PROCEEDINGS
of the
- t: - a 11 vas •••:, :>:M
ON
WATER POLLUTION RESEARCH
TOXICITY IN THE AQUATIC ENVIRONMENT
Assembled by
Edward F, Eldridge
Research & Technical Consultation
Project
U. S. DEPARTMENT OF HEALTH, EDUCATION & WELFARE
Public Health Service
Region IX
Portland, Oregon
November 1961
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FOREWORD
The Public Health Service in May of 1957 established an
activity in the Portland office known as the Research and
Technical Consultation Project. The objectives of this proj-
ect are to provide a closer contact and better service to those
persons engaged in research in the water pollution and related
fields and to stimulate the interest of others in this field
of study.
As a part of this activity, a series of symposia have been
held where an opportunity is provided for an informal discussion
of subjects relating to water pollution. To date, ten symposiums
have been held in Portland covering the following subjects:
1. Research Relating to Problems of Water Pollution
in the Northwest
2. Financing Water Pollution Research
3. The Slime (Sphaerotilus) Problem
4. Short-term Bio-Assay
5. Siltation - Its Sources and Effects on the Aquatic
Environment
6. Oceanography and Related Estuarial Water Problems
of the Northwest
7. Status of Knowledge of Watershed Problems of the
Northwest
8. Radioactive Waste Problems in the Pacific Northwest
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9. Research in Water Pollution and Other Environmencal Health
Fields
10. Toxicity in the Aquatic Environment
Over a hundred persons representing universities, colleges,
regulatory agencies, and State and Federal departments met in Port-
land on November 14 for a discussion on the "Toxicity in the Aquatic
Environment." Attendance-wise this was the largest symposium to-date
indicating a considerable interest in this subject. This report
contains the proceedings of this meeting.
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TENTH PACIFIC NORTHWEST RESEARCH SYMPOSIUM
SUBJECT: TOXICITY IN THE AQUATIC ENVIRONMENT
DATE: November 14, 1961
PLACE: Hearing Room, Interstate Commerce Commission
410 S. W. Tenth Avenue, Portland, Oregon
AGENDA
A.M.
9:00-9:10 I - Foreword and Introductions - E. F. Eldridge
9:10-9:50 II - Introduction to Toxicity Research — Scope
and Significance of Problem - Dr. C. M.
Tarzwell, Chief, Aquatic Biology, Robert A.
Taft Sanitary Engineering Center.
Ill - Pesticides
9:50 - 10:30 1. Public Health Service Studies - Dr. H. P.
Nicholson, Chief, Pesticide Pollution
Studies, Public Health Service, Atlanta,
Georgia.
10:30 - 11:00 2. Toxicity of Insecticides to Fish in the
Northwest - Dr. Max Katz, Research Asso-
ciate Professor, College of Fisheries,
University of Washington.
11:00 - 11:30 IV - Toxicity of Metals and Their Detection -
Dr. W. Allan Moore, Chief of Laboratory,
Public Health Service, Portland, Oregon.
11:30 - 12:00 V - Pulp Mill Wastes vs. Oysters - Dr. R. E.
Dimick, Professor, Fish and Game Management,
Oregon State University.
P.M.
1:15-1:45 VI - Process Wastes from Atomic Energy Operations -
Mr. P. A. Olson, Biology Operation Section,
General Electric Company, Richland, Washington,
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1:45 - 2:15 VII - Toxic Effects of Organic Deposits - Mr. Charles
Ziebell, Biological Analyst, Washington Pollu-
tion Control Commission.
2:15 - 2:45 VIII - Toxicity vs. Agricultural Plants and Soils -
Dr. C. D. Hoodie, Professor of Soils, Washington
State University.
2:45 - 3:15 IX - The Shellfish Toxin Problem — Effect on Humans -
Mr. John Girard, Sanitarian, Washington Department
of Health.
3:15 - 3:45 X - Toxicity Bioassays vs. Chemical Methods -
Dr. Peter Doudoroff, Fisheries Research, Public
Health Service, Oregon State University.
3:45 - 4:15 XI - Bioassay — The Bi-Valve Larvae Tool - Mr. Charles
E. WoeIke, Fisheries Biologist, Washington Depart-
ment of Fisheries.
4:15 - 4:45 XII - Bioassay Studies at Nanaimo, British Columbia -
Dr. D. F. Alderdice, Fisheries Research Board of
Canada, Biological Station, Nanaimo.
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TOXICITY IN THE AQUATIC ENVIRONMENT
INTRODUCTORY REMARKS
E. F. Eldridge*
Life on planet "Earth" is becoming more and more complex.
There is some question as to whether Communism and the atomic
bomb are our major hazards to life. It may well be that our
own uncontrolled disturbance of natural life cycles will be
our undoing in the final analysis. This is an awesome picture
and may be overemphasized, but it is surely one which should
be given serious consideration.
In recent years a great deal of effort has been placed on
the production of chemicals foreign to those naturally present
in our environment. Many of these are designed to destroy some
form of life. These toxic chemicals are certainly not specific
in their action. If they are toxic to undesirable forms, they
may well be equally toxic to desirable ones. Even the destruc-
tion of the so-called "pests" may have an effect on the environ-
ment.
*Physical Sciences Administrator, U. S. Department of Health,
Education and Welfare, Public Health Service, Division of Water
Supply and Pollution Control Program, Pacific Northwest, Portland,
Oregon.
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This destruction of life may or may not be desirable, but one
thing is certain, it must be subject to control. In order to control
the effects of these substances, it is necessary that we know more
about them.
The major carrier of such substances is "water." Our objective
today is to examine the problems of toxicity in this media and to
delineate areas of further study.
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INTRODUCTION TO TOXICITY RESEARCH—SCOPE
AND SIGNIFICANCE OF PROBLEM
C. M. Tarzwell*
(Note: Dr. Tarzwell*s paper was not available in
time to be included in these Proceedings. In lieu
of this paper, the following abstracts of his state-
ments as taken from the recording of the symposium
have been substituted.)
What is a favorable environment? What is the importance for
the continued survival and well-being of man? What value should
be placed upon a favorable environment and further, what efforts
and costs are justified in maintaining this environment? These
are important questions and questions which have to be faced in
this age of technology.
Men, as well as the aquatic life of our frash and marine
waters, are products of their environment. Through the process
of development they became adapted to the physical and chemical
conditions which were prevalent at that time. Through the course
of time, these environmental conditions have become requirements.
These are the things that are required for the survival, growth,
reproduction, and general well-being of life. That is the reason
why organisms that have evolved in different areas have different
environmental requirements.
*Chief, Aquatic Biology, Robert A. Taft Sanitary Engineering
Center, Public Health Service, Cincinnati, Ohio.
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Man has learned to change his immediate environment to a cer-
tain extent. He has his houses, clothes, and central heating systems
with their smells and gases. He has inside toilets, plumbing and
sewage system, which takes the waste away from homes into our streams
and dumps below town. In these ways, man is controlling his environ-
ment*
This has lead him to be somewhat brash in his approach to the
problem of the total environment. In his pursuit for wealth and
power and the better way of life, he has forged ahead without regard
to the effects of the products and by-products of these actions on
the total environment. If any of the earth's resources may be said
to have some primary importance to continued existence, they are
air, soil, and water. If to these is added sunlight, these four
constitute the basic requirements for all life on this earth. These
are the basic resources.
In this age it seems that man has forgotten his origin and is
blind to the real environmental requirements—the conditions under
which he developed. Consequently, these resources have become the
victims of the modern indifference.
The outstanding problem of the time has become the contamination
of the total environment, with substances of incredible potential for
harm. Substances that not only exist in the air, and persist in the
soil and water, but accumulate in fishes, plants, and animals and
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even penetrate germ cells or alter the factors of heredity.
Ever since the chemist has manufactured substances which nature
never invented, problems have become increasingly complex and
the hazards greater. Man only recently began to perceive the
potential of these materials for harm. Only recently has he
developed an awareness of the consequence of their manufacture
and their widespread use and of the waste products resulting from
their manufacture. These materials are well known—the petro-
chemicals, the synthetic organics, the pesticides, the herbicides,
the insecticides, the detergents, and other synthetic materials.
Many of these compounds are not broken down to bacterial action
and their accumulation adds to the problems. They often defy
detection or identification by methods in ordinary use in our
water treatment plants and other laboratories.
They are unreasonably stable in many instances and conven-
tional methods of waste treatment do not remove them. Once
introduced into natural waters, they are a continuing threat not
only to the aquatic life, but to man and animals which may wish
to drink the water or to use the aquatic animals as food. The
use of these materials is widely dispersed and quantities are
increasing. For instance, about 637 million pounds of pesti-
cides and 500 million pounds of detergents were manufactured in
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1960. These substances are appearing in our surface waters in appre-
ciable quantities and they have also been found in the ground water.
An example is the Colorado experience where the wastes were dis-
charged into the ground and how the effects are creeping over large
areas destroying the vegetation and poisoning animals.
This is the problem that man is facing today. Now what is he
going to do about it? In approaching any problem, the first require-
ment is to define terms in order that everyone is talking about the
same thing. There are several that are very often used loosely.
"Pollution" is one of these. "Pollution" is the addition of any
material or any change in water quality or condition which interferes
with, lessens or destroys the beneficial use of a water. If there
is not an interference with water use, there is not pollution.
Therefore, when a person speaks of pollution in general, he very
often uses the term loosely.
Another term which should be defined is "toxicity." What is
"toxicity"? What determines "toxicity"? Many of the food elements
needed for growth, such as sulphur, potassium, calcium, iron, magne-
sium, and iodine, can become toxic if they are present in large enough
quantities or are not buffered. The trace elements especially such as
silicone zinc, copper, manganese, molybdenum and boron, can become
quite toxic if they are in large enough amounts. However, these
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elements also are required for good growth. It is the amount that
is of importance. Therefore, a requirement such as "nothing toxic
shall be added to a water," is not adequate or reasonable. We
need to specify the amount that can be added without causing
unsatisfactory conditions*
Bio-assays are used for measuring toxicity, especially for
acute toxic effects. They also can be used to show long-term
effects through continuous exposure. However, for the long-term
effects some physiological tests will eventually have to be used.
These will involve the effects on growth, activity, and certain
other physiological functions.
Now what are the sources, the nature, and extent of our
pollutants? How do they effect the aquatic environment and what
are the considerations which should be taken into consideration
in approaching the problem? The source of toxicants are the
wastes. They may be present in municipal wastes, industrial
wastes, or agricultural wastes.
Agriculture should be considered to be an industry with
certain wastes. Crops, gardens and forests are treated with mate-
rials that have very high toxicity, and very persistent. They
reach surface and ground water by runoff or seepage.
•-4
In the handling of toxic wastes, consideration must be given
to the source. If they originate at a point such as a sewer
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outfall, they may be amenable to treatment. There are those that
come from land and may be contributed from an entire watershed.
These cannot be collected and treated. This means, therefore, that
in meeting these problems we must control the material at the source
of use. This, of course, applies to silt, fertilizers, and radio-
active material, as well as to pesticides and other toxic substances
used on vegetation and soils. One exception is the removal of these
materials from domestic and industrial water supplies by water treat-
ment.
Among the factors to be considered in a study of toxicity are
precipitation, amount and rate of runoff, the season of the year,
temperature, the dilution available and, above all, the quality of
the receiving water. This latter, of course, is determined by the
geology of the area, the type of soil, the vegetation, etc. The
quality of the receiving water can be all important in determining
the toxicity of materials that was added to it.
Next, it must be recognized that there are great variations in
the toxicity of certain materials. For instance, it is found that
the organic phosphorus compounds vary greatly in the toxicity to
different species of organisms. The greatest variation experienced
is that of Delnab, which is 940 times more toxic to bluegills than it
is to the goldfish. This may be an extreme range, but among the
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organic phosphorus compounds we find wide ranges, such as mala-
thion is about 500 times more toxic to salmon than it is to fathead
minnows, and about 250 times more toxic to bluegills. Also, there
are differences in tolerance between fish and the aquatic organisms.
Endrin is very toxic to fish, whereas some other materials are more
toxic to some aquatic insects than they are to fish. Toxicity also
varies in the different age groups, and with activity. These
variations must be taken into consideration. The most sensitive
forms must be protected.
Some substances are acutely toxic, whereas some have an
accumulative effect. The time of exposure is another variable.
It is also necessary to consider sub-lethal effects. The
species can be eliminated even if the fish or an aquatic organism
is not killed. The organism may be so weakened physiologically
that its predators and other organisms will overcome it and the
species may eventually disappear. All aquatic life is in compe-
tition. If the ability of an organism to meet its environment
and to compete with other organisms is reduced, it can be effec-
tively wiped out as if it were killed.
This problem of toxicity has not been met effectively to
date. There is a lack of knowledge of the amount of each toxic
•"••li
substance which is allowable. The levels of concentrations, both
upper and lower, which are required for life must be determined.
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The levels of concentration which are acutely toxic and those which
are toxic under conditions of continued exposure must be known. To
insure growth, survival, reproduction, general well-being, and the
production of fish as a crop requires that the most favorable levels
of concentration in which to get the best production be maintained.
This is not an easy task and will require research and investi-
gation by many organizations and groups. It will require coordina-
tion of effort and the exchange of information. It must be done if
the problems present and future are to be met.
In short, water quality criteria is needed--that defines the
allowable amounts of certain materials that may be present in the
stream. Without this knowledge, pollution cannot be effectively and
efficiently detected and evaluated. Neither can a determination be
made of the amount and nature of waste treatment required. The
quality of water required for each beneficial use must be known.
This knowledge is lacking in many areas and especially in the area
of toxicity.
It is necessary, therefore, that serious consideration be given
to the contamination of the environment. Water is only one source of
exposure--there is air, food, etc. The materials from manufacturing
processes and their by-products and the products of advanced tech-
nology which are leading to what is considered a better way of life,
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may in reality be the evil products which can adversely affect
the total environment. "Toxicity,'1 therefore, is now a problem
which concerns all.
DISCUSSION
STATEMENT: I would like to give several examples of the effect
of toxicity. Arsenic is used in herbicides, for insect control,
and in the South, in cotton and tobacco fields (Recently new
organic insecticides have taken its place in the tobacco field
application). From 1932 to 1952 the arsenic in tobacco contin-
uously increased--as much as 300 to 600 per cent. The use of
arsenic, therefore, is widespread.
Arsenic was found in a water supply in Argentina. Cancer
was very widespread. When the arsenic was removed cancer de-
creased.
Arsenic was used in the vineyards of Germany for about 20
years (1920 to 1940). Records now show that workers have cancer
of the skin, liver, etc. This is a long time effect.
Effects that were not suspected are now turning up. We do
not know the effects of such substances in water where they are
present for long periods.
Q, Is the Robert A. Taft Sanitary Engineering Center actively
r-1.
working on water quality criteria?
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A. We are putting all our eggs in one basket and that is the basket.
Yes, we are doing all we can with the funds available. We are
now going ahead with physiological studies. We are trying to
determine sub-lethal effects. We feel that the physiological
studies will be of outstanding importance. It is hoped that these
studies will disclose the required water quality criteria for
those substances investigated. They will not, of course, specify
the criteria to be used in enforcement activities. These must be
established by regulatory agencies using the study as a base.
Q. Do you feel that the research in this particular phase of the
pollution problem can keep ahead of the development of new
chemicals?
A. It hasn't. We need much more research in the biological phase
and we might as well face up to it.
Q. Who in your opinion should do this research?
A. Actually, the industry should do this research before the chemical
is marketed. I realize that this will cost the industry money and
may interfere with the production of new chemicals. Before these
chemicals are spread in the environment, we should learn much more
about their effects.
DDT, for instance, and some of the insecticides are known to
effect the hormones and other physiological functions of humans
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and animals. These chemicals are having implications which
you never dreamed of. The problem is a product of our civ-
ilization and we must face it. Otherwise} we may destroy
the very environment in which we originated.
Q. Is there any estimate as to how much we would have to increase
our research in order to catch up with the developments to
date?
A. Any statement in this regard would be purely a guess, since
it is impossible to estimate the time involved in doing
research. We cannot just study the simple effects, we have
to study the multiple effects in the environment and when we
get into that it will take a long time, many people, and
considerable money. There is plenty of work for all of us--
those that work in the universities, in the states, in the
various fisheries departments and in all other interested
agencies. It is important, also, that we get together and
coordinate as much as possible our efforts in order to avoid
duplication. It will take millions of dollars in research
in order to protect our aquatic life from the effects of these
toxicant materials.
STATEMENT: More biologists working in4the water pollution are
required, if this research is be done adequately. There are few
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universities or colleges in the country making any effort to train
biologists in water pollution research. One of the outstanding
schools where such training is available is Oregon State University.
Q. In the past there seems to have been money made available in the
form of grants for medical and engineering research* Is it now
possible that these grants will be more directed toward the
biological field?
A. The Public Health Service through the Research and Training Grants
Branch now has training and research funds for any person in any
school and in any department of those schools, including biology,
who will work on water supply and water pollution research. The
funds were recently made available by the Congress. The funds
are available and we are presently in the process of determining
how best to use the money.
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PESTICIDE POLLUTION STUDIES IN THE
SOUTHEASTERN STATES
H. P. Nicholson*
Technological advances during the past twenty years have
resulted in the development of new types of pesticides and
vastly expanded use. United States production now exceeds 300,000
tons annually of about 200 basic compounds (1,2). It has been
estimated that pesticides are applied annually to over 100,000,000
acres in the United States (2). This usage has generated special
concern among those persons and agencies responsible for water
pollution control and conservation because
1. Pesticides are poisons by nature even though their
destructive qualities are valued when properly controlled.
2. Their effectiveness often demands widespread applica-
tion to land, forest and water where ultimate disposition cannot
always be pre-detetrained.
3. Destructive side effects in the form of unwanted
deaths of aquatic and upland animals, and other incidents,
have attracted special attention and marshalled public opinion.
*Chief, Pesticide Pollution Studies, Division of Water
Supply and Pollution Control, Public Health Service, Region IV,
Department of Health, Education, and Welfare, Atlanta, Georgia.'
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4. Their development has been so rapid that evaluation of side
effects has not kept pace.
Dr. Mark D. Hollis, Assistant Surgeon General and Chief Sanitary
Engineer of the United States Public Health Service, warned in 1959,
"The treatment of large acreages with weedicides, insecticides, and
(other) pesticides results in contamination of water supplies, both
surface and ground waters, through percolation and runoff" (3).
He pointed out the need to learn more about behavior of chemical
exotics in streams, their relations to maximum water reuse, and
effects singularly and in combination on aquatic life.
Certain pesticides have occurred in our waters. Middleton and
Lichtenburg (4) reported detection of DDT in April, May, and June
1957 in the Mississippi River at Quincy, Illinois, and at New
Orleans; in the Missouri River at Kansas City, Kansas; and in the
Columbia River at Bonneville, Oregon. Previously it was reported
from Lake St. Clair and the Detroit River. They also reported
aldrin in a single sample collected at Pullman, Washington, from
the Snake River. Sources of these insecticides and circumstances
connected with their appearance were not determined.
Herbicides also have been a source of trouble. Cottam (2)
reported a brief industrial discharge of 2,4-D in 1945 in California
that penetrated the underground aquifers and contaminated a city
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water supply for a period of four or five years. He also dis-
cussed an instance where 2,4-D apparently was generated from a
combination of industrial wastes lagooned near Henderson, Colo-
rado. First noticed in 1951 when crop damage resulted from use
of well water for irrigation, some 60 square miles of ground water
were at one time said to be contaminated.
More recently the Basic Data Branch of the Public Health
Service's Division of Water Supply and Pollution Control assem-
bled data on pollution caused fish kills that occurred in 1960
throughout the United States (5). These data show that 81 of
305, or 27 percent of all fish kills reported, were attributed
to agricultural poisons.
Stimulated by a growing awareness of the potentialities
for water quality degradation and loss of water resources
associated with large scale production and use of pesticides,
the Public Health Service began studies in 1959 in the South-
eastern States to evaluate the occurrence of pesticides in both
surface and ground waters. It was desired to learn if use of
these biologically active chemicals was creating more than
sporadic water pollution readily detectable by fish kills, crop
damage, or the creation of undesirable taste. We wished to
*vL
know, in brief, how general was the occurrence of pesticides in
surface and ground waters, what were the less obvious effects
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on organisms living in the aquatic environment, and what factors
relate to the presence or absence of selected pesticides in water?
These questions have many ramifications involving multiple disci-
plines. The pesticide pollution studies staff in Region IV includes
persons trained in chemistry, entomology, sanitary engineering,
limnology, agricultural engineering, and microbiology. As the need
arises we borrow the assistance of persons trained in other disci-
plines from sister agencies such as Geological Survey, Fish and
Wildlife Service, Tennessee Valley Authority, and various State
Agencies.—
The first of our studies was begun in June 1959 and still con-
tinues in a 400 square mile cotton growing area drained by a single
river system (6). A municipal water treatment plant using water
from the stream is located in the lower portion of the watershed.
An estimated 15,000 acres of cotton are grown annually in the basin,
mostly in small plots averaging about 10 acres. Principle insec-
ticides employed are toxaphene (65% of total by weight of active
ingredient), DDT (257.) and the gamma isomer of BHC (57.). In 1959
an estimated 79,000 pounds (technical) were applied and in 1960
about 59,000 pounds were used.
J7 Chemical analyses and bioassays in connection with these
studies are done by the Department of Agricultural Chemistry, Clemson
College, Clemson, South Carolina, that is under contract to the Public
Health Service.
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Nearly continuous water sampling has been carried on at
the water plant by adsorption of insecticides on activated
charcoal. Companion samples are taken from both raw and treated
water. Gamma BHC was recovered in amounts ranging from 150 to
760 parts per trillion over a period of two months during the
late summer of 1959, but not in 1960. A substance, identified
as toxaphene on the basis of very strong circumstantial evidence,
was recovered with intermittent absences throughout the first 22
months of study. Later samples remain to be analyzed. Recovered
concentrations ranged from less than 5 to 150 p.p.t, with larg-
est recoveries occurring during and just after the late summer
period of intensive insecticide application.
Available evidence indicates that gamma BHC and toxaphene
were present in solution in river water, not adsorbed on sedi-
ment; and that they probably reached the river in surface runoff.
Furthermore, the contribution was from all parts of the water-
shed, not just a few strategically located fields. The fact that
toxaphene can and does appear in water throughout the year is
deemed highly significant and relates to the large quantities of
this insecticide applied to the land, its persistence in soil,
and its solubility in water. DDT, that is as persistent but not
as soluble, was not recovered. The municipal water treatment
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process did not remove these insecticides or reduce the quantities
present. Current studies are directed toward removal by use of
activated carbon in water treatment.
Studies of fish populations, macro-invertebrates and zooplank-
ton were conducted during 1959 and 1960 to determine effects of
exposure to these insecticides (7). No adverse results were demon-
strated at the concentrations to which these organisms were exposed.
A second study recently completed involves factors related to
parathion contamination of a farm pond located in a peach orchard,
persistence of that insecticide and the biological effects of that
contamination (8,9). It was determined that parathion contamina-
tion of water in the pond (0.02 p.p.b.) and pond bottom mud
(1.9 p.p.m.) occurred in the spring of 1960 before any use of para-
thion that year. It was found that the contamination probably
occurred in March 1960 during a period of accelerated soil erosion,
and that the source of the parathion was the orchard soil where the
insecticide had persisted for at least nine months. This changed
our opinion of parathion which had been thought to decompose much
more rapidly.
Not all problems are related to use of pesticides in agriculture
or forestry. The discharge of parathion bearing wastes in May 1961
by an industry manufacturing insecticides resulted in an extensive
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local kill of fish and is suspected of affecting fish over 100
miles of stream.
Formulating plants and storage houses also are sources of
potential trouble. Most states have a number of these estab-
lishments. In the Southeast we had 223 registered formulating
or manufacturing establishments in 1957-1958. The explosion and
subsequent destruction by fire of one such place in South
Carolina in 1961 awakened us to the realization of their poten-
tial as sources of water contamination.
Other studies under way in the Southeast involve use of
endrin in sugar cane culture, possibilities of ground water
contamination in areas of intensive pesticide usage, and studies
of selected herbicides persistence in natural waters. As in
most such cases, more questions seem to arise than are answered.
However, it is believed that a basic need for knowledge is
being filled, and that knowledge will have increasing value
and application as time passes.
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BIBLIOGRAPHY
1. Shepard, H. H., J. H. Mahan, and C. A. Graham, 1961. The Pes-
ticide Situation for 1960-1961. Agricultural Stabilization,
U. S. D. A.
2. Cottam, C., 1960. Pesticides and Water Pollution. Proceedings
the National Conference on Water Pollution, Washington, D. C.
pp 222-235. Dec. 12-14.
3. Hollis, Mark D., 1950. Water Pollution as Related to the Prob-
lem of Water Resources. Lecture presented at 33rd Annual
Meeting of the Ohio Sewage Treatment Conf., June 18, Cincinnati,
Ohio.
4. Middleton, F. M. and J. J. Lichtenburg, 1960. Measurement of
Organic Contaminants in the Nation's Rivers. Indust. and Eng.
Chem. 52:99A-102A.
5. Anonymous. Pollution-caused Fish Kills in 1960. Basic Data
Branch, Div. of Water Supply and Pollution Control, Public
Health Service, Department of Health, Education, and Welfare.
Public Health Service Publication No. 847.
6. Nicholson, H. P., H. J. Webb, G. J. Lauer, R. E. O'Brien, A. R.
Grzenda, D. W. Shanklin, L. E. Priester, Jr., and D. H. Smith.
(In press). Water Pollution by Insecticides in an Agricultural
River Basin. Part I. Occurrence in River and Treated Munici-
pal Water. Submitted to Jour. Econ. Entomology.
7. Lauer, G. J., A. R. Grzenda, H. F. Nicholson, and D. W. Shanklin.
(In press). Water Pollution by Insecticides in Agricultural
River Basin. Part II. Effects on Aquatic Animals. Sub-
mitted to Jour. Econ. Entomology.
8. Nicholson, H. P., H. J. Webb, G. J. Lauer, R. E. O'Brien, and
A. R. Grzenda (In press). Insecticide Contamination in a
a Farm Pond. Part I. The Origin and Duration of Insecticide
Contamination. Submitted to the Trans. Amer. Fisheries Soc.
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9. Grzenda, A. R., G. J. Lauer, and H. P. Nicholson. (In
press). Insecticide Contamination in a Farm Pond.
Part II. The Biological Effects of Insecticide Con-
tamination. Submitted to the Trans. Amer. Fisheries
Soc.
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TOXICITY OF INSECTICIDES TO FISH
IN THE NORTHWEST
Max Katz*
The material presented in this paper has appeared in a
recent issue of the journal of the American Fisheries Society
under the title "Acute Toxicity of Some Organic Insecticides
to Three Species of Salmonids and to the Threespine Stickleback."
The information presented here is contained in the eight tables
which follow this discussion. References are also shown.
These tables contain a record of tests for toxicity of a
number of chlorinated insecticides to Northwest cold water fish.
The estimated median tolerance limits (TLm) for chinook and coho
salmon and rainbow trout fingerlings to the insecticides tested
are listed in Table 1 and are expressed in parts per billion
(p.p.b.). Table 2 compares the 96-hour TI^ of chinook and
coho salmon and rainbow trout with results reported by Henderson
and Pickering for fathead minnows, bluegill, goldfish, and guppy.
The Tiro for marine threespine stickleback are shown in Table 3.
Endrin was consistently the most toxic of all of the
compounds tested. Co-Ral is the least. Cohos and rainbows
*Research Associate Professor, College of Fisheries, University
of Washington, Seattle, Washington.
28
-------
were generally more tolerant than chinooks except in the case
of endrin where the difference was slight. The differences
in tolerance of the three species, however, were not large in
most cases.
Comparison with the results of Henderson and Pickering
shows the salmonids, especially the chinook salmon, to be
somewhat more sensitive than the warm-water fish.
The fact that some insecticides are toxic only in
relatively high concentrations indicates that useful chemicals
can be developed and used that will not be highly toxic to fish.
Table 4 shows endrin to be extremely toxic and that the
96-hour TLn, does not vary greatly for the six species of fish
tested.
Table 5 shows that there is little difference in the
toxicity of endrin in waters varying in salinity from 5,000
to 27,000. (Sticklebacks were used for the salinity experi-
ment.)
It was found that the volume of water containing the
insecticides had an effect on the tolerance of fish. For
instance, ten fish in a specific concentration of the chemical
contained in one liter died more rapidly than the same number
of fish in the same concentration contained in a total volume
of 10 liters.
29
-------
This factor was also demonstrated by the results in Table 6
where the number of fish in similar volumes of water containing
the chemical was varied from 5 to 20. This experiment indicated
that the fish died in the container containing the largest number,
This is important in setting up aquaria experiments, but is not
of much practical significance.
Experiments were run using small bluegills and varying
the temperature from three to 25 degrees centigrade. Table 7
shows that the toxicity increased with the temperature--being
about 30 times that at 25 degrees as it was at 3 degrees centi-
grade.
Table 8 shows the results of a test for the effects of
endrin on embryonic developments. The table indicates that
endrin concentrations up to 4.2 p.p.b. did not effect the
fertilization and hatching of stickleback eggs. After the eggs
hatched the pattern of mortality started to develop. The test
indicates that the larvae are more tolerant than the adult fish.
This finding substantiates the results of other research and
indicates that the egg and larvae stages are not as sensitive to
toxicants as are the adults.
30
-------
TABLE 1
ESTIMATED MEDIAN TOLERANCE LIMITS (TLm) OF SOME INSECTICIDES FOR
CHINOOK SALMON, COHO SALMON, AND RAINBOW TROUT
(Expressed In Parts Per Billion Active Ingredient)
Chinook
Insecticide
Toxaphene
Aldrin
Dieldrin
DDT
Lindane
Methoxychlor
Heptachlor
Chlordane
Endrin
Guthion
Malathion
Co-Ral
Sevin
24
7.9
12.4
7.9
38.0
56.0
28.0
32.4
59.0
2.0
6.8
24.5
-
-
Salmon
Hours
48 72
3.3
10.6
6.7
17.0
42.0
27.9
26*6
59'. o
1.2
6.2
23.9
-
-
2.7
8.7
6.1
14.0
42.0
27.9
23.0
57.0
1.2
4.3
23.6
-
-
96
2.5
7.5
6.1
11.5
40.0
27.9
17.3
57.0
1.2
4.3
23.0
-
-
24
13.0
-
17.5
66.0
60.0
66.2
61.9
100.0
1.3
7.0
-
22,000.0
1,330.0
Coho
48
10.5
61.0
15.3
46.0
56.0
66.2
60.4
86.0
0.8
5.0
-
20,000.0
997.0
Salmon
Hours
72
10.0
48.6
14.4
44.0
56.0
66.2
60.4
82.0
0.52
4.8
-
18,000.0
997.0
Rainbow Trout
96
9.4
45.9
10.8
44.0
50.0
66.2
59.0
56.0
0.51
4.2
-
15.000.0
997.0
24
11.5
42.4
15.7
42.0
42.0
62.6
36.7
56.0
0.79
4.7
-
-
-
Hours
48 72
8.4
23.9
13.0
42.0
41.0
62.6
33.8
44.0
0.58
3.8
-
1,800.0
1,600.0
8.4
20.3
9.9
42.0
39.0
62.6
25.9
44.0
0.58
3.8
-
1,500.0
1,350.0
96
8.4
17.7
9.9
42.0
38.0
62.6
19.4
44.0
0.58
3.2
_
1,500.0
1,350.0
-------
TABLE 2
THE 96-HOUR MEDIAN TOLERANCE LIMITS OF SOME INSECTICIDES FOR RAINBOW TROUT,
CHINOOK SALMON, COHO SALMON, FATHEAD MINNOW, BLUEGILL, GOLDFISH, AND GUPPY
(Expressed In Parts Per Billion Active Ingredient)
u>
1X3
Insecticide
Toxaphene
Aldrin
Dieldrin
DDT
Lindane
Methoxychlor
Heptachlor
Chlordane
Endrin
Malathion
Co-Ral
Sevin
Guthion
Chinook
Salmon
2.5
7.5
6.1
11.5
40.0
27.9
17.3
57.0
1.2
23.0
_
_
4.3
Coho
Salmon
9.4
45.9
10.8
44.0
50.0
66.2
59oO
56.0
0.51
_
15,000.0
997.0
4.2
Rainbow
Trout
8.4
17.7
9.9
42.0
38.0
62.6
19.4
44.0
0.58
_
1,500.0
1,350.0
3.2
Fathead
Minnow^
7.5
33.0
16.0
32.0
62.0
64.0
94.0
52.0
1.0
12, 500. O2
18, 000. O3
6,700.03
93.03
Bluegill1
3.5
13.0
7.9
16.0
77.0
62.0
19.0
22.0
0.6
95. 03
180. O3
5,300.03
5.23
Goldfish1
5.6
28.0
37.0
27.0
152.0
56.0
230,0
82.0
1.9
_
_
_
-
Guppy1
20.0
33.0
22.0
43.0
138.0
120.0
107.0
190.0
1.5
_
_
_
—
J7 Henderson, C., Q. H. Pickering and C. M. Tarzwell, 1959. Relative toxicity of ten chlori-
nated hydrocarbon insecticides to four species of fish. Trans. Amer. Fish. Soc. 88:23-32.
2f Henderson, C., and Q. H. Pickering, 1958. Toxicity of organic phosphorous insecticides to
fish. Trans. Amer. Fish. Soc. 87:39-91.
JJ/ Henderson, C., Q. H. Pickering and C. M. Tarzwell, 1959. The toxicity of organic phosphorous
and chlorinated hydrocarbon insecticides to fish in "Biological Problems in Water Pollution."
Trans, of the 1959 Seminar, R. A. Taft, San. Eng. Center. Tech. Rept. W60-3, 76-88.
-------
TABLE 3
ESTIMATED MEDIAN TOLERANCE LIMITS FOR STICKLEBACKS OF SOME
INSECTICIDES IN WATERS OF 5 AND 25 PARTS PER THOUSAND SALINITY
(Expressed In Parts Per Billion Active Ingredient)
u>
5 parts per thousand salinity
Insecticide
Toxaphene
Aldrin
Dieldrin
DDT
Lindane
Methoxychlor
Heptachlor
Chlordane
Endrin
Guthion
Ma lath ion
Co-Ral
Sevin
Hours
24
—
-
20.7
22.0
50.5
93.6
120.8
118.0
-
15.8
96.9
-
-
48
12
48.3
18.9
21.0
45.0
86.4
111.9
118.0
0.45
15.8
94.0
2,254.0
16,625.0
72
9.8
41.5
18.0
18.5
45.0
86.4
111.9
90.0
0.45
14.9
94.0
1,862.0
6,175.0
96
8.6
39.8
15.3
18.0
44.0
86.4
111.9
90.0
0.44
12.1
94.0
1,862.0
3,990.0
25_parts per thousand salinity
24
—
-
-
18.0
66.0
-
111.9
180.0
-
6.9
76.9
-
-
Hours
48
•»
44.2
-
15.0
51.0
69.1
111.9
170.0
-
5.0
76.9
1,764.0
10,450.0
72
8.8
27.4
13.5
14.5
50.0
69.1
111.9
170.0
0.58
4.8
76.9
1,568.0
4,940.0
96
7.8
27.4
13.1
11.5
50.0
69.1
111.9
160.0
0.50
4.8
76.9
1,470.0
3,990.0
-------
TABLE 4
TOXICITY OF AN ENDRIN FORMULATION AT 20°C TO RAINBOW TROUT,
COHO AND CHINOOK SALMON, GUPPIES, BLUEGILLS, AND GAMBUSIA
T^
24 hr
48 hr
72 hr
96 hr
Rainbow
Trout
2.17
1.45
1.12
0.90
Coho
Salmon
12.0
0.56
0.30
0.27
Chinook
Salmon
1.50
1.01
0.92
Guppies
1.10
0,90
Bluegills
0.95
0.60
Gambusia
1.80
1.05
0.75
TABLE 5
TOXICITY OF AN ENDRIN FORMULATION IN WATERS OF VARIOUS
SALINITIES AT 20°C TO THE MARINE THREE SPINE STICKLEBACK
Salinity (part per thousand)
TLm
48 hr"
72 hr
96 hr
27
_..__..._
1.71
1.65
22
2.40
1.57
1.20
15
2.25
1.71
1.57
5
2.62
1.95
1.57
TABLE 6
TOXICITY OF AN ENDRIN FORMULATION AT 20°C TO VARIOUS NUMBERS OF
BLUEGILLS IN THE SAME VOLUME OF WATER. TOTAL VOLUME 15 LITERS.
5 £ish 10 flsh 20 fish
72 hr
96 hr
0.95
0.60
2.25
0.86
2.70
1.10
TABLE 7
TOXICITY OF AN ENDRIN FORMULATION TO SMALL BLUEGILLS
AT TEMPERATURES RANGING FROM APPROXIMATELY 3° TO 25°C
Temperature (degrees C.)
TLm
72 hr
96 hr
3
(1.6-4.5)
8.25
6
(3.5-8.0)
23.2
5.5
10
2.4
15
1.95
1.65
20
2.17
0.86
25
0.36
0.33
34
-------
TABLE 8
EFFECTS OF AN ENDRIN FORMULATION ON THE EMBRYONIC DEVELOPMENT, HATCHING, AND SUBSEQUENT
SURVIVAL OF MARINE THREES?INE STICKLEBACKS IN WATER OF 10 PARTS PER THOUSAND SALINITY AT 20°C
ppb
active
ingredient
Control
0.75
1.35
1.8
2.4
3.2
4.2
no. of
eggs
38
32
21
27
; 22
24
26
% eyed
eggs 4 days
after
fertilization
63
78
43
63
63
58
67
eyed fish
hatching in
8-9 days
85
52
44
82
36
71
54
no. of
fish
hatched
20
13
4
13
5
10
13
7, Survival
Days after beginning of
hatching
3 4 5" eT " 7~~8
100
100
100
84
80
90
61
100
69
75
69
40
90
31
85
61
75
61
20
10
0
75
45
50
30
20
0
0
20
15
50
7
0
0
0
5
0
0
7
0
0
0
-------
REFERENCES
1. Katz, Max, 1961. "Acute Toxiclty of Some Organic Insecti-
cides to Three Species of Salmonids and to the Threespine
Stickleback." Trans. Amer. Fish. Soc. Vol. 90
No. 3:264-268.
2. Katz, Max and George G. Chadwick, 1961. "Toxicity of Endrin
to Some Pacific Northwest Fishes." Trans. Amer. Fish. Soc,
Vol. 90, No. 4:394-397.
36
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TOXICITY OF METALS AND THEIR DETECTION
W. Allan Moore*
The problem of the toxicity of metals to the biota of
streams has raised many questions and has been the subject of
many papers appearing in the biological field. In relation to
these toxicity problems, there is the necessity to differentiate
between the physical and chemical characteristics of such waters
and the analytical methods necessary to determine the chemical
entities which may contribute to or be wholly responsible for
the toxicity results obtained.
It is impossible to review here the effects of the various
metal ions and their analytical determination to which have been
ascribed toxic effects to stream biota. I further believe that
it is necessary to point out that no one parameter, whether in
the field of biology, bacteriology, chemistry or engineering,
can be used to evaluate such toxic effects.
As mentioned previously a great number of metallic ions
have been investigated in relation to their toxic effects. In
this group may be mentioned copper, chromium, nickel and zinc,
*Chief, Laboratory, U. S. Department of Health, Education
and Welfare, Public Health Service, Division of Water Supply and
Pollution Control program, Pacific Northwest, Portland, Oregon.
37
-------
since these four metals resulting from industrial processes would
be expected to appear more frequently and in greater concentra-
tions.
Of the four metals mentioned, copper has received the most
attention. In fact, "Standard Methods" established in the 10th
edition the maximum concentration of copper at 0.01 rag. per liter
in their "Standard" dilution water used for the BOD test. Per-
haps this concentration may be too high or low, but the experi-
mental results so far published have not presented any conclu-
sive evidence that this is the minimum or maximum copper concen-
tration which will affect the BOD test. There can be a vast
difference between the amount of copper ion added and its ionic
concentration in solution to which may be ascribed toxic effects.
The physical and chemical characteristics of the particular
substrate used, will control to a large extent the concentration
of the metal ion present.
It would seem natural, therefore, that a difference in
toxicity can be obtained between waters having a high pH value
(soft waters) and those having a lower pH value (hard waters).
The concentration of organic components present may also effect
the toxic limits of metallic ions. Of those industrial pro-
cesses involving the plating of copper, the plating solution
38
-------
used is usually the copper cyanide complex in an alkaline solu-
tion. However, in pilot plant studies using the activated sludge
process, the removal efficiency of BOD showed little difference
when the copper was fed as the sulfate or as the complexed cyanide
in equivalent copper concentrations. This immediately brings up
the question of the analytical determination of the various
physical states in which copper may be present. In a given sub-
strate copper may be present as the insoluble, colloidal, or as
the soluble metal ion. The total copper can be determined by
first destroying the organic material, adjustment of pH and
development of color by using either the cuprethol or the
cuproine methods. In using the former reagent, it is necessary
to adjust the pH to 5.2 t 0.1. This is best accomplished by
using a phthalate buffer instead of the acetate buffer recom-
mended in "Standard Methods."
In using the '•cuproine" method less rigid control of the
pH is necessary and this reagent is specific for copper and can
be used in the presence of nickel, and chromium, whereas, with
the cuprethol method the presence of either of these two ele-
ments may cause interference. For high concentration of copper
greater than 10 mg. per liter, a volumetric method is used. In
this procedure the iron is complexed as the fluoride, potassium
iodide added to the acidified solution and the liberated iodine
39
-------
titrated with standard thiosulfate. When the soluble and colloi-
dal copper concentrations are to be determined, the sample is
first filtered through a millepore filter. The soluble copper
may be determined by either the cuprethol or cuproine methods
directly. The total copper in the filtrate (colloidal plus
soluble) is determined by wet combustion and the difference
between this and the soluble copper is equal to the colloidal
copper present. It is the concentration of soluble copper to
which may be ascribed the toxic properties of this metal.
In plating solutions in which chromium is used, the metal
in these solutions is in the hexavalent state. The waste dis-
charged from such operations will not only contain the excess
hexavalent chromium, but also the trivalent form. When such
acid wastes are discharged to a sanitary sewer, the excess
acidity is neutralized and the trivalent chromium is precipi-
tated as a hydra ted oxide (C^O-j.X F^jO) and rendered innocuous.
It is, therefore, with the hexavalent form that we are particu-
larly concerned. A portion of this hexavalent chromium will be
reduced to the trivalent state by the organic material present
in the sewered waste. It has been found that concentrations of
hexavalent chromium as high as 50 mg. per liter will not appre-
ciably affect the activated sludge process when fed on a con-
40
-------
tinuous basis. Even when a slug dose of 500 mg. per liter of
hexavalent chromium was fed, the pilot plant recovered after
four days.
The analytical determination of chromium in various sub-
strates is not particularly difficult. However, if chlorides
are present (concentrations greater than 100 mg. per liter)
the chromium must first be reduced with sulfite during the
oxidation of the organic matter present. The reduced chromium
is oxidized to the hexavalent state with KM^ and reacted with
diphenylcarbazide. Soluble chromium (hexavalent) may be deter-
mined by filtering the sample through a millepore filter and
determining the chromium in the filtrate directly.
It has been found that the toxicity of zinc (a commonly
occurring metal) is about the same regardless of whether the
metal is fed as the sulfate or as the complexed cyanide. Its
analytical determination, however, is not as simple as that of
copper and chromium. Many analytical methods have been proposed,
but the comparatively new method in which "zincon" is used has
been found to be satisfactory in many cases. However, in cer-
tain substrates this method leaves much to be desired and the
rather tedious polarographic method has been utilized.
41
-------
Nickel has not proven to be as toxic as some investigators
have claimed. As with many of the other heavy metals, the physi-
cal and chemical characteristics of the substrate will determine
the concentration of nickel ion in solution. For example, in
the primary settler of the activated sludge system anaerobic
conditions are encountered with the subsequent formation of sul-
fides. The presence of such sulfides will remove a portion of
the nickel in solution and render it non-toxic.
The colorimetric determination of nickel in the past was
carried out by using dimethyIglyoxime. Due to the fact that
wide variations can be obtained by this method, a more sensitive
and reliable reagent was sought. The compound, «<- furil dioxime
was found to meet the necessary criteria and today is the reagent
of choice. High concentrations of nickel are best determined
volumetrically with "versenate" using verichrome Block-T as an
internal indicator. In this method the nickel must be precipi-
tated twice as the glyoxime, dissolved and titrated with the
above reagent.
DISCUSSION
Q. In the determination of zinc was the test made directly on
the water or was it necessary to concentrate before testing?
A. It was necessary to concentrate.
42
-------
Q. At what concentration can a direct polarographic reading be
made for zinc?
A. I think you can get down to less than a part per million if
you use concentration, but I do not know how low a concentra-
tion can be determined by a direct reading.
Q. You mentioned that it was possible to distinguish between
dissolved and colloidal copper. I presume by "dissolved"
you mean "ionic" copper. How do you separate the dissolved
from the colloidal?
A. First, we test for total copper (after destroying the organic
material). Then we filter another aliquot portion of the
sample through a millepore filter which will take out all
of this suspended copper. The colloidal and soluble copper
pass through the filter. The colloidal copper will not
react with cuprethol or cuproine. Therefore, the amount of
soluble copper can be determined in the filtrate from the
millepore filter. Total copper is determined using the
same filtrate. You can get the colloidal copper by dif-
ference.
Q. Was this colloidal copper in the form of a carbonate?
A. No, it was in the form of cupreous oxide.
43
-------
Q. What about precipitating copper sulfide from a cyanide
complex?
A. Most textbooks will say that the sulfide cannot be precipi-
tated from the copper complex. Unfortunately, this is not
true. At a pH of 10 no sulfide is obtained from a complex,
but, if the pH is lowered to 9, a small amount of sulfide
is precipitated, and at a pH of 7.5 the copper will preci-
pitate in the form of a sulfide. Therefore, the cyanide
complex is broken up in a pH above 7.0.
44
-------
PULP MILL WASTES VERSUS OYSTERS
R. E. Dimick*
There is need for standard methods by which to evaluate
the effects of pulp mill wastes on oysters, particularly in
the range of concentrations which may be slowly lethal or sub-
lethal. Variations in the results of bioassay reported by
different sources may be due to differences in techniques,
characteristics of waste samples employed, and to other factors,
Bioassay procedures with oysters should encompass the
several life-history stages or phases such as spawning, ferti-
lization, embryonic development, pelagic larvae, growth and
general condition of the adults. It is generally recognized
that oyster bioassay methods should provide for the evaluation
of injurious effects of waste concentrations far below those
causing rapid injuries or mortalities in other test animals.
Recent investigations in several laboratories indicate that
the embryonic stages of some bivalves are particularly suscep-
tible to a variety of toxic substances. These animals, there-
fore, present a promising possibility as test animals for
short-term (2 to 48 hours) bioassay assessments of toxic or
non-toxic properties of wood processing wastes.
*Professor, Fish and Game Management, Oregon State University,
Corvallis, Oregon.
45
-------
Some of the results obtained at the Yaquina Bay Laboratory
indicate that variations in the characteristics of the pulp mill
waste samples employed may have resulted in significant dif-
ferences in the apparent damages to oyster larvae. This sug-
gests that consideration be given to the use of waste samples
which are similar in characteristics to the wastes encountered
in receiving waters, especially those occurring on or near
oyster grounds.
Rates of flow may cause variations in bioassay. In case
of long-term (8 to 9 months) continuous flow exposures to spent
sulfite liquor concentrations in which the effect on adult
oyster mortalities was not clearly demonstrated, it was found
that two liters per minute solution flow was not adequate to
maintain 100 oysters per test tray in approximately equal
condition. Condition factors decreased from front to rear in
the test trays. Solution flows in current bioassays have been
increased to four liters per minute per 100 test oysters
(Natives and Kumamotos) and five liters per minute for 25
Pacifies. (Food supply is undoubtedly involved in the above
variation.)
Another problem in long-term oyster bioassays which has
been encountered is the high mortalities occurring with Native
46
-------
oysters in the control groups. These mortalities range in
different years from 10 percent in 6 months to 24 percent in
one year. On the other hand, mortalities in Kumamotos and
Pacifies have been extremely low (0.0 to 7.0 percent), usually
about two percent in 8 months. A reliable method should be
devised to assess significant differences in Native-oyster
mortalities occurring in SSL concentrations ranging from 10 to
200 p.p.m. during testing periods of 8 months to one year.
There appears to be no problem with this species in high SSL
concentrations (500 to 2000 p.p.m.) for differences in mor-
tality rates in relation to increases in SSL concentrations
become evident within 70 to 80 days.
In the case of Kraft mill effluents, the main problem is
conducting long-term oyster bioassays appears to be that mill
waste samples even from the same mill vary in toxicity from
time to time, and in the same sample with time and differences
in storage conditions. Perhaps there is need for a prepared
or synthetic standard Kraft mill effluent for test and compara-
tive purposes.
DISCUSSION
Q. Did you test any of the components in the kraft liquor?
A. No, however, the mill did give us a list of some of their
47
-------
chemicals, along with other information regarding the waste
that is discharged.
48
-------
GRANTS PROGRAM OF THE PUBLIC HEALTH SERVICE
Ralph H. Holtje
(Note: Mr. Holtje of the Research and Training
Grants Branch of the Public Health Service, Wash-
ington, D. C., described briefly the new arrange-
ments for administering research and training
grants through the Division of Water Supply and
Pollution Control.)
Congress within the last few months has made moneys avail-
able for fellowships and training grants to individuals, train-
ing grants to institutions, and for demonstration grants. A
description of each of these types of grants can be obtained by
a request to Research and Training Grants Branch, Division of
Water Supply and Pollution Control, Public Health Service,
Washington 25, D. C.
The details of the procedures to be used in administering
this grant program are presently being developed in the Washing-
ton office. Appropriations have been made for research grants
for 1962 in the amount of $2.67 million. This is for research
in universities and other organizations outside of the Federal
Government. Appropriations for fellowship grants amount to
$100,000, which will take care of about 20 fellowships during
this year; for training grants $900,OQ0, which are used for
adding to the faculty and facilities and strengthening the
-------
curricula of colleges and universities; and for demonstration
grants the amount is $400,000, The latter type of grant is to
bridge the gap between the obtaining of new knowledge and
putting it into use.
50
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FISH TOXICITY STUDIES ON ATOMIC PROCESS WASTES*
P. A. Olson**
At the Hanford Works large volumes of Columbia River water
are passed through the nuclear reactors as a coolant and are
ultimately returned to the river. This effluent constitutes a
potential hazard to aquatic life since it contains certain
toxic chemicals, is heated and is mildly radioactive. Investi-
gations of the effect of this effluent on aquatic life is one
of the research activities of the Biology Laboratory.
This presentation reviews the results of some of the past
laboratory experiments. Young salmon were the primary test
animals for these studies since they represent a species of
economic value and also appear to be among the forms most
sensitive to the effluent water. Because of easier availability
most of the stocks tested were of Puget Sound origin and these
are somewhat more sensitive to the effluent than those of
Columbia River origin (1). The studies were designed to deter-
mine the effluent concentrations at which adverse effects were
*Work performed under Contract No. AT(45-1)-1350 between the
Atomic Energy Commission and General Electric Company.
**Biology Laboratory, Hanford Laboratories, General Electric
Company, Richland, Washington.
51
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apparent and to evaluate the separate toxic components involved.
The studies were run on a chronic basis characteristically cover-
ing the early life period of the salmon in fresh water from the
time of fertilization of the eggs until the seaward migration of
the fingerlings. Experimental levels or conditions in these
laboratory tests causing adversity to the young fish exceed
existing river conditions by a substantial margin.
Effluent Monitoring
Table 1 summarizes the results of a test exposing young
Chinook salmon to effluent water from November when the eggs
were fertilized until June when the young fish were ready to
migrate. Concentrations of 5 per cent and greater resulted
in increased toxicity.
Table 1
Mortalities and growth of young chinook salmon exposed
to different concentrations of reactor effluent
Condition
Control
17. reactor effluent
27. reactor effluent
37. reactor effluent
57. reactor effluent
107. reactor effluent
Per Cent Mortality
Egg
12.2
11.9
12.0
13.0
13.1
17.0
Fry
4.0
2.9
2.4
2.0
5.0
46.6
Finger ling
2.5
4.0
2.6
4.6
8.1
29.5
Total
18.7
18.8
17.0
19.6
26.2
93.1
Average
WeiRht (g)
1.8
2.3
2.8
3.2
4.2
5.3
52
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Far more young salmon encounter the Hanford Section of the
Columbia as brief transients (upriver fingerlings on their ocean
migration) than as progeny arising from spawning in this immediate
section. Young fish which are exposed to the effluent for the
first time as fingerlings are far more effluent resistant than
when exposed at younger life stages. This has been demonstrated
by experiments in which both chinook and blueback fingerlings
have been exposed to concentrations as great as 10 per cent
effluent for several months without demonstrating any adverse
effect (1,2,3).
Until recently the concentration of effluent in the river
remained relatively stable throughout the day but, of course,
fluctuated seasonally with changes in river flow. However,
since the completion of a power dam just upriver from the Hanford
Project, the river flow is no longer stable throughout the day;
it may be 2.5 times greater during the afternoon and evening
when the power load is highest than during the early morning
when the load is minimal (4). Studies carried out to determine
whether or not these diurnal fluctuations would increase the
toxicity of the effluent to Columbia River fish involved exposing
young chinook salmon to three types of flowing water conditions
'4
between mid-November and early June (5).
53
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No effluent was added to the water supplied to the control
group; in the second case enough effluent was added to make a 4
per cent strength solution since this was expected to produce
some toxic effect; and in the third case, which simulated fluc-
tuating river flow, effluent was added according to the following
pattern:
Per Cent Effluent
6.2
3.8
3.0
3.8
Time of Day
3 a.m. to 9 a.m.
9 a.m. to 11 a.m.
11 a.m. to 11 p.m.
11 p.m. to 3 a.m.
Avg. for 24-hour period
The results are summarized in Table 2.
Table 2
Comparative toxicity of reactor effluent under fluctuating
conditions
4.0
iondition
iver water
% effluent--steady
3 to 6.27. effluent--
fluctuated
Per Cent Mortality
Egg
2.4
2.7
2.3
Fry
0.9
1.4
2.1
Fingerling
3.1
10.4
8.3
Total
6.4
14.5
12.7
Average
Weight (g)
1.0
1.8
2.0
The increased toxicity of the 4 per cent effluent over that
of the river water was evident. The fluctuating concentration of
effluent did not increase the toxicity over that of the 4 per cent
effluent administered at a steady rate.
54
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Toxicity of Hexavalent Chromium
The toxic effect to fish from high concentrations of efflu-
ent has been attributed in part to the presence of sodium dichro-
mate. Chronic exposure tests of young salmon and trout to
hexavalent chromium have demonstrated slight toxic effects at
concentrations approaching 0.02 ppm and marked toxicity at 0.08
ppm (6).
In addition to such chronic tests as above, a test was run
to determine if intermittent exposures involving the same total
amount of dichromate over a period of time would affect fish
more or less than the chronic exposure (7). Since chronic
exposures to fish of 0.08 ppm Cr(VI) are quite toxic (6)
similar exposure levels were used to study the relative effect
of intermittent exposures compared to chronic. Newly spawned
chinook salmon eggs were placed in each of three troughs under
the conditions shown below. The experimental design was such
that each lot was exposed to the same total amount of Cr(VI)
over a two weeks1 period. The experiment was continued for
seven months until the young fish were of sufficient size and
age for migration to the ocean.
55
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ppm Cr(VI) Added
0.07 (continuous)
0.14 (alternate weeks)
0.49 (one 24-hour period per week)
None (control)
The following summarizes the results of the test:
Table 3
Mortalities and growth of young chinook salmon
exposed to chronic or intermittent levels of hexavalent chromium
ppra Cr(VI) Added
0.07 (continuous)
0.14 (alternate weeks)
0.49 (one 24-hour period
per week)
None (control)
Per Cent Mortality
Egg
6.5
6.4
7.3
6.9
Fry
4.5
7.9
1.9
2.5
Finger ling
43.3
35.3
27.4
3.2
Total
54.3
49.6
36.6
12.6
Average
Weight (g)
0.76
0.89
0.94
1.96
No Significant difference in mortality occurred during the
egg stage. After hatching, approximately six weeks passed
before the death rate began to increase. At the end of the fry
stage, poorest survival was in the lot which received 0.14 ppm
(alternate weeks) and best survival was in the lot which received
0.49 ppm (one 24-hour period per week). At the conclusion of the
test, significantly fewer fish survived in the 0.07 ppm (continu-
ous), and best survival was in 0.49 ppm (one 24-hour period per
week).
56
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The growth results are in agreement with the mortality data,
At the end of the experiment the smallest fish were in the chron-
ically exposed lot and the largest were in the lot exposed for
24 hours each week.
For this experimental design the chronic release of dichro-
mate was slightly more toxic to aquatic life than if the same
quantity were released intermittently.
Temperature Tolerance Study
A study was made on the temperature tolerance of eggs and
young of chinook salmon which spawn in the main stem of the
south central portion of the Columbia River (Priest Rapids
locality) (8). This section supports a fall run which spawn
between the middle of October and the middle of November. In
general, the river temperatures range from the mid to high
fifties during major spawning.
A pair of ripe chinook salmon (local stock) was captured
on the spawning grounds. Eggs were taken and the progeny sub-
jected to a temperature test from time of capture of October 26
until the following May. The control temperature followed a
seasonal trend typical for the locality. It started at 57F,
reached a minimum of 36F and increased to A7F at the end of the
test. Other experimental lots averaged-2F, 4F and 8F warmer
57
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than the control throughout the greater part of the test.
presents the summary of results.
Table 4
Mortalities and growth of Columbia River chinook
exposed to different temperature conditions
Table 4
Initial
Temperature
57F
59F
6 IF
65F
Per Cent Mortality
EKK
7.9
6.2
8.7
10.8
Fry
1.8
1.0
1.6
40.9
Finger ling
6.3
2.8
0.0
27.2
Total
16.0
10.0
10.3
78.9
Average
Weight (g)
1.0
1.3
2.2
4.1
Consistent mortality above that of the control occurred only
in the warmest lot. The results of this single experiment indi-
cated that the eggs could begin incubation at temperatures as
high as 61F without significant loss.
Radioactivity
The quantities of the various radionuclides which accumulate
in the different species of fish is dependent primarily on the
feeding habits of the fish and the metabolism of particular
nuclides and not so much on their abundance in the water. The
adult salmon which do not feed after leaving the ocean on their
spawning migration remain virtually uncontaminated. Suckers, which
58
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feed directly upon such material as algae, usually contain higher
concentrations of nuclides than species such as bass which obtain
the isotopes third or fourth hand via other food organisms.
Although a variety of radionuclides have been found in
Columbia River fish, over 90 per cent of the radioactive mate-
32
rial is P (9). There is a seventy-five fold difference in
concentration of radioactive materials in fish between winter
and late summer (10). The lower levels during the cold season
of the year are attributed to reduced consumption of radioactive
food organisms and slower metabolic rates.
Although a number of tests have been run to follow the uptake
and deposition of several isotopes in both marine and fresh-water
fish, there is little direct information on the concentrations of
deposited isotopes which produce significant radiation damage to
fish. Some local laboratory data are available on the concentra-
tion of radiophosphorus in fish which is tolerable. Damage was
not detected in cichlid fish with an average concentration of
32
0.09 ;ic P /g fish after nine months' exposure to the isotope.
Another experiment showed slight radiation damage in trout tested
for six months and having concentrations of about 0.42 uc P^/g
fish (11). From these data one might infer that the threshold
32
concentration of PJ causing some damage lies somewhere between
59
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these two values. The lower value exceeds that of sampled Colum-
bia River fish by an order of 100.
REFERENCES
1. Olson, P. A. and R. F. Foster, "Effect of reactor effluent
water on chinook salmon fingerlings," in "Hanford Biology
Research - Annual Report 1954," Document HW-35917. 19-23
(1955) (OFFICIAL USE ONLY).
2. Olson, P. A., Jr. and R. F. Foster, "Reactor effluent
monitoring with young chinook salmon," in "Hanford Biology
Research - Annual Report 1953," Document HW-30437. 24-35
(1954).
3. Olson, P. A. and R. F. Foster, "Effect of reactor area
effluent water on migrant juvenile blueback salmon," in
"Hanford Biology Research - Annual Report 1954," Document
HW-35917, 24-27 (1955) (OFFICIAL USE ONLY).
4. Harza Engineering Company, Chicago, "Priest Rapids Hydro-
electric Development, Columbia River, Washington. Volume
II, Hydroelectric Projects Report, Public Utility District
No. 2 of Grant County, Washington," June 1955.
5. Olson, P. A., "Effects of variable river flow on the toxi-
city of reactor effluent," in "Hanford Biology Research -
Annual Report 1958," Document HW-59500. 135-137 (1959).
6. Olson, P. A. and R. F. Foster, "Effects of chronic exposure
to sodium dichrornate on young chinook salmon and rainbow
trout," in "Hanford Biology Research - Annual Report 1955,"
Document HW-41500. 35-47 (1956).
7. Olson, P. A, and R. F. Foster, "Further studies on the effect
of sodium dichrornate on juvenile chinook salmon," in "Han-
ford Biology Research - Annual Report 1956," Document
HW-47500. 214-224 (1957).
8. Olson, P. A. and R. F. Foster, "Temperature tolerance of eggs
and young of Columbia River chinook salmon," Transactions
of the American Fisheries Society. Vol. j85, 203-207 (1957).
60
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9. Foster, R. F. and R. L. Junkins, "Off-project exposure from
Hanford reactor effluent," Documen" HW-63654. (1960),
10. Davis, J, J. and R. F. Foster, "Bioaccumulation of radio-
isotopes through aquatic food chains," Ecology 39. 530-535
(1958).
11. Watson, D. G., L. A. George and Patricia L. Hackett, "Effects
of chronic feeding of Phosphorus-32 on rainbow trout," in
"Hanford Biology Research - Annual Report 1958," Document
HW-59500. 73-77 (1959). "
61
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AN EXAMPLE OF THE TOXIC EFFECTS OF ORGANIC DEPOSITS
Charles D. Ziebell*
Introduction
Today we have heard some fine presentations pretaining to
pesticides, insecticides, metals, and pulp mill wastes, and the
part they play in creating toxic conditions to various fish and
shellfish. My specific topic, AN EXAMPLE OF THE TOXIC EFFECTS
OF ORGANIC DEPOSITS, does not lend itself to specific evaulation
as some of the previously mentioned toxicity studies have done.
However, a relationship does exist between the aforementioned
toxic substances and organic deposits even though it is not
always evident on the surface.
Controlled laboratory experiments are excellent guide
lines from which to derive certain conclusions. In an ever-
changing situation in the field, as in an estuary, one must be
cautious in order not to make precarious or erroneous conclu-
sions since results of experiments are not always as defined as
in controlled situations.
*Senior Pollution Analyst, Washington State Pollution
Control Commission, Olympia, Washington.
62
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With this in mind, let us begin to consider briefly what
organic deposits we might normally expect in a marine environ-
ment. Bader (1954), in his study of marine sediments, found
that a series of bottom samples revealed essentially dark mud
with variable amounts of organic matter ranging from about 1 to
8 per cent. He also found up to a certain point, organic de-
posits were beneficial to pelecypods. Many pelecypods actually
utilize organic material from detritus for food. He also states
that above 3 per cent organic content the products of decompo-
sition and/or the decline in available oxygen become limiting
variables and thus population densities decrease.
So, speaking in terms of environmental suitability--in
this case for pelecypods--a certain amount of organic material
deposited on the bottom can be considered normal and beneficial.
However, excessive organic deposits can create situations that
have deleterious rather than beneficial effects.
Microbiological decomposition of organic matter in sediment
occurs in marine environments. Waksman and Starkey (1931) have
shown that natural decomposition of organic matter can produce
aldehydes, hydrogen sulfide, and many other toxic products.
Waldichuk (1959) stated that the build-up of materials
which settle out from pulp mill wastes contributes to a change
63
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in the bottom ecology. The organic wastes blanket the bottom of
marine waters and render them unsuitable for the development and
growth of desirable marine benthic organisms. Populations of
bottom dwelling animals gradually shift from the sensitive species
to more tolerant forms, and as conditions deteriorate further even
these forms may not be able to survive. In general, this gives us
some background information with which we can basically differen-
tiate between normal and abnormal organic deposits and the
conditions pertaining thereto.
Origin of Abnormal Organic Deposits
There are several ways in which abnormal organic deposits
get into waterways to produce sludge beds. One common source is
that of municipal sewage. Wood fibers lost from pulp and paper
making processes cause sludge beds in many areas as these fibers
sink to the bottom. Sawdust from log pond saws, without proper
collection units, are another means by which sludge beds can be
formed. Cannery and frozen food waste solids, if not properly
collected, can cause abnormal organic deposits in a waterway.
These are just a few of the more common sources of organic waste
materials which can create sludge bed problems in waterways in
our constantly growing and developing civilization.
-------
Sludge Bed Formation. Decomposition, and Effect Upon Salmonid Fishes
With the introduction of extraneous organic deposits, eventu-
ally sludge beds are formed and changes in the environment are
inevitable. Aerobic decomposition of the organic material takes
place followed by anaerobic decomposition when the dissolved oxy-
gen supply is exhausted. When anaerobic decomposition occurs
organic material is reduced and eventually sulfides, mercaptans
and methane gas are the products.
Sulfides have been demonstrated to be toxic to fish at very
low concentrations, particularly fish in the family salmonidae.
In addition to toxicity, other factors must be considered when
decomposing organic deposits are involved. In a fairly recent
study which the Pollution Control Commission made, a problem was
encountered that was not as clearly defined as an ordinary toxi-
city problem. A report was received that downstream migrant
steelhead were seen in distress in a small estuary near a particu-
lar industry. Initial investigation substantiated this report
and our first observations during low tide led us to suspect that
excessive wood fiber deposition would be a factor in this partic-
ular problem. Subsequent water quality analysis confirmed, in
part, our suspicions. Sulfides above 1 ppm were detected in
several areas of the estuary.
65
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The next thought was to test the fish in live boxes in areas
above and below the industry to determine whether or not an acute
toxicity problem actually did exist. These live box tests were
conducted at different stages of the tide, A site to place a live
box was chosen in a narrow passageway below the industry. All
fish migrating seaward would have to go through this area in order
to get to Puget Sound. The live box was anchored to the bottom
of the channel and migrant size steelhead were placed in it.
Three hours and 20 minutes later all of the fish were dead. Tests
above the industry revealed no mortalities.
Taking into account the rapid mortalities demonstrated in
the live box experiment below the industry, there was some doubt
that sulfide was the only toxic substance involved.
Consequently, polyethylene test aquaria were brought to the
estuary and water containing industrial effluent that had seeped
through exposed sludge beds was collected from the main channel.
This time the location was approximately 200 yards closer to
the outfall of the industry concerned. Test fish were placed in
the test aquaria shortly after the sample had been taken. Within
three minutes the fish in this aquarium passed through a state
of equilibrium loss and lay immobile on the bottom. In 15 minutes
all of the test fish were dead. Temperatures were checked and
66
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dissolved oxygen determinations were made concurrently. Both
were within tolerable ranges.
This rapid mortality came as somewhat of a surprise. The
difference in the length of time until total mortality of fish
in the live box test and those in the aquarium bio-assay was much
greater than had been anticipated.
Our next move was to make in-plant investigations and then
conduct controlled bio-assays with effluent from the industry.
While investigating in the plant it was learned that bactericides
containing chlorinated phenols and mercury were being used, and
that these materials made up a portion of the effluent. Bio-
assays using the industry waste water demonstrated an acute
toxicity problem. But again, these results were not comparable
to any other tests, since these test fish died in 42 minutes.
After this series of experiments we felt we had determined the
origin of the toxic materials, and the stage of the tide when we
could expect rapid mortalities. We also decided that the possi-
ble reason for the difference in time until death between fish
in the aquaria and live boxes was neutralization of some of the
reducing compounds, a function of dilution, mixing, and aera-
tion, and dilution of the mill waste water. There was no doubt
at this point that the source of a toxic substance had been
67
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located but one question was not answered. It was still not
definitely known why the time until death between the bio-assays
with the industry waste water and the tests with estuary water
below the outfall were so incomparable.
After a review of the test data and after further observa-
tions of the physical situation were made, it was assumed that a
change was experienced as the industrial effluent flowed over the
decomposing sludge beds. As the already acutely toxic waste water
channeled its way over and through the decomposing wood fibers
during a low tide, a chemical change occurred to produce a more
highly toxic water. One possibility could be the presence of
phosphine or its compounds had leached from recently deposited
fibers.
Circumstances such as these are quite often overlooked.
There are times when both effluent and receiving water analysis
may not indicate trouble, but at the bottom of a river or estu-
ary something else may be taking place. Unseen synergistic
effects may put a different light on many problems.
Conclusions
There could be many situations such as was experienced here
that are not always evident or even detectable through chemical
analysis or other means. There could be many of these situa-
68
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tions existing right now where on the surface everything is appar-
ently under control. Effluent analysis may reveal that conditions
should propose no problem. But are we on firm ground when we make
conclusions based on these analyses without actually knowing what
happens under the surface? I sometimes wonder. Is it possible
that there are more instances where synergism or chemical changes
have created problems of acute or chronic toxicity? Have sludge
beds and decomposition of organic material with their resulting
production of sulfides and other products coupled with a syn-
ergistic affect, created problems unseen on the surface? Have
our salmonoid fishes, which by the way, have a pneumatic duct
to the air bladder and sink when they're dead, been affected
without our seeing or knowing it? Gentlemen, these are some of
the things to think about. Too often we have a tendency to over-
look potential problems. Honestly sometimes, but nevertheless
unwisely. Perhaps we should try to project our thinking toward
the future. I'm sure we will uncover many things that have been
overlooked in the past,
REFERENCES
Bader, Richard G., The Role of Organic Matter in Determining the
Distribution of Pelecypods in Marine Sediments. Journal of
Marine Research, Colume 13, No. 1, 1954.
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Fair, Gordon M. and Geyer, John C., Water Supply and Waste-Water
Disposal. John Wiley & Sons, Inc., New York, 1956.
Wagner, R. A., Livingston, A., and Ziebeil, C. D., An Investiga-
tion of Water Quality Conditions In Chamber Creek Estuary.
Technical Bulletin 24, Washington Pollution Control Commission,
1958.
Waksman, S. A. and Starkey, R. L., Soil and the Microbe. John
Wiley & Sons, Inc., New York, 1931.
Waldichuk, Michael, Effects of Pulp and Paper Mill Wastes on
the Marine Environment. Technical Report W 60-3, Robert A.
Taft Sanitary Engineering Center, Cincinnati, Ohio, 1959.
Washington Department of Fisheries, Toxic Effects of Organic and
Inorganic Pollutants on Young Salmon and Trout. Research Bulle-
tin No. 5, September, 1960.
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TOXICITY VERSUS AGRICULTURAL PLANTS AND SOILS
C. D. Hoodie*
Introduction
Terrestrial plants use the soil as their root hold and
as the main source of all nutrients except carbon which comes
from the atmosphere. The soil must provide water for growth
and as a medium from and through which other nutrients are
absorbed. Plant growth is possible only when the active por-
tion of the plant's root system is bathed in water. It can
easily be demonstrated that terrestrial plants operate entirely
in an aquatic environment. This aquatic environment is repre-
sented by soil solution—the solution which occupies the voids
in the soil. The soil scientist concerned with plant nutrition
studies the soil solution in much the same way as the limnolo-
gist or oceanographer studies the waters of streams, lakes,
and the ocean. The chemistry of the soil solution is somewhat
more complex than the chemistry of stream, lake, or sea waters
because of the powerful influence of the soil solids, but the
basic concepts do not differ greatly. It is worth noting that
*Professor of Soils, Washington State University, Pullman,
Washington.
71
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range of ionic concentrations in the soil solution overlaps the
range of ionic concentrations in waters of interest to both the
limnolegist and the oceanographer.
The Soil Solution as the Aquatic Environment of Plants
The soil is a three-phase system—solid, liquid and gas.
The liquid and gaseous phases occur in the pores of the solid
matrix, and are reciprocally related. As the moisture content
(soil solution) is depleted by plant use or evaporation, the air
content of the soil increases. The soil solution exists as films
around the soil particles, microorganisms and plant roots in the
soil. The higher the moisture content, the thicker are these
films. The soil solution always contains dissolved solids.
The quantity and composition of the salts in the soil solution
depends upon a host of factors: the input of salts by rain or
irrigation water, the weathering rate of soil minerals, the
extent of removal of nutrients by cropping, and the leaching and
drainage conditions in the soil are prominent examples. The con-
centration of salts thus depends upon the net input-outgo balance
and upon the water content of the soil.
Plant roots ramify through the soil and are bathed by the
soil solution. Plants obtain water, oxygen and all of the 12
essential mineral elements from or through the soil solution.
72
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As the moisture content of the soil decreases, plant growth slows
and finally ceases because of inability to meet evapotranspiration
requirements for water and because of restricted nutrient uptake.
Microbiological processes are similarly affected. The soil solu-
tion thus plays a central role (Fig. 1), and its composition
reflects the current status of a wide variety of forces.
The composition of the soil solution can be used to indicate
proportions among exchangeable cations on the clay and humus
colloids (22), and to predict existing nutrient conditions for
plants with respect to nitrogen, sulfur and phosphorus (7, 8,
15).
Soil chemists have used many techniques to study the soil
solution and its role in plant nutrition. There is no satisfactory
means for obtaining reasonable volumes of the soil solution as it
exists under field conditions; the amounts extractable, even under
high pressures, are small (22). As a compromise, extracts are
made of the soil after water has been added to the saturation
point, that is when the air has been displaced and the pore
space is full of water. When moistened to the saturation per-
centage, all soils are about equally wet in the sense that the
moisture content will be about twice the moisture content of the
soil at field capacity--the condition which obtains after 1-3
73
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Fig. 1 — The Central Role of the Soil Solution
Exchangeable cations sorbed on the negatively-charged
soil and plant roots equilibrate with the same cations
in solution. Plant roots and microorganism's select
certain nutrients and reject others. The distribution
of ions within the soil solution is heterogeneous;
cations are positively adsorbed on negatively-charged
surfaces whereas anions are negatively adsorbed.
74
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days of free drainage following a rain or an irrigation. The con-
centration and composition of the saturation extract serve as
indexes of conditions in field-moist soils which are significant
to growing plants and provides a basis for comparing soils of
very different moisture-holding capacities (22).
The concentration of ions in the saturation extract, as in
irrigation waters, is simply indicated by conductivity (22).
The unit of conductance most widely used in soils work is the
millimho/cm.; in this paper, the micromho/cm. will be used
because of its wide employment in water quality studies.
For most soils, the concentration of the saturation extract
is usually less than 1000 micromhos/cm.; above this point con-
centration and composition begin to adversely affect plant
growth.
Concentrated soil solutions occur generally under two condi-
tions: (1) where drainage is restricted and (2) where saline
waters are used for irrigation. For the diagnosis of detrimental
effects of soil salinity or the potential detrimental effects of
irrigation waters on soil properties and crop growth, three
attributes of the saturation extract of the soil or of an irri-
gation water are generally considered to be "important:
1. Total Concentration (Salinity Hazard) -- expressed in
75
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terms of conductivity or meq./l.
2. Proportion of Sodium to Other Cations (Sodium Hazard) —
expressed in a number of ways: soluble sodium percentage
(SSP), sodium absorption ration (SAR), and residual sodi-
um carbonate (RSC) are examples.
3. Specific Toxic Effects — commonly expressed as p.p.m.
or meq./l.
Toxicities Caused by Salinity and Excessive Sodium
The Salinity Hazard is a function of salt concentration and
operates in one of two ways: (1) to prevent germination and
emergence of seedlings (1,2,3,5,12) and (2) to increasing total
moisture stress and hence to reducing the amount of moisture
available for uptake by plants (5,12,13,20).
Where salinity is high in the seed bed, erratic germination
is common; if salinity is extreme, the land may be barren. Most
plants are more sensitive to salinity during germination than at
later growth stages. The usual result, therefore, of soil salin-
ity is a partial stand of the crop plant and a much reduced
yield. Through proper placement of irrigation furrows with
respect to seeds rows (4, 13) or through bed design (2,3,4),
it is possible to irrigate in such a way as to reduce the salin-
ity hazard during germination and emergence and thereby ensure
76
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a good stand of plants and a reasonable crop yield.
The diagnosis £f salinity hazard is based on the measured
conductivity of the saturation extract of the soil or of irriga-
tion water. From numerous observations of crop behavior a scale
of salinity can be established. Table 1 is the scale most widely
used in the United States. Figure 2 illustrates one scheme for
interpreting the conductivity of irrigation waters. It should
be emphasized that Table 1 and Figure 2 represent interpretive
guides (19,22,23) which, although widely used, are subject to
modification. Other diagnostic schemes and guides have been pro-
posed and used. The experienced diagnostician uses all the
information at hand and considers other modifying factors such as
soil characteristics (texture, structure, etc.), soil and irriga-
tion management, and drainage when assessing hazards from salinity
or any other source.
The Sodium Hazard operates in a variety of ways. Cations in
solution equilibrate with exchangeable cations on the soil col-
loids (clays and humus). In normal, productive soils the cation
exchange system is dominated by Ca*4" and Mg"1^ and the soil
physical condition is such that water enters and moves readily
in the root zone and gaseous interchange is 'tfapid; plants are
able to develop profuse root systems capable of active respiration
77
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TABLE 1
Scale of Conductivity of the Saturation
Extract of Soils in Relation Crop Response (22)
Conducttvity
of Sat. Ext.
Crop Response
/mhos/cm.
0 - 2,000
2,000 - 4,000
4,000 - 8,000
8,000 - 16,000
16,000 - ?
Salinity effects mostly negligible
Yields of very sensitive crops may
be restricted
Yields of many crops restricted
Only tolerant crops yield
satisfactorily
Only a few very tolerant crops yield
satisfactorily
78
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100
3 4567 6 1,000
3 4 5,000
100 eso 750
CONDUCTIVITY— MIOROMHO8/6M.
SALINITY HAZARD
Fig. 2 — Classification of Irrigation Waters
Based on Conductivity attd Sodium
Adsorption Ratio (22.23)
79
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and nutrient absorption. Upon the addition of moderate amounts
of salt (salinization), desirable physical conditions are main-
tained as long as Ca"^" salts dominate. When the proportion of
Na+ salts in the soil solution increases markedly, Ca"*~*" and
Mg"*~*" are displaced from the exchange complex by Na+, soil phys-
ical condition deteriorates, and a structure adverse to plant
growth results. Soils with much exchangeable Na+ (sodic or
alkali soils) may become dispersed and puddled and, as a result,
poorly aerated. Under these conditions both water absorption
and nutrient uptake by the plant are restricted. If the exchange
complex becomes more than 40-50% saturated with Na+, nutritional
disturbances are evident. Under most circumstances, high alka-
linities (pH 8.5-10.0) are associated with high levels of
exchangeable Na+. The end result is a severe nutrient imbalance;
extremely low levels of available Ca"*^" may occur in sodic soils
which contain as much as 207. CaCO-j. Other deficiencies are
also notes: Fe deficiencies are common and P and Zn deficiencies
have been observed.
Crop failures on sodic soils are chiefly due to germina-
tion and emergence failures—as is the case for saline soils.
Most sodic soils are also saline (saline-sodic). However, germi-
nation and emergence failures on saline-sodic are more often due
80
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to poor physical condition than to salinity. Because of the dis-
persed nature of the sodic soil, water does not infiltrate readily
with the result that the seeds may not get sufficient water to
permit germination. A common hazard with sodic soils is the forma-
tion of harsh surface crusts with sufficient mechanical strength
to prevent emergence of seedlings; these crusts form as the soil
surface dries following a rain or an irrigation.
The diagnosis of Sodium Hazard via analysis of the soil solu-
tion or the irrigation water is effected in several ways. On
soil solutions, two ion ratios are widely used to indicate the
hazard: the soluble sodium percentage (SSP) and the sodium
absorption ration (SAR). The SSP is simply the percentage ratio
of Na to other cations in solution.
Na x (meq./l.)
% M
-
Na -I- K + Ca + Mg
The SAR is a more useful value because it accounts for the
differences in behavior of monovalent and divalent cations in
the soil-water system. The U. S. Salinity Laboratory (22) has
proposed this value:
SAR - Na* (meq./l.)
Mg"f+/2
because it can be used with considerable reliability to predict
the degree to which Na+ saturates the exchange complex of the
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soil—a matter of importance in predicting soil behavior. The K+
ion is not considered chiefly because the amount of K*" in the soil
solution is seldom appreciable even where much exchangeable K*"
exists. Although the SAR concept is not fully accepted by all soil
scientists, there is no doubt that the SAR of a soil solution in
equilibrium with the exchange complex has a tremendous practical
value. It is readily obtained, is sufficiently accurate for
diagnosis and its interpretation in terms for the probable status
of exchangeable Na+ in the soil is relatively simple. The inter-
pretation of soil behavior in terms of crop response is too
complex for treatment here; interested readers should consut
references 6, 8, 14, 17, 18, 19 and 22.
For determining the suitability of water for irrigation pur-
poses, the SSP and the SAR are augmented by a third concept, that
of "residual sodium carbonate" (RSC). The SSP and the SAR of an
irrigation water are readily computed from the water analysis but
their interpretation is complicated because irrigation water is
altered as it becomes soil solution. If the irrigation water
became soil solution simply by loss of water through evapotran-
spiration, no problem would exist. Unfortunately exchange re-
actions between cations occur, plants take out some constituents
and add others (HC03) and precipitates such as CaCO^ and CaS04
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form as solubilities are exceeded. The RSC concept, first pro-
posed by Eaton (10,11) helps interpret some of these changes.
When solutions containing Ca4"1" and HC03 are concentrated by
evaporation, CaC03 often precipitates.
Ca++ + HC03" CaC03 + rf1"
The equation indicates that alkalinity would favor precipi-
tation of CaCC>3. Many irrigated soils in the Western States are
alkaline, and some are strongly so. The consequences of such a
selective precipitation in a solution containing mixed salts are
important; it should be obvious that removal of Ca44" from solu-
tion will alter the Na+/Ca-H' ratio in solution. As the Na^/Ca44"
ratio (shown either as the SSP or the SAR) increases, the Sodium
Hazard increases. The RSC concept is a measure of potential
maximum effect of precipitation of Ca and Mg carbonates.
RSC « (C03" + HC03") - CCa44" + Mg44") in meq./l.
When the RSC is positive, it would be possible to have a
solution virtually free of Ca4*. In point of fact, one actually
finds little or no Ca44" in soil solutions expressed from sodic
soils of pH 8.5 - 10 where appreciable C03" + HC03" exist.
The extent to which the precipitation of Ca44" 4- Mg44" by
HC03" actually occurs is still an unsolved problem. The irrigation
regime, evapotranspiration loss, drainage characteristics of the
83
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soil and many other factors all bear of this question. The RSC is,
nonetheless, a valuable aid in interpreting the hazards to the use
of a given irrigation water.
Figure 2 presents the system of interpreting quality of irri-
gation water based on Salinity Hazard and Sodium Hazard proposed by
Wilcox (22,23). Wilcox (23) considers RSC as another criterion.
Doneen (9) has proposed the use of "effective salinity," a scheme
by which total salinity is reduced by the amounts of Ca-H- and Mg*"*"
which may be precipitated as CaCC>3, MgC03 or CaSO, * Other classi-
fication schemes for irrigation waters have been proposed which
include waters far more concentrated (> 5000 micromhos/cm.) than
those considered by Wilcox (Fig. 2). The requirements for
handling more concentrated waters are discussed by Wilcox (23).
Specific Ion Effects
Effects of ions which are not attributable osmotic effects
on water availability are described, for convenience, as toxic
effects of the ion or salt in question. Under this definition
toxicity may be either a direct effect of the ion on some process
or an indirect effect on uptake or metabolism of essential ele-
ments.
The effects of high concentrations of specific ions or salts
on plant growth is too complicated to present here. Bernstein
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and Hayward (5,12) have reviewed the literature on this subject
in some detail. Wide differences in plant response to a given
ion or condition is the rule. The brief review which follows
is chiefly to illustrate the range of toxicities encountered.
Specific ion effects are broadly of two types: (1) nutrient
deficiencies caused by interference with the uptake process and
(2) ion accumulations in injurious amounts.
The effects of high HC03" and high Na+ are examples of
nutrient deficiencies. Continued use of high HC03 waters
(>200 p.p.m.) has consistently caused chlorosis in deciduous
fruits in the Pacific Northwest (16). The chlorosis can be
cured by iron injection or by substituting water of lower HC03
content. These same waters may be used with no adverse effects
j j
on most field crops. On sodic soils, deficiencies of Ca ,
Zn*"1" and Fe*"1" have been observed.
Injurious effects are more common. Toxicity of Na has been
noted for a few select plants; almonds, and avocado are injured
by Na+ at levels so low as to be regarded as negligible for
most crops. Some plants (peaches and other stone fruits, pecans,
avocados, grapes and citrus) are sensitive to Cl at levels which
do not affect most truck and field crops. The case of B is
probably worked out in the greatest detail. Many data are
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available on the differences in B requirements among plants,
their tolerance for excess B and on diagnosis of deficiencies
and excesses. The concentrations of B necessary for the growth
of plants of high B requirement may actually be toxic for plants
very sensitive to B. Wilcox (23) lists the permissible level
of B in irrigation waters according to crop sensitivity and the
relative B tolerance of many crop plants.
Injuries to plants from excess As, Cu, Zn, Mo, and Mn are
known. The aquatic environment, i.e., the soil solution, is
not the point of diagnosis, however. It is simple enough to
demonstrate injury to plants grown in sand or solution culture
from Cu, Zn, etc., (21) but the data derived from such experi-
ments are essentially meaningless when it comes to diagnosis
of toxicities under field conditions. The solid phase of the
soil, mineral and organic, absorbs most of these elements so
strongly that their concentrations in the soil solution is often
negligible and well below toxic limits set in solution or sand
culture. Attempts to correlate plant uptake of such elements
with soil supplies (8) usually use salt solutions, dilute
acids, or chelating agents as extractants. There presently
exists no reliable diagnostic tool for predicting injury to
plants from these elements. Much remains to be done to develop
sound diagnostic techniques for assessing deficiencies and
86
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excesses of many elements.
Injuries to plants from F, Pb or Se arising from soil sources
are rare or unknown. Plants do absorb these elements from soils
but do not appear to be injured thereby. Fluoride injury to
plants appears to be due primarily to atmospheric pollution.
Animals, however, may be injured by consuming forages and waters
containing large amounts of F, Pb, Se, and NO-j .
REFERENCES
1. Bernstein, L., "Salt Tolerance of Grasses and Forage Legumes,"
USDA Agr. Inf. Bui. 194 (1958).
2. Bernstein, L., "Salt Tolerance of Vegetable Crops in the
West," USDA Agr. Inf. Bui. 205 (1959).
3. Bernstein, L., "Salt Tolerance of Field Crops',1 USDA Agr.
Inf. Bui. 217 (1960).
4. Bernstein, L., and Fireman, M., "Laboratory Studies on Salt
Distribution in Furrow-Irrigated Soil with Special Refer-
ence to the Pre-emergence Period," Soil Sci. 83:249-263
(1957).
5. Bernstein, L., and Hayward, H. E., "Physiology of Salt
Tolerance," Ann. Rev. Plant Physiol. 9:25-46 (1958).
6. Bernstein, L., and Pearson, G. A., "Influence of Exchangeable
Sodium on the Yield and Chemical Composition of Plants:
II. Wheat, Barley, Oats, Rice, Tall Fescue, and Tall
Wheatgrass," Soil Sci. 86:254-261 (1958).
7. Bingham, F. T., "Soil Test for Phosphate," Cal. Agric.
3(8):11, 14 (1949).
8. Chapman, H. D., "Leaf and Soil Analysis in Citrus Orchards,"
University of California, Division of Agricultural Sciences
Manual 25 (1960).
87
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9. Doneen, L. D., "Salinization of Soils by Salts in the Irriga-
tion Water,1' Trans. Am. Geophys. Union 35:943-950 (1954).
10. Eaton, F. M., "Irrigation Agriculture Along the Nile and the
Euphrates," Sci. Monthly 69:34-42 (1949).
11. Eaton, F. M., "Significance of Carbonates in Irrigation Waters,"
Soil Sci. 69:123-133 (1950).
12. Hayward, H. E., and Bernstein, L., "Plant Growth Relationships
on Salt-Affected Soils," Bot. Rev. 24:584-635 (1958).
13. Heald, W. R., Hoodie, C. D., and Learner, R. W., "Leaching and
Pre-emergence Irrigation for Sugar Beets on Saline Soils,"
Washington Agr. Exp. Sta. Bui. 519 (1950).
14. Kelley, W. P., "Alkali Soils; Their Formation Properties and
Reclamation,11 Reinhold Publ. Corp., New York (1951).
15. Leggett, G. E., "Relationships Between Wheat Yield, Available
Moisture, and Available Nitrogen in Eastern Washington Dry
Land Areas," Washington Agr. Exp. Sta. Bui. 609 (1959).
16. Harley, C. P., and Linder, R. C., "Observed Response of
Apple and Pear Trees to Some Irrigation Waters of North
Central Washington," Proc. Amer. Soc. Hort. Sci.
46:35-44 (1945).
17. Pearson, G. A., and Bernstein, L., "Influence of Exchangeable
Sodium on the Yield and Chemical Composition of Plants: I.
Green Beans, Garden Beet, Clover, and Alfalfa," Soil Sci.
82:247-258 (1956).
18. Pearson, G. A., "Tolerance of Crops to Exchangeable Sodium,"
USDA Agr. Inf. Bui. 216 (1960).
19. Richards, L. A., Bower, C. A., and Fireman, M., "Tests for
Salinity and Sodium Status of Soil and of Irrigation
Water," USDA Cir. 982 (1956).
20. Richards, L. A., "Availability of Water to Crops on Saline
Soils," USDA Agri. Inf. Bui. 210 (1959).
88
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21. Stiles, W., "Trace Elements in Plants," 3rd Ed., Cambridge
University Press (1961).
22. U. S. Salinity Laboratory Staff, "Diagnosis and Improvement
of Saline and Alkali Soils," USDA Agr. Handbook 60 (1954).
23. Wilcox, L. V., "Classification and Use of Irrigation Waters,"
USDA Circ. 969, Washington, D. C. (1955).
DISCUSSION
STATEMENT: I presume that there is some question in the mind of
some here as to why this type of discussion was included in this
agenda. The quality of water for agricultural use is just as
much a water pollution problem as any other use discussed today.
One of the outstanding differences between this discussion of the
agricultural phases of the water toxicity problem and the pre-
vious biological discussions, is that Dr. Hoodie talked with
much more assurance regarding the effect of toxic elements on
plant. There is considerable in common between land and water
plants and land and water environment, if these areas of common
interest can be found. We in water pollution could learn con-
siderable from the agricultural and soil scientists and much of
the knowledge they have could be applied to some degree to our
water quality problems. Actually, we are dealing with the water
quality in our studies of soils because water is involved,
although it may be soil water. There is one advantage that the
agriculturists have over the fisheries biologist and that is
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that his crop is always there when he wishes to study it, while
fish are transient and may be many miles away.
Q. How common are the damages to which Dr. Hoodie alluded in
his talk?
A. In regard to the number of acres effected by excess concen-
trations of salt, there are thousands of such acres in the
State of Washington. This is one of the hazards of irriga-
tion development. There have been as many acres of land
ruined by excess salt accumulation, due to a lack of under-
standing, as there have been acres developed for irrigation.
This is a problem associated with improper drainage. It
occurs under natural conditions in poorly drained soils and
it occurs during irrigation when adequate drainage is not
provided.
Q. In view of the problems arising from the use of water for
irrigation and in view of the present surplus of food,
shouldn't there be some restriction on the use of water for
this purpose?
A. This is a question which always arises; however, the facts
are that surpluses exist only for certain crops and not for
others. As our population expands, it is estimated that by
1975 these surpluses will largely disappear. In addition,
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the present food surplus Is one of the most important national
resource that we have against disaster. If we divert this
water from irrigation to other purposes, we will be in a very
difficult position when these surpluses disappear and we need
this food for our expanding population. While agriculture is
an industry, we have to remember that the individual operator
in this industry is the farmer. He has no spokesman and as
an individual operator must exist on his wages. You cannot
simply divert water from this man as you can where you can
juggle it from one use to another in order to find a better
use for it.
Q. Did you say the farmer was an individual without a spokesman?
A. He doesn't have a spokesman in the area we are discussing.
The farmer does not understand the implications of such
things as the use of insecticides and pesticides as a part
of a calculated risk to national health. Usually he does
not know what you are talking about any more than the
operator in an industrial plant knows about the consequences
of the material he uses. He takes a calculated risk every
year of his life--whether he's going to make a living or
^«.
not. Agricultural production is in the hands of individuals
who have to make a living.
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Agricultural scientists know fairly well what the quality
of water required for agricultural purposes must be. But they
know far less about what agriculture does to the quality of
water as it affects other people. There is a potential among
the agricultural scientists to find this out, but they have
no directive nor money to do this. I suggest that it would
advance this problem of water pollution a great deal if use
were made of the agricultural scientists on some of these prob-
lems. Some of their programs can be directed toward this area
if there is support for it.
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PARALYTIC SHELLFISH POISONING
John G. Girard*
Paralytic shellfish poisoning results from the ingestion of
any of various species of molluscs that have significant amounts
of a naturally occurring toxin. McFarren, ej: £^., (1957) enu-
merate over 600 cases of such poisoning with 69 deaths through-
out the world from 1793 to 1954.
The first recorded cases and deaths attributed to the
poisoning on this continent were reported by Captain Vancouver
in his journal published in 1798. In 1793, while on Vancouver
Island, British Columbia, four members of his crew, one of whom
died, became ill from eating roasted mussels. The fact that
Vancouver wrote that his crew had some idea of treatment of those
stricken indicates strong possibility that these were not the
first cases of the illness. Since that time cases of the
poisoning have been reported in California, Oregon, Washington,
British Columbia, and Alaska on this coast and Nova Scotia,
New Brunswick, Quebec, and Maine on the Atlantic Coast. The
most recent outbreak on the Pacific Coast of any magnitude
occurred in British Columbia in October, 1957 4i when 50 cases of
*Shellfish Sanitarian, Washington State Department of Health,
Seattle, Washington.
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the illness resulted from the consumption of toxic Pacific
oysters (Davies, ej: _al., 1958).
It is difficult to conclude that from the incidence of
poisoning, this can be considered to be a major public health
problem. Nevertheless, the fact that man can receive a fatal
dose of the toxin from a single serving of highly toxic shell-
fish and that there is no known antidote requires our strict
attention. At the present time, there are no means for pre-
dicting the occurrence of the toxin within shellfish. Preven-
tion of the illness is based on a control program which identi-
fies toxic shellfish before they reach the consumer. This
requires submitting lots of shellfish from suspected areas to
a laboratory assay procedure to ascertain the level of toxin
within the shellfish.
McFarren, ejt a±,, (1960) writes "The first symptoms of
poisoning (Meyer, et al_., 1928 and Medcof, et al_., 1947), in
humans are associated with peripheral paralysis which may pro-
gress from a slight tingling and numbness about the lips to a
complete loss of strength in the muscles of the extremities and
neck and even to death by respiratory failure. In a moderately
severe case, the tingling, stinging sensation around the lips,
gums, and tongue develops in about five to thirty minutes after
-------
consumption of the mussels. This is regularly followed by a numb-
ness or a prickly feeling in the fingers and toes and, within 4
to 6 hours, the same sensations may progress to the arms, legs,
and neck, so that the voluntary movements, as for example,
raising the head, can be made only with great difficulty,"
In reviewing the writings of the early workers on this prob-
lem, it is interesting to observe that they concluded that the
cause of the poisoning was due to something the shellfish ingest.
Probably the main difficulty confronting them was the unpredict-
able occurrence of the toxin in the shellfish. The fact that
human cases appeared after eating shellfish, shellfish, became
toxic very suddenly and evidently became safe to eat shortly
thereafter, required investigation to be accomplished during
an outbreak. Beginning in 1927 and continuing into 1937,
Sommer and Meyer were able to ascertain the cause of toxic shell-
fish in California (Sommer, e_t aJL., 1937). These workers estab-
lished that the toxin in the mussel Mytilus californianus was
associated with the occurrence of the dinoflagellate Gonyaulax
catenella and that these organisms were poisonous. They fur-
ther established that the mussels were toxic only when G.
*"•*
catenella was in the overlying water and that the toxicity of
the mussel was proportional to the number of G. catenella in the
95
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water and in the stomach of the mussels. Sommer also fed cultures
of G. catenella to mussels, thus making them toxic and after
removing them from the organisms and placing them in sea water
free from the organism, found the mussels to be free of toxin
within a short time.
In studying the shellfish poisoning problem in the Bay of
Fundy on the East Coast, Needier (1949) showed the causative
organism to be Gonyaulax tamarensis. At the present time, the
causative organism for the occurrence of paralytic shellfish
poison in waters of the State of Washington is not known. In
1960, Dr. Sparks and associates initiated a study on the shell-
fish toxicity problem in Washington with one of the objectives
being to attempt to ascertain the causative organism (Sparks,
et aU, 1961).
The present method used for determining the toxin in shell-
fish is a modification of one described by Sommer and Meyer in
1937. The collected meat of a representative lot of shellfish
is washed, allowed to drain and then ground until a homogeneous
mixture is obtained. One hundred grams of the material is
introduced into a beaker into which is added 100 ml of 0.1 N
hydrochloric acid. The mixture is stirred thoroughly, heated to
allow gentle boiling for 5 minutes, and allowed to cool to room
temperature. The pH of the mixture is tested and if necessary,
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adjusted to 3.0. The volume is then brought back to 200 ml and
an adequate portion of the supernatant liquid is decanted off for
the test. One ml portions are introduced intraperitonaeally into
3 mice weighing between 18 and 22 grams each and the median death
time for the mice is determined. If the death time is less than
5 minutes, dilutions are made to obtain death time between 5 and
7 minutes. Applying the death time into a table and formula
developed by Sommers, the amount of toxin in the extract is
expressed in numbers of mouse units per 100 gram of meat. By
definition a mouse unit is the minimum amount of poison required
to kill a 20 gram mouse in 15 minutes when 1 ml of solution is
injected intraperitonaeally.
The most important of early studies on the chemistry of
the toxin was published by Sommer, et al., (1948). It was deter-
mined that detoxification occurs with increases in pH and tempera-
ture. At pH 5.0 there is a loss in toxicity of only 6% in 6 days
at 25°C., but at a pH of 6.6 this loss increases to 35% and 747.
at a pH of 11.5. Likewise, an increase in the temperature from
25° to 100° at 5.0 causes the loss in toxicity to increase from
67. in 6 days to 8570 in one day. Recently Shantz, et al., (1957)
'•-ii
concluded that both the clam and mussel toxin must be the same or
nearly the same with a molecular formula of C10H1704N7.2HCL.
97
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Beginning in 1944, personnel from Northwestern University,
University of California, and the Chemical Corps Biological Lab-
oratories at Fort Detrick were successful in the purification of
the toxins from the California mussel M. ca1ifornianus and butter
clams Saxidomus giganteus (Shantz 1960). It was found that the
toxins are among the most potent known to man and that the
purified toxin from mussels is the main cause of "mussel poisoning"
described by Meyers, ejt al,. , (1928). An interesting aspect of the
work on the butter clam is that in some cases it was required to
process up to 8 tons of the clams to obtain one gram of the toxin.
For many years various workers recognized some of the
inadequacies of the use of the mouse unit as a means of expressing
the amount of the toxin present in shellfish. This is primarily
related to the varying tolerance of the toxin by different strains
of mice. In 1955, research was initiated to implement the use of
the purified toxin in the standardization of animal assay. This
was successful and presently, by using the CF value for the mouse
colony used, the amount of toxin is expressed by micrograms of
toxin per 100 gram of meat.
At the present time, work is being done in attempting to
develop a chemical method for the quantitative determination of
the toxin in shellfish. This test may be of particular value in
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areas where mice are difficult to obtain, but its usefulness must
be further studied (McFarren, et_ ajL., 1960).
The phytoplankton of concern possess the ability to reproduce
themselves into huge populations in a very short time, thus caus-
ing shellfish feeding on them to become toxic. Although each
species of shellfish appear to concentrate the toxin at a rate
different from other species, all bivalves should be considered
suspect in critical areas during periods when the causative
organism is present. Of particular interest is the fact that the
butter clam evidently may retain the toxin for long periods of
time, thus complicating this aspect of the problem.
On this coast, all reported cases have come from the con-
sumption of mussels, butter clams and Pacific oysters. In Cali-
fornia and Oregon, seasonal closure of coastline areas is
evidently the primary means for providing the necessary protec-
tion. In Alaska, high levels of toxin in butter clams occurring
throughout the year prohibit the commercial exploitation of vast
shellfishery resources. In Washington, a monitoring program is
provided, in addition to excluding shellfishery utilization in
certain areas, between April 1, and October 31. As in other
jurisdictions, shellfish areas are closed when the amount of the
toxin in shellfish exceeds 80 micrograms per 100 ml of the meat.
99
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Regarding the question of the minimal amounts of the toxin
required to produce intoxication in humans, Medcof, et a1. (1947)
concluded that there is wide variation of severity of reaction to
nearly equal doses amongst the people involved and that some
people have a natural tolerance to the toxin.
For many years, it was believed that fatality to humans would
occur only upon ingestion of approximately 40,000 mouse units. At
the present time, it is generally believed that the minimal lethal
dose may be far below this level.
Probably the most perplexing facet of the toxicity problem
has been the attempt to correlate shellfish toxicity with such
ecological factors as meteorological and oceanographic informa-
tion. It appears that on this coast, the highest shellfish
toxicities occur in the summer. There are indications that along
the coast there are upwellings of colder water producing water
temperatures 3 or 4 degrees lower than the average to be expected
for the latitude. The average summer water temperature in areas
on this coast when toxicity appears ranges from 10° to 14°C.
(McFarren, et al., 1960). In studies of the Bay of Fundy,
Needier (1949) has been able to show that the organism G.
tamarensis may appear after the surface water reaches a temperature
of 10°C. This information has proven to be quite useful to the
100
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control authorities in their monitoring program for that area.
Our present understanding of the toxicity problem, as it
occurs in Washington, is essentially limited to knowledge of levels
attained in various species of shellfish, frequency of occurrence,
and areas associated with the toxin. Unfortunately, little has
been done in studies relating to the identification of the causa-
tive organisms, the ecological conditions which govern the inci-
dence of toxin, or the means for predicting the occurrence of toxin
in shellfish. Sparks, _et £l. (1961) have recently released a sta-
tus report on their attempts in gaining information on some of
these aspects of the problem and we are looking forward to further
developments.
In closing, may I state that these discussions are not to be
considered a complete delineation of the problem, but only to pro-
vide some understanding of the basic elements of the problem as
it exists in this area.
REFERENCES
Davies, F. R. E., Edwards, H. I., Kitchen, W. L. H., and Tomlinson,
H. 0. 1958. Shellfish toxin in cultivated oysters. Can. J.
Public Health. 49,286.
Medcof, J. C., Lein, A. H., Needier, A. 3., Needier, A. W. H.,
Gibbard, J., and Naubert, J. 1947. Paralytic shellfish poisoning
on the Canadian Atlantic Coast. Bull. Fisheries Research Board
Can, 75, 1.
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Meyer, K. F. , Sommer, H. and Schoenholz, P. 1928, Mussel poisoning.
J. Preventive Med. 2,365.
McFarren, E. F., M. L. Schafer, J. E. Campbell, K. H. Lewis, E. T.
Jensen and E. J. Schantz 1957. Public health significance of
paralytic shellfish poison. Proc. Natl. Shellfisheries Assoc.
47:114.
McFarren, E. F., M, L, Schafer, J. E. Campbell, Keith Lewis, E. T.
Jensen and E. J. Schantz. 1960 Public health significance of
paralytic shellfish poisoning. Advances in Food Research. Vol.
10, Academic Press: 135-177.
Needier, A. B. 1949. Paralytic shellfish poisoning and Gonyaulax
tamarensis. J. Fisheries Research Board Can. 7,490.
Schantz, E. J. 1960. Biochemical Studies on Paralytic Shellfish
Poisons. Annals of the New York Academy of Sciences. Vol. 90
Art. 3:843.
Schantz, E. J., J. D. Mold, D. W. Stanger, J. Shavel, F. J. Riel,
J. P. Bowden, J. M. Lynch, R. W. Wyler, B. Riegel and H. Sommer.
1957. Paralytic shellfish poison. VI. A procedure for the
isolation and purification of the poison from toxic clam and
mussel tissues. J. Am. Chem. Soc. 79:5230.
Sommer, H. and Meyer, K. F. 1937. Paralytic shellfish poisoning.
Arch. Pathol. 24, 560.
Sommer, H., Riegel, B., Stanger, D. W., Mold, J. D., Wikholm,
D. M., and McCaughey, M. B. 1948. Paralytic Shellfish Poison,
J. Am. Chem. Soc. 70.
Sommer, H., Whedon, W. F,, Kofoid, C. A., and Stohler, R. 1937.
Relation of paralytic shellfish poison to certain plankton
organisms of the Genus Gonyaulax. Arch. Pathol. 24,537.
Sparks, A. K., and A. Sribhibhadh. 1961. Status of Paralytic
Shellfish Toxicity Studies in Washington. Fisheries Research
Institute, College of Fisheries, University of Washington.
Circular No. 151. (To be presented at the Fourth National
Shellfish Sanitation Workshop, Washington, D. C., November
28-30, 1961.)
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DISCUSSION
Q. Does cooking effect the toxicity of shellfish toxin?
A. The average cooking time will destroy between 35 and 60% of
the toxic properties depending upon the method of cooking,
Q. How are you monitoring the oyster industry in Washington as
regards shellfish toxin?
A. We are running samples from a number of areas every two
weeks.
Q. If you find the toxin present, will this be in time to stop
the shipment of the oysters to the market?
A. We are not testing actual shipments but are making our tests
in the actual growing areas and hope to close these areas
before the toxin reaches a critical level. Of course, there
are other types of shellfish involved such as molluscs and
clams. Those areas must also be controlled. Actually, most
of the oysters are grown in areas outside of those where the
toxin has been found.
Q. Isn't this problem usually associated with the outer waters
of the sea, rather than those of bays and estuaries?
A. Yes.
Q. In what part of the shellfish is the toxin usually found?
A. Largely in the digestive tract of the oyster. In the butter
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clam about 607. of toxin is in the siphon of the clam. I think
this is the only species studied where it is outside of the
digestive tract.
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TOXICITY BIOASSAYS VERSUS CHEMICAL ANALYTICAL
METHODS - THEIR ADVANTAGES AND LIMITATIONS
Peter Doudoroff*
Toxicity bioassays of industrial wastes are necessary, in
addition to chemical analyses, for the following reasons:
1. The substances that are toxic to aquatic life and that
can occur as important components of complex industrial efflu-
ents are very numerous. It is often impossible to predict just
which toxic components will be present and important in a
particular effluent, and so need to be tested for or measured
chemically. Indeed, some of the most important toxic constit-
uents of common wastes, such as pulp mill effluents, have yet to
be chemically identified.
2. No chemical analytical methods have as yet been devised
for the rapid determination of low yet dangerous concentrations
of many toxic substances in waste waters. Those analytical
methods that have been developed for the routine examination
of polluted waters often fail to distinguish between highly toxic
*Supervisory Fishery Research Biologist, In Charge, Fish
Toxicology and Physiology Studies, Robert A. Taft Sanitary
Engineering Center, Public Health Service, and Professor,
Department of Fish and Game Management, Oregon State University,
Corvallis, Oregon.
105
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constituents and other chemically related components that are
relatively or entirely harmless.
3. The toxicity of the many toxic water pollutants to
various aquatic organisms, in different natural waters, and in
various possible combinations, has not yet been adequately
investigated. Therefore, the toxicity of a complex waste to the
important organisms present in the receiving water often cannot
be reliably predicted even when all of the waste components have
been identified and accurately measured chemically. It must be
determined empirically by biological test.
Routine toxicity bioassays of industrial effluents cannot,
however, provide all of the information concerning the prop-
erties of these effluents that may be required in connection
with a toxicity investigation. Some of the reasons why chemical
analyses may be essential in addition to such routine toxicity
bioassays follow:
1* In order to devise appropriate corrective measures, it
may be necessary to know whether the observed toxicity of a
waste or receiving water is or is not ascribable to some partic-
ular chemical component known or believed to be present. Routine
bioassays demonstrate only the toxicity and not the cause thereof,
although the appearance and behavior of the test animals may
106
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suggest the nature of the principal toxicant involved. Only
chemical analysis can definitely establish the presence of a
particular toxicant in concentration sufficient for producing
the observed toxic effects on the test animals.
2. Relatively short-term, routine, acute toxicity bioassays
are capable of demonstrating only the presence of rapidly lethal
or obviously harmful concentrations of toxicants. They are
unsuitable for detecting in polluted waters low concentrations
of toxicants that can produce only chronic effects, such as
long delayed mortality and sublethal impairment of functions or
performance of organisms. Only sensitive chemical tests and
relatively difficult and prolonged biological tests may be
capable of revealing- the presence of these chronically harmful
concentrations of toxicants.
3. Even acutely toxic concentrations of pollutants in waste
waters may be demonstrable by bioassays of several days' duration
only. Continual or frequent renewal of the test media in the
course of the bioassays may be necessary, because many toxic
pollutants do not persist long in dilute solutions, being sub-
ject to bacterial decomposition, chemical oxidation, etc. Only
chemical analytical methods thus may be appropriate for rapid
and easy detection even of acutely toxic concentrations of some
107
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pollutants in waste waters, which may be essential to proper control
of waste discharges.
4, Some waste constituents that are not themselves demon-
strably toxic to aquatic life are nevertheless potentially harm-
ful, being potential sources of highly toxic substances. For
example, complex metallocyanide ions are not markedly injurious
to fish, but hydrocyanic acid formed through their dissociation
or decomposition (e.g., when the hydrogen-ion concentration is
increased, or under the influence of light) is extremely toxic.
Thus, ordinary bioassays may not reveal the latent acute toxi-
city of certain wastes whose dangerous though nontoxic compo-
nents can be detected by appropriate chemical analytical
methods.
It has been explained why neither toxicity bioassays nor
chemical tests alone can be relied upon in seeking to evaluate
the actual and potential toxicity to aquatic life of wastes and
polluted waters and to regulate discharges of toxic wastes.
Both of these complementary approaches to toxicity problems
clearly are necessary. There is need, however, for further
development and refinement of both of these approaches.
Attempts must be made to develop toxicity bioassay methods
suitable for detecting as promptly as possible the presence of
108
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slowly lethal and sublethal concentrations of toxicants in polluted
waters. Low concentrations of toxicants that impair the appetite
and growth of fish, interfere with their normal activity or swim-
ming performance, or seriously alter their behavior patterns can
be eventually as destructive to valuable fish populations as
those that are rapidly fatal. The components of complex wastes
likely to have these effects on aquatic life in receiving waters
may not even be the same components as those that cause death at
high concentrations of these wastes. The former components may
still be present in harmful concentrations when the concentrations
of the latter components have been reduced by dilution to harm-
less levels. Bioassay methods suitable for detecting injurious
effects of wastes at concentrations far below those that cause
rapid mortality of the test animals thus can be even more
instructive than those now in common use.
One may not assume that any given measurable response of
an organism to low concentrations of a toxicant is necessarily
evidence of significant harm to that organism. Nor may one
assume that a response observed at harmful concentrations of some
toxicants will occur also at harmful concentrations of all other
toxicants. Some of the responses, such as changes of breathing
rate, can be transient or essentially adaptive. Only when the
demands placed upon an organism by a change of its environment
109
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overburden its capacity to adapt itself to such changes, so that
its ability to survive, grow, and reproduce is impaired, can the
change be said to be truly harmful. The proper selection of
responses to be observed and measured in seeking to detect harm-
ful sublethal concentrations of toxic pollutants by bioassay,
and also the interpretation of the observations, thus are not
simple matters.
Increased standardization of toxicity bioassay methods is a
desirable objective, and continued efforts are being made in this
direction. However, excessive or premature standardization can
impair the usefulness of these methods and may tend to discourage
experimentation which is essential to progress.
Chemical analytical methods also need much improvement.
New methods especially suitable for application to toxicity
problems must be developed. Unfortunately, most chemists working
in the field of water pollution in the past have concerned them-
selves very little with the utility of their analytical methods
in connection with problems in the toxicology of aquatic organ-
isms. They have largely neglected the question whether a
particular water pollutant being measured (because of its toxi-
cological importance) is or is not actually present in a form
that is toxic to aquatic life. For example, precise and sensitive
110
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methods have been developed for the determination of cyanide in
polluted waters without seeking to distinguish between cyanide
present as free, molecular hydrocyanic acid and that present in
the form of complex metallocyanide ions. Likewise, available
analytical methods for the determination of various toxic heavy
metals fail to distinguish between simple metallic cations and
any metal present in the form of complex ions or colloidal pre-
cipitates (insoluble carbonates, hydroxides, etc.)- Yet,
it is well known that free, molecular hydrocyanic acid and most
of the heavy metal cations are exceedingly toxic to fish, whereas
complex metallocyanide ions and precipitated insoluble metallic
compounds (including the cyanides) are relatively or entirely
harmless. The available analytical methods, involving serious
disturbance of ionic equilibria in water samples, can be useful
to the biologist, but obviously they are not entirely satis-
factory or adequate for the investigation and evaluation of the
toxicity of polluted waters. Nevertheless, surprisingly little
interest has been shown by water chemists in the past in the
development of more appropriate analytical methods that would
yield toxicologically more meaningful results. Some apparently
find it difficult even to understand the need for such new
methods.
Ill
-------
Cooperative studies at Oregon State University recently have
been directed toward filling the need for new analytical methods
directly applicable to toxicity problems and for clear demonstra-
tion of the utility of such methods. Working with Professor Harry
Freund of the Chemistry Department, C. R. Schneider has recently
devised a highly satisfactory, as yet unpublished method for the
determination of free, molecular hydrocyanic acid in complex
cyanide solutions, without undue disturbance of ionic equilibria.
This has made possible reliable prediction of the acute toxicity
to fish due to cyanide of waters containing cyanide in combina-
tion with various heavy metals. It is to be hoped that other
chemists will in the future take an increasing interest in simi-
lar problems. There is a clear need and opportunity for closer
collaboration of chemists with biologists in the water pollu-
tion field.
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BIOASSAY - THE BIVALVE LARVAE TOOL
Charles E. WoeIke*
Problem;
Those of us working with bioassays sooner or later feel the
need for better tools for measuring toxicity. A bioassay method
which is economical to conduct, yields reproducible results,
detects either chronic or acute toxicities or both, can be used
either in the laboratory or field any season of the year, and
utilizes a non-migratory commercially important organism would
seem to approach the ideal situation. Work being conducted in
a number of areas indicates that bioassays using bivalve larvae
may fulfill these requirements. I am speaking primarily of the
larvae of clams and oysters.
Materials and Methods:
The procuring of bivalve larvae is not difficult and a
trained biologist in a marine laboratory can fairly quickly
and easily learn the techniques. During the normal reproductive
season, adult clams and oysters are available from their native
habitats for spawning. Other months of the year these molluscs
*Biologist, Washington State Department of Fisheries,
Brinnon, Washington.
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can be "conditioned" for spawning by placing them in flowing warm
sea water for three to four weeks. Adult clams and oysters with
ripe gonads are naturally spawned by increasing the water tempera-
ture 5-10° C. and adding sperm to the water. The fertilized eggs
are added to water containing the material bioassayed, at a
density of 15-20,000 per liter by some workers, less by others.
After 48 hours at 20° C. the eggs normally develop to free
swimming straight hinge larvae. At this time aliquot samples
are taken and microscopically evaluated to determine the effect
of the material on the embryonic development of the eggs. Bio-
assays may be terminated at this point, or the larvae fed on
phytoplankton and grown in the variable. Often materials which
have little or no effect on embryonic development at a given
concentration will have marked effects on either the growth of
the larvae or their ability to metamorphose from larvae to
juveniles. This is particularly true of some of the insecti-
cides and herbicides. These bioassays can be conducted in the
laboratory with dilutions of materials you wish to test. Bio-
assays can also be conducted using water from the field in which
the variable is known to occur.
Some workers have conducted shorter bioassays by observing
the effects of a material on the swimming of the larvae over a
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four hour interval and the subsequent recovery of swimming ability,
Other workers have conducted longer term bioassays with young
oyster spat which have attached to glass slides. In these studies
it is possible to see the physiological response of the oyster,
since the slide acts as a window through which internal organs of
the oyster spat may be observed.
Technique Problems;
From this brief summary of the method one might assume there
are no control or technique problems with the bivalve larvae
bioassay. This, of course, is not true; the important point how-
ever is that most of these problems can be either solved or con-
trolled without undue difficulty.
Among the problems associated with the larval bioassay, a
major one is to procure larvae whenever desired. Earlier workers
sacrificed adults with ripe gonads and washed the eggs free from
the female. Due to the frequency with which unsatisfactory
results were achieved from apparently immature eggs, this practice
was dropped. Larvae are now procured only from naturally liber-
ated eggs. This method has resulted in consistent reproducible
results. The drawback to this procedure is the ever present
danger of being unable to achieve a spawning when desired. It
has been our experience that a stock of 30-40 thermally condi-
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tioned adults is adequate to assure a spawning for bioassay pur-
pose at any planned time plus or minus 30 minutes.
"Age of the water11 used, i.e., time elapsed between removal
from the natural environment and introduction of the eggs to the
water, has an effect on the results. We have found that water
held two hours or less in one gallon containers has no measurable
effects on bioassay results. Water from 3 to 6 hours old may have
an effect, water which is 24 hours old has very definite effect on
the results of larval bioassays. In our work we make every effort
to utilize water less than "3 hours old."
The "quality" of control water is also of concern, since
metal ions, bacteria, temperature, plankton and other suspended
material may affect results. To minimize the danger of prob-
lems from these sources we utilize water drawn through non-
metallic, non-toxic lines with a hard rubber pump. This water
is heated to a constant temperature of 20° C., filtered through
a 5 micron filter and treated with ultraviolet light. While
much of this seemingly elaborate treatment is not necessary at
all times, we prefer to use control water of as near a constant
quality as possible.
We have found that the size of containers used and the type
(glass or plastic) have slight statistical effects on our results--
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not great enough to be of concern but nevertheless great enough to
indicate the merit of utilizing the same size and type of container
in any given series of bioassays.
The age of developing embryos when introduced to the variable
bioassayed must be controlled. With many materials response
changes (generally declines) with increasing age, therefore, to
achieve comparable results we introduce developing embryos to the
material being bioassayed between one and two hours after fertili-
zation. This practice is satisfactory where we are concerned with
relative toxicities only. Where minimal toxicity levels are
desired, fertilization must occur in the variable.
Where low level effects are being considered, problems of
sampling variation can mask results; however, this problem can
be minimized by increasing the number of cultures in the controls
and variables to increase the statistical reliability. In our
work a given bioassay will usually be made up of 60 cultures,
with at least 107. of them controls and duplicate or triplicate
cultures of each variable.
Our 48-hour bioassays are generally carried on in one liter
polyethylene beakers seeded with 10-15,000 eggs; however, tech-
nique evaluation studies with densities up to 60,000 per liter
have been conducted. At 60,000 per liter there is some indica-
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tion of adverse effect from "over seeding."
The 48-hour bioassays are evaluated on the basis of percent
abnormal larvae. Normal larvae are those which are fully shelled.
Some of those called normal are often of irregular shape and
undersized; however, using this "full shelled" criterion elimi-
nates much of the variability which arises when several people
are evaluating the larval samples. It has been our experience
that those larvae termed abnormal will not feed and grow, and as
a result die within a few days. A further basis for considering
the abnormals undesirable is the absence of abnormal larvae in
plankton samples from nature. We, therefore, use percent abnormal
larvae as an index of effect and consider a material which induces
abnormality in excess of the controls to be adversely effecting
the larvae. Where over 507, of the larvae are abnormal it is very
probable that the lethal threshold has been reached. Where ab-
normals reach 90% we consider the variable to be clearly lethal
to the larvae.
Throughout the entire method every precaution is taken to
ensure a healthy group of larvae for use in bioassays, however,
occasional inferior larval lots are encountered. These are
readily detected by the percent abnormality of the controls--we
totally reject any bioassay where our controls have in excess of
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107. abnormals; between 5-10% we are extremely cautious in making
firm conclusions. We prefer to deal with larval groups where the
controls average less than 2.5% abnormals.
In addition to the aforementioned problems, feeding rates and
infestation of the cultures with other organisms--especially small
marine worms and crustaceans--must be solved where growth, survival
and setting of the larvae are studied. The problem of feeding the
larvae in these longer studies materially increases the complexity
and cost of a larval bioassay program. As a general rule, we do
not attempt to feed and grow larvae unless we have reason to sus-
pect effects which are not detected in the 48-hour bioassays.
Depending on the type of work being done, all of these prob-
lems, part of them, or additional ones may be encountered when
using the bivalve larvae bioassay. It is sufficient to reiterate
that while problems exist, they are not insurmountable.
Results from Larval Bioassays:
Among those who have been working with bivalve larvae as a
bioassay tool in other areas are Dr. Loosanoff and his associates
on the East Coast, and Okuba and Dr. Imai and his associates in
Japan. In the Pacific Northwest, Willie Breese of the Oregon
State Yaquina Bay Laboratory, John Denison of the Rayonier Research
Laboratory, and the Washington State Department of Fisheries, have
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done varying amounts of bioassay work utilizing bivalve larvae.
While in many respects this bioassay tool is in early stages of
development, the costs in terms of equipment, outlay and time re-
quired to achieve results make it an extremely attractive one,
especially when dealing with concentrations of materials which
appear to act in a chronic rather than acute manner on the adult
organisms.
Thus far these workers have reported studies with industrial
wastes, salinities, temperatures, insecticides, weedicides, chemi-
cals, silt and effects of materials such as stainless steel, plas-
tics, metals, etc., on water quality.
Toxicity information reported from larval bioassays is not
extensive as yet; however, I will mention a few of the results I
am aware of. In general, water which has been oftentimes only
briefly in contact with metals, especially some types of stain-
less steel, is lethal. Many plastic materials, notably pure
polyethylene, are non-toxic; many of those which contain plasti-
cizers are lethal. Glass is non-toxic. Potassium cyanide is
lethal at 0.014 p.p.m., mercuric chloride at 0.027 p.p.m.,
copper sulphate at 0.04 p.p.m., sodium sulfide at 2.44 p.p.m.,
sulphuric acid at 33.11 p.p.m., Roccal at 1 p.p.m., Dowicide A
about 10 p.p.m., Dowicide G at 0.25 p.p.m., NABAM at 0.50 p.p.m.,
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allyl alcohol at 2.5 p.p.m., DIURON at 5 p.p.m., NEBURON at 2.4
p.p.m., Sevin at 5 p.p.m., toxaphene at 10.0 p.p.m., Guthion at
1.0 p.p.m., Dicopthion at about 2 p.p.m., and one of our big
local problems, S.W.L., has been found lethal at 13 p.p.m. when
the eggs are fertilized in the S.W.L. It cannot be stated that
all of these values were derived under exactly the same procedures
outlined in this report; however, the general techniques would
appear to be the same, and I would expect the same general param-
eters would be reached by any of the workers mentioned. In
general, where differences of results have been encountered, these
differences have been due to either technique or interpretation
differences.
In Operation:
At our Point Whitney Laboratory we routinely use 48-hour
development of Pacific oyster larvae as a bioassay tool. New
materials such as plastics, paints, chemicals and even wood
(treated and untreated), which will come in contact with our labo-
ratory water supply are bioassayed before we use them. We also
have been bioassaying the natural waters within 4 miles of our
laboratory for nearly a year to collect data on the statistical
validity of the method, its reproducibility, and the range of
variation found in the larval response to "field water." This
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past summer we conducted extensive "field water bioassays" with
marine water from a number of areas other than Hood Canal. At
present, we are statistically analyzing these data.
Future Work:
We expect in the immediate future to conduct bioassays of
water samples from the field as easily as we now make chemical
analysis of these same samples. We plan extensive use of larval
bioassays in areas where shellfish problems exist to ascertain
whether adult mortalities, poor growth, reduced fatness or repro-
ductive failures are related to poor water quality as measured
by the larvae. Finally, we hope to be able to bioassay frac-
tions of complex wastes where often only a few milliliters of
extracted material is available for bioassay purposes.
Application and Interpretation;
One major stumbling block to the general acceptance of this
bioassay tool by lay and technical people has been that it deals
with only one phase of the life cycle of the organism. The con-
tention often put forth is that while biologically interesting,
the larval approach is not realistic, since it does not neces-
sarily reflect the effect on the adult, which after all is the
item of economic interest. I must object to this rather fuzzy
thinking on the part of these persons and point out that if a
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material breaks the life cycle of the organism considered, very
soon there will be no adults to worry about. In my opinion any
material which breaks the life cycle of an organism at the repro-
ductive stage represents an adverse effect on the adult. A more
valid objectiion, however, deals with lower concentrations of a
material which have an adverse effect on the larvae but are not
necessarily lethal. These critics maintain that we cannot validly
infer "effect" on the larvae as evidence of long-range effect on
the adults. This objection does have merit. Work is currently
being carried on by several agencies which should provide data
on the relationship of effect on larvae versus effect on the
adult. In the interim, I prefer to take the stand that an
adverse effect on the larvae most probably will be reflected
in undue stress on the adult. Under stress th'e adult's normal
resistance to disease, changes in ecology, changes in tempera-
ture, salinity, dissolved oxygen or other environmental variables
may be adversely affected.
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BIOASSAY STUDIES AT NANAIMO, BRITISH COLUMBIA
Don F. Alderdice*
When an investigator is faced with a practical problem in
water quality control, the prevailing approach entails his attempt
to provide an estimate, based on some suitable response metameter,
representing the maximum alteration which may be permitted in
terms of a particular additive or change. Moreover, the estimate
is usually derived by consideration of the response of a particu-
lar species and age of fish selected because of its availability
or association with the situation being examined. The dosage-
response relationship is normally obtained where the response
metameter is a function of the concentration or level of the
variable in question, and of the time of exposure required for a
sample of test animals to respond.
The question now arises: How representative is the estimate
so provided of the dose-response relationship if the ancillary
conditions under which the estimate was made are allowed to vary?
In effect, variation in the environment is the rule, and esti-
mates of effect in terms of a change in one variable in the
*Associate Scientist, Biological Station, Fisheries Research
Board of Canada, Nanaimo, British Columbia.
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environment, obtained under constrained experimental conditions,
may not be expected to apply without error to combinations of
ancillary variables at levels other than those under which the
estimate was obtained.
The question of the possible variability of a response meta-
meter, upon which practical estimates are based, is one that is
being considered at Nanaimo. The basis for the studies lies in
multivariate analysis. The experimental approach is based on the
work of Box (e.g., Biometrics, JX)(1) : 16-60, 1954). An example
of the method is outlined for the response of coho salmon smolts,
in terms of median resistance time, to 3 mg/L of sodium penta-
chlorophenate over levels of salinity, temperature and dissolved
oxygen as ancillary variables.
The response of coho smolts was examined over the combina-
tions of a composite factorial design for estimation of the slope
coefficients in the polynomial
222
b33x3 "*"
b!2xlx2 + b!3xlx3 + b23x2x3
where
Y «= response time, minutes
xl = f (salinity)
X2 = f (temperature)
X3 « f (dissolved oxygen)
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Analysis provided
Y = 51.0867 + 9.0469xL - 10.2603x2 - 0.0068x3 +
- 1.9932x2 - 2.6706 x| - 2.5000x^X2+ 2.5000x x
Reduction of the polynomial to its canonical form, involving the
transformation of axes to a set conforming to the geometric con-
figuration of the response domain, provides an equation capable
of more straightforward interpretation. Thus
Y - Yc = 5.8356X1 - 2.0648X? -
s 1 f.
where Ys - the response time at the centre of the domain and the
canonical variables
Xl = 0.9779(x1 - xls) - 0.1539(x2 - x2s) + 0.1418(x3 - x3g)
X2 - 0.0907(x1 - xlg) + 0.9221(x2 - x2s) + 0.3763(x3 - x3s)
X3 = 0.1883(x1 - xls) - 0.3557(x2 - x2s) + 0.9155(x3 - x3g)
where x^s = the value of x at the domain centre.
In this particular case the response domain is of elliptical
hyperboloid configuration with the optimum response time remote
from the domain centre along th X* axis.
Having defined the 3-factor domain, a further series of
tests were conducted along the Xj^ axis to find the response
optimum.
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Results of this study are as follows: Over all levels of
salinity between 0-27% S, temperatures between 0-14° C, and
dissolved oxygen levels between 3 and 8 mg/L, the unique combina-
tion of these factors providing maximum resistance time to 3
mg/L, the unique combination of these factors providing maximum
resistance time to 3 mg/L of sodium pentachlorophenate was at
157, S
2.57° C
5.31 mg02/L
The geometric centre of the response domain was at
0.50% S
4,28° C
4.27 mg02/L
Over the levels of the three ancillary variables considered, the
median resistance time corresponding to the set of levels pro-
viding the smallest resistance time was approximately one-half
of that provided by the set of levels providing the greatest
resistance time. Variations in response time were such that
maximum tolerance was provided at a unique locus approximately
at 15% S, 2.57°C and 5.31 mg02/L.
Interpretation of the results may be premature at this
time. One interesting consideration deserves some comment: The
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oxygen level at the response optimum. Observation indicated
that the velocity of the response was activity-dependent. At
high oxygen levels the test fish were considerably more active
than those tested at low oxygen levels. However, below about
5 mg02/L the effect of reduced oxygen concentration appears to
influence resistance in spite of reduced activity in the low-
oxygen tests. It appears that a compromise is reached in this
instance between hypoxial and activity-induced stresses at
about 5 mg02/L.
The method employed in this study is capable of quite
general application to n quantitative variables. The experi-
mental designs employed are standard or composite complete or
fractional factorials. Although there are rigorous computa-
tions associated with the method, the amount of experimental
time involved in the use of composite designs is considerably
less than that required for complete factorial designs.
The method of multivariate analysis outlined is considered
to be a useful technique in considerations of the manner in
which the response of an animal may be altered under the varia-
tions of environmental conditions to which, at one time or
another, such an animal may be subjected.
(MS in preparation for submission to Journal of Fisheries
Research Board of Canada, 1961.)
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DISCUSSION
STATEMENT: A book which covers fairly well the material presented
in the previous paper is that of Turnbull and Eakin, entitled
"Introduction to the Theory of Canonical and Matrices," published
by Dover Publications, Inc., New York,
Q. How many individual bioassays were required to come out with
those three numbers (salinity, temperature, and dissolved
oxygen)?
A. Thirty.
Q. This seems to be considerably fewer than would be necessary
if you were to hold two of these items constant and vary
the third. That is, you could run experiments with constant
salinity and temperature and vary the oxygen.
A. You could do this, but this will not satisfy the situation
that we set up in the first place. If you are considering
that certain constants are zero, this might apply. This is
not the case, however, since all of these items vary. The
point is, therefore, you cannot make the assumption that
these variables are zero.
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ATTENDANCE AT THE TENTH SYMPOSIUM
November 14, 1961
James L. Agee
Don F. Alderdice
Herman R. Amberg
Aven M. Andersen
E. R. Anthony
Leo L. Baton
William J. Beck
Donald J. Benson
Thomas P. Blair
R. 0. Blosser
W. P. Breese
James E. Britton
Charles T. Bryant
William E. Bullard
John V. Byrne
Richard J. Callaway
Andrew Carey
Dale A. Carlson
Henry P. Carsner
George G. Chadwick
David B. Charlton
R. M. Chatters
William D. Clothier
J. F. Cormack
R. E. Dimick
Peter Doudoroff
Donald P. Dubois
Gilbert H. Dunstan
Leonard B. Dworsky
Edward F. Eldridge
Dorothy McKey Fender
Harry Freund
Herbert F. Frolander
John Girard
Dolores Gregory
Robert A. Gresbrink
George H. Hansen
Eugene P. Haydu
D. K. Hilliard
U. S. Public Health Service
Fisheries Research Board of Canada
Crown Zellerbach Corporation
Washington Dept. of Fisheries
U. S. Geological Survey
Oregon State Board of Health
Shellfish Sanitation Laboratory
Oregon State Sanitary Authority
Oregon State Board of Health
Nat'l Council for Stream Imprvm't
Oregon State University Fish Lab.
U. S. Public Health Service
U. S. Geological Survey
U. S. Public Health Service
Oregon State University
U. S. Public Health Service
Oregon State University
University of Washington
Northwest Weed Service
U. S. Public Health Service
Charlton Laboratories
Washington State University
Oregon State Fish Commission
Crown Zellerbach Corporation
Oregon State University
U. S. Public Health Service
U. S. Public Health Service
Washington State University
U. S. Public Health Service
U. S. Public Health Service
Portland State College
Oregon State University
Oregon State University
Washington State Health Department
Portland State College
Oregon State Board of Health
Washington Pollution Control Comm.
Weyerhauser Timber Company
U. S. Public Health Service
Portland
Nanaimo, B.C.
Camas
Brinnon
Portland
Portland
Gig Harbor
Portland
Portland
Corvallis
Newport
Portland
Portland
Portland
Corvallis
Portland
Corvallis
Seattle
Tacoma
Corvallis
Portland
Pullman
Portland
Camas
Corvallis
Corvallis
Portland
Pullman
Portland
Portland
Portland
Corvallis
Corvallis
Seattle
Portland
Portland
Yakima
Longview
Anchorage
130
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Dale A. Hoffman
Ralph H. Holtje
G. LaMar Hubbs
Gary W. Isaac
Bryan Johnson
C. R. Johnson
D. C. Joseph
Max Katz
Stanley Knox
L. B. Laird
Robert E. Leaver
Bob Lewis
Alfred Livingston
Donald Lollock
Jim McCarty
James E. McCauley
R. K. McCormick
Robert McHugh
E. A. McKey
James A. Macnab
Robert J. Madison
Nick Malueg
Harold E. Milliken
Al Mills
C. D. Moodie
W. Allan Moore
H. P. Nicholson
Phillip A. Olson
G. T. Orlob
Eben L. Owens
Kilho Park
Maynard W. Presnell
Edison L. Quan
Robert L. Rulifsen
J. F. Santos
Carl R. Schneider
William B. Schreeder
Robert W. Seabloom
Dean L. Shumway
Albert K. Sparks
J. D. Stoner
John C. Stoner
Philip N. Storrs
Arporna Sribhibhadh
U. S. Public Health Service
U. S. Public Health Service
U. S. Public Health Service
Rayonier, Incorporated
Washington pollution Control Comm.
Portland State College
California Fish & Game Department
University of Washington
Washington Pollution Control Comm.
U. S. Geological Survey
Washington State Health Department
California Fish & Game Department
Washington Pollution Control Comm.
Oregon State University
University of California
Oregon State University
Washington State Health Department
Oregon State Board of Health
Portland State College
U. S. Geological Survey
U. S. Public Health Service
Oregon State Board of Health
Washington Pollution Control Comm.
Washington State University
U. S. Public Health Service
U. S. Public Health Service
General Electric Company
University of California
Nat'l Council for Stream Imprvm't
Oregon State University
Shellfish Sanitation Laboratory
Oregon State Sanitary Authority
Oregon State Fish Commission
U. S. Geological Survey
Oregon State University
U. S. Public Health Service
University of Washington
Oregon State University
University of Washington
U. S. Geological Survey
Lane County Health Department
U. S. Public Health Service
University of Washington
Portland
Wash., D.C.
Anchorage
Shelton
Olympia
Portland
Sacramento
Seattle
0lymp ia
Portland
Seattle
Redding
Olympia
Corvallis
Berkeley
Corvallis
Seattle
Portland
Portland
Portland
Portland
Portland
Olympia
Pullman
Portland
Atlanta
Richland
Berkeley
Corvallis
Corvallis
Gig Harbor
Portland
Portland
Portland
Corvallis
San Francisco
Seattle
Corvallis
Seattle
Portland
Eugene
San Francisco
Seattle
131
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Clarence M. Tarzwell
John D. Thorpe
Roger Tollefson
W. W. Towne
R. A. Wagner
D. T. Walsh
Donald 6. Watson
E. J. Weathersbee
Bill Webb
Donald R. Well
Henry 0. Wendler
W. Q. Wick
Don Wilson
John N. Wilson
Ruth Winchell
Charles E. Woe Ike
Charles D. Ziebell
U. S. Public Health Service
Lane County Health Department
Consulting Biologist
U. S. Public Health Service
University of California
Washington Department of Fisheries
General Electric Company
Oregon State Board of Health
Idaho Department of Fish & Game
Washington Department of Fisheries
Washington Department of Fisheries
OSU Extension Service
Washington Pollution Control Comtn.
U. S. Public Health Service
Portland State College
Washington Department of Fisheries
Washington Pollution Control Comm.
Cincinnati
Eugene
Toledo
Portland
Berkeley
Brinnon
Richland
Portland
Boise
Brinnon
Vancouver
Tillamook
Olympia
Portland
Portland
Brinnon
Olympia
132
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