THE STABLE MANGANESE
CONTENT
OF MOLLUSCS
FROM LAKE MAGGIORE
DETERMINED
BY
ACTIVATION
ANALYSIS
Margaret
Merlir#
F. Girardi,2 R. Pietra,2 and A. Braxxeh’
EURATOM,
C.C.R. Ispra, Italy
Determinations of stable manganese in four species of freshwater molluscs were made by
activation analysis. Significant differences were found between the two species of snails and
between the univalvcs and bivalves. There is a correlation between the accumulation of
fallout “Mn and stable Mn in the different organs of the bivalves. In the clam shell, Mn
appears to bc nonhomogencously distributed and to increase in concentration with size.
There was a difference in the Mn content of tissues of bivalves collected from different sites.
INTRODUCTION
Manganese-54 is formed by neutron activation of iron in the shells of bombs detonated during nuclear tests; as a result, it
is part of radioactive fallout. Although it
was detected in marine plankton, molluscs,
and fish after the Eniwetok
explosions
(Lowman, Palumbo, and South 1957; Lowman ISSO), there has been little in the literature concerning its appearance in organisms until very recently (Ravera and
Vido 1961; Folsom et al. 1963; Malvicini
et al. 1963; Scheffcr and Ludweig 1963).
This radionuclide
first appeared in bivalves of Lake Maggiore in Northern Italy
in September 1960. The soft tissues of two
species of lamellibranchs, U& sp. ( Ravera
and Vido 1961) and Anodonta sp. ( Merlini,
unpublished data), revealed high quantities
of ““Mn, although thcrc was no evidence of
it in the soil, the water of the lake, aquatic
plants, or other animals at that time. The
question of the quantity and distribution
of stable manganese in these animals,
which accumulate large amounts of the
radionuclide,
arose. The information
in
Vinogradov
(1953) indicated
lamellibranchs were the molluscs with the highest
content of stable Mn in their tissues. We
were able to confirm these data for Unio
with activation analysis (Merlini 1962; Girardi and Merlini 1963).
The desire to extend the investigation of
stable Mn to other species of molluscs from
l Biology Service.
2 Nuclear Chemistry.
the same environment resulted in simplifying the technique to facilitate the determination of a large number of samples.
This study concerns a technique for the
continued study of stable manganese in a
large number of diversified samples and
the results obtained on four species of molluscs from Lake Maggiore: Unio mancus
elongatulus (Pfeiffer)
and Anodonta cygnea ( bivalves); Viv@arm a&r ( Cristofori
and Jan) and Lyrnnuea ovata ( univalves ) .
The data permit a correlation between the
stable element and 64Mn from fallout. In
addition, they provide more up-to-date information on the quantity and distribution
of Mn in freshwater molluscs.
The technical assistance of Mr. Hcnk
Vissers is gratefully acknowledged.
MATERIALS
AND
METHODS
All of the univalves and some of the bivalves were collected from the same area
of the lake, Angera, about 10 km south of
Ispra. For comparison, Unio and Anodonta
were selected from another area in the lake,
Lisanza, where the bottom material is mud
rather than sand. Viviparus were taken from
a muddy bottom and a rocky one, while
Lymnaea were found on the stones at the
edge of the water at the Port of Angera.
The snails were divided into size classes
and their tissues were pooled for analysis,
U&o and Anodonta were separated into
shell and soft tissues and the latter further
dissected as follows: gills plus palps, mantle, visceral sac, and adductor muscles.
371
372
MARGARET
Choice of analytical
MERLINI,
F. GIRARDI,
method
An analytical method may be chosen for
its sensitivity, precision, rapidity, simplicity
of analysis, cost, and so on. In the case of
manganese, activation analysis was used for
the following reasons:
1) Reliability. The reliability of a chosen
method for biological materials is dependent upon the homogeneity of the sample.
In our case, acid dissolution introduces errors from impure reagents. Therefore, homogenization by powdering in an agate
mill is done on pooled samples when it is
necessary for sampling. The usual method
is to dry, weigh, and seal the samples in
polyethylene bags. Thereafter, contamination by Mn is impossible. After irradiation,
the sample is counted at least three times
to be sure that the activity decays with the
half-life of “Mn. Under these circumstances
the only possibilities for chance errors are
from dust containing
Mn or erroneous
weighings. With a minimum of care, these
errors can be reduced to obtain completely
reliable data.
2) Rapidity. The analyses are done with
the semi-automatic method described by
Girardi and Merlini ( 1963). The operator
needs only to prepare the samples in polyethylene bags for irradiation, put them on
the sample changer after irradiation,
and
prepare the machine for the automatic run.
For rapidity, the method can compete with
any other analytic technique for determining manganese in biological materials.
3) Applicability.
Samples of animal, plant,
or mineral materials, detritus, and so on,
can be determined with the same technique
for concentrations of Mn ranging from less
than one ppm to several per cent.
4) Practicability.
Activation analysis is
often considered a sophisticated technique,
but the method as described is so automatic that any properly equipped laboratory
can carry it out.
Nuclear reactions
Manganese has only one stable isotope,
“Mn, which is transformed to 60Mn by the n,
7 reaction. Manganese-56 is a beta-gamma
R. PIETRA,
AND
A. BRAZZELLI
emitter with a half-life of 2.56 hr. The activation cross-section is 13.3 barns. Other
isotopes are formed from Mn by means of
neutron capture, but the cross-sections and
half-lives are such that they are never used
for the analysis of stable Mn.
The determination
of stable manganese
by this method is an ideal example of analysis by means of activation. The cross-section is large and the half-life of the radioisotope is short enough to give a good
saturation with a short irradiation time. Its
short half-life also permits a check on the
purity of the radiations emitted by following the decay of the gamma spectrum.
The only other nuclear reaction which
produces 60Mn is the reaction:
The cross-section of the reaction is 48 millibarns for a fission neutron spectrum. The
totality of the interference was measured
for our irradiation position by irradiating
highly pure iron and measuring the 6uMn
formed. One gram of iron gives 1.2 ppm of
Mn, and this amount can bc overlooked in
most biological samples, except when the
sample is rich in iron and the level of manganese is below one ppm.
PrepaTation of sample
Preliminary trials showed that the concentration of Mn in our samples was from 10
to thousands of ppm. These data helped
decide the choice of the irradiation
and
counting technique, because the quantity
of 60Mn is such that it appeared more desirable to reduce it rather than increase it.
Avoiding contamination is the prime factor governing the preparation of samples
for activation analysis. Therefore, the animals were dissected on Plexiglas and dried
at IOOC. Individual
pieces of tissue were
sealed in plastic bags without previous
treatment. Pooled samples were powdered
with an agate mortar and pestle or in an
agate homogenizer, and duplicate samples
were prepared. In both cases the weight of
the sample never exceeded 200 mg to keep
within the desired range of activity.
SlYABLE
TABLE
1
2
3
2
1. Precision ancl accuracy
105
105
4,850
4,850
145,000
5.28
4.97
5.89
5.55
x
x
x
x
MANGANESE
of the cleternaination
10’
IO0
10’
loo
IO6
Average
5.43 x loo
Relative standard deviation = -t-5
* Irradiated
IN
MOLLUSCS
373
of manganese
Iron wise
by neutron irradiation
0.345
0.369
0.370
0.366
0.365
12 determinations
0.363
0.365
in a flux of 1013 neutrons cm-3 set-1.
Irradiations
The irradiations were #done in the PI1
position (pneumatic
Rabbit)
or in the
4DHl position of the Ispra I reactor. The
thermal neutron flux is of the order of 2 X
1013 neutrons cm-” see-l in both positions.
The samples were irradiated in groups of 8
to 10 at a time. The Mn content of the polyethylene bags proved to be negligible
( about 0.07 ppm ) . An iron wire containing
a known concentration of Mn (0.365%) was
irradiated with the samples as a standard.
The space occupied by the samples and the
standard was rather large (a cylinder 20
mm in diamctcr and 100 mm long in the
case of position PII; and a sphere 35 mm
in diameter in the case of position 4DHl).
But in both cases, the errors due to the
lack of an even distribution
of flux are
negligible (less than 3% difference from the
center of the capsule to the radial position
farthest away, with no lack of homogcncity
axially). The duration of irradiation varied
according to the nature of the samples to
be analyzed but, in general, it was short
( about 10 set ) .
Measurement of activity
The problem of diminishing the counting
rate of the ‘“Mn formed was simply solved
by the particular setup used. An integral
line assembly of a NaI thallium activated
crystal 3 inches (7.6 cm) in diameter with
a 3-inch (7.6 cm) photomultiplier
tube
was placed in the center of a cubic lead
shield 80 X 80 X 80 cm, (inside dimensions ), 10 cm thick, and lined on the inside
with a degraded shield of 1 mm of Cd and
0.25 mm of Cu.
A sample changer allowed for the automatic counting of a series of eight samplcs. Since the counting time was the same
for all eight samples, the sample changer
was made so that one could choose the distance between dctcctor and sample according to five different positions, thus
avoiding large variations in counting rates.
The output pulses were analyzed with a
Labcn 512 channel analyzer and recorded
on punched tape. Punched tapes and other
information
regarding the irradiation
and
counting parameters were sent to the Scientific Data Processing C’entre for automatic calculation of weights and concentrations by an IBM 7090 computer. The
computer program (Borclla
and Guzzi
1964) was coded in Fortran. The analysis
rcquircd approximately 0.5 set (computer
time) per spectrum.
Sensitivity
The sensitivity depends on the nature of
the sample, as is always the case when
measuring a mixture of radionuclides without chemical separation after activation.
For
the present study the sensitivity never
constituted a problem. When necessary, the
detection limit was lowered to 0.1 ppm or
less without any important
modification
of the method.
Precision and accuracy
The precision was estimated by irradiating known quantities of solutions of manganous salts of various concentrations. The
374
MARGARET
TABLE 2.
The average
MERLINI,
amount
I?‘. GIRARDI,
R.
of stable manganese
PIETRA,
AND
A.
BRAZZELLI
in different pofrtions of male and female Viviparus
samples are shown with the average
ater, and in Lymnaca ovata. The results of replicate
Molluscs
Gastropoda
Mn,
No. of
animals
Shell
Soft
mm
tissues
Operculum
Embryos
Prosobranchia
Viviparus
ater
Mu&sand
bottom
Rock bottom
899
3.84
899
3.78
899
3.28
8QQ
3.26
888
3.15
888
3.11
899
3.32
888
97
94
95
89
81
80
83
81
103
96
87
90
94
97
110
118
3.12
39
95
38
::
92
38.5
43.5
36
35
38
36
36
34
30
31
50
56
46
42
80.5
82.0
99.5
88.5
95.5
114.0
35.5
37.0
35.0
30.5
53.0
44.0
181
165
138
132
220
*
131
*
125
*
109
*
*
210
231
*
173
48
49
48.5
50
47
48.5
50
50
50
135
220
131
125
109
210
231
Pulmonata
Lymnaea
ovata
Group I
45
Group 2
* Sample
I
9000 -
37
37
33
33
1.63
45
1.63
I
I
98.0
activity produced was then measured and
referred to a weight unit. The relative
standard deviation of five ,determinations
was k 5%. The accuracy was measured by
comparing the results obtained by activation analysis of the iron wire used as a
manganese standard with the results obtaincd by chemical analysis, No positive or
negative bias was found within the limits
of experimental error. The results are summarized in Table 1. The precision of activation analysis determinations is always influenced by the presence of other radionuelides in the gamma spectrum. Since ““Mn
was the predominant source of gamma activity in all the irradiated specimens (Fig.
1 ), the precision of the actual analysis was
about that quoted for the pure standards.
I
56M”
0.85,
7000 6000 6 5000 8
4000 3000 2000 56Mn
0.5
1,
I
0
33
88.5
lost.
8000 -
0
95
82
100
95
37
1.0
4.5
2.0
RESULTS
MeV
saml+G. 1. A typical spectrum of an irradiated
ple of gills from Unio ( 0.07 grams). Irradiation
time: 10 set; decay time: 5 hr; counting time: 1
min.
Gastropoda
Viviparus
analyses
ater (Cristofori and Jan). The
in Table 2 can be summa-
shown
STABLE
MANGANESE
TABLE 3. The manganese content of shells of Unio
collected from two different areas of Lake Maggiore, Angera and Lisanxa. Calculations are based on
dry weight (1OOC). Average values are for different size classes
Lisanza
Angcra
Sherczyth
4.30
5.15
5.45
5.95
Mn, mm
216
222
336
293
Average : 267
6.00
413
6.00
371
6.00
252
6.00
319
6.05
593
6.10
388
6.15
354
6.20
318
6.35
268
6.40
479
6.60
184
Average: 337
7.35
632
7.40
575
7.50
838
7.90
766
Average: 689
Shdf;gth
Mn, pm
4.60
4.80
5.00
5.00
5.25
5.50
5.65
5.70
5.70
5.75
5.90
181
206
252
335
356
276
204
261
345
440
524
Average : 307
6.00
517
6.00
353
6.00
264
6.05
568
6.05
374
6.10
299
6.10
267
6.10
350
Average : 374
7.00
429
7.00
577
7.15
650
7.20
745
7.25
603
Average : 600
rized as follows :
1) On an equal weight basis, the operculum has the highest Mn content. It
shows, however, greater variation
than
either the shell or soft tissues.
2) The shell contains more than twice
the Mn found in the soft tissues,
3) The whole embryo is slightly richer
in Mn than the other soft parts of the female.
4) Viviparus living on a rocky bottom
appear to have more Mn than snails living on mud and sand.
5) There seems to be no significant
difference in Mn content related to sex or
size of the animal.
Lymnaea ovata. For L ymnaea the order of
manganese enrichment is opposite of that
found for Viviparus. The soft tissues of
IN
375
MOLLUSCS
TADLE 4.
Size classes,
cm
4.5-5.95
6.0-6.6
7.35-7.90
The amount of Mn in the soft tissues of
Unio of different size classes
Mn, mm
Gills
Mantlc
8,874
10,782
21,666
3,106
5,465
18,427
Visceral sac Muscle
774
975
3,207
752
971
3,554
Lymnaea contain more than twice the stablc manganese found in their shells (Table
2). This is as much as in the shells and
twice as much as in the soft parts or embryos of Viviparus. On an equal wedght
basis, Lymnuea contains less ( 50 ppm ) Mn
than Viviparus ( 81 ppm ), despite the high
content of the element in its soft tissues.
Pdecy podu
The soft tissues of Unio are rich in stable
manganese ( Merlini 1962 ) , but the question
arises as to whether or not the Mn content
of the shells and soft tissues increase with
the size of the animal, and if there is a
difference in the quantity of the element in
animals from different collection sites.
Unio mancus elongatulus (Pfciffer) . Unio
were collected at random from two locations in the lake that vary considerably in
type of sediment. The sublittoral zone of
Angera is very sandy, whereas the mud bottom at Lisanza is richer in organic matter
( Table 3). Although the number of samples
was limited, there does not appear to bc
any significant difference in the Mn content
of the shells from Angera and those from
Lisanza. Despite the great variation, it
appears that Mn content increases with
size.
The soft tissues of the different size
classes of Unio from Angera were pooled
and analyzed. The averages indicate that
Mn increases with size in each segment of
the soft tissues (Table 4).
Taking only one size class in consideration from each area, the organs and tissues
which seemed more likely to show a diffcrence in the quantity of the element due to
the environment were analyzed. On the
average, the animals from Lisanza appear
to have significantly more Mn in their soft
376
TABLE
MARGARET
5.
Manganese
MERLINI,
6.20
6.05
R. PIETRA,
level in soft tissues of Unio collected
Average
lygk$
Anger-a
Lisanza
F. GIRARDI,
AND
A. BRAZZELLI
from two different
areas of Lake Maggiore
Mn, wm
Gills
12,974 -c- 1,245
15,382 Z!I 598
tissues, particularly the mantle and visceral
sac (Table 5). This was confirmed by pooling the content of the organs and tissues of
a smaller size class.
To determine whether manganese is distributed
homogeneously
throughout
the
shell, Mn was measured in different parts
of badly eroded shells of Unio (Fig. 2). The
exposed layer of mother-of-pearl was easily
removed and the shell was broken into
different pieces. The highest values were
obtained for the mother-of-pearl
stratum
from the umbo arca, and the lowest for the
hinge ligament. The edge of the shell with
all layers ( conchiolin, prismatic, and nacreous) has less Mn than the nacreous layer
a1one.
Anodonta cygnea. Anodonta, unlike Unio,
require sediment rich in organic matter and
so are less frequently encountered in the
Angera area where Unio is found in abundance. Therefore, the results given in Table
6 arc for animals of the Lisanza area only.
The amount of Mn in the soft tissues of
Anodonta is less than that found for Unio
examined over the same range of shell sizes
( Girardi and Mcrlini 1963). The rank order
of Mn concentration is: gill, mantle, visceral sac, and muscle, as in Unio. There is one
obvious difference between the two spccics
-the
mantle of Unio contains more Mn
than that of Anodonta. This has been confirmed by other determinations not reported
here.
DISCUSSION
It has often been stated that if the elemental composition of certain organisms
and their environment were known, one
could calculate, for equilibrium conditions,
the extent of accumulation of radionuclides.
The uptake of 54Mn and the quantity of the
stable clement in the tissues of molluscs
illustrates this point. One would not expect
Mantle
7,032 +
737
11,842 k 1,702
Visceral sac
Muscle
888 -t- 92
3,599 I+ 649
1,009 -I 220
1,697 + 204
to find much 64Mn in gastropods like Vivipaurs and Lymnaea having small amounts
of the stable clement. In fact, 64Mn has not
been detected in these animals. In Unio,
instead, it has been possible to correlate
the preferential sites of 64Mn accumulation
with that of stable Mn. The distribution of
“‘Mn among the ,diffcrent parts of Unio
( Gaglione and Ravcra 1964) and that of
the stable clement, determined by us, expressed as per cent of content in the whole
animal, is as follows:
Per cent
stable Mn
pedant
Shell
Soft tissues
34.4
65.6
35.0
65.0
Gills
Mantle
Visceral
23.6
25.4
16.6
18.0
38.0
8.7
sac
The correlation is even more striking
when one considers that the animals were
collected at different times and sites in the
lake. Furthermore,
Ravera and Gaglione
( 1962) found an increase in “Mn concentration with age and size in Unio, a fact in
agreement with the present results obtained
on stable manganese.
Although the “Mn content of the mantle
of Anodonta was high (Merlini, unpublished
data), it was less than that of Unio in
1960. Recently, Gaglione and Ravera ( 1964)
noted that it was one-third that in Unio.
These observations fall in line with the
data reported here on stable Mn.
In addition to the above, much has been
learned concerning manganese in molluscs.
We concur with Vinogradov’s ( 1953) conclusion from earlier data that the amount
of the element in gastropods varies with the
site of collection and the type of nutrition.
The difference in alimentation between
Viviparus and Lymnuea might explain the
diffcrcnce in the Mn content of their soft
tissues. Lymnaea live on stones at the edge
378
MARGARET
MERLINI,
F. GIRARDI,
small part of the difference can be accounted for by the fact that the shells of
Viviparus contain 10% calcite whereas the
shells of Lymnuea from the same area are
100% aragonite
( Merlini,
unpublished
data). However, an examination of the dissccted embryos of Viviparus shows that the
shell contains twice the amount of Mn
found in the soft tissues. Obviously the preferential deposition of Mn in the shell goes
back to its formation in utelro.
Two facts concerning stable Mn in shells
of Unio are in agreement with Nelson’s
(1963) report for strontium in shells of
freshwater clams from the Tennessee and
Clinch rivers : 1) the amount of the element is at least partially dependent on the
age of the organism; and 2) the distribution
is nonhomogeneous. Nelson noted an increase in strontium in the nacreous layer
with age and related this to a decreasing
surface-volume
relationship. Such a relationship might well be the cast in Unio
for Mn, but the data so far obtained on the
nacreous layer are not sufficient to confirm
A complete study of the radioecology and
biogeochemistry of manganese in an aqueous environment such as Lake Maggiorc requires the investigation of certain parameters on a statistical basis:
1) Individual variation within the same
size class.
2) Changes in Mn content of soft tissues with season, size, and physical state
( reproductive or nonreproductive
period )
of the animal.
3) The influence of the immediate environment upon the quantity of Mn found
in the animals through an investigation of
the manganese level in water, sediment,
and plants of the lake.
This study answers only parts of these
questions. The perfect functioning
of the
method of activation analysis, combined
with computer calculation of the data as
described, should supply the necessary
answers.
R. PIETRA,
AND
A. BRAZZELLI
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