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STUDIES IN ANIMAL AGGREGATIONS : MASS

PROTECTION AGAINST COLLOIDAL


SILVER AMONG GOLDFISHES
W. C. ALLEE AND EDITH S. BOWEN
Whitman Laboratory of Ezperimental Zoology, The University of Chioago

SURVIVAL O F GROUPS VERSUS ISOLATED ANIMALS


Drzewina and Bohn ( '21 et seq.) demonstrated clearly the
protective value of numbers of animals as compared with
single individuals when exposed to equal volumes of colloidal
silver suspension of the same concentration. Allee and
Schuett ('27) confirmed and extended these experiments.
Mass protection from the toxic effects of colloidal silver has
been demonstrated by these workers for a wide range of ani-
mals, including, among others, various Protozoa, planarians,
leeches, ophuroid starfishes, crustaceans, and frog tadpoles.
Drzewina and Bohn originally interpreted their results as
being due primarily to the more rapid production of some
autoprotective substance by the group than by the isolated
individuals, which acted by some other means than the fixa-
tion of the toxic materials. The experiments of Allee and
Schuett indicated that, under the conditions of their experi-
ments, a major part of the protection furnished by the mass
was due to the more rapid adsorption of the toxic colloidal
silver either directly on the surface of the animals or upon
the slime which the animals had shed into the surrounding
medium, but their experiments did not prove conclusively that
the observed protection was due entirely to such adsorption.
The present investigation waa aided by a grant to The University of Chicago
from the Rockefeller Foundation. W e are indebted to Messrs. E. A. Rosenbaum
and Edward Haeniach, of the local Department of Chemistry, for nearly all the
chemical analysea.
185
THE JOURNAL OF EXPIBIYLNTAL ZO6I&QY, VOL. 61. XO. 9
rzEaUABY, 1esa
186 W. C. ALLEE AND EDITH S. BOWEN

Carpenter ('27, '30) approached this problem from a dif-


ferent angle and found good evidence that fishes exposed to
lead nitrate in a processionary series remove a part of the
toxic materials and gradually render the solution less toxic.
Her conclusions are supported by admittedly imperfect
chemical analyses.
sllee ( '28, '29)' working with carefully controlled salt con-
tent, was able to demonstrate a protection for the marine
planarian Procerodes when isolated into fresh water in which
other animals had lived. Drzewina. and Bohn ('28) and
others (see Allee, '31) have produced evidence that mass
protection may be due to bio-electrical phenomena. Recently,
Fowler ( '31) has shown that the mass protection or the lack
of it in the case of Daphnia exposed to toxic concentrations
of various electrolytes is related primarily to the faster pro-
duction of CO, by the group which slows down their rate of
metabolism and produces longer survival for members of the
group if the concentration of the toxic material is relatively
high, and has the opposite result if the concentration of
electrolytes is relatively low. The action in this respect is
in line with the effect of rate of metabolism upon survival
in Child's ( '15) susceptibility experiments.
I n view of the fact that groups of animals may produce
ii protective effect by the adsorption of toxic substances such
as colloidal silver, and of the further fact that other mecha-
nisms for group protection have been demonstrated, it is
important to know whether all the group protection against
colloidal silver can be explained by the fixation of the silver
by adsorption on slime or on other organic materials, or
whether a part of the protection is furnished by other
mechanisms.
Accordingly, experiments were run to test the survival
value of groups of goldfish (Carassius auratus) as compared
with an equal number of fishes isolated into the same volume
and concentration of colloidal silver suspensions. Sample
solutions were then subjected to quantitative analyses to
determine the effect of the grouped and of the isolated fishes
STUDIES IN A N I M A L AGGREGATIONS 187

upon the concentration and distribution of the colloidal silver.


The stock suspensions of colloidal silver used in these experi-
ments were made by dissolving 4 grams of commercial
dextrine in 100 ml. distilled water, and then adding 4 grams
of stick sodium hydroxide. To this alkaline dextrine solu-
tion, 3 grams of silver nitrate dissolved in 20 ml. water were
added. After about thirty minutes, the color of the precipitate
changed from black to reddish-brown. Then 100 ml. of 95
per cent alcohol were added and the mixture was stirred.
After allowing thirty minutes for settling, the supernatant
liquid was decanted and the silver was dispersed by adding
1 liter of water and shaking. The definitive suspensions were
then made by diluting a given number of milliliters of the
stock suspension with distilled water to give approximately
the required concentration. Since the stock suspensions
varied in amount of dispersion-hence in toxicity also-the
actual number of milliliters per liter is not particularly signifi-
cant. The whole supply for a given experiment was made
up at once in a large glass bottle and mixed thoroughly. A
liter of such a suspension was placed in each of the crystalliz-
ing dishes to be used in the experiment. These dishes were
24 cm. in diameter and 7 cm. deep. They were left uncovered
to allow a free exchange of carbon dioxide and oxygen with
the air. This treatment allowed a slight amount of evapora-
tion, but distilled water was added at the end of the experi-
ment to bring each suspension up to the original volume
before analysis.
Goldfish which averaged about 3 cm. in length, exclusive of
the tail, were used as experimental animals. Their physi-
ological condition probably varied, depending upon the length
of time in the laboratory, temperature, etc., but the fishes
used in any one experiment were from the same general stock
and had been under identical conditions for several days
previous to the experiment. Each fish was placed in dis-
tilled water for about three minutes to dissolve electrolytes;
it was removed upon filter-paper and then placed into the
appropriate dish. Although the stock fishes selected for a
188 W. C. G E E AND EDITH S. BOWEN

given experiment were fairly uniform in size and general


behavior, pairs of these fishes, as nearly identical as possible,
were selected, one of which was placed in the dish containing
the group and the other was isolated. Hence the grouped and
the similar number of isolated fishes were as nearly com-
parable as could be selected by the methods used.
The first two experiments were run at room temperatures,
but, beginning with experiment 3, survival tests were carried
on in a room with controlled temperature (72F.) and con-
trolled humidity (70 per cent). Observations were made at
intervals of two and five minutes, respectively, in experiments
1and 2, and at thirty-minute intervals in later experiments.
The gills of the fishes swimming about in the colloidal silver
suspension became coated with a dark substance. At intervals
these coatings were shed and settled to the bottom; there
were noticeably more such in the dish containing the group.
The death point was somewhat difficult to determine exactly,
as the fishes gradually became quiet and all apparent activity
ceased. When no respiratory movements were perceptible
and a fish gave no reaction to pinching the tail with tweezers,
it was considered dead and removed immediately.
Two suspensions in which a single fish had died, the fifth
and sixth of the series of ten in each experiment, were
analyzed for free and for adsorbed silver. These were com-
pared with similar analyses from the suspensions in which
the group had died. Control lots of colloidal silver suspen-
sions which had been standing in crystallizing dishes, but
without fish, were bottled at the same time as the experimental
suspensions and were analyzed for any effect upon the sus-
pension other than that produced by the fishes. The whole
liter of colloidal suspension was kept in each case and was
thoroughly mixed before definitive samples of 200 ml. were
taken for analysis. The dark shreds of mucus with their load
of adsorbed silver largely dispersed upon shaking, so that
the suspensions approached homogeneity.
STUDIES IN A N I M A L AQQREQATIONS 189

Each fish when removed at death was placed in 25 per cent


(by volume) solution of nitric acid for three minutes and
then in distilled water for ten minutes to remove surface
silver. The fish was then placed in 25 per cent nitric acid
for analysis of internal silver, if any; no dependable indica-
tions of internal silver were found. The sample for analysis
was pipetted, dissolved in dilute nitric acid, and made just
alkaline with sodium-hydroxide solution. It was found neces-
sary to avoid excess alkali. The precipitated silver oxide
was dissolved by the addition of a small amount of potassium
cyanide. The solution was then electrolyzed between platinum
electrodes. The suspensions which had contained many fishes
for a considerable period of time, called hereafter strongly
conditioned suspensions, showed a residue which was insolu-
ble in dilute nitric acid, even when warmed. A quantitative
analysis showed it to consist of silver and organic material.
The quantity of silver in this residue was obtained by ignit-
ing it and weighing. A correction was made for the weight of
the filter ash. Some of the very strongly conditioned suspen-
sions contained lumps of organic material and adsorbed silver
which made the obtaining of a representative sample some-
what di5cult and less certain. The analytical procedure
adopted has its most important source of error for inorganic
analyses in the weighings, which should be accurate to within
-+ -0003 gram. The presence of organic materials in some
of these suspensions may invalidate the results to a greater
extent, but the totals check to within .0010gram.
EXPERIMENTAL RESULTS
The biological results obtained in these experiments are
indicated in table 1, which shows in detail the survival of
the different individual fishes which composed the groups,
and the accompanying set of isolated fishes for each of the
three experiments for which complete chemical analyses were
made are given in table 2. Since the fishes are listed in order
of their death, one can follow the progress of the death of the
isolated and grouped fishes. Doing so, we find, for example,
190 W. C. ALLEE AND EDITH S. BOWEN

that when the last isolated animal died, there were three,
seven, and six grouped fishes, respectively, still living. Seven
such experiments were run in all, with varying concentrations
of colloidal silver ; the other four, however, were not subjected
to chemical analyses. Without exception the mean survival
time of the group exceeded that of the accompanying isolated
fishes. The mean survival time f o r the seven sets of ten
isolated fishes was 182 minutes; for the seven accompanying
groups of ten each, 507 minutes. The difference, 325 minutes,
is 64 per cent of the survival time of the group. When ex-
TABLE 1
Surcival time of fishes in collotdal silver suspensions
---__ _ _ _ _ _ __ _ _ ~ _
Experiment ' I I11
-
Temperature 1 ~- 70F.
__
72 F.
ORDER OF DEATH I ~ O ~ T E ID QBOUP IROLATED amUP

45 I 45 150 240
50 I 90 180 390
210 390
210 420
I GO 1 355 210 570
6 157 240 780
7 205 270 780
8 1 100 279 I 90 420 270 810
I I
9
10 1
157
207
280
337 I
130
150 I 470
560
300
450
810
840

amined statistically by Student's method for paired experi-


ments, using percentage differences because of the different
concentrations of colloidal silver in the pairs of experiments,
the probability is found to be less than 0.001. Such results
are according to expectation, since the more slime secreted,
the greater would be the adsorption of colloidal silver. I n
some of the trial experiments where very weak suspensions
were used, the fishes survived for longer periods than those
given above, and it was found that if an individual lived for
much over twenty-four hours, it usually lived for several days.
Results of the chemical analyses made on these suspensions
are given in table 2. These are all the analyses that were
STUDIES IN ANIMAL AWREOATIONS 191

made upon this range of concentrations of colloidal silver.


The amount of silver in each case totals approximately, as
nearly as can be expected with the unavoidable errors in tech-
nique, the amount in the original suspension, indicating that
all of the silver was recovered in the analyses. The amount
of free colloidal silver, the electrolyzed silver of the analy-
ses, is much less in each instance for the group than for the
TABLE a
Besults of chemical analyses for silver in ezperiments I , I I , and 111; figures are
given in grams for 30O-mZ. samples
EXPEXIYENT XI
. -~
BXPEBIYENT I
__ - ~. -
EXPEBIYENT I11
~

BUSPENSION Clectro- Slectro- Electro-


lJZ0d tesidue Total lpad Residue Total Reiiduc Total
nilver silver silver
lyzed
__ __ ___ -__ __ -~ ~

Stock .0117 .0103 .0151 .0156


.0161
Control, .0119 .0109 .0161 .0164
isolated .0168
Control, ,0119 .0114 ,0164 .0163
group .0163
Isolated A .0117 .0107 .0166 .0163
.0160
Isolated B .0119 .0105 .0161 .0159
.0157
Group .0071 .0052 .0123 .0043 .0068 ,0111 .0054 .0109 .0163
.0053 .0115 .0168
Surface A .0002 .oooo .oooo
Surface B .0001 .0001 .oooo
Surface .0002 .0003 .oooo
group ~

accompanying isolated fishes; the difference is found to be in


the residue.
These experiments demonstrate that the increased survival
of the group over that of isolated individuals is accompanied
by a marked decrease in the amount of dispersed colloidal
silver in the suspension which contained the group to be con-
trasted with a slight decrease in the suspensions containing
individual fishes-a finding which strongly suggests that this
decrease is the mechanism of protection for the groQp.
192 W. C. ALLEE AND EDITH S. BOWEN

EFFECT O F VOLUME UPON THE SURVIVAL O F GROUPED AND


ISOLATED FISHES
If it is true that slime production and adsorption thereon
are the protective mechanisms responsible for the longer sur-
vival of the group, this effect should not be present when the
volume and the silver dosage per fish are identical for both
grouped and isolated individuals, as under the simplest con-
ditions the silver adsorbed by the slime secreted by one fish
would be the same whether the fish were isolated or a member
of a group. These relations would not hold if the fishes by
close aggregation could maintain a low silver concentration
in a small portion of the large volume, or if the group stimu-
lates or retards slime secretion or has some other effect, such
as affecting the rate of metabolism, which could result in
differential survival. To test this situation with goldfish a
series of ten paired experiments was run. I n each experi-
ment ten fishes of the same size were used, five of which were
placed together in 1 liter of colloidal silver suspension in a
crystallizing dish, while the other five were placed separately,
each in 200 ml. of suspension in a finger-bowl. Observations
were made at thirty-minute intervals. The results, given in
table 3, show practically no difference in length of survival
time under the two conditions. I n some cases the average
was greater for the grouped, in other cases for the isolated
fishes. The mean difference for all is only four minutes
longer for the grouped fishes. Calculated according to
Students method, there are 79 chances in 100 for as great
variation by random sampling. This is a good indication that
no difference existed with the fifty fishes observed under each
condition.
These experiments clarify the situation by showing that
the enhanced resistance of grouped fish to colloidal silver
demonstrated in the preceding section in which grouped and
isolated fishes were exposed to equal volumes and equal con-
centrations is not due to the more rapid production of slime
or other protective material per individual of the group, but
rather to a direct and simple mass relation. Even so, these
STUDIES IN ANIMAL AQGREQATIONS 193

experiments do not demonstrate that adsorption of the silver


on slime is the sole protective mechanism acting.
ISOLATED FISH PLACED SUCCESSIVELY IN THE SAME SUSPENSION
Earlier workers using a variety of toxic conditions have
reported mass protection where the animals were not present
in the solution at the same time, but where an individual was
TABLE a
Survival of groups v e r m isolated fiehes with volume per indioidwl the same

A. Ieolated fiehes
120 150 60 120 150 150 150 120 120
120 180 120 180 150 240 180 180 150 150
180 180 180 180 240 270 180 180 180 150
210 180 180 210 270 270 180 240 180 210
240 210 210 390 300 300 240 240 240 240
Mean 174- 1 180 I 150 I 216 240 1 246 1 186 1 198 1 174 1 174
B. Groups
1 180 150 150 150 120 ' 60 180 180 150 150
2 180 150 180 180 180 150 180 180 150 150
3 180 180 180 300 210 180 180 180 150 150
4 210 210 210 300 210 210 210 210 180 180
5 330 210 330 390
____-__
240
__
Mean
Mean
difference
216 180 210 264

42 0 60 48 1 1
Average mean diff erenee, -4 minutea. Probability,
198

0 1-12
-.786
I -8

Snapenmion strength, 9.05 per cent.


Buspanuion atrength, 7.5 per cent.

placed in a solution in which one or more animals had previ-


ously died. Ten experiments were run simultaneously to try
this out for goldfishes in colloidal silver suspensions. Five
fishes were placed successively in a liter of suspension in the
crystallizing dishes described previously ; observations were
1

made at fifteen-minute intervals, and as each fish died it was


replaced by another of the same size.
194 W. C. ALLEE AND EDITH S. BOWEN

The results are given in table 4. There is considerable


variation in the survival time of different fishes of the same
size just as there was with all other tests with isolated fishes.
This variation is particularly evident here, where it is seen
that in eleven cases of the fifty tested a fish survived a shorter
time than its immediate predecessor. I n the series as a whole
R distinct tendency toward an increase in survival time is
evident as the experiment proceeded, and, although the mean
difference between any two successive sets is not significant,
the fifth fishes survived on the average seventy-five minutes
TABLE 4
Suroianl in minutes of five isolated fishes placed successively in a liter of 10 per
cent colloidal silver suspension
.
~. ____
EXPERIYENT 1ST FI8R 2 N D FISH 3RD FISH 4l'H FISH 5TR FISH
- -______._ ~ _ _ _ _ _ . __
1 75 105 105 105 195
2 75 150 120 150 120
3 165 195 240 2 70
4 195 165 180 210
5 135 180 240 150
6 150 255 195 195
7 210 165 255 285
8 150 195 285 240
9 180 195 270 285
10 210 255 300 345
~___.-

ILL LV,VVV UA uuba~u~ue


uu ~ A G ~ a. V ~ I I ~ U U L L
L uy uam-
I ~ I I ~ U L L I

pling. These results are to be expected if the protection


is due to the adsorption of silver on the successive slime
secretions, and indicate a progressive lessening of the amount
of colloidal silver present-an indication which is supported
by data to be reported in the following section.
SURVIVAL O F ISOLATED FISHES I N CONDITIONED VERSUS
UNCONDITIONED SUSPENSIONS
I n this part of the work the effect of the conditioning of
the suspension of colloidal silver upon survival time was
tested. The survival time of isolated fishes was ascertained
STUDIES IN ANIMAL AOGREQATIONS 195

in unconditioned suspensions, in conditioned suspensions, and


also in conditioned suspensions to which a double amount of
the colloidal silver had been added.
Preliminary experiments were run with ten fishes in a liter
of colloidal silver suspension to determine the optimum dilu-
tion which would give the best conditioning effect. A 3 per
cent suspension of the colloidal silver stock on hand gave by
chemical analyses the following results for two such trials
run simultaneously after the last of the group had died:
A B
YO. Yg.
Electrolyzed silver, .0017 .oooo
Residue, .0066 .0093
Total silver, .0083 .0093
Original suspension, .0086

For the experiment 30 liters of a 3 per cent colloidal silver


suspension were made up in two large bottles and mixed
thoroughly by pouring back and forth so that the concentra-
tion was the same throughout. Ten goldfishes were placed
into each of twenty dishes containing 1 liter of this suspen-
sion. Ninety-five were removed at death, and at the end of
twenty-four hours the five fishes still living were also re-
moved. The 20 liters of goldfish-conditioned suspensions
were mixed thoroughly by pouring from one bottle to another,
and divided into two equal parts ; to one of these the original
amount of colloidal silver stock was again added. This made
the total concentration 6 per cent, double that of the initial
suspension, provided none had been removed in the condition-
ing process. The experiment was set up with, A) ten separate
liters of unconditioned suspension, B) ten of conditioned
suspension, and, C) ten of conditioned suspension to which
the original amount of colloidal silver had been added again.
A single fish was now placed in each liter. Two complete
samples from each part of the experiment were kept for
analysis.
The results given in table 5 are quite conclusive. The mean
survival time of 2.0 hours in the unconditioned suspension
(A) was increased to 4.5 hours by the conditioned suspensions
196 W. C. ALLEE AND EDITH S. BOWEN

even with double the concentration of colloidal silver ( C ) ,


and when no new colloidal silver was added after the con-
ditioning (B), the survival time was increased to 9.1 hours.
These results lend themselves to statistical analysis by
TABLE 6
Isolated fishes in liters 01 different types of colloidal silver szcspeneione. Stock,
.0086 gram per ZOO ml.
A. 3 PEB OENT
B. 3 PEB CENT 0. 3 Pan arm
UNOONDITIONED CONDITIONED SUSPENSION
COHDITIONED BUSPlNSION - 3 PEB CENT = 6 PEB OENT
SUSPENSION
-~ -___
3urvival :hemica1 analysis lurvival :hemica1 analysis .
n hours ~~~$~~>hemica1analysia n hours
___ __ -
1 2 No.3 12 No. 3 6 No.3
2 3 S,.0062 10 s, .oooo 6 8,.0077
3 2 R,.0003 11 R, .0089 4 R,.0115
4 1.5 T,-0065 6 T,.0089 4 T,.0192
5 3 8 4 (6.7per cent)
6 1 No.6 8 No.6 4 No.6
7 2 5, .0088 11 8,.0002 4 S,.0078
8 1.5 R,.OOOO 9 R,.0087 4.5 R,.0126
9 2 T,.0088 9 T,.0089 3.5 T,.0204
10 2 ~.
7 5
__
(7.1per cent)
Mean I 2.0 I I 9.1 I I 4.5
8 = electrolyzed d v er ; R = reeidue; T = total.

Students method and are shown thereby t o be statistically


significant :
Mean diflarmkce. ProbabQity
HOW#?
A versus B, 7.1 .0136
B versus C, 4.6 .0162
A versus C, 2.5 .0156

The chemical results show that, although there is a decided


color in the 3 per cent conditioned suspension, actually the
free colloidal silver has been entirely removed, probably by
adsorption upon slime. The doubly charged suspension has
less free silver than the unconditioned 3 per cent suspension,
indicating that some of the newly added silver is adsorbed
upon the slime already present. The result under A , line 2
in table 5, giving only 0.0062 for the electrolyzed silver, is
probably incorrect. Due to unavoidable circumstances, it
STUDIES IN ANIMAL AGGREGATIONS 197

was impossible to run a duplicate experiment to check this,


but the results for the single fishes in the first section of the
present paper together with the second set of results under A
leave little doubt as to the results to be expected here.
The possibility of free carbon dioxide furnishing the pro-
tective mechanism, as shown in Fowler's ( '31) work with
Daphnia, is decidedly diminished by this experiment. The
mixing of the conditioned solutions by pouring and shaking
must have given an opportunity for elimination of any carbon
dioxide produced by the group and yet the results show fully
as great protection as in the group and single comparisons.
The adsorption of the toxic silver on slime is again demon-
strated and again apparently accounts for the observed
protection from colloidal silver.
An explanation of the fact that the goldfishes are killed
in the 3 per cent conditioned solution (B), in which, accord-
ing to the chemical analyses, all of the silver has been
removed, is not clear. There are various possibilities. In the
first place, the chemical analyses give the final results of
adsorption after the solutions have stood, sometimes for
several days. These results may indicate a greater amount
of adsorption than actually exists at the close of the experi-
ment. I n the second place, the silver, even after adsorption
upon slime, may still be toxic, especially since the slime
remains in part dispersed. This may produce death, but only
after a definitely longer time than the unadsorbed silver
particles. Finally, the fishes may have received sufficient
initial injury to produce delayed death despite the steadily
decreased toxicity.
pH AS A POSSIBLE FACTOR
Tests showed that the pH of the colloidal suspensions used
varied considerably on both sides of neutrality. Accordingly,
experiments were planned to determine directly the impor-
tance, if any, of the hydrogen-ion concentration in fish sur-
vival in colloidal suspensions. The pH determinations of the
colloidal silver suspensions were made electrically and those
198 W. C. ALLEE AND EDITH S. BOWEN

~ _ ~ _ _ _ _ _ _ ~ _ ~ ~ ~ _ _ _
1
.___

-~ - -
5 PER CENT
SUSPENSION
10 PER CENT
SUSPENSION 1 5 PER CENT
SUSPENSION

'1
~ ~~

PH ~ PH PH
Stock 4.3 9.6 ~ 7.0
Control, isolated 4.4 8.9 ~ 6.8

Isolated A 5.8 ' 9.1 6.6

1
Isolated B 5.8 9.0 6.5
Group 5.6 7.2 6.6
Mean survival 10 isolated (minutes) 249 135 223
Mean survival group of 10 (minutes) 603 1.59 528

_______
7 hours ' 24 hours
_____.
7 hours 24 hours

Control, isolated 8.0 7.5 4.5 4.5


Control, group 8.0 7.5 4.5 4.5
Isolated A 7.5 7.1 4.9 6.1
Isolated B
Group I 7.5
6.9
7.1
7 .O
5.0
6.2
6.1
6.7
-

5 per cent suspensions with an initial pH of 4.3 and of 7.0.


Group protection was also in evidence in the stronger suspen-
sion, which was initially strongly alkaline, though, as was to
he expected, this stronger suspension killed more rapidly and
did not show so great a difference between the survival of
grouped and of isolated fishes. Under these conditions of
high alkalinity there was a decided drift toward neutrality
in control vessels as well as in those containing isolated and
grouped fishes. The changes in this instance were more com-
STUDIES IN ANIMAL AOGIREQATIONS 199

plete for the latter. I n acid suspensions there was a similar


drift toward neutrality which proceeded further in the vessel
with the grouped fishes, although the end point showed rela-
tive equality in the comparable sets. With suspensions which
were originally n'eutral, the drift toward acidity was common
to controls that contained no fishes as well as in the experi-
mental lots.
Since a colloidal silver suspension is a fairly complicated
system the toxicity of which might be affected in various
ways, experiments were performed to find whether an initial
pH of these ranges is toxic per se, apart from any effect of
the colloidal suspension itself. Accordingly, grouped and
isolated fishes were placed in distilled water raised to pH
9.4 by NaOH in one experiment and lowered to pH 4.5 by
HC1 in another. The fishes were apparently quite normal at
the end of twenty-four hours, and the pH results were as
shown in table 7. The same change toward neutrality is evi-
dent here. This is probably due to reactions incident to the
formation of carbonates or t o other buffering by excretory
products.
EXCRETORY PRODUCTS
Fishes give off other products than slime and GO, into the
medium surrounding them. We have been unable to obtain
analyses of such excretions except for certain marine fishes.
Basing estimations on the analyses of twenty-four-hour
samples of urine of such fishes as given by A. No11 ( '24), it
was calculated that a maximum production from ten fishes
averaging 2 grams in weight, which is slightly more than the
fishes used actually weighed, would include per liter : crystal-
line urea, 4 mg. ;NaCl, 6 mg. ;Na,S -9H,O, 3 mg., besides other
compounds not present in appreciable amounts in the analyses
reported by Noll.
Two lots of colloidal silver suspension were made as nearly
alike as possible. The first was kept as a control, the second
received the solution just indicated before final dilution with
distilled water. The effect of such treatment was tested as
recorded in table 8. In experiment 9 five pairs of fishes,

T H E JOURNAL OF EXPERIYENTAL ZO6LOGY, V O L . 61, NO. 2


200 W. C. ALLEE AND EDITH S. BOWEN

equal in size, were isolated, one into the usual colloidal silver,
and the other into the chemically conditioned colloidal silver
suspension. I n experiment 10 ten such pairs were used and
in addition the survival of a group of ten fishes in uncondi-
tioned suspension was tested simultaneously.
TABLE 8
Effect of excretory products upon survival o f goldfish in colloidal silver suspensions
Ii ISOLATED FISJHEB
__-_____
I- QROUPED FISIIES
-
I Unconditioned suspension 1 I
s " ~ p ~ ~ ~ ~ , ~ Unconditioned
r e " * suspension
~______-- I---____- --___
-~
Experiment 9
____________
1 1
210 I 330 1
180 300 1
240 I 330 1
I I
210 240
I 300 1
210 ___I - ~- -

210 I 300

'
________
Experiment 10
_ _ _________ ~ ______ _ _ ~-_____
- ___
150 330 I 150
180 300 I 210
210
I 300 I 240
I
240 I 420 1 330
360 1
240 390
120
I
I 270 i 390
180 1 3 60 I 390
240 i 360 1 450
120 I 300 1 570
_- 150
1 P?
__-,____----I
I 120
I
720
Mean 719 I R A

The fishes in the suspension containing urea, NaC1, and


N h S have a longer survival time than those in a straight
colloidal silver suspension with an average difference of 129
minutes. This gives a probability of less than 0.000001. These
products of excretion act, therefore, as a protective mecha-
nism, possibly by antagonizing the toxic effect of the silver
ions in solution, as NaCl has been shown to do. I n a suspen-
sion made in exactly the same way as the colloidal silver
except for the addition of the AgNOs, the grouped and iso-
STUDIES IN ANIMAL AGQREGATIONS 201

lated fishes were apparently perfectly normal at the end of


twenty-four hours, indicating that the silver is the toxic ele-
ment concerned.
Some of the fishes in the group died before any of those in
the suspension containing urea and inorganic salts, but some
of the group outlived all of the isolated fishes. This occurred
in spite of the fact that a full twenty-four-hour amount of
excretory products was added. This indicates that with the
grouped fishes some mechanism other than the protection by
excretory products is acting, the more so since the mean
survival of the grouped fishes exceeds that for the fishes
isolated into colloidal silver plus excretory products, even
though the group could be expected to excrete only about one-
fourth of the excretory products that were placed in the other
solution at the beginning of the experiment. This supports
the work reported earlier in this paper concerning group
protection as a result of adsorption of the colloidal silver
on slime secreted by the fishes during the progress of the
experiment.
DISCUSSION
As indicated in the introduction, there has been for some
years no need for further experimentation concerning the
possibility of securing mass protection for animals from the
toxic effects of colloidal silver. There has existed a need for
a careful analysis of the mechanism or mechanisms involved
in the readily demonstrated group protection. The pertinent
possibilities suggested to date include the following, which are
by no means mutually exclusive:
1. The group more effectively produces an autoprotective
substance (Drzewina and Bohn, '21, '28).
2. The larger number of animals more rapidly adsorbs or
exhausts the toxic agent, or, on the other hand, each animal
receives a smaller dose (Bresslau, '24; Allee and Schuett,
'27 ; Carpenter, '27).
3. The grouped animals tend to show a reduction in the
general rate of metabolism, as measured by 0, consumption
or C02 production, as compared with the isolated animals
202 W. C. ALLEE AND EDITH S. BOWEN

during the early stages of their association (Allee, '20, '26,


'28a); this reduction favors survival in toxic solutions at
certain concentrations (Fowler, '31).
4. The group may be protected by altering theeelectrical
conditions (Drzewina and Bohn, '26), or, as these workers
state in 1928, animals may be mutually influenced by the
presence of other animals without the diffusion of any sub-
stances-a sort of catalysis by contact.
If we limit the discussion concerning which of these sug-
gested mechanisms has been working in the protection of
grouped goldfishes from the toxic action of colloidal silver
to the evidence to be obtained from the experiments dealing
with these fishes, which have been summarized in the earlier
parts of the present report, it is apparent that unless slime
and the salts contained in the secretions are regarded as con-
stituting an autoprotective secretion, there is no evidence for
the first assumption just given. For, in review, the evidence
shows that: 1) All the silver has been accounted for in the
chemical analyses with the different treatments, and that the
suspensions containing grouped animals have had signifi-
cantly more silver removed than have the suspensions which
contained isolated fishes. 2) With volume and silver dosage
identical per individual fish, there is no significant difference
in survival; i.e., there is no significant evidence of group
protection. 3 ) Although the data from the processionary
series might be interpreted as supporting the autoprotective
secretion hypothesis in the absence of chemical analyses, yet
the observed increase in survival in later members of such
a series is also entirely consistent with the assumption of
decreased toxicity of solution which has been demonstrated
for other types of experiments. 4) The results of the experi-
ments in which the colloidal suspensions were conditioned by
having &he8 live in them for twenty-four hours or until death
showed in the chemical analyses that by the time the analyses
were made all the free colloidal silver had been removed from
a 3 per cent conditioned suspension, and that a part of the
silver newly added to make a doubly charged suspension was
STUDIES IN A N I M A L AGGREGATIONS 203

also removed, whereas practically no free silver was lost


from the suspensions that contained only a single fish.
All the evidence against the hypothesis of an autoprotective
secretion that acts other than by adsorbing or otherwise
removing toxic silver from suspension actively favors the
second hypothesis that group protection does exist in these
cases because of the removal of the toxic element. The evi-
dence from the chemical analyses appears conclusive and is
not opposed by other experiences in this laboratory concern-
ing group protection from colloidal silver. Again, as in the
work of Allee and Schuett ( '27), the only question concerns
whether or not all the observed protection.can be attributed
to the working of this factor, but here the answer is appar-
ently definite, namely, that no effect other than the observed
removal of the toxic colloidal silver need be assumed t o
account for the demonstrated group protection.
There remains another mechanism over and above the re-
moval of colloidal silver which would have group protective
value. Schuett has discovered (unpublished), and one of us
has independently confirmed, the discovery that grouped gold-
fishes have a lower rate of oxygen consumption than have
isolated fishes in a similar volume of water. This is in keep-
ing with previous work on the initial effect of grouping upon
oxygen consumption, and, as Child ('15) and others (Allee,
'14) have demonstrated, animals with a lower rate of general
metabolism tend to survive longer in toxic solutions, when
other conditions are equal and the toxicity is such that death
occurs before acclimatization can be a factor. Such condi-
tions obtain in these experiments, and this may well be a
contributing factor to the lengthened survival of the group.
Our experiments do not demonstrate the mechanism produc-
ing this decreased oxygen consumption of the group, but they
do indicate that it is not due to the accumulation of COz,which
Fowler ('31) found to be effective with Daphnia exposed to
toxic concentrations of many electrolytes. The reasoning
follows : The carbon-dioxide relationships are greatly dif-
ferent in alkaline and in acid media, yet group protection
204 W. C . ALLEE AND EDITH S. BOWEN

occurs in colloidal silver suspensions in which the toxicity is


not associated, in so far as our experiments are concerned,
with the pH of the media. I n the suspensions with a pH of
less than 7.0, free CO, might accumulate which would have
a depressing action. I n the suspensions with the same
volumes used for grouped fishes and for each isolated fish the
surface ratio per grouped and isolated individual was 45 : 450,
which should allow a marked accumulation of CO, in the
vessel containing the group. But when the surface relations
per fish were reversed in the experiments with equal volumes
per individual and became 90: 78, the grouped fishes still
averaged a larger. survival time, although the difference was
not statistically significant. The difference in survival can
more readily be accounted for on the different ratios of col-
loidal silver per individual than by any CO, surface relations
which, if exact, should give an indication of greater survival
in the vessel with the smaller surface area per fish; and this
is not the case. Further evidence that CO, accumulation is
not the protective mechanism with these goldfishes exposed
to colloidal silver has been given above.
The group protection is not to be attributed to the
hydrogen-ion concentration of the suspensions acting directly
on the fishes or indirectly on the toxicity of the colloidal silver
suspensions. While the experiments reported concerning pH
relations to colloidal silver toxicity do not prove the relations
exhaustively, they do show that fishes introduced into water
approximately as unbuffered as are the colloidal silver sus-
pensions survive despite the exposure to solutions with an
initial pH of 9.4 and of 4.5. Again with colloidal silver pres-
ent, mass protection occurs with the initial pH at 4.3, 7.0,
and 9.6.
Of the two mechanisms which may be acting, the greater
removal by the grouped fishes of toxic colloidal silver and
the lower rate of general metabolism, it would appear that
the former is much the more important in these experiments.
This is indicated by our experience with different concentral
tions of colloidal silver acting as determined by varying the
STUDIES IN ANIMAL AGGREGATIONS 205

initial concentration or by removal through slime or other


agencies. It accordingly is pertinent to examine the different
substances given by the fishes into the surrounding medium
which might affect this result. There are four main groups of
these: slime, CO,, urinary secretions, and faeces. The slime
is clearly effective in fixing colloidal silver. The visual evi-
dence of dark coatings that appear over the gills and body of
the fishes which may be shed, causing discrete string-like
masses in the previously clear medium, is now happily sup-
ported by chemical analyses. The CO, relations have been
discussed and do not appear to be s i g a c a n t under the con-
ditions of these experiments. The principal salt s contained
in urinary secretions in amounts comparable with twentp-
four-hour conditioning of the medium have distinct protective
value, but apparently are not so effective a protective agent
as is the slime. The exact mechanism of the protection fur-
nished by these salts is obscure and discussion on this point
is reserved for the present. Faeces probably act both by
adsorbing and by releasing inorganic salts. They are always
present in the experiments, but their relative importance as
a part of the protective mechanism has not been quantitatively
investigated; our experience indicates that the faeces are of
less importance as a protective meohanism than is slime.
SUMMARY
1. Other things being equal and in the absence of over-
crowding, a group of goldfishes survives longer than do fishes
isolated into similar volumes and concentrations of colloidal
silver.
2. There is no positive evidence for group protection when
grouped and isolated fishes are exposed to the same volume
of colloidal silver per individual.
3. In a processionary series of fishes in one and the same
suspension of colloidal silver, the later individuals live
significantly longer than those first exposed.
4. Chemical analyses show that grouped fishes remove
much more colloidal silver from suspension than do isolated
206 W. C. ALLEE AND EDITH S. BOWEN

fishes, so much so that this alone will account for the greater
survival value of the group.
5. Suspensions of colloidal silver in which fishes have died,
or have lived twenty-four hours without dying, are definitely
less toxic than unconditioned suspensions and contain less
unadsorbed silver even when double the amount is added to
the experimental solution as to the control.
6. Chemical analyses show that the depth of color of a
colloidal silver suspension is not an adequate criterion of
the amount of silver present.
7. Fishes give off slime, GOz,urinary excretions, and faeces
into their surrounding medium. Of these, CO, does not ap-
pear important in protecting the group from colloidal silver.
All the others are effective, with slime the most important
of the three.
8. Grouped goldfishes use less oxygen than do fishes iso-
lated into the same volume. Such decreased metabolism
has a survival value in the presence of toxic conditions that
do not permit acclimatization; however, this does not appear
to be an important protective mechanism against colloidal
silver.
9. There is no evidence of the action of a special autopro-
tective secretion with a differential protective action that
favors the grouped fishes which acts without decreasing the
concentration of the toxic agent.
10. There is nothing in the present experiments to suggest
that such an autoprotective secretion may not afford differ-
ential group protection with other toxic agents or to suggest
that group protection is always due to the more effective
removal, fixation, or more tenuous distribution of the lethal
factor.
11. The general biological importance of the phenomena
here reported is discussed at length elsewhere (Allee, '31).
LITERATURE CITED
ALLEE,W. C. 1914 Certain relations between rheotaxia and resistance to
potaaaium cyanide in Isopoda. Jour. Exp. Zool., vol. 16, pp. 397-412.
1920 Animal aggregations. Anat. Rec., vol. 17, p. 340.
STUDIES IN ANIMAL AGGREGATIONS 207
ALLEE, W. C. 1926 Studies in animal aggregations: cawea and effecta of
bunching in land bopods. Jour. Exp. Zool., vol. 45, pp. 255-277.
1928 Studies in animal aggregations: mass protection for Planaria
from ultra-violet radiation. Phymol. Zool., vol. 1, pp. 509-530.
1928 a studies in animal aggregations: maaa protection from fresh
water for Procerodea, a marine turbellarian. Jour. ESP. Zool., vol. 50,
pp. 61-84.
1929 MWE protection from hypotonic sea water for Proeerodes, a
marine turbellarian. Jour. Exp. Zool., vol. 54, pp. 349-379.
1931 Animal aggregations. Univ. of Chicago Press, Chicago.
ALLEE, W. C., AND J. F. S C H 1927 ~ Studies in animal aggregations: the
relation between mass of animals and resistance to colloidal silver.
Biol. Bull., vol. 53, pp. 301-317.
BBE~~LAU, E. 1924 Die Ausscheidung von Schutestoffen bei einzelligen Lebe-
ween. Ber. Senckenberg. Naturf. Ges., Bd. 54, 8. 49-67.
CABPENTEB, K. c. 1927 The lethal action of soluble metallic salts on hhes.
Brit. J. Exp. Zoiil., vol. 4, pp. 378-390.
1930 Further researches on the action of metallic salts on fishes.
Jour. Exp. Zool., vol. 56, pp. 407-422.
CHILD, C. M. 1915 Senescence and rejuvenetwence. Univ. of Chicago Press,
Chicago.
DMEWINA,A., AND G. BOHN 1920 Variations de la eeneibilite B leau douce
dee Convoluta suivant les Btats phyeiologiquea et le nombre des anhaux
en expbrience. Comp. Rend. Acad. Sci., T. 171, pp. 1023-1025.
1921 Variations de la aueeeptibilit6 aux agents nociPs avec le
nombre dee animaux traitbs. Ibid., T. 172, pp. 485487.
1921 a La defense dea animaux g r 0 ~ p 6v~i ~ - h - dee
~ i ~agents nocifs.
Ibid., T. 172, pp. 779-781.
1921b Variations dans le temps de la Asietance a w agents
physiques et chimiquee chez Rana fnsca. Comp. Rend. Soc. de biol.,
T. 84, pp. 963-965.
1921 c Sur des ph6nomanea d auto-protection et 1auto-destruction
chez dee animaux aquatiques. Comp. Rend. Acad. Sci., T. 173, pp.
107-109.
1926 Action de largent metallique Bur le sperme e t les larves
dOnr&. Ibid., T. 182, pp. 1651-1652.
1926a Action antagoniste de largent e t de 1Btain metallique
Bur les Btres vivants. Ibid., T. 183, pp. 571-572.
1926 b Activation par la lumiare den effets de largent BUT Con-
voluta. Ibid., T. 183, pp. 677-679.
1927 Influence des parois des vaaes sur les rBactione dea animaur.
Ibid., T. 185, pp. 875-877.
1928 Les Convoluta. Ann. Sci. Nat. Zool., T. 10, dr. 11, pp. 294-
398.
FOWLEB, J. F. 1931 The relation of numbera of aaimnll to wrvival in toxic
concentrations of electrolytes. Phymol. Zoiil., rol. 4.
N o u , A. 1924 Die Exkretion (Wirbeltiere). Handbuch der Vergleichenden
Physiologic von Hans Winterstein, Bd. IIb, 8. 799-801.