Studies on the Structure, Development, and
Physiology of the Nephridia of Qligochaeta.
VI. The Physiology of Excretion and the Significance of the Bnteronephric Type of Nephridial
System in Indian Earthworms.
By
Kara Narayan Bahl, D.Sc. (Panj.), D.PML, D.Sc. (Oxon),
(Merton College, Oxford)
Professor of Zoology, University of Lucknow, India.
With 7 Text-figures and 11 Tables.
CONTENTS.
PAGE
1. INTRODUCTION
344
2. T H E NATURE AND MASS OF EXCRETORY SUBSTANCES EVACUATED
B Y THE EARTHWORM ( P H E R E T I M A
POSTHUMA).
.
.
348
(a) ESTIMATION OF AMMONIA AND U R E A I N W A T E R CONTAIN.
ING EARTHWORMS
348
(6) ANALYSIS O F THE U R I N E O F EARTHWORMS, AND ESTIMATION OF AMMONIA AND U R E A THEREIN
.
.
3. T H E EXCRETORY SUBSTANCES I N THE COELOMIC F L U I D
4. T H E EXCRETORY SUBSTANCES I N THE BLOOD
.
.
.
.
351
.
.
.
354
356
5. T H E INITIAL PLACES O F E X C R E T I O N — T H E BODY-WALL AND THE
INTESTINAL W A L L
6. T H E R O L E
.
.
.
OF T H E N E P H R I D I A
.
.
.
.
.
358
I N EXCRETION AND OSMOTIC
361
REGULATION
(A)
T H E OSMO-REGULATORY FUNCTION OF T H E N E P H R I D I A
(i) VOLUME-REGULATION
363
(ii) T H E R A T E OF EXCRETION OF U R I N E
(iii) T H E OSMOTIC
PRESSURE
.
.
.369
OF BLOOD,
COELOMIC
O F BLOOD,
COELOMIC
OF BLOOD,
COELOMIC
371
F L U I D , AND U R I N E
(iv) T H E P R O T E I N
CONTENTS
372
F L U I D , AND U R I N E
(v) T H E CHLORIDE
CONTENTS
375
F L U I D , AND U R I N E
(vi) CONCLUSIONS
.
363
.
.
.
.
.
.
376
344
K. N. BAHL
PAGE
(B)
THE
EXCRETORY
INCLUSIONS
OF
THE
'CILIATED
M I D D L E T U B E ' (ATHEOPHAGOCYTIC S E C T I O N ) O F T H E
NEPHRIDIA
377
7. E X C E E T O E Y ORGANS O T H E E THAN N E P H R I D I A
.
.
.
.
385
8. SUMMARY
387
9. R E F E R E N C E S
388
I. INTRODUCTION.
OUE knowledge of the physiology of excretion in the Oligochaeta is still very incomplete. Stephenson (19) gave an
exhaustive account of published work up to 1930, while Stolte
(£§) has admirably summarized the latest accounts up to 1938.
But, as remarked by Stolte, each one of the number of contributory processes involved in excretion has been investigated
with varying results, so that it is not possible as yet to have a
complete picture of the whole process. Similarly, Heidermanns
(14) states: 'The findings about the excretory material of
worms vary greatly even to-day. Apart from the non-unanimity
of accounts about the material which appear in the inner
metabolism and the materials of the purin-group which are
stored in the cells of the chloragogen tissue, several authors
also differ from one another in their published results with
regard to the nature and mass of the excretory substances
eliminated as a result of nitrogen metabolism, as also with
regard to ammonia and urea.' It seems hard to believe, but
nevertheless it is a fact, that we do not yet completely understand the physiology of excretion in the common earthworm
studied by every elementary student of zoology.
Broadly speaking, 'the function of the excretory system is
to keep constant the internal environment of the body, or in
other words, the fluid content of the body as a whole. To this
end, the excretory organs eliminate or segregate unwanted
substances, and retain or reabsorb constituents needful to the
organism' (Wigglesworth, 21). In an earthworm there are two
circulating fluids, i.e. the blood and the coelomic fluid, which
remain completely separated from each other, but together
form the greater part of the internal environment of the body.
EXCRETION IN INDIAN EARTHWORMS
345
How does an earthworm keep these two fluids constant, and
how do its excretory organs or nephridia eliminate or segregate
unwanted substances ?
The nephridia have, on the one hand, a fairly rich bloodsupply1—Benham's diagram of the blood-supply of the nephridium of L u m b r i c u s (9) shows a copious blood-supply, and
so does a diagram of the blood-supply of a septal nephridium
of P h e r e t i m a p o s t h u m a (Text-fig. 1)—and, on the other,
they not only float within the coelomic fluid but are in open
communication with it through their nephridiostomes. • Some
workers have, therefore, assumed that there is a division of
functions between these two fluids. Eogers (17 a), for example,
says: 'The coelomic 'fluid receives from the gut the foodmaterials as they diffuse through the walls and conveys these
food-materials to the various cells of the body. It also receives
from the active cells of the body the various metabolic wastes
and conveys them to the nephridia, through which they are
eliminated. The blood or haemolymph in the closed system of
tubes, on the other hand, is a solution of haemoglobin and
serves as the carrier of oxygen to the various cells and tissues
of the body.' This division of functions between the coelomic
fluid and blood, in which blood has nothing to do with excretion, is an assumption which has never been tested and proved
so far.
The problem of excretion in the earthworm may be resolved
under the following heads: (1) Where do the metabolic wastes
first originate and what is their chemical nature ? (2) Does the
coelomic fluid contain nitrogenous waste products in solution
and, if so, in what form ? (3) Does the blood take any part in
carrying excretory products, or is it merely a carrier of oxygen ?
(4) What exactly is the role of the nephridia ? Are they only
excretory or do they have any other function besides? Are
there any other excretory organs besides nephridia? (5) Are
any excretory products stored within the body of the earthworm ? If so, in what form and where ? (6) What are the
1
Willem and Minne's (22) statement that 'in many Oligochaeta the.
nephridia have no blood-supply' does not hold for the nephridia of earthworms.
,
NO. 340
Aa
346
K. N . BAHL
characters of the excretory fluid as it is finally evacuated from
the body? An attempt has been made in this memoir to deal
with all these aspects of the problem, and to present, as far as
possible, a complete picture of the whole process of excretion.
Almost all previous workers on the physiology of excretion
TEXT-ITC. 1.
A septal nephridium of P h e r e t i m a p o s t h u m a showing the
course of the blood-vessels and capillaries in it. affnv, afferent
nephridial vessel; ce, connecting loop-capillaries between the
afferent and efferent vessels; commv, commissural vessel; sb,
septo-nephridial branch of the ventro-tegumentary vessel.
(x dr. 120.)
in the Oligochaeta have studied the process in the European
type genus L u m b r i c u s ; little attention has been paid to
other forms, particularly the tropical earthworms, which present
EXCRETION IN INDIAN EARTHWORMS
347
important differences in their nephridial system from that of
L u m b r i c u s and would thus be likely to throw considerable
light on the physiology of the different processes involved in
excretion. Another unfortunate defect in the publications of
previous workers, with a few notable exceptions, is that they
seldom give a complete account of the biochemical methods
they have employed in arriving at their results. This makes
the task of a subsequent worker difficult, as he cannot adequately check their results by using the same methods as they
employed. I have, therefore, tried to give, as far as possible,
full details of the methods employed by me in all my biochemical estimations.
I have studied the process of excretion mainly in the earthworm P h e r e t i m a ( P e r i c h a e t a ) p o s t h u m a (2), which
possesses three types of nephridia: (1) open septal nephridia,
80-100 in each segment, which discharge their excretory products through an elaborate system of canals into each segment
of the i n t e s t i n e and not to t h e e x t e r i o r ; (2) closed
integumentary nephridia, about 200 in each segment, which
open to t h e e x t e r i o r on the body-wall; and (3) closed
pharyngeal tufted nephridia, which number several hundreds
in each of the three segments (IV-VI) where they occur, and
which open i n t o t h e b u c c a l c a v i t y and p h a r y n g e a l
l u m e n . All these three types of nephridia are extremely
minute and lack the terminal bladder of the nephridium of
Lumbricus.
I am deeply indebted to my friend and colleague Dr. S. M.
Sane of the Department of Chemistry who has readily helped
me in this work at all times. and without whose active cooperation this work would not have been possible. My best
thanks are due to Dr. M. L. Bhatia for his kind help in the
preparation of illustrations and to Mr. L. N. Johri for his painstaking assistance in the greater part of this work. I am also
very thankful to Dr. N. K. Panikkar of the University College,
Trivandrum, for reading through my manuscript and making
several useful suggestions.
348
K. N. BAHL
2. THE NATURE AND MASS OF EXCRETORY SUBSTANCES
EVACUATED BY THE EARTHWORM ( P ' H E R E T I M A P O S T H U M A ) .
Before determining the place of origin and the course of
elimination of the excretory products, it is necessary to find out
the nature and mass of excretory substances as they are finally
evacuated by the earthworm, as that will provide the necessary
clue to the proceeding contributory processes of excretion. The
first step, therefore, was to analyse and estimate the nitrogenous excretory products as they are finally evacuated by the
earthworm.
(a) E s t i m a t i o n of A m m o n i a and U r e a in
Water containing Earthworms.
Lesser and Delaunay (as quoted by Stolte, 20) investigated
the nitrogenous contents of the excretory fluid voided by the
nephridia of the earthworm L u m b r i c u s . Lesser found only
ammonia, while Delaunay found ammonia, urea, and also
nitrogen as amins, but no -uric acid. Lesser kept earthworms in
distilled water for twenty-four hours, while Delaunay kept
them for eight days, since he found that evacuation of the
contents of the terminal bladder of a nephridium took place
only once in three days. This observation of Delaunay does not
hold in the case of P h e r e t i m a because its nephridia do not
possess a terminal bladder to retain the excretory fluid for any
length of time and must therefore go on discharging the urine
all the time. I may state at once that although keeping earthworms in distilled water is a convenient method of obtaining
their nephridial fluid, the water is bound to contain in addition
substances defaecated through the anus as well as those discharged through the mouth. Even an earthworm whose gut
has been cleaned of its earthy contents by keeping it in water
for several days, gives out watery drops from the mouth as well
as anus, after it is wiped dry with a piece of cloth. Lesser and
Delaunay did not take worms with clean guts and, therefore,
all their samples of water containing earthworms must have
contained not only the nephridial fluid but also faecal matter
discharged through the anus, as well as the watery discharge
EXCRETION IN INDIAN EARTHWORMS
349
given out through the mouth. They, therefore, tested not only
the nephridial fluid but also the discharges from the two ends of
the gut.
I have repeated the experiments of Lesser and Delaunay
with the earthworm P h e r e t i m a p o s t h u m a . Only worms
with clean guts were kept in distilled water for twenty-four
hours, and the water was tested for ammonia, urea, and uric
acid. While ammonia and urea were present, there was no
trace of uric acid. The criticism made above on the experiments
of Lesser and Delaunay holds equally in the case of my experiments, but there is an important difference. While L u m b r i c u s possesses only funnelled nephridia opening directly to the
exterior through comparatively large nephridiopores, the innumerable integumentary nephridia of P h e r e t i m a (2) opening to the exterior on the integument are all closed nephridia
with no open communication with the coelomic fluid. The
numerous funnelled septal nephridia of P h e r e t i m a (2) do
not open to the exterior but into the intestine all along its length,
so that their excretory fluid must necessarily pass out through
the anus. Further, the closed pharyngeal nephridia of P h e r e t i m a must discharge their fluid through the mouth. In the
case o f . P h e r e t i m a , therefore, one must include the discharges through the mouth and anus along with the nephridial
fluid voided through integumentary nephridiopores, so as to
estimate correctly the total quantity of the excretory substances voided by the earthworm.
For quantitative estimations the water containing the earthworms was collected every twenty-four hours; it was clear and
contained hardly any proteins. Ammonia and urea were
estimated and the results of the estimations are given in
Table I.
It will be seen from this table that the quantity of ammonia
excreted is two to four times that of urea. Delaunay (12 a)
gives the percentage of ammonia-nitrogen and urea-nitrogen
in L u m b r i c u s as 46 to 48 per cent, and 6 to 12 per cent,
respectively; on converting these figures into ammonia and
urea, I find that my results (proportions between ammonia and
urea) are more or less in accord with those of Delaunay (12 a).
350
K. N. BAHL
TABLE I. Ammonia and Urea voided in Water
in Twenty-four Hours.
Serial
number.
of
worms.
Weight
in
grams.
N(urea and
ammonia) in
milligrams
(per 100 gm.
of bodyweight).
1.
2.
3.
4.
5.
average
20
20
20
20
20
20
21-68
28-48
30-98
27-82
28-20
27-43
4-97
6-11
5-33
4-67
5-02
5-22
Number
Ammonia in
milligrams
(per 100 gm.
of bodyweight).
Urea in
milligrams
(per 100 gm.
of bodyweight)
(by diff.).
5-24
6-54
4-87
4-30
5-52
5-29
1-39
1-65
2-82
2-29
0-88
1-80
These estimations, therefore, confirm Delaunay's conclusions:
(1) that nitrogenous excretory substances in the earthworms are
eliminated as ammonia and urea only, and that no uric acid is
excreted by the earthworm; (2) that there is more of ammonia
than of urea.
The procedure followed for these estimations was as follows:
Earthworms were kept in water for several days till their guts
were cleaned of all their earthy contents. Sets of twenty earthworms
each were kept in 50 c.c. of distilled water in several glass dishes for
twenty-four hours. At the end of twenty-four hours worms were
wiped dry and weighed, and the water in each dish was divided into
two equal parts. From one part ammonia was estimated directly by
Foreman's method of alcoholic distillation.1 The other part was
treated with urease2 to convert urea into ammonia and the total
ammonia produced was estimated also by Foreman's method: this
ammonia included both ammonia excreted as such, as well as ammonia from urea. Since free ammonia had been estimated separately
from the same water, the difference between the two estimations
gave the ammonia obtained from urea, and from this figure the
percentage value of urea was calculated.
1
The estimations and their calculations were made according to the
method described for urine by S. W. Cole in his 'Practical Physiological
Chemistry' (Heffer and Sons, Cambridge, 1942), pp. 334-5.
2
In. all estimations of urea tabloids of urease supplied by the British
Drug Houses were used.
EXCRETION IN INDIAN EARTHWORMS
351
(&) A n a l y s i s of t h e U r i n e of E a r t h w o r m s , a n d t h e
E s t i m a t i o n of A m m o n i a a n d U r e a t h e r e i n .
Although keeping earthworms in distilled water is a convenient method of collecting their excretory fluid, it is at best
an indirect method, giving urine only in a highly diluted condition. As far as I have been able to find out from literature, no
worker has so far succeeded in collecting a sufficient quantity
of pure urine of an earthworm. Eustum Maluf (16) gives the
osmotic pressure of the blood, but puts a query mark against
that of the urine. Adolph (1) gives the osmotic pressure of the
body juice, but not that of the urine. Carter (10) says that in "
the earthworm 'no analyses of the urine have yet been made'.
The difficulty has, of course, been to collect a sufficient quantity
of urine for either qualitative or quantitative work.
I have successfully made use of the following simple method
of collecting the urine of earthworms:
Freshly collected earthworms were brought into the laboratory
and kept in water for three or four days to remove most of the earth
from their guts. A clean dry glass dish with high walls was divided
into two parts by a closely fitting vertically placed glass plate. The
worms were taken out one by one, wiped dry with a piece of cloth,
and kept in one half of the glass dish. On keeping the dish in a
slanting position, worms collected against the vertical glass plate in
a cluster; and droplets of colourless urine oozed out of the integument, as also from the mouth and anus, and collected in the lowest
part of the other half of the dish. In about twenty minutes, 4 to
5 drops of urine were excreted by forty earthworms. A fresh lot of
forty worms was treated similarly and another 4 to 5 drops were
obtained. This process was repeated several times until about 8 c.c.
of urine was collected in 4 to 5 hours.1
In order to prevent evaporation from the body surfaces of the
worms and of the excreted urine itself in dry weather, the dish containing earthworms was placed in a larger dish containing water,
and both dishes were covered with a large bell-jar so that the atmosphere for the worms was as humid as possible.
1
With practice and improvement of collecting technique I have been
able to collect 25 c.c. of urine in two and a half hours from 105 earthworms
in wet weather.
352
K. N. BAHL
A qualitative analysis of earthworm's urine revealed the
presence of basic radicles like sodium, potassium, calcium, and
magnesium, as well as acid radicles like chlorides and phosphates, and the absence of sulphates. The reaction with litmus
earfhwormi
bell-Jo.
small ejlass-dish
larcje qlass-dish
wooden block
water
TEXT-FIG. 2.
Apparatus used for collecting the urine of earthworms.
was markedly alkaline, the pH being 8-3. Xanthoproteic test
(Cole, 1942, p. 80) showed the presence of a merest trace of
protein.
Ammonia and urea were estimated quantitatively by Foreman's alcoholic distillation method and the results obtained are
as follows:
TABLE
II. Ammonia and Urea in the Urine of Earthworms.
Serial
number.
N(urea and ammonia)
in milligrams per
100 c.c.
Ammonia in
milligrams
per 100 c.c.
Urea in milligrams
per 100 c.c.
{by diff.).
1.
2.
3.
4.
5.
Average
3-61
3-61
3-61
4-04
3-61
3-69
2-19
2-80
2-19
2-80
3-35
2-66
3-87
2-80
3-87
3-72
1-95
3-24
The test for uric acid was negative.
In this table it is noteworthy that the percentage of ammonia
EXCRETION IN INDIAN EARTHWORMS
353
and urea n i t r o g e n is almost constant in all the five estimations; only it is distributed in slightly different proportions
between ammonia and urea. Delaunay also found variation in
the distribution of nitrogen between ammonia and urea, and
thought that it was possible that a part of excreted urea was
transformed rapidly into ammonia. That this is probable is
seen by comparing the proportions of ammonia and urea in
Tables I and II. In Table I ammonia is, on an average, three
times that of urea, since the urine was in water for twenty-four
hours, while in Table II the average percentage of ammonia
is about 18 per cent, less than that of urea, since the urine was
kept only for four to five hours and at a fairly low temperature.
The important conclusion is that in the earthworm, as in
most aquatic invertebrates, urea and ammonia form the main
bulk of the nitrogenous excretion, and that no uric acid is
excreted by the earthworm.
All previous workers collected urine in water and consequently it was a very dilute urine on which they made their
estimations. The percentages of ammonia and urea had to be
calculated n o t on the volume of urine, but either on the
weight of earthworms excreting urine or on the total nitrogen
excreted, because the exact volume of urine was never known.
This is how I have myself calculated the figures in Table I.
But on obtaining urine as such, it has now become possible to
estimate the percentage of urea and ammonia in relation to the
volume of urine excreted, as given in Table II, and thus to make
a parallel comparison with similar percentages in the coelomic
fluid and blood (Tables III and IV). But it must be realized
that, as stated in chapter 6 a (i) ( v i d e i n f r a ) , the urine
collected is that of earthworms living like freshwater animals,
and not as terrestrial animals, the urine of which would presumably be more concentrated.
The method adopted for estimating urea and ammonia was
as follows:
6-5 c.c. of urine was diluted with 15 c.c. of distilled water and
25 c.c. of absolute alcohol was added to precipitate the proteins,
which were present in a fine colloidal state. The liquid was centrifuged and filtered. The nitrate was divided into two equal parts:
354
K. N. BAHL
from one part ammonia was estimated directly by Foreman's
alcoholic distillation method, while the other part was treated with
urease to convert urea into ammonia, and distilled by Foreman's
alcoholic method. The difference between the two estimations gave
the value for urea.
3. THE EXCRETORY SUBSTANCES IN THE COELOMIC FLUID.
Having ascertained that the chief nitrogenous excretory products voided by the earthworm are urea and ammonia, the next
step is to find out where these excretory products come from.
All the three kinds of nephridia—septal, integumentary, and
pharyngeal—float in the coelomic fluid; further, the septal
nephridia are in open communication with the coelomic fluid
through their nephridiostomes; at the same time all the three
kinds are copiously supplied with blood. We must presume,
therefore, that urea and ammonia in solution must come to the
nephridia either from the coelomic fluid or from the blood or
from both. Taking the coelomic fluid first, we know that it fills
the entire coelomic cavity and keeps moving back and forth in
a live earthworm. The eoelomic fluid of P h e r e t i m a is milkwhite in colour and contains as many as five kinds of corpuscles
(Kindred, 15), of which the most numerous are the phagocytes.
What the respective functions of these five kinds of corpuscles
are, is not yet known with certainty, but there is little doubt
that the phagocytes engulf bacteria, dead chloragogen cells,
and other solid waste matters present in the coelomic fluid. It
has been generally assumed that the coelomic fluid also contains
metabolic wastes in a dissolved state; for example, Stephenson
(19) writes: 'A certain amount of coelomic fluid c o n t a i n i n g
e x c r e t o r y s u b s t a n c e s in s o l u t i o n (the spaced words
are mine) passes through the nephrostome and is propelled
down the nephridial tube by ciliary action.' But there is no
mention in literature of any worker having tested and estimated
the nitrogenous excretory substances in s o l u t i o n in the
coelomic fluid. The only statement I have come across is: that
'the chief function of the coelomic fluid consists in the distribution of fluid food-materials. Besides there is an excretory
function through the nephridia, about which up till now v e r y
355
EXCRETION IN INDIAN EARTHWORMS
l i t t l e is k n o w n ' 1 (Stolte, 80)/ There is no mention of
ammonia, urea, uric acid, or any other nitrogenous excretory
substance in solution having been found in the coelomic fluid.
Even Heidermanns (14), who estimated the ammonia and urea
contents of the gut and the body-wall, did not think of estimating ammonia and urea contents of the coelomic fluid or of blood.
I, therefore, estimated the ammonia and urea contents of the
coelomic fluid and the results obtained are given in the following
table:
TABLE
Serial
number.
III. Ammonia and Urea in Coelomic Fluid.
N(urea and ammonia)
Ammonia
in milligrams
in milligrams
(per 100 ex.).
(per 100 ex.).
Foreman's
method.
1.
2.
3.
4.
5.
4-01
3-98
501
5-47
4-50
3-38
4-0
4-24
4-94
3-9
Average
4-79
4-13
JM essierization
method.
6.
7.
—
—
—
—
Urea in milligrams
(per 100 ex.)
(bydiff.).
Ammonia+Urea
3-5 mgm.
4-5
2-29
1-67
3-02
30
2-61
2-52
—
—
The test for uric acid was negative.
It will be seen from this table that, on an average, every
100 c.c. of coelomic fluid contain 4-13 mgm. of ammonia and
2-52 mgm. of urea. By comparing these figures with those in
Table II, it will be seen that while the percentage of ammonia
voided by the earthworm in its urine is, on an average, slightly
lower than that contained in the coelomic fluid, the percentage
of urea voided is slightly higher than that contained in the
coelomic fluid. It is likely, therefore, that the nephridia derive
their urea Lorn some other source as well, and that other source
to be tested is obviously blood.
1
The spaced words are mine.
356
K. N. BAHL
The method followed for estimating ammonia and urea in the
coelomic fluid was as follows:
Forty to fifty live earthworms were cut open and yielded about
10 c.c. of coelomic fluid.1 The coelomic fluid obtained was very
nearly pure; only a very small quantity of blood came with it. The
fluid was immediately centrifuged, and the corpuscle-free fluid was
decanted off and measured, and then diluted with an equal amount
of distilled water. Treatment with eight times its volume of absolute
alcohol precipitated all proteins.2 After filtration the clear colourless
fluid obtained was measured and divided into two equal parts. From
one part ammonia was estimated directly by Foreman's alcoholic
distillation method, while from the other part total ammonia (free
ammonia+urea ammonia) was estimated by the same method after
treatment with urease. The difference between the two estimations
gave the value for urea.
Urea in the coelomic fluid was also estimated by the UreaseNesslerization method. The fluid was incubated with urease at
37° C. for half an hour to convert urea into ammonium carbonate.
Proteins of the fluid were precipitated by NaOH and zinc sulphate
solutions. Part of the supernatant fluid was treated with Nessler's
reagent. Standard solutions of an ammonium salt were also treated
with Nessler's reagent, and compared with the Nesslerized coelomic
fluid colorimetrically by the Klett-Bio Colorimeter and the amount
of urea in 100 c.c. of the fluid was calculated. This is the routine
method followed for estimation of urea in human blood in the
Pathological Department of the Lucknow University Hospital and
I am indebted to Dr. V. S. Mangalik of the Pathology Department
for kindly making colorimetric estimations of coelomic fluid and
blood at my request.
4. T H E EXCRETORY SUBSTANCES IN THE BLOOD.
On looking through the literature I found that although
some authors like de Bock and Freudweiler had ascribed an
1
Care was taken not to let the intestinal contents get mixed with the
coelomic fluid; any worms in which the intestine got punctured were
promptly rejected.
2
Various reagents, like trichlor-acetie acid, sodium tiingstate and
sulphuric acid, and absolute alcohol were tried for precipitating proteins
in the coelomic fluid before estimating the amount of urea. Of these
absolute alcohol proved the most convenient and most efficient.
357
EXCRETION IN INDIAN EARTHWORMS
excretory function to the amoebocytes of the blood, no worker
had so far made a blood-urea estimation of earthworm's
blood, although it is so commonly done of human blood. In view
of the assumption commonly made and expressed by Eogers
(17 a) it is clearly important to know whether blood takes any
part in the excretion of ammonia and urea. I, therefore,
estimated the amounts of these two substances in the earthworm's blood, and my results are given in the following table:
TABLE
IV. Ammonia and Urea in Blood.
Urea in milligrams
(per 100 c.c.)
Serial
number.
N(urea-\-ammonia) in
milligrams
(per 100 c.c).
Ammonia, in
milligrams
(per 100 c.c).
1.
2.
3.
4.
3-08 )
2-98 Foreman's
4-01 method.
2-98 .
2-49
2-49
3-06
1-81
2-207
1-97
3-19
3-18
3-26
2-71
2-638
Average
5.
Nesslerization 1
method.
/ ""*"
(by diff.)-
Ammonia-)-Urea
5-8 mgm.
By comparing the figures in this table with those in Table III
(for coelomic fluid), it is readily seen that while the average
percentage of urea in the blood is about the same as that in the
coelomic fluid, the average percentage of ammonia is distinctly
lower. Since the nephridia are copiously supplied with blood,
the conclusion is irresistible that the nephridia eliminate
ammonia and urea from the blood as well as from the coelomic
fluid. The assumption of Eogers (17 a) that coelomic fluid is
concerned with the distribution of food-materials and excretion,,
and that blood is a mere carrier of oxygen is, therefore, clearly
untenable. We must conclude that the blood collects the
metabolic wastes from the tissues of the body just in the same
wpy as the coleomic fluid.
Circulating human blood has an ammonia value of zero or
below analytical level. But after shedding, ammonia appears
almost immediately, which in the rabbit is said to amount to
358
K. N. BAHL
1 mg. per 100 c.c. My estimations were carried out on shedded
blood of earthworms, and it is quite possible that part of the
ammonia is a post-mortem product and that the actual percentage is much lower in the circulating blood.
At first I thought it would be difficult to collect enough blood to
carry out estimations of ammonia and urea in the blood; in fact, my
attempts at taking out blood from the hearts and dorsal vessel by
means of an injecting needle and syringe were not successful. But
while collecting the coelomic fluid, I found that a quantity of blood
oozed out of the cut blood-vessels when dissected worms were left in
a glass dish. In order to obtain a sufficient quantity of blood in as
clean and pure a condition as possible, earthworms were dissected
and as much of coelomic fluid removed as possible; the wall of the
intestine was cut through to remove its contents, and then the
hearts were cut open and such cut worms were left in a petri dish.
The dish was kept in an inclined position in order to let the oozing
blood collect at the lowest part of the dish. In this way about 5 c.c.
of blood could be obtained in about two hours by cutting open
thirty-five to forty earthworms.
At first a few crystals of potassium oxalate were added to prevent
coagulation, but it was soon found that the blood of an earthworm
does not coagulate,1 so that after the first collection, no oxalate was
added for any of the subsequent estimations. Although all care was
taken to remove as much of the coelomic fluid as possible, the blood
obtained did contain a very small quantity of coelomic fluid.
For estimations 3 c.c. of blood was diluted with 21 c.c. of distilled
water, and then 40 c.c. of absolute alcohol were added to precipitate
the proteins. The fluid was then centrifuged and the supernatant
fluid filtered. As in the case of the coelomic fluid, ammonia was
estimated directly by Foreman's alcoholic distillation method, while
ammonia and urea together were estimated by the same method after
treatment of the fluid with urease. The difference between the two
estimations gave the value for urea.
5. T H E INITIAL PLACES OF EXCRETION : T H E BODY-WALL
AND THE INTESTINAL W A L L .
Having found that both the coelomic fluid and blood contain
ammonia and urea, the next question was to find out as to
where these excretory products came to the coelomic fluid and
1
It indicates the absence of fibrinogen.
EXCRETION IN INDIAN EARTHWORMS
359
blood from. Bearing in mind the fact that the body of an earthworm is made up essentially of two tubes, the body-wall and the
alimentary canal, the body-wall being concerned primarily with
locomotion and the alimentary canal with assimilation of food,
the natural presumption is that these would be the two main
seats of metabolism in the earthworm, and that excretory
products would be first formed at these two places. With
regard to the gut-wall, we may also bear in mind that the
intestine is thickly covered all over with chloragogen cells which
have been credited by Schneider (18) and others with hepatic
structure and function. Both the body-wall and the alimentary
canal are richly supplied with blood-vessels, and both of them
are also in immediate contact with the coelomic fluid; the
excretory products of metabolism formed in the gut-wall and
the body-wall would, therefore, be discharged either into the
blood or into the coelomic fluid or into both—it must be into
both, since ammonia and urea are constantly present in both
the fluids. It does not preclude the possibility that small
amounts of urea and ammonia may be formed as metabolic
wastes within the coelomic fluid and blood themselves.
Ammonia and urea were, therefore, estimated in both the
intestine and the body-wall, and the results obtained are given
in the following table:
TABLE
V. Comparative Amount of Ammonia and Urea
in the Intestine and Body-wall.
Intestine.
Body-wall.
•§• ft.
i,
fe-S
1.
2.
\
4.
5.
Average
3-0
3-0
3-0
3-0
3-0
2-49
3-60
4-76
6-12
4-76
4-01
4-41
4-39
6-00
4-39
3-0
4-34
4-64
fe-S
30
30
3-0
30
30
30
3-60
3-60
2-49
4-76
3-60
3-61
201
2-01
1-99
2-40
2-01
2-08
360
K. N . BAHL
It will be seen from this table that while the percentage
amount of ammonia excreted by the intestine is only slightly
higher than that excreted by the body-wall, urea excreted by
the intestine is more than twice that excreted by the body-wall.
Heidermanns (14) carried out estimations of ammonia and
urea in the intestine and body-wall of L u m b r i c u s and his
results are given below (Table VI) for comparison with my
results in Table V.
TABLE
VI. Comparative Amount of Ammonia and Urea in the
Intestine and Body-wall (After Heidermanns).
Intestine.
Ammonia
milligrams
per cent.
5-2
9-8
7-1
9-2
7-5
Urea
milligrams
per cent.
13-2 TT
28-8 U l e a S e
11-6 m e a 9-8/
16-5 ] Xanthydrol
12-0 /
urea.
Body-wall.
Ammonia
milligrams
per cent.
4-6
2-1
Urea
milligrams
per cent.
0-8
1-9
1-4
2-3
lUrease
/urea.
\ Xanthydrol
/urea.
It should be noted at once that Heidermanns's figures for
ammonia and urea in the intestine are very high indeed as
compared with mine.
Heidermanns's estimations show that urea formed in the
intestine is, on an average, t e n times as much as that formed
in the body-wall, while in my estimations the proportion is
2-2 : 1 . This discrepancy may be partly due to the fact that
P h e r e t i m a is a much more active worm than L u m b r i c u s
so far as body movements are concerned and hence the metabolism in the body-wall would be greater in P h e r e t i m a than
in L u m b r i c u s . L u m b r i c u s is a comparatively sluggish
worm, while P h e r e t i m a keeps moving about restlessly all
the time. This may account for the higher percentage of
ammoma and urea in the body-wall of P h e r e t i m a as compared with that of L u m b r i c u s , but it is difficult to explain
why the percentage of ammonia and urea is higher in the
EXCRETION IN INDIAN EARTHWORMS
361
intestine of L u m b r i c u s than in that of P h e r e t i m a .
Either Heidermanns did not precipitate the proteins completely or he allowed autolysis to take place before he made his
estimations. On looking through his figures for ammonia and
urea in the intestine, one cannot fail to notice that while his
figures for ammonia are more or less constant, those for urea
show a very wide variation indeed.
Heidermanns, on the basis of his estimations, held that ' the
chloragogen tissue is the c e n t r a l o r g a n of u r e a m e t a b o l i s m ' . According to my estimations also, the percentage
of urea is highest in the intestine (chloragogen tissue) as compared with that in the body-wall, blood, coelomic fluid, and
urine. There is no doubt, therefore, that the chloragogen tissue
is an important place, if not the central organ, of urea metabolism.
For estimation of ammonia and urea in the intestine and body-wall,
twenty-five to thirty fresh earthworms were dissected and the bodywall and intestine separated and washed. 3 gm. of each was weighed
and pounded with quartz sand with pestle and mortar. The paste
was mixed with a sufficient quantity of distilled Water (21 c.c), and
then 40 c.c. of absolute alcohol were added to precipitate all proteins.
The fluid was then centrifuged and filtered. Ammonia and urea were
estimated by Foreman's alcoholic distillation method as before.
6. THE E O L E OF THE NEPHRIDIA IN EXCRETION AND
OSMOTIC EEGULATION.
Having come to the conclusion that ammonia and urea are
first formed in the intestinal wall and the body-wall, that they
pass therefrom into the coelomic fluid and blood, and are
thence eliminated to the exterior, we shall now consider the part
played by the nephridia in eliminating these excretory substances from the coelomic fluid and blood, and in regulating
the osmotic relations of these internal fluids.
As already stated, P h e r e t i m a has two kinds of nephridia:
the closed integumentary and pharyngeal nephridia, and
the o p e n septal nephridia. Both kinds possess a long winding
intracellular canal which runs throughout the body of each
nephridium. In a septal nephridium (Text-fig. 3), the two
NO. 340
B b
362
K. N . BAHL
limbs—the straight and the twisted—are 225 //. and 480 \K
respectively in length, while the intracellular canal is as long
as 4-45 mm., i.e. more than six times the length of the two
limbs put together. Further, the canal has four ciliated tracts
b.c.t.
e.
a.'
TEXT-FIG. 3.
A septal nephridium of P h e r e t i m a p o s t h u m a showing the
course of the intracellular canal and its ciliated tracts, a-a', the
first ciliated tract; 6-6', the second; c-c', the third; aaAd-d', the
fourth ciliated tract; bet, the brown ciliated tube (phagocytic
section); /, funnel; si, straight lobe; tl, twisted loop with its
two limbs. (x dr. 120.)
in its course, and one can easily see in a live nephridium under
the low power of the microscope that liquid is driven down the
tube by the beating of the cilia of the nephridiostome and the
four ciliated tracts. In the closed nephridia there are only two
ciliated tracts (&-&' and G-C') and no nephridiostome, but the
BXCEBTION IN INDIAN EARTHWORMS
363
cilia in these ciliated tracts keep beating, as in an open nephridium, and drive the fluid down the tube. There seems little
doubt that the nephridia derive the fluid in their intracellular
canals from the coelomic fluid and blood. In the closed integumentary and pharyngeal nephridia the movement of the
cilia in the ciliated tracts probably sets up a slight pressure
which is enough to draw liquid by a process of nitration from
the blood and coelomic fluid, through the exceedingly thin walls
of the nephridium into the lumen of the intracellular canal. In
the open septal nephridia, however, the coelomic fluid plasma
passes directly through the nephridiostome into the intracellular canal of the nephridium, but the blood-plasma can be
extracted by filtration alone even by the septal nephridia.
In vertebrates and even in some of the higher invertebrates
the mechanism of renal secretion has been analysed into processes of filtration, reabsorption, tubular excretion, and chemical
transformation (23). We have to find out how far these processes can be detected and demonstrated in the nephridial
secretion of an earthworm.
We have referred above to the extremely long and much
coiled intracellular canal in the nephridium of an earthworm:
the glandular cells of the nephridium may remove something
from the blood and coelomic fluid, and add it to the liquid
contained within the lumen of the intracellular canal, or as
Kogers (17 a) has pointed out, the long nephridial canal ' may
serve as a means of conserving water which might otherwise
be lost to the organism'.
(A) The O s m o - r e g u l a t o r y F u n c t i o n of t h e
Nephridia.
In order to regulate the osmotic relations of the coelomic
fluid and blood, the excretory organs of the earthworm must
(1) keep the v o l u m e of these internal fluids more or less
constant, and (2) eliminate most of the basic and acid
r i d i c l e s formed in the body (23).
(i) V o l u m e - E e g u l a t i o n . Although an earthworm is a
terrestrial animal, it is still aquatic in its respiratory habit, as
we know that a certain amount of moisture is always necessary
864
K. N. BAHL
to keep its skin moist, or else the skin becomes desiccated and
the animal dies of asphyxia. Besides it must require an adequate
amount of water for its metabolic needs, considering that it
has two circulating fluids—blood and coelomic fluid—in its
body. An earthworm does not drink water through its mouth,
but it absorbs all the water it needs through its skin. It is well
known that earthworms can remain in water for months without
any harmful effects. Darwin quotes Perrier who kept large
worms alive for nearly four months completely submerged.
I myself kept seven sets, each of fifteen earthworms (P h e r e t i m a p o s t h u m a ) , submerged and starved in tap-water for
twenty-two days with only twelve casualties in all. Eustum
Maluf (16) also kept earthworms in water for several days, and
concluded from his experiments that earthworms are generally
capable of living indefinitely in fresh water. That water enters
the body of the earthworm by osmosis through its integument
has been conclusively proved by experiment. Adolph (1) found
that a group of large worms which were dug from wet ground
on a very warm day gained 15 per cent, in weight in 100
minutes after immersion in tap-water at 28° C. Eustum Maluf
(16) repeated Adolph's experiment and confirmed his observation. I have also repeated Adolph's experiment and found that
earthworms gained 7 to 16 per cent, in weight in tap-water in
seven hours, as shown in the graph on p. 365. Actually the gain
in weight must be more, since the earthworms defaecated a certain amount of earth during these seven hours which was not
taken into account in my weighings. In a second lot of five sets
of earthworms, with twelve worms in each set, I found that the
gain in weight was as much as 11-12 to 26 per cent, on keeping
them in tap-water for five hours. The defaecated earth was
again not taken into account.
In order to keep the volume of the internal fluids more or less
constant, these large quantities of absorbed water must be
eliminated, or else the internal fluids would become highly
diluted and the earthworm would burst through continual
absorption of water. Such a fatal result, however, seldom
occurs; in fact, the volume of the internal fluids as indicated
by the weight of the worms is kept constant, as is shown by the
Xpoq jo
TEXT-FIG. 4.
A graph showing changes in body-weight following immersion of
earthworms in tap-water. The initial weight of worms is taken as
100. During the first six or seven hours there is an increase of
weight by 7 to 16 per cent. It is worth noting that after the
first five or six days the worms keep a more or less constant
weight, varying only by 1*7 to 2-8 per cent, around the mean or
average weight.
366
K. N. BAHL
fact that when worms have been in tap-water for four to five
days and all the earth has been defaecated, they keep up more
or less a constant weight for the succeeding eight or nine days,
the weight varying only by 1-7 to 2-8 per cent, around the mean
or average weight, thus setting up, so to speak, a new equilibrium in tap-water (Text-fig. 4), wherein the water absorbed
and the water excreted balance each other. The question arises
as to how the large quantity of absorbed water is eliminated by
the earthworm. When an earthworm ( P h e r e t i m a ) , which
has been in tap-water for several days and whose gut has been
thoroughly cleaned, is mopped with a dry towel and examined
under a binocular dissecting microscope, it is seen that the skin
soon becomes wet on account of the secretion of urine through
the innumerable nephridiopores of the integumentary nephridia,
a watery drop is ejected through the anus and next a drop from
the mouth, and that water is ejected more frequently and therefore more copiously through the anus than through the mouth.
Adolph (1) could not see the fluid ejected through the nephridiopores of L u m b r i c u s but could see the watery discharge
through the anus; Eustum Maluf (1@) could see clear colourless
liquid spurting and oozing out of the nephridiopores and
flowing into intersegmental furrows, and inferred the discharge
of water through the anus and mouth from his weighing experiments only; but I have been able to see water being discharged
through all these three openings. By Iigating the earthworms
at one or both ends and finding an increase as well as a decrease
in weight, Eustum Maluf concluded that 'when a worm is first
introduced into tap-water, its gut is of paramount importance
in osmo- and volume-regulation, but that there is a definite
volume-regulative tendency on the part of the kidneys (nephridia) also, which, in t h e n o r m a l w o r m , 1 is however, completely masked by such a function on the part of the alimentary
tract'.
I have repeated Eustum Maluf's experiment of Iigating
ends of the worms and have confirmed bis observation
there is a distinct decrease in weight, but my conclusion
this observation is different. It must be remembered
1
The spaced words are mine.
both
that
from
that
EXCRETION IN INDIAN EARTHWORMS
367
immersion in water is not the normal environment for an earthworm. Adolph rightly concluded that 'in their usual environment, moist ground, earthworms are partially desiccated'.
Bustum Maluf did not observe the exudation of fluids in earthworms freshly taken from the soil. If an earthworm (P h e r e t i m a p o s t h u m a ) is taken direct from the soil, washed and
mopped with a dry towel, and then observed under a binocular
microscope, it is seen that although the skin becomes moist on
account of exudation from the numerous nephridiopores, and
there is a small watery discharge from the mouth as the worm
protrudes its buccal chamber, there is no w a t e r y d i s c h a r g e
at all from the anus—only more or less solid faecal pellets being
defaecated at intervals. The discharge on the skin is apparently
through the integumentary nephridia, and that from the mouth
through the pharyngeal nephridia and the 'salivary glands',
but the copious discharge through the anus is completely
absent. Thus it is clear that in an earthworm living in the soil
the gut does not excrete water and so takes practically no part
in osmo- and volume-regulation. My conclusion, therefore, is
that in its usual environment, the nephridia of the earthworm
function adequately as volume- and osmo-regulatory organs as
they have to deal only with a small quantity of metabolic water
and water normally absorbed by the skin for respiratory and
general metabolic needs of the body. But when an earthworm
is placed in water for several hours, the skin absorbs large
quantities of water which cannot be eliminated by the nephridia
alone, and then the gut comes to their rescue, so to speak, and
takes a prominent part in osmo- and volume-regulation by
eliminating through the anus the large quantity of water
absorbed through the skin.
In its normal environment the amount of water absorbed
by an earthworm is small: the worm is never fully hydrated or
as Adolph puts it, it is partially desiccated; in a form like
L u m b r i c u s , with open exonephric nephridia, the water of the
coelomic fluid plasma passing freely into the nephridial canal
and that of the blood filtering into it are probably reabsorbed
by the glandular cells of the nephridium and also-by the wall
of its terminal bladder. Delaunay's observation that urine is
868
K. N. BAHL
excreted from the terminal bladder only once in three days
lends support to my conclusion. But in a form like P h e r e t i m a with enteronephric nephridia the loss of water is completely reduced, as the greater part of the nephridial fluid is not
discharged to the outside but passes into the gut which effectively absorbs a large part of the water. The part of the
nephridial fluid which is discharged to the outside directly is
excreted by the integumentary nephridia which have no
terminal bladders. By estimating the percentage of moisture
in the fresh 'castings' (faeces) of P h e r e t i m a and E u t y p h o e u s and always finding the percentage higher in E u t y p h o e u s than in P h e r e t i m a , I have already (4) shown that
the gut of P h e r e t i m a is much more efficient in absorbing
water than the gut o f E u t y p h o e u s which possesses exonephric
nephridia like those o f L u m b r i c u s .
We have already seen that ammonia and urea form the main
bulk of nitrogenous excretion of the earthworm and that these
are excreted in low concentration (Table II). A certain minimum amount of water must be excreted to eKminate even these
low amounts of excretory products. In a form like L u m b r i c u s or E u t y p h o e u s , therefore, in its normal environment, the necessary amount of water is excreted and the rest
conserved by the nephridia, and the gut takes practically no
part in water-conservation, as is shown by the fact that its
vermicelli-like loosely semi-solid castings contain a large percentage of water. But in a form like P h e r e t i m a , the
nephridia of which lack a terminal bladder, the task of waterconservation is taken over largely by the gut into which the
nephridial fluid is discharged and which gives off solid pelletlike castings, with a comparatively small percentage of water.
It seems, therefore, that an earthworm, when submerged in
water, can live like a freshwater animal, like its freshwater
allies, absorbing water through its skin and eliminating it
largely through its gut and partly through its-nephridia. As
there is abundance of water, there is no need of conservation of
water. But in its normal environment, moist earth, an earth-,
worm is partially desiccated, and conservation of water is of
great importance to it as it is to a terrestrial animal; the water
EXCRETION IN INDIAN EARTHWORMS
369
is conserved by the nephridia and their bladders in L u m b r i c u s , and by the nephridia and the gut together in
Pheretima.
(ii) The E a t e of E x c r e t i o n of Urine.—Considering
that the rate of excretion of urine would throw light on volumeregulation, I measured the rate at which urine is excreted by
earthworms, as is shown in Table VII.
It will be seen from this table that I could collect 3-7 c.c. to
7-2 c.c. of urine in three and a half hours. When it was raining
and the humidity in the atmosphere was high, the quantity of
urine was very much more than that obtained on a dry day,
because there was little evaporation from the body-surface of
earthworms and the urine itself. In the first ten minutes, the
quantity of urine excreted is at its maximum, but it goes on
decreasing in successive ten minutes. In experiment 3, the
volume of urine collected was 6-5 c.c, while the weight lost by
earthworms was 10-16 gm., which is accounted for by the
evacuation of faeces and the evaporation of moisture from the
body-surface of earthworms during the period of collection of
the urine. The volume of urine c a l c u l a t e d for twenty-four
hours comes to 49-2 c.c, i.e., about 45 per cent, of the weight of
the body. In the experiment the excretion of urine slows down
considerably after an hour and a half, and earthworms have to
be kept in water again for some time before we can get urine
out of them a second time. But there is little doubt that when
earthworms are continually kept in water, the intake and outflow of water in twenty-four hours must be much more than
45 per cent, of the body weight. It is clear that earthworms in
water excrete urine very largely from the water absorbed by
them through their skin, and that the quantity of metabolic
water is very small indeed. From experiment 8, one can
calculate that a fully hydrated earthworm will excrete at least
0-82 c.c. of urine in twenty-four hours, the average weight of
an earthworm being 1-6-1-8 gm.
Earthworms for collection of urine had been kept in water for
three to four days to get rid of most of the earth from the gut. Fresh
earthworms from the soil excrete very little urine and even that is
difficult to collect as it gets mixed with the faeces. It would be
72
70
72
70
65
60
65
60
75
1.
2.
3.
4.
5.
6.
7.
8.
9.
—
—
—
0-3
0-2
Nil.
Nil.
—
—
"Will
IN 11.
—
—
1-7
0-8
0-55
0-5
one drop
Volume of
urine exNumber Number creted in
of
of 10 minutes
Expt. worms.
in c.c.
4-8
4-1
5-1
3-7
7-2
6-9
4-3
4-0
6-5
Total
volume
collected
in c.c.
3
3
4
3
3
3
Hours
3
2
3
30
30
30
30
30
45
minutes
35
50
10
32-9
26-6
27-2
25-3
49-37
44-16
28-8
33-8
49-2
Excreted
volume calculated for 24
hours in c.c.
104-71-96-49
100-18-89-87
93-27-82-97
98-99-89-01
Not recorded.
Not recorded.
108-26-97-06
106-86-98-99
109-36-99-20
27-5-29
28-30
28-8-32
28-5-31-0
27-29
26-5-28-5
25-5-28
Weight of earthworms before and
after collection Temperature
in gm.
in°C.
VII. Bate of Excretion of Urine.
Period in
which collected
in hours.
TABLE
Cool rainy
day.
Morning
cool and
humid due
to a little
shower.
Remarks.
O
OS
EXCRETION IN INDIAN EARTHWORMS
371
interesting if one could collect a sufficient quantity of urine from
worms fresh from the soil and compare this quantity with that
collected from worms progressively subjected to exposure and
desiccation.
(iii) T h e O s m o t i c P r e s s u r e of B l o o d , Coelomic
F l u i d , a n d Urine.—In order to form an idea of the role of
the nephridia in regulating the osmotic relations of the internal
Z2S&.
C
O
^ : ^ ^
BLoT^SSEL
.ux»*.O^-O*fc,
MVPOTONIC URINE
(A»O-O5ff-OOS8*C)
TEXT-ETG. 5.
A diagrammatic representation of the body of P h e r e t i m a
p o s t h u m a showing the depression of the freezing-point of
the different fluids of its body (plan adapted from Bustum Maluf).
fluids, I determined the osmotic pressure of blood, coelomic
fluid, and urine by measuring the depression of the freezingpoint of each by Beckmann's method, and my results are as
follows:1
VIII. Depression of the Freezing-point of the
posthuma.
Fluids of Pheretima
1. Blood-plasma
A = 0-40° 0.-0-50° C.
2. Coelomic fluid-plasma A = 0-285° C.-O-310 0.
3. Urine .
.
. A = 0-050° 0.-0-065° C.
TABLE
These figures are diagrammatically represented in Text-fig. 5.
The depression of the freezing-point (A) is a measure of the
molecular concentration, and therefore of the osmotic pressure
of a solution. Bustum Maluf (16) gives 0-45° C. as thefigurefor
tne depression of freezing-point of the blood of L u m b r i c u s ,
1
I am indebted to Mr. M. Raman Nayar of the Chemistry Department
for determining the depression of the freezing-point of these fluids for me.
372
K. N. BAHL
while Adolph (1) gives 0-31° 0. as the figure for the depression
of freezing-point of the body juice o f L u m b r i c u s . My figures
are in agreement with theirs. The water content of the body of
P h e r e t i m a i s a variable factor and this is apparently reflected
in the slight variation in the osmotic pressures of the three
fluids. As far as I know, no worker has so far estimated the
depression of the freezing-point of the urine of an earthworm, as
no one was able to obtain it in sufficient quantity. From the
figures of the depression of the freezing-point given above, it
will be seen that the coelomic fluid is h y p o t o n i c to the blood,
and that the urine is markedly h y p o t o n i c to both the
coelomic fluid as well as the blood.
The difference in the depression of the freezing-point between
blood and coelomic fluid is very striking and forms an interesting osmotic problem by itself. How is the blood maintained
hypertonic to the coelomic fluid, and what are the factors
responsible for it? As we shall see presently (vide
infra),
the blood has a higher protein content but a lower chloride
content than the coelomic fluid, so that these two factors
cannot account for the whole story. It seems that a detailed
chemical analysis of blood as well as coelomic fluid is called for
to solve the question of the difference in osmotic pressure
between these two fluids.
Samples of blood and coelomic fluid for the determination of the
depression of freezing-point were obtained in as pure a condition as
possible. Samples of urine were taken from worms which had been
in water for some days and had been, so to speak, living like freshwater animals, eliminating water in large quantities through the gut
as well as through the nephridia.
(iv) The P r o t e i n C o n t e n t s of B l o o d , C o e l o m i c
F l u i d , a n d U r i n e . — It has been proved conclusively that in
the amphibian kidney the fluid passing from the glomerular
capillaries into the Bowman's capsule is a protein*free filtrate
practically identical with the blood-plasma except for its
colloids (i.e. proteins and fats), and that it passes out of the
capillaries as a result of the purely physical process of filtration.
It was, therefore, considered advisable to estimate the protein
373
BXCBBTION IN INDIAN EARTHWORMS
contents of the blood, coelomic fluid, and urine of the earthworm
to find out if its urine was really a protein-free nitrate. My
estimations of proteins are as follows:
TABLE
IX. Protein Contents of the Fluids of Pheretima.
Blood-plasma.
Coelomic fluidplasma.
Urine.
gm. per 100 c.c.
gm. per 100 c.c.
gm. per 100 c.c.
1.
2.
3.
4.
3-457
3-949
3-757
3-376
0-550
0-479
0-458
0-429
0-025
0-029
0-036
Average
3-643
0-479
0-030
In man the blood-plasma protein concentration is 6-5-8-5 gm.
per 100 c.c, i.e. about double that of the earthworm.
It will be seen from this table that the protein content of the
blood-plasma is seven to eight times that of the coelomic fluidplasma and that the urine is not protein-free, since it contains
measurable traces of colloidal proteins. Picken (17) has found a
similar condition in the urine of Arthropods studied by him and
says: ' An examination of the urine has shown almost certainly
in C a r c i n u s and possibly in P o t a m o b i u s and P e r i p a t o p s i s , that it contains a little protein.' He traces these
proteins in the urine of C a r c i n u s either to proteins derived
from blood, or to cell breakdown in the kidney, or to mucus.
In the earthworm the mucus secreted by the skin, and the
mucin and the proteolytic enzyme of the saliva discharged
through the mouth may account for the traces of proteins found
in the urine. If this supposition be correct, then we can hold
that the urine is really a protein-free nitrate, but that traces of
protein find their way into it from these sources during the
process of collection of the urine.
In an earthworm the nephridia must derive their urinary
f lid both from the blood and the coelomic fluid. On an analogy
with what has been proved in the case of the amphibian kidney
we may presume that the part of the urine derived from the
blood is really a protein-free filtrate, filtered from the blood-
374
K. N. BAHL
capillaries of the nephridia, as through a semi-permeable membrane, but this cannot be true of the part of the urine derived from
the coelomic fluid. As the coelomic fluid-plasma passes directly
and freely into the septal nephridia through their open funnels, it
must contain colloidal proteins in the concentration of about 480
mg. per 100 c.c, and since proteins are too valuable to the earthworm to be allowed to be lost with the urine, we must presume
that the cells of the nephridia keep on reabsorbing the proteins
of the coelomic fluid-plasma as it passes through the nephridia
as urine.
In a form like P h e r e t i m a in which the urine of the septal
nephridia is discharged into the intestine, it is probable that any
proteins still left over in the urine after their reabsorption by
the nephridia would be reabsorbed by the intestine, but in a
form like L u m b r i c u s in which the urine passes out directly
through the nephridia, the nephridia alone must be reabsorbing
efficiently all the proteins passing into them in the coelomic
fluid-plasma. The protein estimations were made on fluids
collected from earthworms which had been in water for several
days and were living like freshwater animals, eliminating as
much water as they were absorbing. The fact that protein
concentration of the coelomic fluid-plasma is about 16 t i m e s
that of the urine strongly supports the conclusion that proteins
are reabsorbed by the nephridia.
The average percentage proportion of the protein contents of
the three fluids works out as—blood plasma 100: coelomic fluidplasma 13-1: urine 0-82. The difference between the protein
contents of the blood-plasma and coelomic fluid-plasma is considerable, but there is no doubt that there is a large amount of
protein matter contained in the corpuscles of the coelomic fluid,
while the blood has very few corpuscles in it, and all its proteins
are suspended in a colloidal state. The differences in the osmotic
pressures of the three fluids cannot be due to their protein
contents, as the protein osmotic pressure in any case must be
very small indeed. In man it is 25 mm. Hg; in the earthworm it
will be only about 12 mm. Hg.
Each fluid (blood, coelomic fluid, or urine) was centrifuged and
10 c.c. of the supernatant fluid was treated with 70 c.c. of strong
EXCRETION IN INDIAN EARTHWORMS
375
alcohol to precipitate all the proteins. The liquid wasfilteredthrough
a previously weighed filter-paper and the precipitate was washed
several times with warm distilled water. The precipitate on the
filter-paper was then dried and weighed several times till the weight
was constant. This weight minus the original weight of the filterpaper gave the weight of the proteins in 10 c.c. of each fluid, from
which the weight per cent, was calculated.
(v) The C h l o r i d e C o n t e n t s of B l o o d , Coelomic
F l u i d , a n d Urine.—Since a qualitative analysis had shown
the presence of chlorides in all the three fluids, it was thought
that a quantitative estimation of the chloride contents of the
blood, coelomic fluid, and urine would throw light on the differences in the osmotic pressures of the three fluids. Further, a
comparison of the chloride contents of the three fluids would
give us an idea of the reabsorption of chlorides, if it occurs,
within the nephridia. In order to form as accurate an idea as
possible of the reabsorption of chlorides, worms were kept in
oxygenated distilled water for six days, so that all the earth in
the gut had been defaecated and the worms were living like
freshwater animals, eliminating as much water as they were
absorbing. In such worms there was no question of conservation
of water by its reabsorption by the nephridia and the gut, and
therefore a comparison of the concentration of chlorides in the
three fluids would indicate directly the amount of reabsorption
of chlorides. The results of chloride estimations are as follows:
TABLE
X. Chloride Contents of the Fluids of Pheretima.
Blood-plasma.
Coelomic fluid-plasma.
Urine.
(mgm. per 100 c.c.)
(mgm. per 100 c.c.)
(mgm. per 100 c.c.)
79-26
77-23
81-30
79-26
3-556
3-862
1.
2.
3.
45-80
50-82
51-42
Average
49-35
3-7
In terms of NaCl the proportions work out roughly to blood
82 : coelomic fluid 132 : urine 6. From these figures two important conclusions can be readily drawn: (1) that there is a
reabsorption of chlorides on a large scale by the nephridia, and
376
K. N. BAHL
(2) that the chloride content cannot account for the higher
osmotic pressure of the blood, since the chloride content of
blood is decidedly lower than that of coelomic fluid. It is evident
that some other factor or factors are involved which need
further investigation.
The method followed for chloride estimations is that recommended
by Cole, 1942 (p. 378). 10 c.c. of each fluid was diluted with 70 c.c.
of distilled water, and treated with 10 c.c. of 10 per cent, sodium
tungstate and then with 10 c.c. of 2/3 normal sulphuric acid to
precipitate the proteins. The filtrate (coloured yellow in the case of
blood, pale yellow in the case of coelomic fluid, and colourless in the
case of urine) was titrated with acid silver nitrate (N/50) and ammonium thiocyanate (N/50) as directed by Cole. The results obtained
by this method were confirmed by the ignition method as described
by Skinner.1 10 c.c. of eachfluidwas treated with 20 c.c. of 5 per cent,
sodium carbonate and evaporated to dryness in a platinum dish and
then ignited at dull red heat. The product was extracted with hot
water andfilteredthrough ashlessfilter-paperand the residue ignited
a second time. The ignition product was extracted with dilute
nitric acid and added to the main nitrate. The combined filtrate
was titrated with acid silver nitrate and thiocyanate as before.
I am deeply indebted to Mr. M. Eaman Nayar of the Chemistry Department who has taken great pains in making these
estimations for me.
(vi) Conclusions.—Taking into account the observations
and their interpretations as recorded under the preceding five
sub-headings, the conclusions arrived at may now be summarized
as follows: (1) That the part of urine which is excreted from the
blood is probably a protein-free filtrate, but that the coelomic fluidplasma entering the nephridia through funnels must contain
proteins suspended in a colloidal form and these proteins are reabsorbed by the nephridia. (2) That minute traces of protein
present in the urine as finally collected probably come from the
mucus secreted by the skin, and the proteolytic enzyme secreted
by the salivary gland. (3) That the urine as finally excreted and
collected is h y p o t o n i c to both the blood and the coelomic
fluid. (4) That there is a reabsorption of the chlorides on a large
1
Skinner and others—' Official and Tentative Methods of Analysis of the
Association of Official Agricultural Chemists' (Washington, 1935).
EXCRETION IN INDIAN EARTHWORMS
377
scale from the initial nephridial filtrate during its passage
through the nephridia. (5) That in the normal environment, in
a form like E u t y p h o e u s or L u m b r i c u s , the nephridia
are concerned with nitrogenous excretion, water-conservation,
and protein and salt reabsorption, but when the worm is kept
in water the gut takes a prominent part in the regulation of
water exchange by excreting water through the anus and the
mouth. In enteronephric forms, like P h e r e t i m a , however,
the nephridia and the gut together are normally concerned with
water-conservation. (6) That the osmotic relations of the
coelomic fluid and blood are regulated (a) by keeping the volume
of these fluids more or less constant through the nephridia when
worms are in soil, and through the nephridia and the gut when
the worms are in water, and (b) by eliminating acid radicles
like chlorides and phosphates, and basic radicles like sodium,
calcium, potassium, and magnesium through the nephridial
fluid. (7) That the higher osmotic pressure of the blood as
compared with that of the coelomic fluid cannot be accounted
for by the chloride content alone, since it is actually lower in
the blood than in the coelomic fluid. Further investigation on
this point is called for.
(B) The E x c r e t o r y I n c l u s i o n s of t h e ' C i l i a t e d
Middle T u b e ' ( A t h r o p h a g o c y t i c Section) of
the Nephridia.
It is well known that in the nephridium of L u m b r i c u s the
so-called 'ciliated middle tube' and the adjoining ampulla have
a brownish semi-opaque appearance due to the presence of
brownish granules within the nephridial cells surrounding the
ciliated tract. Even in preserved specimens of L u m b r i c u s ,
which alone have been available to me, one can easily see the
opaque brown colour of the ciliated middle tube. Schneider
(18), on the basis of bis injection experiments, called this area
the p h a g o c y t i c s e c t i o n of the nephridium. Cuenot (IE)
confirmed Schneider's observation and found that the middle
tube alone was stained by physiological injections. Schneider
recognized this phagocytic section in the nephridia of all the
Oligochaeta investigated by him except in P h e r e t i m a , and
NO.
340
cc
378
K. N . BAHL
'Phagocytare Abschnitte in den Nephridien fehlen bei
P e r i c h a e t a ( P h e r e t i m a ) ' . Unfortunately Schneider
made a mistake here, as the phagocytic section is as clearly
present in the septal nephridia of P h e r e t i m a (Text-fig. 6)
as in those of other Oligochaetes. I have found that the phago-
A
TEXT-ITG. 6.
A. A microphotograpb. of a few septal nephridia of P h e r e t i m a
p o s t h u m a (X cir. 50). The elongated black areas mark the
heavy deposits of brownish yellow granules in the 'ciliated
middle tube' (athrophagocytic section). B. A section passing
through two nephridia, showing deposits of brownish yellow
granules in the ciliated middle tubes ( x cir. 275). nph, nephridium; peg, deposits of pigmented excretory granules.
cytic ciliated tract is present also in the septal nephridia of
E u t y p h o e u s (6), L a m p i t o (3), H o p l o c h a e t e l l a (6),
and T o n o s c o l e x (5), and is yellowish-brown or even blackish
in colour. The granules are stored within the cytoplasm of the
cells of the nephridia surrounding the ciliated canal and are
apparently harmless storage excretory products; they can be
clearly seen in whole mounts (Text-fig. 6 A) as well as in sections of septal nephridia (Text-fig. 6 B). It is noteworthy that
EXCRETION IN INDIAN EARTHWORMS
379
the deposits in P h e r e t i m a and T o n o s c o l e x are much
heavier than in L u m b r i c u s . In P h e r e t i m a the deposit
is heaviest at the beginning of the ciliated canal and becomes
progressively lighter towards the distal end of the canal, thus
forming a gradient of thickness of the deposit. Further, the
deposit appears in the form of a linear series of cylindrical rings,
appearing like the nodes' and internodes of a bamboo stem.
The determination of the chemical nature of these excretory
granules has been a subject of great difficulty and I have spent
a considerable amount of time and labour on it. I am stating
my conclusions as follows:
(1) T h a t t h e P i g m e n t e d E x c r e t o r y G r a n u l e s are
not Guanine.
Willem and Minne (22) who tested these brownish granules
in the nephridia of L u m b r i c u s came to the conclusion that
they were granules of g u a n i n e . This statement has never
been questioned but has been implicitly accepted by subsequent
workers, and has been incorporated as such both by Stephenson
(19) and Stolte (20). Willem and Minne relied exclusively on
solubility tests, and say that 'the granules resist the action of
alcohol, ether, chloroform, and ammonia, but are soluble in
potash and hydrochloric acid—these chemical c h a r a c t e r s c o r r e s p o n d to g u a n i n e ' . (The spaced words are
mine.) I have repeated these solubility tests of Willem and
Minne, and find that my results are greatly at variance with
theirs. For these solubility tests I have used the nephridia of
P h e r e t i m a as well as those of L u m b r i c u s , and am giving
my results in Table XL
Taking Willem and Minne's second statement first, that the
granules are soluble in potash and hydrochloric acid, I find that
although the granules are soluble in 2 per cent, caustic soda or
potash, they are a b s o l u t e l y i n s o l u b l e in h y d r o c h l o r i c
a c i d . I have tried all strengths of this acid, even concentrated, in cold and also boiling 5 per cent, and 2 per cent,
hydrochloric acid—still the granules will not dissolve. If the
granules were of guanine, they would dissolve in 5 per cent,
boiling hydrochloric acid forming guanine-hydrochloride. I
Sol. in
11 days.
Insol.
Sol.
(within 24
hours).
HOI
6
Solubility in
5
KOH
2
7
Sol. in
5 days.
Glacial
5
Acetic
Ammonia. per cent. per cent. Acid.
Sol.
Insol.
Sol. in
Sol. in
11 days. (within 24
5 days.
hours).
4
9
10
Glycerine
Acetone. per cent.
Insol.
Insol.
although
the colour
fades.
Do.
Insol.
8
* The granules fade in colour after fifteen days, but are not dissolved even after twenty-six days.
Insol. * Sol. in
32 days.
Chloroform.
Sol. in
32 days.
Lumbricus. Sol. in
30 days.
of the
3
Ether.
Insol,*
Alcohol
Inclusions
2
(Rectified
'Ciliated
Middle Tube.' spirits).
Pheretima. Sol. in
12 days.
1
TABLE XI.
Sol. (within
24 hours).
Pyridine.
Sol. (within
24 hours).
10
EXCRETION IN INDIAN EARTHWORMS
381
tried pure guanine as a control and found that it readily dissolved in 5 per cent, boiling hydrochloric acid. Coming now to
Willem and Minne's first statement that the granules resist the
action of alcohol, ether, chloroform, and ammonia, I am afraid
I find that even this statement of theirs is not quite correct.
Although the pigmented granules are insoluble in ether, they
dissolve, though very slowly, in ammonia, alcohol, and chloroform as shown in the table on p. 380. Further, they also
dissolve slowly in glacial acetic acid. If the granules were of
guanine, they would be precipitated by both ammonia and
acetic acid instead of being dissolved in them.
As a further test I took a very large number of the nephridia
of P h e r e t i m a and kept them in 5 per cent, hydrochloric acid
at 65° C. for twenty-four hours. The pigmented granules, of
course, showed no sign of solution, but in order to make sure if
there was even a trace of guanine dissolved in the hydrochloric
acid, I filtered the material and allowed the filtrate to evaporate.
If there had been any guanine in the filtrate (hydrochloric
acid) I should have got needle-like crystals of guanine hydrochloride. But no such crystals were formed, showing that there
was no guanine in the pigmented granules which could dissolve
in hydrochloric acid.
These reactions, therefore, with hydrochloric acid, ammonia,
and acetic acid prove that the pigmented granules in the middle
tube of the nephridia of L u m b r i c u s and P h e r e t i m a are
n o t of g u a n i n e .
(2) T h a t t h e P i g m e n t e d E x c r e t o r y G r a n u l e s are
n o t U r i c Acid or U r a t e s .
The fact that these excretory inclusions form an apparently
amorphous pigmented deposit, soluble in alkalies but insoluble
in hydrochloric acid, led me to suspect that they may be deposits of uric acid or urates. In fact, the deposit closely
resembles the deposit of amorphous urates as figured in books
of clinical medicine (13). I therefore tried again and again the
murexide test for uric acid or urates not only on the nephridia
on the slide, but also on the caustic soda and sodium carbonate
solutions of the excretory material. In none of these trials
882
K. N. BAHL
could I get a positive murexide test, showing that the deposits
are n o t of uric acid or urates.
For a time I thought that possibly the quantities are too
small for giving the murexide test. I, therefore, tried with a
large quantity of nephridial material with the same negative
result. Bearing in mind the fact that a small crystal of uric
acid is enough to give the murexide test on a cavity slide, I have
no doubt that, had there been any uric acid in the nephridial
granules, I should have got the murexide test with the large
quantities of material I used time after time for this test.
(3) T h a t t h e P i g m e n t e d E x c r e t o r y G r a n u l e s are
of B l o o d - p i g m e n t ( h a e m o c h r o m o g e n ) .
Besides alkalies like caustic soda and sodium carbonate,
pyridine1 quickly dissolves the excretory granules. Pyridine is
a colourless liquid solvent, and I found that when nephridia of
P h e r e t i m a with heavy deposits of pigmented granules were
kept in it, the deposits were dissolved within twenty-four hours,
giving pyridine a brownish red colour. Large quantities2 of
nephridial material were used to get a satisfactorily intense
colour of pyridine. The coloured pyridine was then subjected
to spectroscopic examination. Two clear bands could be seen.
The band near the D line (the a-band) is narrow with its centre
about 5579 A.U., i.e., about mid-way between the D and E
lines. The /2-band is broad and has its centre about 5267 A.U.
The actual figures are:
AA 5659-5500; centre 5579 A.U.
First Absorption Band:
Second Absorption Band: AA 5405-5130; centre 5267 A.U.3
These figures are identical with those of the two bands of
h a e m o c h r o m o g e n . A photograph of the absorption spectrum of the pyridine solution of the granules is shown in Textfig. 7. These spectroscopic bands, therefore, conclusively prove
1
I am indebted to Dr. S. B. Dutt of the University of Allahabad for
suggesting this solvent for a spectroscopic examination of the material.
2
I am thankful to Messrs. V. G. Jhingran and S..D. Misra for dissecting
out and collecting a large quantity of septal nephridia for making a pyridine
solution.
3
I am indebted to my friends Dr. D. B. Deodhar and Dr. P. N. Sharma
for making these spectroscopic examinations for me.
EXCRETION IN INDIAN EAETHWOHMS
that the excretory granules consist of the blood-pigment, as
their pyridine solution gives the two bands characteristic of
haemochromogen.
The pyridine solution was made from septal nephridia taken
from earthworms ( P h e r e t i m a ) preserved in formalin. It
was at once suspected that pyridine may have dissolved the
blood-pigment from the minute capillaries and blood-vessels
TEXT-FIG. 7.
A photograph of the absorption spectrum of a pyridine solution of
the pigment granules of the septal nephridia of P h e r e t i m a
p o s t h u m a , showing the two typical absorption bands of haemochromogen. The spectrum of iron arc accompanies that of
pigment granules in order to locate the exact position of the bands.
that necessarily accompanied the nephridia, when they were
kept in pyridine. But examination under the microscope
clearly revealed that while the pigmented excretory granules
had been completely and quickly dissolved, the blood-vessels
and capillaries retained their full red colour and had apparently
suffered no change. But in order to make the assurance doubly
sure, I kept pieces of blood-vessels only from the same material
for a m o n t h in pyridine and examined this pyridine solution
spectroscopically. Although a very faint band (the a-band)
could be seen, nothing of the /3-band was visible. The septal
nephridia had never been kept in pyridine for more than
twenty-four hours. As soon as the pigment granules got dissolved, this lot of septal nephridia was taken out and a fresh lot
put in, and so on, until the pyridine had an orange-red colour.
I have no doubt in my mind, therefore, that the haemochromogen in the pyridine solution of septal nephridia came from
384
K. N. BAHL
the pigmented excretory granules and n o t from the bloodvessels.
The question naturally arises whether these pigmented granules
are only of haemochromogen or whether there is some other
substance accompanying them. There seems no doubt that
haemochromogen must ultimately come from blood, and it leads
one to the conclusion that the glandular cells surrounding the
ciliated tract abstract haemoglobin from the blood, transform it
to haemochromogen, and store it there. There is one important
consideration which makes it possible that there is some other
substance along with haemochromogen in the pigmented excretory granules, a substance which probably comes from the
coelomic fluid. It has been noticed by me time after time that
the pigmented granules are completely absent in the closed
integumentary and pharyngeal nephridia, and are present
o n l y in the open septal nephridia, into which alone the plasma
of the coelomic fluid enters directly through their nephridiostomes. The possibility is that as this plasma passes through
the nephridiostome into the intracellular canal of the nephridium, it carries with it extremely minute solid granules which
may be very finely divided broken bits of corpuscles or yellow
cells—in fact, any particles which can go through the very fine
and efficient sieve of the cilia of the nephridiostome; these
particles are taken up by the ciliated cells of the middle tube
and stored there along with haemochromogen extracted from
the blood-capillaries. In this connexion it is pertinent to recall
the injection experiments of Cordier (11 a) who injected colloidal
fluids of varying degrees of dispersion into the coelomic fluid
and found that they collected in the walls of the ciliated middle
tubes of the nephridium. Cordier believed that, like his artificial
colloidal fluids, natural colloidal solutions and solid particles
are absorbed by the ciliated tract and are deposited in the form
of brown granulations, and he, therefore, recognized the
function of the cells of the ciliated middle tube as a t h r o p h a g o c y t o s i s (Gk. a t h r o i s , to collect). Cordier says that
particles taken up by the athrophagocytic cells are retained for
two months and even longer; I have no doubt that once they
are taken in, they are retained throughout life. Cordier pro-
EXCRETION IN INDIAN EABTHWORMS
385
bably did not continue his observations longer than two months.
The fact that blood-pigment granules are present only in the
septal nephridia and not in the integumentary and pharyngeal
ones can also be explained by the possibility that the ' ciliated
tract' is athrophagocytic only in the septai nephridia and not in
the integumentary and pharyngeal ones.
I have carefully observed that the pigmented granules are
very sparse in the nephridia of young earthworms, but the
deposit becomes heavier and heavier as the earthworm grows
in age. In fact, one would be justified in saying that the septal
nephridia of P h e r e t i m a and other earthworms function as
'storage kidneys' which go on storing these brownish yellow
granules throughout life. Further, the fact that pigmented
granules dissolve so quickly in pyridine while the blood takes
a very long time indicates that the pigment in the granules is
haemoglobin which has already been alkalined and reduced, and,
therefore, dissolves quickly in pyridine to give the absorption
bands of haemoehromogen. It is probable that pyridine dissolves out only haemoehromogen and leaves the other substance,
if there is any, behind in the nephridia.
We have already discussed the processes of nitration and
reabsorption as they occur in the nephridial secretion of
the earthworm (Chapter 6 A); the storage of blood-pigment
granules with or without another substance in the walls of the
nephridial tubules demonstrates the process of chemical transformation (p. 363), whereby the products of blood destruction
are rendered innocuous and stored within the nephridial cells.
7. EXCRETORY ORGANS OTHER THAN NEPHRIDIA.
Just as the athrophagocytic section of the nephridium stores
up the destruction products of blood, it is reasonable to expect
a similar organ in the body of the earthworm to deal with
similar products of coelomic fluid, which contains several
kinds of innumerable corpuscles. Schneider (18) found in all
the species of Oligochaeta investigated by him d o r s a l phagocytic organs with the function of cleansing the coelomic fluid
of dead particles. P h e r e t i m a possesses these phagocytic
organs as white fluffy bodies situated on either side of the
386
K. N. BAHL
dorsal vessel, and attached to it, from the twenty-sixth segment
backwards. I have recently (8) described paired v e n t r a l
phagocytic organs in M e g a s c o l e x t e m p l e t o n i a n u s ;
this earthworm has no funnelled nephridia at all in its body and
its closed integumentary and pharyngeal nephridia do not
possess a phagocytic section in them. But it has very large
ventral phagocytic organs which are richly supplied with blood;
it is possible that these phagocytic organs or 'septal sacs' of
this earthworm deal with the destruction products both of
blood and coelomic fluid.
The nephridia of P o n t o s c o l e x c o r e t h f u r u s (7) possess
very large funnels with the largest nephrostomial opening I
have seen, through which the coelomic corpuscles can pass
easily; immediately behind the funnel each nephridium possesses a 'receptacle', which is filled with coelomic corpuscles in
all stages of degeneration. Towards the distal end of the
nephridium, immediately preceding the terminal bladder, there
is a thick-walled glandular duct which contains yellowish brown
granules in its walls. It would seem, therefore, that the nephridium of P o n t o s c o l e x has two phagocytic sections, one for
dealing with the destruction products of coelomic fluid and
another with those of blood.
In mammals phagocytic cells are scattered throughout the
body and are grouped together as the r e t i c u l o - e n d o t h e l i a l system. It comprises mainly the endothelial cells of the
spleen, certain branched cells in the bone marrow, the Kupffer
cells of the liver, and the reticulum cells in lymph glands. The
r e t i c u l o - e n d o t h e l i a l system is concerned with blood
destruction and carries the disintegration of haemoglobin as far
as bilirubin (23). In the earthworm it would seem that it is
similarly scattered and comprises the dorsal and ventral phagocytic organs, the phagocytic section or sections of the septal
nephridia, and the aggregates of phagocytic cells within and
above the typhlosole. It is concerned with the disintegration
of both the coelomic fluid and blood, the disintegration of
haemoglobin being carried as far as h a e m o c h r o m o g e n only
in the septal nephridia.
EXCRETION IN INDIAN EARTHWORMS
387
8. SUMMARY.
1. In an earthworm, as in most aquatic invertebrates, urea
and ammonia form the main bulk of nitrogenous excretion and
there is no trace of uric acid. These excretory products are first
formed in the body-wall and gut-wall, pass therefrom into the
coelomic fluid and blood, and are thence eliminated to the
exterior by the nephridia. In P h e r e t i m a urea and ammonia
pass out from the nephridia to the exterior either directly
through the skin or through the two ends of the gut.
2. Ammonia and urea have been estimated for the first time
in the blood, coelomic fluid, and urine of the earthworm. It has
been shown that blood is not a mere carrier of oxygen, as
Eogers believed, but that it also takes part in carrying urea and
ammonia from the body-wall and gut-wall to the nephridia.
The blood of the earthworm does not coagulate, indicating
absence of fibrinogen.
3. The role of the nephridia in excretion and osmotic regulation has been determined. A comparison of the osmotic pressures of blood, coelomic fluid, and urine shows that the coelomic
fluid is h y p o t o n i c to the blood, and that urine is markedly
h y p o t o n i c both to the blood and coelomic fluid. The protein
and chloride contents of the blood, coelomic fluid, and urine
have been determined with a view to elucidate the differences
in their osmotic pressures. It has been found that the urine
contains the merest trace of protein, but that the amount of
proteins in the blood is about eight times that contained in the
plasma of the coelomic fluid. On the contrary, the chloride
content of the coelomic fluid-plasma is about 60 per cent,
higher than that of the blood-plasma.
4. The part of urine which is excreted from the blood is
probably a protein-free filtrate, but the nephridia reabsorb all
the proteins passing into them with the coelomic fluid-plasma.
Similarly, there is a reabsorption of chlorides on a large scale
from the initial nephridial filtrate during its passage through
the nephridia.
5. A convenient method has been devised for collecting
urine of the earthworm, which has made it possible to collect as
much as 25 c.c. of urine in two and a half hours. The rate of
388
K. N. BAHI,
excretion of the urine has been determined and it has been
found that in an earthworm living in water the outflow of urine
in twenty-four hours must be more than 45 per cent, of its
body-weight.
6. It seems that an earthworm, when submerged in water,
can live like a freshwater animal, and its gut acts as an osmoregulatory organ in addition to the nephridia, but in the soil it
lives like a terrestrial animal and the osmo-regulatory function
is adequately discharged by the nephridia alone which reabsorb
chlorides and proteins, and are also active in the conservation
of water. In P h e r e t i m a and other earthworms with an
enteronephric type of nephridial system, the gut takes a prominent part in reabsorbing the water of the nephridial fluid and
conserving water to its maximum extent.
7. The phagocytic section (ciliated middle tube) believed by
Schneider to be absent in the nephridia of P h e r e t i m a has
been shown to be distinctly present; it is also present in the
nephridia of L a m p i t o , B u t y p h o e u s , and T o n o s c o l e x .
The brownish yellow granules characteristic of this phagocytic
section form a heavy deposit in the septal nephridia of P h e r e t i m a p o s t h u m a , heavier than that described in L u m b r i c u s. The deposit increases with the age of the earthworm and
forms a ' storage excretory product'.
8. Spectroscopic examination has revealed that these brownish yellow granules, so far believed to be of guanine, are really
blood-pigment granules, since a pyridine solution of them
shows the two characteristic bands of h a e m o c h r o m o g e n .
With regard to the blood-pigment, the nephridia function as
' storage kidneys'.
•
9. The mechanism of nephridial excretion of the earthworm
can be analysed into processes of filtration, reabsorption, and
chemical transformation.
10. It, is probable that the dorsal and ventral phagocytic
organs of earthworms are additional excretory organs.
9. REFERENCES.
1. Adolph, Edward F., 1927.—"Regulation of volume and concentrations
in the body fluids of earthworms ", 'Journ. Exptl. Zool.', 47.
EXCRETION IN INDIAN EARTHWORMS
389
2. Bahl, K. N., 1919.—"New Type of Nephridial System found in . . .
Pheretima", 'Quart. Journ. Micr. Sci.', 64.
3.
1924.—"Enteronephric Type of Nephridial System in Lampito",
ibid., 68.
4.
1934.—"Significance of Enteronephrie System in Indian Earthworms", ibid, 76.
5.
1941.—"Enteronephric nephr. syst. in Tonoscolex", ibid., 82.
6.
1942.—"Nephridia of the sub-family Octochaetinae", ibid., 83.
7.
1942.—"Nephridia of Pontoscolex", ibid., 84.
8.
1945.—"Nephridia of Megascolex etc.", ibid., 86.
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Paris', 113.
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Biol.', 15.
12 a. Delaunay, H., 1931.—"L'excretion azotee des invertebres", 'Biol.
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14. Heidermanns, C, 1937-8.—"tlber die Harnstoffbildung beim Regenwurm", 'Zool. Jahrbuch.', 58.
15. Kindred, J. E., 1929.—"Leucocytes and Leucocytopoietic Organs of
an Oligochaete", 'Journ. Morph.', 47.
16. Maluf, N. S. Rustum, 1939.—"Volume- and Osmo-regulative Functions of the Alimentary Tract of Lumbricus terrestris", 'Zool. Jahrbuch.', 59.
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17 a. Rogers, C. G., 1938.—'Text-Book of Comparative Physiology'.
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18. Schneider, G., 1896.—"PhagocytSre Organe und chloragogenzellen
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22. Willem, V., and Minne, A.,—1900.—"Rech. sur l'excretion chez
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23. Wright, Samson, 1942.—'Applied Physiology'. Oxford.