—
R e p rin te d fr o m S u b le th a l E ffe c ts o f T o x ic C he m icals o n A q u a tic A n im a ls — E d ite d b y
J .H . K o e m a n a n d J .J .T .W .A . S trik
O 1 9 7 5 E lse vie r S c ie n tific P u b lis h in g C o m p a n y — A m s te rd a m /P rin te d in T h e N e th e rla n d s
15
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SUBLETHAL PHYSIOLOGICAL STRESS INDUCED BY CADMIUM AND MERCURY IN THE WINTER S
FLOUNDER, PSEUDOPLEURONECTES AMERICANUS
CO
A. CALABRESE, F. P. THURBERG, M. A. DAWSON and D. R. WENZLOFF
s
a,
a
N a tio n a l M arine F is h e r ie s S e r v i c e , M id d le A t l a n t i c Coastal F is h e r ie s Center,'
M i l f o r d L a b o r a t o r y , M i l f o r d , C o n n e c tic u t U. S. A.
SUMMARY
«g
c'
5
W in te r f lo u n d e r ( Pseu dopleuronectes am eric anus) were exposed t o 5 and
[
10 ppb cadmium o r mercury f o r 60 days t o de term in e changes in oxygen con- m '
sumption r a t e s and hematology, as w e ll as metal u p ta ke .
Flo u nd er exposed
t o cadmium r e s p i r e d a t a lo w e r r a t e than t h e c o n t r o l s , w h i l e tho se exposecfv
t o 10 ppb m ercury r e s p ir e d a t a h ig h e r r a t e .
No s i g n i f i c a n t hematological;
d i f f e r e n c e was found between c o n t r o l s and cadmium-exposed f i s h .
In
m ercury-exposed f i s h , however, t h e r e were d i f f e r e n c e s i n plasma p r o t e i n
o
l e v e l s , plasma o s m o l a l i t y , h e m a t o c r i t , hemoglobin and mean c o r p u s c u la r
hem oglo bin .
No d e t e c t a b l e l e v e l s o f cadmium were found i n b lo o d and g i l l _ *
t i s s u e s , b u t c o n s id e r a b le amounts o f m ercury were accum ulated.
o»
INTRODUCTION
R e l a t i v e l y l i t t l e i s known about t h e e f f e c t s o f lo w doses o f heavy
m e ta ls on t h e normal p h y s i o l o g i c a l f u n c t i o n s o f marine f i s h o v e r extended
p e r io d s .
U n t i l r e c e n t l y , most s t u d ie s conducted on a q u a t i c organisms have
been concerned w i t h d e te rm in in g the c o n c e n t r a t i o n s t h a t cause m o r t a l i t y
b u t r e c e n t s t u d i e s have shown d e l e t e r i o u s p h y s i o l o g i c a l e f f e c t s o f sub­
l e t h a l l e v e l s o f m etals on marine f i s h [ 1 , 2, 3, 4, 5 ] .
The gradual
e l i m i n a t i o n o f a valued m arine specie s by low c o n c e n t r a t i o n s o f p o l l u t a n t s
may be no le s s s e r io u s than t h e i r r a p i d d e ath.
I n a sense, i t i s more
s e r i o u s because i t i s le s s l i k e l y t o be obvio us and t o be t r a c e d t o i t s
source in t i m e t o p e rm it re c o v e ry o f the enviro nm en t.
The p r e s e n t s t u d y was undertaken t o d e term in e any p h y s i o l o g i c a l damage
caused by low l e v e l s o f i n o r g a n i c cadmium and mercury on th e co m m e rc ia lly
v a l u a b le w i n t e r f l o u n d e r ( Pseudopleuronectes am e ric an us ) a f t e r 60-day
exposures t o the se m e ta ls .
The parameters examined were oxygen consump­
t i o n r a t e , some aspects o f hematology and chemical up take i n t o the b loo d
and g i l l s .
METHODS AND MATERIALS
Exposure
W in te r f l o u n d e r were c o l l e c t e d by o t t e r t r a w l i n Long I s la n d Sound
near M i l f o r d , C o n n e c t ic u t , and h e ld in t h e l a b o r a t o r y i n f l o w i n g , sandf i l t e r e d s eawate r f o r one t o two weeks p r i o r t o m ercury o r cadmium expo­
s u r e . The f i s h were f e d chopped clams (S p i s u l a s o l i d i s s i m a ) d u r in g
h o l d i n g and th r o u g h o u t t h e exposure p e r io d .
T e s t a n im a ls were exposed in
2 8 5 - l i t e r f i b e r g l a s s tanks f i l l e d t o 228 l i t e r s w i t h s a n d - f i l t e r e d sea­
w a te r (24 -26 pp t s a l i n i t y ) by a p r o p o r t i o n a l - d i l u t i o n apparatus [ 6 ] .
T his
d i l u t e r c o n t r o l l e d the i n t e r m i t t e n t d e l i v e r y o f t o x i c a n t - c o n t a i n i n g w a te r
and c o n t r o l w a t e r a t a f l o w r a t e o f 1 . 5 l i t e r s t o each ta n k e ve ry 2.5
m in utes t h r o u g h o u t the t e s t p e r io d . T h is f lo w p r o v id e d a p p r o x im a t e l y 4
com ple te d a i l y exchanges o f w a te r i n each t a n k .
Cadmium, as cadmium
ÍS
16
C h lo r i d e ( CdC 1 2 - 2>á H2 O), and m e rcu ry, as m e r c u ric c h l o r i d e ( HgCl 2 ) » were
added a t c o n c e n tr a ti o n s o f 5 and 10 ppb.
Background c o n c e n t r a t i o n s o f
the se two m etals in the in com ing seawater were le s s than 1 ppb.
Each ex­
p e rim e n t c o n s is te d o f 18 f i s h pe r c o n c e n t r a t i o n p e r metal (6 f lo u n d e r in
each t a n k ) , ave rag ing 219 g in w e ig h t (range 98 t o 465 g) and 282 mm in
t o t a l le n g th (range 219-390 mm). Tests were conducted in d u p l i c a t e , f o r
a t o t a l o f 108 f lo u n d e r exposed t o each o f t h e two m e t a ls . Water tem pera­
t u r e ranged from 3 t o 6 °C d u r in g cadmium exposure and from 7 t o 11 °C
d u rin g mercury exposu re. A f t e r 60 da ys' exposure the f i s h were removed
and examined f o r s ig n s o f s u b le t h a l s t r e s s .
R e s p ir a t io n
A s i n g l e g i l i was d i s s e c t e d from each f i s h , t h e bony arch removed, and
th e g i l i placed in a 1 5 -m l, W arbu rg-type f l a s k .
Each f l a s k c o n t a in e d 5 ml
o f m e t a l - t r e a t e d w a te r from the t a n k in which the f i s h had been exposed.
Oxygen consumption was m o n ito re d o v e r a 4 - h o u r p e r io d a t 20 °C u sing a
G ils o n D i f f e r e n t i a l R e s piro m eter.
Oxygen consumption r a t e s were c a l c u ­
l a t e d as m i c r o l i t e r s o f oxygen consumed per hour pe r gram dry w e ig h t o f
g i l i t i s s u e ( y l / h r / g ) c o r r e c t e d t o m i c r o l i t e r s o f d r y gas a t s tan dard
te m p e ra tu r e and p re s s u re .
Hematoloqy
Blood was c o l l e c t e d by c a r d ia c p u n ctu re u s ing a 5-ml p l a s t i c s y r in g e
and a 20-gauge need le .
Red blood c o r p u s c le (RBC) c ounts were made i n a
hemocytometer using EDTA t r e a t e d b lo o d . Blood samples were d i l u t e d 1:200
w i t h Hendricks s o l u t i o n u sing b loo d d i l u t i n g p i p e t s .
Hemoglobin concen­
t r a t i o n s were determined on h e p a r in i z e d blood u s ing the cyanmethemoglobin
method w i t h Hycel c h e m ic a ls . Absorbance was read on a Bausch and Lomb
S p e c t r o n i c 20 a t 540 nm. H e m a to c r it was measured u sing m ic r o h e m a t o c r it
tubes and h e p a r in iz e d b lo o d .
Plasma p r o t e i n was determ in ed i n a Bausch
and Lomb 3L R e fra c to m e te r u sing th e plasma o b t a in e d i n the h e m a t o c r i t
d e t e r m in a t io n .
Plasma f o r o s m o l a l i t y rea dings was o b t a in e d from h e p a r in ­
iz e d bloo d samples pooled fro m 2 t o 3 f i s h .
These samples were c e n t r i ­
fuged a t 1700 X g f o r 20 m in utes and read on an Advanced 3L Osmometer.
The e f f e c t o f th e h e p a rin on the o s m o l a l i t y o f the bloo d was n e g l i g i b l e .
The mean c o r p u s c u la r volume (MOV) was c a l c u l a t e d by d i v i d i n g the hemato­
c r i t X 10 by the number o f red bloo d c e l l s i n m i l l i o n s pe r miri3. The mean
c o r p u s c u la r hemoglobin (MCH) i n picograms per c e l l was c a l c u l a t e d by
d i v i d i n g t h e hemoglobin in grams % by t h e RBC c o u n t. The mean c o r p u s c u la r
hemoglobin c o n c e n t r a t i o n (MCHC), o r th e hemoglobin i n grams per 100 ml o f
packed red c e l l s , was c a l c u l a t e d by d i v i d i n g hemoglobin in grams % x 100
by the h e m a t o c r i t.
Meta! Uptake
Blood and g i l i samples were analyzed f o r metal c o n t e n t .
Blood was
drawn by h e a r t pu ncture and analyzed as whole b lo o d .
G i l l s were d is s e c te d
from each f i s h , the bony arch d i s c a r d e d , and t h e g i l l s r in s e d i n seawater
and f r o z e n p r i o r t o a n a l y s i s . A l l samples were wet ashed w i t h 70% n i t r i c
a c id on a hot p l a t e , take n up in 25 ml o f 10% n i t r i c a c i d , then read d i ­
r e c t l y on an a to m ic a b s o r p ti o n s p e c tro p h o to m e te r ( P e r k in - E lm e r Model 403).
17
RESULTS
R e s p ir a t io n
G i l i - t i s s u e oxygen consumption i n w i n t e r f l o u n d e r was s i g n i f i c a n t l y r e ­
duced (P < .0 5 ) a f t e r 60 days' exposure t o cadmium a t 5 and 10 ppb.
Flo u n­
de r exposed t o 10 ppb m e rcu ry , however, r e s p i r e d a t a s i g n i f i c a n t l y h i g h e r
r a t e (P < .0 5 ) than the c o n t r o l s , w h ile f i s h exposed t o 5 ppb r e s p i r e d a t
the same r a t e as c o n t r o l f i s h ( F i g . 1).
700
O)
^ -6 5 0
3C
o
o. 6 0 0
E
3
OJ
0
M ercu ry
550
c
0)
o>
1
500
0
M eta l C o n c e n tra tio n (ppb)
F ig . 1. E f f e c t s o f mercury and cadmium on oxygen consumption o f w i n t e r
flo u n d e r.
Each p o i n t re p r e s e n t s mean g i l l - t i s s u e oxygen consumption
v a lu e o f 32 f i s h .
Bars r e p r e s e n t stan dard e r r o r s .
The d i f f e r e n c e i n oxygen consumption r a t e between t h e two c o n t r o l groups
is a t t r i b u t e d t o seasonal r e p r o d u c t iv e c o n d i t i o n .
The cadmium group was
t e s t e d in F e b ru a ry when t h e f i s h were g r a v i d , a c o n d i t i o n r e q u i r i n g a
high m e t a b o lic r a t e and a consequent in c r e a s e i n oxygen consumption.
The
m ercury group was t e s t e d in May a f t e r t h e f i s h had spawned and no lo n g e r
possessed t h e m e t a b o l ic burden o f r e p r o d u c t i o n . Beamish [ 7 ] r e p o r t e d
s i m i l a r l y h ig h r a t e s o f oxygen consumption i n brook t r o u t ( S a l v e l in u s
f o n t i n a l i s ) and o t h e r f i s h e s d u r in g spawning season.
Hematology
There was no s i g n i f i c a n t d i f f e r e n c e between c o n t r o l s and cadmiumexposed f i s h f o r any h e m a to lo g ic a l t e s t , b u t s i g n i f i c a n t d i f f e r e n c e s were
noted in m ercury-exposed f i s h (T able 1 ) .
Plasma p r o t e i n rose from 5 .4 t o
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19
6.3% in f i s h
The n o r m a lly
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from c o n t r o l
Umminger and
exposed t o 5 ppb mercury w i t h a decrease i n plasma o s m o l a l i t y .
hyposmotic bloo d became even more so.
Exposure t o 10 ppb
r e s u l t e d i n a number o f changes: Plasma p r o t e i n rose s i g n i f i ­
h e m a t o c r i t , hemoglobin and MCH decreased.
H e m atological data
f i s h were s i m i l a r t o those observed in w i n t e r f lo u n d e r by
Mahoney [ 8 ] and B rid ge s e t a l . [ 9 ] .
Meta! Uptake
S i g n i f i c a n t amounts o f mercury b u t no d e t e c t a b l e amounts ( < 0 . 2 - 0 . 3 ppm)
o f cadmium were found i n bloo d and g i l i t is s u e s o f f l o u n d e r exposed t o
the se m e ta ls f o r 60 days. Mean l e v e l s o f 20.6 and 4 2 .8 ppm m e rcu ry , wet
w e ig h t b a s i s , were found i n g i l l s o f f i s h exposed t o 5 and 10 ppb m ercu ry,
re s p e c tiv e ly .
A lth o u g h b loo d accumulated c o n s id e r a b ly le s s mercury than
g i l l s , t h e l e v e l s o f 2 . 9 and 3 .8 ppm found i n t h e exposed f i s h were s i g ­
n i f i c a n t l y h i g h e r than t h e c o n t r o l s (T able 2 ).
Table 2
L e vels o f cadmium and m ercury in g i l l s and bloo d o f w i n t e r f lo u n d e r ex­
posed t o th e s e m etals f o r 60 days.
Values given are t h e mean o f 4 samples.
Each sample was a pool from 3-6 f i . s t i .
MEAN CONCENTRATION (ppm, wet wt ■)
Exposure Levels
Cadmi um
Mercury
G ili
BÍ ood
G ili
0
<0.3
<0.3
5
<0.3
<0.2
2 0 . 6 ( S . E . , 0 .7 2 )
2.9 (0 .55)
10
<0.3
< 0.2
42 .8(2.53 )
3.8 (0 .7 7 )
< 0.14
BÍ ood
< 0.04
DISCUSSION
Oxygen consumption i s a v a l u a b le i n d i c a t i o n o f s u b le t h a l s t r e s s .
Any
s i g n i f i c a n t v a r i a t i o n from t h e normal o r c o n t r o l v a lu e m ig h t r e f l e c t an
a l t e r a t i o n i n the m e t a b o lic demand o f the f i s h o r damage t o the r e s p i r a ­
t o r y system.
I n t h i s s t u d y , cadmium depressed oxygen consumption o f
w i n t e r f l o u n d e r a t a c o n c e n t r a t i o n as low as 5 ppb, a l e v e l found in c e r ­
t a i n p o l l u t e d e s t u a r i n e w a te rs [ 1 0 ] .
T hurberg and Dawson [ 1 1 ] noted a
s i m i l a r d e p re s s io n i n t h e cunner ( T a u to g o la b ru s ad spe rsus) , an e s t u a r in e
f i s h , exposed t o s u b le t h a l l e v e l s o f cadmium; Newman and MacLean [12].
noted g i l i t i s s u e a b n o r m a l i t i e s in these same f i s h .
Ledgerv/ood and Brown
[ 1 3 ] r e p o r t e d cadmium-induced aneurysms i n t h e g i l i la m e ll a e o f s t i c k l e ­
backs (G aste ro ste u s a c u l e a t u s ) .
The f a c t t h a t no g i l i p a th o lo g y was ob­
served [ 1 4 ] and no d e t e c t a b l e amounts o f cadmium were found i n g i l i t i s ­
sues o f f lo u n d e r exposed t o t h i s metal suggests cadmium-induced d i s r u p t i o n
elsewhere in t h e f i s h .
Mercury-exposed f lo u n d e r e x h i b i t e d e le v a te d g i l i - t i s s u e r e s p i r a t i o n
r a t e s , a p o s s ib le i n d i c a t i o n o f in cre a s e d m e t a b o lic demand due t o mer­
c u r y - in d u c e d p h y s i o l o g i c a l o r b ioc h em ic a l a l t e r a t i o n s w i t h i n the f i s h .
20
S t r i p e d bass (Morone s a x a t i l u s ) exposed t o t h e same l e v e l s o f m ercury ex­
h i b i t e d s l i g h t l y depressed oxygen consumption a f t e r a 60-day exposure, but
e le v a te d r a t e s a f t e r a 120-day exposure [ u n p u b lis h e d d a t a ] .
The high
m e t a b o lic r a t e o f f l o u n d e r exposed t o m ercury may have c o n t r i b u t e d t o the
h ig h l e v e l s o f mercury found in t h e g i l i t i s s u e s .
MacLeod and Pessah [ 1 5 ]
r e p o r t e d high m ercury r e s id u e s i n t h e g i l l s o f ra in b o w t r o u t ( Salmo
g a i r d n e r i ) h e ld i n m ercu ry-conta m in a te d w a te r a t h ig h te m p e ra tu re s and
a t t r i b u t e d t h i s t o t h e in crea sed r a t e a t which m e rcu ry-c o n ta m in a te d water
was pumped over the g i l l s .
H e m atological t e s t s have been an im p o r t a n t d i a g n o s t i c t o o l i n m ed ic in e
f o r many y e a r s , and re c e n t s p e c u l a t io n has in d i c a t e d t h a t t h e y may be
e q u a l l y v a lu a b le as i n d i c a t o r s o f d is e a s e o r s t r e s s i n f i s h [ 1 6 ] .
Hema­
t o l o g i c a l changes i n f i s h have been r e l a t e d t o te m p e ra tu r e and season [ 9 ] ,
d i e t [ 1 7 ] , p e s t i c i d e s t r e s s [ 1 8 ] and metal s t r e s s [ 1 9 ] .
In t h i s s t u d y ,
cadmium-exposed f lo u n d e r showed no h e m a to lo g ic a l changes and no d e t e c t a b le
up take o f cadmium. Mercury-exposed f i s h , however, showed high t i s s u e
l e v e l s o f mercury and several h e m a to lo g ic a l a l t e r a t i o n s .
The r e s u l t s o f t h i s s tud y are v a l i d o n l y under t h e c o n d i t i o n s o f the
e x p e r im e n t. These data would alm ost c e r t a i n l y change i f such f a c t o r s as
t e m p e r a t u r e , season, r e p r o d u c t iv e s t a t e o r s a l i n i t y were changed.
In te r­
a c t i o n o f seve ral p o l l u t a n t s a n d / o r n a t u r a l f a c t o r s can a ls o a l t e r the
c h a r a c t e r o r degree o f a p o l l u t a n t e f f e c t [ 2 0 , 2 1 ] ,
Such f i n d i n g s empha­
s iz e t h e need f o r a b ro ad er rese arch e f f o r t t o o b t a i n a comprehensive
u n d e rs ta n d in g o f the r o l e o f heavy metal p o l l u t a n t s i n t h e m arine en­
v iro n m e n t.
N o te : The use o f t r a d e names is to f a c i l i t a t e d e s c r i p t i o n and does not
im p ly endorsement by the N a tio n a l M arine F i s h e r i e s S e r v ic e .
ACKNOWLEDGEMENT
The au th o rs tha nk Miss R i ta S. R i c c i o f o r he r c r i t i c a l
t y p i n g o f t h i s m a n u s c rip t.
r e a d in g and
REFERENCES
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21
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