AN ANALYSIS OF NITROGEN WASTE PRODUCTS
IN

LORINA PLANAXIS
Ann Louise Cox
May 30, 1964
Biology 175h
Few studies have been made of the nitrogen waste products
of the littorines. Spitzer in 1937 (see Fretter and Graham, 1962)
studied the nitrogenous excretory products of Littorina littorea,
a gastropod located in the low intertidal of Europe. Needham
(1935) studied the uric acid content of four species of littorines,
but did not analyze the kidney for other waste products. The
purpose of this investigation, therefore, was to obtain data
on the total non-protein nitrogen, NPN, and the various nitrogenous
constituents of the kidney of Littorina planaxis, a gastropod
with wide vertical distribution in the intertidal of California.
These earlier studies note that excretory products are a function
of the environment but fail to take into account the fact that
this gastropod is subjected to a wide daily variation in water
availability which could cause variation in the kidney contents.
In the present study attention was paid to the variation in the
tidal cycle and its possible effect on the nitrogenous consti¬
tuents of the kidney.
METHOD
Two areaswere chosen from which to collect snails: one
population was located at the lower limits of the species in the
intertidal where there is water present 90% of the time: The
snails here measured from 7-9 mmin size. The second population
occurred at the upper limits of the intertidal where the snails
are exposed to air, wind, desiccation, and such approximately
95% of the time and range in size from 10-12 mm. Collections
were made at various periods through five tidal cycles. Two
series of observations two and one-half weeks apart were carried
out on small numbers of snails from both areas. The second,
less complete, was intended as a check on the first. Five samples
of ten snails each were examined at each point in the tidal cycle
in an effort to obtain an accurate estimation of the concentration
range of these products. At each of eight different times in
the tidal cycle fifty snails were gathered from one area and the
kidneys dissected out immediately. The dissections were trouble-
some, because kidney tissue is intermingled with surrounding
body tissue and difficult to extract whole. Ten kidneys were
weighed together after all excess water had been removed. This
tissue was ground and deproteinized with dilute sulfuric acid
and sodium tungstate at ph 6-7. There resulted five protein¬
free filtrates from each point in the cycle.
The total NPN was determined by the Kjeldahl method of
digestion with acid followed by Nesslerization. Ammonia content
was also determined by Nesslerization, as was urea, after
hydrolysis with urease (Baker and Adamson). Uric acid was
determined by the method of Benedict and Franke (1922). All
colorimetric measurements were made with a Klett-Summerson
Photoelectric Colorimeter and converted to milligrams of nitrogen
with a standard curve prepared on the same machine.
RESULTS
It can be observed from Figure 1, which shows the variation
in the nitrogenous excretory products of snails in the low
intertidal, that there is a definite drop in NPN at Low Low Water
when the snails were receiving a minimal amount of wave action.
(Hereafter, all tides will be referred to with the appropriate
capitals, eg. LLW) Another low point occurred at mid-HHW-HLW
D
when the water was receding. The largest amount of NPN
(3.45-5.58 mg NPN/ gm. kidney) in the kidney of snails found
at this level was at HLW with a secondary peak at mid-LLW-HHW.
Because no reading was taken at mid-HLW-LHW, it is inaccurate
to state unequivocally that HLW was a definite high point with
a gradual decline thereafter. Figure 2 showing the various
nitrogenous excretory products through a second tidal cycle,
suggests that, on the contrary, there was a peak reached at
mid-HLW-LHW. Again, in Figure 1 ammonia seemed to vary directly
with the total NPN at each point in the tide, with the one ex¬
ception of HHW when ammonia content fell but NPN did not.
During this period snails were being submerged once every 30
seconds and splashed continuously. The urea content curve was
approximately the same as that of ammonia, but urea usually seemed
to be in smaller quantities. However, at LLW there was more urea,
12-427 of NPN, than ammonia, 9-197, and quite a lot of undeter¬
mined nitrogen, 46.47-77.122. Uric acid seemed to be present in
only trace amounts. (Refer to Table 1)
Observations made in the second tidal cycle, taken two
and one-half weeks later, on low L. planaxis, show that the least
amount of NPN occurred again at LLW (See Figure 2). Here there
was only one measurement, but it is significantly lower than
any point on either side. There was also a decrease at HLW,
but none at mid-HHW-HLW as in the first series of test. As for
the constituents, ammonia seemed to follow the same curve as that
in the first cycle, with low points at LHW, LLW, and HHW. Urea
again showed the same correspondence with ammonia except at LLW
when it constituted 46.77 of NPN and ammonia 40.5%. In this
O-



DADN
series of tests, more urea was present (average,25.47) than in
the first tidal cycle (average 22.137). The ammonia-N content
was also increased from 33.34% to 41.7%. Here, as in the first
run, uric acid was present in only trace amounts, except at
irregular times, at LLW, when it amounted to as much as 3.13%.
Generally those snails from higher in the intertidal showed
a greater variation in total NPN at any one time in a tidal cycle
than the lower snails tested simultaneously. From Figure 3
it can be observed that snails in higher areas did not show the
low NPN at LLW that snails from lower areas did. On the contrary,
NPN was at its lowest at HHW and immediately after LHW, the only
periods in the cycle when these snails received water directly
in the form of splash. At LHW a wide range of NPN was seen,
2.83-6.36 mg/ gm. of kidney. This could be the result of the
lower snails collected having been splashed but the higher ones
remaining dry. Ammonia did not show quite the same correspondence
with NPN as it did in the lower areas. There seemed to be less
drastic decreases. The most noticeable one occurred at mid-LHW-LLW.
This corresponded to the drop in NPN evidenced at the same time.
There was a rise after HHW and also a peak at LLW. Overall, the
percentage of ammonia-N in NPN was less, 24.927, and showed less
variation than that of ammonia-N found in snails in lower in the
intertidal (33.34%) Urea, as in the lower areas, corresponded
to the ammonia, always remaining less and falling quite a bit
at HHW. Again uric acid was found in negligible amounts.
The second series of testin the high area involved only
a small part of the tidal cycle, so little can be said about
the total picture. Two interesting observations can be made,
O
O


HETTHNTE
e
however: uric acid appeared in large quantities fro the first
time in the course of this investigation; up to 20.97 of the NPN
at mid-LHW-LLW consisted of this waste product. Simultaneous
with this sudden increase in uric acid, urea and ammonia decreased
and subsequently rose again when uric acid nitrogen fell.
DISCUSSION
In general it can be stated that there was variation in
NPN as well as constituent products in the kidney of Littorina
planaxis within a tidal cycle. Needham's assumption, an important
basis for his analysis, that the kidney retains the renal waste
products for very long periods did not seem to hold for the snails
studied here.
More specifically, kidneys of snails located at the lower
limits of the species' distribution showed an interesting
anomaly at LLW. One might have expected a greater amount of NPN
at that time than others,because there was no water available
for diffusion of urea and ammonia into the environment; however,
the contrary was seen to occur. The amount of ammonia-N averaged
2.25 mg/gm of kidney at this time, whereas an average of all the
other measurements was 3.54 mg/gm of kidney. If this is correlated
with the fact that the ammonia-N percentage at LLW, 9-197, was
very much lower than that normally found, 33.34%, it might be
hypothesized that ammonia is being eliminated as a gas. Or,
because ammonia is toxic and requires much water for elimination,
perhaps less ammonia is produced by the snail at this time in the
tidal cycle. At any rate, all snails tested seemed to be exhibitig
the same tendency toward very low NPN and ammonia at this time.
Not including the LLN percentages, it would seem that the
average percentage of ammonia-N, 33.34%, in this littorine
corresponds to the 39.9% given by Spitzer (1937) for L.littorea,
a snail occurring lower in the intertidal. Only three ammonia
measurements were over Spitzer's maximum of 50%.
Concerning the other constituents of NPN in the low areas,
it seems that less urea than ammonia was found in the kidneys,
except at LLW. This latter observation could be a result of
the fact that urea had no way of leaving as a gas (as ammonia might),
so it contiued to be stored in the kidney until enough water
was available for elimination by diffusion. The average, as
compared to Sptizer's figure of 12.6% was 22.13%. This
increased slightly to 25.4% in the second tidal cycle. As for
the very low uric acid content in the snails low in the intertidal,
it might be argued that since these L. plandis are around water
90% of the time, they have no need of utilizing a water con-
servation mechanism; the uric acid present might merely represent
the degradation of nucleic acids. However, the second series of
analyses showed slightly greater amounts of uric acid being
produced at irregular intervals. This at least indicates a
capability of synthesizing uric acid from ammonia.
Snails from the upper extremes in the intertidal also
showed variation in kidney contents with tidal changes. These
results seem to be more easily explained: urea and ammonia
diffuse into the water when it is available to the snail, at
LHW and HHW; in the interim, there is storage of the products
and gradual increase, with one drop at HL, which is perhaps due
to ammonia gas elimination. It would appear that there was more
NPN/gm of kidney in snails from the higher location than in
those found lower. It is possible that a certain minimal amount
of waste is continually diffusing into the water available to
the lower snail, but those in the higher regions have no water
for this elimination. This difference in NPN might also be due
to the ease of obtaining kidney free of other tissues in the
larger snails from the high area. This separation is more
difficult in the smaller snails from lower in the intertidal.
This other tissue contains no NPN but contributes to the weight
of the sample.
Along with the greater amounts of NPN there seemed to be
less ammonia-N (24.927) in snails from the higher areas. Perhaps,
because ammonia is more toxic than other waste products and re¬
quires more water for excretion, a snail exposed to air for 957
of the time can't tolerate such high percentages of ammonia as
snails in the lower, wetter areas (33.347). The one time,LLW,
when the lower snail was exposed to an environment similar to
that of the higher snails there was a remarkable decrease in
ammonia-N.
A large percentage of undetermined -N was found in the
first series of tests, 63.587, and much less two and one-half
weeks later, 45.76%. This same decrease in undetermined-N was
found in snails from the lower regions, with 46.717 in the first
cycle and 31.06% in the second. Perhaps, this observation could
be correlated with the finding that urea was in much smaller
quantities in the first run, 11.07%, than in the second,21.87.
When added together, ureatundetermined-N =- 74.65% NPN in the
first series and 67.56% in the second. These percentages seem
to be very similar. The "equation" does not hold for snails
in the lower regions. Perhaps, a change has taken place in the
system of the snail with the onset of the summer tides, warmer
weather, and more nutrient growth. This suggestion implies that
urea and undetermined-N are products, either terminal or
intermediate, of the same pathway. Further investigations to
determine what these unknown materials are, as well as studies
of this correlation with urea, could prove interesting. Such
analyses might reveal the pathway by which urea is formed.
In the second abbreviated series of tests on snails from
high in the intertidal, uric acid was found to be in vast quantities
at irregular intervals in the tidal cycle. With each increase
in uric acid there was a simultaneous decrease in urea, and vice
versa. Although urea varied at other times when only trace amounts
of uric acid were found, this inverse relationship might prove
interesting for further study. Perhaps uric acid and urea are
a part of the same metabolic pathway.
The problem of uric acid production is an interesting one.
Needham (1935), in proof of his hypothesis that limited access to
water results in an increase in the formation of uric acid,
presents definite ranges of concentrations of uric acid for the
four species he tested. Littorina rudis is located a little
below L. planaxis in the intertidal and it contained 5.1 mg uric
acid/ gram dry kidney weight. If the data obtained in the present
study is calculated in the above manner, the largest figure for
L. planaxis is 3.5 mg uric acid/ gm wet kidney weight; this
large amount was found in L. planaxis only once in the tidal
cycle, with smaller amounts occurring at other times but only
sporadically. Certainly these quantities were not constant, in
contrast to Needham's findings. It is suggested, therefore, that
L. planaxis has no prolonged increase or retention of uric acid
but that production is spasmodic and elimination rapid.
Ideally, the analyses of the kidney contents of L. planaxis
should have been carried on through one day's tidal cycle. Also,
an examination of the food eaten by the snails of these two
extreme areas in the intertidal might, to some extent, explain
the differences in the amounts of the various nitrogenous excretory
products found between the low and high snails in this investigation.
SUMMARY
1. There was found to variation in the total NPN as well as
constituent products in the kidney of Littorina planaxis.
2. Snails from low in the intertidal contained more NPN, ammonia,
and urea than those in the high regions.
3. Snails in the low area contained very little MPN and ammonia
at LLW.
4. Urea was found in smaller quantities than ammonia in snals
from both regions; in both areas, however, the average amount
of urea increased from the first tidal cycle observation to the
second, carried out two and one-half weeks later.
5. There was less undetermined-N in the kidneys of low area snails
than high. These averages decreased over a period of two and
one-half weeks.
6. Uric acid-N was negligible until the second tidal cycle
observations, where it then constitued up to 20.77 NPN in the
kidneys of higher snails and 3.137 NPN in those lower. This
production was very spasmodic with rapid elimination.
58
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TIDE
HLW
mID
LAW
mID
LLW
mID
HHW
m1D
HLW
mID
LHW
m1D
LLW
MID
H HW
m1D
HLU
mID
LHW
m1D
LLW
mID
HHW
LAW
miD
RID
LEW
HHW-LLO
Von- Peors Mreocee Perrn
URIC ACID OTHEES
m6. NPN
NA,
UREA
6M. KIDNEY
% N
% N
% N
% N
1/90 - 575
1, 1ou Besa
3.45-5.58
0.382-0.599 46.85- 63.56
1.85- 16.05
26.3- 43.3
NO - DATA
0.276- 1.02 45.5 - 65.48
6.56 - 13.6
2.73 - 4.16
19. 9 - 43.4
0.262- 1.02
63.49-90.24
2.35 - 3.87
9.57- 35.5
1.25 -4.16
12.4 - 41.6
46.4 -77.12
0.233- 1.07
9.25 - 18.0
2.92 -4.35
30.26 - 45.5
11.94 - 42.4
O.262-0.182 26.5 -48.16
O.343-0.866 31.2 -64.7
2.57 -3.8
13.0 -40.8
9.6 - 33.
0.336-0.770 14.8 -47.75
2.15 - 3.1
15.5 - 56.7
8.75 - 44.7
5119
1A. LouRee
5 122
5.6 - 7.2
40.0
55.0-65.2
0.322
4.68
43.0 -73.8
21.5 -43.2
O. 300-0.533
1.4 -31.8
6.68 - 8.42
5.24-8.0
26.2 -35.5
1.61 - 1.97
19.2 -24.5
38.39-52.63
20.3 - 25.6
0.705-0.800
17. 3-36.2
37.5-61.6
6.77 -6.9
40.5
4.31
46.7
3.13
9.67
o.61 -1.68
13.4 - 47.3
5.98-6.7
27.3-30.3
24. (2-21.8
o.168-1.76
7.01 - 8.55
20.0-37.0
8.75-13.
52.63-71.88
30.18
1.43
59. 64
8.33
8.75
4130
515
Hen Ress
2.
3.13-4.46
5.3 -16.55
59.75-68.67
14.65-34.7
0128 -0.172
4.89- 6.44
70.18-79.70
6.68-12.35
0.121 - 0.334
7.61 -21.7
o.181 - 0.275
2.83-6.36
15.95-35.4
4.0 - 25.6
38.72 -74.70
2.44-9.13
7.58 -27.8
O.150-0.280
45.48-68.81
17.9 -26.5
6.6 - 25.
23.7- 49.8
O.141 -0.332
3.51 - 5.65
25.77-69.86
O.108 -0.156
5.01 -7.49
13.8 -22.8
2.09-13.9
69.56 -76.49
2.31 -4.02
21.4-61.0
0.150 -0.42
4.06 - 9.95
33.70 -70.8
2-h Hieneea
5119 - 5 122
17.0- 32.2
12.8 -15.2
18.3 - 38.9
5.53-7.53
36.7-38.9
28.5-29.8
15.9 - 29.3
O.409-0.608
40.49 -55.0
10.45 -11.5
6.04 - 9.51
11.0-22.4
0.823-20.9
24.5-37.8
30.3 - 52.3
O.113-0.135
22.1 -25.0
49.0 - 55.82
28.8-29.05
8.25 -9.6
21.6 - 73.62
0.127-0.213
3.9-8.85
17.5-45.8
8.75-32.4