By
(Merton College, Oxford)
Professor of Zoology, University of Lucknow, India.
With 7 Text-figures and 11 Tables.
C O N T E N T S .
PAGE
1. INTRODUCTION 344
2. T H E N A T U R E AND M A S S OF EXCRETORY SUBSTANCES EVACUATED
B Y THE EARTHWORM ( P H E R E T I M A P O S T H U M A ) . . . 348
(a) ESTIMATION O F 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 ESTIMA-
TION OF AMMONIA AND U R E A THEREIN . . . 351
3. T H E EXCRETORY SUBSTANCES I N T H E COELOMIC F L U I D . . 354
4. T H E EXCRETORY SUBSTANCES I N T H E BLOOD .
.
.
. 356
5. T H E INITIAL P L A C E S O F E X C R E T I O N — T H E BODY-WALL AND T H E
INTESTINAL W A L L .
.
.
.
.
.
.
. 358
6. T H E R O L E OF T H E N E P H R I D I A I N E X C R E T I O N AND OSMOTIC
REGULATION 361
(A) T H E OSMO-REGULATORY F U N C T I O N OF T H E N E P H R I D I A 363
(i) VOLUME-REGULATION 363
(ii) T H E R A T E OF E X C R E T I O N OF U R I N E . . . 3 6 9
(iii) T H E OSMOTIC P R E S S U R E O F B L O O D , COELOMIC
F L U I D , AND U R I N E 371
(iv) T H E P R O T E I N CONTENTS O F B L O O D , COELOMIC
F L U I D , AND U R I N E 372
(v) T H E CHLORIDE CONTENTS OF B L O O D , COELOMIC
F L U I D , AND U R I N E 375
(vi) CONCLUSIONS .
.
.
.
.
.
. 376

344 K. N. BAHL
PAGE
(B) T H E E X C R E T O R Y I N C L U S I O N S O F T H E ' C I L I A T E D
M I D D L E T U B E ' ( A T H E O P H A G O C Y T I C S E C T I O N ) O F T H E
N E P H R I D I A 377
7. E X C E E T O E Y O R G A N S 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 bloodsupply
1
—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 A a

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 n o t to t h e e x t e r i o r ; (2) closed integumentary nephridia, about 200 in each segment, which o p e n t o 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 o p e n 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
L u m b r i c u s .
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 a n d U r e a in
W a t e r c o n t a i n i n g E a r t h w o r m s .
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.
1.
2.
3.
4.
5.
average
Number of worms.
20
20
20
20
20
20
Weight in grams.
21-68
28-48
30-98
27-82
28-20
27-43
N(urea and ammonia) in milligrams
(per 100 gm.
of bodyweight).
4-97
6-11
5-33
4-67
5-02
5-22
Ammonia in milligrams
(per 100 gm.
of bodyweight).
5-24
6-54
4-87
4-30
5-52
5-29
Urea in milligrams
(per 100 gm.
of bodyweight)
(by diff.).
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 urease
2
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 bell-Jo.
earfhwormi 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.
1.
2.
3.
4.
5.
Average
N(urea and ammonia) in milligrams per
100 c.c.
3-61
3-61
3-61
4-04
3-61
3-69
Ammonia in milligrams per 100 c.c.
2-19
2-80
2-19
2-80
3-35
2-66
Urea in milligrams per 100 c.c.
{by diff.).
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 i n 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

EXCRETION IN INDIAN EARTHWORMS 355 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 III. Ammonia and Urea in Coelomic Fluid.
Serial number.
Foreman's method.
1.
2.
3.
4.
5.
Average
JM essierization method.
6.
7.
N(urea and ammonia) in milligrams
(per 100 ex.).
Ammonia in milligrams
(per 100 ex.).
Urea in milligrams
(per 100 ex.)
(bydiff.).
4-01
3-98
5 0 1
5-47
4-50
4-79
—
3-38
4-0
4-24
4-94
3-9
4-13
2-29
1-67
3-02
3 0
2-61
2-52
— Ammonia+Urea —
— 3-5 mgm. —
— 4-5
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 Urease-
Nesslerization 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.

EXCRETION IN INDIAN EARTHWORMS 357 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.
Serial number.
1.
2.
3.
4.
Average
5.
N(urea-\-ammonia) in milligrams
(per 100 c.c).
3-08 )
2-98
4-01
2-98 .
Foreman's method.
3-26
Nesslerization 1 method. / ""*"
Ammonia, in milligrams
(per 100 c.c).
2-49
2-49
3-06
1-81
2-71
Ammonia-)-Urea
5-8 mgm.
Urea in milligrams
(per 100 c.c.)
(by diff.)-
2-207
1-97
3-19
3-18
2-638
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.
\
1.
2.
4.
5.
Average
•§• ft.
fe-S
3-0
3-0
3-0
3-0
3-0
3-0
2-49
3-60
4-76
6-12
4-76
4-34
4-01
4-41
4-39
6-00
4-39 i,
4-64 fe-S
30
30
3-0
30
30
30
3-60
3-60
2-49
4-76
3-60
3-61
2 0 1
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).
Ammonia milligrams per cent.
Intestine.
Urea milligrams per cent.
5-2
9-8
7-1
9-2
7-5
13-2
28-8
11-6
m e a
-
9-8/
16-5 ] Xanthydrol
12-0 / urea.
Ammonia milligrams per cent.
4-6
2-1
Body-wall.
Urea milligrams per cent.
0-8 lUrease
1-9 /urea.
1-4 \ Xanthydrol
2-3 /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. T H E 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 s i x times the length of the two limbs put together. Further, the canal has f o u r 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
N e p h r i d i a .
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 b a s i c 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. I t 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 both ends of the worms and have confirmed bis observation that there is a distinct decrease in weight, but my conclusion from this observation is different. It must be remembered that
1
The spaced words are mine.

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
P h e r e t i m a .
(ii) T h e 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

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 , C o e l o m i c
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
TABLE VIII. Depression of the Freezing-point of the
Fluids of Pheretima posthuma.
1. Blood-plasma A = 0-40° 0.-0-50° C.
2. Coelomic fluid-plasma A = 0-285° C.-O-31
0
0.
3. Urine . . . A = 0-050° 0.-0-065° C.
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 the figure for 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) T h e 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

BXCBBTION IN INDIAN EARTHWORMS 373 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.
gm. per 100 c.c.
1. 3-457
2. 3-949
3. 3-757
4. 3-376
Average 3-643
Coelomic fluidplasma.
gm. per 100 c.c.
0-550
0-479
0-458
0-429
0-479
Urine.
gm. per 100 c.c.
0-025
0-029
0-036
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 was filtered through 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) T h e C h l o r i d e 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 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.
(mgm. per 100 c.c.)
1. 45-80
2. 50-82
3. 51-42
Average 49-35
Coelomic fluid-plasma.
(mgm. per 100 c.c.)
79-26
77-23
81-30
79-26
Urine.
(mgm. per 100 c.c.)
3-556
3-862
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 each fluid was 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 and filtered through ashless filter-paper and 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 S e c t i o n ) of t h e N e p h r i d i a .
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 c c

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 n o t G u a n i n e .
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 c h e m i c a l 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

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

8 8 2 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 a r e 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, pyridine
1
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 quantities
2
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:
First Absorption Band: AA 5659-5500; centre 5579 A.U.
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 h a e m o c h r o m o g e n .
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 3 8 7
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.
9. Benham, W. B.,- 1891.—"Nephridium of Lumbricus and its Blood-
Supply ; with Remarks on Nephridia in other Chaetopoda ", ibid., 32.
10. Carter, G. S., 1940.—'General Zoology of the Invertebrates'. London.
11. Cole, S. W., 1942.—'Practical Physiological Chemistry'. Cambridge.
11 a. Cordier, R., 1933.—"Sur les phenomenes d'athrophagocytose dans le segment cilie de la nephridie du Lombric", 'C. R. Soc. Biol.
Paris', 113.
12. Cuenot, L., 1898.—"Etudes Physiol. sur les Oligochetes", 'Arch.
Biol.', 15.
12 a. Delaunay, H., 1931.—"L'excretion azotee des invertebres", 'Biol.
Rev.', 6.
13. Harrison, G. A., 1937.—'Chemical Methods in Clinical Medicine'.
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