influenced
by developmental work conducted during
this period. Some of the reasons for this
are:
1 Movement of the universities from
"academic biology" to applied
pollution programs.
2 Entrance of the federal government
into enforcement aspects of water
pollution control.
3 A rising economy and the development
of federal grant systems.
4 Environmental Protection Programs
are a current stimulus.
IV WHY THE BENTHOS?
A It is a natural monitor
B The community contains all of the
components of an ecosystem.
1 Reducers
a bacteria
b fungi
2 Producers (plants)
3 Consumers
a Detritivores and bacterial feeders
b Herbivores
c Predators
C Economy of Survey
1 Manpower
2 Time
3 Equipment
D Extensive Supporting Literature
E Advantages of the Macrobenthos
1 Relatively sessile
2 Life history length
3 Fish food organisms
4 Reliability of Sampling
5 Dollars/information
6 Predictability
7 Universality
F "For subtle chemical changes,
unequivocal data, and observations
suited to some statistical evaluation will
be needed. This requirement favors the
macrofauna as a parameter. Macro-
invertebrates are easier to sample
reproductively than other organisms,
numerical estimates are possible and
taxonomy needed for synoptic investi-
gations is within the reach of a non-
specialist.'' (Wuhrmann)
G "It is self-evident that for a multitude of
non-identifiable though biologically active
changes of chemical conditions in rivers,
small organisms with high physiological
differentiation are most responsive.
Thus the small macroinvertebrates
(e. g. insects) are doubtlessly the most
sensitive organisms for demonstrating
2-2
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Using Benthic Biota In Water Quality Evaluation
V
unspecified changes of water
chemistry called 'pollution' .
Progress in knowledge on useful
autecological properties of
organisms or of transfer of such
knowledge into bioassay practice
has been very small in the past.
Thus, the bioassay concept
(relation of organisms in a
stream to water quality) in
water chemistry has brought not
much more than visual demon-
stration of a few overall chemical
effects. Our capability to derive
chemical conditions from biological
observations is, therefore, almost
on the same level as fifty years ago.
In the author's opinion it is idle to
expect much more in the future because
of the limitations inherent to natural bio-
assay systems (relation of organisms
in a stream to water quality). " (Wuhrmann)
REACTIONS OF THE BENTHIC MACRO-
INVERTEBRATE COMMUNITY TO
PERTURBATION
A Destruction of Organism Types
1 Beginning with the most sensitive
forms, pollutants kill in order of
sensitivity until the most tolerant form
is the last survivor. This results in a
reduction of variety or diversity of
organisms.
2 The generalized order of macro-
invertebrate disappearance on a
sensitivity scale below pollution
sources is shown in Figure 2.
Water
Quality
Deteriorating
Stoneflies
Mayflies
Caddisflies
Amphipods
Isopods
Midges
Oligochaetes
Water
Quality
improving
As water quality improves, these
tend to reappear in the same order.
B The Number of Survivors Increase
1 Competition and predation are reduced
between different species.
2 When the pollutant is a food (plants,
fertilizers, animals, organic materials).
C The Number of Survivors Decrease
1 The material added is toxic or has no
food value.
2 The material added produces toxic
conditions as a byproduct of decom-
position (e.g., large organic loadings
produce an anaerobic environment
resulting in the production of toxic
sulfides, methanes, etc. )
D The Effects May be Manifest in Com-
binations
1 Of pollutants and their effects.
2 Vary with longitudinal distribution
in a stream. (Figure 1)
E Tolerance to Enrichment Grouping
(Figure 2)
Flexibility must be maintained in the
establishment of tolerance lists based
on the response of organisms to the
environment because of complex relation-
ships among varying environmental
conditions. Some general tolerance
patterns can be established. Stonefly
and mayfly nymphs, hellgrammites,
and caddisfly larvae represent a grouping
(sensitive or intolerant) that is generally
quite sensitive to environmental
changes. Blackfly larvae, scuds, sow-
bugs, snails, fingernail clams, dragon-
fly and damselfly naiads, and most
kinds of midge larvae are facultative
(or intermediate) in tolerance.
Sludge-worms, some kinds of midge
larvae (bloodworms), and some leeches
2-3
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Using Benthic Biota In Water Quality Evaluation
cc
u
m
2
LJ
>
Ul
&' \
i \
B.
C.
CO
in
D.
TIME OR DISTANCE
..NUMBER OF KINDS
..NUMBER OF ORGANISMS
.SLUDGE DEPOSITS
Four basic responses of bottom animals to pollution.
A. Organic wastes eliminate the sensitive bottom animals
and provide food in the form of sludges for the surviving toler-
ant forms. B. Large quantities of decomposing organic wastes
eliminate sensitive bottom animals and the excessive quanti-
ties of byproducts of organic decomposition inhibit the tolerant
forms; in time, with natural stream purification, water quality
improves so that the tolerant forms can flourish, utilizing the
sludges as food. C. Toxic materials eliminate the sensitive
bottom animals; sludge is absent and food is restricted to thai
naturally occurring in the stream, which limits the number ol
tolerant surviving forms. Very toxic materials may eliminate
all organisms below a waste source. D. Organic sludges with
toxic materials reduce the number of kinds by eliminating
sensitive forms. Tolerant survivors do not utilize the organic
sludges because the toxicity restricts their growth.
Figure 1
are tolerant to comparatively heavy loads
of organic pollutants. Sewage mosquitoes
and rat-tailed maggots are tolerant of
anaerobic environments for they are
essentially air-breathers.
F Structural Limitations
1 The morphological structure of a
species limits the type of environment
it may occupy.
a Species with complex appendages
and exposed complicated respiratory
structures, such as stonefly
nymphs, mayfly nymphs, and
caddisfly larvae, that are subjected
to a constant deluge of setteable
particulate matter soon abandon
the polluted area because of the
constant preening required to main-
tain mobility or respiratory func-
tions; otherwise, they are soon
smothered.
b Benthic animals in depositing zones
may also be burdened by "sewage
fungus" growths including stalked
protozoans. Many of these stalked
protozoans are host specific.
2 Species without complicated external
structures, such as bloodworms and
sludgeworms, are not so limited in
adaptability.
a A sludgeworm, for example, can
burrow in a deluge of particulate
organic matter and flourish on the
abundance of "manna. "
b Morphology also determines the
species that are found in riffles, on
vegetation, on the bottom of pools,
or in bottom deposits.
2-4
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Using Benthic Biota In Water Quality Evaluation
VI SAMPLING PROCEDURES
A Fauna
1 Qualitative sampling determines the
variety of species occupying an area.
Samples may be taken by any method
that will capture representatives of the
species present. Collections from
such samplings indicate changes in the
environment, but generally do not
accurately reflect the degree of change.
Mayflies, for example, may be re-
duced from 100 to 1 per square meter.
Qualitative data would indicate the
presence of both species, but might not
necessarily delineate the change in pre-
dominance from mayflies to sludge-
worms. The stop net or kick sampling
technique is often used.
2 Quantitative sampling is performed to
observe changes in predominance.
The most common quantitative sampling
tools are the Petersen, Ekman, and Ponak
grabs and the Surber stream bottom or
square-foot sampler. Of these, the
Petersen grab samples the widest variety
of substrates. The Ekman grab is limited
to fine-textured and soft substrates, such
as silt and sludge, unless hydraulically
operated.
The Surber sampler is designed for
sampling riffle areas; it requires
moving water to transport dislodged
organisms into its net and is limited
to depths of two feet or less.
Kick samples of one minute duration will
usually yield around 1, 000 macroinvert-
ebrates per square meter (10. 5 X a one
minute kick= organisms/m^).
Manipulated substrates (often referred to
as "artificial substrates") are
placed in a stream and left for a specific
time period. Benthic macroinvertebrates
readily colonize these forming a manipu-
lated community. Substrates may be con-
structed of natural materials or synthetic;
may be placed in a natural situation or
unnatural; and may or may not resemble
the normal stream community. The
point being that a great number of envi-
ronmental variables are standardized and
thus upstream and downstream stations
may be legitimately compared in terms of
water quality of the moving water column.
They naturally do not evaluate what may
or may not be happening to the substrate
beneath said monitor. The latter could
easily be the more important.
REPRESENTATIVE BOTTOM-DWELLING MACROANIMALS
Drawings from Geckler, j., K. M. Mackenthun and W. M. Ingram, 1963.
Glossary of Commonly Used Biological and Related Terms in Water and
Waste Water Control, DHEW, PHS, Cincinnati, Ohio, Pub. No. 999-WP-2.
A
B
C
D
E
F
G
H
Stonefly nymph
Mayfly nymph
Hellgrammite or
Dobsonfly larvae
Caddisfly larvae
Black fly larvae
Scud
Aquatic sowbug
Snail
(Plecoptera)
(Ephemeroptera)
(Megaloptera)
(Trichoptera)
(Simuliidae)
(Amphipoda)
(Isopoda)
(Gastropoda)
KEY TO
I Fingernail clam
J Damselfly naiad
K Dragonfly naiad
(Sphaeriidae)
(Zygoptera)
(Anisoptera)
L Bloodworm or midge
fly larvae
M Leech
N Sludgeworm
O Sewage fly larvae
P Rat -tailed maggot
FIGURE 2
( Chironumidae)
(Hirudinea)
(Tubificidae)
(Psychodidae)
(Tubifera-Eristalis)
2-5
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Using Benthic Biota in Water Quality Evaluations
B X^ C
SENSITIVE
f—
F G
INTERMEDIATE
M
TOLERANT
-------
Using Benthic Biota In Water Quality Evaluation
Invertebrates which are part of the B Flora
benthos, but under certain conditions
become carried downstream in
appreciable numbers, are known as
Drift.
Groups which have members forming
a conspicuous part of the drift
include the insect orders Ephemeroptera,
Trichoptera, Plecoptera and the
crustacean order Amphipoda.
Drift net studies are widely used and
have a proven validity in stream
water quality studies.
The collected sample is screened with
a standard sieve to concentrate the
organisms; these are sorted from
the retained material, and the number
of each kind determined. Data are then
adjusted to number per unit area,
usually to number of bottom organisms
per square meter.
Independently, neither qualitative not
quantitative data suffice for thorough
analyses of environmental conditions.
A cursory examination to detect damage
may be made with either method, but
a combination of the two gives a more
precise determination. If a choice must
be made, quantitative sampling would
be best, because it incorporates a
partial qualitative sample.
Studies have shown that a significant
number and variety of macroinverte-
brates inhabit the hyporheic zone in streams.
As much as 80% of the macroinverte-
brates may be below 5 cm in this
hyporheic zone. Most samples and
sampling techniques do not penetrate
the substrate below the 5 cm depth.
All quantitative studies must take this
and other substrate factors into account
when absolute figures are presented on
standing crop and numbers per square
meter, etc.
Direct quantitative sampling of natu-
rally growing bottom algae is difficult.
It is basically one of collecting algae
from a standard or uniform area of the
bottom substrates without disturbing
the delicate growths and thereby dis-
tort the sample. Indirect quantitative
sampling is the best available method.
Manipulated substrates, such as wood
blocks, glass or plexiglass slides,
bricks, etc., are placed in a stream.
Bottom-attached algae will grow on
these artificial substrates. After two
or more weeks, the artificial sub-
strates are removed for analysis.
Algal growths are scraped from the
substrates and the quantity measured.
Since the exposed substrate area and
exposure periods are equal at all of
the sampling sites, differences in the
quantity of algae can be related to
changes in the quality of water flowing
over the substrates.
VII ANALYSES OF MICROFLORA
A Enumeration
1 The quantity of algae on manipulated
substrates can be measured in several
ways. Microscopic counts of algal
cells and dry weight of a algal mater-
ial are long established methods.
2 Microscopic counts involve thorough
scraping, mixing and suspension of
the algal cells. From this mixture
an aliquot of cells is withdrawn for
enumeration under a microscope.
Dry weight is determined by drying
and weighing the algal sample, then
igniting the sample to burn off the
algal materials, leaving inert inorganic
materials that are again weighed.
The difference between initial dry weight
and weight after ignition is attributed to
algae.
3 Any organic sediments, however,
that settle on the substrate along
with the algae are processed also.
2-7
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Using Benthic Biota In Water Quality Evaluation
Thus, if organic wastes are present
appreciable errors may enter into
this method.
Autotrophic Index
Ash-free Wgt (mg/m )
Chlorophyll a (mg/m^)
B Chlorophyll Analysis
1 During the past decade, chlorophyll
analysis has become a popular method
for estimating algal growth. Chloro-
phyll is extracted from the algae and
is used as an index of the quantity of
algae present. The advantages of
chlorophyll analysis are rapidity,
simplicity, and-vivid pictorial results.
2 The algae are scrubbed from the
artificial substrate samples, ground,
then each sample is steeped in equal
volumes, 90% aqueous acetone, which
extracts the chlorophyll from the algal
cells. The chlorophyll extracts may
be compared visually.
3 Because the cholorophyll extracts fade
with time, colorimetry should be used
for permanent records. For routine
records, simple colorimeters will
suffice. At very high cholorophyll
densities, interference with colori-
metry occurs, which must be corrected
through serial dilution of the sample
or with a nomograph.
C Autotrophic Index
The chlorophyll content of the periphyton
is used to estimate the algal biomass and
as an indicator of the nutrient content
(or trophic Status) or toxicity of the water
and the taxonomic composition of the
community. Periphyton growing in sur-
face water relatively free of organic
pollution consists largely of algae,
which contain approximately 1 to 2 percent
chlorophyll a by dry weight. If dissolved
or particulate organic matter is present
in high concentrations, large populations
of filamentous bacteria, stalked protozoa,
and other nonchlorophyll bearing micro-
organisms develop and the percentage
of chlorophyll is then reduced. If the
biomass-chlorophyll a relationship
is expressed as a ration (the autotro-
phic index), values greater than 100
may result from organic pollution
(Weber and McFarland, 1969; Weber,
1973).
VIII MACROINVERTEBRATE ANALYSES
A Taxonomic
The taxonomic level to which animals are
identified depends on the needs, experience,
and available resources. However, the
taxonomic level to which identifications are
carried in each major group should be
constant throughout a given study.
B Biomass
Macroinvertebrate biomass (weight of
organisms per unit area) is a useful
quantitative estimation of standing crop.
C Reporting Units
Data from quantitative samples may be used
to obtain:
1 Total standing crop of individuals, or
biomass, or both per unit area or unit
volume or sample unit, and
2 Numbers of biomass, or both, of individual
taxa per unit area or unit volume or sample
unit.
3 Data from devices sampling a unit area
of bottom will be reported in grams dry
weight or ash-free dry weight per square
meter (gm/m ), or numbers of indi-
viduals per square meter, or both.
4 Data from multiplate samplers will be
reported in terms of the total surface
area of the plates in grams dry weight
or ash-free dry weight or numbers of
individuals per square meter, or both.
5 Data from rock-filled basket samplers
will be reported as grams dry weight
or numbers of individuals per sampler,
or both.
2-8
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Using Benthic Biota In Water Quality Evaluation
IX FACTORS INVOLVED IN DATA INTER-
PRETATION
Two very important factors in data evalua-
tion are a thorough knowledge of conditions
under which the data were collected and a
critical assessment of the reliability of the
data's representation of the situation.
A Maximum-Minimum Values
The evaluation of physical and chemical
data to determine their effects on aquatic
organisms is primarily dependent on
maximum and minimum observed values.
The mean is useful only when the data are
relatively uniform. The minimum or
maximum values usually create acute
conditions in the environment.
B Identification
Precise identification of organisms to
species requires a specialist in limited
taxonomic groups. Many immature
aquatic forms have not been associated
with the adult species. Therefore, one
who is certain of the genus but not the
species should utilize the generic name,
not a potentially incorrect species name.
The method of interpreting biological
data on the basis of numbers of kinds
and numbers of organisms will typically
suffice.
C Lake and Stream Influence
Physical characteristics of a body of
water also affect animal populations.
Lakes or impounded bodies of water
support different faunal associations
from rivers. The number of kinds
present in a lake may be less than that
found in a stream because of a more
uniform habitat. A lake is all pool,
but a river is composed of both pools
and riffles. The nonflowing water of
lake exhibits a more complete set-
tling of particulate organic matter that
naturally supports a higher population
of detritus consumers. For these
reasons, the bottom fauna of a lake or
impoundment, or stream pool cannot be
directly compared with that of a flowing
stream riffle.
D Extrapolation
How can bottom-dwelling macrofauna data
be extrapolated to other environmental
components? It must be borne in mind
that a component of the total environment
is being sampled. If the sampled com-
ponent exhibits changes, then so must the
other interdependent components of the
environment. For example, a clean stream
with a wide variety of desirable bottom
organisms would be expected to have a
wide variety of desirable bottom fishes;
when pollution reduces the number of bottom
organisms, a comparable reduction would
be expected in the number of fishes. More-
over, it would be logical to conclude that
any factor that eliminates all bottom organ-
isms would eliminate most other aquatic
forms of life. A clean stream with a wide
variety of desirable bottom organisms
would be expected to permit a variety of
recreational, municipal and industrial uses.
E Expression of Data
1 Standing crop and taxonomic composition
Standing crop and numbers of taxa (types
or kinds) in a community are highly
sensitive to environmental perturbations
resulting from the introduction of contam-
inants. These parameters, particularly
standing crop, may vary considerably in
unpolluted habitats, where they may range
from the typically high standing crop of
littoral zones of glacial lakes to the
sparse fauna of torrential soft-water
streams. Thus, it is important that
comparisons are made only between truly
comparable environments.
2-9
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Using Benthic Biota In Water Quality Evaluation
2 Diversity
Diversity indices are an additional tool
for measuring the quality of the environ-
ment and the effect of induced stress on
the structure of a community of macro-
invertebrates. Their use is based on
the generally observed phenomenon that
relatively undisturbed environments
support communities having large
numbers of species with no individual
species present in overwhelming
abundance. If the species in such a
community are ranked on the basis of
their numerical abundance, there will
be relatively few species with large
numbers of individuals and large
numbers of species represented by only
a few individuals. Many forms of stress
tend to reduce diversity by making the
environment unsuitable for some species
or by giving other species a competitive
advantage.
3 Indicator-organism scheme (rat-tailed
maggot studies)
a For this technique, the individual
taxa are classified on the basis of
their tolerance or intolerance to
various levels of putrescible wastes.
Taxa are classified according to
their presence or absence of
different environments as deter-
mined by field studies. Some
reduce data based on the presence
or absence of indicator organisms
to a simple numerical form for ease
in presentation.
t;
b Biologists are engaging in fruit-
less exercise if they intend to make
any decisions about indicator
organisms by operating at the
generic level of macroinvertebrate
identifications." (Resh and Unzicker)
4 Reference station methods
Comparative or control station methods
compare the qualitative characteristics
of the fauna in clean water habitats with
those of fauna in habitats subject to stress.
Stations are compared on the basis of
richness of species.
If adequate background data are avail-
able to an experienced investigator,
these techniques can prove quite useful—
particularly for the purpose of demon-
strating the effects of gross to moderate
organic contamination on the macro-
invertebrate community. To detect
more subtle changes in the macroinver-
tebrate community, collect quantitative
data on numbers or biomass of organisms.
Data on the presence of tolerant and
intolerant taxa and richness of species
may be effectively summarized for evalu-
ation and presentation by means of line
graphs, bar graphs, pie diagrams,
histograms, or pictoral diagrams.
X IMPORTANT ASSOCIATED ANALYSES
A The Chemical Environment
1 Dissolved oxygen
2 Nutrients
3 Toxic materials
4 Acidity and alkalinity
5 Etc.
B The Physical Environment
1 Suspended solids
2 Temperature
3 Light penetration
4 Sediment composition
5 Etc.
XI AREAS IN WHICH BENTHIC STUDIES
CAN BEST BE APPLIED
A Damage Assessment or Stream Health
If a stream is suffering from abuse the
biota will so indicate. A biologist can
determine damages by looking at the
"critter" assemblage in a matter of
minutes. Usually, if damages are not
found, it will not be necessary to alert
the remainder of the agency's staff,
2-10
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Using Benthic Biota In Water Quality Evaluation
pack all the equipment, pay travel and per
diem, and then wait five days before
enough data can be assembled to begin
evaluation.
B By determining what damages have been
done, the potential cause "list" can be
reduced to a few items for emphasis and
the entire "wonderful worlds" of science
and engineering need not be practiced with
the result that much data are discarded
later because they were not applicable to
the problem being investigated.
C Good benthic data associated with chemi-
cal, physical, and engineering data can
be used to predict the direction of future
changes and to estimate the amount of
pollutants that need to be removed from
the waterways to make them productive
and useful once more.
D The benthic macroinvertebrates are an
easily used index to stream health that
citizens may use in stream improve-
ment programs. "Adopt-a-stream"
efforts have successfully used simple
macroinvertebrate indices.
E The potential for restoring biological
integrity in our flowing streams using
macroinvertebrates has barely been
touched.
REFERENCES
1 Hynes, H. B. N. The Ecology of Running
Waters. Univ. Toronto Press. 1970
2 Keup, L. E., Ingram, W. M. and
Mackenthun, K. M. The Role of
Bottom Dwelling Macrofauna in
Water Pollution Investigations. USPHS
Environmental Health Series Publ. No.
999-WP-38, 23pp. 1966.
3 Keup, L. E., Ingram, W. M. and
Mackenthun, K. M. Biology of Water
Populations: A Collection of Selected
Papers on Stream Pollution, Waste
Water, and Water Treatment.
Federal Water Pollution Control
Administration Pub. No. CWA-3,
290 pp. 1967.
4 Mackenthun, K.M. The Practice of
Water Pollution Biology. FWQA.
281pp. 1969.
5 Stewart, R.K., Ingram, W.M. and
Mackenthun, K. M. Water Pollution
Control, Waste Treatment and Water
Treatment: Selected Biological Ref-
erences on Fresh and Marine Waters.
FWPCA Pub. No. WP-23, 126pp. 1966.
6 Weber, Cornelius I., Biological Field
and Laboratory Methods for Measuring
the Quality of Surface Waters and
Effluents. U.S. Environmental Pro-
tection Agency, NERC, Cincinnati,
OH . Environmental Monitoring Series
670/4.73.001 July 1973
7 Keup, L. E. and Stewart, R. K. Effects
of Pollution on Biota of the Pigeon River,
North Carolina and Tennessee. U. S. EPA,
National Field Investigations Center. 35 pp.
1966. (Reprinted 1973, National Training
Center)
8 Wuhrmann, K., Some Problems and
Perspectives in Applied Limnology
Mitt. Internat. Verein Limol. 20:324-402.
1974.
9 Armitag, P. D., Machale, Angelu M., and
Crisp, Diane C. A Survey of Stream
Invertebrates in the Cow Green Basin
(Upper Teesdale) Before Inundation.
Freshwater Biol. 4:369-398. 1974.
10 Resh, Vincent H. andUnzicker, John D.
Water Quality Monitoring and Aquatic
Organisms: the JWPCF 47:9-19. 1975.
11 Macan, T. T. Running Water. Mitt.
Internat. Limnol. 20:301-321. 1974.
This outline was prepared by Lowell E.
Keup, Chief, Technical Studies Branch,
Div. of Technical Support, EPA, Wash-
ington, D.C. 20460, and revised by
R. M. Sinclair, National Training Center,
MOTD, OWPO, USEPA, Cincinnati, Ohio
45268.
Descriptors: Aquatic Life, Benthos, Water
Quality, Degradation, Environmental Effects,
Trophic Level, Biological Communities,
Ecological Distributions
2-11
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STATISTICS AS AN ECOLOGICAL TOOL
I SAMPLING
A Introduction
The sampling problem is seldom fully
appreciated in spite of the fact that the
ecologist relies heavily on sampling ex-
periments as a source of information for
decisions.
1 Conditions necessary
Two main considerations should be
satisfied:
a Avoid bias
Bias often arises from particular
experimental conditions which al-
most never represent the exact
model of the situation about which
inferences are desired.
b The sample should yield information
on its own accuracy.
To obtain a valid estimate of sam-
pling error, each batch of material
must be so sampled that two or more
sampling units are obtained from it.
2 Advantages over complete enumeration.
These include: reduced cost, greater
speed, greater scope, greater accuracy.
B Definitions
We refer to sampling from finite populations.
These are populations from which the ex-
perimental units can be enumerated, and
consequently, randomly sampled. In sam-
pling finite populations, there are several
ways in which a selection may be made.
These include:
1 Simple random sampling
The population is listed and the plan
and sample size fixed. The important
criterion is that each possible sample
has the same probability of being
selected. In the actual process random
number tables are used, and the sam-
pling units are drawn independently.
2 Systematic sampling
In systematic sampling the samples
are equally spaced throughout the area
or population to be sampled. There
are analytic difficulties connected
with this procedure.
3 Stratified random sampling
The material or area is divided into
uniform groups or strata and a number
of observations are taken from each
stratum.
Figure 1 illustrates random, systematic
and stratified random sampling along a
line.
C Simple Random Sampling - Notation
Generally, guides for using simple random
sampling are: a) when the population is
not highly variable, b) when sampling for
proportions, use simple random sampling
when the true value lies between 20 and 80%.
1 Let YI be the i th observation in the
population.
Population size: N
Sample size: n
Population mean:
Y =
N
= -JTJ., for a continuous
variable
P = ~:T , for a proportion
BI. MET. stat.1.2.79
3-1
-------
Statistics as an Ecological Tool
For a proportion, Y. = 0 or 1. Replace
S.Y. by A.
Sample mean:
A S Y
— i i v
Y = v = = — . for a continuous
n n . , ,
variable
P = P = —, for a proportion
For a proportion, replace SY. by a.
Population variance:
N- 1
Population variance for a mean:
2
s.2
y
o , N - n
IT l~N~'
Sample variance:
(Y - y)
—;— an unbiased estimate
n - 1
or S
For computational purposes:
„ SY2 - (SY.)2/ n
n - 1
Sample variance of a mean:
g2 , .L .„...
y n
N
n - 1
N - n
N
when q = 1 - p
(N - n) /N is the finite population correlation
factor or Fpc. It is also written as 1 - n/N
and n/N is the sampling fraction. If it is
small, say 5%, it is usually neglected.
The confidence interval for a mean:
CI = y+ t
An approximation to the confidence interval
for a proportion:
CI = p +
/pq N - n
/ n N - 1
Note: In the formula above, estimated stan-
dard deviation is comparible to population
quantity, not sample quantity. Above more
commonly used.
Example: Attribute sampling. Of 1000
lobsters, 400 are sampled and 120 harbor a
certain parasite. The estimated proportion
with the parasite is a/n = 120/400 = 0. 30.
The 95% confidence interval is . 30 + . 048
using the formula for CI above with
t
.05,
= 1.96.
.05, 10
Example: Continuous variable. The follow-
ing 11 measurements represent a random
sample from a set of 50 objects. Their
measurements are: 3, 6, 6/ 12, 9, 12, 10,
9, 16, 14, 17. The mean is 10 and the stan-
dard deviation is 4. The 95% confidence in-
terval for the mean is 10 +_ 2. 34, t
is 2.228.
D Stratified Sampling. Notation.
To increase precision we may increase n
or reduce population variance. A good way
to decrease population variance is to con-
struct relatively homogeneous strata from
the sampling units. Variations in strata
means in the population do not contribute to
the sampling error of the estimate of the
population mean.
Let Y, . be the ith observation in the kth
lei
stratum, k = 1, ... , s. Strata sizes,
means and variances are described by
N
. ,
or P^, and S with corresponding
sample values of n, , y or p, and s, .
rC it K. K
3-2
-------
Statistics as an Ecological Tool
Stratum mean and variance:
Nk
S Y. . v
| _ i = 1 kl _ Yk
k Nk Nk
r - Ak
k Nk
Nk
K -2
S (Yki-Yk>
.2 _ i = 1 kl k
Nk - 1
Sample mean and variance for k stratum:
\
2 Y, .
£ . i-1 kl \
k ^ nk \
£ - o - &k
Pk - Pk - nk
"k
2 .S, (Yki-^2
k nk- 1
Let N = 1 Nk and n = \ "k and Nk/N = Wk
k K
where W stands for weight.
Population mean (st mean stratified):
f N Y 2 w. Y
- _ k k k _ kk
st " N k
N P -
_ 2 iNk *k _ 2 W P
st " k N k k k
Estimate of population mean:
£ . L Nk^k S W, y,
"st ^st ~ k N k k k
S
ft k k Pk 2 W, p,
st st N k
(sample means are y = 2n y /n and
k k
P = 2 nkpk/n.)
Variance of estimate of population mean:
„ „ N, S.2 N - n,
2 . _ . 2 k k w k
" (yst' ~ k N n, N,
k k
- ^ 1ST ^ 1ST nN
" KT2 " Nk ( Nk nk} " n,
N k
2 , . 2 Nw2 Pk^k Nk - "k
* ( Pst' k 2 n, N. - 2
N k k
Sample variance of the estimate of the
population mean;
2
9 1 St
c^/-\_ -1 S ivr /TVT ,,\ K
S \y i' 0 k ^1^1 ni '
J st !VT2 k k k n.
N k
2
_ 2 2 Sk 1S W s 2
' k Wk n, N k k k
k
In formulas where pfc occurs it is ignore
if small when computing confidence inter
Note: Stratified sampling may be used with
proportional allocation or with a variable
sampling fraction.
-------
Statistics as an Ecological Tool
II EXPERIMENTATION
A Planning Steps
1 Decide and define what the experiment
is intended to do. Specify population
to which results are intended to apply.
2 Gauge the probable accuracy of the
results likely to be obtained.
This is usually done by:
a Estimating the coefficient of variation,
the precentage variation in the ob-
servations that cannot be accounted
for by experimental factors.
b Specifying a value for the accuracy
desired in the treatment effect, ex-
pressed as a percentage of the over-
all mean.
c Specifying the probability for the
true value of the difference to fall
within assigned limits.
B Experimental Design
Definition includes:
1 the set of treatments selected for com-
parison,
2 the specification of the units (animals,
plants, plots, samples) to which the
treatments are to be applied,
3 the rules by which the treatments are
allocated to experimental units,
4 specification of measurements or other
records to be made on each unit.
C Choosing the design includes deciding:
1 whether the design is unifactor or
factorial - unifactor implies one
treatment (factor) to be tested holding
others constant. Factorial implies all
combinations of the different treatments
or factors.
2 whether to group observations
3 whether the number of treatments or
conclusipns is too large to allow full
replication - i. e. <•>•"•*—'
incomplete bl
D Classification of
signs are shown :
III DATA INTERPRE
A Associated Measuicmems
Ecologists frequently obtain data which
may look something like that shown be-
low, and wish to perform statistical tests
with these data.
x
X X
B Procedure
1 Guess the nature of the relationships
from the graph. That is: we may
have
y = bx
y = a + bx
b
y = ax etc.
2 Test the guess by graphical inspection.
3 Perform the appropriate computations
on the transformed data, and make the
appropriate statistical tests. Any
equation which can be converted to the
form:
y = a + bx
can be handled by ordinary regression
computing techniques, described in any
standard statistical text.
3-4
-------
Statistics as an Ecological Tool
C Examples of Linear Relationships
1.
At x • 0, y = 0.
Thus, there is no
intercept, and
y = bx
3.
-a
At x = 0, y = -a.
Thus
y •= -a+bx
2.
a-
At x = 0, y - a.
There is an inter-
cept.
y = a + bx
Negative slope,
thus
y = a - bx
D Other graphs, postulated relations, and
graphical tests include the following:
Raw Data
1.
x
X
xxxx
3.
X
X
X
XX*
XXXXXXXx
Possible Equation
y = ac
-bx
y = ax
Test giving linearity if
equation describes data
log y = log a-bx
plot y against x on
semi log paper
log y • log a + b log x
Plot y against x on log-log
paper
Plot y against _
x
3-5
-------
Statistics as an Ecological Tool
Figure 1. METHODS OF SAMPLING
-x
-X
-X
-X
-X
-X
-X
-X
-X
-X
random
systematic
stratified random
COMPLETE,
BLOCK
one grouping
two groupings
Figure 2
UNIFACTOR
FACTORIAL
random blocks
Latin squares
INCOMPLETE
BLOCK
one grouping
two groupings
Balanced incomplete
blocks
Lattice designs
Lattice squares
Youden squares
Confounded designs
Fractional replications
Split-plots
Quasi-latin squares
3-6
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Statistics as an Ecological Tool
E Calculations in Regression and Correlation
1 The basic observations are in pairs of
associated observations represented by
(x, y). We assume x and y follow, at
least approximately, a bivariate normal
distribution.
2 A convenient desk calculator procedure
is to find the sums, and sums of squares
of products as follows:
(1)
n
Z X
zx2
Z Y
Z Y
ZXY
Next, calculate the three quantities in
the line below:
(2) -- ( Z X)
— (Z Y)2, — (ZX) (ZY)
n n
The variance of the deviations of y from
the regression line is estimated by:
n - 2
Sx
The correlation coefficient r is cal-
culated as:
r =
Zxy
(Zx)(Zy
REFERENCES
Elementary
Snedecor, G. W. Statistical Methods.
Fifth edition. Iowa State College Press,
1956.
Subtract each of these from the last
line of set (1) to give the corrected sums
of squares and cross-products.
Sampling
Cochran, W. G. Sampling Techniques.
New York: John Wiley & Sons, 1953.
(3) Zx2 = ZX2 - - (ZX)2, Zy2 = ZY2 - - (ZY)2,
n n
Zxy = ZXY - (ZX) (ZY)
Experimental Design
Cochran, W. G. , and Cox, G. M. Experi-
mental Designs. Second edition. New
York: John Wiley & Sons, 1957.
The estimate of the true regression co-
efficient is given by:
(4) b =
Zxy
2~
x
and the constant a is given by
a = y - bx.
Bioassay
Finney, N. J. Statistical Method in Bio-
logical Assay. London: Charles Griffin
and Company, 1952.
This outline was prepared by S. B. Saila,
Associate Professor of Oceanography,
Graduate School of Oceanography, University
of Rhode Island, Kingston, Rhode Island.
Descriptors: Statistics, Statistical Methods,
Data Processing and Ecology
3-7
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THE INTERPRETATION OF BIOLOGICAL DATA
WITH REFERENCE TO WATER QUALITY
I INTRODUCTION
Sanitary engineers like to have data
presented to them in a readily assimilable
form and some of them seem a little
impatient with biologists who appear unable
to provide definite quantitative criteria
applicable to all kinds of water conditions.
I think the feeling tends to be that this is
the fault of biologists, and if they would
only pull themselves out of the scientific
stone-age all would be well. I will try to
explain here why I believe that biological
data can never be absolute nor interpret -
able without a certain amount of expertise.
In this respect biologists resemble medical
men who make their diagnoses against a
complex background of detailed knowledge.
Anyone can diagnose an open wound but it
takes a doctor to identify an obscure
disease; and although he can explain how
he does it he cannot pass on his knowledge
in that one explanation. Similarly, one
does not need an expert to recognize gross
organic pollution, but only a biologist can
interpret more subtle biological conditions
in a water body; and here again he can
explain how he does it, but that does not
make his hearer a biologist. Beck (1957)
said something similar at a previous
symposium in Cincinnati in 1956.
II THE COMPLEXITY OF BIOLOGICAL
REACTIONS TO WATER CONDITIONS
A Complexity of the Aquatic Habitat
The aquatic habitat is complex and
consists not only of water but of the
substrata beneath it, which may be
only indirectly influenced by the quality
of the water. Moreover, in biological
terms, water quality includes such
features as rate of flow and tempera-
ture regime, which are not considered
of direct importance by the chemist.
To many animals and plants, maximum
summer temperature or maximum
rate of flow is just as important as
minimum oxygen tension. The result
is that inland waters provide an
enormous array of different com-
binations of conditions, each of which
has its own community of plants and
animals; and the variety of species
involved is very great. Thus, for
example, Germany has about 6000
species of aquatic animals (lilies 196 la)
and probably at least as many species
of plants. Yet Europe has a rather
restricted fauna because of the
Pleistocene ice age; in most other
parts of the world the flora and fauna
are even richer.
Distribution of Species and Environ-
mental Factors
We know something about the way in
which species are distributed in the
various habitats, especially in the
relatively much studied continent of
Europe, but we have, as yet, little
idea as to what factors or combination
of factors actually control the individual
species.
1 Important ecological factors
Thus, it is possible to list the
groups of organisms that occur in
swift stony upland rivers
(rhithron in the sense of lilies,
1961b) and to contrast them with
those of the lower sluggish reaches
(potamon). Similarly we know,
more or less, the different floras
and faunas we can expect in
infertile (oligotrophic) and fertile
(eutrophic) lakes. We are, however,
much less informed as to just what
ecological factors cause these
differences. We know they include
temperature and its yearly
BI.EN. Id. 10.75
4-1
-------
The Interpretation of Biological Data with Reference to Water Quality
amplitude; oxygen, particularly at
minimal levels; plant nutrients,
such as nitrate, phosphate, silica,
and bicarbonate; other ions in
solution, including calcium, chloride,
and possibly hydrogen; dissolved
organic matter, which is necessary
for some bacteria and fungi and
probably for some algae; the nature
of the substratum; and current.
2 Complexity of interacting factors
We also know these factors can
interact in a complex manner and
that their action on any particular
organism can be indirect through
other members of the biota.
a Induced periphyton growths
Heavy growths of encrusting
algae induced by large amounts
of plant nutrients, or of
bacteria induced by ample
supplies of organic matter,
can eliminate or decimate
populations of lithophile insects
by simple mechanical inter-
ference. But the change does
not stop there: the growths
themselves provide habitats
for the animals, such as
Chironomidae and Naidid worms,
which could not otherwise live
on the stones.
b Oxygen levels and depositing
substrates
If oxygen conditions over a
muddy bottom reach levels
just low enough to be intolerable
to leeches, tubificid worms,
which the leeches normally
hold in check, are able to build
up to enormous numbers
especially as some of their
competitors (e. g. Chironomus)
are also eliminated.
c Oxygen levels and non muddy
substrates
One then finds the typical
outburst of sludge worms, so
often cited as indicators of
pollution. This does not
happen if the same oxygen
tension occurs over sand or
rock, however, as these are
not suitable substrata for the
worms. Many such examples
could be given, but they would
only be ones we understand;
there must be a far greater
number about which we know
nothing.
d One must conclude, therefore,
that quite simple chemical
changes can produce far-
reaching biological effects;
that we only understand a
small proportion of them; and
that they are not always the
same.
Classic examples
This seems like a note of despair,
however, if water quality deviates
too far from normal, the effects
are immediately apparent. Thus,
poisonous substances eliminate
many species and may leave no
animals (Hynes 1960); excessive
quantities of salt remove all
leeches, amphipods, and most
insects and leave a fauna con-
sisting largely of Chironomidae,
caddis worms, and oligochaetes
(Albrecht 1954) and excessive
amounts of dissolved organic
matter give rise to carpets of
sewage fungus, which never occur
naturally. Here no great biologi-
cal expertise is needed, and there
is little difficulty in the
communication of results. It is
when effects are slighter and more
subtle that biological findings
4-2
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The Interpretation of Biological Data with Reference to Water Quality
III
become difficult to transmit
intelligibly to other disciplines.
THE PROBLEMS IN PRESENTATION
OF BIOLOGICAL RESULTS
Because of these difficulties various
attempts have been made to simplify the
presentation of biological findings, but to
my mind none of them is very successful
because of the complexity of the subject.
Early attempts at systematization developed
almost independently on the two sides of
the Atlantic, although they had some
similarities.
A Early Studies in the United States
(Richardson and the Illinois River)
In America, there was a simple division
into zones of pollution, e. g. degradation,
septic, and recovery, which were
characterized in broad general terms.
This simple, textbook approach is
summarized by Whipple et al. (1947),
and serves fairly well for categorizing
gross organic pollution such as has been
mentioned above. It was, however,
soon found by Richardson (1929) during
his classical studies on the Illinois
River that typical "indicators" of foul
conditions, such as Tubificidae and
Chironomus, were not always present
where they would be expected to occur.
This was an early indication that it is
not the water quality itself that provides
suitable conditions for "pollution faunas, "
but other, usually associated, conditions -
in this instance deposits of rich organic
mud. Such conditions may, in fact, be
present in places where water quality
in no way resembles pollution, e. g.,
upstream of weirs in trout streams
where autumn leaves accumulate and
decay and cause the development of
biota typical of organically polluted
water. Samples must therefore be
judged against a background of biological
knowledge. Richardson was fully aware
of this and was in no doubt about the
condition of the Illinois River even in
places where his samples showed few
or no pollution indicators.
B The European Saprobic System
In Europe, the initial stress was
primarily on microorganisms and
results were first codified in the
early years of the century by
Kolkwitz and Marsson. In this
"Saprobiensystem, " zones of organic
pollution similar to those described
by the American workers were defined
and organisms were listed as charac-
teristic of one or more zones;
TABLE 1
SAPROBIENSYSTEM - A European system
of classifying organisms according to their
response to the organic pollution in slow
moving streams. (22)
Alpha-Mesosaprobic Zone - Area of
active decomposition, partly aerobic,
partly anaerobic, in a stream heavily
polluted with organic wastes.
Beta-Mesosaprobic Zone - That reach
of stream that is moderately polluted
with organic wastes.
Oligosaprobic Zone - That reach of a
stream that is slightly polluted with
organic wastes and contains the
mineralized products of self-
purification from organic pollution,
but with none of the organic pollutants
remaining.
Poly saprobic Zone - That area of a
grossly polluted stream which contains
the complex organic wastes that are
decomposing primarily by anaerobic
processes.
A recent exposition of this list is
given by Kolkwitz (1950). It was then
claimed that with a list of the species
occurring at a particular point it was
possible to allocate it to a saprobic
zone. This system early met with
criticism for several reasons. First,
4-3
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The Interpretation of Biological Data with Reference to Water Quality
TABLE 2
SAPROBICITY LEVELS ACCORDING TO THE TROPHIC
STRUCTURE OF THE COMMUNITIES OF ORGANISMS
Saprobicity Level
Structure of the Communities of Organisms
I /3-oligosaprobic
Balanced relationship between producers, consumers
and destroyers; the communities of organisms are
poor in individuals but there is a moderate variety of
species, small biomass and low bioactivity.
II a-oligosaprobic
Balanced relationship between producers, consumers
and destroyers; communities of organisms are rich in
individuals and species with a large biomass and high
bioactivity.
Ill /3-mesosaprobic
Substantially balanced relationship between producers,
consumers and destroyers; a relative increase in the
abundance of destroyers and, accordingly, of the con-
sumers living off them; communities of organisms are
rich in individuals and species with a large biomass and
high bioactivity.
IV a-mesosaprobic
V /3-polysaprobic
Producers decline as compared with an increase in
consumers and destroyers; mixotrophic and amphitrophic
forms predominate among the producers; communities of
organisms rich in individuals but poor in species with a
large biomass and extremely high bioactivity; still only
few species of macro-organisms; mass development of
bacteria and bacteria-eating ciliates.
Producers drastically decline; communities of organisms
are extremely rich in individuals but poor in species with
a large biomass and high bioactivity; macrofauna represented
only by a few species of tubificids and chironomids; as in
IV these are in great abundance; mass development of
bacteria and bacteria-eating ciliates.
VI a -polysaprobic
Producers are absent; the total biomass is formed
practically solely by anaerobic bacteria and fungi;
macro-organisms are absent; flagellates outnumber
ciliates amongst the protozoa.
Saprobicity - "Within the bioactivity of a body of water, Saprobicity is the sum
total of all those metabolic processes which are the antithesis of
primary production. It is therefore the sum total of all those
processes which are accompanied by a loss of potential energy. "
Part I, Prague Convention.
4-4
-------
The Interpretation of Biological Data with Reference to Water Quality
all the organisms listed occurred in
natural habitats--they were not evolved
in polluted water--and there was much
doubt as to the placing of many of the
species in the lists. The system, how-
ever, did serve to codify ecological
knowledge about a long list of species
along an extended trophic scale. Its
weaknesses appeared to be merely due
to lack of knowledge; such a rigid
system took far too little account of the
complexity of the reaction of organisms
to their habitats. For instance, many
organisms can be found, albeit rarely,
in a wide range of conditions and others
may occur in restricted zones for
reasons that have nothing to do with
water quality. We often do not know if
organisms confined to clean headwaters
are kept there by high oxygen content,
low summer temperatures, or inability
to compete with other species under
other conditions. In the swift waters of
Switzerland the system broke down in
that some organisms appeared in more
polluted zones than their position in the
lists would indicate. Presumably here
the controlling factor was oxygen, which
was relatively plentiful in turbulent cold
water. In a recent series of experiments,
Zimmerman (1962) has proven that
current alone has a great influence on
the biota, and identically polluted water
flowing at different speeds produces
biotic communities characteristic of
different saprobic levels. He finds this
surprising, but to me it seems an
expected result, for the reasons given
above.
C Recent Advances in the Saprobic System
1 Perhaps Zimmerman's surprise
reflects the deeply rooted entrench-
ment of the Saprobiensystem in
Central Europe. Despite its obvious
shortcomings it has been revised
and extended. Liebmann (1951)
introduced the concept of consider-
ing number as well as occurrence
and very rightly pointed out that the
community of organisms is what
matters rather than mere species
lists. But he did not stress the
importance of extrinsic factors,
such as current, nor that the
system can only apply to organic
pollution and that different types
of organic pollution differ in their
effects; e.g., carbohydrate solu-
tions from paper works produce
different results from those of
sewage, as they contain little
nitrogen and very different sus -
pended solids. Other workers
(Sladecek 1961 and references
therein) have subdivided the more
polluted zones, which now, instead
of being merely descriptive, are
considered to represent definite
ranges of oxygen content, BOD,
sulfide, and even E. coli populations.
Every water chemist knows that
BOD and oxygen content are not
directly related and to assume that
either should be more than vaguely
related to the complexities of
biological reactions seems to me
to indicate a fundamental lack of
ecological understanding. I also
think it is damaging to the hope of
mutual understanding between the
various disciplines concerned with
water quality to give the impression
that one can expect to find a close
and rigid relationship between
water quality measurements as
assessed by different sets of
parameters. Inevitably these
relationships vary with local con-
ditions; what applies in a sluggish
river in summer will certainly not
apply to a mountain stream or even
to the same river in the winter.
Correlation of data, even within
one discipline, needs understanding,
knowledge, and judgment.
Gaspers and Schulz (1960) showed
that the failure of the system to
distinguish between waters that are
naturally productive and those
artifically enriched can lead to
absurd results. They studied a
canal in Hamburg, which because
of its urban situation can only be
regarded as grossly polluted.
Yet it develops a rich plankton.
4-5
-------
The Interpretation of Biological Data with Reference to Water Quality
the composition of which, according
to the system, shows it to be
virtually clean.
D Numerical Application of the Saprobic
System
Once the Saprobiensystem was accepted
it was logical to attempt to reduce its
findings to simple figures or graphs for
presentation of results. Several such
methods were developed, which are
described by Tumpling (1960), who also
gives the original references. In all
these methods, the abundance of each
species is recorded on some sort of
logarithmic scale (e.g. 1 for present,
3 for frequent, 5 for common, etc.).
The sums of these abundances in each
saprobic level are plotted on graphs,
the two most polluted zones showing as
negative and others as positive. Or, the
various saprobic levels are given
numerical values [1 for oligosaprobic
(clean), 2 for j3-mesosaprobic, etc.]
and the rating for each species is
multiplied by its abundance number.
The sum of all these products divided
by the sum of all the frequencies gives
a "saprobic index" for the locality.
Clearly the higher this number, the
worse the water quality in terms of
organic pollution. In a similar way the
so-called "relative Belastung" (relative
load) is calculated by expressing the
sums of all the abundances of organisms
characteristic of the two most-polluted
zones as a percentage of the sum of all
abundances. Then 100 percent is
completely polluted water, and clean
localities will give a low number.
E Weaknesses of the Saprobic System
There are various elaborations of these .
methods, such as sharing of species
between zones and taking account of
changes in base-line as one passes
downstream. None of them, however,
eliminates the basic weaknesses of the
system nor the fact that, as Caspers
and Schulz (1960) point out, there is
little agreement between the various
authors in the assignment of species to
the different levels. Therefore, one
gains a number or a figure that looks
precise and is easily understood, but
it is based on very dubious foundations.
F Comparative North American Systems
Similar systems are indigenous to
North America, but were independently
evolved.
1 Wurtz (1955) and Wurtz and Dolan
(1960) describe a system whereby
animals are divided into sensitive-
to-pollution and non-sensitive
(others are ignored), and also into
burrowing, sessile, and foraging
species (six classes).
BSFP BSFP BSFP BSFP BSFP BSFP BSFP BSFP BSFP
13
M-6
M-5
M-4 8/ZTA6
8/2648 RECOVERY
EPTC
14
M-8
8/28/48
RECOVRY DEGRAD.
\OOJ-
Figure 1. Histograms, based on selected organisms, illustrating stream
reaches of clean, degradation, septic, and recovery conditions [after
Wurtz] 1(22)
Numbers of these species rep-
resented are plotted for each station
as six histograms on the basis of
percentage of total number of
species. If the constitution of the
fauna from control stations or from
similar localities is known, it is
possible to express numerically
"biological depression" (i.e.,
percentage reduction in total
4-6
-------
The Interpretation of Biological Data with Reference to Water Quality
number of species), "biological
distortion" (changes in pro-
portions of tolerant and non-tolerant
species), and "biological skewness"
(changes in the ratios of the three
habitat classes). Such results must,
of course, be evaluated, and the
definition of tolerance is quite
subjective; but the method has the
advantages of simplicity and depend-
ence on control data. Like the
Saprobiensystem, however, it can
have no universal validity. It also
suffers from the fact that it takes
no account of numbers; a single
specimen, which may be there by
accident, carries as much weight
as a dense population.
Patrick (1949) developed a similar
system in which several clean
stations on the water body being
investigated are chosen, and the
average number of species is deter-
mined occurring in each of seven
groups of taxa chosen because of
their supposed reaction to pollution.
These are then plotted as seven
columns of equal height, and data
from other stations are plotted on
the same scale; it is assumed that
stations differing markedly from the
controls will show biological
imbalance in that the columns will be
of very unequal heights. Number is
indicated by double width in any
column containing species with an
unusual number of individuals.
I have already questioned the use-
fulness of this method of presentation
(Hynes 1960), and doubt whether it
gives any more readily assimilable
data than simple tabulation; it does
however, introduce the concept of
ecological imbalance.
200
150
250
200
£
SEMI-HEALTHY
III..
_n
3
VERY POLLUTED
Figure 2. Histograms, based on selected organisms, illustrating healthy,
semi-healthy, polluted, and very polluted stations in Conestoga
Basin, Pa. [ after Patrick ] (22)
TABLE 3 —Classification of Groups
of Organisms Shown in Figure2
I Blue-green algae; green algae of the genera Stigeoc/onium, Spi-
rogyra, and TVibonema; the bdelloid rotifers plus Cepha/odelfa
mega/ocephala and Proales dec/piens
II Oligochaetes, leeches, and pulmonate snails
III Protozoa
IV Diatoms, red algae, and "most of the green algae"
V All rotifers not included in Group I, clams, gill-breathing snails,
and tricladid ftatworms
VI All insects and Crustacea
VII All fish
Beak (1964), another author,
recognized the need for a concise
expression of pollution based on
biological information. Toward
this end, he developed a method of
biological scoring which is based on
the frequency of occurrence of
certain macroscopic invertebrates
obtained from 6 years of study on
one river. It will be noted that the
Biological Score is a modification
and expansion of Beck's Biotic
Index.
-------
The Interpretation of Biological Data with Reference to Water Quality
The indicator organisms are
divided into three categories:
Group I contains the pollution -
tolerant species; Group II comprises
those species which are facultative
with respect to pollution; and
Group III contains the pollution-
intolerant forms. Each group is
assigned a weighted score that can
be allotted to field samples on the
following basis:
a Normal complement of Group III
scores 3 points.
b Normal complement of Group II
scores 2 points.
c Normal complement of Group I
scores 1 point.
The scores are additive; thus an
unpolluted stream will have a
Biological Score of 6. If only
pollution-tolerant forms are found,
the score will be 1. If no organisms
are found, the score will be zero.
Furthermore, a score of, 1 or 2
points could be allotted to Group III
when less than the normal com-
plement is present. Group II could
be treated in a similar manner.
This scoring device correlated well
with the biological oxygen demand,
dissolved oxygen, and solids content
of the receiving water. Beak also
related his scoring device to the
fisheries potential. This relation-
ship is shown in Table 4]
TABLE 4
TENTATIVE RELATIONSHIP OF THE BIOLOGICAL SCORE TO THE FISHERIES
POTENTIAL (after Beak, 196-1) (30)
It has long been known that
ecologically severe habitats con-
tain fewer species than normal
habitats and that the few species
that can survive the severe con-
ditions are often very abundant as
they lack competitors. Examples
of this are the countless millions
of Artemia and Ephydra in saline
lakes and the Tubifex tubifex in
foul mud. This idea has often been
expressed in terms of diversity,
which is some measure of numbers
of species divided by number of
specimens collected. Clearly,
such a parameter is larger the
greater the diversity, and hence
the normality of the habitat.
Unfortunately, though as the
number of species in any habitat
is fixed, it also decreases as
sample size increases so no index
of diversity has any absolute value
(Hairston 1959). If a definite
sample size is fixed, however, in
respect to numbers of organisms
identified, it is possible to arrive
at a constant index.
20
f
10
Pollution status
Biotic index
Fisheries potential
o 10 ?o
Miles from source
Figure 3. Zooplankton species diversity
per thousand individuals encountered in
marine systems affected by waste waters
from petrochemical industrial wastes.
The vertical lines indicate seasonal
variations. (30)
Unpolluted
Slight to moderate pollution
Moderate pollution
Moderate to heavy pollution
Heavy pollution
Severe pollution, usually toxic
6 All normal fisheries for type of
water well developed
5 or 4 Most sensitive fish species re-
duced in numbers or missing
3 Only coarse fisheries maintained
2 If fish present, only those with
high toleration of pollution
1 Yen- little, if any, fishery
0 No fish
4-8
-------
The Interpretation of Biological Data with Reference to Water Quality
Patrick et al. (1954) in effect used
this concept in a study of diatom
species growing on slides suspended
in water for fixed periods. They
identified 8000 specimens per
sample and plotted the results as
number of species per interval
against number of specimens per
species on a logarithmic scale.
This method of plotting gives a
truncated normal curve for a wide
variety of biotic communities.
In an ordinarily diverse habitat the
mode is high and the curve short;
i.e., many species occur in small
numbers and none is very abundant.
In a severe habitat the mode is low
and the curve long; i. e., there are
few rare species and a few with
large numbers. This, again, seems
to me to be an elaborate way of
presenting data and to involve a lot
of unnecessary arithmetic.
30 -
__
15
1-2 2-4 4f 3-lf, 16-32 ~32-~5 C-1-12S 1?8- •<•:,
?55 512 !
Number of ! vjvijvj •':, ;,rr $j:2(:;?~
Figure 4. A graphic comparison of diatom
communities from two different environ-
ments. (After Patrick et al., 1954) (30)
6 Diversity indices vs tabulated data
Allanson (1961) has applied this
method to the invertebrate faunas
of streams in South Africa and has
shown, as has Patrick for diatoms,
that the log normal curve is flatter
and longer for polluted stations;
the difference, however, is not so
apparent that it does not need
exposition. Here, again, I would
suggest that tabulated data are just
as informative. Indeed I would go
further and say that tabulated data
are essential in the present state
of our knowledge. We are learning
as we go along and if the details of
the basic findings are concealed by
some sort of arithmetical manip-
ulation they cannot be re-interpreted
in the light of later knowledge, nor
are they preserved in the store of
human knowledge. This point
becomes particularly clear when
one examines some of the early
studies that include tables.
Butcher (1946) requotes a con-
siderable amount of data he
collected from studies of various
English rivers during the thirties;
they are not only clear and easy to
follow, but they are also informative
about the generalities of pollution
in a way that data quoted only
within the confines of some
particular system are not.
Examples of tabulated data(Table 5)
Simple tabulation of biological data
in relation to water quality, either
in terms of number of organisms,
percentage composition of the biota,
some arbitrary abundance scale,
or as histograms, has been
effectively practiced in many parts
of the world: in America (Gaufin
and Tarzwell 1952, Gaufin 1958),
Africa (Harrison 1958 and 1960,
Hynes and Williams 1962), Europe
(Albrecht 1954, Kaiser 1951,
Hynes 1961, Hynes and Roberts
1962), and New Zealand (Hirsch
1958) to cite a few. These tabu-
lated data are easy to follow, are
informative to the expert reader,
and conceal no facts. Although the
non-biologist may find them tedious,
he need only read the explanatory
paragraphs. It is a delusion to
think that it is possible to reduce
biological data to simple numerical
levels. At best, these can only be
produced for limited situations and
4-9
-------
The Interpretation of Biological Data with Reference to Water Quality
TABLE 5
ORGAH1SM3
DeodroBoma
Spongllla fragills
Trochoapongilla leidyi
Unidentified Sponge
Cordylopbora lacustrls
Dugeala tigrina
Urnatella gracllis
Psludlcella articulata
Fredericella sultana
Prlstina
Hals coanunls
ParanaiB
Unidentified leech
Unidentified Beetle
Chaoborus punctipennls
HydrobaenuB sp. A
Cricotopiia bicinctuo
Unidentified Tendlpedlnl
Harnischia ep. A
Tendlpes oervoaua
Tendlpea, uodestus
Polypedllun sp. B
Calopsectra exigua
Trycorythodes
Stenoneaa
Agraylea
Athripsodes
Pataayia flava
Bydropsyche orrlfl
Cheumatopsyche
Paychonyildae Genua A
Lithasia verrucoBa
Ferrisala shlmekll
QuadrulA sp.
Quadrula tuberculata
Corbicula fluminea
TOTAL
left
F
C
C
5
C
C
31
59
1
51
152
mid
F
5
Ik
1
22
1
90
298
right
2
1
5
2
16
2
2
21
5
56
left
C
p
k
F
1
9
7
5
1
1
2
1(1
141
1 It
30
i*
11
261
aid
F
A
k
F
1
117
29
k
12
3
5
229
671
right '
A
6
F
1
68
1
2
15
7
1)8
17
92
33
1
2
1
381
left
14
F
y
i
i
i
i
3
1
1
19
2
11
7
1
77
11*1
1
mid
A
A
7
26
1
8
2
169
in
.LJA
30
193
5
575
,153
right
F
19
F
F
1
1
3
1
101
1
23^
11
21
1
19
7
M7
F - rev C -
A - abundant
Benthos from Pickwick Tailwater (35)
even then they need verbal exposition;
at worst, they give a spurious im-
pression of having absolute validity.
8 Comparison of stations
My final point in this section con-
cerns comparisons. It is claimed
that the German system, in effect,
measures an absolute state, a
definite level of water quality. We
have seen that this is not a tenable
claim. In the other systems, by
and large, the need to establish
local control stations at which to
measure the normal or "natural"
biotic conditions is accepted, and
then other areas are compared with
this supposed norm. This is, of
course not always possible as there
may remain no unaffected area, or
no unaffected area that is, with
respect to such factors as current,
nature of substratum, etc.,
sufficiently similar to act as a
base-line for data. Nevertheless,
basically, these systems can be
used to compare stations and thus
to assess changes in water quality.
In doing this, they can all be used
more or less successfully, but I
maintain that a table is just as use-
ful as an elaborate analysis, and
I believe that the table should be
included witn whatever is done.
For a particular situation, however,
it is often possible to distill the data
into a single figure as a measure of
similarity between stations.
Coefficients of similarity
Burlington (1962) and Dean and Bur-
lington (1963) have recently proposed
an entirely objective means of doing
this, which involves simple arith-
metical manipulation. In his system,
a "prominence value" is calculated
for each species at each station.
This is a product of its density and
some function of its frequency in
samples, but the details of this
calculation can be altered to suit
any particular situation. Then a
coefficient of similarity between
each pair of stations can be calcu-
lated by dividing twice the sum of
the lower prominence values of taxa
that the two stations have in common
by the sum of all the prominence
values of both stations. Identical
stations will then have a coefficient
of similarity of 1:00; this coefficient
will be lower the more the stations
differ from one another. This is an
easy way to compare stations in an
entirely unbiased way and as such
may satisfy the need for numerical
exposition; however, it tells one
nothing about why the localities are
different and like all the other more
or less numerical methods of pre-
senting data has no absolute value.
Moreover, it still leaves unanswered
the fundamental question of how
different is "different?"
10
-------
The Interpretation of Biological Data with Reference to Water Quality
TABLE 6
•^ Clean
TY (high multiple use indicated^
Order of Tendency to Disappear as |N
gree of Pollution Increases '
DB
CO
OT-(
ci
cO
00
a o
1
•{^Polluted
Types of
Organisms
Present
Plecoptera
nymph
present
Ephemeroptera
nymph
present
Trichoptera
larvae
present
Gammaridae
present
Asellus and/or
Lirceus
present
Tubificid worms,
Tendipes , and
Cricotopus
bicinctus
(one or more of
these groups)
All above types
absent
BIOTIC INDEX
Variety Present
More than one species
One species only
\>
More than one species
JL>
One species only
More than one species
One species only *
All above species absent
All above species absent
All above species absent
Some organisms such as
Eristalis tenax not requiring
dissolved oxygen may be present
•\/ Total Number of Groups
Present
0-1
~
—
—
--
--
4
3
2
1
0
2-5
Bio
7
6
6
5
5
4
4
3
2
1
6-10
Inde
8
7
7
6
6
5
5
4
3
2
11-15
9
8
8
7
7
6
6
5
4
~
16+
10
9
9*
8
8
7
7
6
—
*Stenonema nepotellum excluded 10^. main stream reservoirs and west Tennessee streams
* Stenonema nepotellum (Ephem.) is counted in this section for the purpose of classification.
V QKE FOR EACH KNOWN SPECIES IN THESE GROUPS:
Platyhelminthes
Hirudinea
Mollusca
Crustacea
Plecoptera
Diptera (excluding specific ones listed below)
Coleoptera
Neuroptera
V ONE FOR EACH GROUP. REGARDLESS OF NUMBER OF SPECIES, ETC.:
Annelida excluding Naididae
Naididae
Each Mayfly genera (excluding Stenonema nepotellum)
Stenonema nepotellum
Each Trichoptera family
Chironomidae (excluding specific ones listed below)
Chironomus riparius and plumosus and Cricotopus bicinctus.
Family Simuliidae
adapted from Trent River Board - Tennessee Stream Pollution Control Board 8/66 RMS
11
-------
The Interpretation of Biological Data With Reference to Water Quality
IV THE PROBLEMS OF SAMPLING
The systems outlined above are all based on
the assumption that it is possible to sample
an aquatic habitat with some degree of
accuracy; this is a dubious assumption,
however, when applied to biological data.
From what has been said about the com-
plexity of biological reactions to the various
factors in the environment, and from the
obvious fact that rivers especially are a
mosaic of microhabitats, it is clear that to
achieve numerical accuracy or even some
limits of confidence considerable numbers
of samples need to be taken. Indeed, even
in so apparently unvaried a habitat as a
single riffle, Needham and Usinger (1956)
showed that a very large number of samples
would be necessary to give significant
numerical data.
A Representative Sampling
There is a limit to the number of sam-
ples that can reasonably be taken and,
anyway, it is desirable to sample many
different types of habitat so as to get
as broad as possible an estimate of the
biota. This is the more recent approach
of most of the workers in Central Europe,
who have been content to cite abundances
on a simple relative but arbitrary scale
and to convert this to figures on some C
sort of logarithmic scale for use in
calculations. An alternative is to ex-
press the catch in terms of percentage
composition, but this had the disadvantage
that micro- and macro-organisms cannot
be expressed on the same scale as they
are obtained by different collecting tech-
niques. Also, of course, implicit in
this approach is the assumption that
the sampling is reasonably representa-
tive. Here again we run into the need for
knowledge and expertise. In collection as
well as in interpretation, the expert is
essential. Biological sampling, unlike
the simple, or fairly simple, filling of
bottles for chemical analysis or the
monitoring of measuring equipment, is
a highly skilled job and not one to be
handed over to a couple of vacationing
undergraduates who are sent out with
a Surber sampler and told to get on
with it. This point has also been made
by other biologists, e.g., Patrick (1961)
who stresses the need for skilled and
thorough collecting even for the deter-
mination of a species list.
B Non-Taxonomic Techniques
Alternatively we can use the less
expert man when concentrating on only
part of the habitat, using, say, micro-
scopical slides suspended in the water
to study algal growth. This method
was extensively used by Butcher (1946),
and Patrick et al. (1954) who studied
diatoms in this way. This gives only
a partial biological picture, but is
useful as a means of monitoring a
stretch of water where it is possible
that changes might occur. It is a
useful short-hand method, and as such
is perhaps comparable to studying the
oxygen absorbed from potassium
permanganate instead of carrying out
all the usual chemical analyses on water.
A short method of this kind may serve
very well most of the time, but, for
instance, would not be likely to detect an
insecticide in concentrations that could
entirely eliminate arthropods and hence
fishes by starvation.
Monitoring
It is possible to work out biological
monitoring systems for any specific
purpose. The simplest of these is the
cage of fish, which, like a single type
of chemical analysis, can be expected
to monitor only one thing — the ability
of fish to live in the water — with no
information on whether they can breed
or whether there is anything for them
to eat. Beak et al. (1959) describes a
neat way in which the common con-
stituents of the bottom fauna of Lake
Ontario can be used to monitor the
effluents from an industrial site.
Obviously there is much room for such
ingenuity in devising biological systems
for particular conditions, but this is
perhaps outside the scope of this meeting.
4-12
-------
The Interpretation of Biological Data with Reference to Water Quality
V
CONCLUSIONS
It may appear from the previous sections
that my attitude to this problem is entirely
obstructionist. This is far from being so.
Water quality is as much biological phenom-
enon as it is a chemical or physical one;
often what we want to know about water is
almost exclusively biological - - will it smell
nasty, is it fit to drink, can one bathe in it,
etc? I suggest, therefore, that it is desirable
to organize water monitoring programs that
will tell one what one wants to know. There
is no point in measuring everything biolog-
ical, just as there is no point in performing
every possible chemical analysis; what is
measured should be related to local conditions.
It would be a waste of time to measure
oxygen content in a clean mountain stream;
we know it to be high, and it becomes worth
measuring only if we suspect that it may
have been lowered by pollution. Similarly,
there is little point in studying the plankton
in such a stream; we know it only reflects
the benthic flora. In a lake or in a slow
river, on the other hand, if our interest in
the water lies in its potability, records of
the plankton are of considerable importance
as changes in plankton are, in fact, changes
in the usability of the water.
A Periphyton and Benthos Studies
For long-term studies, especially for
the recording of trends or changes
induced by pollution, altered drainage,
agricultural poisons, and other havoc
wrought by man, one can expect in-
formative results from two principal
techniques: First, we can study
microscopic plant and animal growth
with glass slides placed in the water for
fixed periods; second, we can obtain
random samples of the benthic fauna.
The algae and associated microfauna
tell one a good deal about the nutrient
condition of the water and the changes
that occur in it, and the larger benthic
fauna reveal changes in the trophic
status, siltation due to soil erosion,
effects of insecticides and other poisons,
etc.
B Varying Levels of Complexity
The study of growths on glass slides is
reasonably skilled work, but can easily
be taught to technicians; like chemical
monitoring, such study needs to be
done fairly often. Sampling the benthos
is more difficult and, as explained
above, needs expert handling; unlike
most other monitoring programs,
however, it need be done only in-
frequently, say, once or twice a year.
Inevitably sampling methods will vary
with type of habitat; in each case, the
question will arise as to whether it is
worth looking at the fish also. It is
here that the biologist must exercise
judgment in devising and carrying out
the sampling program.
C Data Interpretation
Judgment is also needed in the inter-
pretation of the data. It is for this
reason I maintain that it should all be
tabulated so that it remains available
for reassessment or comparison with
later surveys. If need be, some sort
of numerical format can be prepared
from the data for ad hoc uses, but it
should never become a substitute for
tabulations. Only in this way can we
go on building up our knowledge.
Perhaps some day we shall be able to
pass all this information into a com-
puter, which will then be able to
e^rcise better judgment than the
biologist. I hope this will happen, as
computers are better able to remember
and to cope with complexity than men.
It will not, however, pension off the
biologist. He will still be needed to
collect and identify the samples.
I cannot imagine any computer wading
about on rocky riffles nor persuading
outboard motors and mechanical grabs
to operate from the unstable confines
of small boats. We shall still need
flesh and blood biologists long after the
advent of the hardware water chemist,
even though, with reference to my
earlier analogy, a Tokyo University
4-13
-------
The Interpretation of Biological Data with Reference to Water Quality
computer recently outpointed 10 veteran
medicals in diagnosing brain tumors and
heart disease. It should be pointed out,
however, that the computer still had to be
fed with information, so we are still
a long way from the hardware general
practitioner. I believe though that he is
likely to evolve before the hardware
biologist; after all, he studies only one
animal.
REFERENCES
Albrecht, M. L. Die Wirkung der
Kaliabwasser auf die Fauna der
Werra and Wipper. Z. Fisch. N.
3:401-26. 1954.
F.
2 Allanson, B. R. Investigations into the
ecology of polluted inland waters in
the Transvaal. Part I. Hydrobiologia
18:1-94. 1961.
3 Bartsch, A. F. and Ingram, W. M.
Biological Analysis of Water Pollution
in North America. Verh. Internat.
Verein. Limnol. 16:788-800. 1968.
4 Beak, T. W., de Courval, C. and
Cooke, N. E. Pollution monitoring
and prevention by use of bivariate
control charts. Sew. Industr.
Wastes 31:1383-94. 1959.
5 Beck, W. M. , Jr. The Use and Abuse of
Indicator Organisms. Transactions
of a Seminar on Biological Problems
in Water Pollution. Cincinnati. 1957.
6 Burlington, R. F. Quantitative Biological
Assessment of Pollution. J. Wat.
Poll. Contr. Fed. 34:179-83. 1962.
7 Butcher, R. W. The Biological Detection
of Pollution. J. Inst. Sew. Purif.
2:92-7. 1946.
8 Cairns, John, Jr. et al. A Preliminary
Report on Rapid Biological Information
Systems for Water Pollution Control.
JWPCF. 42(5):685-703. 1970.
9 Caspers, H. and Schulz, H. Studien
zur Wertung der Saprobiensysteme.
Int. Rev. ges. Hydrobiol. 45:535-65.
1960.
10 Dean. J. M. and Burlington, R. F.
A Quantitative Evaluation of Pollution
Effects on Stream Communities.
Hydrobiologia 21:193-9. 1963.
11 Ferdjingstad, E. Taxonomy and
Saprobic Valency of Benthic Phyto-
microorganisms. Inter. Revue der
Ges. Hydrobiol. 50(4):475-604. 1965.
12 Ferdjingstad, E. Pollution of Streams
Estimated by Benthal Phytomicro-
organisms. I. A System Based on
Communities of Organisms and
Ecological Factors. Int. Revue ges.
Hydrobiol. 49:63-131.
13 Gaufin, A. R. The Effects of Pollution
on a Midwestern Stream. Ohio J.
Sci. 58:197-208. 1958.
14 Gaufin, A. R. and Tarzwell, C. M.
Aquatic Invertebrates as Indicators
of Stream Pollution. Pub. Hlth.
Rep. 67:57-64. 1952.
15 Hairston, N. G. Species Abundance and
Community Organization. Ecology
40:404-15. 1959.
16 Harrison, A. D. The Effects of Sulphuric
Acid Pollution on the Biology of
Streams in the Transvaal, South
Africa, Verh. Int. Ver. Limnol.
13:603-10. 1958.
17 Harrison, A. D. The role of River Fauna
in the Assessment of Pollution.
Cons. Sci. Afr. Sud Sahara Pub.
64:199-212. 1960.
18 Hirsch, A. Biological Evaluation of
Organic Pollution of New Zealand
Streams. N.Z. J. Sci. 1:500-53.
1958.
19 Hynes, H. B. N. The Biology of
Polluted Waters. Liverpool. 1960.
20 Hynes, H. B. N. The Effect of Sheep-
dtp Containing the Insecticide BHC
on the Fauna of a Small Stream.
Ann. Trop. Med. Parasit.
55:192-6. 1961.
21 Hynes, H. B. N. and Roberts, F.W.
The Biological Effects of Detergents
in the River Lee, Hertfordshire.
Ann. Appl. Biol. 50:779-90. 1962.
22 Hynes, H. B. N. and Williams, T. R.
The Effect of DDT on the Fauna of
a Central African Stream. Ann. Trop.
Med. Parasit. 56:78-91. 1962.
23 lilies, J. Die Lebensgemeinschaft des
Bergbaches. Wittenberg-Luther stadt.
1961a.
4-14
-------
The Interpretation of Biological Data with Reference to Water Quality
24 lilies, J. Versuch einer allgemeiner
biozonotischen Gliederung der
Fliessgewasser. Int. Rev. ges
Hydrobiol. 46:205-13. 1961b.
25 Ingram, W. M., Mackenthun, K. M., and
Bartsch, A. F. Biological Field
Investigative Data for Water Pollution
Surveys. USDI, FWPCA Pub. WP-13,
139 pages. 1966.
26 Kaiser, E. W. Biolgiske, biokemiske,
bacteriologiske samt hydrometriske
undersogelser af Poleaen 1946 og
1947. Dansk. Ingenforen. Skr.
3:15-33. 1951.
27 Keup, Lowell E., Ingram, W. M. , and
Mackenthun, K. M. Biology of Water
Pollution. USDI. FWPCA CWA-3,
290 pages. 1967.
28 Kolkwitz, R. Oekologie der Saprobien.
Uber die Beziehungen der Wasser-
organismen zur Ummelt. Schr.
Reihe ver Wasserhyg. 4:64 pp. 1950.
29 Liebmann, H. Handbuch der Frischwasser
und Abwasserbiologie. Munich. 1951.
30 Maciel, Norma C. Levantamento
hipotetico de um rio com rede
Surber. Inst. de Engenharia Sanitaria,
Rio de Janeiro, Brazil. Pub. No. 58,
96 pages. 1969. (Zones of pollution
in a Brazilian river.)
31 Mackenthun, K. M. The Practice of
Water Pollution Biology. USDI.
FWPCA. 281 pp. 1969.
32 Needham, P. R. and Usinger, R. L.
Variability in the Macrofauna of a
Single Riffle in Prosser Creek,
California, as indicated by the Surber
Sampler. Hilgardia 24:383-409. 1956.
33 Olson, Theodore A., and Burgess, F. J.
Pollution and Marine Ecology. Inter-
science Publishers. 364 pages. 1967.
34 Patrick, R. A Proposed Biological
Measure of Stream Conditions, based
on a Survey of the Conestoga Basin,
Lancaster County, Pennsylvania.
Proc. Acad. Nat. Sci. Phila.
101:277-341. 1949.
35 Patrick, R. A Study of the Numbers and
Kinds of Species found in Rivers in
Eastern United States. Proc. Acad.
Nat. Sci. Phila. 113:215-58. 1951.
36 Patrick, R., Hohn, M. H. and Wallace,
J. H. A New Method for Determining
the Pattern of the Diatom Flora.
Not. Nat. Phila. Acad. Sci. 259.
12 pp. 1954.
37 Patrick, Ruth. Benthic Stream Com-
munities. Amer. Sci. 58:546-549.
1970.
38 Richardson, R. E. The Bottom Fauna of
the Middle Illinois River, 1913-1925;
Its Distribution, Abundance, Valuation
and Index Value in the Study of Stream
Pollution. BuH. 111. Nat. Hist. Surv.
17:387-475. 1929.
39 Sinclair, Ralph M., and Ingram,
William M. A New Record for the
Asiatic Clam in the United States--
The Tennessee River. Nautilus
74(3):114-118. 1961. (A typical
benthos faunal list for a large inland
unpolluted river, with an eroding
substrate.)
40 Sladecek, Vladimir. Water Quality
System. Verh. Internat. Verein.
Limnol. 16:809-816. 1966.
41 Sladecek, V. Zur biologischen
Gliederung der hoheren Saprobi-
tatsstufen. Arch. Hydrobiol.
58:103-21. 1961.
42 Sladecek, Vladimir. The Ecological and
Physiological Trends in the Sapro-
biology. Hydrobiol. 30:513-526.
1967.
43 Tumpling, W. V. Probleme, Methoden
und Ergenbnisse biologischer
Guteuntersuchungen an Vorflutern,
dargestellt am Beispiel der Werra.
45:513-34. 1960.
44 Whipple, G. C. , Fair, G. M. and
Whipple, M. C. The Microscopy of
Drinking Water. New York. 1947.
45 Woodiwiss, F. S. The Biological System
of Stream Classification used by the
Trent River Board. Chem. and Ind. ,
pp. 443-447. March 1964.
46 Wurtz, C. B. and Dolan, T. A Biological
Method Used in the Evaluation of Effects
of Thermal Discharge in the Schuylkill
River. Proc. Ind. Waste Conf. Purdue.
461-72. 1960.
4-15
-------
The Interpretation of Biological Data with Reference to Water Quality
47 Zimmerman, P. Der Einfluss auf die
Zusammensetzung der Lebensgemein-
schaften in Experiment. Schweiz. Z.
Hydrol. 24:408-11. 1962.
48 Hynes, H. B. N. The Ecology of Flowing
Waters in Relation to Management.
JWPCF. 42(3):418-424. 1970.
49 Hynes, H. B. N. The Ecology of Running
Waters. Univ. of Toronto Press. 555 pp.
1970.
50 Scott, Ralph D. The Macro-invertebrate
Biotic Index - A Water Quality Measure-
ment and Natural Continuous Stream
Monitor for the Miami River Basin.
17 pp. The Miami Conservancy District,
Dayton, OH 45402. 1969.
51 Cooke, Norman E. Stream Surveys
Pinpoint Pollution. Industrial Water
Engineering, p. 31-33. Sept. 1970.
This outline was prepared by Dr. H.B.N.
Hynes, Chairman, Department of Biology,
University of Waterloo, Ontario, Canada.
Reprinted from: Symposium Environmental
Measurements Valid Data and Logical
Interpretation, July 1964, PHS Publication
No. 999-AP-15, pp. 289-298.
Figures, tables, additional references, and
headings are editorial changes by R. M.
Sinclair, Aquatic Biologist, National Training
Center, MPOD, OWPO, USEPA., Cincinnati,
Ohio 45268.
Descriptors: Aquatic Life, Benthos, Water
Quality, Environmental Effects, Biological
Indices
4-16
-------
APPLICATION OF BIOLOGICAL DATA
I ECOLOGICAL DATA HAS TRADITIONALLY
BEEN DIVIDED INTO TWO GENERAL
CLASSES:
A Qualitative - dealing with the taxonomic
composition of communities
B Quantitative - dealing with the population
density or rates of processes occurring
in the communities
Each kind of data has been useful in its own
way.
II QUALITATIVE DATA
A Certain species have been identified as:
1 Clean water (sensitive) or oligotrophic
2 Facultative, or tolerant
3 Preferring polluted regions
(see: Fjerdinstad 1964, 1965; Gaufin
& Tarzwell 1956; Palmer 1963, 1969;
Rawson 1956; Teiling 1955)
B Using our knowledge about ecological
requirements the biologist may compare
the species present
1 At different stations in the same river
(Gaufin 1958) or lake (Holland 1968)
2 In different rivers or lakes (Robertson
and Powers 1967)
or changes in the species in a river or/lake
over a period of several years. (Carr
& Hiltunen 1965; Edmondson & Anderson
1956; Fruh, Stewart, Lee & Rohlich 1966;
Hasler 1947).
C Until comparatively recent times taxonomic
data were not subject to statistical treat-
ment.
Ill QUANTITATIVE DATA; Typical
Parameters of this type include:
2
A Counts - algae/ml; benthos/m ;
fish/net/day
3
B Volume - mm algae/liter
C Weight - dry wgt; ash-free wgt.
D Chemical content - chlorophyll;
carbohydrate; ATP; DNA; etc.
E Calories (or caloric equivalents)
F Processes - productivity; respiration
IV Historically, the chief use of statistics
in treating biological data has been in the
collection and analysis of samples for these
parameters. Recently, many methods have
been devised to convert taxonomic data into
numerical form to permit:
A Better communication between the
biologists and other scientific disciplines
B Statistical treatment of taxonomic data
C In the field of pollution biology these
methods include:
1 Numerical ratings of organisms on the
basis of their pollution tolerance
(saprobic valency: Zelinka & Sladecek
1964)
(pollution index: Palmer 1969)
2 Use of quotients or ratios of species in
different taxonomic groups (Nygaard
1949)
BI.EN. 3a. 6.76
5-1
-------
Application of Biological Data
3 Simple indices of community diversity:
a Organisms are placed in taxonomic
groups which behave similarly under
the same ecological conditions. The
number of species in these groups
found at "healthy" stations is com-
pared to that found at "experimental"
stations. (Patrick 1950)
b A truncated log normal curve is
plotted on the basis of the number
of individuals per diatom species.
(Patrick, Hohn, & Wallace 1954)
c Sequential comparison index.
(Cairns, Albough, Busey & Chanay
1968). In this technique, similar
organisms encountered sequentially
are grouped into "runs".
SCI =
runs
total organisms examined
d Ratio of carotenoids to chlorophyll
in phytoplankton populations:
°D430/°D665(Margalef 1968)
OD670(Tanaka, et al 1961)
e The number of diatom species present
at a station is considered indicative
of water quality or pollution level.
(Williams 1964)
number of species (S)
number of individuals (N)
number of species (S)
square root of number of individuals (>J N)
" r~
log^
j Information theory:
The basic equation used for
information theory applications was
developed by Margalef (1957).
N!
N ! N, !. . . N !
where I - information/in dividual;
N , N . . . N are the number of
individuals in species a, b, ...
s, and N is their sum.
This equation has also been used
with:
1) The fatty acid content of algae
(Mclntire, Tinsley, and Lowry
1969)
2) Algal productivity (Dickman 1968)
3) Benthic bio mass (Wilhm 1968)
N
(Menhinick 1964)
d =
En. (n. - 1) (Simpson 1949)
where n. = number of individuals
belonging to the i-th species,
and
N = total number of individuals
REFERENCES
1 Cairns, J., Jr., Albough, D.W.,
Busey, F, and Chaney, M.D.
The sequential comparison index -
a simplified method for non-biologists
to estimate relative differences in
biological diversity in stream pollution
studies. J. Water Poll. Contr. Fed.
40(9):1607-1613. 1968.
2 Carr, J.F. and Hiltunen, J.K. Changes
in the bottom fauna of Western Lake
Erie from 1930 to 1961. Limnol.
Oceanogr. 10(4):551-569. 1965.
3 Dickman, M. Some indices of diversity.
Ecology 49(6):1191-1193. 1968.
5-2
-------
Application of Biological Data
4 Edmondson, W.T. and Anderson, G. C.
Artificial Eutrophication of Lake
Washington. Lininol. Oceanogr.
l(l):47-53. 1956.
5 Fjerdingstad, E. Pollution of Streams
estimated by benthal phytomicro-
organisms. I. A saprobic system
based on communities of organisms
and ecological factors. Internat'l
Rev. Ges. Hydrobiol. 49(1):63-131.1964.
6 Fjerdingstad, E. Taxonomy and saprobic
valency of benthic phytomicro-
organisms. Hydrobiol. 50 (4):475-604.
1965.
7 Fruh, E.G., Stewart, K.M., Lee, G.F.
and Rohlich, G.A. Measurements of
eutrophication and trends. J. Water
Poll. Contr. Fed. 38(8):1237-1258.
1966.
8 Gaufin, A.R. Effects of Pollution on a
midwestern stream. Ohio J. Sci.
58(4):197-208. 1958.
9 Gaufin, A.R. and Tarzwell, C.M. Aquatic
macroinvertebrate communities as
indicators of organic pollution in Lytle
Creek. Sew. Ind. Wastes. 28(7):906-
924. 1956.
10 Hasler, A.D. Eutrophication of lakes by
domestic drainage. Ecology 28(4):383-
395. 1947.
11 Holland, R.E. Correlation of Melosira
species with trophic conditions in Lake
Michigan. Limnol. Oceanogr.
13(3):555-557. 1968.
12 Margalef, R. Information theory in
ecology. Gen. Syst. 3:36-71. 1957. .
13 Margalef, R. Perspectives in ecological
theory. Univ. Chicago Press. 1968.
14 Mclntire, C.D., Tinsley, I.J. and
Lowry, R.R. Fatty acids in lotic
periphyton: another measure of
community structure. J. Phycol.
5:26-32. 1969.
15 Menhinick, E.F. A comparison of some
species - individuals diversity indices
applied to samples of field insects.
Ecology 45:859. 1964.
16 Nygaard, G. Hydrobiological studies in
some ponds and lakes. II. The
quotient hypothesis and some new or
little-known phytoplankton organisms.
Klg. Danske Vidensk. Selsk. Biol.
Skrifter 7:1-293. 1949.
17 Patten, B.C. Species diversity in net
plankton of Raritan Bay. J. Mar.
Res. 20:57-75. 1962.
18 Palmer, C.M. The effect of pollution on
river algae. Ann. New York Acad.
Sci. 108:389-395. 1963.
19 Palmer, C.M. A composite rating of
algae tolerating organic pollution.
J. Phycol. 5(l):78-82. 1969.
20 Patrick, R., Hohn, M.H. and Wallace,
J.H. A new method for determining
the pattern of the diatom flora. Not.
Natl. Acad. Sci., No. 259.
Philadelphia. 1954.
21 Rawson, D.S. Algal indicators of trophic
lake types. Limnol. Oceanogr.
1:18-25. 1956.
22 Robertson, S. and Powers, C.F.
Comparison of the distribution of
organic matter in the five Great Lakes.
in: J. C. Ayers and D. C. Chandler,
eds. Studies on the environment and
eutrophication of Lake Michigan.
Spec. Rpt. No. 30, Great Lakes Res.
Div.,Inst. Sci. &Techn., Univ.
Michigan, Ann Arbor. 1967.
23 Simpson, E.H. Measurement of diversity.
Nature (London) 163:688. 1949.
24 Tanaka, O. H., Irie, S. Izuka, and Koga, F
The fundamental investigation on the
biological productivity in the Northwest
of Kyushu. I. The investigation of
plankton. Rec. Oceanogr. W. Japan,
Spec. Rpt. No. 5, 1-57. 1961.
5-3
-------
Application of Biological Data
25 Teiling, E. Some mesotrophic phyto-
plankton indicators. Proc. Intern.
Assoc. Limnol. 12:212-215. 1955.
26 Wilhm, J. L. Comparison of some
diversity indices applied to populations
of benthic macroinvertebrates in a
stream receiving organic wastes. J.
Water Poll. Contr. Fed. 39(10):1673-1683.
1967.
27 Wilhm, J. L. Use of biomass units in
Shannon's formula. Ecology 49:153-156.
1968.
28 Williams, L.G. Possible relationships
between diatom numbers and water
quality. Ecology 45(4):810-823. 1964.
29 Zelinka, M. and Sladecek, V. Hydro-
biology for water management.
State Publ. House for Technical
Literature, Prague. 122 p. 1964.
This outline was prepared by C.I. Weber,
Chief, Biological Methods Section, Analytical
Quality Control Laboratory, NERC, EPA,
Cincinnati, Ohio, 45268
Descriptors: Analytical Techniques, Indicators
5-4
-------
OPTICS AND THE MICROSCOPE
I OPTICS
An understanding of elementary optics is
essential to the proper use of the microscope.
The microscopist will find that unusual pro-
blems in illumination and photomicrography
can be handled much more effectively once
the underlying ideas in physical optics are
understood.
A Reflection
A good place to begin is with reflection at
a surface or interface. Specular (or
regular) reflection results when a beam
of light leaves a surface at the same angle
at which it reached it. This type of
reflection occurs with highly polished
smooth surfaces. It is stated more pre-
cisely as Snell's Law, i. e., the angle of
incidence, i, is equal to the angle of
reflection, r (Figure l) • Diffuse (or
scattered) reflection results when a beam
of light strikes a rough or irregular sur-
face and different portions of the incident
light are reflected from the surface at
different angles. The light reflected from
a piece of white paper or a ground glass is
an example of diffuse reflection.
Figure 1
SPECULAR REFLECTION - SNELL'S
LAW
BI. MIC. 18.2.79
Strictly speaking, of course, all reflected
light, even diffuse, obeys Snell's Law.
Diffuse reflected light is made up of many
specularly reflected rays, each from a
a tiny element of surface, and appears
diffuse when the reflecting elements are
very numerous and very small. The terms
diffuse and .specular, referring to reflection,
describe not so much a difference in the
nature of the reflection but rather a differ-
ence in the type of surface. A polished sur-
face gives specular reflection; a rough
surface gives diffuse reflection.
It is also important to note and remember
that specularly reflected light tends to be
strongly polarized in the plane of the reflect-
ing surface. This is due to the fact that
those rays whose vibration directions lie
closest to the plane of the reflection surface
are most strongly reflected. This effect is
strongest when the angle of incidence is
such that the tangent of the angle is equal
to the refractive index of the reflecting sur-
face. This particular angle of incidence is
called the Brewster angle.
B Image Formation on Reflection
Considering reflection by mirrors, we find
(Figure 2) that a plane mirror forms a
virtual image behind the mirror, reversed
right to left but of the same size as the
object. The word virtual means that the
image appears to be in a given plane but
that a ground glass screen or a photographic
film placed in that plane would show no
image. The converse of a virtual image is
a real image.
Spherical mirrors are either convex or con-
cave with the surface of the mirror repre-
senting a portion of the surface of a sphere.
The center of curvature is the center of the
sphere, part of whose surface forms the
mirror. The focus lies halfway between the
center of curvature and the mirror surface.
6-1
-------
Optics and the Microscope
Object
Virtuol
Image
Mirror
Figure 2
IMAGE FORMATION BY PLANE MIRROR
Construction of an image by a concave
mirror follows from the two premises
given below (Figure 3):
Figure 3
IMAGE FORMATION BY CONCAVE MIRROR
1 A ray of light parallel to the axis of
the mirror must pass through the
focus after reflection.
2 A ray of light which passes through the
center of curvature must return along
the same path.
A corollary of the first premise is:
3 A ray of light which passes through the
focus is reflected parallel to the axis
of the mirror.
The image from an object can be located
using the familiar lens formula:
JL
P
1
q
where p = distance from the object to
the mirror
q = distance from the image to
the mirror
f = focal length
C Spherical Aberration
No spherical surface can be perfect in its
image-forming ability. The most serious
of the imperfections, spherical aberration,
occurs in spherical mirrors of large
aperture (Figure 4). The rays of light
making up an image point from the outer
zone of a spherical mirror do not pass
through the same point as the more central
rays. This type of aberration is reduced by
blocking the outer zone rays from the image
area or by using aspheric surfaces.
Figure 4
SPHERICAL ABERRATION BY
SPHERICAL MIRROR
6-2
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Optics and the Microscope
D Refraction of Light
Turning now to louses rather than mirrors
we find that the most important character-
istic is refraction. Refraction refers to
the change of direction and/or velocity of
light as it passes from one m.-dium to
another. The ratio of the velocity in air
(or more correctly in a vacuum) to UK-
velocity in the medium is called tin-
refractive index. Some- typical values of
refractive index measured with mono-
chromatic light (sodium D line) are listed
in Table 1.
Refraction causes an object immi-rsed in
a medium of higher refractive index than
air to appear closer to the surface than it
actually is (Figure 5). This effect may
into focus and the new micrometer reading
is taken. Finally, the microscope is re-
focused until the surface of the liquid appears
in sharp focus. The micrometer reading
is taken again and, with this information,
tho refractive index rmy be calculated from
the simplified equation:
refractive index =
actual depth
apparent depth
Table 1. KKFRACTIVK INDICES OF COMMON
MATERIALS MKASURED WITH SODIUM LIGHT
Vacuum
Air
CO,
Water
1. 0000000
1. 0002918
1. 0004498
i. :mo
Crown glass
Rock salt
Diamond
Lead sulfide
1.48 to
1. 5443
2.417
3.912
1. 61
Actual
depth
Apparent
depth
Air
Medium
Image
Object
When the situation is reversed, and a ray
of light from a medium of high refractive
index passes through the interface of a
medium of lower index, the ray is refracted
until a critical angle is reached beyond which
all of the light is reflected from the interface
(Figure 6). This critical angle, C, has the
following relationship to the refractive indices
of the two media: {
sin C = —2 , where no
-------
Optics and the Microscope
£ Dispersion
Dispersion is another important property
of transparent materials. This is the
variation of refractive index with color
(or wavelength) of light. When white light
passes through a glass prism, the light
rays are refracted by different amounts
and separated into the colors of the
spectrum. This spreading of light into
its component colors is due to dispersion
which, in turn, is due to the fact that the
refractive index of transparent substances,
liquids and solids, is lower for long wave-
lengths than for short wavelengths.
Because of dispersion, determination of
the refractive index of a substance re-
quires designation of the particular wave-
length used. Light from a sodium lamp
has a strong, closely spaced doublet with
an average wavelength of 5893A, called
the D line, which is commonly used as a
reference wavelength. Table 2 illustrates
the change of refractive index with wave-
length for a few common substances.
F Lenses
There are two classes of lenses, con-
verging and diverging, called also convex
and concave, respectively. The focal
point of a converging lens is defined as
the point at which a bundle of light rays
parallel to the axis of the lens appears to
converge after passing through the lens.
The focal length of the lens is the distance
from the lens to the focal point (Figure 7).
Table 2. DISPERSION OF REFRACTIVE
INDICES OF SEVERAL COMMON MATERIALS
Refractive index
F line D line C line
blue (yellow) (red)
4861A 5893A 6563A
Carbon disulfide
Crown glass
Flint glass
Water
1.
1.
1.
• 1.
6523
5240
6391
3372
1.
1.
1.
1.
6276
5172
6270
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 (£lue) . n (red)
n (593mn) - 1
n (486mji) - n(656mjx)
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.
6-4
-------
Optics and the Microscope
Figure.' 8
IMAGE FORMATION BY A CONVEX l.ENS
The magnification, M, of an image of an
object produced by a lens is given by the
relationship:
image size _ image distance _ q
object size object distance p
where q = distance from image to lens
and p = distance from object to lens.
H Aberrations of Lenses
Lenses have aberrations of several types
which, unless corrected, cause loss of
detail in the image. Spherical aberration
appears in lenses with spherical surfaces.
Reduction of spherical aberration can be
accomplished by diaphragming the outer
zones of the lens or by designing special
aspherical surfaces in the lens system.
Chromatic aberration is a phenomenon
caused by the variation of refractive index
with wavelength (dispersion). Thus a lens
receiving white light from an object will
form a violet image closer to the lens and
a red one farther away. Achromatic
lenses are employed to minimize this
effect. The lenses are combinations of
two or more lens elements made up of
materials having different dispersive
powers. The use of monochromatic light
is another obvious way of eliminating
chromatic aberration.
Astigmatism is a third aberration of
spherical lens systems It occurs when
object points are not located on the optical
axis of the lens and results in the formation
of an indistinct image. The simplest
remedy for astigmatism is to place the
object close to the axis of the lens system.
I Interference Phenomena
Interference and diffraction are two phe-
nomena which are due to the wave character-
istics of light. The superposition of two
light rays arriving simultaneously at a given
point will give rise to interference effects,
whereby the intensity at that point will vary
from dark to bright depending on the phase
differences between the two light rays.
The first requirement for interference is
that the light must come from a single
source. The light may be split into any
number of paths but must originate from
the same point (or coherent source). Two
light waves from a coherent source arriv-
ing at a point in phase agreement will
reinforce each other (Figure 9a). Two
light waves from a coherent source arriv-
ing at a point in opposite phase will cancel
each other (Figure 9b).
Figure 9a.
Two light rays, 1 and 2, of
the same frequency but dif-
ferent amplitudes, are in phase
in the upper diagram. In the
lower diagram, rays 1 and 2
interfere constructively to give
a single wave of the same fre-
quency and with an amplitude
equal to the summation of the
two former waves.
6-5
-------
Optics and the Microscope
o+b
Figure 9b. Rays 1 and 2 are no.v 180°
out of phase and interfere
destructively. The resultant,
in the bottom diagram, is of
the same frequency but is of
reduced amplitude (a is
negative and is subtracted
from b).
The reflection of a monochromatic light
beam by a thin film results in two beams,
one reflected from the top surface and one
from the bottom surface. The distance
traveled by the latter beam in excess of
the first is twice the thickness of the film
and its equivalent air path is:
2 nt
where n is the refractive index and
t is the thickness of the film.
The second beam, however, upon reflection
at the bottom surface, undergoes a half
wavelength shift and now the total retard-
ation of the second beam with respect to
the first is given as:
retardation = 2 nt + -^-
where X is the wavelength of the light
beam.
When retardation is exactly an odd number
of half wavelengths, destructive interfer-
ence takes place resulting in darkness.
When it is zero or an even number of half
wavelengths, constructive interference
results in brightness (Figure 10).
6-6
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^MFigure 11).
Mirror/^
-Cover slip-
Spccimen
\
- * ..
•i
\ /\ / x"IOO%
V X?-1 /Mirror
f *
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.
-------
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:
d =
nm(l
ex
2.44 fx
D
where f is the focal length of the lens,
X the wavelength, and D the diameter
of the lens.
It is seen that in order to maintain a
small diffraction disc at a given wave-
length, the diameter of the lens should
be as large as possible with respect to
the focal length. It should be noted,
also, that a shorter wavelength produces
a smaller disc.
If two pin points of light are to be distin-
guished in an image, their diffraction discs
must not overlap more than one half their
diameters. The ability to distinguish such
image points is called resolving power and
is expressed as one half of the preceding
expression:
resolving power =
360t
1. 22 f X
D
where ns = refractive index of the
specimen
n - refractive index of the
surrounding medium
6 = phase shift of the two
beams, degrees
X = wavelength of the light
t = thickness of the specimen.
J Diffraction
In geometrical optics, it is assumed that
light travels in straight lines. This is not
always true. We note that a beam passing
through a slit toward a screen creates a
bright band wider than the slit with alter-
nate bright and dark bands appearing on
either side of the central bright band,
decreasing in intensity as a function of
the distance from the center. Diffraction
describes this phenomenon and, as one of
its practical consequences, limits the
lens in itF 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
6-7
-------
Optics and the Microscope^
Ey«
VIRTUAL IMAGE FORMATION BY
CONVEX LENS
the object appears to be 10 inches from
the eye, where it is best accommodated.
B Magnification by a Single Lens System
The magnification, M, of a simple magni-
fying glass is given by:
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
Ob)*cttv*
V Mud
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.
6-8
-------
Optics and the Microscopy
Table 3. NOMINAL CHARACTERISTICS OF USUAL
Nominal
focal length
mm
56
32
16
8
4
4
1.8
Nominal
magnif.
2. 5X
5
10
20
43
45
90
N. A.
0. 08
0. 10
0. 25
0.50
0. 66
0. 85
1. 30
Working
distance
mm
40
25
7
1. 3
0. 7
0.5
0.2
Depth
focus
^
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, JJL
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,
SOX
2 OX
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 objectiva on the nosepiece need be
centered only once to the fixed rotating
stage. The insides of objectives are
better protected from dust by the rotating
nosepiece. This, as well as the incon-
venience of the so-called "quick-change"
objective holder, makes it worthwhile
to have one's microscope fitted with
rotating nosepiece.
D The Ocular
The eyepiece, or ocular, is necessary in
the second step of the magnification process.
The eyepiece functions as a simple magni-
fier viewing the image formed by the
objective.
There are three classes of eyepieces in
common use: huyghenian, compensating
and flat-field. The huyghenian (or huyghens)
eyepiece is designed to be used with
achromats while the compensating type is
used with fluorite and apochromatic
objectives. Flat-field eyepieces, as the
name implies, are employed in photo-
micrography or projection and can be used
with most objectives. It is best to follow
the recommendations of the manufacturer
as to the proper combination of objective
and eyepiece.
6-9
-------
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 eyepoint. The
most useful eyepieces lie in the 10 to 20X
magnification range.
E Magnification of the Microscope
The total magnification of the objective-
eyepiece combination is simply the product
of the two individual magnifications. A
convenient working rule to assist in the
proper choice of eyepieces states that the
maximum useful magnification (MUM) for
the microscope is 1, 000 times the numeri-
cal aperture (N. A.) of the objective.
The MUM is related to resolving power
in that magnification in excess of MUM
gives little or no additional resolving
power and results in what is termed empty
magnification. Table 4 shows the results
of such combinations and a comparison
with the 1000XN.A. rule. The under-
lined figure shows the magnification near-
est to the MUM and the eyepiece required
with each objective to achieve the MUM.
From this table it is apparent that only
higher power eyepieces can give full use
of the resolving power of the objectives.
It is obvious that a 10X, or even a 15X,
eyepiece gives insufficient magnification
for the eye to see detail actually resolved
by the objective.
F Focusing the Microscope
The coarse adjustment is used to roughly
position the body tube (in some newer
microscopes, the stage) to bring the image
into focus. The fine adjustment is used
after the coarse adjustment to bring the
image into perfect focus and to maintain
the focus as the slide is moved across the
stage. Most microscope objectives are
parfocal so that once they are focused any
other objective can be swung into position
without the necessity of refocusing except
with the fine adjustment.
The student of the microscope should first
learn to focus in the following fashion, to
prevent damage to a specimen or objective:
1 Raise the body tube and place the speci-
men on the stage.
2 Never focus the body tube down (or the
stage up) while observing the field
through the eyepiece.
3 Lower the body tube (or raise the stage)
with the coarse adjustment while care-
fully observing the space between the
Table 4. MICROSCOPE MAGNIFICATION CALCULATED
FOR VARIOUS OBJECTIVE-EYEJflECE 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
SOX
100X
200X
400X
900X
Eyepiece
15X
45X
75X
150X
300X
600X
135 OX
20X
60X
100X
200X
40 OX
800X
1800X
25X
75X
125X
25 OX
500X
1000X
2250X
MUMa
(1000 NA)
SOX
100X
250X
500X
660X
1250X
aMUM = maximum useful magnification
6-10
-------
Optics and the Microscope
objective and slide and permitting the
two to come close together without
touching.
4 Looking through the microscope and
turning the fine adjustment in such a
way as to move the objective away from
the specimen, bring the image into
sharp focus.
The fine adjustment is usually calibrated
in one- or two-micron steps to indicate
the vertical movement of the body tube.
This feature is useful in making depth
measurements but should not be relied
upon for accuracy.
G The Substage Condenser
The substage holds the condenser and
polarizer. It can usually be focused in a
vertical direction so that the condenser can
be brought into the correct position with
respect to the specimen for proper
illumination. In some models, the conden-
ser is 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 Lens-
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
6-11
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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. micromanipulation.
Ill ILLUMINATION AND RESOLVING POWER
Good resolving power and optimum specimen
contrast are prerequisites for good microscopy.
Assuming the availability of suitable optics
(ocular, objectives and substage condenser)
it is still of paramount importance to use
proper illumination. The requirement for a
good illumination system for the microscope
is to have uniform intensity of illumination
over the entire field of view with independent
control of intensity and of the angular aperture
of the illuminating cone.
A Basic Types of Illumination
There are three types of illumination
(Table 5) used generally:
1 Critical. This is used when high levels
of illumination intensity are necessary
for oil immersion, darkfield, fluores-
cence, low birefringence or photo-
micrographic studies. Since the lamp
filament is imaged in the plane of the
specimen, a ribbon filament or arc
lamp is required. The lamp must be
focusable and have an iris diaphragm;
the position of the filament must also
be adjustable in all directions.
2 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
6-12
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Optics and the Microscope
Table 5. COMPARISON OF CRITICAL,
KOHLER AND JfOOR MAN'S ILLUMINATION
Critical
Kohler
Poor man's
Lamp filament
Lamp condensing lens
Lamp iris
Ground glass at lamp
Image of light source
Image of field iris
Image of substage iris
ribbon filament
required
required
none
in object plane
near object
plane
back focal plane
of objective
any type
required
required
none
at substage
iris
in object
plane
back focal plane
of objective
any type
none
useful
present
none
near object
plane
back focal plane
of objective
the entire field of view is always
illuminated. If the surface structure of the
ground glass becomes apparent in the field
of view the substage condenser is very
slightly defocused.
Critical Illumination
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 lamp filament in the object plane. The
substage condenser iris will now be found
imaged in the back focal plane of the ob-
jective; it serves as a control over con-
vergence of the illumination. Although the
substage iris also affects the light intensity
over the field of view it should most decid-
edly not be used for this purpose. The
intensity of illumination may be varied by
the use of neutral density filters and, unless
color photomicrography is anticipated, by
the use of variable voltage on the lamp
filament.
u
Kohler illumination (Figure 14) differs
from critical illumination in the use of the
lamp condenser. With critical illumination
the lamp condenser focuse^ 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.
It
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
6-13
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Optics and the Microscope
Critical
Kohler
Eye
Eyepoint
Ocular
Focal plane
Focal plane
Objective
Preparation
Substage
condenser
Substage —•
iris
Lamp iris —j
Lamp
condenser
Light source
Poor man's
6-14
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Optics and the Microscope
substage condenser opening. If it
does not, move the lamp away from
the wall to enlarge the filament image;
refocus.
d Turn the lamp and aim it a I the micro-
scope mirror so as to maintain the
same 18 inches (or adjusted lamp
distance).
e Place a specimen on the microscope
stage and focus sharply with a l(i-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 buib base and
slide it up or down. If the base is
fixed, tilt the lamp housing in a
vertical arc with the field diaphragm
as the center of movement (again
endeavoring to keep the lamp dia-
phragm in the centered position).
If you have mastered this step, you
have accomplished the mast difficult
portion. (Belter microscope lamps
have adjustments to move the bulb
independently of the lamp housing to
simplify this step. )
j Put the specimen in place, replace
the eyepiece and the desired objec-
tive and re-focus.
k Open or close the field diaphragm
until it just disappears from the field.
1 Observe the back aperture of the
objective, preferably with the Bertrand
lens or the.auxiliary telescope, and
close the aperture diaphragm on the
substage condenser until it is about
four-fifths the diameter of the back
aperture. This is the best position
for the aperture diaphragm, a posi-
tion which minimizes glare and maxi-
mizes the resolving power. It is
instructive to vary the aperture dia-
phragm and observe the image criti-
cally during the manipulation.
m If the illumination is too great,
insert an appropriate neutral density
filter between the illuminator and
the condenser. Do not use the con-
denser aperture diaphragm or the
lamp field diaphragm to control the
intensity of illumination.
Poor Man's Illumination
Both critical and Kohler illumination re-
quire expensive illuminators with adjust-
able focus, lamp iris and adjustable lamp
mounts. Poor man's illumination requires
a cheap illuminator although an expensive
illuminator may be used if its expensive
features are negated by inserting a ground
glass diffuser or by using a frosted bulb.
Admittedly an iris diaphragm on the lamp
would be a help though it is not necessary.
a The illuminator must have a frosted
bulb or a ground glass dif?user.
6-15
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Optics and the Microscope
It should bo possible to direct it in
the general direction of the substage
mirror, very close thereto or in
place thereof.
b Focus on any preparation after
tilting the mirror to illuminate the
field.
c Remove the top lens of the condenser
and, by racking the condenser up or,
more often, down, bring into focus
(in the same plane as the specimen)
a finger, pencil or other object placed
in the same general region as the
ground glass diffuser on the lamp.
The glass surface itself can then be
focused in the plane of the specimen.
d Ideally the ground glass surface will
just fill the field of view when centered
by the substage mirror; adjustment
may be made by moving the lamp
closer to or farther from the micro-
scope (the position might be marked
for each objective used) or by cutting
paper diaphragms of fixed aperture
(one for each objective used). In this
instance a lamp iris would be useful.
e Lower the condenser just sufficiently
to defocus the ground glass surface
and render the field of illumination
even.
f Observe the back aperture of the
objective and open the substage con-
denser iris about 75 percent of the
way. The final adjustment of the
substage iris is made while observing
the preparation; the iris should be
open as far as possible, still giving
good contrast.
g The intensity of illumination should
be adjusted only with neutral density
filters or by changing the lamp voltage.
Proper illumination is one of the most im-
portant operations in microscopy. It is
easy tojwdge a microscopist's ability by
a glance at his field of view and the objec-
tive back lens.
H llcsolving Power
The resolving power of the microscope is
its ability to distinguish separate details
of closely spaced microscopic structures.
The theoretical limit of resolving two
discrete points, a distance X apart, is:
„ • • •~«*rx
X 2 N.~A~.
where X - 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. 3, 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 microscope 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
—
where n - the lowest index in the space
between the object and the
objective.
Angular aperture
Object
Figure 15
ANGULAR APERTURE OF
MICROSCOPE OBJECTIVE
6-16
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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 b^e 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 cpurse, 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.
6-17
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Optics and the Microscope
IV PHOTOMICROGRAPHY
A Introduction
Photomicrography, as distinct from micro-
photography, is the art of taking pictures
through the microscope. A microphoto-
graph is a small photograph; a photomicro-
graph is a photograph of a small object.
Photomicrography is a valuable tool in
recording the results of microscopical
study. It enables the microscopist to:
1 describe a microscopic field objectively
without resorting to written descriptions.
2 record a particular field for future
reference,
3 make particle size counts and counting
analyses easily and without tying up a
microscope,
4 enhance or exaggerate the visual micro-
scopic field to bring out or emphasize
certain details not readily apparent
visually,
5 record images in ultraviolet and infra-
red microscopy which are otherwise
invisible to the unaided eye.
There are two general approaches to photo-
micrography; one requires only a plate or
film holder supported above the eyepiece
of the microscope with a light-tight bellows;
the other utilizes any ordinary camera with
its own lens system, supported with a light-
tight adaptor above the eyepiece. It is
best, in the latter case, to use a reflex
camera so that the image can be carefully
focused on the ground glass. Photomi-
crography of this type can be regarded
simply as replacing the eye with the camera
lens system. The camera should be focused
at infinity, just as the eye is for visual
observation, and it should be positioned
close to and over the eyepiece.
The requirements for photomicrography,
however, are more rigorous than those
for visual work. The eye can normally
compensate for varying light intensities,
curvature of field and depth of field. The
photographic plate, however, lies in one
plane; hence the greatest care must be
used to focus sharply on the subject plane
of interest and to select optics to give
minimum amounts of field curvature and
chromatic aberrations.
With black and white film, color filters
may be used to enhance the contrast of
some portions of the specimen while mini-
mizing chromatic aberrations of the lenses.
In color work, however, filters cannot
usually be used for this purpose and better
optics may be required.
Photomicrographic cameras which fit
directly onto the microscope are available
in 35-mm or up to 3-1/4 X 4-1/4 inch sizes.
Others are made which accommodate larger
film sizes and which have their own support
independent of the microscope. The former,
however, are preferred for ease of handling
and lower cost. The latter system is pre-
ferred for greater flexibility and versatility
and lack of vibration. The Polaroid camera
has many applications in microscopy and
can be used on the microscope directly but,
because of its weight, only when the micro-
scope has a vertically moving stage for
focusing rather than a focusing body tube.
B Determination of Correct Exposure
Correct exposure determination can be
accomplished by trial and error, by relating
new conditions to previously used successful
conditions and by photometry.
With the trial and error method a series of
trial exposures is made, noting the type of
subject, illumination, filters, objective,
eyepiece, magnification, film and shutter
speed. The best exposure is selected. The
following parameters can be changed and
the exposure time adjusted accordingly:
1 Magnification. Exposure time varies
as the square of the magnification.
Example: Good exposure was obtained
with a 1/10-second exposure
and a magnification of 100X.
If the magnification is now
6-18
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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 = ,/in /200v2 =
\>ld magnification ' ~J/1U1100'
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
v ( )2 = 1/10 (-^—;r)2 = 1/40 or,
vnewN. A. 0.50
say, 1/50 second.
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 Tri-X film at
1/100 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
6-19
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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 birefringent crystals.
One of the best solutions to this problem
is to measure the photometer reading with
no preparation on the stage. A first-order
red compensator or a quartz wedge is in-
serted when crossed polars are being used
to illuminate the entire field.
An alternative is to place the cell on the
ground glass where the film will be
located. However, although all variables
except film speed are now taken into
account, measurements in the image plane
have the disadvantage of requiring a more
sensitive electronic photoelectric apparatus.
No matter what method is used for placing
the photocell, the exposure time can be
determined by the general formula:
exposure time = meter 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 = 40 X 1/5 = 8.
Assume now that a new picture
is to be taken at another
magnification (but with the
same film and projection
distance) and that the new
meter reading is 16; therefore:
exposure time = k/meter
reading = 8/16 = 1/2 second.
V MICROMETRY
A Particle Size Determination
Linear distances and areas can be
measured with the microscope. This
permits determination of particle size
and quantitative analysis of physical
mixtures. The usual unit of length for
microscopical measurements is the micron
(1 X 10-3mm or about 4 X 10~5inch).
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
6-20
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Optics and the Microscope
particle size. There are, however, a
variety of approximate and useful proce-
dures as well.
1 Methods of particle size measurement
a Knowing the magnification of the
microscope (product of the magni-
fication of objective and eyepiece),
the size of particles can be esti-
mated. For example, with a 10X
eyepiece and a 16-mm (or 10X)
objective, the total magnification
is 100X. A particle that appears to
be 10-mm at 10 inches from the eye
has an actual size of 10 mm divided
by 100 or 0. 10 mm or 100 microns.
This is in no sense an accurate
method, but it does permit quick
estimation of particle size; the error
in this estimation is usually 10-25
percent.
b Another approximate method is also
based on the use of known data. If
we know approximately the diameter
of the microscope field, we can
estimate the percentage of the
diameter occupied by the object to
be measured and calculate from
these figures the approximate size
of the object. The size of the micro-
scope field depends on both the objec-
tive and the ocular although the latter
is a minor influence. The size of
the field should be determined with
a millimeter scale for each objective
and ocular. If this is done, esti-
mation of sizes by comparison with
the entire field diameter can be quite
accurate (5-10%).
c The movement of a graduated mechan-
ical stage can also be used for rough
measurement of diameters of large
particles. Stages are usually gradu-
ated (with vernier) to read to 0. 1
millimeter, or 100 microns. In
practice, the leading edge of the
particle is brought to one of the lines
of the cross hair in the eyepiece and
a reading is taken of the stage position.
Then the particle is moved across the
field by moving the mechanical stage
in an appropriate direction until the
second trailing edge just touches the
cross-hair line. A second reading is
taken and the difference in the two
readings is the distance moved or the
size of the particle. This method is
especially useful when the particle
is larger than the field, or when the
optics give a distorted image near the
edge of the field.
d The above method can be extended to
projection or photography. The image
of the particles can be projected on a
screen with a suitable light source or
they may be photographed. The final
magnification, M, on the projection
surface (or film plane) is given approxi-
mately by
M = DXO. M. XE. M. 125
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
6-21
-------
Optics and the Microscope
5 (0.01)
= 120 times.
This magnification figure can be
used to improve the accuracy of
method 4 above.
f The simplest procedure and the most
accurate is based on the use of a
micrometer eyepiece. Since the
eyepiece magnifies a real image
from the objective, it is possible
to place a transparent scale in the
same plane as the image from the
objective and thus have a scale
superimposed over the image. This
is done by first placing an eyepiece
micrometer scale disc in the eyepiece.
The eyepiece micrometer has an
arbitrary scale and must be cali-
brated with each objective used. The
simplest way to do this is to place
the stage micrometer on the stage
and note a convenient whole number
of eyepiece micrometer divisions.
The value in microns for each eye-
piece micrometer division is then
easily computed. When the stage
micrometer is removed and replaced
by the specimen, the superimposed
eyepiece scale can be used for accu-
rate measurement of any feature in
the specimen by direct observation,
photography or projection.
2 Calibration of eyepiece micrometer
Each micrometer stage scale has
divisions lOOn (0. 1 mm) apart; one
or two of these are usually subdivided
into 10u (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 microns per
eyepiece scale division. To make the
comparison as accurate as possible, a
large part of each scale must be used
(see Figure 17). Let's assume that
with the low power 16-mm objective
6 large divisions of the stage scale
(s. m. d.) are equal to 38 divisions of
the eyepiece scale. This means that
38 eyepiece micrometer divisions (e.m. d.)
are equivalent to 600 microns. Hence:
1 e. m. d. = 600/38
= 15. 8n.
Figure 17
COMPARISON OF STAGE MICROMETER
SCALE WITH EYEPIECE MICROMETER SCALE
Thus when that micrometer eyepiece
is used with that 16-mm objective each
division of the eyepiece scale is equivalent
to 15. 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 135u in
diameter.
Each objective on your microscope must
be calibrated in this manner.
A convenient way to record the necessary
data and to calculate u/emd is by means
of a table.
6-22
-------
Optics and the Microscope
Table 7
Objective
No. smd = ^i = \i -
no. emd no. emd 1 emd
32-mm 18 = 44 1800 = 44 40. 9^
16-mm 6 = 38 600 = 38 15. 8ji
4-mm 1 = 30 100 = 30 3. 33|ji
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).
I-P
'&
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^', page 26.
The averages^ with respect to number,
dj; surface, d$; and weight or volume,
d4, are calculated as follows for the
data in Table 9.
6-23
-------
Optics and the Microscope
TmbU 8. PARTICLE SIZE TALLY FOR A SAMPLE OF STARCH GRAINS
Six* CUM
(emd»)
Number of particles
Toul
rt-*j
rtHJ 1
n-u ri-w rr+j rt-u rt-*j
n-*4 n~u r*-»j r*-*a
rt-u r*-u 111
16
M
no
rt-u rt-*A rtna i-t-*a rt-u rt-tj
rtsu
rt-*u rtnw rtna it-w rt-u ri-*-i r-na
rt-*u rt-w rtn-i rt-w rt-*-» I-KU
rt-w
107
1 1
rt-w
r-»-*a
«-*a rt-w r»-*4 I-I-M ITHJ
rt-*j
1 1
4$
SI
s
470
*emd • excniece micremeter.dixi«iaa*
dj = Znd/Sn = 1758/470
= 3.74 emdX 2.82*= 10. 5>i
d3 = End3/End2 = 37440/7662
= 4.89 emdX 2.82 = 13.8,1
d4 = Znd4/Znd3 = 199194/37440
= 5.32 emdX 2.82 = 15. Op.
*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
for the cumulative weight or volume
curve, are plotted against d. Finally,
the specific surface, Sm, in square
meters per gram, m. may be calculated
if the density, D, is known; the surface
average d%, is used.
If D
1.1. Sm= 6/d3D= 6/13.8(1.1)
0. 395m2/g.
6-24
-------
Optics and the Microscope
Table 9. CALCULATIONS FOR PARTICLE SIZE AVERAGE
d
(Aver. diam. n
in emd)
1
2
3
4
5
6
7
8
16
98
110
107
71
45
21
2
nd
16
196
330
428
355
270
147
16
nd*
16
392
990
1712
1775
1620
1029
128
-»
16
784
2970
6848
8875
9720
7203
1024
nd4
16
1568
8910
27392
44375
58320
50421
8192
470 1758 7662 37440 199194
B Counting Analysis
Mixtures of particulates can often be
quantitatively analyzed by counting the
total number of particulates from each
component in a representative sample.
The calculations are, however, compli-
cated by three factors: average particle
size, particle shape and the density
of the components. If all of the compon-
ents were equivalent in particle size,
shape and density then the weight per-
centage would be identical to the number
percentage. Usually, however, it is
necessary to determine correction factors
to account for the differences.
When properly applied, this method can
be accurate to within _+ 1 percent and,
in special cases, even better. It is often
applied to the analysis of fiber mixtures
and is then usually called a dot-count
because the tally of fibers is kept as the
preparation is moved past a point or dot
in the eyepiece.
A variety of methods can be used to
simplify recognition of the different
components. These include chemical
stains or dyes and enhancement of optical
differences such as refractive indices,
dispersion or color. Often, however, one
relies on the differences in morphology.
e. g. counting the percent of rayon fibers
in a sample of "silk".
Example 1: A dot-count of a mixture of
fiberglass and nylon shows:
nylon
fiberglass
262
168
Therefore:
nylon = 262/(262 + 168)X 100
= 60. 9% by number.
However, although both fibers are smooth
cylinders, they do have different densities
and usually different diameters. To
correct for diameter one must measure
the average diameter of each type of fiber
and calculate the volume of a unit length
of each.
aver. diam. volume of
H l-(i slice, \i?
nylon
fiberglass
18.5
13.2
268
117
The percent by volume is, then:
262X 268
nylon =
(262 X 268)+(168X 117)
78. 1% by volume.
X 100
Still we must take into account the density of
each in order to calculate the weight percent.
6-25
-------
Optics and the Microscope
If the densities are 1. 6 for nylon and 2. 2
for glass then the percent by weight is:
„ . _ 262X268X1.6 _ _
% nylon - (262 X 268 X 1.6)+(1G8X 117X2.2) * luu
= 72% by weight.
Example 2: A count of quartz and
gypsum shows:
quartz 283
gypsum 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:
_ 283 X nd4/6XDq _
% quartz = irg4/ 6 x Dq + 467 x w dJ/6 X 0. 80 X Dg X 10°
where Dq and Dg are the densities of quartz
and gypsum_ respectively; 0. 80 is the shape
factor and d4 and d| are the average parti-
cle sizes with respect to weight for quartz
and gypsum respectively.
ACKNOWLEDGMENT: This outline was 2 Loveland. R. P., J. Roy. Micros. Soc.
prepared by the U. S. Public Health Service. 79. 59. (1960).
Department of Health. Education and Welfare.
for use in its Training Program. 3 Chamot. Emile Monnln. and Maaon. '
Clyde Walter. Handbook of Chemical
REFERENCES Microscopy. Vol. 1. third ed.
1 n, r* ,T7 ^ . , „ John Wiley and Sons. New York (1959).
1 Bunn. C. W. Crystal Growth from
Solution. Discussions of the Faraday DESCRIPTORS: Microscope and Optical
Society No. 5.132. Gunery and Jackson, properties
London. (1949).
6-26
-------
ARTIFICIAL SUBSTRATES
I INTRODUCTION: THE NATURE OF
ARTIFICIAL SUBSTRATES
A Artificial substrates are anything
deliberately placed in the water for the
purpose of providing a place for benthic
or attached (sessile, sedentary, etc.)
organisms to grow on or in. This is in
contrast to "bait" which is used as an
attractant.
B Their origins for commercial use, or human
food production are rooted in antiquity.
Some examples are:
1 Ropes, poles, brush, concrete
structures, and other objects thrust
into the bottom, or suspended in
estuarine waters to catch and grow
oysters and mussels (cultural techniques),
known virtually around the world.
2 Straw or reed tepees planted in shallow
alkaline lakes (in Mexico for example)
to catch the eggs of Corixids (Insecta:
Order Hemiptera, back-swimmers).
Eggs are harvested by drying and
brushing them off onto white sheets.
Used for human food.
C The fouling of ships bottoms, piling, etc.
by barnacles and other marine life is an
"artificial substrate in reverse".
D The use of aggregate to support a zoogloeal
mass of micro-biota in a trickling filter,
thus simulating a riffle area in a surface
stream, is a modern concept to harness
and make use of "consumer" and "reducer"
elements of a community in order to
dissipate the energy (oxidize, exhaust the
food value) contained in sewage.
II ECOLOGICAL BASIS
A Artificial substrates are based on the
"laws of organismal distribution. "
1 Any given kind of organisms tends to be
present (inhabit) in all available suitable
habitats.
*A community which has achieved a point of no further change, under a given set of
environmental conditions. Time scale may vary with circumstances.
NOTE: Mention of commercial products and manufacturers does not imply endorsement
by the Environmental Protection Agency.
2 Any given habitat tends to be inhabited
by all suitably adapted kinds of
organisms.
B A "substrate" being an object (or group
of objects) constitutes a habitat suitable
for sessile or attached organisms, and
also those that naturally burrow in, crawl
over, or otherwise live associated with
objects. Natural objects here could mean
the bottom, stones, sticks (floating or
sunk), etc.
C Organisms that would not be attracted to
substrates would be plankton and nekton
(fish and larger swimming invertebrates).
D Ecological Succession
Colonization is rapid in a biologically
productive water, and normally reaches
a stable climax* community in about a
month. A typical outline of successive
forms to appear in a freshwater situation,
for example, might be as follows.
1 Periphyton (slime forming) stage
(see also below)
a Bacteria - within an hour
b Diatoms - within the first day
c Other micro-algae - within the first day
d Protozoa - within the first day
2 Macroinvertebrate dominated stage
(see also below)
a Primary attached or sedentary
colonizers - second to third day
1) Net caddisflies
2) Bryozoa
3) Cordylophora caspia
4) Hydra
BI.MET.fm.7d. 2.79
7-1
-------
Artificial Substrates
b Primary foragers
1) Mayflies
1) Stoneflies
3) Midges
c Secondary attached or sedentary
colonizers:
o
1) Sponges
2) Filam entous alga e
d Adventitious forms
1) Crustaceans
2) Flatworms °
3) Leeches
4) Snails
5) Other
3 Artificial substrates in a marine
environment proceed through similar
stages, except that the macroinvertebrate
0 stage may be more subject to variation
in the attachment of broods of barnacle,
oyster, and other larvae resulting from
o greater numbers of types present, tidal
current variation, meteorological
conditions, etc.
Ill ARTIFICIA L SUBSTRATES A S SCIENTIFIC
COLLECTING EfeVICES
A A review of the history of artificial
substrates for collecting microorganisms
(aufwuchs) (Cooke, 1956) indicates that
glass microscope slides were first used
for this purpose about 1915. Wood or
metal panels appear, however, to have been
deliberately exposed for the scientific
collection of larger organisms at least
since approximately the turn of the century,
and probably long before that (Visscher, 1928).
B Biological Applications
The principles of the artificial substrate
remain the same, regardless of the
community sampled. Two general types
of communities and associated samplers
have been employed:
1 Periphyton (or aufwuchs) samplers
Periphyton is the community of slime
forming microorganisms which is the
first to attach to objects newly exposed
under water. This community is
generally considered to provide an
anchor layer to which other higher
forms of life can more readily attach.
It tends to persist until overgrown or
displaced by larger organisms, and
then in turn can be found spreading over
the surfaces of these same larger
plants and animals.
c
2 Periphyton has been widely studied as
it appears on 1 X3 glass microscope
slides which are equally convenient to
expose in the field and to study in the
laboratory.
3 Particular studies have included:
a The original bacterial and fungal
slime
b Diatom identification and counts
c Identification and counts of other
microscopic algae °
d Protozoans
e Primary productivity
O
0
4 The macroinvertebrate community is
sampled by a great variety of devices
such as those cited below. The
organisms are usually removed from
the substrate for study. Applications
have included the following:
a General study of the macroinverte-
brate community
7-2
-------
Artificial Substrates
b Estimates of productivity
c Studies of the life cycle of particular
species
d Studies of the influence of the sub-
strate on the attachment of sessile
forms
1) The influence of toxic paints for
the prevention of fouling organisms
2) Wood panels to study the pene-
tration of boring molluscs and
crustaceans
C Effect of type of device on what is collected
1 Wood boring organisms like teredo
worms (Mollusca, Pelecypoda) or
gribbles (Arthropoda, Isopoda)would
obviously be attracted primarily to
wood (although some are known to bore
in other materials).
2 Delicate forms and crawling forms
would be most likely to be collected on
devices having a shape to protect
against strong currents.
3 Those with strong attachments could
endure swift currents; often, surpris-
ingly, even during periods of original
attachment (ex. byssus attached clams
which are also benthic forms).
4 Bottom burrowers would be most likely
collected in artificially contained
portions of bottom material.
D Effect of Location
1 The depth at which a sampler is sus-
pended may influence the organisms
attracted.
2 Location in or out of a current, direct
sunlight, etc., will influence the take.
E Some Types of Devices
1 Cement plates, panels, and blocks
2 Ceramic tiles
3 Wood blocks
4 Metal plates
5. Glass slides -1X3 inch micro slides
are used by many workers. Numerous
devices are employed to hold them.
They are generally either floated
(Weber and Raschke 1966) or sus-
pended in racks, anchored to
submerged bricks or other objects.
6 Plastic petri dishes
Burbanck and Spoon utilized an
ordinary 50 X 12 mm plastic petri
dish for collecting sessile protozoa.
Sickle modified this by using a
styrofoam cup (6 oz. size) with the
bottom third being cut off. The
lower unit of the plastic dish is
easily wedged into place in the cup
and the device is simply held by a
nylon line on a rope held in place by
an appropriate anchor and float.
The cup which tends to float is so
held that the petri dish bottom is in
a horizontal position and bottom side
up.
7 Multiple plate (Hester and Dendy, 1962)
a Common current procedure
utilizes 3-inch squares of 1/4
inch thick Masonite separated
by 1-inch square spacers.
These may be:
b Threaded on an eye bolt or long
rod.
c Suspended by a loop of nylon cord.
8 Baskets or trays of bottom-type
material
a Trays of bottom material sunk in
the surface layer of the bottom.
b Baskets of stones suspended in
the water (Anderson and Mason,
1966).
9 Boxes, cages, bundles, etc., of
brush, reeds, or artificial material.
7-3
-------
Artificial Substrates
10 Polyethylene tapes
11 Plastic webbing
Minnesota Mining and Manufacturing
Company conservation web no. 200.
12 Styrofoam
13 Glass cover slips
Small slips are floated on the surface
of the water. Highly useful for protozoa
and rotifers. Remove and place on a
micro slide. Examine as a wet mount.
F Retrieval is an acute problem with all of
these samplers.
1 Physical factors
a Relocation
b Floods and drift
c High water
2 Well marked samplers or floats are
naturally vulnerable to the public,
resulting in disturbed, damaged, or
destroyed sample gear.
a This has been overcome by an
ingenious submerged float and
recovery line device. The weak
link in a submerged recovery line
is a modified flash bulb. An
electronic device actuated by an
underwater gun breaks the bulb
allowing the float and attached
line to surface. (Ziebell,
McConnell, and Baldwin)
b This unit has been further modified
by Fox (University of Georgia
Cooperative Fishery Unit) who
used an inexpensive detonator,
"Seal Salute". The latter is an
inexpensive fused charge designed
for underwater explosion.
IV ARTIFICIAL SUBSTRATES OR SAMPLERS,
AND WATER QUA LITY
A Artificial substrates provide a habitat
("place to live"). It follows from the
laws of distribution (II A I and 2 above),
that the community which inhabits a
device will be governed by the physical
nature or structure interacting with the
characteristics of the surrounding water
(velocity, temperature, chemical
characteristics, etc.). Since the nature
of the sampler is controlled, it is evident
that the characteristics of the water
constitute the variable factor.
B Water Quality Surveillance
1 Similar substrates suspended side by
side in the same water tend to accumulate
(essentially) the same communities and
quantities of organisms.
2 Similar substrates suspended in different
waters accumulate different communities
and quantities.
3 Ergo: different communities and
quantities collected from similar
substrates at different places and times,
probably indicate different water qualities.
a These may be natural (seasonal,
diurnal, etc.)
b Or they may be a result of human
influences (pollution)
c A series of samplers the length of a
stream, lake, or estuary can suggest
"steady state" differences in water
quality.
d A series of samplers exposed over a
period of time at a given site can
suggest changes of water quality in
time.
4 The artificial substrate thus essentially
constitutes an in-situ bioassay of the
water.
7-4
-------
Artificial Substrates
Interpretation and Significance of
Collections
1 The unit of comparison is most
appropriately taken as "the sampler".
The artificial substrate by definition is
not the natural local bottom material,
and unless it consists of a portion of
that bottom which has been actually
removed and replaced in an artificial
container (III-D-7)the composition and
magnitude of the community it contains
may or may not bear a definitive
relationship to the actual natural
problem. The take of the artificial
substrate thus may have relatively
little relationship to the take of a
Peter sen or an Ekman grab (dredge).
2 Comparisons between different types
of samplers are likewise hazardous.
Each is what it is, and if they are
different they are not identical; thus
the biota each collects cannot be
expected to be identical (CF: II A).
3 Artificial substrates should generally
be compared on a "sampler vs sampler"
basis, or for periphyton, "unit area
vs unit area".
REFERENCES
1 Anderson, J. B. and Mason, William T. Jr.
A Comparison of Benthic Macro -
invertebrates collected by Dredge and
Basket Sampler. Jour. Water Poll.
Cont. Fed. 40(2):252-259.
2 Arthur, John W. and Horning, W. B. , II.
The Use of Artificial Substrates in
Pollution Surveys. Amer. Midi. Nat.
82(l):83-89.
3 Besch, W., Hoffman, W., and Ellenberger,
W. Das Macrobenthos auf
Polyatchylensubstraten in Fliessgs-
wasseren. Annals de Limnologic.
3(2):331-367. 1967.
4 Burbanck, W.D. 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.
5 Cooke, William B. Colonization of
Artificial Bare Areas by Microorganisms.
Bot. Rev. 22(9):613-638. Nov. 1956.
6 Fox, Alfred C. Personal Communication.
1969.
7 Hester, F.E. and Dendy, J.S.
A Multiple-Plate Sampler for Aquatic
Macroinvertebrates. Trans.Am.
Fish. Soc. 91(4):420-421. April 1962.
8 Hilsenhoff, William L. An Artificial
Substrate Device for Sampling Benthic
Stream Invertebrates. Limnology and
Oceanography. 14(3):465-471. 1969.
9 Mason, W.T., Jr., Anderson, J.B., and
Morrison, G.E. A Limestone-Filled,
Artificial Substrate Sampler Float Unit
for Collecting Macroinvertebrates in
Large Streams. Prog. Fish-Cult.
29:74. 1967.
10 Ray, D. L. Marine Boring and Fouling
Organisms. University of Washington
Press, Seattle. pp 1-536. 1959.
11 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.
12 Sladeckova, A. Limnological Investigation
Methods for the Periphyton ("Aufwuchs")
Community. Bot. Rev. 28(2):286-350.
1962.
13 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.
7-5
-------
Artificial Substrates
14 Visscher, J. Paul. Nature and Extent ___
of Fouling of Ships Bottom. Dept. _,. .,. , , TT .„ _ ,
_ „ „ -rn u T-L This outline was prepared by H. W. Jackson
Comm. Bur. Com. Fish. Doc. _,. r _. , . . K. J, ,, _. . '
wo 10^1 nn 1Q^! 252 1928 Chief Biologist and R. M. Sinclair, Aquatic
No. 1031. pp 193-252. 1928. Biologist. National Training Center, Water
-ic TTT T- /-. T-, j T, ui « T Programs Operations, EPA, Cincinnati,
15 Weber, C. E. and Rauschke, R. L. ohi 4"i268
Use of a Floating Periphyton Sampler
for Water Pollution Surveillance DESCRIPTORS: Artificial Substrates.
Water Poll. Sur. Sept Applications Bentho Bottom ga
and invertebrates.
T -
FWPCA-USDI, Cincinnati, Ohio.
September 1966.
16 Wene, George and Wickliff, E. L.
Modification of a Stream Bottom and
its Effect on the Insect Fauna.
Canadian Entomologist. Bull. 149,
5 pp. 1940.
17 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.
7-6
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BENTHIC INTEGRITY AND MACRO INVERTEBRATE DRIFT
I Significant number of benthic macro
invertebrates under certain conditions
join stream drift.
A This phenomenon was only discovered
in mid century. The organisms as well
as the phenomenon is termed
collectively, Drift.
B Macroinvertebrates which drift
include are the insect orders,
Ephemeroptera, Trichoptera, Plecop-
tera and the crustacean order
Amphipoda.
C Other invertebrate groups exhibit drift
patterns.
II FOUR BASIC TYPES OF DRIFT ARE
RECOGNIZED
A Catastrophic Drift
Floods wash numerous benthic organisms
downstream. Application of pesticides
may also cause such drift.
B Constant Drift (Incidental or Adventitious)
Organisms are constatly being dislodged
from the substrate during normal
activities and carried downstream.
C Periodic (Diel) Drift
In contrast to the other categories, this
is a specific behavior pattern and related
to circadian activity rhythms. Periodic
or diel drift occurs in peaks for successive
24-hour periods.
D Seasonal Drift (Related to Life History
Development)
Seasonal drift occurs, for example, in
some maturing stoneflies which drift
downstream for emergence. This is
another reason for a serious consider-
ation of drift in bottom fauna sampling
since such presence of stoneflies could
easily be misinterpreted.
1 Night-active. Light intensity is the
phase-setting mechanism.
2 Day-active. Water temperature
appears to be the phase-setter.
Ill DIEL DRIFT
A Diel activity rhythms generally include
two peaks during the 24-hour period;
one major and the other minor.
1 The bigeminus type in which the
major peak occurs first (after sunset).
§
O
FIGURE 1
2 The alternans pattern with the major
peak occurring last.
$-,
X!
1200 1800 2400
Time
0600
Sunset Sunrise
FIGURE 2
B Drift Rate and Density (Waters, 1969)
1 Drift rate defined is ".. .the quantity
of organisms passing a width transect
or portion thereof, per unit time;
BI. ECO. 22a. 2.79
J-l
-------
Benthic Integrity and Macro Invertebrate Drift
it is a measure of displacement or
the movement of organisms from one
place to another."
2 Drift density"... is the quantity of
organisms per unit volume of water,
in much the same way as plankton
density can be defined. "
IV DETERMINING BENTHIC INTEGRITY
THRU DRIFT SAMPLING
A The drift from productive upstream
reaches may support a fish population
existing in relatively barren stream
sections.
B Drift will colonize artificial substrates,
such as suspended rock baskets, when
placed in such habitats.
C A bottom sampler such as the Surber,
could also be sampling drift when only
resident benthic organisms are intended
to be collected. This would depend on
the hour of collection and length of time
the Surber sampler is in the water.
D Application of drift studies have been
widely used in pesticide related studies
and routine monitoring. Dimond
concluded that:
1 The status of drift is a much better
indicator of the steady state and of
total productivity than is the status
of the bottom fauna.
2 Bottom sampling, however, is
superior when analyzing survival
and recovery of the quality of popula-
tion.
3 A combination of both in such a
sampling program would be most
likely to yield the most useful data.
E Drift sampling techniques have been
useful for recovery of large numbers of
sand-dwelling mayflies, which were
once rarely collected.
V MAJOR TAXA INVOLVED IN DRIFT
A The crustacean order Amphipoda
1 Gammarus species
2 Hyalella azteca
B The Insect Orders
1 Ephemeroptera
Baetis species (apparently universal)
2 Plecoptera
3 Trichoptera
4 Diptera
Simuliidae
5 Elmidae
C The main groups exhibiting very high
drift rates include: Baetis, some
Gammarus species, and some Simuliidae.
REFERENCES
1 Anderson, N. H. Biology and Down-
stream Drift of some Oregon
Trichoptera. Can. Entom. 99:507-
521. 1967.
2 Dimond, John B. Pesticides and
Stream Insects. Bull. 23, Maine
Forest Service, 21 pp. 1967.
3 Dimond, John B. Evidence that drift of
Stream Benthos is Density Related.
Ecology 48:855-857. 1967.
4 Pearson, William D., and Kramer,
Robert H. A Drift Sampler driven
by a Waterwheel. Limnology and
Oceanography 14(3):462-465.
5 Reed, Roger J. Some Effects of DDT on
the Ecology of Salmon Streams in
Southeastern Alaska. Spec. Sci.
Report-Fisheries 542: 1-15. U.S.
Bureau Comm. Fisheries. 1966.
-------
Benthic Integrity and Macro Invertebrate Drift
6 Water, Thomas F. Interpretation of
Invertebrate Drift in Streams.
Ecology 46 (3):327-334. 1965.
7 Waters, Thomas F. Diurnal Periodicity
in the Drift of a Day-active Stream
Invertebrate. Ecology 49:152. 1968.
8 Waters, Thomas F. Invertebrate
Drift-Ecology and Significance to
Stream Fishes. (T. G. Northco e,
Ed. ) Symposium Salmon and Trout
in Streams. University of British
Columbia, Vancouver, pp. 121-134.
1969.
This outline was prpared by R. M. Sinclair,
National Training Center, MOTD, OWPO,
USEPA, Cincinnati, Ohio 45268.
DESCRIPTORS: Aquatic Life, Aquatic Drift,
and Invertebrates
8-3
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EFFECTS OF THERMAL POLLUTION ON THE BENTHOS
I INTRODUCTION
A Fish may be obviously important but they
are not the only important organisms or
the only ones being studied at present.
The food organisms are just as important
in the long run. Lose the organisms which
convert the energy of the primary pro-
ducers to a form usable by the fish and
we lose the fish themselves.
B In a river, most of the microscopic popu-
lation are benthic organisms, since the
plankton have difficulty maintaining position
in the stream flow. The benthos is a
stationary community which should reflect
the action of the temperature in the area
of influence. Of course, bottom debris
may serve to protect benthic organisms
to some extent from full exposure to the
heated water.
II SUBLETHAL EFFECTS ON AQUATIC
INSECTS
A In most western streams the stoneflies,
caddisflies and mayflies are the primary
fish food organisms. At the same time,
these organisms have definite environ-
mental requirements and cold, well-
oxygenated water is a prime factor.
1 Preliminary work at the Duluth
Laboratory indicates that temperatures
would probably become lethal to any
cold water fish like trout before the
insects would die.
a According to Usinger (1956), the
heat tolerance of macroscopic
invertebrates is well above that of
fish.
b For example, soldier fly
(stratiomyidae) larvae were found
living in thermal waters at tempera-
tures up to 120°F.
96 hour TL values
Table 1 shows
determined for some insect species by
Nebeker and Lemke of the Duluth
Laboratory.
a These temperatures are well above
the 12°C suggested as the maximum
limit for spawning and egg develop-
ment in salmon and trout.
b This doesn't tell the whole story
because the insects may be harmed
in other ways.
B Gaufin, formerly of Utah, and also Nebeker
of the Duluth Laboratory, have demonstrated
that temperature increases can cause pre-
mature emergence.
1 A 10°C rise from ambient winter tem-
perature caused one species of stonefly
to emerge in January instead of May.
"One must imagine how perplexed these
organisms must be as they expect nice
warm spring weather only to freeze to
death as they emerge. "
2 Nebeker found that a temperature in-
crease for another species caused the
males to emerge as much as two months
ahead of the females!
C Either situation would prevent reproduction
and would be fatal to the species although
not fatal to individuals prior to emergence.
D Even without lethal effects we may find
changes in community due to variation in
optimum temperatures between species.
This has not been studied enough in the
field to really determine the overall effect
on a natural system but it is something
which we will have to know more about in
the future.
Ill SUBLETHAL EFFECTS ON SHELLFISH
A Most shellfish, such as clams, oysters,
crabs and lobsters, which are directly
beneficial to man as a food source, are
marine, stenothermal organisms. Some
species are stenothermal for one develop-
mental stage and eurythermal for another.
Generally, however, breeding and spawning
requirements are stenothermal.
1 The time of mollusc, e. g. clams,
oyster, etc. , spawning is temperature
dependent.
2 Most molluscs with specific temperature
breeding relationships spawn in the spring
and summer, and many do not spawn until
a certain temperature is reached.
B The American oyster Crassostrea virginica
spawns at temperatures between 15 and 34°C
BI.ECO. he. 5. 2.79
9-1
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Effects of Thermal Pollution on the Benthos
TABLE 1
Temperatures at which 50% of the test species died after 96 hours
exposure (TLm96) when acclimated at 10°C for one week.
Species Tested
Taeniopteryx maura (winter stonefly)
Ephemerella subvaria (mayfly)
Isogenus frontalis (stonefly)
Allocapnia granulata (winter stonefly)
Stenonema tripunctatum (mayfly)
Brachycentrus americanus (caddisfly)
Pteronarcys dorsata (stonefly)
Acroneuria lycorias (stonefly)
Paragnetina media (stonefly)
Atherix variegata (true fly)
Boyeria vinosa (dragonfly)
Ophiogomphus rupinsulensis (dragonfly)
TLm
21
21.
22.
23
25.
29
29.
30
30.
32
32.
33
96 (°Celcius)
o
5°
5°
o
5°
0
5°
o
5°
o
5°
o
12°c (55°F) Maximum temperature recommended in Water Quality
Criteria for spawning and egg development of salmon and trout.
From: Nebeker, Alan V. and Armond E. Lemke, 1968. Preliminary
studies on the tolerance of aquatic insects in heated waters. Journal
of Kansas Entomological Society 41: 413-418. July, 1968.
9-2
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Effects of Thermal Pollution on the Benthos
(59 and 93. 2°F) depending on its condition,
and spawning is usually triggered by a
rise in temperature.
C Many species tolerate temperatures in
excess of those at which breeding occurs.
1 For example, the shore crab Carcinus
maenas thrives, but does not breed, at
temperatures of 14 to 28°C (57. 2 to
82.4°F).
2 In this case, temperature limits the
population, but migration of organisms
can occur from outside the heated area.
D Physiology, metabolism and development
are all affected by temperature.
1 The American oyster C_. virginica
ceases feeding at temperatures below
7°C (44. 6°F).
a Above 32°c (89. 6°F) ciliary activity,
which is responsible for water move-
ment, is decreased.
b At 42°C (107. 6°F) almost all body
functions cease, or are reduced to a
minimum.
2 The European oyster Ostrea lurida
tends to close its shell as temperatures
drop.
a At 4 to 6°C (39. 2 to 42. 8°F) the
oyster's shell remains closed most
of the time.
b At 6 to 8°C (42.8 to 46.4°F) the
shell opens for about 6 hours per day.
c At 15°C (59°F) the shell stays open
for 23 hours a day.
E Very little is known about prolonged effects
of temperatures above 32 to 34°C (90 to
94 F) on oysters; however, long exposure
to such temperatures may impede the
oyster's normal rate of water circulation.
When either low or high temperatures cause
shells to close or ciliary action to cease,
oysters cannot feed and subsequently lose
weight. Thus, temperature changes can
produce an effect similar to chronic
toxicity.
F The distribution of benthic organisms is
temperature dependent.
1 The American oyster (T. virginica
is present in Gulf Coait waters that
that may vary between 4 and 34 C
(39. 2 and 93. 2°F), but the European
oyster O. edulis is restricted to water
temperatures of 0 to 20°C (32 to 68°F).
The opossum shrimp Neomysis
americana is not often found at tempera-
tures above 31°C (87. 8°F) in the
Chesapeake estuary.
IV LETHAL EFFECTS
A Studies of particular species of benthic
macroinvertebrates have indicated that
lethal temperatures vary considerably
with the type of organism.
1 Laboratory investigations on the fresh-
water snail Lymnaea stagnalis showed
a lethal temperature of 30. 5UC (89. 6°F),
while the species Viviparous malleatus
did not succumb until the temperature
reached 37. 5°c (99. 5°F).
2 Agerborg (1932) observed a freshwater
snail, Physa gyrina, living and repro-
ducing nicely in zones up to 91. 4°F in
heated wastewater.
3 Hutchinson (1947) reported that
Viviparous malleatus, a freshwater
snail, was not killed until the tempera-
ture reached 37. 5°C (99°F).
B Several snails, including Australorbis
glabratus, suffered heat damage at 105. 8°F
(Von Brand, et. al. 1948).
C Other examples show that the limpet,
Ancylus fluviatilus, was not hurt by a
temperature of 96. 8°F while 87. 8*% was
lethal to Acrolexus lacustris (Berg, 1952).
D When an unidentified species of crayfish
was acclimated to 45°F, it had a lethal
temperature of 93°F (Trembly 1961).
E Sprague (1963) reported a 24-hour lethal
temperature of 94. 3°F for a freshwater
sowbug, Asellus intermedius, and a scud,
Gammarus fascTatus. Another scud,
Hyalella azteca, was killed at 91.8°F.
F Field work on rivers has indicated that
benthic organisms decrease in number
when water temperature exceeds 30°C
(86°F).
1 The macroinvertebrate riffle fauna of
the Delaware River has decreased due
to heated water discharges.
9-3
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Effects of Thermal Pollution on the Benthos
B
a At 35°C (95°F) many caddisfly,
Hydropsyche, were dead, and those
which remained alive were extremely
sluggish.
b This study suggests that there is an
upper tolerance level near 32.2°C
(90°F) for a variety of different
benthic forms with extensive losses
in numbers and diversity accompany-
ing a further increase in temperature.
POPULATION SHIFTS CAUSED BY HEAT
ADDITION
Trembley (1960) studied the bottom fauna
of the Delaware River at the Martins Creek
Power Plant.
1 In the zone of maximum temperature rise
rise just below the outfall, there was
obvious reduction of species and
individuals.
2 In the cool water unaffected by the
thermal overflow, there was no reduction
in macroinvertebrates.
3 During the cooler seasons there was
repopulation of the areas affected during
the hot months by thermal discharge.
4 Even during the summer, there was a
significantly higher standing crop at the
downstream site in comparison to the
normal river control station.
Coutant (1962) followed Trembley's
Martins Creek research with a study of
the macroinvertebrate bottom fauna of
the riffle areas of Big Kaypush and Little
Kaypush Rapids.
1 He confirmed Trembley's conclusions.
From July through October, there was
substantial reduction in the number,
diversity, and biomass of benthic
organisms in the path of the heated
water.
2 At a distance of one mile downstream
from the point of discharge, he found
a normal population structure.
3 In his traverse studies, he observed
an increase in both variety and number
of organisms as he progressed from
hot to cool water, demonstrating the
effect of temperature as the primary
limiting factor.
4 The work also showed the restricted
effect of heated discharges in changing
the biological communities. The data
suggest a tolerance limit near 90°F for
a normal population structure with
extensive loss in numbers and diversity
of organisms accompanying further rise.
Wurtz and Dolan (1960) reported a study on
bottom organisms in the Schuylkill River
at the Cromby Power Plant.
1 These authors gave no temperature data;
however, the subcommittee of the
Pennsylvania Electric Association (Mason,
1962) showed severe temperature altera-
tion in this reach of river since the plant
used 85% of the river flow as cooling water.
2 The river showed a very elevated tem-
perature and slow recovery. Wurtz and
Dolan evaluated the effects of heated dis-
charges in terms of biological depression,
biological distortion, and biological
skewness.
3 Station 10 at Phoenixville Pumping Station,
0. 5 miles below the plant, showed the
greatest deviation.
4 At Station 13, six miles below the power
plant, the river biology had recovered.
This case illustrates ultimate recovery
from an extreme condition.
VI SUMMARY
It is clear from the valid biological data pre-
sented that increased temperature of the water
does alter the species and individual composi-
tion of the benthic population which, of course,
being generally sessile, is unable to avoid
exposure.
A CK NOW LEDG ME NT S
Material for this outline was taken from
The Industrial Waste Guide, Bruce A. Tichenor
and Alden G. Christiansen, authors; Thermal
Pollution: Status of the Art, Frank L. Parker
and Peter A. Krenkel, authors; and Technical
Seminar Paper, Biological Effects, Dr. Ronald
Garton, author.
REFERENCES
1 Agersborg, H. P. K. The Relation of
Temperature to Continuous Reproduction
in the Pulmonate Snail. Nautilus, 45.
121. 1932.
9-4
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Effects of Thermal Pollution on the Benthos
2 Berg, K. On the O2 Consumption of
Ancylidae (Gastropoda) from an
Ecological Point of View.
Hydrobiologia. 4. 225. 1952.
3 Coutant, C. C. The Effect of a Heated
Water Effluent Upon the Macroinverte-
brate Riffle Fauna of the Delaware
River. Penn. Acad. Science. 37. 58.
1962.
4 Hutchinson, L. Analysis of the Activity of
the Freshwater Snail, Viviparous
malleatus (Reeve). Ecology 28. 335.
1947.
5 Sprague, J. B. Resistance of Four Fresh-
water Crustaceans to Lethal High
Temperatures and Low Oxygen. Journal
Fisheries Research Board, Canada. 20.
387. 1963.
6 Trembley, F. J. Research Project on
Effects of Condenser Discharge Water
on Aquatic Life. Progress Report.
1960. The Institute of Research Lehigh
University. 1961.
7 Usinger, R. L. Aquatic Insects of
California. University of California
Press. 1956.
8 VonBrand, T., Nolan, M. O., and Man,
E. R. Observations on the Respiration
of Australorbis glabratus and Some
Other Aquatic Snails. Biology Bulletin.
95. 199. 1948
9 Wurtz, C. B., and Dolan, T. A Biological
Method Used in the Evaluation of Effects
of Thermal Discharge in the Schuylkill
River. Proc. 15th Industrial Waste
Conference. Purdue University. 461.
1960.
This outline was prepared by John F. Wooley,
Biologist, Manpower and Training Branch,
Pacific Northwest Water Laboratory, Federal
Water Quality Administration.
DESCRIPTORS: Aquatic Life, Benthos,
Benthic fauna, Cooling water, Invertebrates,
Power plants, and Thermal pollution.
9-5
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