&EPA
Researc
jelopmeo!
Bioaccumulation of
Heavy Metals by
Littoral and Pelagic
Marine Organisms
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EPA-600/3-79-038
March 1979
BIOACCUMULATION OF HEAVY METALS
BY LITTORAL AND PELAGIC MARINE ORGANISMS
by
John H. Martin
Moss Landing Marine Laboratories
Moss Landing, California 95039
Grant NO. R802350
Project Officer
Donald K. Phelps
Environmental Research Laboratory
Narragansett, Rhode Island 02882
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND 02882
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
Narragansett, U. S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U. S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
11
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FOREWORD
The Environmental Research Laboratory of the U. S. Environmental
Protection Agency is located on the shore of Narragansett Bay, Rhode Island.
In order to assure the protection of marine resources, the laboratory is
charged with providing a scientifically sound basis for Agency decisions on
the environmental safety of various uses of marine systems. To a great
extent, this requires research on the tolerance of marine organisms and
their life stages as well as of ecosystems to many forms of pollution stress.
In addition, a knowledge of pollutant transport and fate is needed.
This report describes a three-year study to measure concentrations of
various metals by atomic absorption spectrophotometry in important littoral
and pelagic marine organisms.
ill
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ABSTRACT
Marine organisms appear to be useful indicators of heavy metal pollu-
tion in the marine environment. In order to test this concept, research
was performed to determine the levels of heavy metals in selected indicator
organisms. Several approaches were used. The first was to select inter-
tidal invertebrates that are widely distributed and are readily accessible
for collection. Tests with the limpet Acmaea scabra proved inconclusive,
while those with the turban snail (Tegula funebralis) showed anthropogenic
silver input. The experience gained from these studies indicated that
serious problems could exist when using organisms as monitors. As a result,
a study on pooling of individuals for monitoring studies was performed and
the results are reported here.
A second approach was to transplant oysters and mussles from clean to
polluted environments in order to see if these organisms reflected ambient
environmental levels. Significant increases in selected elements were ob-
served in both bivalves and the general approach appears promising.
In addition to the monitoring research, we also studied several poten-
tial heavy metal problems. For example, we studied premature pupping in
the California sea lion (Zalophus califomianus) and found that normal
mothers had molar ratios of 1 Hg:l Se:l Br, while the mothers having pre-
mature pups had Hg:Se ratios near unity, but Br concentrations were always
depressed in these individuals. Whether these findings are directly re-
lated to premature pupping was impossible to ascertain. We also report
that squid (Loligo opalescens) have extermely high Cu levels in their
livers (up to 15 mg/g dry) and that the Cu is highly correlated with Ag. A
study was also performed on cadmium in sea otters (Enhydra lutris). These
mammals eat invertebrates almost exclusively; as a result, older indivi-
duals accumulate large amounts of this element especially in the kidney
(up to 960 ug Cd/g dry weight). The relationship between Cd and other
elements was also studied and the results are discussed.
Elevated Cd levels were also found in a study of plankton collected
from Baja California waters. Subsequent research has revealed that the Cd
is of natural origin and not from anthropogenic sources. As is the case
with many other pollutant studies, the general conclusion drawn from this
study and the others mentioned above is that many marine organisms have
high concentrations of heavy metals, but whether the metals are adversely
affecting the organisms cannot be determined on the basis of measuring
amounts alone.
iv
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TABLE OF CONTENTS
Disclaimer ii
Foreword iii
Abstract iv
Section 1: Introduction 1
Section 2: The Use of Organisms as Pollutant Monitors 3
Section 3: General Studies on Metals in Marine Organisms 25
A. Premature Pupping in California Sea Lions 25
B. Silver, Cadmium, Cooper, Zinc and Iron in Squid Livers 27
C. Abalone Copper Toxicity Experiments 28
D. Cadmium in Plankton: Elevated Levels off Baja, California ... 28
E. Cadmium in Sea Otters 30
Section 4: Methods 49
Publications and Theses Based on Research Supported by
EPA Grant # R 802350 51
References
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SECTION 1
INTRODUCTION
The toxicity of heavy metals has been the subject of a vast amount of
research, in large part because of the threat posed by these elements to human
health. For example, several disastrous cases of mass poisonings by mercury
compounds have occurred in recent years; e.g., between 1953 and 1960, 121 cases
of Hg poisoning were recorded in Minamata, Japan; 46 people died. In Iraq, mass
Hg epidemics occurred in 1956 (100 cases; 14 deaths), in 1960 (1,000 cases) and
in 1972 (6,530 cases; 459 deaths). (See Eyl, 1971; Bakir, et al_, 1973.)
Several other heavy metals are also toxic enough to represent human health
hazards. Cadmium-induced decalcification of the skeleton (Itai Itai disease)
has taken 56 lives in Japan (Nilsson, 1970); human lead poisoning has been known
since antiquity (Patterson, 1965).
In addition to the threat to man himself, agricultural scientists have long
been concerned with the toxicity of certain trace elements to valuable domestic
animals. This is best exemplified by selenium, an element whose toxic effects
were apparently first described in 1295 by Marco Polo during his travels through
China (Trelease and Beath, 1949). This element is concentrated by certain plant
species; herbivores grazing on these plants exhibit symptoms commonly called
"alkali disease" and "blind staggers". These conditions can lead to mass mor-
talities; i.e., in 1906 and 1908, 15,000 sheep died of selenium poisoning in the
state of Wyoming (Trelease and Beath, 1949).
Mass mortalities caused by heavy metals have also been observed in the
marine environment. Approximately 2,000 abalone (Haliotis rufescens, H^.
cracherodii) died from copper poisoning when a power plant cooling system was
tested (Martin, et al_, 1977). The copper had leached out of copper-nickel
tubing into sea water which had been standing in the system for several weeks.
When the water was released, it contained approximately 1,899 ppb Cu, a concen-
tration that resulted in the deaths of almost all of the abalone living in the
immediate discharge area.
However, in comparison to the terrestrial environment, instances of this
nature rarely have been reported in the marine environment. Nevertheless,
research efforts on metals in the sea have been increasing in recent years
because of a series of interrelated events. The well-publicized Minamata Bay
disaster was caused by the consumption of marine food items that were contami-
nated with large amounts of methyl mercury. A few years later, tremendous
advances in analytical capabilities for trace elements were being made, in-
cluding the development of a very sensitive technique for Hg (Hatch and Ott,
1968). This led to the finding that important marine food items such as tuna
and swordfish had relatively large quantities of Hg. In conjunction with these
events, man was also becoming increasingly concerned not only with the quality
of food items from the sea, but also with the quality of the marine ecosystem
-------
itself. The combination of these developments resulted in a greatly increased
emphasis on heavy metal research in the marine environment.
In order to learn more about these processes, we began a study on the
bioaccumulation of heavy metals by littoral and pelagic marine organisms in
January 1973. Most of our results have been published in the open literature
and repeating these papers' contents in the text is not warranted. Abstracts
of the papers are included in the sections that follow. In most cases, re-
prints can be obtained by writing the author. We will comment on the papers
and discuss them in light of additional information made available after various
portions of this research were published.
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SECTION 2
THE USE OF ORGANISMS AS POLLUTANT MONITORS
The use of organisms as monitors of various pollutants is attractive for
several reasons: Selected sessil organisms will continuously "sample" pollu-
tants in their environment and thus smooth out large-scale fluctuations such as
pulse input associated with some outfalls. They will also be subjected to
materials from non-discrete point sources, which would be missed in human moni-
toring studies. In most cases, organisms will concentrate selected metals both
from food sources and directly from water. Since some organisms concentrate
large amounts of a given metal, the chances for sample contamination are mini-
mized. In addition, it is easier and less costly to analyze biological tissues
than water samples.
Organisms selected as biological monitors should have a wide range, occur
in large quantities, be easily collectable from shore (eliminating the need for
boats and diving equipment), be easily identified, have a simple life history
and, in order to eliminate complex food web relationships, should also be of low
trophic level. In addition to these biological factors, a monitor organism
should, of course, concentrate metals of interest, preferably in the whole
organism, rather than in a specific organ which can necessitate tedious dis-
sections. Chances for sample contamination are also reduced when dissections
can be avoided.
The following abstracts represent our research on the use of organisms as
pollutant monitors:
Flegal, A.R. and J.H. Martin, 1977. Contamination of biological samples
by ingested sediment. Mar. Poll. Bull. 8:90-92.
Abstract: An inorganic residue, presumed to be ingested sediment, was
found in the rocky intertidal gastropods Tegula funebralis and Acmaea
scabra and the estuarine copepods Acartia tonsa and A_. clausi. When ex-
pressed as a percentage of the sample weight, this residue fraction often
correlated significantly with the elemental concentrations measured in
the organisms.
Flegal, A.R. Trace element concentrations of the rough limpet, Acmaea
scabra. in California. (Accepted by Bulletin of Environmental Contami-
nation and Toxicology).
Abstract: The trace element (Ag, Cd, Cu, Fe, Mn, Ni, Zn) concentrations
of the rough limpet, Acmaea scabra, were determined at twelve locations
along the California coastline. The mean silver concentration of the
organisms was highest at Point LaJolla, but no elemental concentration
exhibited measurable geographic differences. Simple linear correlation
coefficients and multiple analysis of variance statistics indicated a
general independence of the elemental concentrations from each other and
from other biological and geographic variables in the total sample.
Flegal, A.R. The geographic variation of silver in the black turban
snail, Tegula funebralis.
3
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Abstract: The geographic variation of silver concentrations in the black
turban snail, Tegula funebralis, collected from the California-Mexico
coastline indicates an anthropogenic influence. The relative variation
in this species is also consistent with those reported for other organisms.
The experience that we gained in the above studies indicated that serious
problems existed in attempting to use biological organisms as monitors.
For example, animals such as Tegula were very small and detection limits
were pushed to their maximum. Sediment often occurred in association with the
sample, which, in some cases, invalidated the findings for some elements.
Tremendous variability between individuals was observed, even when the samples
were collected in the same area on the same day. It was apparent that improve-
ments could and should be made.
As is the case with almost all research, the state-of-the-art is contin-
ually improving in trace metal analyses. This is especially true since the
completion of our studies. In recent years, the use of graphite furnace atomi-
zers has decreased detection limits to the point where very small animals can
now be analyzed successfully. This opens up a whole realm of species that were
previously too small to serve as successful monitors. With the improved detec-
tion limits, analytical variability between samples will decrease markedly,
provided proper corrections are made for the severe matrix problems that almost
always are associated with the use of furnaces.
Nevertheless, graphite furnaces do nothing to improve variability inherent
within the organisms themselves. Thus, the fact remains that large numbers of
individuals must be analyzed in order to obtain statistically valid differences
between selected areas. This is exemplified by the data in Tables 1 and 2.
Twenty-eight mussels (Mytil us edulis) were collected in San Francisco Bay
for comparison with thirty individuals collected at Moss Landing, California.
The data were then tested to determine the minimum number of replicates needed
to determine valid differences for each element. Very few would be needed for
Pb. The means and standard deviations are so different that only two indivi-
duals from each area would be required. However, in the case of Cd, over 120
individuals would have to be analyzed from each site in order to detect a sig-
nificant difference. Clearly, more than 240 analyses would be unfeasible. This
would be especially true if the analyses were performed using a graphite fur-
nace. This is an extremely time-consuming technique, especially when the method
of standard additions has to be used.
For these reasons, we have begun investigating the feasibility of pooling
samples in order to obtain significant data with a minimal number of analyses.
The following discussion was developed by George Knauer, Mike Gordon and Ann
Hurley of our laboratories.
In order to establish baseline levels of trace metal concentrations in a
given population, the first step must include estimates of the parameters y
(true population mean) and a (true population variance). Since it is not
possible to measure all individuals in a population, it becomes necessary to
sample a portion of the population of interest in an attempt to estimate the
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TABLE 1: Concentrations of trace elements observed in
mussels (Mytilus edulis) collected in an
unpolluted environment (Moss Landing, California)
ng/g
Length
(mm)
57
57
56
56
49
52
50
51
52
47
46
60
48
43
45
54
50
46
48
52
49
55
52
48
56
54
59
50
47
44
Mean
Std Dev
Pb
.38
.27
.90
.31
.51
.58
.23
.16
.32
.12
.48
.01
.16
.36
.16
.27
.29
.17
.27
.63
.84
.49
.26
.39
.71
.23
.17
.29
.32
.45
.36
.21
Cd
6.
9.
13.
11.
14.
11.
13.
9.
3.
10.
12.
9.
9.
6.
8.
17.
9.
14.
7.
9.
14.
23.
15.
17.
12.
12.
13.
5.
5.
6.
11.
4.
9
9
3
0
3
5
7
7
8
7
8
4
8
4
0
0
9
0
0
2
0
9
8
2
8
5
8
3
2
4
2
3
Dry Weight
Cu
17.5
12.4
20.3
20.9
25.2
19.2
20.3
20.4
14.9
24.2
15.4
17.3
15.2
23.1
19.6
19.5
17.4
13.7
16.4
23.1
22.5
29.2
15.7
23.8
13.8
22.2
19.5
18.4
12.6
16.0
18.8
6.0
Zn
46
64
302
102
126
279
122
142
114
105
232
98
47
138
280
195
61
75
118
101
266
162
170
166
98
240
499
138
159
193
161
96
Mn
4.5
3.3
7.5
5.3
8.3
6.3
7.0
7.2
4.4
9.1
4.7
2.8
4.7
11.4
5.2
6.8
5.7
3.8
5.6
9.0
8.1
12.4
5.0
8.4
4.2
6.5
6.7
6.0
7.5
5.9
6.6
c 2.7
Fe
230
200
690
370
570
480
410
510
190
670
260
220
260
810
430
400
360
210
370
750
440
920
340
720
300
300
410
300
490
340
430
200
Al
120
100
480
200
400
340
280
360
120
460
no
10
120
590
240
260
230
90
300
560
290
640
180
480
160
180
200
160
360
220
270
160
Ni
1.0
.9
3.4
2.1
3.0
1.9
2.1
2.8
1.0
3.0
1.1
.9
1.0
3.7
1.8
2.0
1.1
.8
1.1
3.1
1.7
3.2
1.1
3.3
1.4
.4
2.0
1.7
1.6
.7
1.8
1.0
Ag
.22
.12
.13
.19
.23
.06
.19
.19
.11
.21
.13
.21
.18
.20
.20
.16
.20
.15
.16
.16
.25
.31
.14
.19
.13
.19
.24
.10
.20
.11
.18
.05
Cr
1.0
1.0
3.0
2.1
1.4
1.7
1.9
2.4
.7
2.4
1.1
1.2
1.0
3.6
1.8
1.2
2.0
1.9
1.7
2.0
2.1
3.4
1.5
2.4
.6
1.1
2.4
1.2
1.9
1.3
1.7
.8
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TABLE 2: Concentrations of trace elements observed in
mussels (Mytilus edulis) collected in a pol-
Length
(mm)
47
54
56
48
48
61
43
55
59
53
58
47
65
46
60
53
51
56
60
59
50
61
53
52
48
63
60
53
Mean
Std Dev
1
uted environment (San Francisco
yg/g Dry Weight
Pb
2.
2.
4.
3.
2.
2.
4.
1.
.
2.
.
9.
•
2.
1.
•
3.
3.
.
.
1.
1.
2.
7.
7.
.
3.
4.
2.
2.
05
17
98
19
71
59
23
53
16
92
44
40
84
66
09
39
33
90
73
21
86
37
52
48
36
72
70
36
82
31
Cd
3.2
10.0
11.4
7.8
12.5
11.8
8.9
23.2
15.7
10.4
11.2
11.0
11.2
7.7
10.4
19.7
21.4
23.1
20.2
17.8
10.4
8.4
10.4
10.9
13.0
9.5
12.4
13.4
12.8
4.2
Cu
19.2
32.4
40.4
45.2
29.7
23.4
39.4
35.9
23.0
22.1
21.9
28.8
26.5
33.0
28.1
27.9
44.4
35.2
35.4
27.2
25.0
22.1
32.8
28.1
28.2
31.8
31.2
33.2
30.4
6.7
Zn
156
570
437
587
380
182
565
184
110
136
62
353
130
382
185
136
274
169
128
124
93
133
252
714
472
165
316
497
281
182
Mn
97.2
131.0
164.8
126.4
22.1
63.2
150.6
124.0
54.8
48.8
41.7
62.2
41.7
86.4
59.1
14.6
68.1
27.8
28.2
85.1
71.4
43.1
18.6
136.8
115.7
66.5
48.3
87.9
74.3
42.4
Fe
360
840
520
670
140
400
700
470
350
340
290
470
340
500
300
150
500
220
310
530
410
140
90
590
420
390
290
520
400
180
Al
340
770
440
560
40
290
620
390
280
280
230
420
280
390
270
120
420
130
230
450
340
70
70
580
350
300
270
500
340
170
Bay)
Ni
6.4
10.4
9.6
14.4
6.7
5.0
10.1
8.5
8.0
6.1
7.8
9.4
11.0
11.0
8.0
11.7
16.2
10.6
10.9
8.9
7.0
5.4
4.4
11.3
7.6
10.8
8.4
6.6
9.0
2.7
Ag
.22
2.33
.56
.38
1.01
.19
.64
1.32
.18
.20
.13
.49
.21
.28
2.46
.63
1.06
.28
.31
.67
.22
.33
.30
.28
.25
.59
.24
1.15
.60
.59
Cr
2.1
3.2
2.2
3.2
.9
1.0
3.0
2.7
1.5
2.0
1.7
2.3
2.1
1.8
1.6
1.4
3.2
2.2
1.4
2.2
1.3
.6
.7
2.2
1.9
2.1
1.8
2.4
2.0
.7
-------
_ 2
above parameters with the statistics X (estimated population mean) and a
(estimated population variance). It is an accepted fact that the larger a
random Cample from a normally distributed population, the better the estimate of
y and a will be. It should be emphasized however, that each variable (i.e.,
Cu, Zn, etc.) has its own true mean and variance.
Clearly, our aim is to establish the best possible estimate of the true
population mean and variance for metals of interest in a selected species with
the expenditure of a reasonable amount of effort. As an illustration of the
process that we intend to follow to estimate y and a , we have used the Cu data
from analyses of samples of Mytilus edulis collected from Moss Landing in 1975
(Table 1). Cumulative means; e.g., X,+X2/2, X,+X2+X3, X,+. ..XW30, (Elliott,
1971), were plotted against numbers of individuals/mean to determine tf^number
of individuals which must be analyzed to give a good estimate of y and a (see
Figure 1). Fluctuations about the calculated mean at n=30 (18.9 yg Cu/g dry
wt.) become minimal around n=18. From this, it is logical to conclude that the
analysis of eighteen individuals should give a reasonable estimate of y. Note
that Figure 1 clearly suggests that analysis of five to ten samples will not
yield an adequate estimate of y. Of course, this is for one element in one
species. Similar tests would have to be performed for other elements and
species.
The detection of a given difference between means will depend on the
estimates of y and a and upon the sampling design chosen. For example, use of
the Cu results obtained from Mytilus can be used to arrive at2a number of dif-
ferent sampling design schemes. Our best estimate of y and a (standard devia-
tion) using the My t i1 us Cu data are 18.9 and 4.14, respectively. We have used
these Mytilus Cu data to calculate the number of samples required to detect a
statistically significant difference (P < 0.05) between two means which differ
by 5 - 100%. The results are presented graphically in Figure 2 and in tabular
form in Table 3. These data can be used to determine the number of individual
analyses needed to detect some desired difference between means; e.g., 20%, or
the number of individuals which can be pooled to yield a similar resolution.
Note that one individual/pool is equivalent to the analysis of n individuals
that would be necessary to achieve some desired difference between means. The
advantage of pooling two or more individuals becomes apparent when the detection
of small differences between means with a minimal amount of effort is desirable.
It may be seen from Table 3 that, as the number of individuals/pool increases,
the number of pooled samples decreases up to the level of 75% difference between
means. At the 75% and 100% difference, three pools are needed regardless of the
number of individuals/pool. Clearly, there are an infinite number of sampling
designs which could be employed from these data; however, the choice of a single
design depends upon many factors. These include the effort required to collect
and dissect the organisms, the analytical time/sample and the total analytical
load. For example, if it is desired to resolve a 20% difference between two
means such as between 10 and 12 ppm Cu, we could analyze twenty-seven indivi-
duals (1 individual/pool, 27 pools), three individuals/pool with twelve pools or
ten individuals/pool with four pools with respect to Cu in Mytilus. It should
be made clear that, when individuals are pooled, more total organisms are re-
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24
23
LJ
2'
§20
(J
19
18
17
16
2
FIGURE I
1O 14 18
INDIVIDUALS/MEAN
22
26
3O
Cumulative Copper Means (With Selected 95% Confidence Intervals)
from Analysis of n=30 MytiI us
8
-------
14O
12O
100
to
880
Q_
6O
4O
20
O
3 5
INDIVIDUALS/POOL
10
FIGURE 2:
Plot Showing Number Pooled Mussel Samples
(Ordlnate) and Number Individuals/Pool
(Abscissa) To Be 90% Certain (I-&, Type II
Error) of Detecting 5, 10, 20, 50 or 1
Difference Between Two Copper Means at
P < 0.05 Using t-Test
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TABLE 3: Number of pools required to be 90% certain
of detecting a significant difference
(P < .05) between two means (Cu in Mytil us)
differing by 5, 10, 20, 50, 75, 100%;
d' = (y1-y2)/V2o (Dixon and Massey, 1969)
Difference in
Means (%)
Individuals/Pool d1 Number Pools
5
5
5
5
10
10
10
10
20
20
20
20
50
50
50
50
75
75
75
75
100
100
100
100
1
3
5
10
1
3
5
10
1
3
5
10
1
3
5
10
1
3
5
10
1
3
5
10
.16
.28
.36
.51
.32
.53
.68
.97
.65
1.09
1.41
1.99
1.62
2.79
3.60
5.10
2.42
4.19
5.40
7.50
3.23
5.60
7.20
10.20
556
146
88
44
106
40
25
13
27
12
7
4
6
3
3
3
3
3
3
3
3
3
3
3
10
-------
quired than when individual analyses are performed. In the above example,
twenty-seven organisms were required when individuals are analyzed, but thirty-
six organisms are required, i.e., three individuals/pool, twelve pools, to yield
the same level of resolution for pooled samples. However, if there is no limit
to the number of individuals available, then pooling may be desirable in that
the analytical load can be reduced considerably.
The above discussion suggests that pooling of large numbers of individuals
with consequent digestions and analyses of a few pools will enhance the chance
for successful monitoring using organisms. However, this concept will have to
be thoroughly tested using actual samples. We are participating in the "Mussel
Watch" (National Marine Pollution Monitoring Program) in cooperation with Dr.
Edward Goldberg of Scripps. The project is funded by EPA and involves the
collection of mussels from the conterminous United States and subsequent analy-
ses by selected investigators for various pollutants. This is a very prodigious
undertaking. Time-saving procedures such as pooling may be particularly appro-
priate to large-scale studies such as these.
Another approach to using marine organisms as marine monitors is that of
transplanting selected organisms to environments of interest and observing
changes in metal concentrations with time. A manuscript using this approach is
presented in its entirety. References mentioned are included in the report
reference section.
11
-------
BIOLOGICAL MONITORING OF TRACE METALS IN THE MARINE ENVIRONMENT
WITH TRANSPLANTED OYSTERS AND MUSSELS
M.D. Stephenson
CALIFORNIA DEPARTMENT OF FISH AND GAME
Monterey, California 93940
R.M. Gordon
J.H. Martin
MOSS LANDING MARINE LABORATORIES
Moss Landing, California 95039
ABSTRACT
Oysters (Crassostrea gigas) and mussels (Mytilus edulis) were
transplanted from a relatively unpolluted area to locations where trace
metal monitoring was desired for a period of thirty days. These species
were most effective in accumulating Ag, Cd, Cu and Ni. Significant
differences between the control sites, Redwood City, Monterey sewage
outfall and Elkhorn Slough, were observed. Transplanting is an ef-
fective method of detecting and monitoring trace metal inputs in marine
waters.
12
-------
INTRODUCTION
Trace metals in the marine environment have been monitored by a vari-
ety of techniques, including analyses of seawater, suspended particulates,
sediments and biological material (Martin, et. §U 1976; Bruland, et al_,
1974; Preston, et al_, 1972; Anderlini, et a]_, 1975). The analysis of bio-
logical material has proven valuable because organisms generally contain
easily detectable concentrations of metals, are relatively easy to collect,
are relatively inexpensive to analyze and can provide an integrated record
of environmental trace metal concentrations over the lifespan of the organ-
ism. The establishment of monitoring baselines for trace metals in marine
organisms, however, has sometimes proven difficult because of the high
variability between individual organisms. Factors that contribute to this
variability include:
1. Individual organisms may vary in metal content with size and age
(Mackay, et_ al_, 1975; Schulz-Baldes, 1973; Romeril, 1971). Organisms of
the same size may not be found at all monitoring sites.
2. Many species are too small for analyses.
3. Many animals are covered by, or contain, sediment in their diges-
tive systems (Flegal, 1978a; Flegal and Martin, 1977), which may contaminate
the sample.
4. Individual tissues vary in metal concentrations. Whole organ-
isms are analyzed instead of individual tissues. In this way, tissues
that concentrate trace metals are combined with tissues that do not (or are
contaminated), thus obscuring results.
5. Organisms may undergo seasonal changes in trace metal concentra-
tions (Bryan, 1973; Phillips, 1976).
6. Intertidal organisms may vary in trace metal concentration in
relation to the tidal heights at which they live (Phillips, 1977).
An approach using transplanted organisms such as oysters and mussels
reduces the influence of many factors which contribute to high sample vari-
ability. The goal is not to establish a baseline, but to detect changes
in trace metals over a given time period. Transplanting organisms has the
following advantages over collecting resident organisms:
1. Large numbers of individuals are available either commercially or
in natural beds.
2. Oysters can be aged in most cases and all organisms can be the
same age.
3. There are few sediment contamination problems with these organisms.
13
-------
4. Large enough individuals can be obtained so that individual tis-
sues are easily dissected and analyzed.
5. The samples can be run inexpensively with flame atomic absorption
spectrophotometry for most metals if large individuals are obtained.
6. The organisms can be easily transplanted to almost any site in
the marine environment.
One transplantation design was evaluated in this study. Oysters
(Crassostrea gigas) and mussels (Mytilus edulis) were collected from rela-
tively unpolluted areas and transplanted to various locations. Uptake
rates were determined and related to the environment to which they were
transplanted.
METHODS
Oysters (C_. gigas) were collected at Johnson's Oyster Company at
Drake's Bay, California on May 17, 1975. They ranged from 9 to 14 cm in
length. The gills of ten individuals were immediately dissected for later
analysis. Groups of ten individuals were placed in nylon net bags (1/2-
inch mesh) and suspended in the water column from a buoyed polypropylene
line. Mussels (M. edulis) were collected from the west side of Tomales
Bay on May 17, 1975 and treated in the same manner as the oysters.
Resident populations of oysters and mussels were analyzed to compare
with the transplants and to determine the tissues to be used. Four and
one-half-year-old C_. gigas and seven-year-old C_. virginica (originally
transplanted to Redwood City as spat) were collected at Redwood City in
April 1975. M. edulis were collected from native populations at Elkhorn
Slough and Redwood City in April 1975.
The control areas, Drake's and Tomales Bays, are located approximately
one hundred miles north of San Francisco and have no major anthropogenic
discharges. Organisms were transplanted to the Port of Redwood City and
Elkhorn Slough (enclosed bays), Monterey sewage outfall (semi-protected
outer coast) and Granite Canyon (exposed outer coast). The Port of Redwood
City pier is located about thirty miles south of San Francisco within San
Francisco Bay. It was selected because it is within the San Francisco
metropolitan area and is near to a major municipal sewage discharge as well
as being near many industrial discharges and therefore should have maximum
exposure to pollutants. Monterey sewage outfall is located approximately
one-half mile east of the harbor at Monterey, California. It was selected
because it is an area with a small municipal sewage discharge (2.2 mgd) and
is without any major industrial discharges. Elkhorn Slough is located
about one hundred miles south of San Francisco. It receives flow from the
Salinas River, which, in turn, receives municipal sewage discharges from
several small communities. The harbor at the mouth of the slough has
14
-------
several hundred boats. In addition, there is a cooling water discharge
from a large fossil fuel power plant. This area was selected because it is
a relatively clean enclosed bay, in contrast to Redwood City.
Tissues from the resident populations of oysters and mussels were dis-
sected from animals that had been frozen, while the gills of transplanted
organisms were dissected fresh. The tissue was dried at 70 °C for 24 hours,
dissolved with 70% quartz redistilled G. Frederik Smith redistilled HNO^,
charred at 350 °C and oxidized with 30% hydrogen peroxide (H^Op). The solu-
tion was diluted to 15 mis with 1% HNOo and analyzed by flame on a Perkin-
Elmer Model 603 atomic absorption spectrophotometer. The deuterium arc
background corrector was used to correct for non-atomic absorption. Cu and
Ag analysis of M_. edulis gill tissues required the use of the Perkin-Elmer
HGA 2100 graphite furnace. These digestion and analysis techniques were
shown to be accurate by the digestion and analysis of National Bureau of
Standards orchard leaves and bovine livers.
Medians were used instead of means to compute accumulation rates, be-
cause medians are less affected by extreme values (Sokal and Rohlf, 1969).
A non-parametric multiple comparison test for unequal sample sizes was used
to test for significant differences in trace metal concentration between
sites (Hollander and Woolf, 1973).
RESULTS
Metal levels in natural populations of M. edulis, C_. virginica and
C_. gigas^ are found in Table 1. These data were used to determine the
tissues used in the transplant experiment. Oyster gills and whole bodies
showed large differences in metals between the control sites (Tomales Bay
and Elkhorn Slough) and the relatively polluted site (Redwood City). We
decided to use gills because:
1. These data indicated that gills concentrate metals as well as
whole bodies; e.g., oysters.
2. Previous studies have shown whole body tissues to vary seasonally
in trace metal concentrations. This may be due to seasonal changes in gonad
development. Thus, gill tissue would not be expected to vary seasonally as
much as whole bodies. This is particularly important in transplantation ex-
periments in which collections are made when the population had both spawned
and unspawned organisms.
3. There is little problem with sediment contamination of gill tis-
sues.
15
-------
CTi
TABLE 1: Trace metal levels in resident mussels and oysters
(Means in ug/gm dry weight ± standard deviation)
Location
No.
Samples Cd
Cu
Zn
Mn
Fe
Ag
Crassotrea virqinica
gills
whole bodies
Crassostrea gigas
gills
whole bodies
Mytilus edulis
whole bodies
Tomales
Redwood
Tomales
Redwood
Tomales
Redwood
Toma 1 es
Redwood
Elkhorn
Redwood
Elkhorn
Redwood
Bay
City
Bay
City
Bay
City
Bay
City
SI.
City
SI.
City
9
10
10
10
10
10
10
9
30
28
30
28
27. 6± 5.7
49.5±11.4
12. 2± 4.3
39.7±10.9
20. 3± 6.8
56.4±14.2
9.0± 3.9
40. 9± 9.4
11. 2± 4.3
12. 8± 4.2
Pb
.36+ .21
2.8 ±2.3
295±131
1810±372
273±117
1510±702
178± 58
1910±430
86± 30
860+287
19+ 6
30+ 6.7
A!
274±163
336+172
3560±2310
19900±6450
3570±1220
19300±8970
290± 100
8040±1590
442+ 134
3550+1440
161± 96
281+ 182
Ni
1.9+ .95
9.0±2.7
28.
29.
34
13
125+
111±
40±
61±
6.
74.
Cr
1.7+.82
2.0+.72
1+ 5.5
7± 7.3
±18.7
+ 3.3
43.8
38.1
16.0
18.0
6+ 2.7
3+42.4
288±180
159± 51
260±207
148± 27
270± 81
580±511
368+149
668+274
433±195
402±177
5.5± 2
145 +46
6.4± 2
81.5±56
3.7±
189 ±40
1.7+
82 ±36
.18±
.60+
.9
.8
.7
.99
.4
.50
.05
.59
-------
M_. edulis whole bodies showed large differences between Redwood City
and Elkhorn Slough, although these were not always of the same magnitude
as the oyster differences.
Results of the individual analyses of oyster gill tissue from Drake's
Bay and the four transplantation sites are shown in Table 2; individual
analyses of the mussel gill tissue from Tomales Bay and the four transplan-
tation sites in Table 3. Species differences in gill tissue uptake are
evident, with oyster gill accumulating higher concentrations of all metals.
Statistically significant (p < .05) differences between control and
transplantation sites were found for some metals. Both oysters and mussels
at Redwood City contained significantly higher levels of Cu than the con-
trol organisms. In addition, the Redwood City oysters had significantly
higher Ag and Cd levels. The Monterey sewage outfall site had significantly
higher levels of Ag in oysters and of Ag and Cu in mussels, compared to the
control sites. Mussels at Elkhorn Slough had significantly higher Cu levels
than at the control area. Levels of Fe and Ms in oysters (p < .15) were
significantly lower at the Monterey sewage outfall site compared to the re-
spective control site, evidence of significant depuration.
Accumulation rates in terms of concentration increase per month are
shown in Table 4 A. Oysters at every transplantation site accumulated up
to forty times more Ag, Cu and Cd (except for Cd at Monterey sewage outfall)
than mussels. They also had a higher depuration rate for Mn and Fe than
mussels. In addition, Redwood City oysters accumulated more Ag, Cu and Cd
than Monterey sewage outfall oysters. Mussels, however, accumulated
slightly less Ag, Cu and Cd at Redwood City than at the Monterey sewage out-
fall. Redwood City mussels and oysters accumulated high concentrations of
Mn in comparison to the other transplantation sites. Monterey sewage out-
fall oysters accumulated high levels of Ag and Cu, almost no Cd and depura-
ted more Mn and Fe than those at the other stations. Metal accumulation
rates at Elkhorn Slough and Granite Canyon were generally low except for Cu
in mussels at Elkhorn Slough. Zn data did not show significant trends at
any station.
Accumulation rates expressed as percent increase per month are shown
in Table 4 B. They show the same trends as the concentration increase per
month rates in Table 4 A, with the following exceptions: Redwood City did
not always have higher uptake rates than Monterey sewage outfall; for ex-
ample, Monterey sewage outfall mussels had a higher percent increase rate
than Redwood City mussels in Ag and Cu. Also, mussels sometimes had a
higher accumulation rate than oysters. For example, mussels at Monterey
sewage outfall and Elkhorn Slough had higher uptake rates of Ag and Cu than
oysters.
17
-------
TABLE 2: Metal concentrations in oyster (C_. gigas) gills
one month after transplantation
Location
Drake's
Bay
(control)
Granite
Canyon
Monterey
Sewer
Outfall
Elkhorn
Slough
Replicate
Number
1
2
3
4
5
6
7
8
9
10
X±95% cc
Median
1
2
3
4
5
6
X±95% cc
Median
1
2
3
4
5
6
7
8
9
X±95% cc
Median
1
2
3
4
5
6
7
8
9
X±95% cc
Median
Ag
1.8
1.4
1.0
2.2
1.9
2.5
1.6
.9
1.9
1.9
1.7±.4
1.9
2.6
3.0
2.5
2.2
2.2
2.1
2.4±.4
2.0
4.8
2.5
5.4
4.3
5.0
5.3
5.1
5.2
3.9
4.6±.7
5.0
.5
1.9
2.1
.9
2.5
1.2
3.3
1.7
2.7
1.9±.7
1.9
Cd Cu Fe
ygm/gm dry weight
6.4
7.9
10.0
10.6
11.9
14.0
6.2
13.1
10.3
8.7
9.9±1.9
10.1
14.9
11.1
17.6
11.2
9.4
9.5
12.3±3.4
11.1
9.6
12.9
13.2
9.5
9.6
16.3
8.3
23.7
6.8
12.2±4.0
9.6
20.4
12.7
6.8
12.7
12.1
9.8
15.7
9.4
10.7
12.3±3
12.1 10
41.7
43.4
18.5
48.8
42.1
56.1
56.8
32.7
29.6
34.1
40.4±8.6
41
35.1
73.2
54.1
23.2
47.4
50.7
47.3±17.9
49.1
69.1
26.8
62.9
53.9
41.6
67.8
49.1
58.0
48.1
53.0±11.4
53.9
23.9
46.8
62.9
22.6
40.4
41.5
72.7
46.0
80.0
48.5±15.3
46.0
233
271
302
304
344
188
212
354
302
256
277±39
286
218
184
305
260
209
283
243±49
239
154
166
204
178
233
169
129
230
249
190±31
178
183
308
192
339
249
251
213
151
233
235±46
233
Mn
119
44
148
160
53
98
70
86
58
77
91±29
81
47
65
37
21
24
45
40±17
41
12
26
33
56
50
28
16
29
58
34±13
29
89
29
45
42
52
38
64
60
42
51±14
45
Zn
1810
2350
1460
2470
2790
2980
2200
2000
2030
1880
2200±330
2110
1530
3540
2480
1460
1880
2320
2200±810
2097
2330
2260
1830
1950
1840
2250
1500
2790
1820
2060±290
1950
1220
1960
1940
740
2100
1230
1800
1510
3060
1730±510
1955
-------
TABLE 2 (Continued)
Location
Replicate
Number
Ag
Cd Cu Fe
pgm/gm dry weight
Mn
Zn
Redwood
City
1
2
3
4
5
6
7
8
X±95% cc
Median
13.3
38.1
10.5
21.8
12.0
26.6
22.9
22.2
20.9±7.6
22.0
10.8
18.2
15.8
19.6
18.0
20.9
19.9
12.7
17.0±3.0
18.1
190
323
108
209
214
236
167
215
207+51
212
220
244
210
309
252
239
156
227
232±36
233
102
79
180
69
105
79
41
148
100±37
85
3020
4000
1250
2740
4070
3520
2410
2380
2920±790
2880
19
-------
TABLE 3: Metal concentrations in mussel gills one month
after transplantation
Location
Tomales
Bay
(control)
Monterey
Sewer
Outfall
Elkhorn
Slough
Redwood
City
Replicate
Number
1
2
3
4
5
6
7
8
X"+95% CL
Median
1
2
3
4
5
Xi95% CL
Median
1
2
3
4
5
6
Xi95% CL
Median
1
2
3
4
5
6
7
8
9
Xi95% CL
Median
Ag
.04
.02
.06
.26
.03
.18
.03
.49
.14+. 14
.04
.39
.51
1.06
.32
.35
.53i.38
.34
.05
.06
.03
.07
.05
.07
.06i.02
.06
.29
.46
.26
.13
.39
.12
.21
.28
.44
.29+. 10
.28
Cd Cu Fe
pgm/gm dry weight
1.5
1.0
1.6
1.1
4.9
3.7
1.6
.8
2. Oil. 2
1.5
1.4
2.7
2.0
2.9
2.0
2.2i.8
2.0
8.2
4.5
2.3
2.8
.9
1.4
3.4±2.8
2.5
2.1
1.9
1.5
4.6
1.2
3.9
1.4
4.2
6.2
3. Oil. 4
2.1
5.4
4.2
5.5
5.3
7.6
5.2
6.0
8.1
5.9+1.1
5.4
10.2
9.1
10.9
7.6
11.4
9.8il.9
10.2
10.3
10.4
9.5
8.7
9.2
10.7
9.8+. 8
9.9
11.0
10.0
9.5
9.5
10.7
7.4
8.8
9.4
14.1
10.0+1.4
9.5
168
130
126
212
160
161
125
184
158+25
160
90
88
93
76
92
88+9
90
135
321
198
266
174
184
213+28
191
149
133
261
83
182
120
159
163
211
162+40
163
Mn
7.6
6.0
9.1
7.5
10.9
7.1
7.4
6.0
7.7il
7.4
5.2
5.0
5.5
4.5
6.9
5.4il
5.2
9.3
9.3
7.1
9.1
6.8
7.5
8.2il
8.3
46.9
16.3
30.9
15.4
20.8
21.1
21.8
10.0
24.6
23.1+8
21.0
.4
.1
.2
.2
20
-------
TABLE 4 A: Uptake rates in transplanted organisms.
Concentration increase per month
Redwood
City
OYSTERS
Monterey
Sewer
Elkhorn
Slough
Granite
Canyon
Ag
Cu
Cd
Mn
Fe
Zn
20.1*
171.0*
8.0*
4.0
-53.0
764.0
3.1*
13.0
-.6
-52.0
-108.0
-163.0
0
5
2
-36
-53
-158
0.1
8.0
1.0
-40.0
-47.0
-16.0
MUSSELS
Redwood
City
0.24
4.10*
0.60
13.60
3.00
Monterey
Sewer
0.3*
4.8*
0.5
-2.2
-70.0
Elkhorn
Slough
0.02
4.50*
1.00
0.90
31.00
Granite
Canyon
--
--
--
--
_ _
TABLE 4 B: Uptake rates in transplanted organisms,
Percent increase per month
OYSTERS
Ag
Cu
Cd
Mn
Fe
Zn
Redwood
City
1089.0*
417.0*
78.0*
4.9
-18.5
36.0
Monterey
Sewer
170.0*
31.0
-0.5
-64.0
-38.0
-0.8
Elkhorn
Slough
2.7
12.0
19.0
-44.0
-19.0
-7.0
Granite
Canyon
27
20
10
-49
-16
-1
MUSSELS
Redwood
City
600.0
75.0*
40.0
184.0
1.9
Monterey
Sewer
875.0*
88.0*
33.0
-30.0
-32.5
Elkhorn
Slough
37
83*
70
12
19
Granite
Canyon
--
—
--
--
~ ~
*P < .05 that the controls differed significantly from this value.
1
Concentration increase/month = (median ppm of experimental site - median
ppm at control site)/l month.
"Percent increase/month = (median ppm of experimental site - median ppm of
control site)/(median ppm of control site x 1 month).
21
-------
Table 5 shows the percentage of the trace metal content of four-year-
old resident oysters accumulated in one month by transplanted oysters at
Redwood City. The data show 10.5% of the Ag, 16.5% of the Cd, 9% of the Cu,
4% of the Mn and 9.5% of the Zn were accumulated in one month. This indi-
cates that the oysters had only accumulated a small fraction of the metals
found in resident oysters. It also indicates that, if uptake were constant,
it would take less than one year to accumulate a trace metal load equal to
that of natives. Presumably, then, the oysters accumulate trace metals at
diminishing rates which may approach zero, thus establishing an equilibrium.
DISCUSSION
Areas which are relatively high in trace metals can be identified by
using significance tests on transplant and control data. This technique
was used in identifying Redwood City as high in Ag, Cd and Cu, Monterey
sewage outfall high in Ag and Cu and Elkhorn Slough high in Cu. One might
expect Redwood City and the Monterey sewage outfall to be high in several
trace metals because of industrial and municipal waste discharges, and
Elkhorn Slough to be high in Cu because of the proximity of the transplants
to the cooling water discharge from the copper-nickel condensing system in
the fossil fuel power plant (approximately 100 m away). However, because
of the difficulties encountered using conventional monitoring techniques,
the elevated concentrations of these elements have previously gone undetected.
One should use accumulation rates with some caution. If accumulation
rates are assumed to be proportional to the levels of trace metals in the
environment, then the accumulation rates could provide a relative measure
of the degree of trace metal contamination at each site. However, we have
found accumulation rates at a given site to be species-dependent. The dif-
ference in uptake rates between the two species could be due to a variety
of factors, among them:
1. The two species feed on different types of suspended particulates
and, if uptake through ingestion of food is important, the difference in
trace metals in their food may cause the difference. However, Cd uptake
1n £• virgim'ca through ingestion of food is only one-tenth the amount ab-
sorbed directly from solution (Kerfoot and Jacobs, 1976).
2. These two species favor slightly different habitats. The oysters
typically favor quiet waters, while M_. edulis can occur near the mouths of
harbors where the wave action is stronger. This may explain the higher
uptake of some metals by mussels at the Monterey sewage outfall and the
higher uptake of metals by oysters at Redwood City.
3. Mussels may have reached their accumulation capacity at Redwood
City, Monterey sewage outfall and Elkhorn Slough (see Table 4 A). The con-
centrations of Ag and Cd are nearly equal at Redwood City and Monterey
22
-------
TABLE 5: Percent of metal concentrations in resident four-and-one-half-year-
old oysters accumulated by transplanted oysters in one month at
Redwood City.
Median Con-
Median Con- Median Con- centration Median Con- % Accumulation
Ag
Cd
Cu
Fe
Mn
Zn
centration
of Control
Transplants
1.9
10.1
41
286
81
2110
centration
of 1 -Month
Transplants
22
18.1
212
233
85
2880
Accumulation
in 1 Month
(from Table 46)
20.1
8
171
-53
4
764
centration
of 4-Year-Old
Residents
192
48
1891
427
101
8012
in One Month
by Transplan-
tation
10.5
16.5
9.0
—
4
9.5
*Median concentration of 1-month oysters/median concentration of
4-year-old oysters x 100%.
23
-------
sewage outfall. The concentrations of Cu are approximately equal for these
two sites and Elkhorn Slough as well. It may be that the mussels were
accumulating at all sites at a maximum rate and could not accumulate more
at Redwood City if it were available. If this is the case, transplants
should be left out long enough to reach equilibrium with the environment.
In future studies, uptake should be determined over several time intervals.
The rate could then be calculated from a curvilinear function based on
several measurements. Perhaps then, species accumulation rates may be more
accurately compared.
The transplantation technique should be evaluated further using
several species, tissues and time intervals.
CONCLUSIONS
Significant differences in concentrations of Ag, Cd, Cu and Mn be-
tween control and transplanted oysters and mussels were observed. This
indicates that the transplantation method is useful for detecting the pre-
sence of elevated concentrations of these metals. This method can be used
as a valuable monitoring tool to inexpensively survey discharges to deter-
mine whether more expensive conventional monitoring of water quality param-
eters should be initiated or intensified. Species differences in uptake
rates suggest that, in future studies, several species, tissues and time
intervals should be used.
24
-------
SECTION 3
GENERAL STUDIES ON METALS IN MARINE ORGANISMS
A. Premature Pupping in California Sea Lions
Scientists involved in environmental pollution research are often required
to perform detective work; i.e., mortalities or deleterious effects are observed
for a group of organisms and the question arises: Was an environmental pollu-
tant responsible and, if so, which one? Answering such questions is very dif-
ficult because of the complexities that are almost always encountered.
This situation is exemplified by studies on premature pupping in the Cali-
fornia sea lions (Zalophus californianus). Large numbers of premature pups have
been counted on the sea lion rookeries since 1968 (see Gilmartin, et al_, 1976;
Odell, 1970). Research aimed at determining the causes for these events have
revealed that: (1) The mothers of premature pups are usually only six to eight
years old, while the mothers of full-term pups are at least ten years old (Gil-
martin, ejt al_, 1976). (2) Many of the abnormal mothers are infected with Lep-
tospirosis, a virus known to cause abortions (Vedros, et al_, 1971; Gilmartin, et
al, 1976). (3) The abnormal mothers have significantly higher amounts of
polychlorinated biphenyls (PCBs) and DDT compounds (DeLong, et al_, 1973; Gil-
martin, et. aj_, 1976). (4) The normal mothers have significantly higher amounts
of mercury, selenium and bromine (Martin, et al_, 1976). The latter findings
were of interest because each normal mother had equimolar amounts of Se and Hg
in their livers. In addition, excess or equimolar amounts of Br were always
found in conjunction with these elements. In contrast, the mothers of premature
pups had equimolar amounts of Se and Hg; however, Br levels were severely de-
pressed. Perhaps these findings indicate that Br is also involved in the Hg-Se
detoxification mechanisms (see Parizek, ejt al_, 1971) and, for some reason, it
was not functioning in the abnormal mothers. Whether it was responsible for the
premature pupping is unknown. However, these results suggest that absolute
amounts of elements are not as important as the relationship between elements.
In addition to demonstrating the complexities involved with environmental
detective work, the four factors mentioned above also point to the desirability
of simultaneous measurement of different pollutant classes, as well as natural
factors within the same samples. Erroneous conclusions can be reached when only
one pollutant is measured. As the Se-Hg interaction indicates, this is espe-
25
-------
dally true for one heavy metal.
Research of this nature is extremely important in spite of the complexities
involved in attempts to interpret findings. For example, the cause of eggshell
thinning in the brown pelican was suggested when scientists found high DDT
levels in association with these birds. Subsequent research led to the ban of
the use of this pesticide in the United States. On the other hand, the detec-
tive work may yield no firm conclusions. This is the present case with the sea
lions. Nevertheless, reproductive failure (premature pupping) has also been
reported for harbor seals in San Francisco Bay and Puget Sound. Perhaps studies
on these animals will shed more light on the causes for these events. Whenever
deleterious effects are observed, multifaceted research should be performed to
determine whether anthropogenic and/or natural causes were responsible.
An abstract of our sea lion work follows.
Martin, J.H., P.D. Elliott, V.C. Anderlini, D. Girvin, S.A. Jacobs, R.W.
Risebrough, R.L. Delong and W.G. Gilmartin. 1976. Mercury-selenium-bromine
imbalance in premature parturient California sea lions. Mar. Biol. 35:91-
104.
Abstract: High premature birth rates have been observed in the rookeries
of the California sea lion Zalophus caliform'anus since 1968. The reasons
for the premature pupping are complex and, hence, not well understood,
although leptospirosis infection and elevated PCB and DDT residues have
been implicated. We were interested in determining what role trace and
major elements played in these events. Livers and kidneys from ten normal
parturient and ten premature parturient mothers and their pups were anal-
yzed for Hg, Se, Br, Cd, Ag, Cu, Fe, Zn, Mn, K, Na, Ca and Mg in order to
detect differences that might exist between the two groups. A further
objective was to establish how these elements varied in relation to each
other in the normal and abnormal sea lions. Our results revealed that Hg,
Se, Cd and Br levels were significantly higher in the livers of the normal
mothers and that these elements were all in balance (highly correlated)
with each other. This was especially true for Hg, Se and Br. In mothers
with high concentrations of these elements (e.g., Hg greater than 800
pg/g dry weight), atomic ratios of approximately !Hg:lSe:lBr were observed.
Atomic Se:Hg ratios were also near unity in the abnormal mothers; however,
Br concentrations were always severely depressed in these individuals.
Normal full-term pups had higher hepatic levels of Hg and Se, and near-
perfect 1:1 Se:Hg atomic ratios were almost always observed. In contrast,
the livers of the premature pups appeared to be deficient in Hg, and,
consequently, elevated Se:Hg ratios were always found. In almost all
cases, the premature pups had increased concentrations of Na, Ca and Br.
Levels of these elements were correlated with their Se:Hg ratios. Amounts
of Mn and Cu were reduced in the premature pups and negatively correlated
with Se:Hg ratios. The results suggest that balance between elements is of
more importance than absolute concentration when the possible effects of
toxic elements are considered. It also appears that bromine may be impor-
tant in the detoxification process involving Se and Hg and perhaps Cd as
well; i.e., every mother that had Br in balance with Hg, Cd and Se had a
26
-------
normal pup, while every mother that'lacked sufficient Br had a premature
pup. The question of whether Hg detoxifies Se is also raised. All the
normal pups had Se:Hg atomic ratios of less than 2.2, while all the premature
pups had reduced Hg amounts and Se:Hg ratios above 3.4.
B. Silver, Cadmium, Copper, Zinc and Iron in Squid Livers
We were especially interested in squids, not only because of their enormous
importance in the pelagic food web (Clarke, 1966, p. 265), but also because they
concentrate silver (Folsom and Young, 1965; Folsom et aJU 1970), an element well
known for its toxicity to marine organisms (Soyer, 1963; Bryan, 1971; Calabrese,
e_t aj_, 1973). The measurement of this metal in liver tissue, where it is known
to concentrate, was thus the primary objective of our research. In addition, we
wanted to compare silver concentrations with levels of three other closely
related elements: copper, cadmium and zinc. Iron was also determined for
background information.
The most significant finding in the study was the enormous copper concen-
trations in the Loligo opalescens livers and the correlations that exist between
this element and silver and cadmium. The two most plausible explanations would
seem to be: (1) Since squids require copper for the synthesis of their respira-
tory pigment, hemocyanin, these high levels may be entirely natural; in the
process of concentrating Cu, some incidental uptake of Ag and Cd occurs. (2)
Silver and/or cadmium may cause copper to be concentrated in the liver of L_.
opalescens; this process thus reflects a sub-lethal effect caused by these heavy
metals. Both metals are well known for their toxicity and both are known to
affect copper metabolism, at least in birds and mammals (e.g., Anke, et al.
1970). In laboratory studies with rats, Van Campen (1966, p. 129) noted that
the "major effects of silver were a decrease in the relative proportion of Cu
in blood and an increase in the proportion deposited in the liver". In general
however, these processes are only poorly understood. Underwood (1971, p. 76)
states: "The diversity of the physiological responses and the interactions with
copper produced by the four elements, zinc, cadmium, silver and mercury, which
are chemically very similar, is surprising and, at present, inexplicable".
These relationships have also been observed in other molluscs. Windom and
Smith (1972) reported correlations between Cu:Ag, Cu:Zn and Ag:Zn in East Coast
(USA) oysters. Anderlini (1974) also noted a negative correlation between
silver and copper in West Coast abalone. Thus, representatives of three classes
of mollusc are known to have these relationships.
The abstract of our squid livers paper follows.
Martin, J.H. and A.R. Flegal. 1975. High copper concentrations in squid
livers in association with elevated levels of silver, cadmium and zinc.
Mar. Biol. 30:51-55.
Abstract; Livers from 43 Loligo opalescens, 14 Ommastrephes bartrami and 7
Symplectoteuthis oualaniensis were analyzed for their silver, cadmium, cop-
per, zinc and iron contents. Copper concentrations of up to 15,000 yg/g
27
-------
dry weight were found in L_. opalescens in conjunction with significant cor-
relations between this element and Ag, Cd and Zn. The latter elements are
known to affect Cu metabolism in terrestrial organisms; however, whether
the correlations occurring in marine organisms represent casual or cause-and-
effect relationships is as yet unknown.
C. Abalone Copper Toxicity Experiments.
In cooperation with the California Department of Fish and Game, we took
part in copper toxicity studies that demonstrated that adult abalone died when
subjected to levels of 65 and 50 ug Cd/liter. These findings supported the con-
tention that 1,500 abalone died from copper poisoning when the cooling system of
the power plant was tested.
Martin, M., M.D. Stephenson and J.H. Martin. 1977. Copper toxi-
city experiments in relation to abalone deaths observed in a power
plant's cooling waters. Calif. Fish and Game, 63^(2):95-100.
Abstract: Toxicity of copper as copper sulfate and larval red abalone,
Maliotis rufescens, and adult black abalone, Haliotis cracherodii, was
determined by static bioassay in seawater at 14 °C (57 °F). Copper
accumulation studies and histopathological analysis in digestive gland
and gill tissues were conducted. The TL^s for adult red and black
abalone were 65 ppb and 50 ppb copper, respectively. The TL™ for
larval red abalone was 114 ppb copper. Copper was found to accumulate
in gill tissues of red and black abalone at 56 ppb copper concentration.
Histopathological abnormalities in gill tissues occur at concentrations
above 32 ppb.
D. Cadmium in Plankton: Elevated Levels off Baja, California.
On three occasions, we observed elevated cadmium levels in plankton col-
lected off Baja, California. These findings were described in an article
published in Science:
Martin, J.H. and W.W. Broenkow. 1975. Cadmium in plankton: elevated con-
centrations off Baja California. Science 190:884-885.
Abstract; One hundred thirty-five plankton samples were collected in the
northeast Pacific Ocean and analyzed for their cadmium content. Concentra-
tions were generally low (2 to 5 micrograms of cadmium per gram, dry weight)
in all samples except for the plankton collected off Baja California, where
high values (10 to 20 parts per million) were consistently found on two
cruises.
A question familiar to all researchers studying heavy metals in the marine
environment was raised: Is the element in question derived from natural or
anthropogenic sources? In this case, we postulated that the Cd could either be
concentrated naturally in these waters by upwelling processes (Schutz and Ture-
28
-------
kian, 1965; Riley and Taylor, 1972) or it was from some unknown anthropogenic
source. Obviously, the only way to find out was to measure cadmium in sea
water.
Under the auspices of the NSF IDOE program, we began working on this prob-
lem in January of 1975. After perfecting methodology (see Martin, et al,
1976b), we were able to successfully collect sea water at several depths without
contaminating the water with extraneous amounts of Cd.
In our Science article, we postulated that the reason for the high levels
was that Cd concentrations in the water must also be elevated here. However,
when we measured the amounts in the water, we were surprised to find that sur-
face values were extremely low (4.5 ng Cd/liter, Bruland, e_t aU 1978). We can
now tentatively conclude that the Cd we find in the plankton in these waters is
from a natural source; that is, as deep waters upwell to the surface, they will
bring up plant nutrients that stimulate phytoplankton growth and, at the same
time, elevated amounts of Cd will be introduced to the surface. Whether the
phytoplankton also take up amounts of Cd proportional to phosphate and nitrate
is unknown. However, our preliminary data suggests that the Cd is not concen-
trated actively. Phytoplankton collected in Monterey Bay after intense upwell-
ing pulses contained very small amounts of Cd (1 to 2 ppm; see Martin and
Knauer, 1973). Although phosphate was not measured in these samples, they
should be rich in this element, since actively growing phytoplankton usually
have high concentrations of P and N (Parsons and Takahashi, 1973). These re-
sults suggest that Cd uptake would occur after bloom conditions cease; that is,
after a population becomes senescent. When this slow-down occurs, sufficient
time would be allowed for Cd uptake to occur via passive adsorption. With
sufficient time, Cd levels would increase markedly in the plankton and, at the
same time, decrease in the water column. This, then, could explain the levels
we found off Baja in the plankton and water. Most of the Cd had been adsorbed
onto the plankton and associated levels were high (15-20 ppm). Because of this
removal, dissolved amounts become low due to this extraction process (5 ppt).
When this equilibrium is reached, Cd and P in the plankton also become corre-
lated. However, if organic matter decreases, amounts of Cd would also decrease.
This is suggested by two samples collected far offshore that had very little
particulate P and Cd. The surface depletion and deep enrichment of Cd also
suggests that Cd is removed from the surface in association with particles. As
the particles sink and decompose, P and N are also released which results in the
dramatic increases in the levels of these elements in the deep ocean. In concert
with this process, Cd is also released.
With the discovery that Cd is correlated with plant nutrients, we are now
in a position that enables us to detect superfluous amounts of this element in
the water column. That is, when Cd levels lie above the predicted amounts from
the nutrients present, (ng Cd/liter = -3.6 + 34.9 [umol P0d/liter]) this can
indicate anthropogenic .input.
Thus, these findings suggest three things: (1) Cd levels in plankton off
Baja California are apparently due to natural processes. (2) Unlike other ele-
29
-------
merits, Cd is rapidly regenerated in the water column and, consequently, only
limited amounts will be removed from the water column to the sediments. Thus,
in areas with limited mixing, any introduction of Cd may result in increased
levels within a relatively short period of time. (3) With our knowledge on
the distribution of Cd in the water column and its relationship with PO,, we
are now in a position to determine anthropogenic enhancement.
E. Cadmium in Sea Otters
Measurements of heavy metals in top predators are needed for an under-
standing of food-chain amplification processes in the marine environment.
Marine mammals have proved especially useful for mercury and several studies
have been conducted in which levels were estimated in seals; e.g., Freeman and
Horn, 1973; Koeman, et al_, 1975; Gaskin, et a]_, 1972; Martin, ejt al_, 19760., and
whales; e.g., Hall, et a]_, 1971. Since most of these marine mammals feed mainly
on members of the pelagic food chain, it is desirable to compare heavy metals in
species feeding on benthic food items. The California sea otter (Enhydra
lutris) is especially interesting, as it feeds primarily on mollusks (Vandevere,
1969), which are known to readily concentrate cadmium (e.g., Anderlini, 1973;
Brooks and Rumsby, 1965), an element well known for its toxicity (e.g., Nilsson,
1970). The main objective of this study was to determine the cadmium levels now
existent in the California sea otter herd. Since we were interested in relating
levels of this element to other heavy metals and essential elements, Ag, Hg, Cu,
Zn, Fe, Mn, K, Na, Mg, Ca and Sr concentrations were determined as well. The
digestion methods we used are described in Section 4.
Results
As expected, sea otters concentrate large quantities of cadmium, especially
in their kidneys (Tables 4 and 5). A typical otter begins life with about 1
yg/g dry weight in its kidneys. Levels steadily increase as the animal grows
up and eventually amounts as high as 964 ppm can occur in the females; up to 350
ppm in the males (Figure 3). It is also evident that female otters concentrate
more Cd than males, since seven females had amounts exceeding that of the highest
male (Figure 3).
The relationship between Cd and Zn is well known, and an interesting pattern
was observed in the otters (Figure 4). A typical otter has approximately 100
ppm zinc in its kidneys at birth; or, in other words, 100 times as much Zn as
Cd. As Cd concentrations increase, zinc levels also go up, but the rate of
increase is slower than that for Cd. This results in a steadily decreasing
Zn:Cd ratio, until amounts of the two elements become nearly equal (approxi-
mately 300 ppm). Eleven otters had renal Cd levels above 300 ppm; only one of
these animals had over 300 ppm Zn (the female with 964 ppm Cd). Thus, it appears
that Zn uptake responds to increasing renal Cd concentration, but this response
appears to cease after Cd levels exceed those for Zn. This may have important
30
-------
TABLE 4: Metal concentrations in female sea otter kidneys
Animal Length Weight
No. mm Kg
Cd
Zn
Cu
Fe
pg/g Dry Weight
Mn K Na Mg_
Ca
*Not detected
**Animal was decapitated
Sr
1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
461
675
655
734
620
807
806
810
740
863
946
1017
##
1 160
879
928
995
1098
974
836
985
1040
929
1075
1058
1075
910
1040
1035
953
1.2
2.8
3.3
3.4
2.3
6.0
4.8
4.5
3.9
10.3
10.7
12.0
4.4
14. 1
9. 1
6.9
10.0
14.2
9.2
6.4
9. 1
10.0
8.0
14.0
10. 1
1 1.2
6.5
10. 1
8.9
9.8
0.7
1.2
1.4
2.0
2.3
6.9
15.5
17.7
25.0
26.3
26.5
28.2
31.2
32.8
34.8
36.9
49.3
51.6
53. 1
58.9
60.4
64.7
73.8
78. 1
79.3
103.4
104. 1
106.4
133.7
138. 1
75
92
86
1 10
108
87
177
128
138
109
105
107
133
107
1 13
134
165
283
146
370
145
147
145
172
206
143
182
194
232
210
0.09
0.72
0.28
0.54
0. 17
1.33
0.39
1.47
1.05
2.27
1.53
6.00
0.60
14.61
0.66
3. 17
2.92
2.32
8.68
2.84
5.65
3.60
5.42
4.21
1.59
7.59
5.39
5.95
7.64
5.97
21
38
15
38
29
25
45
28
23
42
28
29
120
14
25
37
40
52
29
47
54
48
45
34
40
45
45
30
28
35
503
316
453
587
281
225
321
459
383
341
385
438
622
787
246
816
493
663
901
257
495
713
568
552
561
422
41 1
461
718
388
1.96
3.40
3.42
2.20
1.75
3.38
4.81
5.10
5.94
6.29
3.59
4.69
7.40
3.05
2.39
4.80
5.06
3.95
3.93
3. 19
5.31
3.42
1.70
3. 13
4.77
6.02
4. 16
3.49
3.99
5. 18
5960
6990
9185
6600
8770
4100
8920
5270
9170
9450
6870
6060
8380
7240
6410
8300
6790
6840
8100
5975
6460
4865
6740
6510
6970
6880
6700
5970
8380
7770
4890
7990
9880
16320
10380
12700
10900
8670
9470
5610
6510
13930
16855
6980
5430
6620
15180
10540
6910
17200
9780
10520
9155
10550
9930
6520
13005
13540
10580
1 1 150
437
649
800
917
852
1469
1043
646
655
1 122
1 196
2476
731
505
569
787
1 198
1087
650
812
887
625
412
430
852
538
1560
840
718
986
596
285
182
2750
536
627
670
247
353
1010
620
2149
283
249
369
101
493
667
151
717
217
309
426
618
486
452
312
263
429
281
0.93
1 .20
1.28
22.36
nd*
17.61
4.20
1 .36
1.46
1 1 .26
5. 18
29.44
5.46
0.78
3.88
0.77
4.53
5.96
1.83
5.42
2. 17
1.32
2.27
5.99
3.44
4.59
4.42
1 .92
7.98
2.30
conti nued
-------
TABLE 4 (continued)
to
IV)
Animal Length Weight
No . mm Kg
31 1080 11.4
32 1225 15.6
33
34
177 14.1
120 11.3
35 1327 25.4
36 1216 23.0
37
38
39
40
41
140 10.4
1 32 1 I . 9
178 12.0
250 16.1
192 15.2
42 1280 16.8
Cd
198.2
222.9
268.8
284.5
332.7
373.6
407.4
418.9
426.6
593.9
679.8
964. 1
Zn
292
177
288
305
188
243
283
268
200
253
241
382
Hq
6.24
3. 10
29.58
3.83
1.49
2.50
2.71
3.69
6.81
2.66
2.56
4.20
Cu
46
30
35
45
27
15
59
49
51
31
34
52
yg/g
Fe
549
616
576
401
519
283
650
470
560
464
571
632
Dry We'
Mn
5.92
3.49
4.72
4.66
3.19
3.18
4.88
3.87
5. 18
3.97
3.57
3.99
ght
K
6750
8020
8960
8340
6520
6540
7390
6180
6720
8015
6405
9390
Na
9710
9820
9905
10860
5040
7430
15750
5920
7150
1 1080
8460
8975
Mq
1289
575
719
724
496
588
567
396
444
702
550
550
Ca
708
343
171
252
252
371
307
348
308
424
402
249
Sr
5.92
0.93
2. 12
1.55
1.30
3.32
1.39
1.83
1.73
2.26
1.56
2.99
-------
TABLE 5: Metal concentrations in male sea otter kidneys
GO
GO
An i ma 1
No.
1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Length
mm
455
590
422
584
520
490
759
489
778
694
1010
973
965
1 132
920
1091
1 155
1069
1 130
II 13
1031
1220
1002
1297
1263
1259
1294
1225
945
Weight
Kg
1.2
2. 1
1.0
2.1
1.8
1.5
3.7
1.3
5.0
3.3
16.9
8.8
9.8
18.9
8.5
14.8
20.2
21. 1
17. 1
17.2
9.8
20.4
9.9
29.9
22.9
26.0
30.4
23.9
1 1. 1
Cd
nd*
0.3
0.6
0.7
0.8
0.8
1.0
2.0
5.5
16.8
17.3
29.2
32.5
32.6
34.2
42.3
44.4
• 62.8
64.5
65.7
66.0
68.8
87.3
92.3
107. 1
1 16.0
1 19.8
120.8
120.9
Zn
123
102
72
122
137
91
78
72
106
1 18
1 16
99
137
138
155
126
1 16
105
116
177
155
126
174
134
107
168
170
189
148
H.q
0.59
0.09
0. 12
0.28
0.25
0.25
0.24
0. 14
4.30
1.83
2.51
3.00
1.44
2.98
1. 10
3.23
2.12
25.45
5.31
4. 19
2.90
23.59
7.87
7.69
3.72
7.05
10.81
7.62
5.64
Cu
41
20
13
47
66
28
12
16
31
27
66
25
30
22
51
29
25
57
45
1 13
25
38
42
14
26
18
32
23
85
Fe
623
525
786
468
465
447
648
371
353
240
622
589
353
688
487
284
308
969
291
273
486
541
495
538
748
1083
753
540
260
pg/g
Mn
2.46
2.47
0.55
3.62
4.06
2.70
3.04
2.42
2.93
3.34
4.49
3.83
4.25
3.18
4.55
3.76
4.19
4.73
6.16
8.46
4.24
5.83
5.11
4.05
2.88
3.67
4.40
4.02
5.40
Dry Weight
K Na
8500
6335
1800
7820
81 15
9820
8120
5060
5940
5550
5520
6345
7370
5500
7940
9450
6720
6340
10105
8910
7890
1 1080
7250
3740
5300
8030
7400
8885
8100
16385
10200
18700
10370
8540
12275
5000
4580
34830
7825
6170
5640
1 1550
10180
1 1 160
9130
12330
9290
7370
6860
14050
7120
12700
251 10
5440
7300
6340
5760
6880
Mq
1405
865
454
864
727
141 1
500
506
1426
555
1720
539
927
566
882
1857
1368
2650
1587
1894
2360
653
1387
2350
464
1 160
1414
826
643
Ca
1075
371
1 108
346
376
295
276
361
2560
152
3638
237
266
326
560
9540
456
2817
910
1032
807
263
905
141 1
263
1209
2356
357
386
Sr
1.64
1.24
7.76
1 .98
1.65
3.19
1.44
1 .03
7.68
1.22
32.97
0.60
4.56
nd
6.93
90.76
6.90
20.04
9.28
II. 00
5.84
3.27
6.96
15.87
0.94
15.09
17.70
3. 17
3.86
*not detected
conti nued
-------
TABLE 5 (continued)
GO
An i ma 1
No.
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Length
mm
1225
1269
1323
1228
1090
1075
1220
1232
1340
1 175
1358
1268
1308
1 170
1408
1231
1260
1388
Weight
Kg
26. 1
26.9
21.3
28.3
15.5
1 1.0
21.3
23. 1
18.4
15.0
29.0
23.4
21.3
16.6
22.8
20.9
29.9
31.5
Cd
126.8
129.9
133.4
133.7
135.4
142.2
147. 1
155.2
181.0
181.7
195.5
226.0
238.1
252.3
272.2
302. 1
317.7
350.4
Zn
180
144
202
138
179
193
170
191
305
222
221
246
228
303
258
296
188
251
Hg
5.54
7. 12
13.31
6. 17
5. 16
2.46
5.28
12.44
9.47
4. 1 1
6.21
1 1.31
4. 1 1
22.03
8.03
9. 12
2.97
1.76
Cu
43
27
20
22
45
40
56
19
37
22
26
41
26
48
25
21
24
31
pg/g
Fe
945
721
845
565
602
525
327
599
741
426
924
364
883
605
957
572
370
21 10
Dry We
Mn
5.37
6.50
6.59
4.82
4.70
3.94
3.97
3.30
3.93
4.65
5.55
7.86
3.90
5.47
2.25
5.94
4.20
4.27
ight
K
9310
7605
9700
4925
4960
7875
8690
6720
8260
8530
8090
9170
9235
8410
6240
81 15
7860
5500
Na
12540
8320
8870
7220
8240
9950
10630
6080
7650
6480
6820
7475
6360
8725
91 10
8630
9035
15715
Mg
2655
931
682
854
959
798
960
599
741
643
1023
1058
539
846
675
585
566
702
Ca
1 178
578
599
405
1204
448
298
295
2782
183
1012
471
862
173
870
265
1718
337
Sr
9.45
2.69
2.51
4.01
8.90
3.72
3.60
1.28
9.47
0.66
5.00
2.09
1.85
1.47
3.07
4.83
1 1.60
4.04
-------
1000
600
TJ
CJ
§ 400
200
0
in
C\J
+ o
•*•
-------
400
350
300
cr?
^
ui
3 250
"3 200
c
Q)
150
100
° +
GO
o
o
o o
•*•+%
o o
0 0
50
CSI
C3
CJ
(S3
CO
00
renal Cd (ug/g)
FIGURE 4:
Renal cadmium concentrations versus renal zinc concen-
trations (both expressed in yg/g dry weight) in sea
otters. + = female; o = male.
36
-------
health implications, since Schroeder has reported the occurrence of hypertension
in rats when a renal Cd:Zn weight ratio of .58 is exceeded (ScKroeder and Buck-
man, 1967).
Cadmium was also concentrated in the livers of the otters (Tables 6 and 7),
but levels were almost always lower than those observed in the kidneys. No
relationship was apparent between hepatic Cd and Zn, however. Muscle and heart
tissue samples were also analyzed for this element (Tables 8 and 9). In gen-
eral, low values were found except in the animals that had exceptionally high
renal and hepatic Cd concentrations. For example, Female #42, which had 964 and
665 ppm Cd in her kidneys and liver, had 19.1 and 17.0 ppm Cd in her muscle and
heart tissues. This suggests that the normal detoxification organs were over-
loaded and Cd was allowed to concentrate throughout the animal.
In comparison to Cd, levels of mercury were relatively low in most otters;
the highest amount (61.2 yg Hg/g dry weight) was found in the liver of Male #22.
Hepatic Hg concentrations versus lengths are plotted in Figure 5. As was the
case with Cd, mercury levels were also low in the younger, shorter otters.
However, unlike Cd, adult males appear to concentrate more Hg than the females.
For example, ten out of eighteen males whose length exceeded 1,200 mm, had over
10 ppm Hg, while all five females over 1,200 mm had less than 5 ppm Hg. In
general, otters of intermediate length (sub-adults) appeared to concentrate Hg,
regardless of sex (Figure 5). As expected, animals with high hepatic levels
also had increased concentrations in their kidneys (Tables 4 and 5), muscles
(Table 8) and hearts (Table 9). This is exemplified by Female #33, which had
50.6 ppm in her liver, 29.6 ppm in her kidney, 10.6 ppm in her muscle and 7.37
ppm in her heart.
Silver was found in the livers, but not the kidneys, of almost all otters
and was more highly concentrated in the females than in the males (Tables 6
and 7). In general, younger otters had the highest concentrations. For ex-
ample, Females #20 (41.1 ppm Ag), #28 (22.58 ppm) and #30 (20.70 ppm) were all
sub-adults; the highest male, #13 (13.21 ppm), was immature.
Silver is also highly concentrated in very young otters; Males #1-8 and
Females #1-5 all had relatively low Hg and Cd levels. These data suggest that
the animals had not started feeding and were either newly-born or still suck-
lings. The high Ag concentrations (up to 9.09 ppm) suggest that this element
readily passes across the placenta! barrier (or with the milk).
Why the females concentrate more Cd and Ag and less Hg than the male otters
is unknown. Perhaps it is the result of the basic physiological differences
between the two sexes. For example, all of the victims of the Ouchi-Ouchi
disease in Japan were females over forty years old who had had multiple births
(Shigematsu and Hasegawa, 1971). The difference may also be related to behavior;
the females and a few dominant males usually occupy the larger best feeding
areas, while the majority of the males are crowded into smaller, less desirable
areas. Thus, feeding pressure is greater and the males may have to select food
items containing less Cd and Ag and more Hg.
37
-------
TABLE 6: Metal concentrations in female sea otter livers
Ag Cd Zn Hg
Animal Length Weight
No. mm kg
u>
00
yg/g Dry Weight
Cu Fe Mn
Na
Mg
Ca
*Not detected.
**Animal was decapitated.
Sr
1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
461
675
655
734
620
807
806
810
740
863
946
1017
**
1 160
879
928
995
1098
974
836
985
1040
929
1075
1058
1075
910
1040
1035
953
1.2
2.8
3.3
3.4
2.3
6.0
4.8
4.5
3.9
10.3
10.7
12.0
4.4
14. 1
9. 1
6.9
10.0
14.2
9.2
6.4
9. 1
10.0
8.0
14.0
10. 1
1 1.2
6.5
10. 1
8.9
9.8
0.87
3.72
9.09
6.63
2.33
6.78
2.09
1 1 .47
5.48
1.71
8.60
1 .93
8.26
0. 13
0.85
5.31
3.90
9.22
4.75
41. 10
2. 13
7.91
1.93
7.41
1.47
3.03
7.50
22.58
7.00
20.70
0.2
1.5
1.3
1.2
0.6
14.8
16.6
8.9
19.8
15.7
7.5
15.9
16.0
6.3
31 .3
16.2
21.0
1 1 1.5
24.4
29.9
21.5
28.9
62.9
49.3
71.4
38.2
64.6
43.4
31.9
35.0
181
243
151
169
99
205
287
21 1
203
1 17
1 16
161
168
121
179
168
212
190
190
250
155
263
262
290
325
216
212
298
251
286
0.35
1. 10
0.44
1.41
0. 15
5.37
0.98
4.22
1.69
5.03
2.66
17.77
0.60
25.40
0.87
5.27
4.82
3.50
29.95
4.46
9.52
7.91
15.26
13.77
4.22
9.91
9.01
1 1.02
19. 10
9.25
102
242
70
1 18
107
341
197
223
263
130
73
177
312
54
101
272
56
126
276
140
67
126
172
162
163
1 17
127
198
171
131
852
1380
1424
4237
485
1251
4413
596
946
488
400
855
546
3174
301
1 120
678
1417
2048
275
751
1546
443
920
1325
446
716
516
1623
724
2.56
9.70
8.42
9.75
2.58
10.26
22.38
21 .97
22.50
9.65
7.93
9.87
20.93
10.50
1 1.33
23.69
21 .47
17.95
22.78
29.04
26.56
16.31
1 1.82
18.89
34.36
13.39
28.53
35.37
19.03
23.46
41 10
8265
7075
4970
4220
3770
8530
3740
7420
7350
4640
4440
5820
5950
5470
5180
4360
3030
7205
5665
4760
3670
5000
4460
5580
4690
7135
5090
6025
3780
4760
1 1020
6990
17430
6810
18175
14015
6655
7420
6340
4920
7590
18270
5200
4450
4660
17450
7520
4600
16040
9710
9740
12410
10340
8660
6415
10415
8565
10125
7360
450
840
753
2130
463
2735
1921
701
676
1230
697
1283
950
617
550
673
2310
1645
713
1 180
1 1 18
831
882
670
1066
735
1 140
892
1030
1064
547
640
220
3185
331
2294
4538
133
187
3275
418
1 135
309
169
92
1 13
2056
632
98
1 187
234
1054
832
886
370
1 159
604
294
1 102
994
1.23
1.23
ND*
10.84
ND
7.68
9.21
ND
2.05
12.40
4.85
1 1.58
4.73
1 .47
1.05
0.39
8.96
3.78
0.94
4.20
2.60
2.45
3.68
1.74
3.12
4.94
3.54
ND
5.33
2.39
conti nued
-------
TABLE 6 (Continued)
Animal Length Weight
No. mm kg Ag
CO
10
Cd
Zn
yg/g Dry Weight
Hg Cu Fe Mn
Na Mg
Ca
Sr
31
32
33
34
35
36
37
38
39
40 1
41 1
42 1
1080
1225
177
120
327
216
140
132
178
250
192
280
1 1.4
15.6
14.1
1 1.3
25.4
23.0
10.4
1 1.9
12.0
16. !
15.2
16.8
4.52
2.30
2.44
4.56
1.54
0. II
8.72
9.46
4.05
1.95
7.09
5.50
73.4
78.4
122.0
59.7
121.8
85.0
161.6
175.4
115. 1
239.8
288.5
614.8
287
260
313
305
164
156
354
313
300
218
282
356
12.21
4.56
50.64
7.48
3.39
2.46
5.96
12.99
18.82
3.94
3.09
4.29
238
97
1 16
1 17
121
10
138
174
189
109
104
50
1287
1416
1064
626
676
364
1063
1264
2570
1046
522
988
15.52
15.56
19.25
24.95
12.04
8.33
35.07
27.59
35.72
19.55
19.32
1 1.80
3445
6165
6580
5840
6880
3750
6920
4925
7445
4700
5880
5235
7100
9000
12960
8825
3830
6100
6120
5190
8160
8180
4700
1 1430
2100
700
829
849
556
833
600
689
1000
575
505
550
1362
341
378
299
120
398
161
288
522
153
142
475
5.23
ND
3.62
1.06
0.86
3.87
0.96
0.65
0.26
ND
0.83
3.94
-------
TABLE 7: Metal concentrations in male sea otter livers
Ag Cd Zn Hg
Animal Length Weight
No. mm kg
Cu
yg/g Dry Weight
Fe Mn K
Na
Mg
Ca
*Not detected.
Conti nued
Sr
1
2
3
4
5
6
7
8
9
10
1 1
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
455
590
422
584
520
490
759
489
778
694
1010
973
965
1 132
920
1091
1 155
1069
1 130
1 1 13
1031
1220
1002
1297
1263
1259
1294
1225
945
1225
1.2
2. 1
1.0
2. 1
1.8
1.5
3.7
1.3
5.0
3.3
16.9
8.8
9.8
18.9
8.5
14.8
20.2
21.1
17. 1
17.2
9.8
20.4
9.9
29.9
22.9
26.0
30.4
23.9
1 1. 1
26. 1
4. 19
1.58
ND
5.77
7.26
6.27
1.35
1.43
7. 1 1
3.94
3.58
5. 17
13.21
5.34
1.46
0.81
2.32
5.07
1.00
0.75
ND
2.33
4.54
0.53
0. 18
1. 13
ND
ND
2. 16
2.02
1.0
ND*
ND
0.2
0.2
0.7
ND
O.I
9.6
16. 1
9.4
19.0
12.4
8.8
21.4
18.3
13.8
27.3
24.1
26.3
47.5
31.0
56.6
27.8
33.3
39.9
30.2
62.0
66. 1
44.7
282
221
186
243
258
199
180
213
230
244
139
300
244
179
193
161
166
1 1 1
141
126
275
165
231
122
95
121
153
206
175
143
1. 16
0.29
0.34
0.31
0.48
0.58
0.44
0.33
11.15
2.25
6.00
5.50
1.69
6.19
2. 15
7.53
3.28
23.25
13.76
14. 19
16.55
61.23
15.46
17.64
16.02
4.74
28.77
29.27
10.26
4.81
17]
220
41
225
198
191
135
120
358
162
179
240
58
187
1 19
231
86
179
123
164
256
214
138
45
93
40
120
195
1 13
122
1968
3183
1874
1405
1385
2156
1935
1308
3057
821
720
767
399
943
804
1073
356
2019
410
234
1 190
1568
861
1025
757
3824
1372
1051
142
3732
4.51
13.43
0.26
8.99
9.00
5.65
16.23
3.64
14.81
19.31
3.90
17.20
15.50
8.92
34.84
9.56
10.39
7.90
10.25
10.01
20.94
19. 16
14.87
6.78
6.07
14.39
2.93
12.24
10.49
9.83
6550
4900
2160
4650
6340
8800
6910
4690
3760
6750
3865
6470
6075
6145
3960
5885
6250
3990
6565
4410
5470
8930
51 10
3455
3740
5140
2900
5475
6370
5800
14040
10415
18730
9160
8500
14880
3730
4970
29290
9860
6515
7260
13610
13020
6270
7470
7195
7800
8470
5860
15640
8770
10190
15440
4250
5760
4990
4880
7310
8225
1 170
965
1890
930
854
1850
482
420
3330
829
845
660
915
546
1 104
1243
1020
2034
900
744
1388
1000
1059
2035
383
730
284
644
845
1903
1472
531
2255
353
506
1040
164
357
3720
262
1839
199
1 181
2189
335
3605
380
2660
531
1690
1291
817
2604
885
171
314
192
233
396
680
5.72
4.95
12. 12
3.56
2.28
1.83
1.00
1. 15
8.90
0.83
1 1.00
0.24
6.49
4.52
2. 15
28. 19
2.80
9.05
5.47
5.22
7. 19
1.47
9.58
10.82
0.78
2.26
5.21
1.63
2.31
5.27
-------
TABLE 7 (Continued)
yg/g Dry Weight
An i ma 1
No.
31
32
33
34
35
36
. 37
38
39
40
41
42
43
44
45
46
47
Length
mm
1269
1323
1228
1090
1075
1220
1232
1034
1175
1358
1268
1308
1 170
1408
1231
1260
1388
Weight
kg
26.9
21.3
28.3
15.5
1 1.0
21.3
23.1
18.4
15.0
29.0
23.4
21.3
16.6
22.8
20.9
29.9
31.5
Ag
1.75
0.62
1.73
ND
1.81
2.89
0.90
4.40
1.79
0.51
ND
1.66
3.33
0.76
ND
2.71
4.56
Cd
55.1
30.4
46.0
27.9
60.6
59.9
29.5
74.7
50.7
49.4
83.4
105. 1
71.7
35.5
51.2
109.9
22.3
Zn
172
180
158
163
226
221
201
226
316
153
237
274
488
225
248
205
270
Hg
5.35
22.1 1
5.49
7.6i
4.66
10. 15
25.22
23.00
8.67
3.88
25.32
6.43
23.84
7.08
22.49
2.89
7.67
Cu
75
68
64
95
120
234
134
167
283
65
82
1 16
194
68
58
79
133
Fe
519
2749
589
61 1
798
237
1282
3269
727
1294
754
2843
2789
5878
1744
477
16772
Mn
10.09
12.68
6.48
6.48
20.37
10.54
13.34
19.22
15.46
6.76
7.85
14.78
19.95
16.14
10. 1 1
7.29
12.94
K
6170
7875
4120
3570
4480
5220
7860
5930
6040
4580
5370
7310
6340
5410
3230
5450
4480
Na
4440
4770
8610
9925
9690
12415
5030
6250
7130
6050
5640
6000
7750
8820
7980
10170
14790
Mg
677
580
1557
620
II 18
1082
667
943
755
644
758
587
847
1 170
984
822
2058
Ca
52
176
431
796
447
536
142
444
21 1
147
194
115
358
654
378
683
1432
Sr
ND
0.90
7. 18
3.39
2.73
5.29
1.03
3.54
1.45
3. 14
2.07
ND
1.74
4. 1 1
5.30
6.77
9.49
-------
t\J
TABLE 8: Metal concentrations in sea otter muscles. Part A: Females.
Animal W/9 dry weight
No. Cd Zn Hg Cu Fe Mn K Na Mg
Ca
Sr
2
3
4
5
6
7
8
I 1
13
16
17
19
20
21
22
23
24
25
26
27
28
29
30
32
33
34
35
36
37
39
40
41
42
ND*
ND
ND
ND
0.2
ND
ND
0. 1
0.9
ND
1. 1
ND
3.8
ND
2.5
0.4
0.5
1.4
0.6
ND
ND
ND
1.7
7.2
1.5
1.0
2. 1
7.0
1.4
5. 1
8. 1
12.3
19. 1
168
197
204
87
167
275
189
150
203
160
193
185
190
185
154
164
188
330
105
189
209
186
164
259
213
226
193
221
232
223
179
180
234
0.36
0.26
0.25
0.01
0.63
0.17
1.42
1.10
0.29
1.12
1.52
3.53
0.84
2.61
1.96
2.47
3.14
1.08
3.17
2.65
2.96
2.80
2.32
1.55
10.61
2.02
0.87
1.19
—
3.78
1.82
1.78
2.23
9.9
8.5
14.7
6.7
18.2
14.4
31.7
10.4
28.6
10.0
8.2
9.5
25.7
13.5
13.3
16.4
9.4
10.3
6.2
17.2
1 1.6
9.1
10.7
9.8
3.4
7.9
7. 1
7.2
10. 1
3.9
2.8
6. 1
6.9
250
259
240
143
381
320
418
344
236
557
503
749
338
564
830
388
662
514
420
470
536
829
604
II 10
678
631
480
536
1034
1 153
712
737 '
843
0.75
0.41
0.57
ND
0.66
1. 13
1.26
0.61
0.94
ND
1.39
1.32
2.59
ND
0.58
0.94
2.42
0.56
0.32
ND
ND
1.91
0.97
0.20
2.68
1.00
0.23
0.33
0.84
3. 15
0.67
0.90
0.50
8960
9235
7050
6970
4990
7630
8610
10495
7730
10020
7250
12820
9400
9290
8495
7450
8270
6690
8880
8100
8570
8550
7600
6845
5830
8705
12440
9250
13850
7010
5930
13920
5570
9860
8830
13700
7670
32800
10000
4770
5750
17200
4590
16400
2560
24700
7040
6680
8130
12300
7080
5590
6940
9320
7800
1 1400
11800
9060
4950
2220
6530
3770
6850
9120
3460
5280
813
863
680
627
2320
725
980
1050
4870
864
968
934
967
888
673
1250
486
600
569
956
1020
646
824
623
946
843
838
800
915
963
917
875
71 1
363
234
1490
440
1580
1730
215
332
994
196
474
93
683
197
175
267
376
350
519
320
196
219
322
260
194
89
152
222
102
170
141
155
153
1.57
0.43
14.01
ND
7.84
4. 10
ND
2.92
3.97
ND
3.53
1.23
6.82
ND
0.40
0.39
2.54
2.59
4.92
ND
ND
1.40
ND
1.97
ND
ND
1 .87
2.68
1.60
ND
2.65
1.83
1.07
*Not detected.
-------
TABLE 8
Animal
No.
1
2
3
4
5
6
7
9
10
11
12
13
14
; 16
17
18
19
20
21
22
23
24
25
26
29
31
32
34
35
36
38
39
: Part B:
Cd
ND*
ND
ND
ND
ND
ND
0.2
ND-
ND
0.2
ND
ND
ND
1.1
ND
3.6
0.1
2.1
2.5
0.5
1.3
0.4
0.4
4.8
0.6
0.7
0.8
ND
3.3
0.5
2.4
ND
Males
Zn
109
184
87
173
168
209
247
147
215
119
225
182
111
188
129
116
163
98
149
138
205
95
126
149
114
106
245
167
237
116
184
206
Hg
0.65
0.01
0.19
0.11
0.10
0.15
0.03
3.28
0.92
0.79
1.66
0.91
1.02
0.77
0.94
3.29
2.07
1.67
2.14
6.67
2.01
2.92
1.33
2.42
2.58
2.44
5.31
1.47
1.33
1.37
3.53
0.79
Cu
13.7
9.3
16.1
13.1
17.4
124.7
14.4
14.3
14.7
12.2
14.1
13.1
6.4
7.4
5.0
4.5
4.3
15.4
10.5
4.0
29.8
4.6
6.0
5.9
5.4
4.9
7.1
5.8
10.1
4.7
9.4
9.6
Fe
ug/g dry
391
202
526
723
265
529
303
250
285
429
517
410
565
427
368
543
386
245
595
514
517
448
397
816
381
494
650
492
587
468
1070
693
Mn
weight
2.06
0.54
0.54
22.20
1.08
2.87
1.14
ND
0.46
0.85
0.87
ND
1.28
0.74
ND
0.91
0.62
0.89
1.21
0.74
1.07
ND
0.07
0.54
0.78
ND
1.05
ND
1.05
ND
0.50
ND
K
8510
6910
2120
6790
8040
9390
11080
5930
8440
9050
9590
8580
8850
5830
8260
5220
7290
8860
9640
7480
9920
7590
9130
9700
10600
9920
10800
6600
9160
9530
8280
9130
Na
14300
9870
16700
8900
9930
10500
7950
24100
10500
8580
6890
7880
4750
6500
4140
7420
5180
7000
14100
3400
22500
9200
3690
6880
5940
3530
5130
12200
7550
5160
5120
6320
Mg
1230
784
1550
692
795
993
775
873
730
1190
773
733
875
720
875
783
959
1510
653
693
2070
1500
852
1250
940
904
722
1010
712
1090
782
950
Ca
1040
328
1970
567
580
768
328
1110
376
1290
314
263
135
290
420
315
288
645
543
129
1210
266
333
129
175
94
124
219
276
120
308
94
Sr
1.68
1.91
4.58
1.32
3.64
1.50
2.68
5.48
1.96
15.50
0.69
0.67
ND
1.72
ND
4.19
2.26
4.82
4.11
ND
8.84
3.91
1.83
0.95
1.02
ND
1.14
ND
0.66
ND
1.93
ND
-------
TABLE 8:
Animal
No.
Part B (Continued)
Cd Zn Hg
cu
Na
Mg
Ca
Sr
40
42
43
44
46
47
7.1
4.1
1.5
4.9
2.4
5.0
200
229
262
211
91
191
1.36
3.20
4.78
4.15
1.57
3.67
7.5
8.0
6.0
1.3
4.3
4.2
- ug/g or
721
783
783
1210
550
973
y weignt —
1.01
0.30
0.89
0.35
ND
0.76
6900
8450
6430
6550
8930
5020
4350
5290
6940
8650
3980
11800
2240
785
687
568
862
683
552
138
106
210
82
232
3.13
0.16
ND
ND
ND
ND
* ND = Not detected.
-------
C71
TABLE 9:
Animal
No.
2
13
20
29
30
32
33
34
39
40
42
I
3
6
12
22
26
29
31
39
42
43
47
Metal concentrations in sea otter hearts
FemaIes
Ug/g dry weight
Cd Zn Hg Cu Fe Mn K Na
Mg
Ca
Sr
ND*
4.1
3.6
3.4
3.1
1 1.4
7.0
3.1
6.6
16.5
17.0
ND
ND
ND
0.7
5.4
2.0
1 1.0
1.4
1. 1
2.9
4.9
3.6
108
1 10
! 12
99
101
105
98
100
1 15
109
129
1 14
106
104
91
101
92
104
84
99
79
95
82
0.40
0. 18
0.70
3. 16
1.40
1.30
7.37
1.59
3.75
1. 19
0.03
__
0.22
0.21
1.43
8.24
1.37
2.46
1. 15
0.61
1.83
3.00
2.45
41.0
32.3
31.4
21.8
14.4
12.2
16.6
15.5
19.3
15.2
15.3
36.3
20. 1
25.6
17.6
34.6
12.2
28.0
12.0
14.7
1 1.4
15.6
12.4
371
460
502
871
878
873
666
428
475
521
705
845
716
61 1
445
814
795
339
442
385
585
509
847
1. 10
2. 10
2.46
2.25
3.72
3.51
2.45
3.62
3.70
1.89
1.23
ND
ND
0.71
0.99
2.89
0.77
3.23
1.45
2.62
1.39
3.45
2.59
7190
6770
4900
3870
3740
3580
3930
5380
4230
4130
2730
Males
6270
2860
5180
5070
6500
4290
6480
6880
5460
4840
3920
2660
9870
14600
17100
10200
1 1000
6960
10700
10000
1 1500
12800
12900
13300
24000
9880
6610
8840
7280
12900
7330
9650
7980
12400
24000
825
1220
1300
778
527
617
1010
1 130
788
887
1320
1020
3050
1180
755
552
809
1970
996
942
765
1400
2290
309
704
1 120
492
970
8340
645
432
731
744
1900
839
2150
800
242
562
468
608
340
501
345
593
963
ND
5.91
7.09
ND
4. 18
10.80
3.72
3.23
ND
4.32
8.99
ND
11.10
3.70
ND
3.38
3.98
7.85
2.46
3.93
ND
3.87
13.01
*Not detected.
-------
70
60
50
en
D
40
CD
1C
O
O
Q_
0)
_C
30
20
10
0
in
c\j
o o
o
+ 0
*«*
0+
0+
+
-K)
is
tn
in
is
in
Length (cm)
FIGURE 5:
Length versus hepatic mercury concentrations (yg/g dry
weight) in sea otters. + = female; o = male.
46
-------
If behavior, not physiological reasons, is responsible for the observed
differences in heavy metal content, we can expect changes in the various amounts
of metals concentrated, regardless of sex. In recent years, feeding pressure in
the growing herd has increased to the point where traditionally favored food
items such as sea urchins and abalone are no longer available within the Mon-
terey to Cambria range. This has forced many of the otters to select other prey
organisms. In recent months, they have been observed feeding upon squid and, in
a few cases, fish such as slow-moving cabezon (Scorpaenicthys marmoratus). If
the fish become more important in the otter diet, we can expect to find increas-
ing Hg concentrations and reduced Cd levels in future specimens. This pheno-
menon may already be reflected in the higher Ag and Hg levels found in more
recently collected sub-adult otters of both sexes.
The remaining elements, Zn, Cu, Fe, Mn, K, Na, Mg, Ca and Sr, were measured
in order to determine if concentrations of these essential elements varied in
response to heavy metal levels. Many of these animals had very high Ca, Sr, Mg,
Na and Fe levels in their livers (Tables 6 and 7). In almost all cases, in-
creases in the concentrations of these elements in other organs were also ob-
served. For example, Male #47 had 16,770 ppm Fe in its liver and 2,110 ppm in
its kidney. This animal also had consistently high Na levels in its liver
(14,790), kidney (15,715), muscle (11,760) and heart (24,000 yg Na/g dry weight).
Whether these abnormally high or, in some cases, low, concentrations of
essential elements are the result of natural variability or a pollutant or
disease is presently unknown. We will continue to work with these data in an
effort to better understand their meaning. From our experience with the sea
lions (Section 3-A), we have learned that the absolute concentration of a heavy
metal is not as important as the relationship between that element and another
element that may ameliorate its effect; i.e., Hg and Se. In the sea lions,
SerHg imbalance appears to affect levels of several essential elements. Thus,
we are curious about Se levels in the otters. Since Se has been shown to pro-
tect against Cd as well as Hg toxicity (Parizek, ejt al_, 1971), it will be inter-
esting to find out if the animals with high Cd levels also have high Se concen-
trations.
Because of the relationship we found between Mn and the ratio of Se:Hg in
the aborted sea lion pups, we are especially interested in some of the otter
pups that had low Mn contents. For example, Male #3 had only 0.26 ppm Mn in its
liver; its K concentration was low (2160); its Ca concentration was very high
(2255 ppm). Thus, chemically, this animal was very similar to the aborted sea
lion pups. Department of Fish and Game personnel who collected the otter re-
ported that it appeared to have been born prematurely. In addition to #3, there
are also five males and five female pups whose livers contained less than 10 ppm
Mn.
Obviously, understanding the meaning of the various data presented above is
a very complicated task. Nevertheless, many interesting questions are asked
that deserve answering in future studies. The otters also represent a very
valuable monitoring tool. Since they are an endangered species, otter carcasses
47
-------
are regularly collected and necropsies are performed. Thus, tissues are regu-
larly made available and long-term trends in metal concentration processes can
be determined. We continue to receive tissues from California Fish and Game and
will soon be in a position to compare metal levels in the otter herd in the late
'70s with the data we have gathered in the early '70s. These archived samples
may also prove valuable in studies of other pollutants such as radionuclides and
various anthropogenic hydrocarbons.
48
-------
SECTION 4
METHODS
Almost all of the data presented in this report were generated using flame
atomic absorption following sample digestion. Several scientists have expressed
interest in our digestion procedure and, for this reason, it will be repeated in
detail.
1. Weigh a 30-ml graduated pyrex beaker.
2. Place approximately 5 gm wet material in beaker and determine wet
weight.
3. Place in a drying oven for 48 hours at 70-80 °C.
4. Determine dry weight (optimum weight is 1 gm).
5. Using an automatic pipet, add 5 ml redistilled 70% HN03.
6. Cover beaker with a 39-mm (1.5 in.) watch glass.
7. Leave samples at room temperature for 1-2 hours to allow initial
frothing to subside.
8. Place beakers on a hot plate at low setting. (Watch for possible
frothing; if it occurs, remove from heat and tap bottom to break
large bubbles.)
9. Allow sample to reflux for approximately 30 minutes.
10. Remove watch glass and evaporate to dryness. (When sample is nearly
dry, reduce heat to prevent spattering.)
11. When smoking ceases, increase heat to 340 °C. (Increase heat slowly
to prevent ignition and possible loss of volatiles.)
12. After step 11, cool the sample and add 5 ml 70% HN03-
13. Cover beaker with a watch glass and heat at low temperature until
residue redissolves.
49
-------
14. With hot plate at low temperature, begin adding 30% hUCL, drop by
drop, until solution becomes clear and a pale yellow cofor.
15. After clearing, evaporate solution to about 3 ml. If solution
begins to darken, add additional HpCL.
16. Add distilled FLO until level of solution is at the 25 ml mark on
the graduated beaker. Make sure solution is well mixed.
17. With solution at room temperature, weigh the beaker and solution
for viscosity corrections.
18. Analyze samples by flame atomic absorption as soon as possible.
Mercury analyses were performed using three different methods: cold vapor
atomic absorption, x-ray flourescence and isotope-shift Zeeman-effect atomic
absorption. These methods are discussed in the sea lion paper, Martin, et al,
1976.
50
-------
Publications and Theses Based on Research Supported by EPA Grant #R 802350
Flegal, A. R. 1976. A geographic survey of silver concentrations in the
gastropod Tegula funebralis (A. Adams, 1855). M. A. Thesis, California
State University, Hayward.
Flegal, A. R. 1978. The geographic variation of silver in the black turban
snail, Tegula funebralis. In preparation.
Flegal, A. R. 1978. Trace element concentrations of the rough limpet,
Acmaea scabra. in California. Bull. Env. Contam. and Toxicol. In press,
Flegal, A. R., and J. H. Martin. 1977. Contamination of biological samples
by ingested sediment. Mar. Poll. Bull. 8: 90-92.
Glickstein, N. 1977. The toxic effects of mercury and selenium in marine
invertebrate larvae. M. A. Thesis, San Francisco State University.
Martin, J. H. 1976. Impact of metals on the marine environment. Paper
prepared for joint American-Soviet biological seminar to be published
by the U. S. Environmental Protection Agency.
Martin, J. H., and W. W. Broenkow. 1975. Cadmium in plankton: Elevated
concentrations off Baja California. Science 190: 884-885.
Martin, J. H., and A. R. Flegal. 1975. High copper concentrations in squid
livers in association with elevated levels of silver, cadmium, and
zinc. Mar. Biol. 30: 51-55.
Martin, J. H., P. D. Elliott, V. C. Anderlini, D. Girvin, S. A. Jacobs,
R. W. Risebrough, R. L. DeLong, and W. G. Gil martin. 1976. Mercury-
selenium-bromine imbalance in premature parturient California sea
lions. Mar. Biol. 35_: 91-104.
Martin, M., M. D. Stephenson, and J. H. Martin. 1977. Copper toxicity
experiments in relation to abalone deaths observed in a power plant's
cooling waters. Calif. Fish and Game 63_: 95-100.
Purdue, A. 1978. Mercury in sablefish, Anotlopoma fimbria, and a review
of mercury in marine fish. M. A. Thesis, California State University,
Hayward. In preparation.
51
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56
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-79-038
2.
3. RECIPIENT'S ACCESSIOr*NO.
4. TITLE ANDSUBTITLE
BIOACCUMULATION OF HEAVY METALS BY LITTORAL AND
PELAGIC MARINE ORGANISMS
5. REPORT DATE
March 1979
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
John H. Martin
8. PERFORMING ORGANIZATION REPORT NO.
3. PERFORMING ORGANIZATION NAME AND ADDRESS
Moss Landing Marine Laboratory
Moss Landing, California 95039
10. PROGRAM ELEMENT NO.
1BA819
11. CONTRACT/GRANT NO.
Grant 802350
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Narragansett, RI
Office of Research and Development
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
13. TYPE OF REPORT AND PERIOD COVERED
F-inal
14. SPONSORING AGENCY CODE
EPA/600/05
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Marine organisms appear to be useful indicators of heavy metal pollution in the marine
environment. In order to test this concept, research was performed to determine the
levels of heavy metals in selected indicator organisms. Several approaches were used.
The first was to select intertidal invertebrates that are widely distributed and are
readily accessible for collection. Tests with the limpet Acmaea scabra proved incon-
clusive, while those with the turban snail (Tegula funebralis) showed anthropogenic
silver input. The experience gained from these studies indicated that serious
problems could exist when using organsims as montiors. As a result, a study on
pooling of individuals for monitoring studies was performed.
A second approach was to transplant oysters and mussels from clean to polluted
environments in order to see if these organisms relfected ambient environmental
levels. Significant increases in selected elements were observed in both bivalves and
the general approach appears promising.
As is the case with many other pollutant studies, the general conclusion drawn from
this study and the others mentioned above is that many marine organisms have high con-
centrations of heavy metals, but whether the metals are adversely affecting the
organisms cannot be determined on the basis of measuring amounts alone.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
Animal ecology
Marine biology
Metals
Sea Lions
Copper toxicity
Cadmium in plankton
6F
8A
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (This Report)
UNCLASSIFIED
21. NO. OF PAGES
63
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
7
•'•US GOVERNMENT PUmTING OFFICE 19?9-bS7-ObO/Ib )
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