THERMAL EFFECTS ON THE ACCUMULATION

OF ARSENIC IN GREEN SUNFISH,

LEPOMIS CYANELLUS
ELSIE M. B. SORENSEN
Zoology Department
The University of Texas at Austin
Austin, Texas 78712

The pattern of arsenic concentration in several tissues of Lepomis cyanellus was

measured (by neutron activation analysis) as a function of exposure time at 10~ 20~
and 30~ and 0, 30, 60 ppm of arsenic as sodium arsenate. Individual variability of
arsenic uptake did not override trends of greater uptake with increasing exposure time,
temperature, and arsenic concentration. The mean temperature coefficient of 4.5 for
arsenic uptake in livers was higher than O'Hara's (1968) metabolic figures of 1.6 to
3.0 for Lepomis species. The biological half-life of arsenic in liver and gut of live
specimens exposed to 30 and 60 ppm of arsenic at 10~ was about one week.
Percentage survival decreased and mean arsenic uptake increased slightly as temper-
ature and arsenic concentration increased.

Anticipated increases in the numbers of both nuclear- and coal-fired generating

plants will increase thermal and chemical loading of available surface waters,
possibly inducing or increasing concomitant heat and toxicant stress on aquatic
organisms in such areas. Heat release into surface waters has been advocated as less
detrimental to the indigenous fauna than the consumptive use of water by the
exclusive use of evaporative cooling (Hubbs 1973). Chemical releases into heated
surface waters v a r y - - f r o m emissions escaping from coal-fueled plants (Natusch et
al. 1974), nuclear generating plants (Foster 1972), and other industries.

This study was undertaken to measure the extent to which temperature, heavy
metal concentration, and exposure time influence the direct accumulation of a metal
in specific tissues. A fish species (i.e., Lepomis cyanellus) was selected for ex-
perimentation to represent piscean forms which are frequently the most obvious and
economically the most important aquatic organisms exposed to heat and toxicant
stress. Arsenic, in view of its known bioaccumulation capacity, might be conside~d
representative of a group of metals which concentrate in fish.

Materials and methods

Green sunfish were seined from ponds at the San Marcos Fish Hatchery (Hayes
County, Texas) and transported to the Environmental Research Center at the Indust-
rial Generating Company Big Brown Steam Generating Plant in Fairfield (Freestone
County, Texas). Specimens were treated in formalin and in nitrofurazone (Sorensen
1974) for removal of external parasites; length and weight measurements were taken

Archives of Environmental Contamination

and Toxicology Vol. 4, 8-17 (1976)
'e~1976 by Springer-Verlag New York Inc.
 Thermal Effects on Arsenic Accumulation in Sunfish

after anesthetization of specimens in benzocaine (McErlean 1967, McErlean and

Kennedy 1968). Specimens had a mean weight and standard deviation of 14.2 ___ 3.4
g and a mean length and standard deviation of 9.7 ___ 1.0 cm.

After a 16-day period for the removal of damaged fish, nine groups o f about 20
fish each were placed at random in separate aquaria filled with synthetic lake water
solutions (Sorensen 1974). These synthetic solutions had a bicarbonate concentra-
tion of 169 • I1 ppm, a calcium hardness concentration of 92 ~ 3 ppm, a
magnesium hardness of 48 • 4 ppm, and a sulfate concentration of 30.1 ___ 2.1 ppm
before the addition of arsenic. During a four-day interval two sets of three groups of
fish were each slowly adjusted to 30 ~ and 10~ while the third set of three groups
was maintained at 20~ Specimens were acclimated for 16 days, during which fish
were fed ad libitum on trout chow pellets and kept under constant fluorescent
lighting.

Following acclimation, the nine groups of fish were placed in aerated, fresh
solutions of 0, 30, and 60 ppm of arsenic and the temperature to which the group
had been acclimated. Every third day thereafter, specimens were gently moved to
fresh test solutions. Daily dissolved oxygen concentrations were recorded; the
(percent) saturation of oxygen (in all treatments) ranged from 97 to 104%. Mean
daily conductivity measurements ranged from 546 to 674/zmhos/cm; mean daily pH
values ranged from 8.37 to 8.46. Specimens were fed at 2% biomass every third day
to maintain specimen weight. Arsenic sorbed to pellets was undetectable, thus
indirect uptake from food was assumed to be insignificant.

At weekly intervals for the first five weeks (called the "uptake interval"), two
live fish were collected at random from each of the nine treatments and anesthetized
in benzocaine for evisceration of the entire liver, the right muscle fillet, and guts
(without contents). Tissues were placed individually into clean 2/5-dr polyethylene
vials and dried to stabilization at 64~ and then frozen.

After the five-week uptake interval, fish remained only in test solutions contain-
ing no arsenic at all temperatures and in those with 30 and 60 ppm of arsenic at
10~ Mortalities in the 0-ppm treatments were 5% or less. Mortalities due to
arsenic poisoning were much greater than that anticipated from preliminary experi-
ments; thus, the planned three-week "retention interval" (during which fish were
moved from 30 and 60 ppm of arsenic to no arsenic after the initial five weeks
exposure to arsenic) was limited to the 10~ treatments. Tissues from fish used in
the retention portion of the experiment, as well as tissues from dead fish (i.e., a fish
which had succumbed to arsenic poisoning) were treated in the same manner as live
fish tissues. Incidentally, passive sorpti.on of arsenic into liver following postmor-
tem arsenic exposure did not occur after 12.5 hr postmortem exposure of untreated
or control fish to 30 and 60 ppm of arsenic.

After being dried to stabilization, tissues were sealed in the 2/5-dr polyethylene
vials and double encapsulated in 2-dr vials. Arsenic standards (5.75 /~g, 11.5 /zg,
 10 E.M.B. Sorensen

23.0 /xg, and 34.5 /zg), blanks (i.e., p o l y e t h y l e n e vials which had been cleaned,
dried, sealed, and d o u b l e - e n c a p s u l a t e d in the same manner as the standards and
tissues), and tissues were irradiated at 1500 K W - h r in the rotary s p e c i m e n rack o f
The University o f Texas T R I G A Mark I N u c l e a r Reactor at a flux o f 2 x 1012
thermal neutrons/cmZ/sec. F o l l o w i n g a 40-hr r a d i o a c t i v e decay period to reduce the
activity o f sodium-24, i n d i v i d u a l samples were counted 800 sec each on a lithium-
drifted germanium detector connected to a Nuclear Data multichannel a n a l y z e r
system. The 0.560 M e V arsenic-76 p e a k was used for each o f the peak area
calculations made using the Atkinson et al. (1970) total peak area m o d i f i c a t i o n of
C o v e l l ' s method (Covell 1959). Arsenic concentrations in tissue s a m p l e s were
calculated on The University o f Texas CDC 6600 computer b y c o m p a r i s o n with
k n o w n standards (Sorensen 1974); these tissue concentration data were e x a m i n e d
using c o m p u t e r - g e n e r a t e d graphs and linear regression analyses o f tissue concentra-
tion versus time and other specimen parameters.

1.821
l 0 ppm

1.457 I
I
/ 30~

,5.. /
1.093 !
! ~ 20~ - 30 ppm
:::L
/ / \./
/
/
~ .728 - , /
/ /
/
/ 20~ - 60 ppm
.364 -
/ /'~'.-30oc ~ / ,~,,10~ - 60 ppm
y 30 ppm ,t
/ _ ~ ~ , I " " " , ~ ~, j . lOOC. 30 ppm

.000
0 I 2 3 4 5 6 7 8
Time (wk)

Fig. 1. Liver uptake of arsenic for live L. cyanellus for the initial 5 weeks and retention for
the remaining 3 weeks for 10~ 20 ~, and 30~ and 0, 30, and 60 ppm arsenic as sodium
arsenate. Two fish were taken every week except for one or two instances.
 Thermal Effects on Arsenic Accumulation in Sunfish 11

Results and discussion

Data from all nine treatments for liver (Figure 1), gut (Figure 2), and muscle
(Figure 3) showed increasing arsenic concentration with the three measured
parameters (i.e., temperature, exposure time, and arsenic concentration) despite
variability among individual fish. Generally reduced accumulation per week occur-
red in the sequence: liver > gut > muscle. Correlation of liver uptake with time was
highly significant (p < 0.01) for fish in 10~ ppm and 20~ ppm treatments
and was significant (p < 0.05) for 10~ ppm data. No significant correlation
between exposure time and uptake was observed with the other treatments, probably
due to small sample size. Arsenic uptake, however, generally increased with each of
the three measured parameters. No correlation was observed between arsenic uptake
and exposure time for specimens exposed to no arsenic.

Other metals are accumulated in fish to an'increasing extent as temperature (King

1964, Nelson 1967), metal concentration (Mount 1964, Mount and Stephan 1967),

.302
I O ppm

30"C - 60 ppm
.242 "~'~'1 20~ ppm/ /~
I i,~/. / ~ 20=C- 30 ppm

/' f" \/
ii /
II
.181
E
II / 10~ - 30 ppm

o
30 C- k'
,/ / / / / r
.121 3~ t~, // / // - 7 - ' ' " \ '~, ,0~
/ ,,. / , / "'~/

.060
//I ,,. [

/ss'
li~ ~ ~/30oC_Oppm
.

20;C-Oppm tlOOC.Oppm
\
c..._ " ~ ~ - - - _

.000 ' ~ I 9 T 1 I I
0 2 3 4 5 6 7
Time (wk)
Fig. 2. Gut uptake of arsenic for live L. cyanellus for the initial 5 weeks and retention for
the remaining 3 weeks for 10~ 20~ and 30~ and 0, 30, and 60 ppm arsenic. Two fish were
taken every week except for one or two instances.
 12 E. M. B. Sorensen

.08
30 C 20 oC I0~
0 pp
30 ppm 0
60 ppm ~ 0

.06

E
.04
g

.02

.00
1 2 0 2 3 4 5 0 1 2 3 4 5 6

Time (wk)

Fig. 3. Arsenic uptake in muscle tissue of live L. cyanellus during the 5-week "uptake
interval" and one week of the "retention interval" Circles represent one specimen each.

and exposure time (Hannerz 1968, Matida et al. 1972, Mount and Stephan 1967) are
increased. In addition, the biological variability in heavy metal uptake is well
documented (Lunde 1968, Lovett et al. 1972). Hannerz (1968) found as much as a
tenfold variation in mercury content among the same piscean species given an
identical exposure to mercury.

The temperature coefficient (Qlo) for the rate of arsenic accumulation in liver and
gut during the first two weeks was typically ,high. Graphing log velocity of arsenic
accumulation against temperature (Figure 4) allowed direct reading of Q10 values
from the curve (Prosser and Brown 1961). Temperature quotient values for liver
ranged from 1.41 to 11.42 with a somewhat elevated Qlo mean of 4.47, as was the
case for arsenate uptake in marine yeast (Button and Dunker 1971). Individual
variability in the uptake of arsenic, and hence in Q~0 values, was assumed to be an
expression of genomic variability, giving individuals different abilities in handling
this two-fold stress situation.

The biological half-life of arsenic in gut and liver tissues of green sunfish
appeared to be about one week. Variation in muscle tissue uptake data for fish at
10~ during the uptake interval make datar from the retention interval more ambigu-
ous than for gut and liver.

Computer-generated graphs and linear regression analyses were used to examine

arsenic uptake in liver and gut versus several tissue and specimen parameters ( i . e . ,
dry tissue weight, fish total length, fish fresh weight, and specimen condition or
 Thermal Effects on Arsenic Accumulation in Sunfish 13

K-factor). No obvious patterns between fish weight and metal uptake were observed
in this data, as seems to be the case with other heavy metal uptake research (Lovett
et al. 1972, Mount 1964, Keven 1966).

Because arsenic stress and/or arsenic accumulation in L. cyanellus caused mor-

talities, it was possible to measure arsenic uptake in the tissues o f dead specimens.
If a particular tissue accumulates a toxicant to a fixed level before mortality of the
specimen, one might consider the organ a " r e s e r v o i r " of that specific toxicant, as is

I I

-0.2

-0.4 [-",

%%%% ~6

-0.6

E
-0.8 ,,,
~L
6.82
.J
-1.0

$ -1.2
\

-1.4 9 60 ppm
9 30 ppm
- - 1 wk exposure ~
m 2 wks exposure
-1.6 %11.42

-1.9 I I ii
30 20 10
Temperature (~C)

Fig. 4. Temperature quotients (Qxo) for arsenic uptake in the liver of live L. cyanellus. The
plots of uptake rate (in log /zg arsenic/mg dry wt/week)versus temperature (in ~ allowed
calculation of curve slopes (Qlo) for liver after the first and second weeks of in vivo exposure
to 30 and 60 ppm arsenic.
 14 E. M. B. Sorensen

I' 'I t I u
30~
I '
ppm
I ' I u I u

=-0.42x +102
%o LTso = 124
-~, n = 17
I o%
9 %~
%0

I I9 o r i "] , I , I , I i I i
%,1
30~ - 30 ppm
%9 7(=_0.41x +136
%,0
% LT5o = 209
n=17

%e

'~.1 ' I ' I I

-, r,.,,. 20~ - 60 ppm
9,~,o % 9 ~(= - 0.30 X + 1 1 3
LTso = 210
,.:.. n=14

, I , I , I f l'~ I ' I ' J '

20~ - 30 ppm
- - 0.14 x +124
"~*O
LTso = 527
L n=7

100 , I , I I I , I , I , I , I I
10~ 60 ppm
80-
3 -- - 0.12 x +131
60-- LTso = 678
n=6
40-

20-

0 I n I n I I t I i I t I I I a I
0 80 160 240 320 400 480 560 640 720

Time (hr)

Fig. 5. Percentage survival data for 5 of the 9 treatments; percent survivors is illustrated at
given time intervals. Curves drawn through data points are based on linear regression
analyses, and the LTd. (lethal time for 50% survivorship) values are calculated by straight
line graphic interpolation. The number of specimens (n) is recorded.
 Thermal Effects on Arsenic Accumulation in Sunfish 15

the case for zinc in gill/bone ratios (Mount 1964) and for cadmium in gill tissue
(Mount and Stephan 1967). Since Mount and Stephan (1967) found liver to be a
" m o n i t o r " of past cadmium exposure, one might expect liver to '~monitor" past
arsenic exposure, as welt. This was not the case. Graphic representation and linear
regression analyses of arsenic concentration in liver and gut versus time of death for
the five most toxic treatments (Sorensen 1974) indicated that only treatment 20~
ppm for liver was significant even though arsenic uptake increased with the three
measured parameters. Arsenic uptake values for liver and gut in these, as well as
live specimens, overlap; thus, liver and gut cannot be considered "arsenic reser-
voirs" p e r se.

Percentage survival data for the five most toxic treatments (Figure 5) indicated
that elevated temperature and/or arsenic concentration resulted in decreased survi-
val. Increasing temperature from t0 ~ to 20 ~ and to 30~ at a constant arsenic
concentration (i.e., 60 ppm) decreased the let'hal time for 50% mortality (LTso) from
678 to 210 and to 124 hr, respectively. Similarly, exposure to 30 ppm of arsenic
reduced LTso values from 527 hr at 20~ to 209 hr at 30~

Conclusions

These results generally indicate that the release of both heat and toxic metals into
aquatic habitats near electric generating stations can prove detrimental to piscean
forms under certain conditions. The following conclusions were made from the
study:
a. The general trends of increasing arsenic uptake (in liver, gut, and muscle) with
increasing temperature, arsenic concentration, and time of exposure are appa-
rent despite variability between individual fish. Arsenic uptake in tissues of
both live and dead fish decreases in the sequence liver > gut > muscle; the
trends in data on muscle uptake are less conclusive than those of the other
tissues. In addition, arsenic retention in specimens exposed to 10~ appears to
be short in duration--approximately one week.

b. Temperature quotient (Q10) values for arsenic uptake in liver tissue have a
mean of 4.5. Since typical Q~o values for the genus Lepomis range from 1.6 to
3.0, these data exhibit the high Q10 values consistent with those for a marine
yeast (Button and Dunker 1971). These elevated Q~o values suggest that
elevated heat and high metal concentrations act synergistically in heavy metal
uptake.

C. Lethal times for 50% mortality (LTs0), a measure of survivorship, are calcu-
lated by straight line graphic interpolation of linear regressions on existent
survivorship data; all regressions are highly significant. As temperature in-
creases from 10 ~ to 20 ~ to 30~ at 60 ppm of arsenic, percent survival is
reduced such that LTso values decrease from 678 to 210 to 124 hr, respective-
ly. For an arsenic concentration of 30 ppm and temperatures of 20 ~ and 30~
LTs0 values are 527 and 209 hr, respectively.
16 E. M. B. Sorensen

d. Correlation of uptake with various specimen and tissue parameters proves

inconclusive, as has been the case for the majority of available literature
references.

Acknowledgment

The author is grateful to Dr. Clark Hubbs for his continuing support and gui-
dance; his suggestions during all phases of this research were of considerable help.
Utmost thanks are extended to Drs. E. Linn Draper, G. D. Atkinson, Jr., and D. G.
Anderson; to Messrs. Andrzej H. Pradzynski and Roland Henry; and to Ms. Betty
Foster who have made invaluable suggestions, rendered laboratory assistance, and
generaously permitted the use of The University of Texas Nuclear Reactor Laborat-
ory facilities. Many thanks are extended to Mr. R. L. (Dick) White for numerous
suggestions, laboratory assistance, and the transporation and collection of specimens
for this study. Mr. John Tilton and Dr. C. W. Garrard provided useful discussion of
several aspects of this research. Thanks go to Mr. Alan L. Sorensen, Ms. Ruby
Boecker, Ms. Mary Hibbs, Mr. Harmon Henderson, Mr. J. C. Cox, Dr. Joe Truett,
Mr. Ed Walton, Mr. Steve Skelton, Mr. R. W. Ainsley, Dr. J. J. Morgan, Mr. Ken
W. Thompson, and Mr. T. C. Scanlon. This research was supported in part by
Dallas Power and Light Company, a subsidiary of Texas Utilities.

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Manuscript recieved June 25, 1974; accepted November 30, 1974