of the

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                 Assembled by

               Edward F, Eldridge

       Research & Technical Consultation

             Public Health Service

                   Region IX

               Portland, Oregon

                 November 1961


     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

     6.  Oceanography and Related Estuarial Water Problems
         of the Northwest

     7.  Status of Knowledge of Watershed Problems of the

     8.  Radioactive Waste Problems in the Pacific Northwest

      9.  Research in Water Pollution and Other Environmencal Health

     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.



DATE:     November 14, 1961

PLACE:    Hearing Room, Interstate Commerce Commission
          410 S. W. Tenth Avenue, Portland, Oregon


 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,

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.

 1:15-1:45       VI - Process  Wastes  from Atomic  Energy Operations -
                        Mr.  P. A.  Olson,  Biology Operation Section,
                        General  Electric Company, Richland, Washington,

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.


                       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-

     *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,

     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.


                         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.

     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-


     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


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

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

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.

     In the handling of toxic wastes, consideration must be given

to the source.  If  they originate at a point such as a sewer

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-


     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

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

substance which is allowable.  The levels of concentrations, both

upper and lower, which are required for  life must be determined.


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,

may in reality be the evil products which can adversely affect

the total environment.  "Toxicity,'1 therefore, is now a problem

which concerns all.


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-


     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

    working on water quality criteria?

 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


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

    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


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

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.

                          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.'

      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

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

know, in brief, how general was the  occurrence of pesticides in

surface and ground waters,  what were the less obvious effects


 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


     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.

     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

 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

 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.

 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,

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.

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

                           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.

 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


      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-


      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.

     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-


     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.

                            TABLE 1


       (Expressed In Parts Per Billion Active Ingredient)
48 72
59'. o
Rainbow Trout
48 72

                                                  TABLE 2


                              (Expressed In Parts Per Billion Active Ingredient)
12, 500. O2
18, 000. O3
95. 03
180. O3
       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


                              (Expressed In Parts Per Billion Active Ingredient)
5 parts per thousand salinity

Ma lath ion
25_parts per thousand salinity


                            TABLE 4

24 hr
48 hr
72 hr
96 hr
                            TABLE 5

Salinity (part per thousand)
48 hr"
72 hr
96 hr
                          TABLE 6

                  5 £ish              10 flsh            20 fish
72 hr
96 hr
                            TABLE 7

Temperature (degrees C.)
72 hr
96 hr

                                           TABLE 8

no. of
; 22
% eyed
eggs 4 days
eyed fish
hatching in
8-9 days
no. of
7, Survival
Days after beginning of
3 4 5" eT " 7~~8

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.


                           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.

 since these four metals  resulting  from industrial  processes would

 be expected to appear more  frequently and  in  greater  concentra-


      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

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

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-

 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.

     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.


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.

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-


Q.  Was this colloidal copper in the form of a carbonate?

A.  No, it was in the form of cupreous oxide.

Q.  What about precipitating copper sulfide from a cyanide


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.


                       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.


     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


     Another problem in long-term oyster bioassays which has

been encountered is the high mortalities occurring with Native

 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.


 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

chemicals, along with other information regarding the waste

that is discharged.


                       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.


                           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.

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
17. reactor effluent
27. reactor effluent
37. reactor effluent
57. reactor effluent
107. reactor effluent
Per Cent Mortality
Finger ling
WeiRht (g)

      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

between mid-November and early June (5).

     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

                                     Per Cent Effluent
   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
iver water
% effluent--steady
3 to 6.27. effluent--
Per Cent Mortality




Weight (g)

     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.

               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.

                   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
Finger ling
Weight (g)
     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


     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

 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
6 IF
Per Cent Mortality
Finger ling
Weight (g)
     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.


     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

 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-

 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

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

concentration of PJ  causing some damage lies somewhere between

these two values.  The lower value exceeds that of sampled Colum-

bia River fish by an order of 100.


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

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).

 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

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).                             "


                     Charles D.  Ziebell*


     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.

     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

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.

     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

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

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


     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.


     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-

 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,


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.

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,

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.


                         C. D. Hoodie*


    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,

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.

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,


     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

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.

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


     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

          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

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

                           TABLE 1
           Scale of Conductivity of the  Saturation
       Extract of Soils in Relation Crop Response (22)
  of Sat.  Ext.
           Crop Response

     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

Only a few very tolerant crops yield

3  4567 6 1,000
                                          3  4 5,000
      100      eso         750
                     SALINITY  HAZARD
Fig.  2  — Classification of Irrigation Waters
           Based on Conductivity attd Sodium
           Adsorption Ratio  (22.23)

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

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.)
because it can be used with considerable reliability to predict

the degree to which Na+ saturates the exchange complex of the

 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

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

 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-


     The effects of high concentrations of specific ions or salts

on plant growth is too complicated to present here.  Bernstein

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

 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

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 .


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

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).

 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).

 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).


 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

 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


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,

    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.

   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.


                             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.

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

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,

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)
concluded that  both  the clam and mussel toxin must be the same or

nearly the same with a molecular formula of C10H1704N7.2HCL.

     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

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.

     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

 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


      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.


 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.

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.

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.)


 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


 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

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.


                        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.

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

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

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


     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

 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

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

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


     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.


                      Charles E. WoeIke*


     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.

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

 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-

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--

 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-

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

 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

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


     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.,

 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


 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

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

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.


                        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.

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

                                                    b33x3 "*"
                   b!2xlx2 + b!3xlx3 + b23x2x3


    Y «= response time, minutes

   xl = f (salinity)

   X2 = f (temperature)

   X3 « f (dissolved oxygen)

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

     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

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.)


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


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.


                          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
 Nanaimo, B.C.
 Gig Harbor

 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
Wash., D.C.
0lymp ia

Gig Harbor
San Francisco
San Francisco

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.