T D

4*65

UC-NRLF

LIBRARY

OF THE

UNIVERSITY OF CALIFORNIA.

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GIFT OF

Clay

U. S. DEPARTMENT OF AGRICULTURE.

BUREAU OF PLANT INDUSTRY BULLETIN No. 64.

METHOD OF DESTROYING OR PREVENTING
THE GROWTH OF ALG.E AND CERTAIN
PATHOGENIC BACTERIA IN
WATER SUPPLIES.

GEORGE T.^MOORE.

PIIY>I( >i.< rcisr AND ALGOLOGIST IN CHARGE OF LABORATORY
OF PLANT PHYSIOLOGY,

KARL F. KELLERMAN.

ASSISTANT IN PHYSIOLOGY.

VEGETABLE PATHOLOGICAL ANJD PHYSIOLOGICAL
INVESTIGATIONS.

i) MAY 7, 1904.

WASHINGTON:

<;ov i: KN .M i: NT IMJTNTING OFFICE,
1 1*04.

BULLETINS OF THE BUREAU OF PLANT INDUSTRY.

The Bureau of Plant Industry, which was organized July 1, 1901, includes Vege-
table Pathological and Physiological Investigations, Botanical Investigations and
Experiments, Grass and Forage Plant Investigations, Pomological Investigations,
and Experimental Gardens and Grounds, all of which were formerly separate Divi-
sions, and also Seed and Plant Introduction and Distribution, the Arlington Exper-
imental Farm, Tea-Culture Investigations, and Domestic Sugar Investigations.

Beginning with the date of organization of the Bureau, the several series of bulle-
tins of the various Divisions were discontinued, and all are now published as one
series of the Bureau. A list of the bulletins issued in the present series follows.

Attention is directed to the fact that "the serial, scientific, and technical publica-
tions of the United States Department of Agriculture are not for general distribution.
All copies not required for official use are by law turned over to the Superintendent
of Documents, who is .empowered to sell them at cost." All applications for such
publications should, therefore, be made to the Superintendent of Documents, Gov-
ernment Printing Office, Washington, D. C.
No. 1. The Eelation of Lime and Magnesia to Plant Growth. 1901. Price, 10 cents.

2. Spermatogenesis and Fecundation of Zamia. 1901. Price, 20 cents.

3. Macaroni Wheats. 1901. Price, 20 cents.

4. Kange Improvement in Arizona. 1902. Price, 10 cents.

5. Seeds and Plants Imported. Inventory No. 9. 1902. Price, 10 cents.

6. A List of American Varieties of Peppers. 1902. Price, 10 cents.

7. The Algerian Durum Wheats. 1902. Price, 15 cents.

8. A Collection of Fungi Prepared for Distribution. 1902. Price, 10 cents.

9. The North American Spejgies of Spartina. 1902. Price, 10 cents.

10. Records of Seed Distribution and Cooperative Experiments with Grasses and

Forage Plants. 1902. Price, 10 cents.

11. Johnson Grass. 1902. Price, 10 cents.

12. Stock Eanges of Northwestern California: Notes on the Grasses and Forage

Plants and Range Conditions. 1902. Price, 15 cents.

13. Experiments in Range Improvement in Central Texas. 1902. Price, 10

cents.

14. The Decay of Timber and Methods of Preventing It. 1902. Price, 55 cents.

15. Forage Conditions on the Northern Border of the Great Basin. 1902. Price,

15 cents.

16. A Preliminary Study of the Germination of the Spores of Agaricus Campes-

tris and other Basidiomycetous Fungi. 1902. Price, 10 cents.

17. Some Diseases of the Cowpea. 1902. Price, 10 cents.

18. Observations on the Mosaic Disease of Tobacco. 1902. Price, 15 cents.

19. Kentucky Bluegrass Seed: Harvesting, Curing, and Cleaning. 1902. Price,

10 cents.

20. Manufacture of Semolina and Macaroni. 1902. Price, 15 cents.

21. List of American Varieties of Vegetables. 1903. Price, 35 cents.

22. Injurious Effects of Premature Pollination. 1902. Price, 10 cents.

23. Berseem: The Great Forage and Soiling Crop of the Nile Valley. 1902.

Price, 15 cents.

24. Unferrnented Grape Must. 1902. Price, 10 cents.

25. Miscellaneous Papers: I. The Seeds of Rescue Grass and Chess. II. Saragolla

Wheat. III. Plant Introduction Notes from South Africa. IV. Congres-
sional Seed and Plant Distribution Circulars. 1902-1903. 1903. Price,
15 cents.

26. Spanish Almonds and Their Introduction into America. 1902. Price, 15

cents.

[Continued on p. 3 of cover.]

U. S. DEPARTMENT OF AGRICULTURE,

BUREAU OF PLANT INDUSTRY— BULLETIN No. 64.

B. T. GALLOWAY, Chief of Bureau.

A METHOD OF DESTROYING OR PREVENTING

THE GROWTH OF ALG.E AND CERTAIN

PATHOGENIC BACTERIA IN

WATER SUPPLIES.

BY

GEORGE T. MOORE.

PHYSIOLOGIST AND ALGOLOGIST IN CHARGE OF LABORATORY
OF PLANT PHYSIOLOGY,

AND

KARL F. KELLERMAN,
ASSISTANT IN PHYSIOLOGY.

VEGETABLE PATHOLOGICAL. AND PHYSIOLOGICAL
INVESTIGATIONS.

ISSUED MAY 7, 1904.

WASHINGTON:

GOVERNMENT PRINTING OFFICE
1904.

BUEEAU OF PLANT INDUSTRY.

B. T. GALLOWAY, Chief.
J. E. ROCKWELL, Editor.

VEGETABLE PATHOLOGICAL AND PHYSIOLOGICAL INVESTIGATIONS.

SCIENTIFIC STAFF.

ALBERT F. WOODS, Pathologist and Physiologist.

ERWIN F. SMITH, Pathologist in Charge of Laboratory of Plant Pathology.

GEORGE T. MOORE, Physiologist in Charge of Laboratory of Plant Physiology.

HERBERT J WEBBER, Physiologist in Charge of Laboratory of Plant Breeding.

WALTER T. SWINGLE, Physiologist in Charge of Laboratory of Plant Life History.

NEWTON B. PIERCE, Pathologist in Charge of Pacific Coast Laboratory.

M. B. WAITE, Pathologist in Charge of Investigations of Diseases of Orchard Fruits.

MARK A. CARLETON, Cerealist in Charge of Cereal Investigations.

HERMANN VON SCHRENK,^ in Charge of Mississippi Valley Laboratory.

P. H. ROLFS, Pathologist in Charge of Subtropical Laboratory.

C. O. TOWNSEND, Pathologist in Charge of Sugar Beet Investigations.

P. H. DORSETT, Pathologist.

RODNEY H. TRUE,& Physiologist.

T. H. KEARNEY, Physiologist, Plant Breeding.

CORNELIUS L. SHEAR, Pathologist.

WILLIAM A. ORTON, Pathologist.

W. M. SCOTT, Pathologist.

JOSEPH S. CHAMBERLAIN, Physiological Chemist, Cereal Ivestigations. *

R E. B MCKENNEY, Physiologist.

FLORA W. PATTERSON, Mycologist.

CHARLES P HARTLEY, Assistant in Physiology, Plant Breeding.

KARL F. KELLERMAN, Assistant in Physiology.

DEANE B. SWINGLE, Assistant in Pathology.

A. W. EDSON, Scientific Assistant, Plant Breeding.

JESSE B. NORTON, Assistant in Physiology, Plant Breeding.

JAMES B. RORER, Assistant in Pathology.

LLOYD S. TENNY, Assistant in Pathology.

GEORGE G HEDGCOCK, Assistant in Pathology.

PERLEY SPAULDING, Scientific Assistant.

P. J. O'GARA, Scientific Assistant.

A. D- SHAMEL, Scientific Assistant, Plant Breeding.

T. RALPH ROBINSON, Scientific Assistant, Plant Physiology.

FLORENCE HEDGES, Scientific Assistant, Bacteriology.

CHARLES J. BRAND, Scientific Assistant in Physiology, Plant Life History.

a Detailed to the Bureau of Forestry.

b Detailed to Botanical Investigations and Experiments.

LETTER OF TRANSMITTAL

U. S. DEPARTMENT OF AGRICULTURE,
BUREAU OF PLANT INDUSTRY,

OFFICE OF THE CHIEF,
Washington, D. <?., April 30, 1904.

SIR: I have the honor to transmit herewith a paper entitled "A
Method of Destroying or Preventing the Growth of Algae and Certain
Pathogenic Bacteria in Water Supplies," and to recommend that it be
published as Bulletin No. 64 of the series of this Bureau.

The paper was prepared by George T. Moore, in charge of Labora-
tory of Plant Physiology, and Karl F. Kellerman, Assistant in Physi-
ology, in the Office of Vegetable Pathological and Physiological
Investigations, and was submitted by the Pathologist and Physiologist
with a view to publication. The subject discussed in this bulletin
will be of interest and value to all who have to deal with the problem
of preventing algal and other contamination of water supplies.
Respectfully,

B. T. GALLOWAY,

Chief of Bureau.
Hon. JAMES WILSON,

Secretary of Agriculture.

3

218532

PREFACE.

The necessity of finding some cheap and practical method of prevent-
ing or removing algal contamination of cress beds first led this Office
to undertake the investigations described in this bulletin. The success
of the first experiments in 1901 was so marked that it seemed wise to
extend the work, and authority was, therefore, granted by Congress
"to study and find methods for preventing the algal and other con-
taminations of water supplies."

The progress of the investigation has been noted from time to time
in the annual reports of the Bureau. Though the work is not yet com-
pleted, we have been urged to publish the results already obtained for
the consideration of boards of health and officers in charge of public
water supplies.

Doctor Moore and Mr. Kellerman have shown that it is entirely
practicable to cheaply and quickly destroy objectionable alga? in small
lakes, ponds, storage reservoirs, and other similar bodies of water by
the use of extremely dilute solutions of copper sulphate or of metallic
copper. The fact that an extremely dilute solution (one to one
hundred thousand) will also destroy the most virulent typhoid and
cholera bacteria at ordinary temperatures in three hours is of great
importance and significance. Solutions of copper as dilute as this
are not considered injurious to man or other animals. The value of
copper, especially colloidal, in preventing or treating typhoid and
other related diseases should be carefully investigated by competent
pathologists.

We desire it distinctly understood that, so far as bacterial contami-
nation of water is concerned, the methods here proposed are not to
take the place of, but are simply to supplement the standard methods
of filtration; neither can too much stress be laid upon the importance
of the consumer boiling water to be used for drinking purposes when
taken from a contaminated source.

Upon application to the Department by proper authorities, infor-
mation and assistance will be furnished in determining the organisms
causing the trouble in cases of algal pollution, and the proper treat-
ment will be recommended. Jt is earnestly hoped that no test of the
method described here will be made without first consulting the
Department.

5

6 PREFACE.

As stated in the text of the bulletin —

The treatment of water supplies for the destruction of pathogenic bacteria, or any
application of the copper sulphate method, which has to do with the public health
is not contemplated or indeed possible by this Department. The requests of pri-
vate individuals or of unauthorized bodies for information or assistance can not be
granted. When State or local boards of health consider that the disinfection of a
water supply is desirable and wish information upon the subject, it will be supplied
as fully and freely as possible. All experiments of this kind, however, must be
conducted by boards of health, and the Department can serve only in the capacity
of an adviser.

We are under obligation to Dr. H. P. Wolcott and Mr. X. H. Good-
nough, of the Massachusetts State Board of Health, for facilities in
securing material and a temporary laboratory in the Boston State
House; to the United States Bureau of Fisheries for fish used in
experiments; to Dr. J. J. Kinyoun for typhoid cultures; to Dr. M. J.
Rosenau for Asiatic cholera cultures, and to the Bureau of Animal
Industry for cultures of typhoid and facilities for carrying on pre-
liminary experiments.

ALBERT F. WOODS,
Pathologist and Physiologist.
OFFICE OF VEGETABLE PATHOLOGICAL

AND PHYSIOLOGICAL INVESTIGATIONS,

Washington, D. C., April 30, 1904.

CONTENTS.

Page.

Introduction 1 9

Microscopical examination of drinking water 9

Wide distribution of trouble caused by algse in water supplies 10

Methods in use for preventing bad effects due to algse 13

Desirability of other methods 14

Determination of a physiological method 15

Effect of copper sulphate 15

Method of applying copper sulphate 25

Practical tests of the method 26

Water-cress beds 26

Water reservoirs 27

Effect of copper upon pathogenic bacteria 28

Typhoid 28

Asiatic cholera . '. 34

Comparison of effect of other disinfectants 36

Colloidal solutions 36

Conclusions 40

Necessity of knowledge of organism and condition in reservoir 40

Application of method for destruction of pathogenic bacteria not designed

to replace efficient means of filtration already in use 41

Medicinal use 42

Conditions under which the Department of Agriculture can furnish infor-
mation and assistance in applying this method 42

Cost 43

Summary 43

7

B. P. I.— 108. V. P. P. I.— 118.

A METHOD OF DESTROYING OR PREVENTING THE GROWTH OF
ALG^E AND CERTAIN PATHOGENIC BACTERIA IN WATER
SUPPLIES.

INTRODUCTION.

The necessity and importance of maintaining by every possible
means the purity and wholesomeness of public water supplies have
caused those in authority to welcome a method which would in any
way serve as an additional safeguard against the pollution of reservoirs
or would prevent the bad effects produced by the growth of algae and
similar organisms. Although scientific men have been investigating
the various problems involved for a considerable length of time, it is
feared that the public has not always been in sympathy with these
methods, and that, owing to the uncertainty of and disagreement among
eminent authorities, the whole question of water analysis, both chem-
ical and bacteriological, has come somewhat into disrepute.

MICROSCOPICAL EXAMINATION OF DRINKING WATER.

While the best known cases of water pollution are those due to the
presence of typhoid and other germs which have given rise to serious
epidemics, there are a vastly greater number of water supplies which
are rendered unfit for use, not because they are dangerous to public
health, but on account of the very offensive odor and taste produced
in them by plants other than bacteria. For this reason, in recent
years, the question of whether or not a water was fit to drink has been
submitted to the biologists as well as to the chemists and bacteriol-
ogists, a biological examination being generally understood to mean
the determination of the character and quantity of the microscopical
plants and animals the water may contain as distinct from the bacteria.

The history of this method of examining drinking water is really
confined to the last quarter of the nineteenth century, but only within
ten or fifteen years have we had any accurate knowledge of the effect
of these minute plants upon the water in which they live. It is prob-
able that Dr. Hassall, of London, was the first to publish any adequate
account of a thorough microscopical examination of any water supply,
and this work, which appeared in 1850, was practically the only thing

10 METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

upon the subject for twenty-five years, when "MacDonald's Guide to
the Examination of Drinking Water" was published. In the mean-
time various Germans had carried on investigations relating to the
biology of water supplies, notably Professor Cohn, of Breslau, who,
in a paper entitled the "Microscopical Analysis of Well Waters,"
anticipated much that has since been ascertained in regard to the effect
of environment upon the character and quantity of the organism found
in the water. About the time of the appearance of MacDonald's
book, interest in the effect of algae in drinking water first began to be
aroused in this country, and papers by Farlow" and others called
attention to the fact that these plants were responsible for many of
the disagreeable odors and tastes in water reservoirs. By the year
1878 there was on record a list of over 60 cities and towns in the
United States which had had serious trouble because of the presence
of certain forms of vegetation in their reservoirs, but since then thou-
sands of water supplies throughout the country have been rendered
unfit for use by this cause alone. Early in the year 1891 the special
report upon the examination and purification of water by the Massa-
chusetts State Board of Health was published, this being the most com-
plete treatment of the subject which had appeared up to that time.
This report has been supplemented by further investigations and
experiments, and the work accomplished by this board in perfecting
methods for insuring a pure water supply has established the standard
both in this country and abroad for similar lines of investigation.

WIDE DISTRIBUTION OF TROUBLE CAUSED BY ALG^E IN WATER

SUPPLIES.

In order to demonstrate the very wide distribution of the trouble
caused by algae in water supplies throughout the United States, a
circular letter was sent to about five hundred of the leading engineers
and superintendents of water companies, asking for information in
regard to the deleterious effects produced by plants other than bacteria
in water supplies with which they were familiar. Many instructive
replies were received, indicating that those in authority were extremely
anxious to be provided with some efficient remedy for preventing the
bad odors and tastes in drinking water, and that they considered the

« FARLOW. Reports on Peculiar Condition of the Water Supplied to the City of
Boston. Report of the Cochituate Water Board, 1876.

Reports on Matters connected with the Boston Water Supply. Bulletin
of Bussey Inst., Jan., 1877..

Remarks on Some Algae found in the Water Supplies of the City of Boston,

1877.

• On Some Impurities of Drinking Water Caused by Vegetable Growths.
Supplement to 1st Ann. Rept. Mass. State Board of Health. Boston, 1880.

Relations of Certain Forms of Algae to Disagreeable Tastes and Odors.

Science, II, 333, 1883.

WIDE DISTRIBUTION OF TROUBLE CAUSED BY ALG^E. 11

subject worthy of most careful investigation. Quotations from some
of the letters received are given, but, because there might be some
objection to the naming of towns, only the State in which the trouble
occurred is indicated. This is sufficient, however, to show that the
difficulty is not confined to any one part of the country, and that it is
the algae alone which are responsible for most of the bad odors and
tastes reported.

CALIFORNIA:

Any efforts in the direction of preventing the growth of algae will be gratefully
acknowledged. So long as the growth is healthy it is a benefit, but as soon as
the algae break up then trouble begins.
COLORADO:

We have a reservoir of water that has recently become affected through the
presence of micro-organisms of the algae type that impart to the water a dis-
agreeable fishy odor and render its use objectionable.
DELAWARE:

A fishy taste and odor.
ILLINOIS:

The water tasted and smelled like rotten wood.

Trouble serious enough to cause general complaint by consumers on account
of odor and taste.

People declared that the water was musty. The appearance of the growth is
yellowish-brown, and as nearly as I can describe it the smell is musty. I cer-
tainly think the subject worthy of the best thought and work the Government
can give it.
INDIANA:

The growth increased to such an extent that we were compelled to cement the
bottom and 5 feet up the sides. It was as dense as a field of clover in June.

Taste was said by the people to be woody or fishy, like rotten wood or decayed
fish. At one time the report got out that the body of a missing man had been
found in the reservoir.
IOWA:

After certain stages in the alga's growth it seemed to die and become decom-
posed, thus impregnating the water, giving it a most unpleasant odor and taste.
KENTUCKY:

Fishy odor and taste, rather musty.

The odor was so strong that we had to discontinue sprinkling the streets and
lawns.

Urgency in this case is great, indeed almost imperative, since the condition of
the water during the past two or three summers has culminated in formal action
by the authorities.
MAINE:

Trouble to such an extent as to lead us to consider, without taking definite
action, whether or not the water should be filtered before being distributed.
Odor is reported as exceedingly disagreeable, so that many customers avoid the
use of it as far as possible and believe it injurious to health.
MASSACHUSETTS:

Trouble very serious; some years water is unfit to drink. Present year odor
and taste are not so strong as last year, when it was almost impossible to drink it.

The odor was so bad that it would be almost impossible to take it as far as the
mouth to taste it. Horses refused it at the street watering troughs and dogs fled
from it.

12 METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

MINNESOTA :

Water at times a fishy odor or taste due to decomposed vegetable matter.
Experts claim it is entirely harmless.
NEW JERSEY:

Dark green gelatinous substance in water, causing a stench almost unbearable.

Have seen Uroglena so abundant that an odor could be plainly detected one-
third of a mile away.
NEW YORK:

Water had a very fishy taste and smell.

So very offensive as to alarm all water takers.

It caused such a prejudice that the supply was rejected, although the pollution
was of short duration.

Strong fishy odor and taste; also odor of "smartweed." Popular complaint
was dead fish in water mains.

Odor and taste were fishy, popularly attributed to dead fish; but this is absurd,
as the Odor is that of live fish.

Odor pondy and fishy; bad water; publicly condemned. Board of health
interfered, yet analysis showed that water was notunhealthful.

Very rank, water smelled bad, particularly when warmed. Tasted bad, but
not injurious to health. Looked better than tasted or smelled.

Water became unfit for use, musty or cucumber taste and smell, odor very
strong in hot \vater; water became slimy, making it exceedingly hard to filter.
Odor and taste at times decidedly fishy. A bright green powder seemed to
have been sprinkled on surface.

I am much interested to know that you are taking up an investigation of algae
and organisms, and I very much hope you will favor me with all circulars and
information which you may issue relating to the same. I have not attempted
to fill out the circular on the back of your letter, but so many cases of trouble of
this kind have come to my attention that any listing of them would be very
difficult.

I am devoutly thankful that science in this particular instance has got beyond
the pursuit of science for recreation's sake and is doing good and endeavoring
again directly to be of much use to mankind. 1 believe your work is the first
done in line of either cure or prevention from algye conducted in a rational man-
ner, or so far as I know even attempted, and I have been connected with or well
informed on public water supplies and their management all my professional
life of some thirty-five years. The worst case I know of is at the — — reservoir.
A special commission is at this moment charged with the duty of advising
whether or not property worth some two million dollars is to be abandoned on
account of annual trouble from algse.
OHIO:

Complaint from customers of a fishy taste in water like the slime from fresh-
water fish.

Water had a fishy taste, causing a general kick; consumers laid it to the fish
in the reservoir.

All water drawn from house bibbs had an objectionable and strong odor, the
popular idea being that it was due to dead fish.

The towns A — and B — both have vile water, A — all the year round, B — for
six or eight weeks in the hottest part of the summer. A — 's water has a vile
odor, offensively musty. All vegetables, cereals, coffee, and such edibles and
drinks made with the water are scarcely endurable to the visitor.
PENNSYLVANIA:

Water had a disagreeable fishy odor.

Water smelled and tasted as if dead fish were in it.

METHODS FOE PREVENTING BAD EFFECTS DUE TO ALGuE. 13

PENNSYLVANIA — Continued.

The growth affected the taste of the water on boiling, but was not regarded as
dangerous to health.

A very fishy taste and smell. I have been unable to locate, but had an idea it
came from vegetation.

The water during the autumn is so foul in taste and odor that it was necessary
to shut off the supply. The odor is similar to that of decayed fish.

The first season of using reservoir the water became so fishy that it was almost
unfit for use. Since that, owing to our care of reservoir, we have had no trouble
whatever.
TEXAS:

At this time of the year algae are fierce; some days we are on top and some
days the algae are on top. Costs us an average of $25 a month for cleaning out
algae from two reservoirs.
WISCONSIN:

Universal complaint, caused by the odor and taste due to algae.

METHODS IN USE FOB PREVENTING BAD EFFECTS DTTE TO ALG-ffi.

In order to prevent the odors and tastes above described,' engineers
and those in charge of water supplies have tried various remedies,
none of which has been perfectly satisfactory. Since few of the
algae can develop without sunlight, the most frequent recommenda-
tion has been to cover the reservoir, and this method has proved suc-
cessful in a few instances. However, the expense involved is so great
as to make the remedy prohibitive in most cases, and other methods
have had to be resorted to. One precaution which is now almost uni-
versally recommended as a means of preventing the growth of algae is
to remove all the organic matter possible from the reservoir and to keep
the source of supply as free as can be from dead and decaying animal
and vegetable matter. In one notable instance millions of dollars
have been spent in the removal of earth and the substitution of gravel
at the bottom of an immense new reservoir. It remains to be seen,
however, whether this will be sufficient to insure permanent freedom
from these troublesome plants. It is certain that attempts of this
kind will delay the appearance of algae in quantity, and, wherever it
is possible to do so, every effort should be made not only to clean up
the reservoir at the time of its construction, but to keep it as free as
possible from organic matter after it is filled. In addition to cleanli-
ness a direct pumping system with duplicate, in case of breakdown or
repairs, has often been recommended for use with ground water,
which usually produces a more luxuriant growth of algae and similar
organisms than surface water. Where it has been necessary to store
such water, it has been advisable to limit the capacity of the reservoir,
and frequently this storage is only intended to be used in case of fire.
Even so, the cleansing of the reservoir and the frequent flushing of
the water mains has been considered necessaiy. In storing surface1
water subdividing the reservoir is occasional^ resorted to, and means

14 METHOD OF DESTROYING ALGJE IN WATEK SUPPLIES.

of obtaining frequent agitation are introduced wherever possible.
The pumping of air into water or aerating it by means of a spraying
apparatus is often of considerable value in removing foul gases which
may be in solution, but the effect of aeration upon the growth of algae
in a reservoir has been very much overestimated, in some cases the
quantity being actually increased by this means.

The filtration of water, both mechanically and by sand, which has
proved so effective for the removal of pathogenic bacteria, has been
recommended as a means of removing the odors and tastes caused by
algae, but the results obtained have not given promise of success.
Perhaps the most careful experiments to determine this point have
been conducted by those in charge of the Ludlow reservoir at Spring-
field, Mass. Here the annual trouble from algae for the past fifteen
years has been so great that every possible means has been used which
offered any relief from the effects produced by these plants. On page
4 of the ".Special Report on the Improvement of the Present Water
Supply and an Alternative New, Independent Supply," made by the
board of water commissioners to the city council of the city of Spring-
field, Mass., April 14, 1902, the following statement is made:

We find, as the results of the experiments of filtration, made with the sanction of
your honorable body during the last fifteen months, that to purify the waters of this
source by filtration would be not only doubtful as to the degree of purification, but
so expensive in the cost of construction and perpetual maintenance thereafter as
to make it inexpedient to attempt improvement by such a method. Your board has
given constant and personal attention to the experimental work, and is convinced
that the excessive growths of obnoxious fresh-water organisms, notably the Ana-
baena, impart to the reservoir such rank and persistent tastes and odors as to make
uncertain entire removal by any method of filtration except that of the expensive
kind, applicable only to the filtering of extremely small quantities of water, and
requiring constant attention and adjustment.

The State board of health, in a special report (p. 84) submitted at the
same time, say that the results of the experiments indicate, in the
opinion of the board, that by double filtration it will be possible to
purify the Ludlow reservoir; hence there seem to be differences of
opinion as to the value of this treatment for the removal of odors and
tastes, but on account of the expense involved there is not likely to be
any very extensive use of this method.

DESIRABILITY OF OTHER METHODS.

While each of the above-mentioned methods has been used with
some success, it is generally conceded by engineers that there is no
known remedy which is universally applicable. It is the practice
of some of the highest authorities to recommend that reservoirs f re-
quentty polluted by algae be abandoned, and steps taken to provide an
entirely new system of supply. This is, of course, the last resort, as
in all such cases a large loss of money is involved. One fact is certain.

DETERMINATION OF A PHYSIOLOGICAL METHOD. 15

If any known method of preventing the growth of algae was considered
truly effective, it would under all circumstances be recommended.

Because of the unsatisfactory results or the prohibitive expense of
the present methods recommended for ridding reservoirs of algae, it
seemed advisable that the problem be taken up from an entirely new
standpoint, one that would take into consideration the biological aspect
of the question and perhaps furnish a solution, through a study of the
physiology of the organisms under laboratory conditions. A series of
investigations were therefore undertaken to discover, if possible, some
substance which, because of its extreme toxic effect upon the alga3
involved, would absolutely prevent their growth in water supplies.

DETERMINATION OF A PHYSIOLOGICAL METHOD.

In determining such a physiological method of dealing with reser-
voirs contaminated by algaer two conditions had to be considered: The
remedy should not only be readily available and cheap enough for
practical use in the largest reservoirs and by the poorest communities,
but under the conditions used it must also be absolutely harmless to
man ; the maximum amount necessary to kill the algae being far below
the amount which could in any way affect the consumer of the water.
Of the large number of substances experimented with, few gave en-
couraging results. Free chlorine at a dilution of 1 to 10,000, and sul-
phur dioxide in saturated aqueous solution at 16° C., diluted 1 to 1,000
and to 10,000, will destroy many of the common forms of algae, but sul-
phur dioxide and chlorine are likewise very injurious to animal life.
Silver has a very high toxicity, and were not the expense prohibitive,
would undoubtedly warrant extended tests. Mercury and lead are,
of course, out of the question, and zinc requires too high a concentra-
tion to be practically considered. The ordinary sodium, potassium,
and ammonium salts, are innocuous,* as are most of the acids. Loew6
finds that magnesium sulphate is toxic in pure solution at 0.4 per cent,
and that oxalates are slightly more toxic; of the acids, 0.0001 per cent
oxalic kills most of the cells of Spirogyra majuscula in five days.
Migula c notes the effect of many of the organic acids, but the use of
these substances in the amounts requisite for treating a contaminated
water supply is entirely impracticable.

EFFECT OF COPPER SULPHATE.

Reviewing the experiments carried on in the Laboratory of Plant
Physiology, as well as the results obtained by other investigators, it

«Cf. Richter, Flora, 75: 4.
&Loew, Flora, 75: 368.

c Migula, Ueber den Einfluss stark verduenter Sauren auf Algenzellen, Breslau,
1888. ( Original not consulted. )
28480— No. 64—04 2

16 METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

seems that copper sulphate is the substance best adapted to the work
in question. This salt has a very high toxicity for algae, and experi-
ments with a number of the forms usually found in reservoirs, and
the source of much trouble, have shown that inconceivably small
amounts of copper are poisonous in a high degree. These experiments
demonstrated, however, that all algae and protozoa are not equally sen-
sitive. Among the latter Params&dum is killed in three hours by a 1
to 1,000,000 solution, while A.?nceba, Diffiugia, and Spi/rost&mum die
within two hours. Crustacea are more resistant, some — Cypris and
Daphnia especially — requiring as much as 1 part copper sulphate to
10,000 of water to kill them. Mosquito larvae die at a concentration
varying from 10,000 to 200,000.

Quoting the results of other experimenters, Devauxa found that
both phaenogams and cryptogams were poisoned by solutions of copper
diluted to the ten-millionth part or less; Coupin& that 1 part copper
sulphate to 700,000,000 of water was sufficient to affect the growth of
seedlings when applied to their roots and that this is the most inju-
rious of the heavy metal salts tested by him; Deherain and De Moussy c
that the development of the roots of seedlings was arrested in distilled
water containing the slightest trace of copper, and they conclude from
this that higher plants during germination, as well as fungi and algy?,
are extremely sensitive to copper; Bain's experiments d indicated that
1 part of metallic copper to 25,000,000 of water was fatal to apple seed-
lings in one day; on the other hand, according to Raulin/ copper chlo-
ride does not injure Sterigmatocystis until a concentration of 1 to 240 is
reached, although silver nitrate is toxic at 1 to 1,600,000.

In dealing with algae, the toxic concentration varies greatly for dif-
ferent genera, even for different species in the same genus. Nageli;
demonstrated the extreme sensitiveness of Splrogyra nitida and S.
diibia to the presence of copper coins in the water. Oscillatoria,
Cladophora, (Edog&nium, and the diatoms succumb in six hours
to a copper sulphate solution of 1 to 20,000, and in two days to 1 to
50,000, according to Bokorny.*7 GaleottiA finds that a concentration
between 1 to 6,300,000 and 1 to 12,600,000 is sufficient to kill Spiro-
gyra nitida in two days, and that the so-called colloidal solutions at 1
to 6,300,000 are fatal in the same length of time; while in the experi-

«Devaux, Compt. Rend., 132: 717.
&Coupin, Compt, Rend., 132: 645.
^'Deherain and De Moussy, Compt. Rend., 132: 523.
^Bain, Bull. Agr. Exp. Sta. Tenn., April, 1902.
"Raulin, Ann. des So. Nat. Bot., 5C Ser., II: 93.

/Nageli, Ueber oligodynamische Erscheinungen in lebenden Zellen. Neue
Denkschr. d. schweizerischen Gesellsch. fiir die gesammten Naturwiss., 33: 51.
r/Bokorny, Arch. f. d. ges. Phys. d. Mensch. u. Thiere, 64: 262.
''Galeotti, Biol. Centralbl., 21: 321.

EFFECT OF COPPER SULPHATE. 17

ments of Israel and Klingman a the presence of 60 sq. cm. of copper
foil in 300 cc. of water for twenty-four hours produced plasinal cut-
ting in S. laxa after one and one-fourth hours, in S. crassa after fifteen
minutes, and in S. majuscula after thirty minutes. The work of
Rumm6 shows 1 to 10,000,000 solution still toxic to a few more sus-
ceptible cells of S. longata. According to Ono, c weak solutions of the
salts of most of the metals encourage the growth of algae and fungi.
Mercury and copper, however, at 0.00005 per cent and 0.00001 per
cent, respectively, distinctly inhibit growth. This was the case with
Stigeoclonium, Chroococcum, and Protococcus.

In the experiments conducted in this laboratory it has not been pos-
sible as yet to include all of the organisms known to pollute water
supplies. It is believed, however, that, pending the completion of
more extensive work, the data at hand will be of considerable benefit
to those who have to deal with contaminated reservoirs. The method
of procedure in studying this question was to determine roughly the
death points of the forms under consideration, using Van Tieghem cells.
Accurate solutions were then made, with distilled water, and 200 cc.
of each solution was pipetted into an Erlenmyer flask. The algae, if
filamentous forms, were rinsed; if free-swimming, they were concen-
trated b}^ the Sedgwick-Raf ter d method from 500 cc. to 5 cc. volume,
and this 5 cc. was added to the treated water. The inaccuracy due to
the addition of the 5 cc. of untreated water to the 200 cc. of treated
water was disregarded. Whenever possible, a test of these concen-
trations, determined experimentally, was made under natural conditions
by treating the pool from which the species under consideration was
taken. If this was impracticable, an additional series was carried
through in aquaria of 15 liters capacity, in which were kept goldfish,
frogs, minnows, Crustacea, and rotifers. Since in no case was there
an appreciable difference in the effect of a concentration upon a par-
ticular organism under either natural or artificial conditions, no special
record is made of these gross experiments.

The different species tested may, for convenience, be grouped as (1)
those with death points at higher concentrations than 1 part copper
sulphate to 1,000,000 parts of water; (2) those with death points between
1 to 1,000,000 and 1 to 5,000,000; and (3) those with death points at
greater dilutions than 1 to 5,000,000.

« Israel and Klingman, Virchow's Archiv., 147: 293.

6 Rumm, Beitrage zur Wissenschaftliche Botanik, 1 : 97.

fOno, Journ. of College of Sc., Imp. Univ. Tokyo, 13: 141.

d Whipple, The Microscopy of Drinking Water, New York, 1899, p. 15.

18

METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

Effect of various concentrations of copper sulphate upon different forms ofalgx.
[d=dead; vfa=very few alive; vfd=very few dead; g=in good condition.]

GOROTJIP 1.
CHLAMYDOMONAS PIRIFORMIS Dill.

Date.

One part copper sulphate to water, parts —

Check.

2,000

5,000

10,000

20,000

200,000

1,000,000

October 19-21

id
^d
id

g
vfd
yfd

g
g
g

g
g
g

g
g
g

g
g
g

g
g
g

October 21-24

October 24-27

RAPHIDIUM POLYMORPHUM Fres.

Date.

One part copper sulphate to water, parts—

Check.

25,000

50,000

75,000

100,000

500,000

1,000,000

October 19-29

d
d
d

d
*d
vfa

id
id
id

iVd

&d

vfd

g
g
g

g
g
g

g%
g
g

November 2-6

November 16-20

DESMIDIUM SWARTZII Ag.

Date.

One part copper sulphate to water, parts —

Check.

50,000

75,000

100,000

150,000

200,000

1,000,000

December 2-5

d
d

d
d

id
Id

vfd
vfd

g
g

g
g

g
g

January 4-7

STIGEOCLONIUM TENUE (Ag.) Rabenh.

Date.

One part copper sulphate to water, parts—

Check.

50,000

100,000

300,000

500,000

1,000,000

2,000,000

December 21-24

id
id
id

id
id
id

id
id
id

id
id
id

vfd
vfd
vfd

g
g
g

g
g
g

January 2-5

January 7-11

DRAPARNALDIA GLOMERATA (Vauch.) Ag.

Date.

One part copper sulphate to water, parts—

Check.

50,000

100,000

300,000

500,000

1,000,000

2,000,000

December 1-8

id

id

id

id

vfd

g

g

NAVICULA Sp.

Date.

One part copper sulphate to water, parts —

Check.

100,000

200,000

300,000

400,000

500,000

1,000,000

October 20-25

d
d

d
vfa

id
id

vfd
vfd

vfd
vfd

g
g

id
g

January 4-9

EFFECT OF COPPER SULPHATE. 19

Effect of various concentrations of copper sulphate upon different forms of algse — Cont'd.

GKROTJIP 1— Continued.
SCENEDESMUS QUADRICAUDA (Turp.) Breb.

Date.

One part copper sulphate to water, parts—

Check.

100,000

200,000

300,000

400,000

500,000

1,000,000

September 14-18

d
d
vfa .

d
vfa
vfa

vfa
vfa
vfa

id
id
id

g
g
g

g
g
g

g
g
g

December 7-12

January 11—15

EUGLENA VIRIDIS Ehrb:

Date.

One part copper sulphate to water, parts—

Check.

100,000

200,000

300,000

400,000

450,000

500,000

September 21-25

d
vfa
vfa

vfa
vfa

vfa

vfa
vfa
vfa

Id
*d
id

id
id
id

g
g
g

g
g
g

October 26-30

December 31-January 2

SPIROGYRA STRICT A (E. Bot.) Wille.

Date.

One part copper sulphate to water, parts —

Check.

50,000

75,000

100,000

200,000

500,000

1,000,000

December 26-30

d

vfa

*d

g

g

g

g

GKROTJP 2.

CONFERVA BOMBYCINUM Ag.

One part copper sulphate to water, parts —

50,000

100,000

300,000

500,000

1,000,000

2,000,000

<jnecjt.

October l^i

d

d

d

d

d

g

October 8-11.

d

d

d

vfa

vfa

g

October 13-17

d

d

d

vfa

vfa

g

g

CLOSTERIUM MONILIFERUM (Bory ) Ehrb.

i
Date.

One part copper sulphate to water, parts —

Check.

25,000

100,000

500,000

1,000,000

2,000,000

December 14-18

dl2hrs

d24hrs

d

d

id

g

SYNURA UVELLA Ehrb.

Date.

One part copper sulphate to water, parts—

Check.

250,000

500,000

606,666

750,000 1,000,000

2,500,000

Marchl4

d5-25min
d6-25min

d!5-30min
dl5-30min

d!5-45min
d 15-45min

d 15-60min d 28-60min
d 15-60min d 28-60min

g at Ihr
g at Ihr

gat Ihr
gat Ihr

March 18

20 METHOD OF DESTKOYING ALGJE Itf WATER SUPPLIES.

Effect of various concentrations of copper sulphate upon different forms of algx — Cont'd.

G-ROUF 3— Continued.
ANAByENA CIRCINALIS Raben,

Date.

One part copper sulphate to water, parts —

Check.

50,000

100,000

500,000

1,000,000

8,000,000

5,000,000

December 26-29

d
d

d
d

d
d

d
d

id
id

vfd
vfd

g
g

January 4—7

ANAB^NA FLOS-AQU^E Breb.

Date.

One part copper sulphate to water, parts —

Check.

50,000

100,000 530,000

1,000,000

3,000,000

5,000,000

July 12-14

d 12hrs
d 12hrs

d24hrs d 24hrs
d24hrs d 24hrs

d36hrs
d 36hrs

d 72hrs
d 72hrs

-id
id

g
g

August 27-29

3.

UROGLENA AMERICANA Calk.

Date.

One part copper sulphate to water, parts —

Check.

1,000,000

2,500,000

5,000,000

10,000,000

March 19,

1903

d 3-5min

d IGhrs

vfa IGhrs

yfa 16hrs

g

The foregoing tables clearly demonstrate the effectiveness of copper
sulphate as an agent for the destruction of algae, and as the cost
for an amount of this salt necessary to make the strongest solution
required will not exceed from 50 to 60 cents per million gallons, but
one condition remains to be satisfied — that it shall be absolutely harm-
less to man, domestic animals, and fish under the conditions used.

In general, animal life is less susceptible to injury by copper than is
plant life, though most of the higher plants, some of the fungi, and,
as the preceding tables show, certain alga? will live in concentrations
of copper suFphate that would be fatal in a few hours to fish and frogs.
The critical concentration for game fish is higher than that for such
fish as carp and catfish. Black bass in good condition have endured
concentrations of 1 to 50,000 for many weeks with no apparent discom-
fort, while 1 to 100,000 was sufficient to kill German and mirror carp
in a few hours, and 1 to 500,000 killed the most susceptible in a few
days. Mud catfish are affected at practically the same concentration;
goldfish at slightly greater, while yellow perch are perhaps Jess sus-
ceptible than goldfish. This agrees with the results of Perry and
Adams, a who state that minnows and goldfish live indefinitely in a 1
to 200,000 solution.*

« Perry & Adams, 4th Kept. River Polut. Conn., 2: 377-391.

EFFECT OF COPPER SULPHATE. 21

The effects of copper upon the higher animals have been studied by
a large number of investigators, and the following results may be
appropriately cited:

Metallic copper and its oxides, mixed with sugar, albuminoids, and
fats, had no noticeable effect upon dogs; even 8 grams of fine powder
(4 grains each of copper monoxide and dioxide) caused only a slight sick-
ness. Verdigris in small amounts produced none of the violent results
it is supposed to cause in man. Soluble salts of copper can be given in
quantities up to 1 gram daily, but more than this has a fatal effect.08

Dogs that had eaten half a gram of copper acetate per day for 24
days suffered but slightly; one dog was unaffected by doses as high as
5 grams at a time.6 Similar results were obtained by Du Moulin, c who
gave dogs and rabbits as much as 3 to 5 grams, causing sickness but
in no case death, and Hippolyte Kuborn d states that a dog can take 4
grams of copper sulphate with but slight effect.

Ellenberger and Hofmeister e experimented with sheep, giving them
from 18 to 182i grams of copper in quantities sometimes as large as
2 grams per day, with fatal results. Tschirsch^ deduced from this that
the nontoxicity of weak solutions of copper does not hold for rumi-
nants, but this seems hardly warranted. Two grams per day can
scarcely be considered a small amount, yet one sheep lived 53 days
and the other 128.

Ever since copper compounds have come into general use as fungi-
cides, the question as to their effect upon the human system has
received more or less attention.9' At times there have been vague
and misleading statements in the public press, calculated to alarm
those who are in the habit of using vegetables and fruits which have
been subjected to treatment with Bordeaux mixture. The popular
belief seems to be that copper is a poison, but it is found upon exami-
nation that the very best authorities are by no means agreed upon this
point. It is true that after the question had been discussed for seven
months before the Belgian Royal Academy of Medicine, in 1885, it
was finally decided that copper compounds in foods were harmful, but it
should be remembered that in the whole discussion, where every effort
was made by one side to show that copper was an actual poison, not a

«Burcq & Ducom, Journal de Pharmacie et Chimie, 25: 546, 1877.

& Galippe, Journal de Pharmacie et Chimie, 23 : 298.

c Du Moulin, Journal de Pharmacie et Chimie, 5 : 189.

d Hippolyte Kuborn, Congres Internationale d'Hygiene, 2: 216, 1878.

« Ellenberger and Hofmeister, Archiv fur wissench. u. prakt. Thierheilkunde, 9:
325, 1883.

/ Tschirsch, Das Kupfer vom Standpunkte der gerichtlichen Chemie, Toxicologie
und Hygiene, Stuttgart, 1893.

Q Spraying Fruits for Insect Pests and Fungous Diseases, with a Special Consider-
ation of the Subject in Its Relation to the Public Health. TJ. S. Department of
Agriculture, Farmers' Bulletin No. 7, 1892. See also Bull. No. 6, Div. Veg. Path.,
U. S. Dept. Agric.

22 METHOD OF DESTROYING ALG.E IN WATER SUPPLIES.

single instance was given of injury to health resulting from the daily
absorption of a small quantity of copper. On the other hand, many
instances were cited where foods containing copper in considerable
amounts were used without producing any harmful effect whatever.
It should be noted also that the law prohibiting the use of copper in
regreening fruits was repealed by the French authorities after the dis-
cussion before the Belgian Academy.

According to Thiemann-Gartner,ff chronic copper poisoning has
never been proved. The supposed copper colic was discussed by
Burcq& before the Congres Internationale d'Hygiene in 1878, and
declared by him to have no existence; he even went so far as to assert
an immunity against cholera for the workers in copper during various
epidemics at Paris, Toulon, Marseilles, and elsewhere, but this state-
ment he afterwards modified with reference to the epidemic of 1832.
The good health of copper workers is also noted by Houles and
Pietra-Santa,c though they do not claim for them immunity from
typhoid and cholera. Gautier^ states that persons working in dye
factories, where the hands, faces, and even hair were colored green by
copper, were pl^sically unaffected, which is true also of copper
turners, who remain apparently in the best of health although con-
stantly in an atmosphere highly charged with copper dust.

A considerable number of experiments have been made to determine
the effect of copper upon man when taken into the intestinal tract.
For fourteen months Galippe e and his family used food cooked and
cooled in copper vessels, the amount of copper present in the food
being sufficient to be easily determined. Robert's experiments^ show
that a 60-kg. man can take 1 gram of copper per day with perfect safety.
From his own results Lehmann^ considers that copper to the amount
of 0.1 gram in vegetables may produce bad taste, nausea, possibly
colic and diarrhea, but nothing more serious. He has himself found
peas containing as much as 630 mg. of copper per kilogram not dis-
tasteful, and 200 mg. consumed at a single meal was without effect.
A very careful and thorough series of tests have shown that some
individuals, at least, can take copper even to the amount of 400 to
500 mg. daily for weeks without detriment to their health.

Tschirsch^ finds that 0.01 to 0.02 of copper (0.039 to 0.078 of copper
sulphate) in dilute form have no effect; 0.05 to 0.2 causes only vomit-
ing and diarrhea.

« Thiemann-Gartner, Handbuch und Beurtheilung der Untersuchung der Wasser,
Braunschweig, 1895.

&Burcq, Congres Internationale d' Hygiene, 1: 529, 1878.

« Houles and Pietra-Santa, Journal de Pharmacie et Chimie, 5th Ser., 9: 303.

d Gautier, Le Cuivre et le Plomb, Paris. 1883.

e Galippe, Compt. Rend., 84: 718.

/Kobert, Lehrbuoh der Intoxicationen. (Original not consulted.)

<7Lehmann, Munch. Med. Wochensch., 38: 603.

'' Tschirsch, 1. c.

UNIVERSI!

EFFECT OF COPPER SULPHATE.

The process of regreening legumes is described by Bouchardat and
Gautier, a showing the amount of copper thus introduced into the
vegetables to be too small to produce any injurious effect. The maxi-
mum amount of this metal in regreened peas as given by Gautier b is
125 mg. per kilogram, in connection with which he notes that Chatin
and Personne have given it as 270 mg. According to Gautier, the
amount of copper ordinarily consumed in a full meal is 95 mg.

Lafar c attributes the green color of Lodisan and Parmesan cheese
to the presence of copper, giving the maximum amount for Lodisan
cheese as 215 mg. per kilogram. Chocolate d contains 0.005 to 0.125
gram per kilogram, cafe bourbon e 8 mg. per kilogram, and beef 1 mg.
per kilogram. There is 0.01 gram of copper sulphate in 1£ pounds of
bread/ 0.1 gram of copper oxide has been found in 1 kilogram of pre-
serves, and similar amounts are normally present in a large number of
commodities used for food.

Medicinal uses of copper compounds are cited by Du Moulin.^ He
has prescribed 12 to 15 eg. for scrofulous children, for cases of oph-
thalmia, etc., and found no ill effects. Copper sulphate in doses of 40
to 50 eg. for four or five days has proved beneficial to children with
diphtheria.

Summarizing from a large number of experiments, Bernatzik^ con-
cludes as follows: After entering the stomach only small quantities of
copper are absorbed by the blood, and toxic action occurs only when
the necessary amount can accumulate in the circulation. Silver,
copper, and zinc have almost the same medicinal properties, the dif-
ference being of degree rather than kind. They differ markedly from
other heavy metals, having no harmful effects upon the tissues, and
producing no fatal functional injuries; hence they are not poisons in
the same sense as are lead, mercury, arsenic, antimony, and phos-
phorus. Moreover, in the case of copper, after suspension of the
dose the injured functions return to the normal.

It is evident that there is still a considerable difference of opinion
among eminent authorities as to the exact amount of copper which
may be injurious, but as a very conservative limit we may accept 0.02
gram as the amount that may with safety be absorbed daily. Accord-
ing to Merck's Index, the National Dispensatory, and the United
States Dispensatory, the dose of copper sulphate for tonic and astrin-

« Bouchardat and Gautier, Congres Internationale d' Hygiene, 5: 486.
6 Gautier, 1. c.

c Lafar, Technical Mycology, 159.
<i Duclaux, Bull, de la Soc. Chim. de Paris, 16: 35.
«Sargeau, Jour, de Pharm., 18: 219, 654; 16: 507.
/Tschirsch, 1. c.

ffDn Moulin, Journal de Pharmacie et Chimie, 13: 189.

^Bernatzik, Encyclop. d. ges. Medicin., 11: 429; Encyclop. d. ges. Heilkunde,
11: 429.

24 METHOD OF DESTROYING ALG^E IN WATER SUPPLIED.

gent purposes is one-fourth grain, or 0.016 gram; as an emetic, a dose
of live grains, or 0.33 gram. Thus it is seen that even if the maximum
concentration of copper sulphate necessary to destroy algae in reser-
voirs were maintained indefinitely, the total absorption from daily use
would be very far below an amount that could produce the least
unpleasant effect. Taking a dilution of one to one million, which in
all cases would be sufficient to prevent the growth of a polluting algal
form, it would be necessary to drink something over twenty quarts of
water a da}7 before an amount which is universally recognized as
harmless would be introduced into the system, while more than fifty
quarts would have to be consumed before there would be danger of
producing an unpleasant or undesirable effect. As will be seen from
the preceding tables the use of copper sulphate at this maximum
strength of one to one million would need to be resorted to only in
extreme cases, and for a very short length of time, for, the reservoir
once entirely free from the organisms, a very much weaker solution
would be sufficient should any further application be necessary.

Perhaps the strongest argument in favor of using a chemical treat-
ment of this kind is that even though enough copper should be added
to a reservoir to make a one-millionth solution, nothing like this
amount would appear in the water distributed. A very large percent-
age of the copper is combined with the alga3 and precipitated in other
ways, so that practically none would remain in solution after the first
few hours. a Samples of water taken from a reservoir treated with
sufficient copper sulphate to make a solution of one to one million,
failed to show any reaction for copper after twenty-four hours,
although all the alga1 were killed. It is believed that the process used
of evaporating down the original quantity and testing by the delicate
potassium ferro-cyanide method would certainly have detected copper
had it been present in the proportion of one to fifty million. Other
tests were made by different chemists, but always with negative results.

In addition to the use of copper sulphate in reservoirs containing
water to be used for domestic purposes, there are possibilities of its
application in treating irrigation reservoirs, small pleasure lakes, fish
ponds, oyster beds, etc. Here it may often be desirable to exceed the
strength of solution that would represent the maximum required in a
municipal water supply. This would be done not only to kill all the
algae, but to destroy or drive away reptiles and other pests, leaving
the water perfectly clear and clean. The use of some such method
for the destruction of mosquito larva? also seems worthy of attention.
The mere removal of the great mass of algal growths in stagnant pools
undoubtedly reduces the number of larvre by destroying this source

a Adsorption, according to True and Ogilvie (Science, N. S., 19: 421), would materi-
ally reduce the quantity of copper in solution. See also Bull. No. 9, Veg. Phys. and
Path., U. S. Dept. Agric.

METHOD OF APPLYING THE COPPER SULPHATE. 25

of their food and depriving them of protection from fish and other
enemies. This is probably the explanation of the reported0 decrease
in the number of mosquito larva? after spraying a lily pond with
Bordeaux mixture, although it is possible that the strength of the
solution r«^d may have been partly responsible for their death. It
is belie vi that it will not be impracticable to use the amounts of
copper si ;hate necessary to actually destroy such larva?. Certainly
this method if effective offers considerable advantages over any now in
use, and it should be thoroughly tested. Cooperative experiments
are now under way with the Bureau of Entomology to determine the
strength of solution necessary to kill larvae of different species and ages
under various conditions.

METHOD OF APPLYING THE COPPER SULPHATE.

The method of introducing the copper sulphate into a water supply is
extremely simple. Though aiw plan will suffice which distributes the
copper thoroughly, the one recommended and used b}T the Department
of Agriculture is as follows: Place the required number of pounds of
copper sulphate in a coarse bag — gunny-sack or some equally loose
mesh — and, attaching this to the stern of a rowboat near the surface
of the water, row slowly back and forth over the reservoir, on each
trip keeping the boat within 10 to 20 feet of the previous path. In
this manner about 100 pounds of copper sulphate can be distrib-
uted in one hour. By increasing the number of boats, and, in the
case of very deep reservoirs, hanging two or three bags to each
boat, the treatment of even a large reservoir may^ be accomplished in
from four to six hours. It is necessary, of course, to reduce as much
as possible the time required for applying the copper, so that for
immense supplies with a capacity of several billion gallons it would
probabl}T be desirable to use a launch, carrying long projecting spars
to which could be attached bags each containing several hundred
pounds of copper sulphate.

In waters that have a comparatively high percentage of organic
acid it is sometimes advisable to add a sufficient amount of lime or
some alkali hydrate to precipitate the copper. The necessity for this
will never occur in a limestone region, as in this case there will always
be enough calcium hydrate or carbonate to cause the desired precipita-
tion. The precipitation of copper does not mean the destruction of
its toxicity, for experiments conducted in this laboratory have con-
firmed Rumm's * results that the insoluble salts of copper, such as the
hydrate, carbonate, and phosphate, are toxic only if they are in con-
tact with the cell, but are highly toxic in that case. In this connection
it should be mentioned that Hedrick0 has described a method for con-

«Hedrick, Gardening, 11: 295. & Rumm, 1. c.

26 METHOD OF DESTKOYIHG ALGJE IK WATEK SUPPLIES.

trolling the growth of algal scum in lily ponds by the use of Bordeaux
mixture which seems to have been temporarily effective. However,
the impracticability of using such a mixture is apparent for the
destruction of microscopic algae distributed through a reservoir or a
lake containing millions of gallons.

PRACTICAL TESTS OF THE METHOD.

WATER-CRESS BEDS.

The first practical test of the treatment of water for the purpose of
killing out extensive growths of algae was made in the fall of 1901 near
Ben, Va., in connection with the cultivation of water cress for market.
Water cress is grown there, as well as in other parts of the country,
in large quantities during the winter, it being a valuable crop at that
season of the year. The cress is confined in beds made by construct-
ing dams across a small stream, which maintains a water level not too
high for the growth of the plants and yet permits flooding when there
is danger of a freeze. In the locality where the experiments were
carried on the water was obtained from a thermal spring with a tem-
perature the year around of about 70° F. Such a temperature was
particularly favorable to the development of Spirogyra and similar fila-
mentous algae, so that when the cress was freshly cut they frequently
increased to such an extent as to completely smother out a large part
of the young and tender plants. The only known remedy under such
conditions was to rake out the water cress and algae and reset the entire
bed. This was an expensive method, however, besides being success-
ful only about half the time. Consequently, it was very desirable to
devise some means of preventing the growth of the algae without
injuring the water cress, and the treatment by means of copper sug-
gested itself. At first a strong solution of copper sulphate was used,
spraying it on the algal covered surface of the beds, but this only
destroyed the few filaments with which the copper came in contact,
the large mass of algae being practically unaffected. The method of
applying the copper by means of dissolving it directly in the beds was
next tried, and the success of the treatment was almost immediately
evident. In this case the amount of copper added was about equal to
a strength of 1 to 50,000,000 parts of water, but it is probable that by
the time it reached most of the Spirogyra it was considerably
weakened, as it was impossible to prevent a slight current of fresh
water from passing through the beds at all times.

The success of the copper treatment for eradicating algae from cress
beds has been thoroughly demonstrated, and there is no reason why
growers should have trouble from this cause in the future. The strength
of the solution used for killing the algae is so very much weaker than
that which might affect the cress that there is no possible danger of

PRACTICAL TESTS OF THE METHOD. 27

injuring1 the latter if the solution is used by anyone capable of observ-
ing ordinary care. The question of how long a treatment is effective
must, of course, depend upon conditions, but it is believed that the
application of the proper amount of copper once or twice a year will
in most cases be sufficient to keep down any algal pest. The manager
of the Virginia Cress Company writes, under date of April 12, 1904:

The "moss" has given me no trouble at all this winter. In fact I have for six
months only had to resort to the copper sulphate once. * * * All the conditions
were favorable last fall and early winter for a riot of "moss," but it did not appear
at all until just a few days ago, and then yielded to treatment much more readily
than it did when I first began to use the copper.

WATER RESERVOIRS.

The successful elimination of algae from the cress beds of the South,
under conditions which were particularly favorable to the growth of
these pests, made it desirable that experiments be inaugurated calcu-
lated to demonstrate the possibility of ridding water reservoirs of the
disagreeable odors and tastes caused by similar organisms. While it
was realized that the popular prejudice against any chemical treat-
ment of drinking water was strong, it was believed that the very weak
solution, together with the very rapid disappearance of the salt added,
would not render it a prohibitive method when applied under the
direction of the proper authorities. It was also found that consumers
of a water which possessed a disgusting odor and taste were not so
prejudiced against the use of even a chemical method of extermina-
tion, provided it could be proved that no bodily harm would result.

In the spring of 1903 there was brought to the notice of the Depart-
ment the supply of a water company in Kentucky, which promised to
furnish a most satisfactory test. Ever since the construction of their
reservoir it had given off an unpleasant odor. For the first two sea-
sons this was supposed to be due to decaying vegetation, but later
years demonstrated the well-known "pigpen" odor due to algae, and
this increased from year to year until it was almost unbearable.

In July, 1903, when the trial was begun, the microscopical examina-
tion demonstrated an average of—

Anabsena per cc. . 7, 400

Clathrocystis do 1,100

Eudorina % do 200

There were about 25,000,000 gallons of water in the reservoir at the
time of the experiment, and on account of the great number of blue-
green algae present it was decided to apply the copper at a strength of
1 to 4,000,000. About 50 pounds of copper sulphate was accordingly
placed in a coarse sack and this, attached to a boat, was dragged over
the surface of the reservoir, giving especial attention to the region
which seemed to contain the greatest number of Andbsena filaments.

28 METHOD OF DESTROYING ALG^J IN WATER SUPPLIES.

The decrease in the number of organisms as the result of this treat-
ment during the next twenty-four hours was very decided. In two
days the surface was clear and the water had lost its blue-green color,
becoming brown, due to the dead organisms held in suspension.
There was a slight increase in odor during the first two days after
treatment, but this was followed by a gradual subsidence until it had
entirely disappeared, not to appear again that season. The following
list of counts made from surface examinations at one station illus-
trates what went on throughout the reservoir, and shows the almost
immediate effect of a 1 to 4,000,000 solution of copper sulphate upon
the number of filaments of Anabdena flos-aquaa. The treatment was
made July 9.

Filaments per
cubic centimeter.

July 6 3, 400

July 10 54

July 11 8

July 13 0

July 15 0

July 20 0

It remains to be seen what the condition will be during the coming
summer, but it is believed it can never be any worse than at the
time of treatment, and it is reasonable to suppose that there will be
considerably fewer organisms this }7ear than last. Even though an
annual treatment of the reservoir prove necessary, involving a cost of
from $25 to $50, the alreadj^ great improvement in the quality of the
water will certainly make it justifiable.

Other experiments of a similar character were carried on in different
parts of the country with reservoirs of a capacity of from 10^000,000
to 600,000,000 gallons. While the results were all favorable, it is
deemed best not to publish any detailed account until the effect of the
treatment can be followed through another season. The summer of
1903 was cold and wet, and in some cases the decrease in the number of
organisms may have been due to these factors. However, the several
instances of the very sudden and rapid disappearance of forms which
were present in tremendous quantity, without any reappearance,
indicated that the treatment was most effective. Those in charge of
these water supplies reported that they were well satisfied with the
result.

EFFECT OF COPPER, UPON PATHOGENIC BACTERIA.

TYPHOID.

The value of copper sulphate as an agent for the destruction of
algae polluting reservoirs suggests its use in cases where the organism
is pathogenic. Since this salt is fatal to the algal growths, it seemed

EFFECT OF" COPPER UPON PATHOGENIC BACTERIA. 29

probable that it would also destroy bacteria, and that cholera germs
and typhoid germs might succumb to its action.

The sterilization of public water supplies by chemical means has so
far seemed an impossibility. Nearly every known substance has been
tested, but the high concentrations required to produce the desired
effect, the extreme toxicity of the agents, their cost, or the difficulty
of application, have eliminated all but copper sulphate as a possibility
for the present purpose. According to Semmer and Krajewski,a a
1 to 160 solution of this salt will inhibit action in infected blood, and
septic bacteria can be destroyed with a 10 per cent solution. Bolton b
says that 1 to 500 is toxic, but 1 to 1,000 permits the growth of cholera;
1 to 200 and 1 to 500, respectively, produce the same results with
typhoid, and some of the spore-bearing forms are unaffected at 2 per
cent. Green c gives 2J per cent as the amount necessary to kill
typhoid in two to twenty-four hours, and finds cholera only slightly
less sensitive. Israel and Klingman,^ however, find that almost
infinitesimal amounts of copper in colloidal solution are fatal to
typhoid, cholera, and Bacillus coli. There is considerable literature
upon the use of copper sulphate as a disinfectant for clothing, bed-
ding, cesspools, etc., but it is not necessar3T to review it at this place.
Sternberg^ found that its germicide power was decidedly superior to
the corresponding salt of iron and zinc, and demonstrated that it
destroyed micrococci from the pus of an acute abscess in the propor-
tion of 1 to 200. He says, "This agent (ciipric sulphate), then, is a
valuable germicide and may be safely recommended for the disinfec-
tion of material not containing spores.''

The high percentage of copper sulphate given by most of these
authorities seems to preclude the idea of its practical use for the pur-
pose desired. It should be remembered, however, that these investi-
gators were working for a very different end, namely, to find concen-
trations destructive to bacteria in the presence of large quantities of
albuminoid and fatty matter. Experiments conducted under similar
circumstances have confirmed the above results, but the conditions
obtaining in public water supplies are widely different. Here the
amount of albuminoid matter is so small that the death point of the
typhoid or cholera organism is lowered tremendously and very dilute
solutions of copper are shown to be toxic. The tabulated results on
the succeeding pages demonstrate this fact.

"Semmer and Krajewski, Arch. f. exp. Path. u. Pharmakol., 14: 139.
b Bolton, Rep. of Com. on Disinfectants, Am. Pub. Health Assn., 1888, p. 153.
c Green, Zeit. fur Hyg., 13: 495.
<* Israel and Klingman, Virchon's Archiv., 147: 293.

^Sternberg, Rep. Com. Disinfection, Am. Pub. Health Assn., 1888, p. 38. See also
Infection and Immunity, New York and London, 1903.

30 METHOD OF DESTROYING ALG.E IN WATEK SUPPLIES.

•Effect of copper sulphate upon Bacillus typhi at different temperatures. «
[Determination made in tubes of bouillon. + indicates growth after 48 hours' incubation; - indi-
cates no growth.]

Duration of exposure to action of copper
sulphate.

I

Tempera-
ture.

Check.

1 part cop-
per sul-
phate to
100,000
parts of
water.

1 part cop-
per sul-
phate to
200,000
parts of
water.

1 part cop-
per sul-
phate to
500,000
parts of
water.

f 38

+

+

+

28

+

+

+

+

23.5

_j_

_j.

-1-

-j.

14

+

+

+

+

4

+

+

+

+

38

+

-

+

+

28

+

-

+

+

4 hours ....

23.5

.j.

(?)

_l_

.(-

14

+

+

+

+

4

+

+

+

+

38

+

-

-

+

28

+

-

-f

+

6 hours

23.5

_^_

._

_l_

+

14

+

+

+

+

4

+

-f

+

+

38

+

-

-

+

28

+

-

+

+

12 hours

23.5

•

_

_!_

_l_

14

+

—

+

+

4

+

(?)

+

+

a Experiment conducted in test tubes, each containing 5 cc. of sterilized water, portions of which
had been previously treated with the desired amounts of copper sulphate. All tubes inoculated with
a 3 mm. loop of a 24-hour culture of B. typhi.

Effect of copper sulphate upon Bacillus typhi cultures of various ages.a

[Determination made in tubes of bouillon. + indicates growth after 48 hours' incubation; — indi-
cates no growth.]

Duration of exposure to ac-
tion of solution of 1 part
copper sulphate to 100,000
parts of water.

Culture 36
hours
old.

Culture 24
hours
old.

Culture 18
hours
old.

Culture 12
hours
old.

Culture 6
hours
old.

Culture 3
hours
old.

+

+

-t-

-j-

6 hours

(?)

__

9 hours

_

_

« Experiment conducted in test tubes each con taining 5 cc. of sterilized water, portions of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a culture of B. tyjthi of the proper age.

Effect of copper sulphate on Bacillus typhi at different temperatures.a
[Determination made in Petri dishes.]

Duration of exposure to action of
copper sulphate.

Tempera-
ture.

Check.

One part
copper sul-
phate to
100,000 parts
of water.

One part
copper sul-
phate to
200,000 parts
of water.

One part
copper sul-
phate to
500,000 parts
of water.

2 hours

0 C.
5

Colonies.
720

Colonies.
315

Colonies.
1 440

Colonies.
894

2 hours

38

1 260

o

312

917

5 hours .

5

155

115

495

278

5 hours

38

37

O

g

21

a Experiment conducted in test tubes each containing 5 co. of sterilized water, portions of which
had been previously treated with the proper amounts of copper sulphate. All tubes inoculated with
a 3 mm. loop of an 18-hour culture of B. typhi.

EFFECT OF COPPER UPON PATHOGENIC BACTERIA.

31

Effect of copper sulphate upon Bacillus typhi at room temperature.**
[Determination made in Petri dishes.]

Duration of exposure
to action of copper
sulphate.

Check.

One part copper sulphate to—

100,000 parts
water.

200,000 parts
water.

500,000 parts
water.

1,000,000 parts
water.

5,000,000 parts
water.

5 hour

1,650
1,836
1,566
1,485
999
1,134
1,080
783
270
297

Colonies.
5,481
918
1,026
864
243
180
156
108
0
0

Colonies.
2,376
2,106
1,242
1,296
1,620
1,161
783
972
72
14

Colonies.
2,754
2,403
1,323
2,835
1,485
1,620
918
1,998
405
42

Colonies.
2,646
1,377
2,673
2,430
2,727
1,782
2,079
1,836
324
243

Colonies.
3,645
1,756
2,808
3,024
2,106
756
1,242
1,458
459
405

1 hour

U hours

2 hours

2J hours

3 hours

3f hours

4 hours

8 hours

12 hours

« Experiment conducted in test tubes each containing 5 cc. of sterilized water, portions of which
had been previously treated with the desired amounts of copper sulphate. All tubes inoculated with
a 3 mm. loop of an 18-hour culture of B. typhi.

Effect of copper sulphate upon Bacillus typhi at room temperature."
[Determination made in Petri dishes.]

Duration of expo-
sure to action of
copper sulphate.

No. 1. Check.

No. 2. One part
copper sul-
phate to
200,000 parts
water.

No. 3. One part
copper sul-
phate to
100,000 parts
water.

No. 4. One part
copper sulphate
to 50,000 parts
water.

No. 5. One part
copper sul-
phate to
100,000 parts
water.

Colonies.

Colonies.

Colonies.

Colonies.

Colonies.

ft

4

3

s

Q

u

•F

&

li
I

-

*o
-3

§

c>

1

f*

£,

5^-

3

"o

~.

0

f

r

>>
It

"o
g

«

If

!•*

T. •

.= .=

— z.

i

as

'O

1

C)

1
s*

0 hour

f 144

\ 792
f 14, 634
116,212
f 954
5H
(24,300
119,400
J20,484
|19, 674
f 6,156
121,600

4
2
2
0
0
3
2
0
0
0
0
0

5
4
7
0
2
31
8
0
0
0
33
0

108
90
11
126
0
0
0
0
0
0
0
0

2
1
0
0
t)
0

1
1

0
0
0

0

7
4
5
2
1
0
1
5
2
0
0
0

3
198
72
6
0
0
0
0
0
0
0
0

1
. 1
3
0
0
0
0
0
0
0
0
0

4
5
4
0
0
0

1

0

1

3
0
0

3,672
5,742
0
4
0
0
0
0
0
0
0
0

0

1

0
0
0
0
0
0
0

6

0
0

0

1

0

1

0
3
0
3
0
2
0
0

234
306
6
4
0
0
0
0
0
0
0
0

0
0
0

1

2
0
0

1

0

1

0
0

5
0
. 0
0
0
0
0

1

0
0
0
0

3 hours

4 hours

6 hours

8 hours ... .

12 hours

a Experiment conducted in 12-liter aquaria. No. 1 was untreated; copper sulphate was added to
Nos. 2, 3, 4, and 5. Three cubic centimeters of a mixture of cultures of B. typhi were added to each
jar 18 hours before treating. All small nonliquifying colonies counted as typnoid.

28480— No. 64—04 3

32

METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

Effect of copper sulphate upon Bacillus typhi at low temperature.0
[Determination made in Petri dishes.]

Duration of exposure to action of copper sulphate.

Tempera-
ture.

Check.

One part
copper to
100,000
parts water.

3 hours -

0 C.
5

Colonies.

2,187

Colonies.
1,944

6 hours

5

2 646

881

9 hours

5

1,026

702

12 hours

5

351

98

24 hours

5

37

o

a Experiment conducted in test tubes each containing 5 cc. of sterilized water, part of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a culture of B. typhi of the proper age.

Effect of copper sulphate upon Bacillus coli cultures of various ages. a

[Determination made in tubes of bouillon. + indicates growth after 48 hours' incubation; — indi-
cates no growth.]

Duration of exposure to
action of solution of 1 part
copper sulphate to 100,000
parts water.

Culture 36
hours old.

Culture 24
hours old.

Culture 18
hours old.

Culture 12
hours old.

Culture 6
hours old.

Culture 3
hours old.

3 hours

+

+

+

+

+

6 hours

_•

+

9 hours

+

a Experiment conducted in test tubes each containing 5 cc. of sterilized water, part of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a culture of B. coli of the proper age.

Effect of copper sulphate upon Bacillus coli at different temperatures.a

[Determination made in tubes of bouillon. + indicates growth after 48 hours' incubation; — indi-
cates no growth.]

Duration of exposure to action of
copper sulphate.

Tempera-
ture.

Check.

One part copper sulphate to —

100,000 parts
water.

200,000 parts
water.

500,000 parts
water.

0 C.

38

+

+

+

+

28

+

+

+

+

2 hours

23.5

+

+

+

+

14

+

+

+

+

4

+

+

+

+

38

+

-

+

+

28

+

+

+

+

4 hours

23.5

_(-

-{.

-j-

+

*

14

+

+

+

4

+

+

+

+

38

+

-

+

+

28

+

+

+

+

6 hours

23.5

_l_

+

+

-f-

• 14

+

+

+

+

4

+

+

+

+

a Experiment conducted in test tubes each containing 5 cc. sterilized water, portions of which had
been previously treated with the desired amounts of copper sulphate. All tubes inoculated with a
3-mm. loop of a 24-hour culture of B. coli.

EFFECT OF COPPER UPON PATHOGENIC BACTERIA.

33

Effect of copper sulphate upon Bacillus coli at room temperature. a
[Determination made in Petri dishes.]

Duration of expo-
sure to action of
copper sulphate.

Check.

1 part copper sulphate to—

100,000 parts
of water.

200,000 parts
of water.

500,000 parts
of water.

1,000,000 parts
of water.

5,000,000 parts
of water.

i hour

Colonies.
3,888
3,456
2,592
2,079
3,969
2,457
1,666
1,323
1,107
297

Colonies.
5,697
2,295
2,565
1,971
2,835
1,701
1,404
675
96
5

Colonies.
4,455
1,755
1,755
3,429
2,295
1,242
2,295
1,593
459
43

Colonies.
8,937
2,700
2,403
1,890
3,456
3,834
1,431
2,403
1,026
366

Colonies.
5,490
3,483
1,377
3,267
2,214
2,106
2,025
1,674
513
513

Colonies.
6,426
2,160
1,873
3,942
2,349
3,078
3,240
1,836
1,728
891

1 hour

H hours

2 hours

2£ hours

3 hours

3£ hours

4 hours

8 hours . .

12 hours

« Experiment conducted in test tubes, each containing 5 cc. of sterilized water, portions of which
had been previously treated with the desired amounts of copper sulphate. All tubes inoculated with
a 3 mm. loop of an 18-hour culture of B. coli.

Effect of copper itulphale upon Bacillus coli at low temperature.a
[Determination made in Petri dishes.]

Duration of exposure to action of copper sulphate.

Temper-
ature.

Check.

1 part cop-
per to 100,-
000 parts
water.

3 hours

°a

5

Colonies.
2 700

Colonies.
2 673

6 hours .

5

3 591

1 620

9 hours .

5

2,403

1,215

12 hours

2 106

1 431-

a Experiment conducted in test tubes each containing 5 cc. of sterilized water, part of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a culture of B. coli of the proper age.

Effect oj copper sulphate upon paracolon cultures of various ages. a

[Determination made in tubes of bouillon. + indicates growth after 48 hours' incubation; —indi-
cates no growth.]

Duration of exposure to ac-
tion of solution of 1 part
copper sulphate to 100,000
parts of water.

Culture 36
hours old.

Culture 24
hours old.

Culture 18
hours old.

Culture 12
hours old.

Culture 6
hours old.

Culture 3
hours old.

3 hours

?

6 hours

?

9 hours

a Experiment conducted in test tubes each containing 5 cc. of sterilized water, part of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a culture of paracolon of the proper age.

These tables show that Bacillus typhi is more sensitive to copper
sulphate than is coli, that the para group are about equally sensitive,
and that temperature has a very important bearing on the toxicity of

84 METHOD OF DESTROYING ALGJE IN WATER SUPPLIES.

the copper in solution. At room temperature, which is near the tem-
perature of a reservoir in summer, a dilution of 1 to 100,000 is fatal to
typli i in three to five hours; at 5° it requires twenty-four hours for
complete destruction.

The results obtained were checked in three ways:

(1) Five cubic centimeters of each of the solutions to be tested,
made up with filtered hydrant water and check tubes of the same
water, were sterilized in test tubes. To each of these -was transferred
one 3-mm. loop of a bouillon culture of the bacillus. After the proper
exposure, a 3-mm. loop of the inoculated water from each tube was
transferred to a sterile bouillon tube with a corresponding number.
These bouillon tubes were then incubated forty-six hours at 38°, the
time and concentration of the agent required to prevent growth being
noted.

(2) Instead of transferring to bouillon tubes from the inoculated
water, the transfer was made to gelatine tubes, and plates were poured
in 10-cm. Petri dishes, thus making it possible to estimate the reduc-
tion in the number of bacteria in concentrations not sufficient to pre-
vent growth.

(3) Five 12-liter aquaria, two of which contained a high percentage
of organic matter, also a large quantity of algse and other aquatic
plants, were inoculated, each wdth 3 cubic centimeters of cultures of
Bacillus typhi of different ages, and allowed to stand eighteen hours,
and two poured plates were made from each aquarium, the 3-rnm. loop
being used in all cases. To these aquaria were then added a 1 per
cent solution of copper sulphate in sufficient quantity to produce the
desired concentration. After the proper time had elapsed, another
series of plates was made, this being repeated every two hours for a
period of twelve hours.

The tests were made upon four distinct cultures of Bacillus typhi,
designated respectively Wasserman, Stokes, Say, and Longcope, and
except in the case of the aquaria series, upon Bacillus coll and some
of the para forms. These organisms were obtained from the labora-
tory of H. K. Mulford & Co.

ASIATIC CHOLERA.

The method of procedure in determining the toxic concentration for
Microspira comma (Spirillum choleras) was identical to that .employed
in the case of Bacillus typhi. The tables on the next page show that
the toxic limits of these two pathogenic organisms are very similar and
that Microspira comma is slightly more sensitive to copper sulphate
than is Bacillus typhi. To destroy the cholera germ requires about
three hours in a 1 to 100,COO solution at a temperature above 20°. A
longer exposure or a higher concentration is necessary to produce this
result at lower temperatures.

EFFECT OF COPPER UPON PATHOGENIC BACTERIA.

35

Effect of copper sulphate upon Microspira comma at different temperatures. <*

v

[Determination made in Petri dishes.]

Duration of exposure to action of
copper sulphate.

Tempera-
ture.

Check.

One part copper sulphate to—

100,000 parts
water.

200,000 parts 500,000 parts
water. water.

2 hours

°C.
5
15
26
30.5
5
15
26.5
30.5
5
15
26.5
30.5

Colonies.
1,866
2,500
3,500
4,556
1,533
1,033
1,033
1,466
2,000
3,033
3,600
1,066

Colonies.
1,400
533
3
7
133
21
.0
0
32
9
0
0

Colonies.
566
1,100
100
66
13
72
6
0
9
20
166
0

Colonies.
3,366
1,000
733
1,433
766
95
11
12
700
84
533
90

4 hours

6 hours

a Experiments conducted in test tubes, each containing 5 cc. of sterilized water, portions of which
had been previously treated with the desired amounts of copper sulphate. All tubes inoculated
with a 3 mm. loop of a 14-hour culture of Jf. comma.

Effect of copper sulphate upon Microspira comma at different temperatures.*1

[Determinations made in bullion tubes. + indicates growth after 48 hours' incubation; — indicates

no growth.]

Duration of exposure to action of
copper sulphate.

Tempera-
ture.

Check.

1 part of copper sulphate to —

100,000 parts 200,000 parts
water. water.

500,000 parts
water.

2 hours

°C.

\ 1?
1 24.4

[ 30.5

I 1?
24.4

[ 30.5

f 1?
24.4

I 30.5

+
+
-f
+
4-
+
+
+
+

+

+

+

+
+

+
+

+
+

+
+
+
-f
+
+
+

4 hours

6 hours

a Experiment conducted in test tubes each containing 5 cc. of sterilized water, part of which had
been previously treated with the desired amount of copper sulphate. All tubes inoculated with a
3 mm. loop of a 16-hour culture of M. comma.

It will be seen that the concentration of copper required is consid-
erably greater than the maximum necessary for the destruction of
algae, and would, of course, be injurious to the aquatic animals nor-
mally present in a reservoir if it were allowed to act for any great
length of time. Experiments in this laboratory have demonstrated,
however, that the time necessary to remove Bacillus typhi is from
three to four hours in summer, twent}'-four hours in the coldest
weather, and that under such conditions the solution does not injure fish
and frogs or the common aquatic plants such as Elodea, Myriophyllum,
and Lemna. To remove the copper at the desired time the method

36 METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

suggested in the preceding section in the case of acid and soft waters
may be employed — that is, precipitate the copper by some soluble
hydroxide or carbonate. This somewhat complicates the treatment,
as it will be necessary to determine from the character of the water
the amount of copper necessary to produce a solution of 1 to 100,000,
as well as to estimate how much of the hydroxide or carbonate should
be added. That such work be conducted under the constant and direct
supervision of competent authorities is even more important than when
treating for algal contamination.

COMPARISON OF EFFECT OF OTHER DISINFECTANTS.

A comparison of the efl'ect of copper sulphate with certain other
substances commonly used as disinfectants is instructive, and gives
some idea of the great toxicity of this metal. Mercuric chloride (cor-
rosive sublimate) is slightly more fatal to typhoid and cholera than
copper sulphate acting at a lower temperature and in a shorter length
of time. Carbolic acid, one hundred times as strong as the dilution
found to be effective for copper sulphate, and acting eight times as
long, failed to kill. The same is true of formalin used between fifteen
and twenty times the strength of a 1 to 100,000 solution. Using one
thousand times the amount of citric acid that would be used of copper
sulphate produces death. Thymol is effective in six hours when used
in a solution of 1 to 5,000, and naphthalene is five times weaker.

COLLOIDAL, SOLUTIONS.

The preceding experiments have dealt with copper in solution as the
salt, of some acid. The effect upon water of metallic copper surfaces,
producing the so-called colloidal solution of copper, deserves especial
mention. As Niigeli, Galeotti, and Israel and Klingman have abun-
dantly demonstrated, the slight amounts of copper thus brought into
solution are highly toxic to many forms of algae and bacteria.

The experiments carried on in this laboratory^ show that it is
undoubtedly possible to exterminate Uroglena and some forms of
Spirogyra by suspending in the water copper foil sufficient to give
an area of about 1 sq. cm. to each 100 cc. of water. This would not
be a practicable method of treating a reservoir, but it suggests the
possibility of sheet copper being used as a preventive of pollution.
By suspending large sheets of this metal at the intake of a reservoir,
it is probable that conditions would be rendered sufficiently antago-
nistic to algal growth to maintain the sterility of a reservoir after
it had once been thoroughly cleansed of polluting forms. It would,
of course, be necessary to keep such copper sheets clean in order to
prevent a reduction of the toxic action due to the formation of an
insoluble or slimy coating on its surface. It is possible that some

COLLOIDAL SOLUTIONS.

37

electrical method may be perfected for rapidly obtaining a strong
colloidal solution, which frill furnish a more convenient means of
application than that of the crude salt.

In regard to the bacteria causing cholera and typhoid, the impor-
tance of the specific toxic effect of colloidal copper is probably much
greater than with algae. The following tables show the proportions
of the area of copper to the quantity of water and to the time and the
temperature necessary to produce the complete sterilization of water
containing these pathogenic germs:

Effect upon Bacillus typhi of exposure to colloidal solution of copper at room temperature.0

[Determination made in tubes of bouillon. -f indicates growth after 48 hours' inoculation; — indi-
cates no growth.]

Duration of exposure to action of copper.

Check.

15 sq. mm.
copper foil
in 10 cc. of
water.

lOOsq.mm.
copper foil
inlOcc. of
water.

225sq.mm.
copper foil
in 10 cc. of
water.

10 hours.

+

+

-j-

+

16 hours

-)-

+

-)_

20 hours

+

-f-

50 hours

+

-f

a Experiment conducted in test tubes containing 10 cc. each of sterilized water. The copper foil
was sterilized and added immediately before inoculating the tubes with the usual 3 mm. loop of a
24-hour culture of B. typhi. This experiment was duplicated with three separate strains of typhoid
with identical results.

Effect upon Bacillus typhi of exposure to colloidal solution of copper at room temperature.0
[Determination made in Petri dishes.]

Duration of exposure to action of copper.

Check.

1 sq. cm.
copper foil
to 5 cc. of
water.

4 sq. cm.
copper foil
to 5 cc. of
water.

i hour

Colonies.
1 650

Colonies.
2 241

Colonies.
2 025

1 hour

1 836

1 944

2 349

Hhours

1 566

1 620

1 188

2 hours

1 485

1 674

1 188

2i hours

999

675

1 053

3 hours

1 134

972

918

3£ hours

1 080

1 242

621

4 hours

783

837

360

8 hours

270

216

o

12 hours

097

24

o

« Experiment conducted in test tubes, each containing 5 cc. of sterilized water. The copper foil
was sterilized, and added immediately before inoculating the tubes with the usual 3 mm. loop of a
24-hour culture of B. typhi.

38

METHOD OF DESTROYING ALG^E IN- WATER SUPPLIES.

Effect upon Bacillus coli of exposure to colloidal solution of copper at room temperature.®

[Determination made in tubes of bouillon, -f indicates growth after 48 hours' inoculation; — indicates

no growth.]

Duration of exposure to action of copper.

Check.

15 sq. nun-
copper foil
in 10 cc. of
water.

100 sq. mm.
copper foil
in 10 cc. of
water.

225 sq. mm.
copper foil
in 10 cc. of
water.

10 hours

-)-

-j-

-)-

_l-

16 hours

+

+

+

20 hours . .

+

+

+

50 hours

+

4.

+

a Experiment conducted in test tubes containing 10 cc. each of sterilized water. The copper foil was
sterilized and added immediately before inoculating the tubes with the usual 3 mm. loop of a 24-hour
culture of B. coli.

Effect upon Bacillus coli of exposure to colloidal solution of copper at room temperature. a
[Determination made in Petri dishes.]

Duration of exposure to action of copper.

Check.

1 sq. cm.
copper foil
to 5 cc. of
water.

4 sq. cm.
copper foil
to 5 cc. of
water.

£ hour '.

Colonies.
3,888

Colonies.
2, 241

Colonies.
3,024

1 hour

3,456

1,971

2,025

li hours

2,592

1,512

2,754

2 hours

2,079

1,188

1,846

2* hour-!

3,969

1,242

999

3 hours

2,457

1,242

1,593

85 hours

1,566

1, 026

2,727

4 hours

1,323

1,323

810

8 hours

1,107

702

69

12 hours

297

348

0

a Experiment conducted in test tubes, each containing 5 cc. of sterilized water. The copper foil
was sterilized and added immediately before inoculating the tubes with the usual 3-mm. loop of a
24-hour culture of B. coli.

Effect upon par acolon of exposure to collodial solution of copper at room temperature. a

[Determination made in tubes of bouillon. + indicates growth after 48 hours' inoculation; — indi-
cates no growth.]

Duration of exposure to action of copper.

Check.

15 sq. mm.
copper foil
in 10 cc. of
water.

100 sq. mm.
copper foil
in 10 cc. of
water.

225 sq. mm.
copper foil
in 10 cc. of
water.

5 hours

+

-)-

+

10 hours

+

+

+

16 hours

+

+

+

_

20 hours

-f

-j-

50 hours

+

+

a Experiment conducted in test tubes containing 10 cc. each of sterilized water. The copper foil was
sterilized and added immediately before inoculating the tubes with the usual 3mm. loop of a 24-hour
culture ot paracolon. This experiment was duplicated upon another form of paracolon with exactly
the same results. -

COLLOIDAL SOLUTIONS.

Effect upon paratyphoid of exposure to colloidal solution of copper at room temperature. «

[Determination made in tubes of bouillon. + indicates growth after 48 hours' inoculation; —indi-
cates no growth.]

Duration of exposure to action of copper.

Check.

15 sq. mm.
copper toil
in 10 cc. of
water.

100 sq. mm.
copper foil
in 10 cc. of
water.

225 sq. mm.
copper foil
in 10 cc. of
water.

10 hours ....

+

+

+

+

+

+

+

20 hours

+

+

+

-f

a Experiment conducted in test tubes containing 10 cc. each of sterilized water. The copper foil
was sterilized and added immediately before inoculating the tubes with the usual 3 mm. loop of a
24-hour culture of paratyphoid.

Effect upon Microspira comma of colloidal solution of copper at various temperatures. a
[Determination made in Petri dishes.]

Duration of exposure to action of copper.

Tempera-
ture.

Check.

| sq. cm.
copper foil
to5cc.
water.

2 sq. cm.
copper foil
to 5 cc.
water.

0 C.

Colonies.

Colonies.

Colonies.

5

1,866

833

2,500

2 hours

15

2,500

733

2,433

26.5

3,500

4,600

333

30.5

4,556

1,666

533

5

1,533

52

29

4 hours

15

1,033

633

366

26.5

1,033

200

0

30.5

1,466

8

30

5

2,000

700

10

6 hours..

15

3,033

45

17

26.5

3,600

300

0

30.5

1,066

4

8

« Experiments conducted in test tubes, each containing 5 cc. of sterilized water, portions of which
had been previously treated with the desired amounts of copper sulphate. All tubes inoculated with
a 3 mm. loop of a 14-hour culture of M. comma.

It is evident that the amount of surface exposed in any ordinary
copper tank would far exceed the amount demanded for the above
results, and it is likewise certain that after standing from 6 to 8 hours
at room temperature in a clean copper vessel water becomes safe to
drink even though it may have contained cholera and typhoid germs.
It remains to be seen whether or not the application of these facts to
conditions in the Tropics, where cholera is abundant, will be of any
value. It would seem that the . construction of canteens and other
water vessels from copper might serve as an additional safeguard, if
not an actual preventive of this disease, and would prove of consider-
able value where distillation or efficient filtration apparatus is not at
hand.

40 METHOD OF DESTROYING ALGJ3 IN WATER SUPPLIES.

CONCLUSIONS.

It is believed that the foregoing experiments demonstrate the possi-
bility of the use of copper sulphate for the destruction or prevention
of growths of algae in water supplies, and that when used under the
direction of a competent authority, it is the only practicable remedy
for this trouble capable of universal application which has ever been
proposed. It is, of course, probable that with the experience which
must come from a wider opportunity for testing this salt, many
improvements will be made in the practical application of the treat-
ment to large bodies of water. However, it is hoped that the results
already obtained, together with trials now under way, will make it
possible to begin using this method within a short time upon a large
scale throughout the country.

NECESSITY OF KNOWLEDGE OF ORGANISM AND CONDITION IN

RESERVOIR.

It can not be too strongly emphasized, however, that harmless as
the method undoubtedly is under proper control, it must always require
a certain amount of definite knowledge in regard to the condition of
the reservoir before any treatment can be made, even by those
thoroughly able to conduct such an experiment. This is regarded as
a fortunate requisite, since it will tend to prevent the irresponsible or
careless dosing of reservoirs by incompetents, who are occasionally in
charge of water supplies.

Before the amount of copper to be added can possibly be known, it
is absolutely necessary to ascertain the exact character of the organ-
ism causing the trouble. This will make a microscopical examination
of the first importance. Also, the sooner such an examination reveals
the presence of the polluting form, the more effective will be the treat-
ment. If examinations are made at short intervals during the entire
year, it is possible to detect the troublesome forms at their first appear-
ance and by prompt treatment to destroy the algae before the consumer
is aware of any difficulty. The early detection of the algse will also
make a considerable difference in the expense of the treatment, as it
may require fifteen or twenty times as much copper to clean a reser-
voir after the bad odor and taste are evident than it would could the
application have been made before the organism began to rapidly
multiply. In all cases the use of copper as a preventive rather than
a cure is advocated, and this can not be intelligently applied unless
the microscopical examinations are thorough and frequent at the time
of year the trouble is to be anticipated.

On account of the necessity of determining the nature of the organ-
ism and the time of its appearance as nearly as possible, it will become
as imperative for water companies to employ some one competent to

CONCLUSIONS. 41

make these examinations as it now is to have a chemist or bacteriolo-
gist. In fact, in regions where the difficulty from algae is great, the
microscopical examination must take precedence of everything else as
a means of keeping the water palatable and satisfactory to the consumer.
In addition to the character of the organisms and the earliest possi-
ble determination of their appearance, it has already been pointed out
that the chemical constitution, the temperature, and other special con-
ditions of the water are factors in determining the line of treatment.
No specific instructions are given in this bulletin for the amount of
copper sulphate which is to be used for each species of algae which is
known to affect water supplies, because it is impossible to make a defi-
nite statement without a knowledge of the conditions already men
tioned. Each reservoir must be regarded as an individual case, re-
quiring special knowledge and a particular prescription. It is believed
that the public water supplies of this country are worthy of such spe-
cial care, and it would be a matter of regret if the method proposed
here should ever be regarded as a universal panacea to be used by
everyone, regardless of the organism to be eradicated and the condi-
tion of the water.

APPLICATION OF METHOD FOR DESTRUCTION OF PATHOGENIC BACTERIA
NOT DESIGNED TO REPLACE EFFICIENT MEANS OF FILTRATION
ALREADY IN USE.

The use of copper sulphate in clearing polluted reservoirs of patho-
genic bacteria, such as typhoid and cholera, is regarded as incidental
to the main purpose of the investigation. There already exists a most
efficient mean's of preventing the appearance of these organisms in
water supplies, and under no circumstances can it be considered that
the method as described is expected to replace or supersede slow sand
or any other efficient filtration. There are conditions, however, which
sometimes make it desirable to thoroughly sterilize a reservoir, and
under those circumstances the use of . copper sulphate is believed to
offer a new and adequate way of dealing with the difficulty. Expe-
rience has demonstrated the impossibility of compelling consumers of
what may be an infected water to boil it, or observe other precautionary
measures, and the absence of proper filtration plants in a very great
number of cities and towns in this country makes it necessary that
some efficient method for destroying disease germs in water be employed
until the danger from pollution be past. Up to this time no satisfac-
tory and yet harmless method has been known that would become
effective in the course of a very few hours and the cost of which was
in the reach of every community. It is believed that the results of
the experiments upon typhoid and cholera germs described in this
bulletin indicate that it will be possible under competent direction to
employ copper sulphate with perfect safety in any municipal water

42 METHOD OF DESTROYING ALG^E IN WATER SUPPLIES.

reservoir which may have become infected with some nonspore-
forming disease germ. Its application to barnyard tanks and pools
as a preventive of hog cholera may also prove to be of value. Since
the selective toxicity of this salt renders it fatal to pathogenic forms
peculiar to water, while the common saprophytic or beneficial bac-
teria are unaffected, the method is particularly -well adapted for this
purpose.

MEDICINAL USE.

While it is not within the province of this bulletin to discuss or
recommend any line of medical treatment, reference should be made
to the fact that certain eminent practitioners, after reviewing the
results here published, are of the opinion that the use of copper in
cases of typhoid fever and related diseases should be more thoroughly
investigated than it has been heretofore. It was the testimony of sev-
eral that other intestinal troubles, more recently presumed to be due
to the presence of .certain disease germs in drinking water and milk,
had responded most favorably to copper in one form or another.

CONDITIONS UNDER WHICH THE DEPARTMENT OF AGRICULTURE CAN
FURNISH INFORMATION AND ASSISTANCE IN APPLYING THIS METHOD.

The problem of destroying or preventing the growth of algse by the
method devised in the laboratory of plant physiology in water reser-
voirs, lakes, ponds, water-cress beds, and wherever these plants have
become a pest, is one which distinctly comes within the province of
the Department of Agriculture. Definite instructions as to the treat-
ment to be followed will at all times be furnished to the proper author-
ities who may desire assistance, and in so far as the limited facilities
of the laboratory permit, determination will be made of the organisms
causing the trouble. It is earnestly hoped that no tests of the method
described here will be made without first consulting with the Depart-
ment. Those most intimately connected with this work are constantly
gaining information and experience, and this may prove of consider-
able value, besides a saving of expense, to those who have occasion to
exterminate algal pests.

The treatment of water supplies for the destruction of pathogenic
bacteria, or any application of the copper-sulphate method which has
to do with public health, is not contemplated or indeed possible by this
Department. The requests of private individuals or unauthorized
bodies for information or assistance can not be granted. When State
or local boards of health consider that the disinfection of a water sup-
ply is desirable and wish information upon the subject it will be
supplied as'f ully and freely as possible. All experiments of this kind,
however, must be conducted by the board of health, and the Depart-
ment can serve only in the capacity of an adviser.

SUMMARY. 43

COST.

No definite estimate of the cost of the treatment of a reservoir can
be given, because of the special conditions governing each case. It is
evident, however, that the maximum cost of material for exterminating
algae can not exceed 50 to 60 cents per million gallons, and will often
be less than half this amount. The cost for the copper-sulphate
destruction of bacteria will be from $5 to $6 per million gallons, and
where lime or some soluble hydrate is used in addition the cost would
be increased about one-third. The cost of labor necessary to intro-
duce these substances will be slight, since two men can usually treat
from 10,000,000 to 20,000,000 gallons in less than three hours.

SUMMARY.

The importance of maintaining all public water supplies at the
highest degree of purity and wholesomeness is too well recognized to
require any discussion.

The disagreeable odors and tastes so often present in drinking water
are due almost exclusively to algae, although the economic importance
of studying these plants has not been recognized until recent years.

These algal forms are widely distributed, and reservoirs in many
States have been rendered unfit for use by their presence.

The methods now known for preventing or removing the odors and
tastes caused by algae have proved unsatisfactory, either because of
prohibitive expense or failure to accomplish result.

It is therefore desirable that some new, cheap, harmless, and effective
method be devised for ridding reservoirs of these pests.

It has been found that copper sulphate in a dilution so great as to
be colorless, tasteless, and harmless to man, is sufficiently toxic to the
algae to destroy or prevent their appearance.

The mode of application makes this method applicable to reservoirs
of all kinds, pleasure ponds and lakes, fish ponds, oyster beds, water-
cress beds, etc. It is also probable that the method can be used for
the destruction of mosquito larvae.

At ordinary temperatures 1 part of copper sulphate to 100,000 parts
of water destroys typhoid and cholera germs in from three to four
hours. The ease with which the sulphate can then be eliminated from
the water seems to offer a practical method of sterilizing large bodies
of water, when this becomes necessary.

The use of copper sulphate for the prevention of disease is regarded
as incidental and is not designed in any way to supplant efficient pre-
ventive measures now in use. It is believed, however, that up to this
time no such satisfactory means of thoroughly, rapidly, and cheaply
sterilizing a reservoir has been known. Since the selective toxicity of

44 METHOD OF DESTROYING ALG,E IN WATER SUPPLIES.

copper sulphate renders it fatal to pathogenic forms peculiar to water,
while the saprophytic or beneficial bacteria are unaffected, the method
is particularly well adapted for this purpose.

Definite knowledge in regard to what organisms are -present, the
constitution of the water, its temperature, and other important facts
are necessary before it is possible to determine the proper amount of
copper sulphate to be added. A microscopical examination thus
becomes as important as a bacteriological or chemical analysis.

No rule for determining the amount of copper sulphate to be added
can be given. Each body of water must be treated in the light of its
special conditions.

The cost of material for exterminating algae will not exceed 50 to 60
cents per million gallons and will usually be less. The destruction of
pathogenic bacteria requires an expenditure of from $5 to $8 per
million gallons, not including the cost of labor.

O

-iiuied from >ver.]

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V Soft Rot of the Calla Lily. 1904. [In Press. ]

61. The Avocado: Its Propagation, Cultivation, and Marketing. 1904. [In
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