-------
In another series of studies, experiments were designed to assay Hg
uptake by gill and hepatopancreas tissues of crabs exposed to various
conditions of temperature and salinity (Vernberg and O'Hara, 1972).
Radioisotopes were used in these experiments.
Gill tissue accumulated much greater amounts of mercury than did the
hepatopancreas regardless of the experimental conditions (Fig. 6A).
Over 82% of the mercury accumulation in the gill tissue occurred within
the first 24 hours in all thermal-salinity regimes, with only slight
additions after 48-and 72-hour exposure to all experimental conditions
except 5°C, 5 °/eo. Under a thermal-salinity regime of 5°C, 5 °/eo the
mercury content declined slightly after 72 hours, probably due to
necrosis and sloughing of the gill epithelium.
26-
24-
22-
20-
18-
16-
i
, 14-
i
* 12-
10-
8-
6-
4-
2-
1 Hepatopancreai
Exposed to:
fa
i
,
s
S^M 30*/.i
0.18 ppm
1
r
i
25
mercury
I
!
5%. 30%,
I
I
33
I
'
I
1
|
Exposed to:
0.18 ppm mercury
* 1 ppm cadmium
b
5
Ttmptratur* °C
5%. 30%. 5Vn 30 V,,
EnvirunnwnUt Rcglm*
A
.
i
25
5V.. 30%.
y
,i
33
5VM 30%.
A B
Figure 6. Tissue uptake of Hg by Uca pugilator under different temper-
ature-salinity condition's after 24-hr exposure (from
Vernberg at al., 1974).
13
-------
The amount of mercury accumulated in gill tissue within the first 24
hours was significantly higher (p<0.01) under the thermal-salinity
regime of 5°C, 5 °/oo, than in tissues of crabs maintained in any other
set of experimental conditions. Under experimental conditions of 5°C,
30 °/oo, mercury accumulation in gill tissue was significantly greater
than at 25°C, 30 °/oo, or at 33°C with either 5 °/oo or 30 °/oo. The
least amount of mercury was accumulated in gill tissue of crabs maintained
at 33QC, 30 °/oo. Thus, low salinity increased mercury accumulation in
gill tissue at all temperatures; low temperature further enhanced gill
tissue accumulation at low salinity (Fig. 6A).
In hepatopancreas tissue the concentration of mercury increased through-
out a 72-hour exposure period in all experimental conditions except 5°C,
5 °/oo. In crabs maintained under these low temperature and salinity
conditions, mercury concentration was significantly lower than in crabs
held at any of the other experimental regimes. After 72 hours there
was less mercury in the hepatopancreas of 5°C crabs than in any other
group, regardless of the salinity. Crabs exposed to 33°C, 30 °/oo,
contained 14 times more mercury in the hepatopancreas than crabs exposed
to 5°C, 5 °/oo (Fig. 6A). However, the aggregate burden of mercury in
the gills and hepatopancreas, when calculated as grams metal present in
both tissues, was found to be relatively constant under all experimental
conditions, although the relative amounts in each tissue were very dif-
ferent. There was no sex difference in mercury uptake in these tissues
under either optimal or suboptimal conditions.
Preliminary screening with cadmium indicated that a concentration of
1 ppm Cd was sublethal for prolonged periods of time to crabs maintained
under optimum temperature-salinity conditions (25°C, 30 */oo). In a
series of experiments parallel to the Hg uptake studies, cadmium uptake
was determined for the hepatopancreas and gill tissue of crabs main-
tained under different temperature-salinity ranges (Vernberg et^ al.,
1974).
In gill tissue, cadmium levels were essentially the mirror image found
for mercury levels. Whereas the highest Hg levels were found at low
temperatures, the highest cadmium levels were found at high temperatures
(Fig. 7A). Cadmium was transferred rapidly and in relatively high
amounts to the hepatopancreas. Under optimum environmental conditions,
the amount of cadmium in the gill and hepatopancreas was approximately
equal. But at either high (33°C) or low (5°C) temperatures, relatively
small amounts of cadmium were found in the hepatopancreas of crabs at
optimum salinity.
In another series of experiments, higher concentrations of Cd were used
to determine uptake levels over a period of time (O'Hara, 1972;
1973). In the first 12 hours of exposure, gill tissue accumulated cad-
mium in proportion to the exposure concentration (Fig. 8). Thus, gill
tissue from crabs exposed to 25 ppm Cd contained 110 ppm; gill tissue
from those exposed to 15 ppm Cd contained 59 ppm, while such tissue
from those exposed to 5 ppm Cd contained 18 ppm. Each accumulation in
gill tissue was about four times the concentration of cadmium in the
surrounding water.
14
-------
0Gm
12-
It-
10-
9-
8-
5-
4-
3-
2-
1-
V Hepalopancreas
24 hour exposure to
1 ppm
cadmium
24
hour exposure to
1 ppm cadmium
•O.IBppm mercury
s
I
5 ••*. 30 V«
1
25
5%. 30*4. 5'
33
k.. 2
ft
»'
i
T«mp«r«u>« °C
t-»
1
,"/l
,11
s
>. 30%.
Erwironmtnlil Regjma
i
25
5%, .
1
0*.. 5'
1
33
/,. 30VH
B
Figure 7. Tissue uptake of Cd by Uca pugilator under different tempera-
ture-salinity conditions after 24-hr exposure to cadmium
alone (A), or cadmium plus mercury (B) (from Vernberg et al.t
1974).
a. 100
X
a
Figure 8.
TIME IN HOURS
Concentration of cadmium in gill and hepatopancreas of crabs
in 5, 15 and 25 ppm Cd** at 30°C, 20 °/oo (from 0'Kara,1972).
15
-------
Gill tissues from crabs in 25 ppm Cd did not increase in cadmium con-
centration appreciably over 110 ppm in 24 hours and exhibited a decline
in tissue concentration at 36 hours. High mortality at 48 hours pre-
cluded additional reliable sampling. Gill tissue from crabs exposed to
15 ppm Cd showed an increase in cadmium content between 24 and 48 hours
with a maximum accumulation of 109 ppm. The significance of the value
around 110 ppm is unclear; it could represent a maximum tissue burden
in terms of equilibrium with the external medium. The cadmium concen-
tration in gill tissues from crabs sacrificed at 60 hours showed a
marked reduction in cadmium content. As there was high mortality of
crabs in this concentration, the lower cadmium content in the tissues
might represent reduced binding of the metal and loss due to the destruc-
tion of tissue. Crabs exposed to 5 ppm Cd continued to concentrate
cadmium in their gill tissue, attaining a maximum of 39 ppm after 60
hours.
After 12 hours exposure, the hepatopancreas concentrated cadmium about
two times greater than exposure level; 25 ppm in the medium concentrated
to 50 ppm in tissue, 15 ppm to 32 ppm, and 5 ppm to 11 ppm. After 24
hours the hepatopancreas of crabs exposed to the highest concentration
was almost completely destroyed; it changed from a firm glandular tis-
sue to an amorphous and liquified condition, thus precluding samples
from these specimens. Crabs exposed to 15 ppm Cd for longer periods
showed an increase in hepatopancreas cadmium level to about 116 ppm in
48 hours, followed by a rapid decline. This decline might be associated
with the breakdown of hepatopancreas tissue. Crabs exposed to 5 ppm
showed the same gradual increase in Cd concentration that was evident
in gill tissue, attaining a maximum of 25 ppm after 60 hours.
Cadmium accumulation was highest in green gland tissue (Fig. 9), with
maximum concentrations of 380 ppm in tissue from crabs exposed to 25
ppm, 171 ppm from crabs in 15 ppm, and 118 ppm from crabs in 5 ppm.
These values are 12 to 20 times the exposure concentrations.
£ 300
&
&
Z
200
S
a
< 100
u
24 34
TIME IN HOURS
Figure 9. Concentration of cadmium in green gland tissue of crabs in
5, 15 and 25 ppm Cd4* at 30°C, 20 °/00 (from 0'Hara,1972).
16
-------
Cadmium levels in muscle tissue remained almost constant throughout the
experiment. Cd tissue concentrations were only slightly above the expo-
sure levels, with maximum concentrations of 29.3 ppm in crabs exposed
to 25 ppm, 17.3 ppm from crabs in 15 ppm, and 8.9 ppm from crabs exposed
to 5 ppm.
To quantitatively assay the total body burden of metal in the gills and
hepatopancreas of fiddler crabs, Cd levels were calculated as yg
present in each tissue and added together. It is clear that this aggre-
gate value increased over time for all temperature-salinity regimes
tested. The maximum uptake was 17.44 yg Cd which occurred at 33°C,
10 °/oo (Table 3). The translocation of cadmium from gills to hepato-
pancreas is indicated by the percent of metal in each tissue and was
most pronounced at high temperatures.
Table 3. CADMIUM CONTENT IN GILL (G) AND HEPATOPANCREAS (H) FROM FID-
DLER CRABS EXPOSED TO 10.0 ppm CADMIUM OVER A 72-HR PERIOD
UNDER VARIOUS TEMPERATURE-SALINITY REGIMES. VALUES ARE MEANS
OF CONCENTRATIONS IN TISSUES OF FOUR ANIMALS + STANDARD ERROR
(from O'Hara, 1973).
24 hr
48 hr
72 hr
Temperature-
salinity
regime
10 C, 1056,
10 C, 3056>
25 C, 1056)
25 C, 3056)
33 C, 10J6)
11 /"* it\&
JJ l**t -3UJ00
Total MS
Cd'+in
GandH
.78
.39
2.08
.77
4.98
1.67
%in
G and
H
G-68.8
H-31.2
G-69.0
H-31.0
G-30.6
H-69.4
G-32.9
H-67.1
G-38.0
H-62.0
G-22. 1
H-77.9
ppm Cd**
in G
and H
20.8±3.3
6.8±0.8
9.1±1.1
3.6±0.8
27.9±3.5
34.2±4.0
10.2±1.4
14.5±2.2
65.1±3.6
74.1±2.8
11.2±0.7
30.8±7.0
Total MS
Cd** in
GandH
1.03
.44
3.72
1.34
10.10
2.50
%in
G and
H
G-55.3
H-44.7
G-44.2
H-55.8
G-26.5
H-73.5
G-24.3
H-75.7
G-21.3
H-78.7
G-22. 3
H-77.7
ppm Cd'H"
in G
and H
28. 1± 1.4
16. 2± 0.8
10.6± 0.9
8.8± 1.2
37. 1± 5.6
78. 3± 5.5
12. 8± 1.8
23. 3± 2.4
98.7±11.1
198.2±14.7
26.5± 2.8
88.0±13.7
Total Mg
Cd++in
GandH
1.86
.75
8.65
1.85
17.44
4.90
%in
G and
H
G-29.9
H-70.1
G-38.6
H-61.4
G-19.9
H-80.1
G-20.2
H-79.8
G-16.2
H-83.8
0-11.3
H-88.7
ppm Cd**
in G
and H
25. 1± 3.7
30. 1± 2.2
10.2± 0.8
6.5± 2.9
70. 8± 8.8
133.8±30.2
15. 5± 1.1
33.7± 6.3
92.0±15.1
200.2±22.1
24.1± 3.5
77.5±13.1
Since these studies established that temperature and salinity differ-
entially affected the uptake of cadmium and mercury in tissues of U_.
pugilator . further work was initiated to consider the effect of dual
exposure to these two metals. Mercury was added in the form of
at an initial concentration of 0.18 ppm Hg for a 72-hour period.
Cadmium was added in the form of CdCl2 plus one yCi of
the total initial concentration to 1 ppm Cd.
to bring
In both the gill and hepatopancreas, mercury uptake was more influenced
by the presence of cadmium than was cadmium uptake by the addition of
mercury (Figs. 6B, 7B) . Generally, where a statistically significant
change in uptake did occur, the uptake of each metal was greater in
the gills and showed a decrease in the hepatopancreas.
17
-------
When Hg alone was present in the water, the crabs effectively transported
Hg from the gills to the hepatopancreas. For example, after 72-hr at
33°C, 30 °/oo» the percentages of Hg in the gill and hepatopancreas
were 35.1 and 64.9, respectively but 96.3 and 3.7 at 5°C, 5 °/oo
(Vernberg and O'Hara, 1972). When both Cd and Hg were present, the
crabs seemingly lost this transport ability (Fig- 6B), and the percent
Hg in the gills remained high (90-98%) regardless of the temperature-
salinity regime. Although TLm values were not determined for the crabs,
mortality rates among the experimental animals were considerably higher
than observed mortalities in crabs subjected to only one metal. The
inability of the 'crabs to survive for long periods of time in the pres-
ence of both metals may well be associated with the inability to trans-
port the Hg from the gills to the hepatopancreas. Figure 7B indicates
that the uptake of Cd by the gills was increased by the addition of Hg
only at high temperature and low salinity (30°C, 5 °/0o). At low
temperature and high salinity the rate of gill uptake decreased, but
only to statistically significant levels after 48 hours. There was
one case in which Hg significantly affected Cd uptake in the hepato-
pancreas; this occurred at 25° and salinity was not a factor.
Metabolism
Metabolic base-line rates were first established for adult male and
female fiddler crabs at 25°C in 30 °/00 sea water (Fig. 10). These
rates were essentially the same for both sexes. After the base-line
rates were determined, the same animals were maintained at 25°C in
30 °/oo seawater with the addition of mercury, and metabolism of the
crabs were measured after 1, 3, 7, 14, 21 and 28 days exposure.
Although a low level concentration of mercury was not lethal to the
crabs under optimum environmental conditions, metabolic rates were
affected, especially for males. The rate of oxygen uptake of males
was significantly lower than that of the females after 21 days in this
o
UJ
s
UJ 100
X 80
5 60
«
u
a.
AC
>
M
1 r«nol«i
|
14
DAYS
17
21
Figure 10. Oxygen uptake rates of male and female Uca pugilator main-
tained in 30 °/oo seawater containing 0.18 ppm Hg at 25°C.
The base-line rate is represented by the first set of data
points on the left. The vertical bar through each mean
value is the standard error (from Vernberg and Vernberg,
1972a).
18
-------
Table 4. SUMMARY OF THE METABOLIC RESPONSE OF GILL AND HEPATOPANCREAS TISSUES FROM FIDDLER CRABS
EXPOSED TO 0.18 ppm Hg UNDER DIFFERENT COMBINATIONS OF TEMPERATURE AND SALINITY. RESPONSES
WERE MONITORED AT WEEKLY INTERVALS FOR 21 DAYS.
Acclimation
temperature
Environmental
regime
Determination
temperature Fig.
(°C) no.
Metabolic response of tissues from exposed
crabs in comparison to response of tissues
from control crabs
25
10
5°C, 5 %
5°C, 30 %
25°C, 30 °/oo
33°C, 5 °/oo
5°C, 5 %
5°C, 30 %
33°C, 5 %
25
33
33
(Fig.17) After 7 days, rates of both exposed tissues
significantly depressed (only female crabs
survived).
(Fig.16) No significant difference in metabolism of
gill tissue from female crabs after 7-day
exposure; gill tissue from males markedly
lower than that from control animals. Hepato-
pancreas from both males and females sig-
nificantly lower.
(Fig.13) Rates of both tissues depressed in both sexes.
(Fig.14) Patterns of response generally similar in tis-
sues of control and experimental crabs. Sig-
nificant differences between control and
experimental animals were observed only after
14 and/or 21-day exposure.
(Fig.18) Rates of both tissues depressed initially
(day 7); after 21-day exposure to Hg, markedly
higher than those tissues from control crabs.
(Fig.19) Metabolic rate of gill tissue lower through-
out; hepatopancreas initially lower, but
significantly higher after 21-day exposure
to Hg.
(Fig.15) Patterns of response and actual rates both
different in gill tissue. In hepatopancreas
pattern of response similar, but rates of
experimental hepatopancreas tended to be
lower than control.
-------
sublethal concentration of mercury (Fig. 10). The metabolic rate of
the males had not returned to the base-line level by the end of the 28-
day experimental period. Both males and females, however, continued to
survive for another month under the same mercury regime as before with-
out any significant increase in mortality.
Under conditions of low temperature (5°C) and low salinity (5 °/oe)
stress, females not only survived much longer than males, but also main-
tained a steadier rate of oxygen uptake (Fig. 11). The metabolic rate
and pattern of the experimental female crabs were similar to those of
the control female crabs. The metabolic rate of male experimental crabs
after a 1-day exposure to mercury was not significantly different from
that of the female experimental or male and female control crabs, but
by day 3 the rate dropped markedly.
I-
(9
30
20
UJ
5
4
CC
O
UJ 10
a.
X
O*
a:
EXPERIMENTAL
^ ~~^^«/e«
CONTROL
Females
13 7
DAYS
Figure 11. Oxygen uptake rates of male and female Uca pugilator main-
tained at 5°C in 5 °/00 seawater with and without the addi-
tion of 0.18 ppm Hg. The vertical bar through each mean
value is the standard error (from Vernberg and Vernberg,
1972a).
Oxygen uptake rates of female control crabs maintained in low salinity
water (5 °/oo) and at high temperature (35°C) were relatively constant
over a 28-day period and tended to be higher than that of control male
crabs (Fig. 12). The metabolic rates of mercury-treated female crabs
remained fairly constant for the first 7 days and then declined
rapidly. The uptake rates of experimental male crabs declined steadily
from day 1 and tended to be lower than those of the females throughout
the remainder of the test period.
20
-------
300
o 200
UJ
*
5 100
u 300
IL
E
5
EXPERIMENTAL
ZOO
m
a:
100
CONTROL
14
DAYS
Zl
Figure 12. Oxygen uptake rates of male and female Uca pugilator main-
tained at 35°C in 5 °/oo seawater with and without the addi-
tion of 9 x 10~7 M HgCl2 or 0.18 ppm. The vertial bar
through each mean value is the standard error (from
Vernberg and Vernberg, 1972a).
To further examine why Hg-treated males died sooner than females, a
series of tissue metabolism studies was carried out (F.J.Vernberg and W.B.Vern-
berg, 1976). Crabs were collected in winter or early spring, then
either warm-acclimated at 25°C or cold-acclimated at 10°C in the labora-
tory for a minimum of two weeks. Each group was then subdivided into
temperature-salinity groups:
5°C, 5 Voo
5°C, 30 °/oo
33°C, 5 °/oo
33°C, 30 °/»o
25°C, 30 °/oo (warm-acclimated
crabs only)
Half the animals in each of these groups were exposed to 0.18 ppm Hg and
the other half placed in untreated sea water. From 10-20 metabolic
determinations were made with gill or hepatopancreas of both male and
female crabs after 1, 7, 14, 21 days exposure to the specified condi-
tions. Results of these studies are summarized in Table 4 and Figures
13-19.
Changes in whole animal metabolism induced by cadmium are also indicated
by thermal-metabolic acclimation patterns (Vernberg, 1975). Respiration
rates were determined on cold- and warm-acclimated crabs that had been
exposed to a sublethal concentration of CdClo (1 ppm Cd) for 24 hours
or 14 days. Rates were measured at temperatures ranging from 10°C to
30°C at 5°C intervals; the salinity was maintained at 30 °/eo. After
a 24-hour exposure to cadmium, the pattern of response of the experi-
mental crabs was modified, with metabolism significantly suppressed
in Cd-exposed crabs for most test conditions (Fig. 20). In the
21
-------
10
»
8
7
6
6
(-1
"
V
6111
Hepatopancreas
1
Control 1 3
7 14 21 28
Number days exposure to 0.18 ppm Hg
Figure 13. Metabolic rates of gill and hepatopancreas tissue from warm-
acclimated male and female crabs exposed to 0.18 ppm Hg under
an optimum temperature-salinity regime of 25°C, 30 °/0o. Verti-
cal bars indicate + one standard error.
Figure 14. Metabolic rates of
gill and hepato-
pancreas tissue
from warm-
acclimated crabs
maintained at
33°C, 5 °/00 for
varying lengths
of time with and
without 0.18 ppm
Hg. Vertical
bars indicate
+ one standard
error.
1500
800
00
CM
O
400
200
Uarm-accl1nated crabs
Experimental conditions: 33°C, 5 0/00, 0.18 ppm
•-— Control; Experimental
G111
Hepatopancreas
14
21
22
Days
-------
Figure 15. Metabolic rates of gill and
hepatopancreas tissue from cold-
acclimated crabs maintained at
33°C, 5 °/0o with and without Hg.
Vertical bars indicate +
one standard error.
1500
1000
600
600
•fc 400
S-
200
Cold-acclimated
Experimental Conditions: 33°C, 5 0/00
• - - Control;
Experimental
G111
i
1
1 1
7 K
Days
Hepatopancreas
I
21
1
7 14
Days
21
taro-accllmated
Experimental Conditions: 5 C. 30 0/00
- - - Control; Experimental
200
I
§•100
60
CO
sm 9
o
Days
1
Figure 16. Metabolic rates of gill and hepatopancreas tissue from wann-
acclimated crabs maintained at 5°C, 30 °/oo with and with-
out 0.18 ppm Hg. Vertical bars indicate + one standard error.
23
-------
Warm-acclimated $
Experimental Conditions: 5°C, 5 0/00
- - - Control; Experimental-
303
6111
1.200
-ISO
90
70
SO
30
Hepatopancreas
Days
Days
Figure 17. Metabolic rates of gill and hepatopancreas tissue from warm-
acclimated crabs maintained at 5°C, 5 °/00 with and without
0.18 ppm Hg. Vertical bars indicate + one standard error.
Cold-acclimated $ + <**
Experimental Conditions: 5°C, 5 0/00. O.'i8 i-rn Ht|
•" Control; Experimental
500
300
150
GUI
Vt
"5
ISO
100
89
60
Hepatopancreas
14
Figure 18. Metabolic rates of gill and hepatopancreas tissue from cold- acclimated
crabs maintained at 5°C, 5 °/oo with and without 0.18 ppm Hg. Vertical bars
indicate + one standard error.
24
-------
300
200
100
BO
GUI
5°C, 30 0/00
Cold-acclimated crabs
—--Control; Experimental
200
100
80
Hepatopancreas
S°C, 30 0/00
:^__£ J
I
Days
14
21
Figure 19. Metabolic rates of gill and hepatopancreas tissue from.cold-
acclimated crabs maintained at 5°C, 30 °/0o with and without
0.18 ppm Hg. Vertical bars indicate + one standard error.
warm-acclimated crabs, oxygen consumption appeared to be independent of
temperature above 15°C. Following exposure to cadmium for 14 days, the
pattern of response of cold-acclimated animals was altered to a greater
extent than the patterns of warm-acclimated animals (Fig. 21). Follow-
ing exposure to cadmium' for 14 days rates of the experimental crabs were
significantly depressed over those of controls at all temperatures above
10°C (Fig. 21). Again, there was a period of temperature insensitivity.
Significantly decreased rates in the warm-acclimated experimental crabs
were also noted at the higher temperature.
Behavior
Qualitative observations of adult crabs in sublethal concentrations of
mercury indicated that after several weeks exposure, sluggishness and
lack of responsiveness set in, followed by a state of torpor before
25
-------
10
9
8
7
6
S
2 _
g
t
COLD-ACCLIMATED
I Control
j& Experimental
* ( 1 ppm cadmium)
8
7
6
5
4
2 _
WARM-ACCLIMATED
Control
Experimental
( 1 ppm cadmium)
30°C
10°
15°
20"
25"
Figure 20. Uca pugilator adult metabolism,24 hour exposure.
26
-------
20—1
10.
9
8
7
6
S
4 -
3 -
i
s
I
I
2 -
r
7
6
5
4 .
3 -
2 .
COLD-ACCLIMATED
Control
Experimental
0 PP" CdC12)
WARM-ACCLIMATED
Control
Experimental
(1 ppn CdCl2)
•Ar
•*•
30
Figure 21.
Temperature °C
Uca pugilator adult metabolism, 14 day exposure.
death. Experiments were therefore undertaken using activity to measure
the effects of sublethal HgCl2 on normal function. An actograph pat-
terned after Naylor (1958) was used in conjunction with an operations
recorder and the data processed as chronological daily time scans
(Barnwell, 1966). It seemed possible that both rhythmic pattern of
locomotor behavior as well as daily intensity of activity might reflect
Hg toxicity in the crabs.
In a preliminary series, groups of 10 males and 9 females were measured
under control conditions of temperature and salinity, with a 12L:12D
light schedule. Great variability in pattern and amount of activity
occurred. A typical example is shown in Figure 22.
27
-------
Figure 22. Activity of Uca
pugilator adult in
untreated seawater
30 °/oo, 25°C, 12L:
12D, using a Naylor-
type actograph.
time of mean low tide
12
24 hr
lighting schedule
X of hour active
100
50
light dark
Further modifications of the recording system, experimental conditions,
and methods of data analysis were made to overcome some of the diffi-
culties in the system. For 10 control crabs and for 9 crabs in
0.18 ppm Hg a very high level of background activity was seen in all
crabs but the crabs were predominantly nocturnal, with the greatest
peaks of activity at the light transitions. Activity in the majority
of mercury-treated crabs was reduced compared with control crabs,
especially in the females. Yet the unexpectedly high level of back-
ground activity interferred with processing and interpreting the data,
necessitating the development of a technique to analyze more quantita-
tively the rhythmic pattern and amount of activity. Braked wheels were
subsequently fabricated which permitted movement only when the crab
walked and, furthermore, which counted in direct relationship to distance
traveled.
The rhythmicity and pattern of activity were first examined. Hourly
counts were plotted in daily scans to give a summary of activity. In
addition, 5-minute counts were processed by a computer periodogram
program for frequency of rhythmic components in the range 6-15 hours
and 20.5 ~26.5 hours to detect tidal and circadian rhythmicity,
respectively. Figures 23 and 24 show such data scan-periodograms for
2 crabs under control conditions of LL, 25°C, and 30 °/oo for 8 days.
A strong rhythmic component is seen in the data at 23.9 hrs for Figure
23 and at 24.3 for Figure 24. Although a large amount of background
noise is apparent in the activity scan of Figure 23, the periodogram
was able to detect dominant frequencies. Secondly, 24-hr totals for
activity were obtained in these experiments (Fig. 25). Considerable
differences were noted in the average daily amount of activity of these
28
-------
Activity Scan
Activity Perlodogram
22 23 24 25 26
Period length In hours
27
24 hr.
Time of Day
Figure 23. Activity of 1 Uca pugilator in LL, 30 °/00 control sea
water, 25"C, in a wheel actograph.
Activity Scan
Activity Perlodogram
21 22
I
23 24 25 26 27
Period length 1n hours
12
24 hr
I
Time of Day
Figure 24. Activity of 1 Uca pugilator in LL, 30 °/oe control sea
water, 25°C, in a wheel actograph.
29
-------
A.
7.000
J • 5378 ± 378
B.
7.000
6.000
5,000
4,000
3,000
2,00i
1,00
X - 1891 1 311
1234567
Day*
Figure 25. Consecutive daily activity totals of 2 Uca pugilator in
LL, 25°C, 30 °/oo untreated control sea water in wheel
actographs.
two control crabs: 5378 + 378 contact counts/day and 1891 + 311/day
respectively for the 7-day recording period.
In a related experiment the mean daily activity rate of individual crabs
kept for two weeks in either control sea water or 0.18 ppm Hg was com-
pared (Fig. 26). These results suggest that inter-individual dif-
ferences are high and would possibly mask treatment differences.
\~~\ Control
5000
Figure 26. Mean daily totals of
activity in Uca
pugilator exposed
for 14 days to 30 °/oo |
sea water containing $
0.18 ppm Hg, 25°C, |
12L:12D.
4000
c 3QOO
if
xZOOO
1000
0
Mlmal f
30
Mercury
-------
On the other hand, consecutive daily totals in untreated seawater
seemed stable enough in each individual (Fig. 25) to serve as a control
for the later Hg exposure. Therefore, a series of experiments consist-
ing of 3 parts was initiated. Part A served as premercury control,
Part B as Hg experimental, and Part C as post-Hg control. Data are pre-
sented in Figures 27 and 28 for the periodogram frequency analysis and
daily activity scans for two representative crabs for the Parts A, B, C.
Rhythmicity was not appreciably modified. Mean daily activity totals
are shown for 5 crabs in Figure 29. A clear reduction of activity
occurred. Average change of Hg-treated over control for the crabs ranged
from 5% increase to 38% reduction (Fig. 29), with mean reduction for all
animals of 23%.
Difficulty was encountered with some crabs (Fig. 28) following feeding
and cage cleaning. The high bursts of activity would definitely interfer
with an assay method. In an attempt to further refine the method several
new sources of food were tried to find a way of keeping a constant
food supply without fouling the living and recording compartment. "Biorell"
proved most satisfactory. More feasible methods of using activity as a
bioassay technique are considered in the discussion.
Electronmicroscope Study of Tissue Anatomy
Electron microscope studies of the tissues of adults treated for 42 days
in 0.18 ppm Hg revealed sites of concentration and tissue damage. Mer-
cury was found primarily in the gills, green gland, and hepatopancreas
with highest concentration in the gills. The mercury caused extensive
alteration of the ultrastructure of the gill filaments (Fig. 30B).
Normally the filaments are characterized by tightly packed, interdigitat-
ing epithelial cells. The basal plasma membranes are thrown into folds
that penetrate the cell almost to their apical surfaces. The cells con-
tain numerous mitochondria localized within the folds (Fig. 30A). In
gill tissue from crabs maintained in the sublethal concentration of
mercury, the filaments showed less cytoplasmic protein, disappearance
of membrane folds, and decreased number of mitochondria. Swelling and
loss of the mitochondrial cristae were also observed (Fig. 30B).
Enzymatic Studies
Results of the studies on the influence of Hg on cytochrome £ oxidase
activity are summarized in Table 5. Enzymatic activity of gill tissue
was affected primarily in tissue from warm-acclimated crabs at low
temperatures. At these temperatures activity was reduced to approxi-
mately one half. Gill tissue from cold-acclimated crabs showed essen-
tially no change in activity. In hepatopancreas tissue from both cold-
and warm-acclimated crabs, however, Hg caused a marked decrease in
activity.
31
-------
days
root
mean
square
24 hr
FYriod K-'jjtli In hours
1
2
3
4
S
C \
7
S
9
10
11
12
13
14
15
1C
days
root
mean
square
24 hr
1
2
3 . .
4
5^
C
7 _
8_
9~
10
II
12
13
14
15
10
8
root
mean
square
2
hr
B
?2 24 26
Period length In hours
Period length in hours
Figure 27. Activity of 1 Uca pugilator in LL, 30 °/oo seawater, 25°C
in a wheel actograph. A, Control; B, 0.18 ppm; C, post-
control.
32
-------
d«yi
root
»Mn
s«u*re
24 lir
22
24 26
Period Imgtli
-------
CD Control
d Mercury
(£23 Post-Control
3000
12500
B
8
'2000
SI 500
1000
500
1
-30
-31
3
-23
4
•29
'animal number
percent activity
change: Control-Hg
Control
Figure 29. Locomotor activity of single fiddler crabs in LL at 25°C,
first in control sea water 30 °/0o» followed by a treatment
in sea water containing 0.18 ppm Hg, and a final post-
control period in untreated sea water.
34
-------
B.
Fig. 30A.B.
Ultrastructure of gill tissue of control adult Uca pugilator.
Normal gill filament showing interdigitating epithelial cells
situated on the basal lamina. Mitochondria are localized
within the folds of plasma membrane. A, 9.200X; B, 22.500X.
Courtesy of N. Watabe.
35
-------
Figure 30C.
C.
Ultrastructure of gill tissue of adult Uca pugilator
maintained in 0.18 ppm Hg for 6 weeks. Analysis of the
tissues showed a concentration of approximately 17 ppm
Hg. Note the loss of cytoplasmic protein. Membrane
folds are not evident, and mitochondria are scarce and
pycnotic. 17.500X (from Vernberg et al., 1974).
36
-------
Table 5. CYTOCHROME £ OXIDASE ACTIVITY IN GILL AND HEPATOPANCREAS TISSUE
FROM COLD- AND WARM-ACCLIMATED CRABS EXPOSED TO 0.18 ppm Hg FOR
VARYING LENGTHS OF TIME.
my moles cytochrome £ oxidized/mg protein/sec.
Temperature °C
Tissue
Gill - W.A.
Gill - C.A.
Hepatopancreas-
W.A.
Day
0 (control)*
3
7
14
28
O(control)*
3
7
14
28
0 (control)*
3
7
14
28
15°
0.803
0.600
0.478
0.543
0.455
0.831
0.572
0.782
6.751
0.703
0.558
0.254
0.256
0.243
0.301
20°
1.120
1.040
0.676
0.823
0.649
0.996
1.201
1.052
1.280
1.067
0.651
0.347
0.414
0.338
0.322
25°
1.000
1.130
0.997
1.140
0.908
1.756
2.023
1.652
1.834
1.890
0.934
0.519
0.434
0.514
0.436
35°
1.45
2.04
1.92
2.86
2.15
3.081
4.423
3.786
3.622
3.488
1.330
0.623
0.672
0.708
0.612
Hepatopancreas-
C.A. O(control)* 0.631 0.938 1.180 1.420
3 0.414 0.474 0.889 1.430
7 0.423 0.602 0.757 2.021
14 0.433 0.558 0.834 1.312
28 0.316 0.448 0.615 1.334
*Not exposed to Hg.
LARVAL STUDIES
Larval studies centered on the synergistic effects of Hg and Cd combined
with temperature and salinity stress. Zoeal stages I, III, V, and
megalopa were selected for study. Response parameters included survival,
metabolism and behavior.
Effect of Hg at Three Sublethal Concentrations Under Optimal Conditions
Viability experiments were first carried out with three concentrations
of mercury (DeCoursey and Vernberg, 1972), in an optimum salinity-
temperature regime (25°C, 30 °/oo)« The highest concentration selected,
0.18 ppm Hg, was sublethal to adults exposed for a period of 6 weeks.
The two lower concentrations, 9 x 10~9 M HgCl2 (1.8 ppb Hg) and
9 x 10""^ M HgCl2 (0.018 ppb Hg) are concentrations within the range
37
-------
reported for certain polluted estuarines (Klein and Goldberg, 1970). A
concentration of (9 x 10"' M HgCl2) 0.18 ppm Hg quickly proved fatal to
stage I zoeae; 50% survival time was less than 24 hours (Fig. 31). The
two lower concentrations of mercury also markedly affected viability,
for survival time was considerably reduced in comparison to control
values: 8 days in 1.8 ppb Hg or 11 days in 0.018 ppb Hg, in contrast to
18 days survival in untreated sea water.
Figure 31. 50 percent survival
time values for zoeae
reared in untreated sea-
water or in three concen-
trations of Hg. The
approximate number of
days for each stage of
larval development are
indicated by brackets
(from DeCoursey and
Vernberg, 1972).
I
e "
g 5
s
19
18
17
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
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-
-
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M
c
O
O
ft
00
H
O
o
—
X>
0.
a
00
rH
n
ex
00
rH
O
Additional viability experiments confirm the findings of the mercury-
reared larvae. Experiments with 500 newly-hatched (stage I) zoeae from
different hatches of eggs suggested considerable variability in survival
time in 0.18 ppm Hg, ranging from 5-72 hours. When control-reared stage
III or stage V zoeae were placed in 0.18 ppm Hg, they appeared even more
sensitive. Only a few first and occasional third stage zoeae were able
to survive for the 24-hr period. No stage V larvae were alive after
24 hours, and most died after 6 hours.
38
-------
Rearing success statistics also support the above results. Survival
results showed definitely reduced survival up to megalopa stage for
mercury-treated larvae: 6 of 100 in 1.8 ppb Hg, 3 of 100 in 0.018 ppb
Hg, but 20 of 100 in the control group.
Metabolism Studies
There was no immediate response of stage Izoeae to any of the concentra-
tions of mercury. Oxygen uptake rates of these larvae one hour after
exposure to the three experimental concentrations of HgCl2 were unchanged
from those of control larvae. A 6-hr exposure to 0.18 ppm Hg markedly
depressed respiration rates of all stages tested (Fig. 32). The greatest
decrease in metabolic rate occurred in stage V zoeae,where the rate of
mercury-exposed larvae was approximately one-third that of control larvae.
A.IO.O
8.0
(.0
2.0
HttaboIlM
-62*
11,0
I
c 14.0
3
ffio.o
5 6.0
2.0
Activity
Zotil Stigt
Figure 32. Percent in metabolic rates (A), and swimming activity (B)
of zoeal stages I, III and V after six-hr exposure to a
mercury concentration of 0.18 ppm Hg. • » Control zoeae,
o - Experimental zoeae. Vertical lines indicate + one
standard error (from DeCoursey and Vernberg, 1972).
39
-------
Twenty-four hour exposure to the lowest mercury concentration used was
without effect on metabolic rate in stage I zoeae,while 1.8 ppb Hg
depressed rates. In stages III and V zoeae,there was a general tendency
for mercury to increase metabolic rates (Fig. 33). In contrast, the
oxygen uptake rates of stage V zoeae reared in mercury tended to be
depressed, while the rate of stage III larvae were unchanged (Fig. 34).
Figure 33. Acute exposure: meta-
bolic rates of control
zoeae and zoeae exposed to
low concentrations of Hg
for 24 hr. Vertical lines
indicate + one standard
error (from DeCoursey and
Vernberg, 1972).
10.0
8.0
6.0
4.0
2.0
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Figure 34.
Ill V III V
Zoeal Stage
Chronic exposure: metabolic rates (left) and swimming activ-
ity (right) of control zoeae and zoeae reared in low concen-
trations of HgCl2« Vertical lines indicate + one standard
error (from DeCoursey and Vernberg, 1972).
-------
Behavior Studies
Observations on the normal swimming behavior of stage I, III, and V
served as a baseline for detecting effects of the three mercury test
solutions (DeCoursey and Vernberg, 1972). Using the maxillipeds, the
zoeae usually swam in a fairly straight line (Fig. 35A). This type of
swimming was interspersed with a variable amount of "tail lashing
maneuvers," which resulted in a rapid change of direction, or often a
whirling type of locomotion. Stage I zoeae usually swam in a start
and stop fashion, while stage III zoeae, with a marked increase in size
and complexity of the maxillipeds, were strong, steady swimmers. Stage
V zoeae, which had increased greatly in weight, with little further
development of the maxillipeds, were relatively slow, sluggish swimmers,
often hovering close to the substrate. Such stage dependent differences
are reflected quantitatively in the rate of swimming (Fig. 32B).
When zoeae were first introduced into the mercury, they often remained
motionless for several seconds on the bottom of the dish, then darted
erratically with considerable tail lashing for several minutes before
adopting a characteristic swimming pattern. As the effects became more
pronounced with time, the zoeae manifested marked swimming abnormalities
such as erratic spiral swimming, swimming on their sides, or darting up
from the bottom of the dish, then settling slowly to the bottom followed
by disoriented twitching movements (Fig. 35B,C).
The effect of mercury solutions on normal activity was assayed at
regular intervals after the start of exposure by tracking the actual
swimming path in order to determine rate of activity. Six-hour expo-
sures to 0.18 ppm Hg reduced the swimming rate of all larvae, with a
greater effect on stage V larvae (Fig. 32B). Behavioral effects of
24-hr exposures are summarized in Figure 36; a concentration of 0.18
ppm Hg markedly reduced activity of all groups compared to the controls:
49% reduction for stage I, 79% for stage III, and 100% for stage V. As
with 6-hr values (Fig. 32B), the older stages appear more sensitive to
mercury than the newly-hatched larvae. When exposed to the two lower
concentrations of mercury, the rate of swimming changed relatively
litte; it was depressed to some extent for stage I, and elevated slightly
for the older stages.
The chronic effects of mercury on activity are summarized in Figure 34.
The highest concentration, 0.18 ppm Hg, was fatal to all zoeae, usually
in less than 1 day, and therefore rearing could only be carried out in
the more dilute concentrations. The data suggest that no change in
activity of stage III occurred, but fifth stage swimming rate increased.
This may represent a tendency for a more erratic type of swimming than
in controls, as in the acute studies.
41
-------
B.
Figure 35. Effect of mercury on the
swimming behavior of stage I
zoeae; A. Control in 30 °/00
untreated seawater; B, Experi-
mental zoeae in 30 °/00 seawater
containing 0.18 ppm Hg after six-
hr exposure; C. Experimental
zoeae in 30 °/00 seawater con-
taining 0.18 ppm Hg after 24-hr
exposure. o=outline of test
chamber drawn to the scale of
the track; =horizontal swim-
ming track of one zoea for 60
sec.; X=starting point of test.
(from DeCoursey and Vernberg,
1972).
Figure 36. Acute exposure: swim-
ming activity of control
zoeae and zoeae exposed to
low concentrations of Hg
for 24 hr. Vertical lines
indicate + standard error
(from DeCoursey and
Vernberg, 1972).
10
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0
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V
Zoeal Stage
42
-------
Response to Hg Under Different Temperature-Salinity Regimes
Since the larvae inhabit estuaries where temperature-salinity conditions
are often suboptimal, the next series of experiments considered effects
of temperature-salinity stress within the range encountered in nature,
with and without exposure to a low level concentration of mercury. The
three parameters were survival, phototactic response, and 02 consumption
of the larvae.
After hatching, groups of control and Hg-treated larvae (1.8 ppb Hg)
were reared in each of the following temperature-salinity conditions:
1. 30°C, 30 °/oo
2. 30°C, 20 °/oo
3. 20°C, 30 °/»o
4. 20°C, 20 °/oo
Viability of larvae under various test regimes is indicated by 96-hr
mortality data. A total of 240 larvae were used with 30 in each of the
8 regimes, with mortality checked daily. Percent mortality data were
analyzed by means of a factorial design with three factors: temperature
(T), salinity (S), and mercury (Hg). Specifically, the experimental
design was a 2J factorial with 3 replications of 100 larvae each, making
a total of 24 observations. The two levels of each factor were: tempera-
ture, 20°C and 30°C; salinity, 20 °/00 and 30°/00; and mercury, 0 ppb
and 1.8 ppb. Thus, the 24 observations may reasonably be considered as
continuous responses of a function of the three factors and interactions.
Since the observations are treated as percentage measurements generated
by data from binomial populations, the transformation y = arcsin *^c,
where x is observed percent mortality, is appropriate to stabilize vari-
ances (Mendenhall, 1968).
The 96-hr mortality studies are summarized in Figure 37. There was no
difference between the controls and mercury-exposed zoeae in the 96-hr
survival under conditions of high temperature (30°C) and optimum salinity
(30 °/oo)- However, control larvae maintained at high temperature and
in low salinity (30°C, 20 °/oo) showed a 27% increase in mortality over
larvae not exposed to mercury. At low temperature there was a marked
increase in mortality at both optimal and low salinity with the addi-
tion of mercury. The analysis of variance for these data indicated the
following factors had significant effects at the 5% level:
T, S, Hg, T x Hg, S x Hg
The interaction of T x S was not significant.
Metabolic rates were determined for a total of 90 first-stage zoeae which
had been maintained for 24 hrs under the various suboptimal temperature-
salinity regimes, with and without the addition of 1.8 ppb Hg. Meta-
bolic measurements were made on 55 third-stage larvae reared in 20°C
regimes with and without the addition of mercury. It was not possible
to measure third-stage responses in the 30°C regimes because of the
high mortality at elevated temperatures.
43
-------
9
-------
Firs', stage zoeae
8.0-
7.0-
£ 6.0-
1
* 5.0-
•o
{? 4.0-
.c
£3.0-
* 2.0-
1 .0-
0
•
_ Reared in
• 1 .8 ppb Hg
D Control
1
1
1
25°C-30%oS 30°C-20°/ooS 20°C-20%05
300C-30%oS 200C-30°/ooS
Environmental regime
Figure 38. Metabolic rates of first-stage U. pugilator zoeae reared
under optimal and suboptimal temperature regimes with and
without the addition of Hg. Metabolic rates of larvae
reared under optimal conditions are based on data from
Vernberg et al., 1973.
Third stage zoeaa
7.0-1
Reared in
"1.8 ppb Hg
a Control
.e»
6.0-
5.0-
4.0-
3.0-
1.0-
n
A
T
20°C-207a<5S
20°C-30°/eoS
Environmental regime
Figure 39. Metabolic rates of third-stage U. pugilator zoeae reared
under optimal and suboptimal temperature regimes with and
without the addition of Hg. Metabolic rates of larvae
reared under optimal conditions are based on data from
Vernberg e_t al., 1973.
-------
The phototactlc responses of zoeae were determined in a horizontal
light gradient, using apparatus modified from Ryland (1960). Plexiglas®
U-shaped tubes 4 cm wide x 40 cm long x 2.5 cm deep were filled with
150 cc test solution. After exposure of the larvae to the specified
conditions, groups of 50 or less (stages I, III, or V) or a single larva
(megalopa or crab stages) were transferred to a tube. The tubes were
aligned on a base-plate divided into 10 equal segments, such that
parallel rays from a narrow-beam theater lamp provided a horizontal light
gradient. Intensity ranged from 1600 f.c. at the anterior end (segment
1) to 1300 f.c. at the posterior end. A black housing helped minimize
light scattering. Temperature of the test chamber was maintained at
25°C.
In preliminary tests, the position of the larvae in the light gradient
was noted at 10, 20, 60 minutes, and 2, 4, 6, 8, 10, 12 hours after the
start of the test. Since readings did not change appreciably after 10
minutes this testing time was chosen for all later tests. The tubes
were routinely reversed 180° after the test readings, and a second read-
ing made 10 minutes later. Since reversed readings did not differ
markedly from the first reading, only the initial readings were used in
analyzing the responses of the larvae. Kite diagrams (Bayne, 1964) were
constructed from pooled data for all animals in a specific test condi-
tion. Phototactic responses were first determined for 473 larval and
early crab stages at 25°C, 30 °/0o in untreated seawater. Responses
were next measured for a total of 3,586 first-stage zoea which had been
maintained 24 hours under the various temperature-salinity regimes with
and without the addition of mercury. Tests were made for a total of
200 third-stage larvae reared in 20° regimes, under control or Hg-
treated conditions. As noted above, it was not possible to measure
third-stage responses in 30°C regimes due to high mortality rates at
this temperature. Numbers of individuals used in each category are
indicated in the figures.
The effects of suboptimal temperature-salinity-Hg regimes on phototactic
response were also analyzed statistically. The basic experimental plan
for stage I larvae was a 23 factorial experiment with the same factors
(temperature, salinity, mercury) and levels as described above for the
96-hour mortality study. The same assumptions are reasonable and the
response measured was the percent photopositive. All animals in the
anterior 5 segments of the gradient tube were considered photopositive,
and those in the posterior 5 segments photonegative. The observed
percentages were again transformed by y • arcsin Sx~ , where x was the
observed percentage. For the third-stage larvae, not enough experimental
units were available for a complete analysis. However, the data were
analyzed by a chi-square analysis of the 2x2 table of photopositive
responses for two levels of temperature (20°C and 25°C at 30 °/oo) and
the two levels of Hg (0 and 1.8 ppb). The chi-square value was also
calculated at 20°C for the two levels of salinity (20 %o and 30 °/0o)
and the levels of Hg.
46
-------
All control zoeal stages tested (I, III, and V) were markedly photo-
positive under optimum temperature-salinity conditions. Most megalops
were also photopositive, but early crab stages appeared to be indif-
ferent to light (Fig. 40). The phototactic response of stage I control
larvae reared under the various environmental regimes shows some vari-
ability in the response (Fig. 41). The analysis of variance for these
data indicate the following effects were significant:
At the 5% level: T x Hg, S x Hg
At the 10% level: Hg (and T x Hg, S x Hg)
The interaction S x T was not significant.
Control
Zoeae stage HI Megalops Crab stage
Zoeae stage £ Crab stage I
+ o
1 2
01 6
'! 8
" 10
e.oeae
m
n.
stage i
r T
1
i
349 n « 53
1
1
16 n»16
Figure 40. Positive phototactic response of II. pugilator larvae in the
optimum (25°C, 30 °/oe) regime with untreated seawater
(from Vernberg et al., 1973).
100-1
90-
80-
70-
P
$
i so-
o 40-
0.
30-
20-
10-
O-
_ Kear
• 1 O »•
1 = 349 140 1>8f
50 16
493
36
P Cont
536
350
39
97
Figure 41.
25QC-30%oS SO'C^O'/ooS 20°C-20%oS
30°C-30%oS 20°C-30%oS
Environmental regime
Phototactic response of first-stage U. pugilator zoeae reared
under optimal and suboptimal regimes with and without the
addition of Hg (from Vernberg et al. , 1973) .
47
-------
Larvae reared to the third stage under suboptimal conditions showed
marked changes in phototactic response. At 20°C, in a salinity of
either 20 °/0o or 30 °/oo, the photopositive response of controls was
sharply decreased over controls in optimal conditions (Fig. 42). Mercury-
exposed larvae reared under these regimes were much more photopositive
than control ones. Chi-square analysis for the two levels of tempera-
ture and the two levels of Hg (at a salinity of 30 °/oo) indicates an
interaction between T and Hg at the 5% significance level. At a tempera-
ture of 20° C, however, a repetition of the experiment at the two levels
of Hg (0 ppb and 1.8 ppb) and the two levels of salinity (20 °/oo and
30 °/oo) indicated no significant interaction of S with Hg.
Reared in
1.8 ppb Hg
100 -,
90-
80-
70-
S 50-
Photopositive
*•• O)
0 O
1 1
30-
20-
10-
f
i - 52
* • -1.1
riPrt
LJ \f\J
1
31
5
1
7
25°C-30%0S 2C°C-20%oS
20°C-30°,ix>S
Environmental regime
Figure 42.
Phototactic response of third-stage IJ. pugilator zoeae with
and without the addition of Hg (from Vernberg et al., 1973).
The final part of the larval studies concerned the effect of a sub-
lethal dose of cadmium (1 ppb) on zoeal survival, metabolism and swim-
ming rate.
The synergistic effects of temperature, salinity and cadmium on 96-hour
mortality of zoeae was evaluated in a 5-factor temperature x 5-factor
salinity matrix. One hundred zoeae were tested for each condition in
the matrix. Untreated sea water was used for the control series, and
the experimental series was dosed with 1 ppb Cd. The 96-hr percent
mortality Dumber dead „ ,nn\ was calculated for each condition.
total
x 100)
48
-------
The mortality rates of cadmium-exposed zoeae were greater at 15°C, 20 °/<><>;
20°C, 20 °/o» and 30 °/00; and at 30°C, 15 °/o» and'35 °/oo.
A computer program was written for analyzing the mortality data from the
5x5 factor T, S matrix with and without added Cd. The results are
portrayed graphically as a surface response curve series (Fig. 43).
ZOEA - CADMIUM
% MORTALITY
ZOEA - CONTROL
% MORTALITY
0.0 7.0 14.0 21.0 28.0 35.0 42.0
TEMPERATURE (C)
0.0 7.0 14.0 21.0 28.0 3S.O 42.0
TEMPERATURE (C)
Figure 43. Surface response curves for the 5x5 factorial matrix of
survival (S - T, with and without Cd) (from Vernberg et al.,
1974).
The effects of 1 ppb cadmium upon metabolism of zoeae under optimum
temperature-salinity conditions are summarized in Figure 44A. Cadmium
elevates respiration of stage I and III zoeae but greatly depresses the
rate in stage V zoeae.
Swimming rates of control and Cd (1 ppb) reared zoeae under optimum
salinity and temperature conditions were determined for zoeal stages I,
111, and V (Fig. 44B). Cadmium exposure resulted in a decrease of
activity at all stages but the difference was significant only for
stage I larvae (Table 6).
Table 6. EFFECT OF SUBLETHAL CADMIUM ON SWIMMING ACTIVITY OF ZOEAE.
Stage
I
III
V
Mean
23.5
53.1
42.8
Control
S.E. N*
1.42
5.8
3.5
18
20
20
Sig.
1% level
n.s.
n.s.
Cd-lppb
Mean S.E.
18.2
41.9
37.8
1.62
4.13
5.1
N*
16
20
18
% Red.
22.6
21.1
11.6
*N is mean value/animal based on'4 determinations.
49
-------
ffi
0
10
? 4
METABOLISM
--CD EXPOSED
CONTROL
i i
eo r
ACTIVITY
ZOEAL STAGE
Figure 44. Percent change in metabolic rates (A), and swimming activity
(B) of zoeal stages I, III, and V of Uca pugilator after
rearing in 1 ppb Cd (from Vernberg ej^ a_l., 1974).
Swimming rates in control larvae paralleled the response seen among
controls in the mercury study with an increase in average rate in stage
III,and gradual decrease to stage V as the larvae became heavier. Scale
units in Figures 32 and 44B differ since the mercury studies used a
tracking method of actual path and distance traveled, while Cd studies
utilized a counting chamber and measured number of grid lines crossed.
50
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SECTION VI
DISCUSSION
The sea contains trace amounts of many metals, and some are essential for
normal growth in marine organisms. In higher concentrations, however,
certain metals can be quite toxic, especially in combination with less
than optimal environmental conditions. Adult crabs for example, can tol-
erate relatively high concentrations of mercury and cadmium for long per-
iods of time when temperature and salinity are optimal, but under stress-
ful temperature-salinity regimes, survival time is considerably shortened.
Mercury proved to be more toxic to male than to female crabs. In this
study, fiddler crabs of both, sexes were found to withstand mercury in
combination with high temperature and low salinity better than in combi-
nation with low temperature and low salinity. Mortalities at both low
and high temperatures were greater than under optimum temperature-salinity
regimes (Vernberg and Vernberg, 1972a). Jones (1973) observed similar
results in a study on the response of marine and estuarine isopods to Hg.
He suggested that estuarine species, which are subjected to daily fluctu-
ations of salinity and temperature, could be expected to be more adversely
affected by the same concentration of Hg than open ocean animals living
in a relatively stable environment.
In contrast to mercury, cadmium was most toxic at higher temperatures and
low salinities, and there were no observed differences in toxicity between
males and females. Other studies indicate that the influence of tempera-
ture on toxicity of pollutants is frequently unpredictable. Sprague (1970)
for example, stated that "no assumptions should be made about temperature
effects on toxicity." In a recent review on the effects of temperature
upon the toxicity of chemical pollutants to aquatic animals, Cairns et al.
(1975) have pointed out the widely varying responses of different groups
of organisms to thermal-chemical stresses.
With both Cd and Hg, death of adult fiddler crabs probably is related to
the accumulation of metal in the gills and the subsequent breakdown in
osmoregulatory or respiratory functions. There are, however, major dif-
ferences between the rate of uptake and site of accumulation of these two
metals. Total mercury uptake is independent of the thermal-salinity re-
gimes. However, at low temperatures, mercury is not translocated away
from the gills to the hepatopancreas, thus leaving high mercury residues
in the gill. Similarly, mercury was not translocated from the gills to
the hepatopancreas under any temperature-salinity conditions in crabs
that were simultaneously exposed to mercury and cadmium. Cadmium uptake,
on the other hand, is strongly dependent on temperature-salinity conditions.
At each temperature, crabs in low salinity water accumulated more cadmium
than those exposed to high salinity. This effect is probably due to
osmotic stress.
51
-------
An intriguing puzzle in these studies has been the greater sensitivity of
the male to mercury poisoning in comparison to the female both at high
and low temperature. It cannot be explained on the basis of greater
mercury uptake by tissues in male crabs under stressful environmental
conditions, for these rates were essentially the same in tissues of male
and female crabs (Vernberg and O'Hara, 1972). However, one clue may lie
in the difference in metabolic responses of male and female crabs, both
at the whole animal and the tissue level. Under conditions of thermal and
salinity stress, without the addition of mercury, the metabolic rate of
the female crabs tended to be more stable and less depressed than in male
crabs. The addition of mercury to the already stressful conditions
doubtless accentuated these differences.
One indication of why females survive Hg poisoning better than males may
lie in the metabolic response of gill tissues following exposure to cold.
In crabs that were cold-acclimated, there were no differences between
control males and females in the metabolism of isolated tissues regardless
of the temperature-salinity regimes. The same was also true for Hg-exposed
male and female crabs. In warm-acclimated female crabs transferred to a
low temperature-high salinity regime (5°C, 30 °/oo), the metabolic rate
of gill tissue remained nearly constant through day 7. This was true
regardless of whether or not they were exposed to Hg. In contrast, the
rates of both control and Hg-treated gill tissue from males decreased
sharply under this regime, with the tissue from the treated group showing
an even lower rate than controls.
The basic regulatory mechanism which fails when warm-acclimated crabs are
subjected to temperature-salinity extremes is unknown, but osmoregulatory
failure is probably involved. In a study on osmoregulatory mechanisms in
fishes, Renfro et_ al. (1974) found that HgClo depressed ion transport.
The authors suggested that part of this inhibition was due to interference
with Na-K-ATPase activity; it would seem possible that a similar mechanism
is involved in Hg-poisoned U. pugilator. The cause of the differential
response between males and females to the temperature-mercury-salinity
stress is unknown.
Exposure of ]J. pugilator adults to cadmium quickly altered metabolic ac-
climation patterns. The most striking change was the loss of compensatory
metabolic temperature response to the cadmium-exposed crabs. Thus in
warm-acclimated animals the metabolic response of Cd-exposed crabs re-
mained unchanged over the temperature range 15-30°C whereas in control
crabs rates increased sharply. Since the ability of temperate zone
animals to adjust metabolically to temperature change is vital, loss of
this metabolic ability obviously would lessen their chances for survival.
A number of difficulties were encountered in attempting to use locomotor
activity of adult Uca pugilator as an indicator of stress. The design
of the activity transducer was critical. Friction-damped whells appeared
to give a reliable index of activity. Feeding of the crabs for long-term
recording was a problem. Starvation definitely resulted in depressed activ-
ity levels, while intermittent feeding stimulated bursts of activity
52
-------
during the time course of the experiment. A continuous supply of non-
fouling food partially solved these difficulties. The high variability
of activity in fiddler crabs, which was considerable, and the time and
expense involved in semiautomatic recording raised serious problems for
the development of an adult activity bioassay. In most cases it was
possible to detect a precise frequency of rhythmic activity only by means
of a periodogram computer analysis. When only daily totals of locomotor
activity were used as the assay criterion, inter-individual variations
proved excessive. A more feasible method of activity assay would involve
use of large numbers of crabs. After maintenance in either
control or experimental conditions, activity could be measured for a
relatively short period in the actographs and mean values obtained for
two groups.
Larval stages of IJ. pugilator were more sensitive to mercury by two
orders of magnitude than either the adults or eggs. At a concentration
of Hg sublethal to the adults and eggs, larvae only survived about 24
hours. Sensitivity of the larvae to acute mercury exposure increased as
the larvae developed. A few larvae were able to live and grow in dilute
mercury solutions with only slight changes in activity or metabolism.
However, viability tests showed that the normal mortality rate among
developing control zoeae was greatly accelerated in mercury-stressed
larvae. The small proportion of larvae to reach stage V in 1.8 ppb Hg
or 0.018 ppb Hg doubtless represents highly resistant individuals. Such
results suggest that marine crustacean larvae may be considerably more sus-
ceptible to mercury pollution than previously suspected. Differences in
tolerance to mercuric acetate have been found in life cycle stages of the
fish Oryzias latipes where embryos were more sensitive than larvae, and these
in turn were more sensitive than adults; the least sensitive was the egg
stage (Akiyama, 1970).
Despite the fact that there are wide fluctuations in temperature and
salinity in the estuary, numerous studies have shown that most temperate
zone species of crustacean larvae develop over a rather limited tempera-
ture-salinity range (Costlow and Bookhout, 1971; Costlow jet_ al_., 1962,
1966; F.V.Vernberg and W.B.Vernberg, 1976). Our survival data on larvae of U.
pugilator reared under suboptimal regimes demonstrated that developing
larvae are particularly sensitive to warm water and low salinity. Mor-
tality at high temperature (30°) was greatly increased over 25° (optimum)
values, and only a few IJ. pugilator larvae underwent development to the
crab stage under low salinity regimes regardless of the temperatures.
At low temperatures, mercury sharply increased mortality rates. However,
at high temperatures, control mortalities were so great without mercury
that the added stress of mercury reduced survival only slightly.
Physiological responses of larvae also reflect the stress of suboptimal
conditions. The immediate effect of reduced temperature (20°C) with
optimum salinity was to depress the metabolic rate of the zoeae, although
the animals did acclimate with time to the lower temperature. This
acclimation is evidenced by the fact that the metabolic rate of third
stage larvae reared at 20°C was,the same as the rate in those larvae
reared at 25°C. In a combination of low temperature and low salinity,
53
-------
however, the zoeae did not show metabolic acclimation. U_. pugjLlator
larvae do not tolerate low salinity waters as well as other species of
Uca (F.J.Vernberg and W.B.Vernberg, 1976). Since tJ. pugilator is restricted
to sandy substrate habitats where there is rapid mixing of water with the
more saline waters of the incoming tide, they are not normally exposed
to low salinity waters. The lack of observed metabolic acclimation at
low salinity confirms these ecological observations. The marked alter-
ation of respiration rates in larvae exposed to mercury could appreciably
affect the ability of the larvae to compete in the estuarine environment.
At 20°C, 20 °/oo, for example, metabolic rates of both stage I and stage
III zoeae were lower than those of zoeae reared under optimal conditions.
Similar responses to decreased temperature have been noted for other
organisms, and are generally considered to be adaptative responses (Vernberg
and Vernberg, 1972b). Mercury effectively minimized this adaptive re-
sponse.
The locomotor behavior of larval U_. pugilator also reflected heavy metal
toxicity. With the 3 concentrations of mercury employed, swimming
activity was modified in direct proportion to the concentration and
duration of exposure. Modification of locomotor activity could decrease
the ability to avoid predation or to capture food.
As with many intertidal zone animals, the behavior of the larval stages
is modified by their response to light. Many species are photopositive
throughout larval life. In newly-hatched larvae, this response brings
them into the phytoplankton-rich waters where they grow and develop. In
older stages, positive phototactic responses insure movement to the
surface and eventually into intertidal areas suitable for metamorphosis
and later development (Thorson, 1964). The response for stage I Uca
larvae was strongly photopositive regardless of the salinity or temper-
ature regime. Other workers have found that temperature and reduced
salinity can modify the response to light. These modifications are
thought to have adaptive value for the larvae. In the eyed-veliger
stage of Mytilus edulis, for example, the larvae concentrate toward the
light at temperatures between 7° and 15°C, but when temperatures are
raised to 20°C, the larvae are no longer photopositive (Bayne, 1964).
Thorson (1964) has suggested that the lack of photopositive response at
high temperatures would tend to remove the larvae to deeper, cooler water
where they are metabolically better adapted. The phototactic response of
stage I zoeae was not significantly modified by the addition of mercury
at any of the environmental regimes tested. However, in third-stage
larvae reared at 20°, the photopositive response of the control group was
considerably reduced compared to the mercury treated group. Salinity
apparently was not a critical factor since the phototactic response was
essentially the same in larvae reared at either 30 °/oo or 20 °/oo-
Although cadmium did increase mortality of zoeae over that of control
larvae, it was not as toxic as mercury. Under optimum conditions, the
same number of cadmium-exposed zoeae survived to the megalops stage as
controls; it was only under suboptimal conditions that mortalities in-
creased .
54
-------
The metabolic rates of zoeae reared in cadmium were more affected than
were those of zoeae reared in mercury. The metabolic rates of mercury-
reared zoeae were essentially the same as those of control zoeae (Fig. 36),
In cadmium-reared larvae, however, both stage III and stage V zoeae were
markedly different from that of control larvae (Fig. 47). Such results
suggest again that pollutants can modify normal physiological function,
thereby reducing chances for survival in nature, but the mode of action
of a heavy metal contaminant may well vary with the metal involved.
55
-------
SECTION VII
REFERENCES
Akiyama, A. Acute toxicity of two organic mercury compounds to the
teleost, Oryzias latipes, in different stages of development.
Bull. Jap. Soc. Sci. Fish. 36_: 563-570, 1970.
Barnwell, F. H. Daily and tidal patterns of activity in individual
fiddler crabs(Genus Uca) from the Woods Hole region. Biol. Bull.
130:107, 1966.
Bayne, B. L. The responses of the larvae of Mytilus edulis (L.) to light
and to gravity. Oikos 15:162-174, 1964.
Cairns, J., Jr., A. G. Health, and B. C. Parlcer. The effects of tempera-
ture upon the toxicity of chemicals to aquatic organisms.
Hydrobiologia 4^:135-171, 1975.
Costlow, J. D., Jr. and C. G. Bookhout. The larval development of Sesarma
reticulatus Say reared in the laboratory. Crustaceana 4_: 281-294,
1962.
Costlow, J. D., Jr., C. G. Bookhout, and R. J. Monroe. Studies on the
larval development of the crab, Rhithropanopeus harrisii (Gould).
I. The effect of salinity and temperature on larval development.
Physiological Zoology 39:81-100, 1966.
Costlow, J. D., Jr., and C. G. Bookhout. The effect of cyclic tempera-
tures on larval development in the mud-crab Rhithropanopeus harrisii.
In: Fourth European Marine Biology Symposium, D. J. Crisp (ed.)«
Cambridge University Press (1971).
DeCoursey, P. J. and W. B. Vernberg. Effect of mercury on survival,
metabolism, and behaviour of larval Uca pugilator. Oikos 23;
241-247, 1972.
Holmes, C. W., E. A. Slade, and C. J. McLerrare. Migration and distribu-
tion vs. zinc and cadmium in a marine estuarine system. Environ.
Sci. and Tech. £: 255-259, 1974.
Hyman, 0. W. The development of Gelasimus after hatching. J. Morp. 33:
484-525, 1920.
Jones, M. B. Influence of salinity and temperature on the toxicity of
mercury to marine and brackish water isopods (Crustacea). Estur.
Coast. Mar. Sci. .1:425-431, 1973.
56
-------
Klein, D. H. and E. D. Goldberg. Mercury in the marine environment.
Environ. Sci. and Tech. 4_: 765-768, 1970.
Mendenhall, W. Introduction to Linear Models and the Design and Analysis
of Experiments. Wadsworth Publ. Co., Belmont, California (1968).
208 p.
Naylor, E. Tidal and diurnal rhythms of locomotory activity in Carcinus
maenas (L.). J. Exptl. Biol. 35_:602-610, 1958.
O'Hara, J. The influence of temperature and salinity on the toxicity
of cadmium to the fiddler crab, Uca pugilator. Fish. Bull. 71;
149-153, 1972.
O'Hara, J. Cadmium uptake by fiddler crabs exposed to temperature and
salinity stress. J. Fish. Res. Bd. Canada 30^:846-848, 1973.
Otterlind, G. and I. Linnerstedt. Avifauna and pesticides in Sweden.
Var. Fagelvarld 24:363-415, 1964.
Peakall, D. B. and R. J. Lovett. Mercury: Its occurrence and effects
in the ecosystem. BioSci. 22_: 20-25, 1972.
Renfro, J. L., B. Schmidt-Nielsen, D. Miller, D. Benos, and J. Allen.
Methyl mercury and inorganic mercury: uptake, distribution and
effect on osmoregulatory mechanisms in fishes. In: Pollution and
Physiology of Marine Organisms, F. J. Vernberg and W. B. Vernberg
(eds.). Academic Press, New York, 1974, pp 101-122.
Ryland, J. S. Experiments on the influence of light on the behavior
of polyzoan larvae. Exp. Biol. 3^:783-800, 1960.
Simpson, G. G., A. Roe, and R. C. Lewontin. O^iantative Zoology. Harcourt
Brace Co., New York, 1960. 450 pp.
Smith, L. Spectrophotometric assay of cytochrome £ oxidase. In:
Methods of Biochemical Analysis, Vol. 2, David Click (ed.).
Interscience Publishers, London, 1955. pp 427-434.
Sprague, J. B. Measurements of pollutant toxicity to fish. II. Utiliz-
ing and applying bioassay results. Water Res. 4_:3-32, 1970.
Suter,R. B. and K. S. Rawson. Circadian activity rhythm of the deer
mouse, Peromysous; effect of deuterium oxide. Science 160;1011-
1014, 1968.
Thorson, G. Light as an ecological factor in the dispersal and settle-
ment of larvae of marine bottom invertebrates. Ophelia 1^:167-208,
1964.
57
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Vernberg, F. J. and W. B. Vernberg. The critical environmental parameters
on organisms at the extreme of their distribution. In: Physiological
Ecology of Plants and Animals in Extreme Environments, F. H. Whitehead
(ed.), 1976 (In press).
Vernberg, W. B. Multiple factor interaction in animals. In: Physiologi-
cal Adaptation to the Environment, F. J. Vernberg (ed.). Intext
Publishers, New York, 1975. pp 521-537.
Vernberg, W. B., P. J. DeCoursey, and J. O'Hara. Multiple environmental
factor effects on physiology and behavior of the fiddler crab, Uca
pugilator. In: Pollution and Physiology of Marine Organisms,
F. J. Vernberg and W. B. Vernberg (eds.), Academic Press, New York,
1974. pp 381-425.
Vernberg, W. B., P. J. DeCoursey, and W. J. Padgett. Synergistic effects
of environmental variables on larvae of Uca pugilator (Bosc). Mar.
Biol. £2:307-312, 1973.
Vernberg, W. B. and J. O'Hara. Temperature-salinity stress and mercury
uptake in the fiddler crab, Uca pugilator. J. Fish. Res. Bd.
Canada 2£: 1491-1494, 1972.
Vernberg, W. B. and F. J. Vernberg. Studies on the physiological varia-
tions between tropical and temperate zone fiddler crabs of the
genus Uca. IX. Thermal lethal limits of southern hemisphere Uca
crabs. Oikos 1.8:118-123, 1967.
Vernberg, W. B. and F. J. Vernberg. Studies on the physiological varia-
tions between tropical and temperate zone fiddler crabs of the
genus Uca. X. The influence.of temperature on cytochrome £ oxidase
activity. Comp. Biochem. Physiol. 26_:499-508, 1968.
Vernberg, W. B. and F. J. Vernberg. The synergistic effects of tempera-
ture, salinity and mercury on survival and metabolism of the adult
fiddler crab, Uca pugilator. Fish. Bull. 713:415-420, 1972a.
Vernberg, W. B. and F. J. Vernberg. Environmental Physiology of Marine
Organisms. Springer-Verlag, New York, 1972b. 346 pp.
Vernberg, W. B. and F. J. Vernberg. Synergistic effects of temperature,
salinity and mercury on tissue metabolism in the fiddler crab. In:
Physiological Ecology of Plants and Animals in Extreme Environments,
F. H. Whitehead (ed.),-1976. (In press).
Waddell, W. J. A simple ultraviolet spectrophotometric method for the
determination of protein. J. Lab. Clinical Med. 48_: 311-314, 1956.
Waldichuk, M. Some biological concerns in heavy metals pollution. In:
Pollution and Physiology of Marine Organisms, F. J. Vernberg and
W. B. Vernberg (eds.), Academic Press, New York, 1974. pp 1-58.
58
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TECHNICAL REPORT DATA
(I'lrasc read lunlfiH'iionx on tin1 reverse before completing)
1. HI CO Ml NO.
EPA-600/3-77-024
ANO SUll I IT LE
Effect of Sublethal Metal Pollutants on the Fiddler
Crab Uca pugilator
7. AUTHOR(S>
W. B. Vernberg and P. J. DeCoursey
3. RECIPIENT'S ACCESSIOf»NO.
6. REPORT DATE
February 1977 issuing date
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING OHG "VNIZATION NAME AND ADDRESS
Belle W. Baruch Institute for Marine Biology and
Coastal Research
University of South Carolina
Columbia, South Carolina 29208
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
801455
I?. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Narr., Rl
Office of Research and Development
U.S. Environmental Protection Agency
Narragansett, Rhode Island 02882
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/05
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Studies have been carried out on the synergistic effects of sublethal concentrations
of mercury (Hg) and/or cadmium (Cd) in conjunction with temperature and salinity
stress on larval and adult fiddler crabs, Uca pugilator. Six biological parameters
of the adult organism were monitored including survival, tissue uptake, metabolism,
behavior, microscopic anatomy, and enzymatic activity,using metal concentrations of
0.18 ppm Hg and 1.0 ppm Cd. Studies with larval stages (zoeal stages I, III, V and
megalops) considered survival, metabolism and behavior under conditions of 1.8 ppb
Hg and 1.0 ppb Cd. The effect of mercury or cadmium on Uca pugilator depands upon a
number of factors, including stage of the life cycle, sex, thermal history, and
environmental conditions. Data presented here suggest that the mode of action of the
two metals is not the same.
17.
a.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Crustacea
Contaminants
Larvae
Metals
Oxygen consumption
Behavior
Sublethal dosage
b.lOENTIFIERS/OPEN ENDED TERMS
Fiddler crabs
Physiology
Factor interaction
Heavy metals
c. COSATI Field/Group
06C
14B
I'l. UltinilKUriON STATEMENT
Release Unlimited
10. SECURITY CLASS (Tills Report)
Unclassified
21. NO. OF PAGES
69
20. SECURITY CLASS (Thispage)
.Unclassified-
22. PRICE
EPA Form 2220-1 (9-73)
59
ft U. S. GOVERNMENT PRINTING OFFICE: 1977-757-056/5599 Region No. 5-11
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