EPA-430/1-78-001
BIOASSAY FOR TOXIC AND
HAZARDOUS MATERIALS
TRAINING MANUAL
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF WATER PROGRAM OPERATIONS
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EPA-430/1-78-001
March 1978
BIOASSAY FOR TOXIC AND HAZARDOUS MATERIALS
U.S. ENVIRONMENTAL PROTECTION AGENCY
Office of Water Program Operations
National Training and Operational Technology Center
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DISCLAIMER
Reference to commercial products, trade names, or
manufacturers is for purposes of example and illustration.
Such references do not constitute endorsement by the
Office of Water Program Operations, U.S. Environmental
Protection Agency.
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CONTENTS
Title or Description
The Development of Water Quality Criteria in the United States
The Philosophy of Quality Criteria
Significance of "Limiting Factors" to Population Variation
Global Environmental Quality
Ecology Primer
Classification of Communities. Ecosystems. and Trophic Levels
Bioassay and Biomonitoring
Bioassay Facilities and Equipment
Important Data From Acute Mortality Tests
The Statistics of Toxicity Tests
The Selection of Endpoints for Toxicity Tests
Special Applications and Procedures for Bioassay
Ecological Consideration in Planning Water Quality Surveys
Dilution Table
Nomogram for the Solubility of Oxygen
Bioassay Record Sheet
Use of LC - Paper
LC Paper
LCm Paper
Bioassay Record Sheet
Graph paper
Bioassay Paper (Log-probit)
Outline Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
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THE DEVELOPMENT OF WATER QUALITY CRITERIA IN THE UNITED S\rATES
The genesis of water quality criteria in the United States can be traced to the earr 1900's.
Marsh, 1 in 1907, published on the effects of industrial wastes on fish. Shelford, in 1917,
published effect data on fish for a large number of gas-waste constituents. In this early
publication he reiterated that the toxicity of waste differs for different species of fish and
generally is greater for the smaller and younger fish. Powers, 3 working with Shelford,
experimented with the goldfish as a test animal for aquatic toxicity studies.
A monumental early effort to describe and record the effects of various concentrations of
a great number of substances on aquatic life was that of Ellis4 in 1937. Ellis reviewed the
existing literature for 114 substances and in a 72-page document listed lethal concentrations
found by the various authors. He provided a rationale for the use of standard test animals
in aquatic bioassay procedures and used the common goldfish, Carassius auratus, and the
entomostracan, Daph.{lia~, as test species on which experiments were made in constant
temperature cabinets.
Early efforts to summarize knowledge concerning water quality criteria took the form of
a listing of the concentration, the test organism, the results of the test within a time period,
and the reference for a cause-effect relationship for a particular water contaminant. In
early bioassay efforts insufficient attention was given to the quality of the dilution water used
for the experiment and to the effects of such dilution water on the relative toxicity of the tested
contaminant. As a result, conclusions from citations of such references were, at best, difficult
to formulate and most often were left to the discretion of the reader.
In 1952, the State of California5 published a 512-page book on "Water Quality Criteria" that
contained 1,369 references. This classic reference summarized water quality criteria promulgated
by State and interstate agencies as well as the legal application of such criteria. Eight major
beneficial uses of water were described. Three hundred pages':!;)f the document were devoted to
cause-effect relationships for major water pollutants. The concentration-effect levels for the
pollutant in question were discussed for each of the designated water used.
The State of California's 1952 "Water Quality Criteria" was expanded and tremendously enhanced
into a second edition edited by Jack E. McKee and Harold W. Wolf ~d published in 1963 by the
Resources Agency of California, State Water Quality Control Boo rd. This edition, which included
3,827 cited references, was a monumental effort in bringing together under one cover the world's
literature on water quality criteria. Criteria were identified and referenced for a host of water
quality characteristics according to their effects on domestic water supplies. industrial water
supplies, irrigation waters, fish and other aquatic life, shellfish culture, and swimming and other
recreational uses. Specific concentrations were arranged in ascending order indicating the degree
of damage to fish in the indicated time and under the conditions of exposure. The results of such
a tabulation presented a range of values and, as would be expected by those investigating such
conditions. there was often an overlap in values among those concentrations that had been reported
as harmful by others. Such an anomaly is due to differences in investigative techniques among
investigators, the characteristics of the water used as a diluent for the toxicant. the physiological
state of the test organisms. and variations in the temperature in which the tests were conducted.
Nevertheless, the tabulation of criteria values for each of the water quality constituents has been
helpful in predicting a range within which a water quality constituent would have a deleterious effect
upon the receiving waterway.
WP. POL. 14. 1.78
1-1
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The Development of Water Quality Criteria in the United States
In 1966 the Secretary of the Interior appointed a number of nationally recognized scientists
to a National Technical Advisory Committee to develop water quality criteria for five specified
uses of water: agricultural. industrial. recreational. fish and wildlife. and domestic water
supply. In 1968 the report was published.7 This report constituted the most comprehensive
documentation to date on water quality requirements for particular and defined water uses.
The book was intended to be used as a basic reference by personnel in state water pollution
control agencies engaged in water quality studies and water quality standards setting activities.
In some respects. this volume represented a marriage between the best available experimental
or investigative criteria recorded in the literature and the judgments of recognized water quality
experts with long experience in associated management practices. Its publication heralded a
change in the concept of water quality criteria from one that listed a series of concentration-
effect levels to another that recommended concentrations that would ensure the protection of
the quality of the aquatic environment and the continuation of the designated water use. When
a specific aquatic life recommendation for a particular water pollutant could not be made because
of either a lack of information or conflicting information. a recommendation was made to substitute
a designated application factor based upon data obtained from a 96-hour bioassay using a sensitive
aquatic organism and the receiving water as a diluent for the toxicity test.
The U. S. Environmental Protection Agency contracted with the National Academy of Sciences and
the National Technical Advisory Committee's "Water Quality Criteria" and to develop a water
quality criteria document that would include current knowledge. The result was a 1974 publication
that presented water quality criteria as o~ 1972.8
The Federal Water Pollution Control Act Amendments of 1972 (P. L. 92-500) mandated that the
Environmental Protection Agency publish water quality criteria accurately reflecting the latest
scientific knowledge on the kind and extent of all identifiable effects on health and welfare which
may be expected from the presence of pollutants in any body of water.
Section 304(a) of P. L. 92-500 states:
(1) The Administrator. after consultation with appropriate Federal and State agencies and
other interested persons, shall develop and publish. within one year after the date of enactment
of this title (Oct. 18, 1972) (and from time to time thereafteOr revise) criteria for water
quality accurately reflecting the latest scientific knowledge (A) on the kind and extent
of all identifiable effects on health and welfare including, but not limited to. plankton,
fish, shellfish, wildlife, plant life. shorelines, beaches, aesthetics, and recreation which
may be expected from the presence of pollutants in any body of water, including ground
water; (B) on the concentration and dispersal of pollutants, or their byproducts. through
biological, physical, and chemical processes; and (C) on the effects of pollutants on biological
community diversity. productivity, and stability, including information on the factors affecting
rates of eutrophication and rates of organic and inorganic sedimentation for varying types
of receiving waters.
(2) The Administrator. after consultation with appropriate Federal and State agencies and other
interested persons, shall develop and publish, within one year after the date of enactment
of this title (Oct. 18, 1972) (and from time to time thereafter revise) information (A) on the
factors necessary to restore and maintain the chemical, physical, and biological integrity
of all navigable waters, ground waters, waters of the contiguous zone, and the oceans; (b)
on the factors necessary for the protection and propagation of shellfish, fish, and wildlife
for classes and categories of receiving waters and to allow recreational activi ties in and on
the water; and (C) on the measurement and classification of water quality; and (D) for the
purpose of Section 303 of this title, on and the identification of pollutants suitable for maximum
daily load measurement correlated with the achievement of water quality objectives.
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The Development of Water Quality Criteria in the United States
(3) Such criteria and information and revisions thereof shall be issued to the States
and shall be published in the Federal Register and otherwise made available to
the public.
Section 101(a)(2) of P. L. 92-500 states:
It is the national goal that wherever attainable. an interim goal of water quality which
provides for the protection and propagation of fish. shellfish. and wildlife. and provides
for recreation in and on the water be achieved by July 1. 1983.. . "
The objectives of this volume are to respond to these sections of the Act and thus establish water
quality criteria. The "Quality Criteria for Water" will be expanded periodically in the future to
include additional constituents as data become available. While the NAS/NAE "1972 Water Quality
Criteria" considered aluminum. antimony. bromine. cobalt. fluoride. lithium. molybdenum.
thallium. uranim. and vanadium. these presently are not included in this volume; however. they
should be given consideration in the development of Statewater quality standards and quality criteria
may be developed for them in future volumes of the QCW. In particular geographical areas or for
specific water uses such as the irrigation of certain crops. some of these constituents may have
harmful effects. Until such time that criteria for the 10 aforementioned constituents are developed.
information relating to their effects on the aquatic ecosystem may be found in the NAS/NAE "1972
Water Quality Criteria. "
REFERENCES
1 M. C. Marsh. 1907. The effect of some industrial wastes on fishes.
paper No. 192. U. S. Geol. Sur. pp. 337-348.
Water supply and irrigation
2 V. E. Shelford. 1917. An experimental study of the effects of gas wastes upon fishes. with especial
reference to stream pollution. Bull. Illinois State Lab. for-:Nat. History. 11:381-412.
3 E.13. Powers. 1917. The goldfish (Carassius carassius) as a test animal in the study of toxicity.
Illinois BioI. Mono. 4: 127-193.
4 M. M. Ellis. 1937. Detection and measurement of stream pollution. Bull.
Fisheries. 48:365-437.
U. S. Bureau of
5 Water quality criteria. 1952.
State Water Pollution Control Board. Sacramento. Calif.
6 J. E. McKee and H. W. Wolf. 1963. Water quality criteria. State Water Quality Control Board.
Sacramento. Calif. Pub. 3-A.
7 National Technical Advisory Committee to the Secretary of the Interior. 1968. Water quality
criteria. U. S. Government Printing Office. Washington. D. C.
8 National Academy of Sciences. National Academy of Engineering. 1974.
1972. U. S. Government Printing Office. Washington. D. C. (1974).
Water quality criteria.
DESCRIPTORS:
This outline was extracted from: Quality
Criteria for Water - 1976. The Development
of Water Quality Criteria in the United States
Bioassay. Methodology, Water Quality Criteria
1-3
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THE PHILOSOPHY OF QUALITY CRITERIA
Water quality criteria specify concentrations of water constituents which, if not exceeded,
are expected to result in an aquatic ecosystem suitable for the higher uses of water. Such
criteria are derived from scientific facts obtained from experimental or in situ observations that
depict organism responses to a defined stimulus or material under identifiable or regulated
environmental conditions for a specified time period.
Water quality criteria are not intended to offer the same degree of safety for survival and propoga-
tion.at all times to all organisms within a given ecosystem. They are intended not only to protect
essential and significant life in water, as well as the direct users of water, but also to protect
life that is dependent on life in water for its existence, or that may consume intentionally or un-
intentionally any edible portion of such life.
The criteria levels for domestic water supply incorporate available data for human health protection.
Such values are different from the criteria levels necessary for protection of aquatic life. The
Agency's interim primary drinking water regulations (40 FR 59566 Dec. 24, 1975), as required
by the Safe Drinking Water Act (42 U. S. C. 300f, et seg.), incorporate applicable domestic water
supply criteria. Where pollutants are identified in both the quality criteria for domestic water
supply and the Drinking Water Standards, the concentration levels are identical. Water treatment
may not significantly affect the removal of certain pollutants.
What is essential and significant life in water? Do Daphnia or stonefly nymphs qualify as such
life? Why does 1/100th of a concentration that is lethal to 50 percent of the test organisms (LC50)
constitute a criterion in some instances, whereas 1/20 or 1/10th of some effect levels constitutes
a criterion in other instances? These are questions that often are asked of those who undertake
the task of criteria formulation.
The universe of organisms composing Ufe in water is great inJ~ind and number. As in the human
population, physiological variability exists among individuals of the same species in response to
a given stimulus. A much greater response variation exists among species of aquatic organisms.
Thus, aquatic organisms do not exhibit the same degree of harm, individually or by species, from
a given concentration of a toxicant or potential toxicant within the environment. In estabUshing a
level or concentration of a quality constituent as a criterion it is necessary to ensure a reasonable
degree of safety for those more sensitive species that are important to the functioning of the aquatic
ecosystem even though data on the response of such species to the quality constituent under considera-
tion may not be available. The aquatic food web is an intricate relationship of predator and prey
organisms. A water constituent that may in some way destroy or eliminate an important segment
of that food web would, in all likelihood, destroy or seriously impair other organisms associated with
it.
Although experimentation relating to the effects of particular substances under controlled conditions
began in the early 1900's, the effects of any substance on more than a few of the vast number of
aquatic organisms have not been investigated. Certain test animals have been selected by investi-
gators for intensive investigation because of their importance to man, because of their availability
to the researcher, and because of their physiological responses to the laboratory environment. As
general indicators of organism responses such test organisms are representative of the expected
results for other associated organisms. In this context Daphnia or stoneflies or other associated
organisms indicate the general levels of toxicity to be expected among untested species. In addition,
test organisms are themselves vital links within the food web that results in the fish population in
a particular waterway.
WP. POL. 15. 1.78
2-1
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The Philosophy of Quality Criteria
The ideal data base for criteria development would consist of information on a large percentage
of aquatic species and would show the community response to a range of concentrations for a
tested constituent during a long time period. This information is not available but investigators
are beginning to derive such information for a few water constituents. Where only 96-hour
bioassaydata are available. judgmental prudence dictates that a substantial safety factor be
employed to protect all life stages of the test organism in waters of varying quality, as well as
to protect associated organisms within the aquatic environment that have not been tested and that
may be more sensitive to the test constituent. Application factors have been used to provide the
degree of protection required. Safe levels for certain chlorinated hydrocarbons and certain heavy
metals were estimated by applying an 0.01 application factor to the 96-hour LCSO value for" sensi-
tive aquatic organisms. Flow-through bioassays have been conducted for some test indicator
organisms over a substantial period of their life history. In a few other cases, information is
available for the organism's natural life or for more than one generation of the species. Such
data may indicate a minimal effect level, as well as a no-effect level.
The word "criterion" shoul d not be used interchangeably with, or as a synonym for, the work
"standard." The word "criterion" represents a constituent concentration or level associated
with a degree of environmental effect upon which scientific judgment may be based. As it is
currently associated with the water environment it has come to mean a designated concentration
of a constituent that when not exceeded. will protect an organism. an organism community. or
a prescribed water use or quality with an adequate degree of safety. A criterion. in some cases.
may be a narrative statement instead of a constituent concentration. On the other hand a standard
connotes a legal entity for a particular reach of waterway or for an effluent. A water quality
standard may use a water quality criterion as a basis for regulation or enforcement, but the
standard may differ from a criterion because of prevailing local natural conditions, such as
naturally occurring organic acids. or because of the importance of a particular waterway,
economic considerations. or the degree of safety to a particular ecosystem that may be desired.
Toxicity to aquatic life generally is expressed in terms of acute (short term) or chronic (long
term) effects. Acute toxicity refers to effects occurring in a short time period; often death is
the end point. Acute toxicity can be expressed as the lethal conc"~ntration for a stated percentage
of organisms tested, or the reciprocal. which is the tolerance limit of a percentage of surviving
organisms. Acute toxicity for aquatic organisms generally has been expressed for 24- to 96-
hour exposures.
Chronic effects often occur in the species population rather than in the individual. If eggs fail to
develop or the sperm does not remain viable, the species would be eliminated from an ecosystem
because of reproductive failure. Physiological stress may make a species less competitive with
others and may result in a gradual population decline or absence from an area. The elimination
of a microcrustacean that serves as a vital food during the larval period of a fish's life could
result ultimately in the elimination of the fish from an area. The phenomenon of bioaccumulation
of certain materials may result in chronic toxicity to the ultimate consumer in a food chain. Thus,
fish may mobilize lethal toxicants from their fatty tissues during periods of physiological stress.
Egg shells of predatory birds may be weakened to a point of destruction in the nest. Bird chick
embryos may have increased mortality rates. There may be a hazard to the health of man if
aquatic organisms with toxic residues are consumed.
The fact that living systems, i. e.. individuals. populations, species and ecosystems can take up,
accumulate. and bioconcentrate manmade and natural toxicants is well documented. In aquatic
systems biota are exposed directly to pollutant toxicants through submersion in a relatively efficient
solvent (water) and are exposed indirectly through food webs and other biological. chemical. and
physical interactions. Initial toxicant levels. if not immediately toxic and damaging. may accumu-
late in the biota or sediment and increase to levels that are lethal or sublethally damaging to aquatic
organisms or to consumers of these organisms. Water quality criteria reflect a knowledge of the
capacity for environmental accumulation, persistence. and effects of specific toxicants in specific
aquatic systems.
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The Philosophy of Quality Criteria
Ions of toxic materials frequently cause adverse effects because they pass through the semi-
permeable membrances of an organism. Molecular diffusions through membranes may occur
for some compounds such as pesticides. polychlorinated biphenyls and other toxicants. Some
materials may not pass through membranes in their natural or waste-discharged state. but in
water they may be converted to states that have increased ability to affect organisms. For
example. certain microorganisms can methylate mercury. thus producing a material that
more readily enters physiological systems. Some materials may have multiple effects; for
example. an iron salt may not be toxic; an iron floc or gel may be an irritant or clog fish gills
to effect asphyxiation; iron at low concentrations can be a trace nutrient but at high concentrations
it can be toxicant.. Materials also can affect organisms if their metabolic byproducts cannot be
excreted. Unless otherwise stated. criteria are based on the total concentration of the substance
because an ecosystem can produce chemical. physical. and biological changes that may be detri-
mental to organisms living in or using the water.
In prescribing water quality criteria certain fundamental principles dominate the reasoning process.
[n establishing a level or concentration as a criterion for a given constituent it was assumed that
other factors within the aquatic environment are acceptable to maintain the integrity of the water.
[nterrelationships and interactions among organisms and their environment. as well as the inter-
relationships of sediments and the constituents they contain to the water above. are recognized
as fact.
Antagonistic and synergistic reactions among many quality constituents in water also are recognized
as fact. The precise definition of such reactions and their relative effects on particular segments
of aquatic life have not been identified with scientific precision. Historically. much of the data
to support criteria development was of an ambient concentration-organism response nature.
Recently. data are becoming available on long term chronic effects on particular species. Studies
now determine carcinogenic. teratogenic. and other insidious effects of toxic materials.
Some unpolluted waters in the Nation may exceed designated criteria for particular constituents
There is variability in the natural quality of water and certain organisms become adapted to that
quality which may be considered extreme in other areas. Likewi~~.. it is recognized that a single
criterion cannot identify minimal quality for the protection of the integrity of water for every
aquatic ecosystem in the Nation. To provide an adequate degree of safety to protect against long
term effects may result in a criterion that cannot be detected with present analytical tools. In
some cases. a mass balance calculation can provide a means of assurance that the integrity of the
waterway is not being degraded.
Water quality criteria do not have direct regulatory impact. but they form the basis for judgment
in several Environmental Protection Agency programs that are derived from water quality con-
siderations. For example. water quality standards developed by the States under Section 303 of
the Act and approved by EPA are to be based on the water quality criteria. appropriately modified
to take account of local conditions. The local conditions to be considered include actual and pro-
jected uses of the water. natural background levels of particular constituents. the presence or
absence of sensitive important species. characteristics of the local biological community. tempera-
ture and weather. flow characteristics. and synergistic or antagonistic effects of combinations of
pollutants.
Similarly. by providing a judgment on desirable levels of ambient water quality. water quality
criteria are the starting point in deriving toxic pollutant effluent standards pursuant to Section
307(a) of the Act. Other EPA programs that make use of water quality criteria include drinking
water standards. the ocean dumping program. designation of hazardous substances. dredge spoil
criteria development. removal of in-place toxic materials. thermal pollution. and pesticide
registration.
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The Philosophy of Quality Criteria
To provide the water resource protection for which they are designed. quality criteria should
apply to virtually all of the Nation I s navigable waters with modifications for local conditions
as needed. To violate quality criteria for any substantial length of time or in any substantial
portion of a waterway may result in an adverse effect on aquatic life and perhaps a hazard to
man or other consumers of aquatic life.
Quality criteria have been designed to provide long term protection. Thus. they may provide
a basis for effluent standards, but it is not intended that criteria values become effluent standards.
It is recognized that certain substances may be applied to the aquatic environment with the con-
currence of a governmental agency for the precise purpose of controlling or managing a portion
of the aquatic ecosystem; aquatic herbicides and piscicides are examples of such substances.
For such occurrences. criteria obviously do not apply. It is recognized further that pesticides
applied according to official label instructions to agricultural and forest lands may be washed to
a receiving waterway by a torrential rainstorm. Under such conditions it is believed that such
diffuse source inflows should receive consideration similar to that of a discrete effluent discharge
and that in such instances the criteria should be applied to the principal portion of the waterway
rather than to that peripheral portion receiving the diffuse inflow.
The format for presenting water quality criteria includes a concise statement of the dominat criterion
or criteria for a particular constituent followed by a narrative intro duction. a rationale that includes
justification for the designated criterion or criteria. and a listing of the references cited within
the rationale. An effort has been made to restrict supporting data to those which have either been
published or are in press awaiting publication. A particular constituent may have more than one
criterion to ensure more than one water use or condition. i. e.. hard or soft water where applicable.
suitability as a drinking water supply source. protection of human health when edible portions of
selected biota are consumed. provision for recreational bathing or water skiing. and permitting an
appropriate factor of safety to ensure protection for essential warm or cold water associated biota.
Criteria are presented for those substances that may occur in water where data indicate the potential
for harm to aquatic life. or to water users. or to the consumers of the water or of the aquatic life.
or to water users. or to the consumers of the water or of the aquatic life. Presented criteria do
not represent an all-inclusive list of constituent contaminants. Omtgsions from criteria should not
be construed to mean that an omitted quality constituent is either unimportant or nonhazardous.
DESCRIPTORS:
This outline was extracted from: Quality Criteria
for Water - 1976.
Bioassay. Water Quality Criteria
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SIGNIFICANCE OF IIUMITING FACTORS" TO POPUlATION VARIATION
I
INTRODUCTION
A All aquatic organisms do not react uniformly
to the various chemical, physical and
biological features in their environment.
Through normal evolutionary processes
various organisms have become adapted
to certain combinations of environmental
conditions. The successful development
and maintenance of a population or community
depend upon harmonious ecological balance
between environmental conditions and
tolerance of the organisms to variations
in one or more of these conditions.
B A factor whose presence or absence exerts
some restraining influence upon a population
through incompatibility with species
requirements or tolerance is said to be a
limiting factor. The principle of limiting
factors is one of the major aspects of the
environmental control of aquatic organisms
(Figure 1).
II
PRINCIPLE OF UMITING FACTORS
This principle rests essentially upon two basic
concepts. One of these relates organisms to
the environmental supply of materials essential
for their growth and development. The second
pertains to the tolerance which organisms
exhibit toward environmental conditions.
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,uNLIMiTeD GROWTH
" DECREAse IN
" ,,'- ..mn TIOMNS
, ,"
" ,,/ EQUILIBRIUM WITH
l. " ONMENT
,
-",.., INCREASE IN
'. -TiMT11"IONS
\ POPULATION DetLINf
TIME
Figure 1.
The relationships of limiting factors
to population growth and development.
BI. ECO. 20a. 1.78
A Liebig's Law of the Minimum enunciates
the first basic concept. In order for an
organism to inhabit a particular environ-
ment; specified levels of the materials
necessary for growth and development
(nutrients, respiratory gases, etc.) must
be present. If one of these materials is
absent from the environment or present
in minimal quantities, a given species
will only survive in limited numbers, if
at all (Figure 2).
Copper, for example,
is essential in trace amounts for
many species.
III
V
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Significance of "Limiting Factors" to Population Variation
d Where strontium is abundant, mollusks
are able to substitute it, to a partial
extent, for calcium in their shells
(Odum, 1959).
2 If a material is present in large amounts,
but only a small amount is available for
use by the organism, the amount available
and not the total amount present deter-
mines whether or not the particular
material is limiting (calcium in the form
of CaC03).
B Shelford pointed out in his Law of Tolerance
that there are maximum as well as minimum
values of most environmental factors which
can be tolerated. Absence or failure of an
organism can be controlled by the deficiency
or excess of any factor which may approach
the limits of tolerance for that organism
(Figure 3).
Minimum Limit of
Toleration
Range of Optimum
of Factors
Maximum Umit of
Toleration
A baent
Greatest Abundance
Decreasing
Abundance
Decreasing
Abundance
Figure 3.
Shelford's Law of Tolerance.
1 Organisms have an ecological minimum
and maximum for each environmental
factor with a range in between called
the critical range which represents the
range of tolerance (Figure 2). The
actual range thru which an organism can
grow, develop and reproduce normally
is usually much smaller than its total
range of tolerance.
2 Purely deleterious factors (heavy metals,
pesticides, etc.) have a maximum
tolerable value, but no optimum (Figure 4).
2
Figure 4.
A baent
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CONCENTRA TION
Relationship of purely harmful
factors and the abundance of
organisms.
3 Tolerance to environmental factors
varies widely among aquatic organisms.
a A species may exhibit a wide range
of tolerance toward one factor and a
narrow range toward another. Trout,
for instance, have a wide range of
tolerance for salinity and a narrow
range for temperature.
b All stages in the life history of an
organism do not necessarily have the
same ranges of tolerance. The
period of reproduction is a critical
time in the life cycle of most
organisms.
c The range of tolerance toward one
factor may be modified by another
factor. The toxicity of most sub-
stances increases as the temperature
increases.
d The range of tolerance toward a given
factor may vary geographically within
the same species. Organisms that
adjust to local conditions are called
ecotypes.
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Significance of "Limiting Factors" to Population Variation
e The range of tolerance toward a given
factor may vary seasonally. In general
organisms tend to be more sensitive
to environmental changes in summer
than in other seasons. This is
primarily due to the higher summer
temperatures.
4 A wide range of distribution of a species
is usually the result of a wide range of
tolerances. Organisms with a wide
range of tolerance for all factors are
likely to be the most widely distributed,
although their growth rate may vary
greatly. A one-year old carp, for
instance, may vary in size from less
than an ounce to more than a pound
depending on the habitat.
5 To express the relative degree of
tolerance for a particular environmental
factor the prefix eury (wide) or steno
(narrow) is added to a term for that
feature (Figure 5).
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Significance of "Limiting Factors" to Population Variation
B Because of the complexity of the aquatic
environment, it is not always easy to
isolate the factor in the environment that
is limiting a particular population.
Premature conclusions may result from
limited observations of a particular
situations. Many important factors may
be overlooked unless a sufficiently long
period of time is covered to permit the
factors to fluctuate within their ranges of
possible variation. Much time and money
may be wasted on control measures ~ithout
the real limiting factor ever being dis-
covered or the situation being improved.
C Knowledge of the principle of limiting
factors may be used to. limit the number
of parameters that need to be measured or
observed for a particular study. Not all
of the numerous physical, chemical and
biological parameters need to be measured
or observed for each study undertaken.
The aims of a pollution survey are not to
make and observe long lists of possible
limiting factors but to discover which
factors are significant, how they bring
about their effects, the source or sources
of the problem, and what control measures
should be taken.
3-4
D Specific factors in the aquatic environment
determine rather precisely what kinds of
organisms will be present in a particular
area. Therefore, organisms present or
absent can be used to indicate environ-
mental conditions. The diversity of
organisms provides a better indication of .
environmental conditions than does any
single species. Strong physio-chemical
limiting factors tend to reduce the diversity
within a community; more tolerant species
are then able to undergo population growth.
REFERENCES
1 Odum, Eugene P. Fundamentals of
Ecology, w. B. Saunders Company,
Philadelphia. (1959)
2 Reid, George K. Ecology of Inland Waters
and Estuaries. Reinhold Publishing
Corporation, New York. (1961)
3
Rosenthal H. and Alderdice, D. F.
Sublethal Effects of Environmental
Stressors, Natural and Pollutional,
on Marine Fish Eggs and Larvae.
J. Fish. Res~..Board Can. 33:2047-2065,
1976.
This outline was prepared by John E.
Matthews, Aquatic Biologist, R:0bert S. Kerr
Water Rese~rch Cente.r. Ada. Qklahoma.
pescriptors: Population, Limiting Factors
-------
GLOBAL ENVIRONMENTAL QUALITY
I
FROM LOCAL TO REGIONAL TO qLOBAL
PROBLEMS
A Environmental problems do not stop at
national frontiers, or ideological barriers.
Pollution in the atmosphere and oceans
taints all nations, even those benignly
favored by geography, climate, or natural
resources.
1 The smokestacks of one country often
pollute the air and water of another.
2 Toxic effluents poured into an inter-
national river can kill fish in a
neighboring nation and ultimately
pollute international seas.
B In Antarctica, thousands of miles from
pollution sources, penguins and fish
contain DDT in their fat. Recent layers
of snow and ice on the white continent
contain measurable amounts of lead.
The increase can be correlated with the
earliest days of lead smelting and com-
bustion of leaded gasolines. PC B' s are
universally distributed.
C International cooperation, therefore, is
necessary on many environmental fronts.
1 Sudden accidents that chaotically
damage the environment - such as oil
spills from a tanker at sea - require
international cooperation both for
prevention and for cleanup.
2 Environmental effects cannot be
effectively treated by unilateral action.
3 The ocean can no longer be considered
a dump.
D "One of the penalties of an ecological
education is that one lives alone in a
world of wounds. Much of the damage
inflicted on land is quite invisible to
laymen. An ecologist must either harden
his shell and make believe that the conse-
quences of science are none of his
BI. ECO. hum. 2f. 1. 78
business, or he must be the doctor who
sees the marks of death in a community that
believes itself well and does not want to
be told otherwise." Aldo Leopold
u
CHANGES IN ECOSYSTEMS ARE
OCCURRING CONTINUOUSLY
A Myriad interactions take place at every
moment of the day as plants and animals
respond to variations in their surroundings
and to each other. Evolution has produced
for each species, including man, a genetic
composition that limits how far that
species can go in adjusting to sudden
changes in its surroundings. But within
these limits the several thousand species
in an ecosystem, or for that matter, the
millions in the biosphere, continuously
adjust to outside stimuli. Since inter-
actions are so numerous, they form long
chains of reactions.
B Small changes in one part of an ecosystem
are likely to be felt and compensated for
eventually th~).lghout the system.
Dramatic examples of change can be seen
where man has altered the course of
nature. It is vividly evident in his well-
intentioned but poorly thought out tampering
with river, lake, and other ecosystems.
1 The Aswan High Dam
2 The St. Lawrence Seaway
3 Lake Kariba
4 The Great Lakes
5 Valley of Mexico
6 California earthquake (Scientific
American 3981, p. 333)
7 Everglades and the Miami, Florida
Jetport
8 Copperhill, Tennessee (Copper Basin)
9 (You may add others)
4-1
-------
Global Deteriorarion and Our Environmental Crisis
C Ecosystem Stability
1 The stability of a particular ecosystem
depends on its diversity. The more
interdependencies in an ecosystem, the
greater the chances that it will be able
to compensate for changes imposed
upon it.
2 A cornfield or lawn has little natural
stability. If they are not constantly
and carefully cultivated. they will not
remain cornfields or lawns but will
soon be overgrown with a wide variety
of hardier plants constituting a more
stable ecosystem.
3
The chemical elements that make up
living systems also depend on complex,
diverse sources to prevent cyclic
shortages or oversupply.
4
Similar diversity is essential for the
continued functioning of the cycle by
which atmospheric nitrogen is made
available to allow life to exist. This
cycle depends on a wide variety of
organisms, including soil bacteria and
fungi, which are often destroyed by
pesticides in the soil.
5 A numerical expression of diversity
is often used in defining stream water
quality.
D Biological Pollution
Contamination of living native biotas by
introduction of exotic life forms has been
called biological pollution by Lachner
et ale Some of these introductions are
compared to contamination as severe as
a dangerous chemical release. They
also threaten to replace known wildlife
resources with species of little or un-
known value.
1
Tropical areas have especially been
vulnerable. Florida is referred to as
"a biological cesspool of introduced life. "
4-2
2 Invertebrates
a Asian Clams have a pelagic veliger
larvae, thus, a variety of hydro
installations are vulnerable to sub-
sequent pipe clogging by the adult
clams.
b Melanian snails are intermediate
hosts for various trematodes
parasitic on man.
'3
Vertebrates
a At least 25 exotic species of fish
have been established in North
America.
b Birds, including starlings and
cattle egrets.
c Mammals, including nutria.
4 Aquatic plants
Over twenty common exotic species
are growing wild in the United States.
The problem of waterway clogging has
been especiiilly severe in parts of the
Southeast. .
5
Pathogens and Pests
Introduction of insect pests and tree
pathogens have had severe economic
effects.
III
LAWS OF ECOLOGY
A Four principles have been enunciated by
Qr. Barry Commoner.
1
Everything is connected to everything
else.
2
Everything must go somewhere.
3 Nature knows best.
4 There is no such thing as a free lunch.
8 These may be summarized by the principle.
"you can't do just one thing. "
-------
Global Deterioration and Our Environmental Crisis
IV THE THREE PRINCIPLES OF
ENVIRONMENTAL CONTROL (Wolman)
A You can't escape.
n You have to organize.
C You have to pay.
V LEOPOLD'S PRINCIPLE OF BIOTIC
CAPITAL
"The releases of biotic capital tend to
becloud or postpone the penalties of
violence". Can you apply this to other
parts of this outline?
VI POLLUTION COMES IN MANY PACKAGES
A The sources of air. water. and land
pollution are interrelated and often
interchangeable.
1 A single source may pollute the air
with smoke and chemicals. the land
with solid wastes. and a river or lake
with chemical and other wastes.
2
Control of air pollution may produce
more solid wastes, which then pollute
the land or water.
3
Control of wastewater effluent may
convert it into solid wastes, which
must be disposed of on land, or by
combustion to the air.
4 Some pollutants - chemicals. radiation,
pesticides - appear in all media.
B "Disposal" is as important and as costly
as purification.
VII PERSISTENT CHEMICALS IN THE
ENVIRONMENT
Increasingly complex manufacturing processes,
coupled with rising industrialization, create
greater amounts of exotic wastes potentially
toxic to humans and aquatic life.
They may also be teratogenic (toxicants
responsible for changes in the embryo with
resulting birth defects, ex., thalidomide).
muta,genic (insults which produce mutations,
ex., radiation), or carcinogenic (insults
which induce cancer, ex., benzopyrenes) in
effect. Most carcinogens are also muta-
genic. For all of these there are no thresh-
hold levels as in toxicity. Fortunately there
are simple rapid tests for mutagenicity using
bacteria. Tests with animals are not always.
conclusive.
A Metals - current levels of cadmium, lead,
and other substances are a growing concern
for they affect not only fish and wildlife but
ultimately man himself. Mercury pollution,
for example, has become a serious problem,
y'et ~ercury has been present on earth since
hme lmmemorial.
B Pesticides
1
A pesticide and its metabolites may
move through an ecosystem in many
ways. Hard (pesticides which are
persistent, having a long half-life in
the environment includes the organo.-
chlorines. ex.. DDT) pesticides
ingested or otherwise borne by the
target species will stay in the
environment. possibly to be recycled
.:>1' conce{ltra~ed further through the
natural action of food chains if the
species is eaten. Most of the volume
of pesticides do not reach their target
a tall.
2
Biological magnification
Initially, low levels of persistent
pesticides in air, soil, and water
may be concentrated at every step
up the food chain. Minute aquatic
organisms and scavengers. which
screen water and bottom mud having
pesticide levels of a few parts per
billion. can accumulate levels
measured in parts per million -
a thousandfold increase. The sediments
including fecal deposits are continuously
recycled by the bottom animals.
a Oysters, for instance. will con-
centrate DDT 70,000 times higher
in their tissues than it's concentration
in surrounding water. They can
also partially cleanse themselves
in water free of DDT.
4-3
-------
Global Deterioration and Our Environmental Crisis
b Fish feeding on lower organisms
build up concentrations in their
visceral fat which may reach several
thousand parts per million and levels
in their edible flesh of hundreds of
parts per million.
c
Larger animals, such as fish-
eating gulls and other birds, can
further concentrate the chemicals.
A survey on organochlorine residues
in aquatic birds in the Cnadian
prairie provinces showed that
California and ring-billed gulls were
among the most contaminated.
Since gulls breed 0 colonies, breed-
ing population changes can be
detected and related to levels of
chemical contamination. Ecological
research on colonial birds to monitor
the effects of chemical pollution on
the environment is useful.
C "Polychlorinated biphenyls" (PCB's).
PCB's are used in plasticizers, asphalt,
ink, paper, and a host of other products.
Action has been taken to curtail their
release to the environment, since their
effects are similar to hard pesticides.
D Other compounds which are toxic and
accumulate in the ecosystem:
1
Phalate esters - may interfere with
pesticide analyses
2 Benzopyrenes
E Refractory compounds like pentachloro-
phenal &.l1d hexachlorophene are poorly
removed by both water treatment plants
and wastewater treatment plants.
F It is estimated that 80% to 90% of cancers
are caused ])y chemicals both in the work-
ing environment and total environment.
This is shown by high risk industries and
living areas.
G Most of the problems of persistent and
dangerous chemicals in the environment
are" after-the-fact". The solution
obviously is tied to prevention. This is
extremely complicated by economics,
4-4
ignorance, and decision as to risks
involved. Some advertising slogans now
have more than an intended meaning.
H Wittingly or unwittingly we have all become
a King Mithridates. And even a fish is no
longer a fish!
VIII EXAMPLES OF SOME EARLY WARNING
SIGNALS THAT HAVE BEEN DETECTED
BUT FORGOTTEN, OR IGNORED.
A Magnetic micro-spherules in lake sediments
now used to detect changes in industriali-
zation indicate our slowness to recognize
indicators of environmental change.
B Salmonid fish kills in poorly buffered clean
lakes in Sweden. Over the past years there
. had been a successive increase of S02 in the
air and precipitation. Thus, air-borne con-
tamination from industrialized European
countries had a great influence on previously
unpolluted waters and their life.
C Minimata, Japan and mercury pollution.
D Organochlorine levels in commercial and
sport fishing stocks, ex., the lower
Mississippi River fish kills.
E You may complete the following:
1
2
IX SUMMARY
A Ecosystems of the world are linked
together through biogeochemical cycles
which are determined by patterns of
transfer and concentrations of substances
in the biosphere and surface rocks.
B Organisms determine or strongly influence
chemical and physical characteristics of
the atmosphere, soil, and waters.
C The inability of man to adequately predict
or control his effects on the environment
is indicated by his lack of knowledge con-
cerning the net effect of atmospheric
pollution on the earth I s climate.
-------
Global Deterioration and Our Environmental Crisis
D Serious potential hazards for man which
are all globally dispersed, are
radionuclides, organic chemicals, pest-
icides, and combustion products.
E Environmental destruction is in lock-
step with our population growth.
ACKNOWLEDGEMENT:
This outline has been extracted in part from
the first annual report of the Council on
Environmental Quality: Environmental
Quality. US GPO, Washington, DC.
326 pp. $1.75. 1970
REFERENCES
1 Goldman, Charles R. Is the Canary
Dying? The time has come for man,
miner of the worlds resources, to
surface. Calif. Medicine 113:21-16. 1970
2 Lachner, Ernest A., Robins, C. Richard,
and Courtenay, Walter R., Jr.
Exotic Fishes and Other Aquatic
Organisms Introduced into North
America. Smithsonian Contrib. to
Zool. 59: 1-29. 1970
3 Nriagu, Jerome O. and Bowser, Carl J.
The Magnetic Spherules in Sediments of
Lake Mendota, Wisconsin. Water
Res. 3:833-842. 1969
4 Commoner, Barry. The Closing Circle,
Nature, Man, and Technology. Alfred
A. Knopf. 326 p. 1971.
!; Dansereau, Pierre ed. Challenge for
Survival. Land, Air, and Water for
Man in Megalopolis, Columbia Univ.
Press. 235 p. 1970.
6 Wiens, John A. ed. Ecosystem Structure
and Function. Oregon State Univ. Press.
176 p. 1972.
7 Leopold, Aldo. A Sand CQunty Almanac
with Essays on Conservation from
Round River. Sierra Club! Ballantine
Books. 295 p. 1970.
8 Sondheimer, Ernest B. and Simeone,
John B. Chemical Ecology. Academic
Press. 336 p. 1970.
9 Environmental Quality. Second Annual
Report of the Council on Environmental
Quality. August 1971. Fourth Annual
Report 1973.
10 Toxic Substances. Council on
Environmental Quality. 25 p.
April 1971.
11 The Changing Chemistry of the Oceans;
Proc. 20th Nobel Symposium.
Wiley. 1972.
This outline was prepared by R. M. Sinclair,
National Training Center, MOTD, OWPO.
USEPA, Cincinnati, Ohio 45268.
Descriptors: Environmental Effects,
Ecosystems
4-5
-------
ECOLOGY PRIMER
(from Aldo Leopold's A SAND COUNTY ALMANAC)
I
Ecology is a belated attempt to convert
our collective knowledge of biotic materials
into a collective wisdom of biotic manage-
ment.
II The outstanding scientific discovery of
the twentieth century is not television or
radio. but rather the complexity of the
land organism. .
I I lOne of the penalties of an ecological ed-
ucation is that one lives alone in a world
of wounds. Much of the damage inflicted
on land is quite invisible to laymen. An
ecologist must either harden his shell and
make believe that the consequences of
science are none of his business. or he
must be the doctor who sees the marks of
death in a community that believes itself
well and does not want to be told other-
wise.
IV Ecosytems have been sketched out as
pyramids. cycles. and energy circuits.
The concept of land as an energy circuit
conveys three basic ideas:
A That land is not merely soil.
B That the native plants and animals kept
the energy circuit open; others mayor
may not.
C That man-made changes are of a different
order than evolutionary changes. and have
effects more comprehensive than is
intended or foreseen.
V The process of altering the pyramid for
human occupation releases stored energy.
and this often gives rise. during the
pioneering period. to a deceptive exuber-
ance of plant and animal life. both wild
and tame. These releases of biotic
capital tend to becloud or postpone the
penalties of violence.
BI. ECO. 26.6.76
VI A thing is right when it tends to preserve
the integrity. stability. and beauty of the
biotic community. It is wrong when it
tends otherwise.
VII Every farm is a textbook on animal ecology;
every stream is a textbook on aquatic
ecology; conservation is the translation of
the book.
. VIII There are two spiritual dangers in not
owning a farm:
A One is the danger of supposing that break-
fast comes from the grocery.
B The other. that heat comes from the
furnace.
IX In general. the trend of the evidence
indicates that in land. just as in the human
body. the symptoms may lie in one organ
and the cause in another. The practices
we now call conservation are, to a large
extent. local alleviations of biotic pain.
They are necessary. but they must not be
confused with cures.
X An Atom at large in the biota is too free to
know freedom; an atom back in the sea has
forgotten it. For every atom lost to the sea,
the prairie pulls another out of the decaying
rocks. The only certain truth is that its
creatures must suck hard, live fast, and die
often. lest its losses exceed its gains.
REFERENCES
1 Leopold. Luna B. (ed. ).Round River.
Oxford University Press. 1953.
2 Leopold. Aldo. A Sand County Almanac.
Oxford University Press. 1966.
This outline was prepared by R. M. Sinclair.
National Training Center. MOTD. OWPO. USEPA.
Cincinnati. Ohio 45268.
Descriptor: Ecology
5-1
-------
CLASSIFICATION OF COMMUNITIES, ECOSYSTEMS, AND TROPHIC LEVELS
A COMMUNITY is an assemblage of popu- ,
lations of plants. animals, bacteria. and
fungi that live in an environment and inter-
act with one another, forming together a
distinctive living system with its own com-
position, structure, environmental rela-
tions. development. and function.
11 An ECOSYSTEM is a community and its
environment treated together as a function-
al system of complementary relationships.
and transfer and circulation of energy and
matter. (a delightful little essay on the
odyssey of atoms X and Y through an
ecosystem is in Leopold's. A Sand County
Almanac).
III TROPHIC levels are a convenient means
of classifying organisms according to
nutrition. or food and feeding.
A PRODUCER. the photosynthetic plant or
first organism on the food chain sequence.
Fossil fuels were produced photosynthe-
tically:
B Herbivore or primary CONSU1VlER, the
first animal which feeds on plant food.
C First carnivore or secondary CONSUMER,
an animal feeding on a plant-eating animal.
D Second carnivore or tertiary CONSUME R
feeding on the preceding.
E Tertiary carnivore.
F Quaternary carnivore.
G DECOMPOSERS, OR REDUCERS, bact-
eria which break down the above organisms.
Often called the middlemen or stokers of
the furnace of photosynthesis.
H Saprovores or DETRITIVORES which feed
on bacteria and/ or fungi.
HI. ECO. 25. 1. 78
IV Taxonomic Groupings
A TAXOCENES, a specific group of organ-
isms. Ex. midges. For obvious reasons
most systematists (taxonomists) can
speclalize in only one group of organisms.
This fact is difficult for the non-biologist to
grasp:
B Size. which is often dictated by the
investigator I s sampling equipment
and specific interests.
V Arbitrary due to organism habitat
preferences, available sampling devices.
whims of the investigator, and mesh sizes of
nets and sieves.
A PLANKTON, organisms suspended in a
body of water and at the mercy of currents.
This group has been subject to numerous
divisional schemes. Plants are PHYTO-
PLANKTON, and animals, ZOOPLANKTON.
Those retained. by nets are obviously, NET
PLANKTON. Those passing thru even the
finest meshed nets are NANNOPLANK TON.
B PERIPHYTON, the community of micro-
organisms which grow on submerged
substrates. Literal meaning lito grow
around plants ", however standard glass
micro slides are placed in the aquatic
habitat to standardize results.
C BENTHOS, is often used to mean
MACROlNVERTEBRA TES, although there
are benthic organisms in other plant. animal,
and protist groups. Benthic refers strictly
to the bottom substrates of lakes, streams,
and other water bodies.
D MACROlNVERTEBRA TES, are animals
retained on a No. 30 mesh screen (approx-
imately 0.5 mm) and thus visible to the
naked eye.
6-1
-------
Classification of Communities. Ecosystems and Trophic Levels
E MACROPHYTES, the larger aquatic plants
which are divided into emersed, floating.
and submersed communities. Usually
vascular plants but may include the larger
algae and "primitive" plants. These have
posed tremendous economic problems in the
large man-made lakes, especially in
tropical areas.
F NEKTON, in freshwater. essentially fish,
salamanders, and the larger crustacea.
In contrast to PLANKTON, these
organisms are not at the mercy of the
current.
G NEUSTON, or PLEUSTON, are inhabit-
ants of the surface film (meniscus organ-
isms), either supported by it. hanging
from, or breaking through it. Other
organisms are trapped by this neat little
barrier of nature. The micro members
of this are easily sampled by placing a
clean cover slip on top of the surface
film then either leaving it a specified
time or examining it immediately under
the microscope.
H DRIFT, macroinvertebrates which drift
with the streams current either period-
ically (diel or 24 hour). behaviorally.
catastrophically or incidentally.
I
BIOLOGICAL FLOCS. are suspended
microorganisms that are formed by
various means. In wastewater treat-
ment plants they are encouraged in con-
crete aeration basins using diffused air
or oxygen (the heart of the activated
sludge process).
6-2
J IVIANIPULATED SUBSTRATE COM-
MUNITIES. Like the preceding
community. these are manipulated
by man. Placing artificial or natural
substrates in a body of water will cause
these communities to appear thereon.
K We will again emphasize ARBITRARY,
because organisms confound our neat
little schemes to classify them. Many
move from one community to another for
various reasons. However, all these
basic schemes do have intrinsic value.
provided they are used with reasonl
This outline was prepared by R. M. Sinclair,
Aquatic Biologist, National Training Center,
MOTD, OWPO, USEPA, Cincinnati, Ohio 45268.
Descriptors: Biological Communities
REFERENCES
1
Leopold, Aldo. A Sand County Almanac.
Oxford Univ. Press. 1966
2 Peters. Robert Henry. The Unpredictable
Problems of Tropho-dynamics.
Env. BioI. Fish 2:97-101. 1977.
-------
BIOASSAY AND BIOMONITORING
I
INTRODUCTION
A An assay is an evaluation.
B A bioassay is an evaluation in which living
organisms provide the scale.
1 The scale or degree of response may
be the rate of growth or decrease of a
population, colony, or individual; a
behaviorial, physiological, or repro-
ductive response, or simply a live or
no-live response.
2 All types of bioassays may have a role
to play in water quality evaluation at
one time or another.
3 The particular group of bioassays
discussed below are those which con-
tribute to the evaluation of the effects
of liquid wastes on aquatic environments
in which experimental organisms such
as fish are subjected to a series of
concentrations of a known or suspected
toxicant under adequately controlled
conditions for a stipulated period of
time.
C Historical Highlights
1 Prior to 1940, there was little or no
uniformity in performing or reporting
bioassays of water pollutants.
2 By mid-forties, the need for a
standardized technique was becoming
painfully obvious.
a The A tlantic Refining Company
privately published the first
statement of what is today, basically,
our standard method (Hart, Doudoroff,
and Greenbank, 1945).
b This was refined and accorded wide
(but not universal) industrial,
academic, and governmental
acceptance in 1951 (Doudoroff, et aI,
1951).
BI. BIO. met. 17a. 1. 78
3 This method was first developed for
the use of fishes, but has been found
adaptable to a wide variety of
organisms.
D Other Types or Plans of Bioassay
1 Many other designs for th~ expression
of toxicity have been devised such as
those based on time-concentration
curves. Each has its advantages and
proponents, but the basic Standard
Method design remains the most
widely used.
a In situ exposure of experimental
organisms in cages or live cars,
at selected sites above and below
a suspected point or pollution is an
obvious and time tested procedure,
but lacks the precision of laboratory
tests. It has the advantages of
popular appeal, and of expressing
actual environmental conditions.
b The familiar BOD test is a bioassay
of the organic content of water
subject to biodegradation.
E Biomonitoring
Water quality surveillance or monitoring
by means of observing biota can be con-
sidered from two aspects: field and
laboratory. It differs from bioassay
primarily in the objective: a bioassay
is an attempt to determine a specific
defined value or threshold, whereas a
biomonitoring operation is an attempt to
use living organisms to ascertain whether
or not aquatic life is endangered.
1 Periodic biological field surveys,
samples, or other observation may
demonstrate recent excessive pollution
for example.
2 Organisms in a series of flow,;,through
tanks in a laboratory may dem~>nstrate
the occurrence of an unacceptable
7-1
-------
Bioassay and Biomonitoring
increase in the toxicity of an effluent,
without measuring "how much" or
"what. "
II
THE STANDARD METHOD BIOASSAY
A Introduction
1 This procedure is intended for use by
industrial and other laboratories.
2 Its objective is to evaluate the toxicity
of wastes and other water pollutants
to fish or other aquatic organisms.
3 Potential applications. are numerous.
a Dilution and/ or treatment necessary
to avoid acute toxic effects can be
estimated.
b The efficacy of an existing treatment
can be tested.
c The potential usefulness of a proposed
treatment can be estimated.
4 The design of the test need not involve
a chemical knowledge of the toxicant.
a Synergism, antagonism, and other
interactions of chemical components
cannot always be anticipated, but
are automatically included in the
result.
b All chemical and physical information
available is, however, essential to
the adequate interpretation and
application of test results.
5 The test is best used for local
application. Generalizations should
be made with great caution.
6 Field observations should be made of
results of application over a significant
period of time.
7 Careful distinction should be made
between fish mortality due to a
physiological toxicant, and that due
to lack of 00.
7-2
8 A uniform testing procedure is
essential to effective action in water
pollution control.
B Routine Procedure for Static Tests
1 Test organisms should be fish or other
organisms of local significance.
a The most sensitive species
available should be selected, but:
b They should be species which are
amenable to captivity.
c They should be accurately identified.
d They should be relatively uniform
in size. Individuals less than 3
inches in length are usually most
convenient.
e They should be healthy and
thoroughly acclimated to the
laboratory.
f A careful record should be kept
of their origin, handling, and
condition.
2 ~ ~ should preferably be taken
from the receiving stream just above
the discharge being evaluated or in a
lake or estuary, beyond the influence
of the discharge.
a If this is unsuitable, cleaner but
similar waters from a more
remote station may be substituted.
b Artificial "standard" waters are
not recommended for general use,
although many formulae have been
proposed.
c In estuarine situations, a series of
tests (marine grid) should~,
using waters of high and low
salinities as characteristic of the
region.
3 Other experimental conditions
-------
Bioassay and Biomonitoring
a Temperature. The tests should be
performed at a uniform temperature
in the upper part of the expected
summer range, e. g., 25 t 20 C for
warm water fish, and 15 t 20 C for
cold water species.
b Test containers should be of glass,
widemouthed "pickle jugs" or
battery jars are satisfactory. Five
and one gallon sizes are both useful,
but the larger size is required for
conclusive results.
TABLE I
A Guide to the Selection of Experimental
Concentrations, Based on Progressive
Bisection of Intervals on a Logarithmic
Scale.
I
Col. 1 Col. 2 Col. 3 Col. 4 Col. 5
10.0
8.7
7.5
6.5
5.6
4.9
4.2
3.7
3.2
2.8
2.4
2.1
1.8
1. 55
1.35
1. 15
1.0
c A rtificial aeration should not be
used to maintain the dissolved
oxygen concentration. If this falls
below approximately 4 or 5 ppm at
any time during the test, fewer fish
should be used per container or an
auxiliary oxygenation procedure
invoked that is designed to avoid
undue loss of volatile toxicants.
d The number of test animals should
not be less than 10 per concentration
for reliable conclusions; these may
be distributed between two or more
containers.
Effluents of unknown, mixed, or
variable composition are usually
best expressed as percent by volume;
while pure substances, or specific
analyzab~e components are usually
expressed' as milligrams per liter
(ppm). A control or reference tank
containing dilution water only (with
no toxicant) is essential, to dem-
onstrate that all experimental
organisms would have survived had
it not been for the toxicant being
tested.
e Ratio of fish to solution. There
should be not more than one gram
of fish per liter of test solution.
4 Experimental procedure
a All dilutions for a given run should
be prepared from the same sample.
b Duration. Tests should be run for
at least 48 hours, preferable 96.
f
Expression of results. The measure
of relative toxicity is the tolerance
limit (symbol: TL). The time of
exposure "t" must be shown along
with the percentage of fish surviving
(written as a postscript). For
example, a 96 hour TL50 (optional:
TL50 96 hr) of a toxic substance is
that concentration in which 50% of
the experimental organisms survive
for 96 hours. (Figure 1)
c Dead fish should be removed as soon
as observed. Survivors should be
counted and recorded each 24 hours.
d Feeding during the test should be
a voided.
e Experimental concentrations.
Any appropriate series of concen-
trations may be used. A logarithmic
series such as is suggested in
Table I is very convenient.
7-3
-------
Bioassay and Biomonitoring
1 t) € C I~ I 1 I C a L I~ a n (j E
In aC111€ lOXICIT.y
SUB. LETHAL
CONCEN.
TRATI01iS
CRITICAL RANGE
100
.,"CI .
..
PERCENT
SURVIV AL
~
,
50
--
o
)i(
0-'- INCREASING CONC:NTRA TION OF TOXICANT ~ 100
Figure 1
THE CRITICAL RANGE
X = T 1..5 at Concentration
1) A TLSOt is the equivalent of a
median tolerance limit (TL t).
m
a The toxicant may be volatile.
2) This is analogous to the LCSO
(concentration survived by
SO% of the population) of the
toxicologist, but is more
universally usable with the
parameters encountered in the
water environment, some of
which (such as temperature)
cannot be expressed as
"concentrations. II
b Toxic materials may be masked by
a high BOD.
c The toxicant may be progressively
adsorbed on container walk, fish
slime, metabolized or otherwise
changed so that actual concentrations
in tanks change with time.
2 Standards or requirements other than
those involving toxicity per se may be
involved.
3) TLSO's for 96 hours or less are
arbitrarily referred to as
measures of "acute" toxicity,
while TL50's for longer periods
of time are variously referred to
as sub-acute, chronic, etc.
3 Preliminary and concurrent investi-
gations
a Obtain all available information
about unknown to be tested.
C Special Problems of Static Tests
b Does the material lend itself to this
type of test?
1 Unaerated aquaria with finite quantities
of toxicant are not always satisfactory
(static tests).
c Run feasible on the spot analyses
including 00.
4
-------
d Significant quantities of solutions
removed from test containers for
analysis should be replaced with
similar volume of same dilution.
4 Wastes with a high BOD or COD
a Suggested preliminary tests:
1) Set up two identical exploratory
tests.
2) A era te one but not the other.
3) If great difference develops
between them, special pro-
cedures are indicated.
b Oxygenation or aeration of dilution
water before making dilutions may
help.
c Renewal of solutions at stated inter-
vals (12,24, or 48 hours) is approved.
Fish are not harmed by being care-
fully transferred from one container
to another. It is useful where:
1) Initial DO is adequate but slowly
exhausted.
2) Toxicant is volatile, progressively
adsorbed, precipitated, or other-
wise changed.
D Continuous Flow Procedures
1 Continuous flow procedures imply the
continuous or periodic renewal of the
solutions in the experimental containers,
at the same time maintaining the stated
concentrations (including control). The
variety of devices and flow plans to
accomplish this are almost infinite,
two general principals will be outlined
below: assaying and monitoring.
2 Continuous flow bioassay, general
advantages (Figure 2)
a Materials with moderate oxygen
demands may be tested.
Bioassay and Biomonitoring
b Materials which degrade or are
volatile may be tested.
c Due to the constant removal of
metabolic and other wastes, and
the constant supply of fresh
oxygenated water, fish may be fed
and so maintained over a longer
period of time. Containers must.
of course be maintained in reasonably
clean condition.
E Test Concentrations and End Points for
Continuous Flow Assays
1 Test concentrations are in general less
restricted than for static tests. They
need not be so high as to insure
achieving the desired end point in 48
or 96 hours, although they may be so
set if desired.
2 Geometric type series of concentrations
are still desirable (See Table 1).
3 Sub-lethal levels may be tested over
entire life histories of organisms to
determine long range effects.
4 In general, the setup should be pre-
pared, calibr.ated and operated for
several days, or until the concen-
trations have become chemically and
physically stabilized before introducing
the fish or other experimental
organisms.
F Total Fish Weight and Liquid Volume in
Continuous Flow Assays
In general, the constant renewal of test
solution might appear to make possible
testing more or larger fish in less water.
Actually, flow-through volume and total
weight of fish must be so related that
adequate oxygen is maintained. Further-
more, over the longer periods of time
involved, "lebensraum" (or territory)
must be taken into account. Organisms
must not be crowded to the extent that
aggressive behavior and other ecological
competitive factors are introduced.
7-5
-------
Bioassay and Biomonitoring
CONSTANT HEAD
DILUENT SUPPLY
HEATING OR
COOLING
EQUIPMENT
EFflUENT OR
TOXICANT SUPPLY
IPUMP, MARIOTTE
BOm ,ETC.I
, 0
DILUENT WATER
RESERVOIR
A
I
I
I
I
.-------_!
I
I
I
t
C
I
ACCLIMAT IZING
I
TANK
I \
I \
I \
w~
THERMOSTAT
B
I F
OVERflOW
OVERFLOW TO FLOOR DRAINS
Figure 2
BASIC SETUP FOR CONTINUOUS FLOW BIOASSAY
E to n repreoenta one of several exposure tanks containing 0.
graded serics of dilutions of the toxicant, including one control
with none.
G End Points or Reactions to be Evaluated
by Continuous Flow Assays
3 Water and/ or power failure may
jeopardize an assay experiment after
months of time have been expended.
1 The original and traditional end point
of biological evaluations such as those
discussed here was the death of the
organism. This was simple, direct,
and unequivocal. Current practice,
however, often involves much more
sophisticated reactions such as
reduction in the reproductive capacity,
or a change in the breathing rate
(movement of gills).
4 The expense of a long continued test
may not be justified by the result.
5 The above points demonstrate that in
general, flow through bioassays are
not adapted to day-to-day routine
toxicity determinations.
III
REPORTING INTERPRETATION AND
APPLICATION OF BIOASSAY RESULTS
H Special Problems of Flow Through
Bioassays
A Reporting
1 Due to the physical requirements of
maintaining stated concentrations of
chemicals over long periods of time,
laboratory setups are usually com-
plicated and always require attention
and maintenance.
1 Reports should include an orderly
tabulation of all pertinent data such as:
a The type of setup used and duration
of test
2 The proble~ of disease control
frequently develops in populations held
over a long period of time.
b Identity of experimental animals
6
-------
~,
"
~
c Their source, history, average size
and condition. and number used per
concentration
d Source of. and chemical and physical
analysis of experimental dilution
water
e Experimental temperature
f Volumes of experimental liquid in
each container
g Records of routine analyses such as
DO and pH
h Records of chemiCi'-l analyses of
toxicants in experimental tanks
TLSOt or other end point. and data
from which it was determined.
i
B Interpretation and Application
1 The TL50t is an estimate of the mid-
point of the critical concentration range
the interval between the highest con-
centration at which all test animals
survive. and the lowest at which they
all die (Figure 1).
2 The final step is to extrapolate from
this well established mid concentration
to a safe concentration well below the
"critical concentration range. "
Extrapolating or rather: "application
factors" to accomplish this are still
under development and will probably
not be fully developed for many years.
Available data indicate that these
factors must be variable according to
the toxicant in question acting in com-
bination with the receiving water in
question. and considering the entire
aquatic community.
3 For a more complete discussion, see
FWPCA Water Quality Criteria
(Reference No.9) pp. 55-72.
4 Other considerations
a Radioactive wastes must be evaluated
with regard to their chemical toxicity
as well as their radioactivity.
Bioassay and Biomonitoring
b Sub-acute levels of many toxicants
such as lead, arsenic, cadmium.
etc. may exert a low level chronic
toxicity over a long period of time.
c "Safe levels" of a waste in regard
to toxicity to aquatic life may still
exceed standards of other types such
as color, organic content. suspended
solids. etc.
IV
BIOMONITORING AS COMPARED TO
BIOASSA Y (Figure 3)
A Bioassay is (as stated above) the evaluation
of the effects of stated concentrations of
the test material for given periods of
time.
B Biomonitoring is the use of organisms to
detect change in an effluent (surveillance).
It operates continuously and indefinitely.
C Bioassays typically involve relatively
small flows and employ often especially
prepared (perhaps repeatedly prepared)
batches of experimental material, while
biomonitoring typically involves larger
flows. from operating industries or other
installations.
D Bioassays basically determine:
1 Is the substance deleterious. and if so:
2 How deleterious is it?
E Biomonitoring is useful to
1 Demonstrate the continuous suitability
of an effluent (or a predetermined
dilution thereof) for the survival of the
test organism.
2 Detect a change (usually deleterious)
in the biological acceptability of the
effluent.
3 To detect a change in the effect of a
mixture of the effluent and the receiving
water on the test organism (i. e. to
detect a change in the receiving water).
7-7
-------
Bioassay and Biomonitoring
a
CONSTANT
SOURCE OF
EFflUENT
REGULA TING DEVICE
b
CONSTANT
SOURCE OF
DILUTION WATER
-
d
I
,
EXPOSURE
TANK NO.1
o ~
PROPORTIONING
EXPOSURE
I TANK NO.2
V7
EXPOSURE
9 ~ TANK NO. 3
DEVICE
DEVICE
SCREENED
OVERflOW
OUTlETS
Figure 3
BASIC SETUP FOR BIOMONITORING
F Biomonitoring was originally effective
only with relatively fast acting materials,
or in situations where large changes
might occur rather quickly (as for example,
the accidental (?) dumping of a vat of waste
pickle liquor). Recent developments in
the field of biotelemetry now make it
feasible to "wire" a fish with electrodes
(like the astronauts) and so to immediately
record electronically any sudden or subtle
change in the effluent which affects the
physiological parameters being monitored
on the live fish.
REFERENCES
1 American Public Health Association,
Standard Methods for the Examination
of Water and Wastewater, 12th Ed.
(l3th edition when available).
New York. 1965. (l3th edition: 1970).
2 Doudoroff, P., et al. Bio-Assay Methods
for the Evaluation of Acute Toxicity of
Industrial Wastes to Fish. Sew. and
Ind. Wastes, Vol. 23, No. 11.
November 1951.
8
3 Doudoroff, P. and Katz, M. Critical
Review of Literature on the Toxicity
of Industrial Wastes and Their
Components to Fish. I Alkalies,
Acids and Inorganic Gases, Sew. and
Ind. Wastes, Vol. 22, No. 11, p. 1432.
November 1950.
4 Ellis, M. M., Westfall, B.A. and Ellis,
M. D. Determination of Water Quality.
Research Report 9, U. S. Fish and
Wildlife Service, 122 pp. 1946.
5 Hart, W. B., Doudoroff, P. and Greenbank,
J. The Evaluation of the Toxicity of
Industrial Wastes, Chemicals and
Other Substances to Fresh-Water Fishes.
The Atlantic Refining Company,
Philadelphia, Pa. 317 pp.
6 Hart, W. B., Weston, R. F. and De Mann,
J. F. An Apparatus for Oxygenating
Test Solutions in Which Fish are Used
as Test Animals for Evaluating Toxicity.
Trans. Am. Fisheries Soc. 75:228.
1948.
-------
7 Jackson, H. W. and Brungs, W. A.
Biomonitoring of Industrial Effluents.
Proc. 21st Ind. Waste Conf. Purdue
Univ. (Eng. Extension Bull. No. 121).
p. 117. 1966.
8 Lemke, A. E., W. A. Brungs, and
B. J. Halligan. Manual for
Construction and Operation of
Toxicity Testing Proportional
Diluters. U. S. Environmental
Protection Agency, Environmental
Research Laboratory-Duluth. Ecological
Research Series (in press).
9 Water Quality Criteris - 1972.
10 Water Quality Research Needs in Water
Quality Criteria - 1972.
11 Weber, C.!. (et al) Biological Field and
Laboratory Methods. U. S. E. P. A.
Env. Monitoring Series. 670/4-73-001.
(Revision in progress)
12 Peltier, Bill 1978 Methods for Measuring
the acute toxicity of effluents to aquatic
organisms. EPA Envir. Monit. Series
(in press)
1:-1 Tubb, Richard A. Recent Advances in Fish
Toxicology A Symposium EPA Envir.
Res. Series EPA-600/3-77-085 -
July 1977.
14 Stephen, Charles Methods for Acute Toxicity
Tests with Fish, Macroinvertebrates,
and Amphibiams. EPA Envir. Res.
Series. EPA-660/3-75-009. April 1975.
Bioassay and Biomonitoring
This outline was prepared by H. W. Jackson,
formerly Chief Biologist with the National
Training Center, Water Program Ot:e rations,
EPA, Cincinnati, OH 45268.
Descriptors: Bioassay, Laboratory Equipment.
Laboratory Tests
7-9
-------
BIOASSAY FACILITIES AND EQUIPMENT
I
INTRODUCTION
A Types of organisms and where they can
be obtained are discussed elsewhere.
B Here we are concerned with facilities
and equipment for working with the test
animals.
II
EXTENT OF FACIUTIES AND
EQUIPMENT NEEDS
A Depend on Several Cons.iderations
1
Number and size of test animals
2
Type of study
a
Static vs continuous-flow-through
b
Death- survival vs autopsy- sublethal
effects
c
Laboratory setup vs outdoor setup
3
Space limitations
4
Budget and staff available or planned
5
Extel"'t of bioassay program
III STATIC OR FLOW THROUGH
A Static Studies Suitable for:
1
Screening tests for "ball park" toxicity
values to be used in long-term
testing.
2
Comparative toxicity of compounds
having similar metabolizing qualities.
3
Comparative toxicity of various process
effluents in an industrial operation.
4
Screening organisms for relative
sensitivity to a given toxicant.
HI. HIO. 24a. 1. 78
5
When the available toxicant is in limited
quantity it is sometimes necessary to
use a static bioassay.
B
Advantage of Static Test
1
Simplest to set up
C
Shortcomings of Static Test
1
Animals are bathed in their own waste
materials, some of which are toxic.
2
Many toxicants decay with time, floe,
or precipitate, resulting in lower than
desired concentrations.
3
Some test organisms can absorb mucn
of the toxicant into their tissues and
reduce concentrations in water.
D Situations Which Should be Analyzed by
Using a Flow Through Setup
1
LC determinations in general, excep-
tions only for extremely short-term
tests.
2
Any test in which the size or hardiness
of the test organisms compared to the
volume of the test chamber suggests
problems of waste products buildup
or potential diminishing concentrations
of toxicant.
3
Long-term tests studying effects of
continuous or periodic exposures.
E
Shortcomings of Continuous Flow
1
Requires more space, time, and
equipment.
F
Advantage of Continuous- Flow- Through
1
More accurate results.
IV NECESSARY FACILITIES AND EQUIPMENT
A For Static Bioassay
1
Discussed elsewhere
H-l
-------
Bioassay Facilities and Equipment
B For Flow Through Bioassay
1 Test chambers - glass, fiberglass,
plastic, and stainless steel.
2 Dosage apparatus for adding toxicant,
including gear pumps, constant-level-
float siphons, Mariotte bottles, dipping
bird gadgets, syringe devices, and
various combinations of adjustable-
volume venturi~iphon units.
3
Water flow control devices, including
adjustable headboxes, constant level
float valves, simple shut-off valves,
capillary or tapered glass tubing, and
adjustable-volume venturi-siphon units.
4 Combination units handling both
toxicant and water.
a "Slurp-chamber" apparatus
b Serial diluters
c Simplified automatic dosage
apparatus.
C For Special Application
1 Mount degasser
2 Temperature control devices and
recorders
3 pH controlling unit and recorders
4 Demineralizers and carbon filters
5 Paddle wheel setup
6
Variety of test chambers
7 Swimming ability apparatus
8 Movement detectors and recorde rs
9 Egg collecting and hatching apparatus.
D Additional Items Needed or Useful
1 Necessities include: air pumps or
compressed air system, plastic air
tubing, glass and brass fittings
8-2
for air tubing, air stones, small
clamps, variety of plastic and rubber
tubing, variety of regular and capillary
glass tubing, rubber stoppers, formalin
or alcohol preservative, chemical
laboratory glassware (including pipets,
graduate cylinders, etc.), fish-holding
tanks for reserve specimens, small
dip nets, food, data recording form
stamps, pipet bulbs, water quality
analyzing equipment (for DO, pH hard-
ness, alkalinity, etc.), Toxicant analy-
sis equipment (colorimeter, polarograph,
chromatography setup), portable aerating
equipment, fish and water transporting
containers, fish treating compounds
(antibiotics, parasite control chemicals),
refrigeration facilities.
2
Items with special application: Dissec-
ting equipment, tissue processing ma-
terial (for fixing, embedding, and
staining), microtome, microscopes,
drying and ashing ovens, vacuum pump,
scales and balance, activated carbon
column, plankton counting equipment,
appropriate text books and manuals.
E Space Requirements in General
1 Area for hOlding fish for future tests
2 Area for test chambers
3 Area for diluent water storage
4 Area for dosage apparatus
5 Area for water quality and toxicant
analysis equipment
6
Sink and drainboard space
7 General storage area
8 Additional space for special
equipment.
F Example of a Static Bi.oassay Laboratory
(see Figure 1)
1 Area Number 1, provides space for
holding and acclimatizing fish. Each
-------
Bioassay Facilities and Equipment
large aquarium for holding fish (A) is
adequate for accommodating about 200-
300 average size test speciments. An
adequate air supply must be available
at all times to provide for continuous
aeration. Aquarium filters (C) of the
inside type help in keeping the aquarium
clean but best results come from
continually trickling fresh water into
the holding tank. Ordinary tap water
can often be used for holding fish if
chlorine has been removed, for
example, by passing the water through
an activated carbon column (E) and
temperature changes are not too
abrupt. The smaller aquarium (B)
may be used for acclimating the test
fish to the experimental water or hold-
ing them without food for period
immediately preceding the tests.
2 Area Number 2 provides for storage
and preparation of dilution water.
While containers for hauling and storage
may be of other inert materials, size,
and shape, the polyethylene items
illustrated (L, M) have been very
satisfactory for this use. In addition
1---.'-
IS'
.. ~
I-~
9'
I .. 2' -/...
a'
..j
IR AND _TER
AIR AN
WAT R
pHDp
SINK
ca .
00
o .
. c
A
emC!
o
B
a:: 0
.., all G
!q
-------
Bioassay Facilities and Equipment
to the regular supply it may be desirable
to have a supply of distilled or de-
mineralized water.
3 Area Number 3 can be used for prepar-
ing experimental test concentrations
of the effluent in the dilution water and
for exploratory tests to determine the
approximate toxic range. In these
tests air may be needed, depending on
the nature of the effluents.
4 Area Number 4 supplies bench space
for holding 20 full-scale test chambers
(G) on each side which permits the
carrying on of at least 4 full-scale bio-
assays simultaneously. Air must be
provided. A convenient arrangement
for supplying air or oxygen is through
a system of small tubing and 3-way
air valves so that the supply to each
test aquarium can be regulated in-
dependently. This system may be
attached either to the air supply or to
an oxygen cylinder (H) equipped with
a pressure reduction valve and
regulator.
5 Area Number 5 is for conducting the
necessary chemical tests for oxygen
control and to provide information
.necessary for interpreting bioassay
data. Squeeze-o-matic burettes (N)
have been found quite useful for rapid
performance of certain chemical tests.
H-4
----
V DILUENT WATER
A Type of diluent water depends on the type
of data sought.
1
Fresh, brackish, or salt wate r -
depends on organisms.
2 If interested in the effect of a parti-
cular compound in a particular water
body (stream, lake, estuary, EtC.)
then use water from the location where
this compound would enter the waterway.
3 Generally for other purposes, the best
water is the one you have most readily
available. Tap water can be dechlori-
nated; spring, stream, or pond waters
are usually suitable. Well water is
not generally recommended, however.
4 By mixing water from two sources in
large aerated storage facilities, close
water quality can be maintained.
This outline was prepared by T. O. Thatcher,
Former Aquatic Bib"logist, Research and
Development, Cincinnati Water Research
Laboratory, FWPCA.
Descriptors:
Bioassay, Laboratory Equipment
-------
IMPORTANT DATA FROM ACUTE MORTALITY TESTS
I
MEDIAN LETHAL CONCENTRATION (LC50)
A
Mortality
The major result of an acute mortality test
with a particular toxicant and a certain
species of fish is the LC50, ie, the
concentration that kills half of the fish.
In order to calculate the LC50. one must
know the percent mortality for a series
of concentrations of the toxicant. Deter-
mining the percent mortality merely
involves counting the live and dead fish,
but one should report'the criteria for
determining whether a fish is live or
dead.
B
Concentration of Toxicant
Measuring the concentration of the
toxicant is a more difficult problem. In
some cases researchers do not measure
the concentration of the toxicant. This is
especially true of toxicity tests conducted
with complex mixtures. In these cases,
the results are calculated based on the
amount of toxicant that was supposed to
have been used. (The calculation of an LC50
from the mortality vs. concentration data
will be covered in a later lecture).
In most cases the concentration of toxicant
in the water is measured. However, it
is not always easy to decide what measure-
ment to make. Consider the following
cases:
1
If a small amount of DDT is put
into a jar of water about 70 percent
will absorb to the glass walls of the
jar, about 30 percent will accumulate
at the air-water interface, and about
10 percent will dissolve in the water.
Should one base an LC50 calculation
on the total amount of DDT in the jar
or on the concentration of DDT dissolved
in the water?
B1. BIO. met. l8b.I. 78
2
If a moderate amount of copper sulfate
is put into a jar of water, about 20
percent will dissolve and about 80
percent will become a basic copper
precipitate. Some of the precipitate
will form a scum on the water and the
rest of it will be distributed throughout
the water. probably with most of it on
the bottom of the jar. depending on how
well the fish stir up the water. Should
one calculate the LC50 based on the
total amount of copper in the jar. the
dissolved copper. or the amount of
copper dissolved and suspended in the
water? The answer to this question
must take into account the fact that some
species of fish spend most of their time
near the surface and some spend most
of their time sitting on the bottom.
3
If phenol is placed in the water. it will
exist both as unionized phenol and as
ionized phenate ion. It is possible that
one form is much more toxic than the
other. However. practically all methods
for measuring phenol in water will
measure the total amount of phenol in
the water.
These examples indicate that one must
decide exactly how the sample must be
taken and what analytical methods can
be used. Very often the use of the
results of the toxicity test will determine
the answers to these questions. Usually
for static bioassays the LC50 calculation
is based on the total amount of toxicant
put in the test container at the beginning
of the test. This procedure obviously
has its drawbacks. For continuous-flow
tests. generally the best approach is to
take a sample under the surface of the
water. Because of the constant mixing
in the test chamber. this usually. but
not always. represents the total concen-
tration of toxicant to which the fish are
exposed.
9-1
-------
Important Data from Acute Mortality Tests
The decision as to how the sample should
be collected and/or what measurement
should be made must depend on what
question the test is supposed to answer.
The two common questions are:
1.
How much toxicant must be added to
the water to affect the fish in a
certain way?
2.
How much toxicant must the fish be
exposed to in order to affect them
in a certain way?
C
Calculation of the LC50
In reporting the results of a toxicity
test one should report the LC50 for a
given length of exposure with its confidence
limits, the way the concentration of the
toxicant was determined, and the method
used to calculate the LC50 from the
concentration-mortality data. Some
people report the concentration- mortality
data itself.
II
OTHER INFORMATION
A
About the Fish
There is much other information about an
acute mortality test that should be reported
along witli the LC50 value. This can be
broken down into four categories. One
should report information about the fish,
the test conditions, the toxicant, and the
water used in the test. This information
is important because there are many things
that can affect the LC50, and unless this
information is reported, no one else can
use the data. Under information about
the fish, one should report both the
scientific and common names, the age,
life stage, sex of the fish, and the range of
the lengths or weights or both. Very often
people report where the fish were obtained
and their condition, any treatments used
on them, and the holding and acclimatization
procedures used.
!.J-2
B
About the Physical Setup
Information on the physical setup should
include the type of test chambers used,
the volume of water used, the number of
fish per test chamber, and the average
grams of fish per liter of water and
experimental design. For continuous-fJ,ow
tests one should report the flow rate.
C
About the Toxicant
Information about the toxicant should
identify the source of the toxicant and
its composition. One should also describe
the formulation of stock solutions used
to introduce the toxicant into the test
chambers.
D
About the Water
Information about the water should include
the pH, alkalinity, dissolved oxygen,
hardness, total dissolved solids, and
temperature. Conductance and acidity
may be useful. Calcium, magnesium,
sodium, potassium, chloride, and
sulfate measurements can help characterize
the water. Many people also report the
source of the..water and any pretreatment,
such as aeration, activated charcoal, or
softening. One should also measure and
report any unusual constituents in the
water, constituents present in unusual
amounts, or constituents which are known
to affect markedly the toxicity of the
material under test.
Once you have decided what measurements
to make, it is important to use a good
method for the determination. One is
actually better off having no information
rather than having wrong information. I
would recommend use of methods from the
EP A manual titled "Methods for Chemical
Analysis of Water and Wastes" whenever
possible. The manual has two basic purposes:
1. To identify the simplest possible
legally defensible methods; .
2. To promote standardization so that
results will be comparable from one
laboratory to another.
-------
ill
OTHER TOXICITY TESTS
The same kinds of information should
be reported for any other kind of
toxicity test with any other test
organism.
REFERENCES
1.
~tandard Methods, 13th Edition, 1971.
2.
Methods for Chemical Analysis of
Water and Wastes, 1971
3.
Cope Oliver B., Standards for reporting
fish toxicity test, Prog. Fish- Culturist,
23 (4) 187-189, October, 1961.
Important Data from Acute Mortality Tests
This outline was prepared by C. E. Stephan,
Supervisory Research Chemist, Newtown
Fish Toxicology Laboratory, Newtown,
OH 45244.
Descriptors:
Bioassay, laboratory tests
!J-:{
-------
THE STA TISTICS OF TOXICITY TESTS
I
EXPERIMENTAL DESIGN
In general terms a toxicity test is used
to investigate the effect of a toxic agent
on a test organism. Because of biological
variation between individuals, in order
for the test to be meaningful, the effect
must be studied for a group of individual
test organisms, not just one individual.
In addition, an investigator generally
runs a toxicity test to determine the
level of a toxic agent that will produce
a given degree of effect, i. e., to
determine an endpoint. Therefore, in
practice, a toxicity test is usually run
to determine the level of a toxic agent that
produces a defined endpoint in a population
of test organisms. Usually this is
accomplished by exposing portions of the
population to different levels of the toxic
agent and observing the effect of the toxic
agent on the various portions. In the
Acute Mortality Test, this means observing
the percent that is killed at each level of
the toxic agent and then determining the
level that would kill 50 percent of the
population. For a toxicant, this is called
the median lethal concentration (LC50).
This test procedure imposes certain
requirements on the experimental design
of the test if the results are to valid.
A
Randomization
The first requirement is randomization,
both of the test animals and the test chambers.
Randomization of the test animals is important
so that the portion of the population exposed
to each level of the toxic agent is repre-
sentative of the whole population- - at least
as representative as one can make it.
Randomization of the test chambers is
important to minimize the effects of
external factors on the results of the test.
There are several ways randomization
can be performed, such as by drawing cards
out of a hat or using a table of random
numbers. In general, stratified randomiza-
tion is better than total randomization.
81. 1110. met. 19b. 1. 78
B
Replication
The second requirement for a good toxicity
test is replication of test chambers.
Duplication is about as far as most
investigators will go. Replication is
needed because randomization cannot
overcome all problems. One must get
an idea how much variation exists in the
test, and the only way to do this is
through replication. There are two kinds
of replicates--those run at the same time
and those run at different time. Sometimes
it is said that replicates run at the same
time measure reproducibility, and those
run at different times measure repeatibility.
C
Numbers of Subjects.
A third requirement is to have an adequate
number of test animals in the population
and in the portions of the population. This
is replication of test animals. Five animals
p,::r portion is about a bare minimum; ten
is a good compromise between theoretical
and practical necessity. It is obvious that
if only five animals are used in a portion,
the results for a portion can only be 0, 20,
40, 60, 80, and 100 percent. A difference
of one animal between duplicate portions
means a difference of 20 percent. If there
are ten animals per portion, a difference of
one animal will mean only a 10 percent
difference. Even the best random distribution
cannot make the replicates identical, so
there must be enough animals in each replicate
to minimize the consequences of such differ-
ences.
D
Number of Partial Kills
(1)
A fourth requirement of a good
experimental design for toxicity
tests is that there be enough levels
of the toxic agent tested so that the
level producing 50 percent kill can
be determined accurately. If one
only tests levels that kill either 0
percent or 100 percent of the animals,
all he knows is that the LC50 is
between two of the levels tested.
10- ]
-------
The Statistics o:i: Toxicity Tests
If, on the other hand, the investigator
tests levels that kill 0, 20, 40, 60,
80, and 100 percent of the animals,
he can determine the LC50 rather
accurately. There are biological
and practical limitations on how
accurately one can determine an
LC50. However, if results are to
be very meaningful, as a bare
minimum the test should have two
concentrations that produce partial
kills. With two partial kills, one
can calculate the LC50 and its
confidence limits with fair accuracy.
(2)
The degree to v.;hich an investigator
worries about each of these require-
ments must depend on the confidence
one wants to have in the data. One
can ignore all four requirements and
still get a "ball park" figure, but
"ball park" results are not very
useful. Of course, it is ridiculous
to go into great detail on the statistical
requirements for a good toxicity test
and ignore other things such as the
biological and chemical requirements.
IT
ANALYSIS OF THE DA T A
A
Once the test has been run, the remaining
problem is to calculate the results from
the data collected. However, what can be
done with the data is often limited by what
data were collected and how they were. 2
collected. Thus the design of the experiment
should take into account the ultimate use
planned for the data. The only data collected
from most routine toxicity tests is 3
concentration- mortality data.
B
There are several methods for analyzing 4
concentration-mortality data. It is generally
accepted that the best way is to determine
the median lethal concentration (LC50).
Statistically this is a good endpoint and it
is about as useful as any other one. All
of the methods for calculating an LC50
can be visualized as graphical methods
based on a plot of concentration vs.
percent mortality. What changes from
10-2
method to method are the coordinates
and the means of connecting the points.
Standard Methods describes the commonest
g;aphical ~ethoct: but in general graphical
methods are approximate methods, give
no measure of confidence limits, and are
not useful with certain kinds of data.
Most people who want to use a better
method use the Lit chfie ld- Wilcoxon
method. This is a semi-graphical.
approximate probit method, but this is
rather time consuming unless a computer
can be used. Others use various other
parametric or nonparametric methods,
such as the logistic method of Bergson
or a moving average method. All of these
are discussed by Finney. Some of these
methods assume a particular relationship
between concentration and mortality and
some do not. Generally it is impractical
to try to get enough data to prove whether
or not a spe cific relationship exists.
Fortunately, most often the calculated
LC50 and its confidence limits are about
the same for all computational methods.
REFERENCES
1
Cochran, W. G. and G. M. Cox,
Experimental Desil[l!J, Wiley, New York,
1950.
Finney, D. J., Statistical Methods in
Biological Assay: 2nd Edition, Griffin,
London, 1964.
Kempthorne, 0., The Design and Ana!J.'.sis
of Experiments, Wiley, New York, 1952.
Standard Methods, 13th Edition, 1971.
This outline was prepared by C. E. Stephan,
Supervisory Research Chemist, Newtown Fish
Toxicology Laboratory, Newtown, OH 45244.
Descriptors:
Bioassay, Statistical tests. Laboratory tests
-------
THE SELECTION OF ENDPOINTS FOR TOXICITY TESTS
I
INTRODUCTION
An endpoint in a toxicity test is a
defined magnitude of a specific observed
effect of a toxic agent on a living organism.
Thus the selection of an endpoint involves
both the selection of the effect and the
selection of the magnitude. At one time
"toxic" was generally used to mean "to
cause death" and death was almost the
only effect studied. However, today it
is generally recognized that toxic agents
cause many damaging effects other than
death, and so "toxic" is used to mean
"to cause an adverse effect" and "lethal"
is used to mean "to cause death. "
II
DEATH
A
Death was probably the first effect used
in toxicity tests and is still the most widely
used effect because it possesses four very
useful properties:
(1)
It applies equally well to all
organisms;
(2)
It applies equally well to all
toxic agents;
(3)
Usually it can be detected rather
easily without the use of specialized
equipment;
(4)
It is an obviously important
adverse effect.
Because of these properties, death will
probably always be the basic observed
effect for toxicological studies.
B
If an endpoint is to be defined using death
as the effect, in terms of a group of
subjects, there are several magnitudes
of death that can be used, such as:
(1)
the lowest concentration that kills
all of the subjects (LClOO)
f31. BIO. :Wb. 1. 78
(2)
the concentration that kills 50%
of the subjects (LC50)
(3)
the highest concentration that kills
none of the subjects (LCO).
C It has been found that statistically and
practically the LC50 is the best endpoint.
Although it is sometimes argued that LCO
should be a more useful endpoint, the LCO
is difficult to determine.
III
OTHER EFFECTS
There are many effects other than death
that can be and have been used. Warner
(1967) reviewed many histological,
physiological, biochemical, behavioral,
activity and growth effects and endpoints.
The possibilities are only limited by man's
ingenuity, time, and money.
IV CRITERIA FOR THE SELECTION OF
ENDPOINTS
A From all the effects that can p'Jssibly be
used for toxicity tests, one must choose
the best effect for one's own tests. Death
is obviously widely used effect, but it is
generally not sensitive enough. From the
more sensitive effects one must choose
one that is practical and will meet the
needs of the experiment.
B Generally the first consideration is time
and equipment, and these are obviously
important, but usefulness should be the
primary concern. Water pollution control
is a matter of solving practical problems.
Thus an effect should be useful. For many
of the effect that can be used in short,
sensitive tests, there is no information on
usefulness and some of these require
elaborate equipment.
11-1
-------
The Selection of Endpoints for Toxicity Tests
v
CHRONIC TESTS
Much of the work of the National Water
Quality Laboratory is now centered
around what we call the chronic test.
In these tests the animals are exposed
to the toxic agent before and during
spawning and the eggs and fry produced
are exposed for about thirty days or more.
In these tests, we look for effects on survival,
growth and reproduction, because such
effects are obviously important and are
rather easy to study. Chronic tests
generally last for eleven months or more.
In addition, we are studying the usefulness
of some other sublethal effects by
comparing the results of acute tests
with those of the chronic tests.
11-:!
REFERENCES
1. Warner, R. E. Bioassays for Microchemical
Environmental Contaminants. Bull.
World Health Org., ~~ 181-207 (1967)
2. Sprague, J. B., Measurement of
Pollutant Toxicity to Fish.
Water Res.,!, 793-821 (1969)
Water Res., '!t 3-32 (1970)
Water Res., ~ 245-266 (1971)
3, Mount, D. 1., and C. E. Stephan,
Chronic Toxicity of Copper to the
Fatheat Minnow in Soft Water, ..:h
Fish Res. Bd. Canada, 26, 2449- 2457
(1969) -
This outline was prepared by C. E. Stephan,
Supervisory Research Chemist, Newtown
Fish Toxicology Laboratory, Newtown,
OH 45244.
Descriptors:
Bioassay, :\lethodology, Laboratory Test@
-------
SPECIAL APPLICATIONS AND PROCEDURES FOR BIOASSAY
I
INTRODUCTION
A The report of the Co.mcil on Environ-
mental Quality (1970) repeatedly stresses
the need for the development of predictive,
simulative, and managerial capabilities
to combat air and water pollution. The
last capability depends on the first two.
B The standard static jar fish bioassay,
which uses death as a response, enables
one to predict the toxicity of a particular
waste to fish. One limitation of this
procedure is that it uses a grab sample
which represents the quality of the waste
at only one point in time. The water
used to make the dilut.ions is also taken
at one point in time. At the actual
industrial site, the quality of the waste
and the river water vary through time.
A composite waste sample partially
overcomes this limitation, but may mask
variations that are biologically important.
C One could put fish in a continuous flow of
waste diluted with river water, but then
there is one further limitation of the
standard bioassay: death is used as the
response. In order to prevent damage
to organisms, it is necessary to have an
early warning of dangerous conditions.
so that corrective action can be taken.
In other words, symptoms of ill health,
which occur before death, must be detect-
ed if there is to be time fo[' diagnosis and
treatment.
II
METHODS AND MATERIALS
A Fish Movement Patterns
1 Fish movement patterns can be monitored
using the technique of light beam inter-
ruption described in detail by Cairns,
et al. (1970). Dawn and dusk are
s.imulated by a motor- driven dimming
unit which gradually increases the
intensity of the room lights over a
half-hour period starting at 6 :30 a. m.
and gradually decreases the intensity
to 0 over a half-hour period starting
at 6 :30 p. m. The cumulative movement
BI. BIO.met. 22.3.74
of each of six bluegill sunfish, a
single fish per tank, is recorded
every hour throaghout a test except
during the simulated sunrise and
sunset when an additional record is
made on the half hour. Each day is
divided into fO'ur intervals; first
half day. second half day, first half
night and second half night (Table I).
Bef01"e any statistical analysis can
be performed, recordings for day
1 must be completed. After the
cumulative movement for day 1 is
recorded. statistical ana.lyses are
performed after the completion of
each designated time interval. For
example, the cumulative movement
recorded hourly for each fish during
day 1, first half day values are compared
to the cumulative movement recorded
hourly for each fish during day 2, first
half day values.
2 Based on the results of 20 laboratory
experiments "stress detection" is
defined as the presence of two or
more abnormal movement patterns
recorded during the same time interval.
B Fish Breathing
1 Breathing rat.:-.3 may be determined from
polygraph recordings of breathing signals.
The fish are tested in plexiglas tubes
throagh which dechlorinated tap water
or s':>me toxic solution is metered at
a flow rate of approximately 100 mIl min.
Breathing signals are detected by three
platinum wire electrodes placed in the
water; an active electrod.~, an indifferent
electrode, and a groun:l. The test
chambers and meth::>ds of acclimating
the fish are described in more detail
by Cairns, ~t~. (1970) The photoperiod
is the same as that for the fish movement
study.
2 The fish are placed in test chambers by
6 :00 p. m. and the recordings began a.t
6 :0::> a. m. the next day to allow the fish
to recover overnight from handling.
Toxic solutions are introduced at 10:00 a. m.
after the experimental fish have been
exposed to water containing n::> added
]2-1
-------
.Ee~cial Ap?licati0!ls and Proc~~~ fo!.-Bio~~~~:r
toxicant for periods of oae to sIx days.
Ea ~h experimen~al fish thus serves as
its own con~rol. In addition, one or
two fish are n:::ver exposed to the
toxicant and serve as contrals through~
out each experiment. In one experiment,
using zinc as the toxicant, reparted
in Table VI, six control fis~ were
exposed to water containing no. added
zinc far four days.
a Preliminary exidence suggested that
the data cauld be analyzed by separa-
ting the experimental day into four
periods; a period from 6:00 to 8:00 a. m.
when the breathing rates changed
markedly, a period from 9:0oJ a. m.
to 5:00 p. m. when the rates were
comparatively high, another period
a~ rapid change from 6:00 to 8:00 p. m.,
and a night period from 9 :00 p. m. to
5:00 a. m. when the rates were compar-
atively law (Sparks, e..! al., 1970).
b Bluegills increase their brea~hing
rates when exposed to zinc (Cairns,
et al., 1970~ An individual fish was
thus considered to have shown a
respanse each time its breathing rate
during a time period exceeded the
maximum breathing rate observed
during the corresponding period of the
first day, before any zinc was added. A
response was scored for each value
on the second day that was higher than
the first day maximu:-n for the compar-
able period. The control periods
(before any zinc was aided) and the
experiment where no zinc was added
at all were used to determine haw many
false detections this methad af analysis
wauld praduce. The experimental
periads (after zinc was added) deter-
mined :'1aw quickly the method af
a:1alysis could detect zinc cancentratians
in wate t'.
c
Zinc cancentratians were determined
daily by atomic ahsarption spectro-
photometry.
12-2
------
III
RESULTS
A Fish Movement Patterns
1
Table 1 shaws t.he results 'Jf ane
cantinuous flaw experiment car .cied
aut far 20 days. During this experiment
fish were expased to. zinc an day 7
fram 1:00 p.m. until7:0a p.m. at
which time the flaw was returned to.
normal dilution water. The zinc
cal1centratial1s reached their maximum
at 7:00 p. m. and atamic absarptian
analyses an effluent samples ~allected
at this time shawed the fallawing
con,::entrations: tank ane, 13.32;
tank two., less than 0.03; tank three.
11. 39; tank faur, 12.72; tank five,
13.32; and tank six, 12.59 mg/l Zn++.
The results shaw that these cancentratians
af zinc develaping aver the six haur
interval af exposure were insu.fficient
to cause a d,~tectable change in the
mavement patterns af the fish. By
8: 30 a. m. a~ day 8 the effluent zinc
ca:'1centrations were less than a. 30 in
all cases.
2
To dl~termine the percent survival aid
recavery patterns af the fish ance stress
detectiaaoccurL'ed, zinc flaw was
reinitiated at 1:00 p. m. an day 13 af
this experiment. Between 8:03 and
9:00 p. m. an day 13 the zinc cancentration
in the effluent reached a maximum af:
7.51 far tank one; less than 0.05 far
tank two.; 7.49 for tank three; 7. 52 far
tank faur; 7.49 far tank five; and 7.54 mg/l
for tank six. The cancentratians remained
near the abave values until the statistical
analyses shawed "stress detectian"
during the first half night values an day
14 (Tabla 1). As soan as stress detectian
a~curred the flaw was returned to. normal
dilution water. At 10:00 a. m. an d.3.y 15
zinc analyses showed all efflu-ant cancentra-
tians to. be less than 0.70 mg/l Zn++.
Stress detection continued to. be registered
far two. cO:1secutive time intervals following
the initial detection, but after that n:) stress
detectian was registered and the frequency
of abnormal patterns returned to p::oestress
levels within .i8 haars. In this experiment
-------
__~!:~l- ~..EP.lis.ations and Pro~ed'.lres for Bioassay-
-.------
as with an others in which dilution
water containing zinc was replaced with
dilution water minus zinc at that time
of stress detedion ~!!ish survi\Te~!
3
The results from the series of experiments
at progressively lower zinc concentrations
indicate that the lowest detectable con-
centration is between 3,65 (Table II) and
2.93 mg/l zinc (Table III) for a 96-hour
exposure.
B Fish Breathing
1
Table IV shows the Jreathing rates of
five fish on days 1. 2. and 7 of experiment
8. The first four fish were exposed to a
measured zinc concentration of 4. 16 mg/l.
beginning at 10 a. m. on day 7. The fifth
fish served as a control and was not
exposed to any added zinc. The amplitude
of the breathing signals decreased every
night. and the breathing rates for fish 2.
in particular. could not be determined
during some portions of the dark period
(7;30 p.m. - 7:00 a,m.). The maximum
breathing rates for each fish during each
period of the first day are circled. The
breathing rate of any fish during a time
period of day 2 or day 7. which is greater
than the maximum breathing rate recorded
for that fish during the corresponding time
period of the first day has a rectangle
drawn arotl.'1d it. The total number of
flsh showing increased breathing is
given at the bottom of each column. On
day 2. fi~h 2 showed increased breathing
on just two occasions. In contrast after
zinc was added on day 7. three and four
experimental fish at a time showed
increased breathing.
2
Table V S'.lmmarizes the results of
successive comparisons of the first day
maximal breathing rates to breathing
rates on subsequent days (SCM m~thod
of analysis). for experiment 8. During
the control period before any zinc
was added there were 15 occasions when
a sbgle exp.erimental fish responded.
and three occasions when two experimental
fish responded at the sa~e time. At no
time during the control period did more
than two fish show responses together.
After the zinc was introduced. all
four of the exposed fish showed responses
simultaneo'usly on five occasions. and
three fish showed ~~espoi1ses during the
same time interval on 19 occasions. If
the criterion for detection of water
conditions potentially harmful to fish
were two ot' more responses during the
same time period. then three false,
detections would have occurred before
any zinc was added. and 4.16 mg/l zinc
would have been corcectly detected eight
hours after it was introd~eed. If the
detection criterion were three or more
responses during the same time period.
then no false detectiOJ.1s would have
occurred and the zinc would still have
been correctly detected after eight hours.
3
The lowest zinc con~entration tested was
2.55 mg/l. Using a detection criterion
of si.multaneous responses by three fish.
this cm1centration was detected 52 hours
a£ter the zinc was added, with no false
detections occurring during the four hours
before zinc was added (Table VI). The
responses of six control fish that were
exposed to dilution water containing no
added zinc are also shown for comparison.
Note that there was no tendency toward
increased'?reathing rates through time in
the control fish. and that no more than one
control fish showed an :ncreased breathing
rate during one time period.
4
Table VII su:nmarizes information on
three experiments that indicates the
effectiveness of the SCM method of
analysis when different criteria for
detection are used. Changing the criterion
for detection from one to three responses
per time period generally increases the
lag time and decreases the number of
false detections. The lag time is the
time from the addition of zin::: to the first
detection. A false detection is O:1e O~Cllrr-
ing before any olin,:: is added to the water.
IV DISCUSSION
A The experiments described above show that
the movements and breathing rates of bluegill
12-:~
-------
.2E.1!~i_al_~Rlica.!!~~.!1:! Pr.s>~ures for -~~o~ssay
- ---- -.-- - - _._-----
sunfish can be u.3ed to detect sublethal
co:>.centrations of zinc. The criterio:>.
for dl~tection is a certain number of fish
showing an arbitrarily dl~fined respo:l.;;e
in breathing rate or activity during one
time period.
B In choosing a specific criterion for
detection, the risk of not detecting stressful
conditions soon enough must b.~ weighed
against the risk of false dl~tections, and
the choice would probably be determined
by the nature of the pollutant. If a pollutant
is easily detected by the biological monitoring
system, is slow-acting, and if the toxic
effects are reversible, then the criterion
for detection might be. responses by 3/4
of the test fish, to avoid the false detections
that would necessitate expensive remedial
a ~tion or a temporary shut- down. On the
other hand, an industry that produces an
effluent containing a fast-acting toxicant
whose effects are irreversible would
probably use a criterion that leads to rapid
detection (responses by 1/4 to 1/2 oftne
test fish), and would have to go to the
expense of installing holding ponds 01"
recycling facilities to accommodate a
relatively high nllmber of false detectio.'1s.
Alternatively, a safety factor could be
introdu.::ed by metering proportionally
mor-e waste into the dilution water delivered
to the test fish than is delivered to the
stream. The safety factor could be
determined by growth and reproduction
experiments with fish.
C In a.'1 actual industrial situation water and
waste qualities are apt to vary unpredictably,
and it would certainly be desirable to 11a ve
a redundant detection system. It is conceiv-
able that so.'l1e harmful combination of
environmenhl co..dition,;; and waste quality
would he detected by moni~oring one biological
function, but not by monitoring another.
It is also possible that excessive turbidity
would disrupt the light beams of the movement
m:mitor, and not affect the breathing monitor;
or th3.t an excessive concentration o~ electro-
lytes would affect the electrodes of the breath-
ing monitor, but not affect the activity monitor.
Therefore, the a.::tivity monitol' an:! the
breathing monitor have been combined :.n our
labot'atory fm.- further exp.eriments (Fig. 1).
12-4
D The rate of data a.cquisition and analysis
could be greatly speeded up if the
m.:mito:-:-ing system were automated as
shown in Figure 2. The sampling rate
. would be controlled by a minico:nputer
which could receive data from the
movement monitor and the polygraph
via a multiplexer as often as every minute.
The minicomputer would he programmed
to perform statistical analyses every 10
minutes, for example, and output the
res'~lts on a teleprinter.
E Figure 3 shows how the fish monitoring
units would be used at an actual industrial
site. A mOnitoring unit would he located
on each waste stream in the plant and on
the combined waste stream. The experimen-
tal fish in each writ would be exposed to
waste diluted with water from the river
above the plant, and control fish would be
exposed to upstream water alone (Fig. 4).
The iL1for-mation from ea~h monitoring
unit could be a.nalyzed by a central data
processor, and when there was a warning
response, the industry could tell which
waste stream was at fault. If the problem
was outside the plant, the control fish
would show responses.
F Figure 5 shows how the in..pla:-:t'; monitoring
systems would 'Je integrated into a river
management system. The in-plant monitoring
units are shown as squ'ires, and in addition
to supplying information to each industry,
the monitoring units also infol'm the control
center. In such a system, there are several
alternative damage prevention measures
that could be used, in addition to whatever
measures. such as shunting wastes to a
holding pond or recycling wastes for further
treatment, are available to each industry.
If the monitoring units at Industry 2 indicate
that toxic waste conditions are developing,
then the control center might have Industry 1
hold its waste w1til the danger of combining
wastes from Industry 1 and :~ in the river
were alleviated by control measur(::s at
Indu.3try 2. Alternatively, the control center
might call fo!" a release of water from the
upstream dam to dilute the effluent from
industry.
-------
. -- ------ -~e.£~l.~c~~_t121~"!.£..r~~!or Bioass_ay.-
G It is likely that "fish sensors" in
continuous monitoring units at industrial
sites can warn of developing toxic conditions
in time to forestall acute damage to the
fish populations in streams. In conjunction
with stream water quality standards for
chronic exposure, such biological monitoring
systems should make it possible for healthy
fish populations to co- exist with industrial
water use.
ACKNOWLEDGEMENT
This research was supported by grants 18050 EDP
and 18050 EDQ from the ~ater Quality Office, 10
Environmental Protection Agency.
Literature Cited
1 Brungs, W. A. 1969. Chronic toxicity of
zinc to the fathead minnow, .E!!!lephale!l
~Q!!te~ Rafinesque. Trans. Amer.
Fish. Soc. 98(2):272-279.
2 Cairns, J., K. L. Dickson, R. E. Sparks,
and W. T. Waller. 1970. A preliminary
report on rapid biological information
systems for water pollution control. Jour.
Water Poll. Contr. Fed. 42(5):685-703.
3 Eaton, S. G. 1970. Chronic malathion
toxicity to the bluegill (!=~~~is ~2E.9~~~
Rafinesque). Water Research. 4:673-684.
4 :vIcKim, J. M., and D. A. Benoit. 1971.
Effects of long-term exposures to cooper
on survival, growth. and reproduction
of brook trout (Salvelinus fontinalis)
J. Fish. Res. W-:-Canada 28:65'5--662.
5 Moor'~, J. G., Jr.. Commissioner. 1968.
Wa~ QualiD' ~}teria. Report of the
National Teclmical Advisory Committee
to the Secretary of the InterioC'. U. S.
Govt. Printing Office. 234 pp.
6 Mount, D. I. 1968. Chronic toxicity of
cooper to fathea:l minnows (Pimephales
£romel~ Rafinesque). Water Research.
2:215-223.
7 Mount, D. I. and C. E. Stephan. 1967.
A method for establishing acceptable
toxicant limits for fishu Malathion
and the butoxyethanol ester of 2, 4- D.
Trans. Amer. Fish. Soc. 96(2):185-193.
8 Sakal, R. R. and F. J. RO:'11f. 1969.
BiOI!?-~!£l. w. H. Freeman and Co.
776 pp.
9 Sparks, R. E., W. T. Waller, J. Cairns, Jr.
and A. G. Heath. 1970. Diurnal variation
in the behavior and physiology of blue gills
(~t!lJ!I ~.£E9ch~~ Rafinesque).
The ASB Bull. 17(3):90 (Abstract).
Sprague, J. B. 1969. Measurement of
pollutant toxicity to fish I. Bioassay
methods for acute toxicity. Water
Research. 3:793-821.
11 Cairns, John Jr.; Sparks, R. E.; and
Waller, W. T. "A tentative proposal
for a rapid in-plant biological moni-
toring system. " in Biological Methods
for the Assessment of Water Quality.
ASTMSTP 526, American Society
for Test. and Maf., 1973, pp. 127-147.
Th1soutline wasprej;aI:ed by Job.n -Cairns~Jr-.
and Richard E. Sparks, Center for En',iron~
menhl Studies a~:! Department of Biology,
Virginia Polytechnic Institute and State
University, Blacksburg, VA 24061
Descriptors:
Environmental Control, Bioassay, Toxicity,
Fish Behavior, Fish, Monitoring
12-5
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ECOLOGICAL CONSIDERATION IN PLANNING
WATER QUALITY SURVEYS
I
INTRODUCTION
A Ecologists must be willing to take a
chance and predict the ecological
consequences of alternative schemes for
water resources management if we are to
realize the maximum beneficial use of our
resources. Historically, ecologists
have lagged behind their engineering
colleagues in developing prediction
capabilities for a number of reasons.
1 UnfortWlately, the operational charac-
teristics of ecosystems a!'e poorly
understood when compared to the
engineers systems. Engineers can
predict that with so much concrete,
steel, etc., and with a given amoWlt of
labor and money, a dam can be built
on a river which will enable them to
regulate flow behind the dam. The
flow figures can be predicted with
reasonable accuracy, and when the dam
is built, the performance is generally
within the original estimate. However,
due to the complex chemical, physical
and biotic interactions of an ecosystem
prediction of the ecological consequences
of any activity is more involved. The
ecologist has developed only relatively
recently rather primitive prediction
systems for complex natural environments.
2 Another reason that ecologists have
lagged behind engineers is that appropriate
channels of information exchange between
them have not always been open. Two
contrasting philosophies have existed
in the past which have hindered the
ecological management of our water
resources. Engineers, water resource
economists and industrialists have
generally had a construction philosophy
of life which has conflicted with the
conservationists or protective philosophy.
Those who build power plants, dams,
reservoirs, canals, etc., have used the
technology of the time most economically
appropriate with the expectation that'
,what they are building will have a very
short life span in terms of geologic time,
and that when the structure is outmoded
or uneconomical, it will be torn down or
replaced. Plans are also strongly time
W. RE. 30. 10.75
dependent, and once completed must be
initiated in a relatively short time per-iod
or they will become outdat.ed. Complex
sets of conditions involving techn.:>logy,
financing, land acquisition, power demands,
etc., prevail; thus, the engineer is often
characterized by a time an:l tide wait
for no man attitude. The conservationist,
on the other hand, realizes that once' a
rare species endemic to any area is
lost, it is gone for all time, and that
ecosystems once damaged may be difficult,
if not impossible, to restore to their
original condition.
B FortWlately, a new awareness on the parts
of the advocates of both philosophies that
our life support system on earth has two
components, one industrial and the other
ecological, has forced ecologists and
engineers to work together. We currently
realize that the survival of our present
sooial system depends upon our ability to
develop a harmonious relationship between
these components.
C Those people involved in water resources
management realize that some of the
frustration and public outcry about water
resources projects could have been avoided
if proper consideration was given to
ecological information before CO:lstruction.
Present trends in legislative action combined
with the ever growing concern over the
quality of the environment, the "environmental
impact" of any new water resources develop-
ment will be closely scrutinized. It is our
objective here to briefly present some
ecological information that should be consid-
ered along with other parameters in river
basin planning.
n
PRE CONSTRUCTION ECOLOGICAL SURVEY
A One of the common problems of water
resources development is to select a
project site which allows maximum use
without environmental degradation. In
order to get this type of information, it
is necessary to develop a series of
prediction systems which will allow an
ecologist to rank the potential construction
13-1
-------
Ecological Consideration in Planning Water Quality Surveys
--.--- -
-------------
sites. Perhaps one of the best ways to
obtain important types of ecological
information to be used in river basin
planning is through a preconstruction
ecological survey.
1 A preconstruction survey should be
carried out by a team of chemists,
ecologist, engineers, and taxonomists
to get a complete picture of the chemical,
physical and biological condition above
and below the potential site location.
If adequate background data are to be
generated, the team should consist of
one 0::' more chemists, a bacteriologist,
an algologist, a protozoologist, one or
more invertebrate zoologists (including
an aquatic entomologist), and ichthyologist,
and a sanitary engineer. Since this involves
a number of well-trained people, it can be
moderately expensive.
2
The exact cost would depend on a number
of factors including the size and structure
of the river and the number of species
likely to be encountered. Obviously, the
lower Mississippi is a more difficult
river to survey than a small river that
one can throw a rack across. In addition,
a stream already degraded by PQ~lutiol1 is
likely to have fewer species resulting in
less cost for identifying the various
organisms collected than an unpolluted
stream with a very high number of species.
3
BefoJ.~e such a survey is contemplated, it
is well to have a preliminary survey by
a generalist used to dealing with these
problems who can make a firm estimate
of the costs involved and place reliable
time estimates on completion of the
project.
B A survey of this nature will provide a wide
variety of information valuable in making
a choice between prospective project
locations.
1 It will establish a baseline of biological.
chemical and physical water quality
which can be useful in determining the
waste assimilative capacity and other
beneficial uses of the system. If one
13-2
views the waste assimilative capacity
of a river as a natural resource, then
it is only logical to make use of that
capacity along with other uses such as
water supply, recreation a!'1d aesthetics
to derive the maximum beneficial use
from the system.
2 A preconstruction survey will determine
pre- existing man made or natural stresses
on the receiving system. riiorcter to
a.void blame, there is no better defense
than an aggressive offense. Preconstruc-
tion data which documents the water quality
is extremely valuable, particularly in
receiving systems which are already
partially under stress from other waste
discharges. It is essential to establish
the presence of natural stress on a system
and thus avoid blame after project construc-
tion is completed and operations begin.
Natural stress can take range from siltation
and the introduction of organics from leaf
litter, to thermal changes due to the
introduction of underground aquifers.
3
How many water resources projects do
you know that are located near critical
spawning areas of striped bass, just
above, or in the middle of an important
fishery, in an area where the aquatic
life is particularly vulnerable, and the
like? Many of these situations could
have been avoided through a preconstruction
survey before site selection was made.
Alterations in design of discharges could
have received valuable input information
based on this identification 01' valuable
wildlife resources. For example, in
some cases, it might be desirable to design
waste discharge systems so that the waste
is held against one bank of the stream or
river lea.ving a free channel on the other
side where migratory fish could pass
thro".lgh the area.
4 A preconstruction survey is a convincing
demonstration that the resource developers
are sincere in their efforts to protect the
environment. The information derived from
the survey can often furnish .information
about the ecological history of the area a:1d
mak~ some predictions about future trends,
-------
----- -----
Ecol
-------
Ecological Consideration in Planning Water Quality Surveys
_._----------"-- ---
increasingly important as our population
grows and more intensive use is made of the
finite space available to us. In the past
when we damaged an environment seriously,
we could move on to a new undamaged
environment and avoid most. of the immediate
consequences of poor management. Perhaps
the last big movement of~his sort in the
United States was the exodus from the Dust
Bowl. However, since most of the ecosystems
of the United States are at or near tolerable
stress levels, we no longer can go to virgin
territory and escape our environmental
mistakes. As a consequence, we can afford
fewer mistakes without immediate penalty
than we could in the past 0
B One of the obvious protective measures we
might take to prevent major ecological or
environmental problems is to simulate
prospective new uses in scale or laboratory
models and restrict most of our mistakes to
these. This practice is too common in
engineering (for example, the U. S. Army
Corps of Engineers river models at the
Waterways Experiment Station, Vicksburg,
Miss. ) and industrial circles that it would
hardly need mention were it not for the fact
that ecological scale models or environmental
simulation systems are not now commonly
used.
C However, ecologists now are becoming quite
interested in developing scale models to
simulate various environmental systems
and the practice should become increasingly
common in the future. Of course, these
suffer the weaknesses of all scale models
and are still in primitive stages of develop..
mente They need not be extremely expensive
and may be used to generate data which could
be useful in preventing large scale mistakes.
For example, many 0: the events which havE:
occurred in Lake Erie could probably have
been simulated :in models.
D An example of a scale model we commonly
use in our lahoratory is a model stream to
which we have attached a model steam
condensing system allowing an incremental
increase with a v'3.riable contact time in the
condens.:>r (Figure 1). Water from the model
stream is passed through the condensor
system and then through a series of plexiglass
1 :3-4,
--------------
troughs where we allow algal and protozoan
communities to establish. These experi-
mental troughs are cmnpar,::d to control
troughs, and some predictive in~ormation
on the effects of passage through the
system on downstream community
structure determinl~d.
V ECOLOGICAL QUALITY CONTROL
TECHNIQUES
A Since we are a society almost compulsively
dedicated to change, we are desperately in.
need of adequate prediction systems. The
preconstruction survey, p!'edictive bioassay
and scale models previously discussed
allow the ecologist ot make some of these
predictions and help identify the various
alternative uses which might be made of
the environment and to estimate what the
consequences of these will be. If these
techniques were utilized in prospective
water resource projects, we would be on
our way to having adequate enviro!Lilental
planning. However, environmental
planning alone will not be effective unless
good quality control techniques are developed,
as well as adequate environmental management
practices. ..-
B Just as in an industrial process where we
have a system of checks and balances to
insure product control, we must begin to
develop the capabilities for environmental
quality control provided an equitable
environ::nental use plan can be developed.
1
This will require rtlpid biological,
physical and chemical information
systems, so that we get a continuous
flow of information enabling us to
predict unfavorable changes in our
water resource systems.
2 Of the three types of information
systems pr..:viously mentioned, the
development of rapid biological
monitors providing continuo-~s
information has lagged behind the
other two in develop:nent. We can
continuously monitor in a river many
of the physical and chemical parameters
-------
LOCATION OF
TEST TROUGHS
TIMER FOR PHOTOPERIODIC CONTROL OF
GRO-LUX FLOURESCENT AND
INCANDESCENT LIGHTS
TAP WATER
ACTIVATED CHARCOAL
DECHLORINATOR
CONTROL AND EXPERIMENTAL
TEST TROUGHS
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FIGURE 1
SCALE MODEL SYSTEM FOR SIMULATING THE EFFECTS OF
PA~~A(jE THROUGH A STEAM CONDENSOR ON MICROORGANISMS.
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Ecological Consideration in Planning Water Qual ity Surveys
---- -- - --- ---. ~ ----------- -.-.- - - --- -------
for analyses. However, the development
of rapid biological information systems,
both in-stream and in-plant, is essential
to the maintenance of adequate environ-
mental quality control.
3 We need to know the effects on biological
organisms of a waste discharge before it
enters the receiving stream as well as the
biological effects after it enters the stream,
and this information should be produced
rapidly. Present systems are much too
slow in view of the fact that the constituents
of a waste stream are likely to vary from VI
hour to hour and from day to day. Poten-
tially disastrous materials should be
detected before they enter a receiving
stream if at all possible and at the very
least, before substantial damage has
been done in the receiving stream itself.
4 Several potentially useful methods for
rapid in-plant monitoring are being
explored (Cairns, et a!. 1969) a!1d one
rapid in- stream method is now operational
(Cairns, and Dickson, 1971). The in-plant
methods just mentioned use changes in heart
rate, breathing signal, and movements of
the entire fish within a eontainer to detect
sublethal concentrations of toxicants in a
waste discharge. If successful, these a:1d
other "early warning" in-plant systems
could be used to determine the toxicity of
a waste before it left the plant so that the
appearance of a harmful concentration of
a toxicant would activate a control system
and shunt the waste immediate1y to a
holding pond or recycle it for additional
treatment.
C This continual information about the toxicity
of a waste should enable sanitary engineers
to identify periods of operation likely to
produce the most toxic wastes, as well as
identifying those components of the production
process which contribute most of the toxicity.
D Full development of useful ear1y warning
systems with rapid information feedback
will probably take a ~1umber of years and
will require the close cooperation of a
variety of disciplines. No doubt, the early
developmental period will have its share of
1 :3-6
failures, but it is highly probable that
effective systems can be produced and
that their use will substantially improve
environmental quality control. Since
the ultimate test of the effectiveness
of a waste treatment process should be
in the receiving stream, in- stream
early warning systems also should be
developed to insure a continual flow
of information.
SYSTEMS MANAGEMENT OF WATER
RESOURCES
A Present advances in biological monitoring
combL'1ed with physical and chemical
monitoring capabilitie s indicate that in
the near future we ca.."1. develop and operate
a river basin with varied water resource
uses to maximize beneficial use without
ecological damage.
B Figure 2 illustrates a river basin
management system which includes
reservoirs. agricultural uses, industries
and towns, etc. Conceptually, utilizing
a central control center a!1d rapid physical,
chemical and biological monitoring systems
ecological damage could be prevented.
through the opl:!ration of the system as
a whole rather than each water resource
user being concerned only with his own
discharge.
C If an ind"J.stry in the system had a spill
of toxic material which was rapidly
detected through the continuous monitoring
systems, the following activities might
be coordinated by the control center:
1 Upstream reservoirs could increase
discharges for dilution of toxicants.
2 Municipal water users could curtail
use of water and depend on reserves until
toxicity was dissipated.
3
Downstream industries could shunt to
holding pO~lds to prevent synergistic
interactions.
-------
MIXED INDUSTRIAL AND AGRICULTURAL AREA
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DIAGRAM OF A COORDINATED RIVER BASIN MANAGEMENT SCHEME
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Ecological Consideration in Planning Water Quality Surveys
D Obviously, the water resources management
scheme just outlined is optimistic and
depends on the cooperative activities of
state and Federal government as well as
private users of water resources. However,
we are rapid1y approaching the time when
technology is available to do this job.
Implementation of such a program to
protect and wisely utilize our water resources
now depends on our Si!lCerity.
REFERENCES
Cairns, John, Jr. New Concepts for Managing
Aquatic Life Systems. JWPCF 42(1):77-82.
1970.
Cairns, John, Jr., Kenneth L. Dickson,
Richard E. Sparks, and William T. Waller.
A Preliminary Report on Rapid Biological
Information Systems for Water Pollution
Control. JWPCF, 42(5):685-703. 1970.
Cairns, Jolm, Jr. and Kenneth L. Dickson.
A Simple Method for the Biological Assessment
of the Effects of Waste Discharges on Aquatic
Bottom Dwelling Organisms JWPCF 43(5):
755-772. 1971.
McKee, J. E. and H. W. Wolf. ~!;!..Quali!;y
Criteria. The Water Resources A~enc'y of
California, 1- 548. ] 963. .
13-8
--,--..--
This outline was prepared by John Cairns, Jr.,
Research Professor of Biology and Director,
Center for Environmental Studies, and Kenneth
L. Dickson, Assistant Professor of Biology
and Assistant Director, Center for Environmental
. Studies, Virginia Polyte~hnic .Institp~e, o~St~~~. - '.
University, Blacksburg, Virginia2406i..~ h;; ,.,
Descriptors:
River Basins, Surveys, Planning, Environment,
Balance of Nature, Management
-------
DILUTION TABLE
To prepare solutions of concentration indicated at left, take number
Concentration desired of milliliters of stock solution shown below, and make up to one liter
with suitable dilution water.
ppm ppb Stock sol: Stock sol: Stock sol: Stock sol: Stock sol: Stock sol:
% or or 11'/0 . 10;0 .01% . 00 10;0 .00010;0 .00001 %
mr!/l 1Jf!./1 10 r!m 11 1 ([mIl. 1 gm/l . 0 1 ([m /l .001 ([mIl .0001 r!m/l
100. 1,000,000
10. 100.000
1.0 10,000 1000
.56 5,600 560
.32 3,200 320
.18 . 1, 800 180
.1 I, 000 100 1000
.056 560 56 560
.032 320 32 320
.018 180 18 180
.01 100 10 100 1000
.0056 56 5.6 56 560
.0032 32 3.2 32 320
.0018 18 1.8 18 180
.001 10 1.0 10 100 1000 .
.00056 5.6 5.6 56 560
.00032 3.2 3.2 32 320
.00018 1.8 1.8 18 180
. 000 1 1.0 1000 1.0 10 lQO 1000
.
.000056 .56 560 5.6 56 560
.000032 .32 320 3.2 32 320
.000018 .18 180 1.8 18 180
.00001 .10 100 1.0 10 100 1000
.0000056 .056 56 5.6 56 560
.0000032 .032 32 3.2 32. 320
.0000018 ; 018 18 1.8 18 180
.000001 .010 10 1.0 10 100
,00000056 .0055 5.6 5.6 56
.00000032 .003J 3.2 3.2 32
..-.00000018 .0018 1.8 1.8 18 .
.0000001 . 0010 1.0 1.0 10
.000000056 .00056 .56 5.6
.000000032 .00032 .32 3. ~
.000000018 . 00018 .18 1.8.
00000001 .0001 .10 1 0
Originally prepared for Training by C. Henderson; modified and arranged by H. W. Jackson.
BI. BIO. met. 5d. 6. 65
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BI. ECO. 9d. 2. 58
15-1
-------
USE OF LC - PAPER
NOTE: The LC, Paper" has been especially
designed for use in the Training Program of
Water Programs Operations. (It is not known
to be commercially available.) The same
results can be obtained with conventional
graph paper (two-cycle semi-log is
recommended) .
I
INTRODUCTION
A
Measurements of Toxicity
Bioassay results using fish are expressed
in terms of Tolerance Limits (LC) for time
"t" . The percentage of experimental
animals surviving for the specified period
of time is written as a subscript to the LG
symbol. For example, the "96-hour
LC'50" is that concentration of a substance
which 50% of the experimental fish can
tolerate for 96 hours. The LG50 is
equivalent to the median tolerance limit
(LCm).
The use of LC with the percentage subscript
allows the designation of pe)~(;entage surviv-
als other than 50%; e. g., a LC lOt would
indicate that only 10% of the fish could
tolerate a given concentration for time t,
while a LG90t would indicate a concentration
which could be tolerated by 90% of the fish
for the time specified. The LC 50 is curr-
ently the standard and should always be
determined.
Unless specified to the contrary, the lab-
oratory exercise in this training course
will concentrate on the determination of
LC 50's, and the instructions below are so
written.
B
Preliminary Procedures
Examine the "control" container. A
bioassay test should not be accepted as
reliable unless at least 90% of the control
animals survive. . Death of any of the
HI. BIO. met. lab. Be.!. 78
controls should be clearly explained in
the "Notes" at the right. If control
survival is satisfactory, proceed as
follows (if not, repeat the test).
n
PREPARATIONS FOR CALCULATING
A LC 50
A Fill in preliminary information as called
for on the right side of the sheet, including
the subscript "50" in the title and also in
the box after "Final Results"; and the time
intervals to be employed; e. g., 15 min.,
1 hr., 4 hI's., or 24 hI's., 48 hI's., 96 hI's.,
etc. (These are the Tim~ "t's"). Circle
the term in which the experimental con-
centrations are expressed; fill in the name
of the test species, the temperature range,
and describe the dilution water. Any
number of LCm's may be calculated fF0r:!
a given setup at successive time inter:v;il~.
B Insert decimal points and/ or zeros in the
column of numerals above "Bioassay Con-
centrations" to represent the dilutions
actually used in the test. If the series
used does not fit the lines provided, use
the coordinate LC Paper, or request
further instructions.
III
TO ESTIMATE THE LC50 AT TIME "T"
A Find the "Percent Survival" scale at the
bottom of the graph. Indicate the percent
survival at each of the concentrations
tested. Use a code to mark the points at
successive times as: a tiny circle, a
triangle, a square, or a color code.
B Locate the highest test concentration
showing greater than 50% sut'vival.
COlmect this point to the survival percentage
of the next highest concentration with a
straight line.
16-1
-------
Use of LC- Paper
C
Read on the scale at the left, the value of
the point where the above line and the 50%
survival line intersect. This value is
the LC.50 concentration for the time
interval in question.
D
If there are points below (i. e., at lesser
concentrations) which show less than 50%
survival, an unreliable population of
experimental animals, poor handling, or
other detrimental factor may be indicated.
Re-examine the survival of the controls.
If it is less than 100%, consider the
advisability of repeating the test.
IV
COMMENTARY.
The LC 50 t concentration is that which will
permit half of the experimental organisms
to survive for time "t" under the conditions
16-2
of the test. If by chance one of the experi-
ment~ncentrations happened tn have 50%
survival at time "t" that is the LC50 con-
centration, no further calculatic:.'n is necessary
(provided there is no higher concentration
which showed an equal or higher survival
rate).
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
WPO, EPA, Cincinnati, OH 45268.
-------
SEC 151
(6 -58)
Series:
Technician:
BIOASSAY RECORD SHEET
Company:
Date:
Starting Hour
Material bel ng tested:
Source:
Source of dilution water:
Test species
No. individuals per concentration:
Start
Temp. range:
Dilution: Control
00
pH
Hardness
Other
No surviving
% survival
00 ...
pH
Other
24 hours
48 hours
No sur vi vine;
0;0 survival
00
pH
Other
96 hours
No surviving
% survival
00
pH
Other
..
BI. BIO. met. 9c. 11. 69
-------
SEC 151
(6 -58)
Series:
BIOASSAY RECORD SHEET
Company:
Date:
Starting Hour
Technician:
Material bel. ng tested:
Source:
Source of dilution water:
Test species
No. individuals per concentration:
Start
Temp. range:
Dilution: Control
DO
pH
Hardness
Other
No survivin~
% survival
DO .,.
pH
Other
24 hours
48 hours
No surviving
0/0 survival
DO
pH
Other
No surviving
% survival
DO
pH
Other
..
96 hours
BI. BIO. met. 9c. 11.69
-------
LC
PAPER
1000
900
800
700
'Y.- - if ~ - -~- " -~t 1 00
i . -. -:-~
.:'"f '; I i '
'- tl -,:..L l+ ~ri- '- - ' , c.~; ,. I
F ..;.~. !.l' " .,': .".' .'j-f ' -/
,
, ~!."-l. .- .u- :i=; I~ ' q -L" L
b ~1.." ' ;I~ 1"-1 j.{ j' f; .:£::!. .L '
.-
-L "'--:' -£1. H~ I_f-- -.1; - ..
. ". ,":*"- C
" - '. :E~ ~ ~ , -
- .. ::1::1.-;- -- :t. - -
I+-t:t: 1=1-; - 'P=I- -, -r. =- : 5 60
- =-1=t=I-,:ui -- . -. "
- - : .
-- . . - ..
- ~ -- - -.
--
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=-- -- -- - :1:
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.
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. -
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- -.
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.. 1 0
600
500
400
300
zoo
100
90
80
70
60
50
40
30
20
10
o
Log
Scale
100
50
Percent Survival
Bioa s sa y Sea Ie
(insert decimal points)
Note:
This paper not commercially available.
Sheet No.or Code:
o
Material Tested:
Sta rting Date:
Hour:
Final
Results:
Time ruterv ls
LC
Concentrations Expressed as (circle one):
%. mg/l. other:
Test species:
Temperature Range:
Dilution Water(source & characteristics):
Note~:
Technician:
ill. BIO. met.l5b.6. 73
-------
1000
900
800
700
,~.n. ~ ..ll. ...... ..- -H- -t-i- -H--
'F'r .. t Tl-- Iii I n.r 1 ~'1"
io~'~. rL Tt ~ri ~ +F: ,;~.~ r~ it
:i;~ ~7~ J~ ";~1~ :l~i' i.l~ .> ~ ~i+ ~'. ,
=-. .. 1:::j~:.~~E:. 4:..: -.~
: .. .,. .. .. -" F~:..':-t:.£" - , -0
1+-1=1" I=\-t4=H::J=t: -;:. ~. = + 1,-1'" .. :
600
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. ..
400
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zoo
100
90 "
" . -
80 -
- -
. ~
70 .
: ..
- .~ _.
60 = -
.. ..
50
40
30
zo
10
o
Log
Scale
.... - ::\=-1: -
..
-
-
LC
PAPER
-
..
-
,.
1000
560
320
Note:
This paper not commerc.ially available.
-
-
... ..
..
.
'0
- ---
..
..
..
-
.0
.. -
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-
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..
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'.
.. ..
of
..
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.0
;
.- -.
c
-:
..
'.
"'. f
-
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- : 'I'
o'
- ..
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00
- .
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... ..
..
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..
o.
..
..
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..
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-
.. ..
..
-
'.
..
..
-
-
..
,-
..
..
..
..
50
Percent Survival
100
Bioassay Scale
(insert decimal points)
Sheet No.or Code:
Material Tested:
Starting Date:
Hour:
Final
Results:
Time Interv .1s
LC
Concentrations Expressed as (circle one):
0/0. mg/l. other:
Test species:
Temperature Range:
Dilution Water(source & characteristics):
180
Notes:
100
56
3Z
18
10
Technician:
BI. mo. met.lSb.6. 73
-------
SEC 187
(Rev. 11-69)
LC PAPER
m
( Coordinate)
Sheet .No. or Code.
Material Tested:
Starting Date:
Final
Results:
'LC
Concentrations Expressed as (circle one):
%. mg /1. other:
Test species:
Temperature Range:
Dilution Water (source & characteristics):
Notes:
o
Scale
50
Percent
Survival
100
Bioassay
Concentrations
Technician:
Note: This paper not commercially available:
BI.BIO.met.16a.11.69
-------
SEC 187
(Rev. 11-69)
LC PAPER
m
( Coordinate)
Sheet .No. or Code.
Material Tested:
Starting Date:
Final
Results:
r
LC
Concentrations Expressed as (circle one):
%. mg/l. other:
Test species:
Temperature Range:
Dilution Water (source & characteristics):
Notes:
",
o
Scale
50 100
Percent Bioassay
Survival Concentrations
This paper not commercially available:
Technician:
Note:
BI.BIO.met.16a.11.69
-------
(Extracted from:
ORSANCO 24-Hour Bioassay, January 1974)
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J-
L 24 hr.
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96 hr.
48 hr.
Observer:
BI. AQ. 32. 11.74
I
50
1
100
-------
(Extracted from:
ORSANCO 24-Hour Bioassay, January 1974)
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1~4hr.
J
Final I Time interval
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Observer:
96 hr.
BI. AQ. 32. 11.74
-------
(Extracted from:
ORSANCO 24-Hour Bioassay, January 1974)
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J
~4hr.
48 hr.
96 hr.
Observer:
BI. AQ. 32. 11. 74
-------
(Extracted from:
ORSANCO 24-Hour Bioassay, January 1974)
BIOASSAY PAPER (Log~probit)
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