Introduction
Excretion of nitrogen in some form is necessary
for all animals. The simplest excretion product is
ammonia, a substance of considerable toxicity. For
this reason, comparatively few organisms excrete nitrogen
in this form. Only small aquatic organisms appear to
excrete ammonia because their aqueous environment permits
continuous release and the maintenance of subtoxic
concentrations in their tissues.
While nitrogenous excretory rates and products are
known for several oligochaetes (Needham, 1970), only
a few species of polychaetes have been examined. Hult
(1969) found that ammonia was the primary excretor,
product of the polychaete Cirraformia spirabrancha
(Moore, 1904). He also determined the rate of ammonia
excretion. Delaunay (1913) found ammonia to be the
primary excretion product of another polychaete, Aphrodite.
Since ammonia readily diffuses across biological
membranes (Milne, et. al., 1958 and Guggenheim, et. al.,
1971), it is commonly assumed that simple diffusion is
the mode of excretion in many marine invertebrates (Hoar.
1966). Again, the process of excretion has not been
closely examined in polychaetes.
In this study we determined the ammonia excretion
Whitmore and Blodgett
Excretion in Polychaetes
rates of several Northern California polychaetes, Dorvillea
moniloceras (Moore, 1909), Halosydna brevisetosa (Kinberg,
1855), Orbinia johnsoni (Moore, 1909), and Nerinides
acuta (Treadwell, 1914), and the ammonia content of their
nitrogenous wastes. In addition, we studied the relation-
ship between the rate of excretion and the internal
ammonia concentration of D. moniloceras.
Materials and Methods
Worms were collected from Monterey Bay in Central
California and stored in running seawater in the labor-
atory for periods not exceeding one week. In each test.
worms were matched for total volume and date of collection.
The worms were blotted on paper tissues and their volume
was measured by water displacement in graduated cent-
rifuge tubes. These groups of worms were then placed
in finger bowls containing 50 ml. of Instant Ocean
(Aquarium Systems, Inc., Eastlake, Ohio). The bowls
were maintained at ambient seawater temperature. At
various intervals, 5 ml. samples were taken from the
finger bowls for analysis.
Ammonia Determination. The method of Solarazano (1969)
was used with some modification. 1 ml. of sample was
combined with 1 ml. phenol-alchohol solution, 1 ml.
sodium nitroprusside solution, and 2.5 ml. oxidizing
solution. Ammonia free water was prepared by basifying
distilled water with 1 ml. 1 N NaoH/100 ml. distilled
water, boiling for 5 minutes, then cooling and neutral-
Whitmore and Blodgett
Excretion in Polychaetes
izing with 1 N HCl. A series of ammonium sulfate standards
was prepared with each set of analyses. Linearity of
the relationship between concentration and optical
density was observed between 0.75 and 3.0 ug ammonia.
All measurements were made with reference to an ammonia
free water blank or a reagent blank prepared with Instant
Ocean. Readings were taken on a Klett-Summerson photo-
electric colorimeter with a red filter.
rotal Nitrogen Determination. Total nitrogen was deter-
mined as ammonia by the method described above after
digestion in a mixture of 1 part 5% CuSO,, 6 parts 85%
HgPO,, 2 parts concentrated H,SO,, and 9 parts distilled
water. Only 0.2 ml. of the digestion mixture was required
per 1.0 ml. sample. Prior to the development of color,
the digest was neutralized to approximately pH 6-7 and
made up to 1 ml. with ammonia free water.
Body Concentration of Ammonia. Groups of 5 worms each
were homogenized with 15 volumes of water per volume of
worm. Then 2 volumes of sodium tungstate were added to
the homogenate in a 125 ml. erlenmeyer flask. 2 volumes
of 2/3 N H,SO, were added while swirling. The flask
was immediately stoppered and shaken for 30 seconds, fol-
lowed by filtration through paper.
Reproducibility between replicate ammonia determi-
nations was within + 0.1 ug, and + 0.2 ug for the total
nitrogen determinations.
Excretion in Polychaetes
Whitmore and Blodgett 4
Results
Nature of Excretory Product.
The percentage of total
nitrogen excreted as ammonia was determined for four
species. The results, listed in Table 1, represent mean
values, followed by their standard deviations. Although
ammonia appears to be the major nitrogenous excretory
product of D. moniloceras and H. brevisetosa, N. acuta
appears to excrete a conspicuous part of its waste nitro¬
gen in a form other than ammonia.
Excretion Rate. The rate of ammonia excretion, expressed
as ug ammonia excreted/hour/ml. of worm, was estimated
by measuring the change in ammonia concentration in the
ambient water after four hours. The results are presented
in Table 2. In both Tables 1 and 2, one sample consisted
of 5 worms placed in 50 ml. of Instant Ocean.
The ammonia excretion rates of two species were studied
with respect to the effect of worm volume differences.
See table 3 for results. Since worm volume did appear
to influence the measurement of excretion rates, all com¬
parisons were made using comparable volumes of worms.
when the rate of excretion was measured after inter-
vals of 24 hours or longer, the results suggested lower
rates than those presented in Tadle 2. Therefore, the
ambient ammonia concentration was examined as a rate
effector in one species, D. moniloceras. The rate of
excretion was observed to decrease with increasing ambient
ammonia concentration. At a concentration of between
Excretion in Polychaetes
Whitmore and Blodgett 5
3 and 4 ug/ml. excretion ceased. See Figure 1 for results.
The Pearson product moment correlation coefficient (r)
for this data is -.54, significant at p-.05.
it was also noted that worms placed in pure Instant
Ocean after exposure to an ambient ammonia concentration
of 4.4 ug/ml. for 18 hours excreted at 16 ug/ml. worm/hour,
more than twice their normal excretion rate.
rnal Ammonia Concentrations. The internal ammonia
Inter
concentration of ammonia in D. moniloceras, as measured
from whole animal homogenates, varied between 45 and 55
ug/ml. worm. The internal ammonia concentration increased
when the worms were exposed to ambient ammonia concentra-
tions already shown to be inhibitory to the excretory
process. See Figure 2. The internal ammonia concentra¬
tion continued to increase with longer times of exposure
to high ambient ammonia. See Figure 3. Although ambient
ammonia concentrations known to be inhibitory for four
hours were used, worms kept in 4.8 ug/ml ambient ammonia
for 18 hours showed a rate of excretion of 1.67 ug ammonia/
ml. worm/ hour. While this is a lower than normal excre-
tion rate, it does indicate some resumption of excretion
after prolonged exposure to high ambient ammonia concen¬
tration.
Discussion
Ammonia excretion rates for the polychaetes studied
are variable both within and among species, although com-
parable to the rate of Cirriformia spirabrancha found
Excretion in Polychaetes
Whitmore and Blodgett 6
by Hult (1969). In Dorvillea moniloceras and Halosydna
brevisetosa ammonia comprises 80% or more of the total
nitrogenous wastes. This is in agreement with Delaunay's
(1913) figure of 80% for the ammonia content of the
nitrogenous excretion of Aphrodite. In Nerinides acuta
only 47% of the nitrogenous waste is excreted as ammonia,
The range in this figure is quite large, i.e., 37 to 57%.
but such variability is not uncommon. In earthworms,
fasting causes a large decrease in ammonia output (Bishop
and Campbell, 1965). The remainder of the waste product
in Nerinides acuta was not identified and should be the
subject of further study.
In all cases, high ambient ammonia concentration
caused a decrease in the ammonia excretion rate and an
increase in internal ammonia concentration of D. monilo-
ceras. In addition, worms exposed to a high concentration
of ammonia showed rates of excretion more than twice
normal when removed to water of low ammonia concentration.
Also, worms exposed to high ammonia concentrations par-
tially resumed excretion after long periods of exposure.
These observations would suggest a simple diffusion model
of excretion. However, the increase in internal concen¬
tration can not be explained by reverse diffusion from
the water to the worm. Excretion appears to be inhibited
without a similar inhibition of amino acid catabolism.
The ammonia concentration of the body, 5Oug/ml., was
Excretion in Polychaates
Whitmore and Blodgett 7
found to be approximately 500 times higher than that of
the ambient water. Tillinghast (1967) found that gut
ammonia concentration in Lumbricus was 10-50 times higher
than the coelom ammonia concentration. Possibly this
situation also exists in polychaetes, which could explain
now such a high internal concentration can be maintained.
Summary
1. In Dorvillea moniloceras and Halosydna brevisetosa
ammonia comprises more than 80% of the nitrogenous ex-
cretion product. In Nerinides acuta, ammonia comprises
only approximately 50% of the nitrogenous excretion pro-
duct.
2. Excretion rates in these polychaetes are variable
both within and among species, but generally tend to be
between 3 and 8 ug/ml. worm/hour.
3. In Dorvillea moniloceras ammonia excretion rate was
found to decrease as ambient ammonia concentration in-
creased, with some lessening of the effect with longer
periods of exposure to high ambient ammonia concentration.
4. In Dorvillea moniloceras, worms exposed to high ambient
ammonia concentrations and then placed in pure Instant
Ocean exereted ammonia at much higher than normal rates.
3. In Dorvillea moniloceras, it was found that the
worms' internal body ammonia concentrations increased
in high ambient ammonia concentrations and with longer
times of exposure to these concentrations.
0
Acknowledgements
We would like to express our thanks to Dr. John
Phillips for his advice and encouragement throughout
this project.
Literature Gited
Bishop, S.H. and J.w.Campbell, 1965. Arginine and urea
biosynthesis in the earthworm Lumbricus terrestris.
Comp. Biochem. Physiol. 15:51-7.
H.. 1913. Sur quelques faits particuliers a la repar-
Delaunay,
tition de l'azote dans le liquide cavitaire des
Vers
(Aphrodite aculueata, Sipunculus nudus).
C. R. Soc. Biol. 74:154-156, in Campbell, J.w.
ed.,1970. Comparative Biochemistry of Nitrogen
Metabolism. Academic Press, Inc., London.
S.J., J. Bourgoignie and S. Klahr, 1971.
Guggenheim,
Inhibition by ammoniun of sodium transport
cross isolated toad bladder. Am. J. Physiol.
220:1651-1659.
1966. General and Comparative Physiology,
Hoar, W.S.
Prentice-Hall Inc., Englewood Cliffs, N.J.
1969. Nitrogenous waste products and enzymes
Hult, J.E.
in the marine polychaete Cirraformia spirabrancha.
Biochem. Physiol. 31:15-24.
Comp.
Milne, M.D., B.M. Scribner and M.A. Crawford, 1958. Non-
ionic diffusion and the excretion of weak
acids and bases. Amer. J. Med. 24:709-720
in Prosser, C.L. ed., 1973. Comparåtive Anima
Physiology. W.B. Saunders Co., Philadelphia, Pa.
Needham, A.E., 1970. Nitrogen metabolism in Annelida. in
Campbell, J.W. ed.,1970. Comparative Biochemistr
of Nitrogen Metabolism. Academic Press, Inc.,
London.
Solarazano, L., 1969. Determination of ammonia in natural
waters by the phenolhypochlorite method.
Limnol. Oceanogr. 14:799-801.
Tillinghast, E.K., 1976. Excretory pathways of ammonia
and urea in the earthworm Lumbricus terrestris.
J. Exp. Zool. 166:295-300.
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lable 3.Dependence of excretion rate on body volume
Rate
Species
Volume
Dorvillea moniloceras
3m
5.6uglmi/hr
4.7 ughl/hr
Dorvilled monilocergs
12 m
Halosydna brevisetosg.
3.OuglmUhr
1.3m
4:8 uglmihr
Halosydna brevisetosa
35m
VOLUME OF THE WORM
RATE IN ug NHmI WORM/r
whitmore and Biodgett
Excretion in Polychaetes
Figure 1. Rate of excretion of ammonia in ug/ml. worm/hour
as affected by the ambient ammonia concentration. Each
point is an average rate for 5 worms of the species D.
moniloceras, over four hours.
EXCRETION
RATE
QAN,
/mI WORM,
7



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.. .
- â

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—
2
AMBIENT NH, CONCENTRATION ug
m
Excretion in Polychaetes
Whitmore and Blodgett
Figure 2. Body ammonia concentration of D. moniloceras
as affected by ambient ammonia concentration. Normal
body concentration of ammonia refers to ammonia concen¬
tration in the bodies of worms kept in running seawater.
1.6
1.5
1.4
BODY NH
CONCENTRATION
RELATIVE 13
TO NORMAL
1.2
(2hr EXPOSURE
(D) 4 hr EXPÖSURE
(0) 28 hr EXPOSURE
4
2
AMBIENT NHA CONCENTRATION ug
l
0
Whitmore and Blodgett
Excretion in Polychaetes
Figure 3. Body ammonia concentration of D. moniloceras
as affected by length of time exposed to high ambient
ammonia concentration. Normal body concentration of
ammonia refers to ammonia concentration in the bodies
of worms kept in running sea water. Numbers next to
data points refer to ambient ammonia concentrations, in
ug/ml., at the end of the corresponding exposure times.
1.6
1.5
14
BODYNH
CONCENTRATION
RELATIVE TO 43
SORMAL
1.2

11
19





05.7
04.4
03.6
4
16
20
12
24
8
TIME IN HIGH AMBIENT NHZ CONCENTRATION (hrs)
06.6






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28