Nitrogenous materials released from
Mopalia muscosa (Gould, 1846), an intertidal chiton.
ABSTRACT
A study was undertaken to identify the nitrogenous
waste products released from the intertidal chiton Mopalia
muscosa (Gould, 1846). The Solorzano method was used to test
for the presence of ammonia and/or urea, while ninhydrin
positive nitrogenous compounds were identified by the Spies
method. Large variability within each sample collected from
each of five chitons did not allow a significant comparison
between levels of ammonia and urea, although both nitrogenous
compounds were present. However, in three of the M. muscosa
high levels of ninhydrin positive material were found in
comparison to the ammonia and urea levels. For one chiton
these levels were 220.4+27.2 ug of ninhydrin positive nitrogen,
42.2+4.7 ug of urea nitrogen, and 51.4+12.0 ug of ammonia
nitrogen. The ninhydrin positive nitrogen was released from
this M. muscosa at a rate of 0.48mg/100g/day. The original
paper is on file at the library, Hopkins Marine Station,
Pacific Grove, California 93950.
Nitrogenous materials released from
Mopalia muscosa (Gould, 1846), an intertidal chiton.
ABSTRACT
A study was undertaken to identify the nitrogenous
waste products released from the intertidal chiton Mopalia
muscosa (Gould, 1846). The Solorzano method was used to
attermence-terlment
test for the presence of ammonia andr urea while ninhydrin
positive nitrogenous compounds were identified by the Spies
method. Large variability within each sample collected
from each of five chitons did not allow a significant comparison
between levels of ammonia and urea, although both nitrogenous
compounds were present. However, in three of the M. muscosa
high levels of ninhydrin positive material were found in
comparison to the ammonia and urea levels. For one chiton
these levels were 220.4+27.2 ug of ninhydrin positive nitrogen,
42.2+4.7 ug of urea nitrogen, and 51.4+12.0 ug of ammonia
nitrogen. The ninhydrin positive nitrogen was released
from this M. muscosa at a rate of .48mg/100g/day. To obtain
a copy of the original paper, contact the librarian at
Hopkins Marine Station, Pacific Grove, California 93950.
ale o
Nitrogenous Materials Released from
Mopalia muscosa (Gould, 1940),
an Intertidal Chiton
by
Rich Williams
Hopkins Marine Station
of Stanford University
Pacific Grove, California
sentt
Dossenci o
be
Se
ich Willians
page 2
The organisms of the Phylum Mollusca differ greatly
in their nitrogenous excretory products. For example, the
land snail Helix pomatia (
has uric acid
comprising up to 903 of its excretia (Jezewska et al., 1963),
while in Octopus dofleini (Pickford, 1961) ammonia accounts
3 of the total nitrogen excreted (Emmanuel and Martin,
for
1956). The oyster Modiolus demissus (Dillwyn, 1817) posesses
both ammonia and urea as excretory products, 723 and 28%
respectively (Lum and Hammen, 1961).
Studies of the nitrogenous waste products of the class
Polyplacophora are scarce, with information available only
on Crypto
hiton stelleri (Middendorf, 1346), a large sub-
idal chiton. Myers (1920) found both ammonia and urea to
be present in the body fluids of C. stelleri as did Vasu (1965),
who found the ratio of urea to amino nitrogen in the blood
to be approximately 5:1. However, no studies of Cryptochiton
stelleri urine or urine of other chitons are to be found in
literature.
This study was undertaken in hopes of determining the
itrogenous excretory products of an intertidal chiton.
Mopalia muscosa (Gould, 1940) was chosen for several reasons;
it is a large intertidal chiton readily available for collec-
tion, it is abundant along the coastline of Monterey Bay,
and most importantly, it occupies the high:
nbertidal region
(+3--5 feet) and is therefore subjected to long periods of
xposure during the tidal cycle. Therefore, it represents
a Folyplacophoran which should reveal the capabilities of
Rich Williams page 3
this class for the production of non-toxic nitrogenous waste
products.
Materials and Methods
Waste products were collected by allowing the chitons
to excrete into a known volume of sea water. Rate of ex-
cretion was measured by recording the change in concentra¬
tion in the water over time. This method of collection was
used only after attempts at catheterization failed. Cathe-
terization using surgical tubing inserted into the renal
pore produced too high a mortality and the fluid collected
by this method was frequently contaminated with body fluid.
Mopalia muscosa were collected from the high intertidal
regions of Ft. Pinos and South Carmel Beach. The chitons
were transferred dry to finger bowls containing 50 or 75 ml
of .22 micron filtered sea water. The time period between
removal from their natural substrate and placement in the
finger bowl never exceeded 30 minutes. The finger bowls and
all glassware used in the experiment were washed well, rinsed
with IN HCl, and then rinsed with de-ionized water.
The Solorzano method (1969) of ammonia determination was
used with one modification; de-amonified water was prepared
by adding 1 ml of 1II Naon to every 100 ml of distilled water

and boiling for 10 minutes, and when cool 1 ml of II Hol
was added for each 100 ml of distilled water. This
lich illiams page l
de-ammonified water was used in the preparation of all
reagents and in all dilutions. Triplicate 1 ml samples
were used for ammonia determinations.
Crea conce
trations were also measured by the Solorzano
method after the urea had been broken doum to amonia by
urease. To each 1 ml water sample was added.1 ml of a
urease solution containing 50 mg/ml of Matheson, Coleman and
Bell urease. After 15 minutes of incubation at room tem¬
perature the samples were analyzed and corrected for ammonia
nitrogen.
Hinhydrin positive nitrogenous compounds were measured
by the method of Spies (1957) and corrected for armonia
nitrogen. A standard curve was prepared using leucine.
Aonia produces 983 of the color of this standard on a
nitrogen basis.
sults
For the five Mopalia muscosa whose nitrogenous waste
products were monitored overt
monia.
me, mean values or a
urea, and ninhydrin positive material, excluding ammonia, were
calculated for each l hour period. Within the three samples
collected in each time interval, there was too much vari-
abilit
by to permit an estimation of rate of excretion of
ammonia or urea. However, three of the animals studied pro-
duced ninhydrin positive materials in considerable excess
ich Williams page 5
of the ammonia excreted. Table 1 presents these results.
Figure 1 presents the time course of excretion of this
iinhydrin posit
jive material by one Mopalia muscosa. The
average rate of excretion during the first 12 hours was
18.l mg per hour of this material. The falling off of this
rate after 12 hours:
might indicate that excretion is not
continuous, and the decrease in the nitrogen level is due to
bacterial degradation.
The excretion of ammonia nitrogen shows a similar
time course and is presented in Figure 2.
Discussion
Potts (1967) reports that amino nitrogen is one of the
major components of non-protéin nitrogen lost from marine
molluscs. He suggests that free amino acids are lost mainly
through diffusion across the body wall. Spitzer (1937)
reports tha
tilus edulis (Linnaeus, 1758) loses.
.1-1.0 mg/100g/day.of amino nitrogen, while Modiolus der
ssus
loses 1.1 mg/100g/day.
The Mopalia muscosa in Figure 1 lost .10 mg/100g/day
of what appears to be amino nitrogen, assuming 220 ug of
nitrogen to be the maximum level released over the 21 hour
period. Another M. muscosa with significant ninhydrin
i
pos
sitive results lost .60 mg/100g/day of amino nitrogen.
Rich Williams page 6
The amino nitrogen could be lost either through excretion or
fusion, although diffusion might be expected to give a
constant increase over the 21 hour sampling period. Con¬
stant increase of the ninhydrin positive material was not
observed.
by of the ninhydrin positive excretory pro-
The iden
duct or products has not been determined, and the importance
of this material to excretory non-protein nitrogen will
require further study.
Summa
1) Tests for ammonia, urea, and compounds containing nin¬
hydrin positive amino groups were run on materials released
from the intertidal chiton, Mopalia muscosa.
2) Variability in the data for ammonia and urea did not allow
any conclusions to be made about their rates of excretion
over a 21 hour period. However, sig
nificant quantities of
ninhydrin positive material in excess of amonia were detected
in three Mopalia muscosa. It is uncertain whether this latter
material was lost by dif
fusion or release from the excretor
pore. The identit
y of this material has yet to be deter
mined.
ich Williams page 7
Acknowledgments
Many thanks to Dr. John H. Phillips for his invaluable
advice and criticisms. The encouraging words of Anne Edwards,
Will HcCarth
Chuck Baxter, and Dr. Robin Burnett were very
much appreciated. Thanks to the faculty, staff, and students
of Hopkins Marine Station for creating a stimulating atmo-
sphere in which to work.
Rich lilliams page 3
Literature Cited
Emmanuel, C.F. and A.W. Martin
1956. The composition of octopus renal fluid. I. Inor¬
ganic constituents. Z. vergl. Physiol. 39, 226-34.
Jezewska, M.M., Gorzkowski, B. and J. Heller
1963. Seasonal changes in the excretion of nitrogen
wastes in Helix pomatia. Acta biochim. pol. 10, 309-11.
Lum, S.C. and C.S. Hammen
1961. Ammonia excretion of Lingula. Con
np. Biochem. Phy
siol.
12, 185-90.
Myers, R.C.
1920. A chemical study of the blood of several invertebrate
animals. Journ. biol. Chem. 41, 119-47.
Potts, W.T.W.
1967. Excretion in the molluscs. Biol. Reviews 12, 1-11.
Solorzano, L.
1969. Determination of ammonia in natural waters by the
phenolhypochlorite method.
Limnology and Oceanography 11,
799-801.
Spies, J.R.
1957. Colorimetric procedures for amino acids. Methods
in Enzymology, Vol. 3, Academic Press Inc., New York. 168-71.
pitzer, J.M.
1937. Physiologisch-ökologische Untersuchungen über den
Exkretstoffwechsel der Molluskan. Zool. Jb. 57, 157-96.
e
iams page 9
Vasu, B.S.
1965. Variations in the body fluid nitrogenous constit-
ster ochraceous (Schinodermata) and Crypto-
uents of
zon stelleri (Mollusca) in relation to nutrition and
reproduction. Ph.D. Thesis, Hopkins Marine Station,
Stanford University. pg. 85.
Table
lich Willians page 10
Composition of nitrogenous waste products
released by Mo
alia muscosa. Values are for
one of six readings taken over a 21 hour time period.
1H3-N
Urea-
Winhydrin.
Positive-N

Animal 1
12.2 ug
220.1 ug
51.1 ug
19.5
Animal 2
25.1
179.6
19.3
Animal 3
O.O
62.8

* Values corrected for NH.-N.
Figt
e 1:
Rich Williams page 11
Time course of ninhydrin positive
nitrogenous waste products released
from Mopalia muscosa. Ranges in-
dicate standard deviations.
300-
ug. ot aol
NiaSpa
FGITIVE
NiTROEE100
O

—

16
8
TIME (OORs)
FIGURE
24
Rich Williams page 13
5
Rich Williams page 11
2: Time course of ammonia nitrogen
released from Mo
alia muscosa.
Ranges indicate standards deviations.
50
ug. ot
oo-
HMMONIA
NiTeeen 50-
FIGURE A.
24
16
TME (R6)
Rich Williams page 16