N ORM A L V ALU ES AND C H R O M IU M -IN D U C E D CHANGES IN BLOOD
PHYSIOLOGY O F RAINBOW T R O U T , SALMO

GAIRDNERII

By
R O B E R T HARRY SC H IF F M A N

A THESIS
S u b m itte d to th e School f o r A d v a n c e d G r a d u a te S tu d ie s of
M ic h ig a n S tate U n iv e r s it y of A g r i c u l t u r e a n d A p p lie d
S c ie n c e in p a r t i a l f u lf illm e n t of th e r e q u i r e m e n t s
f o r th e d e g r e e of

DOCTOR O F PHILOSO PHY

D e p a r t m e n t of P h y s io lo g y a n d P h a r m a c o l o g y

1957

P roQ uest Num ber: 10008615

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ABSTRACT

T h is

s tu d y p r e s e n t s a p h y s io lo g ic a l a p p r o a c h to th e to x ic ity

p r o b l e m s e n c o u n t e r e d in s t r e a m

p o llu tio n c o n t r o l .

A lth o u g h th i s

w o r k d e a l s w ith the e f f e c t s of p o t a s s i u m c h r o m a t e on r a in b o w t r o u t ,
th e i d e a s p r e s e n t e d m ig h t be p r o j e c t e d in to th e g e n e r a l stu d y of
c h r o n ic to x i c it y of w a s te p r o d u c ts to fis h .
T he n o r m a l h a t c h e r y - r a i s e d ra in b o w t r o u t w as e x a m in e d in
o r d e r to e s t a b l i s h th e fo llo w in g c o n t r o l v a l u e s :
31.8 m l./lO O m l.;

(b) r e d b lo o d c e l l c o u n t,

(c) h e m o g lo b in , 6.51 g./lO O m l.;
(e) p l a s m a v o lu m e
(g) kid n e y w e ig h t,

1.11 m i l l i o n c e l l / c m m . ;

(d) r e d blood c e l l le n g th ,

2.13 m l./lO O g.;

14.69

p .;

(f) blood v o lu m e , 3.25 m l./lO O g.

0.77 g./lO O g. of body w eight;

1.02 g./lOO g. of body w eight;

(a) h e m a t o c r i t ,

(i) s p le e n w eigh t,

(h) l i v e r w eig h t,
0.24 g./lO O g. of

body w eig h t; a n d (j) t o t a l body w a t e r , 7 7.4 g./lOO g.

In a d d itio n to

th e a b o v e , th e k id n e y , l i v e r a n d s p le e n w eig h ts; t o t a l body w a te r ;
a n d p l a s m a a n d b lo o d v o lu m e s w e r e found to c o r r e l a t e
w ith body w eig h t.

T h e r e g r e s s i o n e q u a tio n s a r e

s ig n if i c a n tl y

g iven.

In o r d e r to o b ta in th e ab o ve r e s u l t s , m a m m a l i a n m e th o d s h a d
to be a d a p t e d to s m a l l f i s h w hich r a n g e d b e tw e e n 10.0 c m . to 17.0
c m . in le n g th w ith a m e a n w eight of 19.0 g r a m s .

A s a re su lt

of

th is,

an

ex ten siv e

and

d e ta ile d se c tio n title d

' 'M a t e r i a l s a n d

M e t h o d s " i s in c lu d e d .
In o r d e r to fix a w o rk in g l e v e l of p o t a s s i u m c h r o m a t e , a
2 4 - h o u r m e d i a n t o l e r a n c e l i m it of 100 m g. C r . / l .

w as d e t e r m i n e d ,

a n d a l l e x p e r i m e n t s w e r e done a t a p o t a s s i u m c h r o m a t e c o n c e n t r a ­
tio n e q u iv a le n t to 2 0 m g. C r . / l .
T racer

e x p e r i m e n t s i m p l i c a t e d th e s p le e n a n d b ile a s c o n ­

c e n t r a t i n g c h r o m i u m a b o v e th e le v e l of th e n o n s p e c if ic t i s s u e
c a u d a l p e d u n c le .
d o th e lia l s y s t e m

of th e

A s a r e s u l t , it w as t h e o r i z e d th a t th e r e t i c u l o e n ­
of th e s p le e n a n d th e l i v e r w e r e i n s t r u m e n t a l in

th e e l i m i n a t i o n of c h r o m i u m f r o m th e body by way of th e b il e .
T h e r e w a s no e v id e n c e of a c h a n g e in w eig h t of th e k id n e y ,
liv e r, or

s p le e n , o r a c h an g e in t o t a l body w a t e r due to e x p o s in g the

f i s h to 2 0 m g . C r . / l .

f o r 24 h o u r s .

F u r t h e r i n v e s t i g a t i o n s sh o w ed th a t th e h e m a t o c r i t r o s e f r o m
a c o n t r o l v a lu e of 31.8 to 43.8 in c h r o m i u m - e x p o s e d fis h in 2 4 h o u r s .
W ith th e u s e of e r y t h r o c y t e c o u n ts a n d h e m a t o c r i t s , it w as c a l c u l a t e d
t h a t a p p r o x i m a t e l y 5 9 p e r c e n t of th e r i s e in h e m a t o c r i t w a s due to
i n c r e a s e in c e l l v o lu m e , and 41 p e r c e n t w as due to i n c r e a s e i n c e l l
n u m b er.

It w a s

show n th a t the s p l e e n did not c o n t r i b u t e

to t h i s i n c r e a s e in c e l l n u m b e r .

s ig n if i c a n tl y

W hen th e h e m a t o c r i t r i s e s , t h e r e m u s t be an i n c r e a s e d blo o d
v o lu m e a n d / o r d e c r e a s e in p l a s m a v o lu m e .

N e i t h e r of t h e s e

e v e n ts

c o u ld be sh o w n to ta k e p la c e u n d e r th e e x p e r i m e n t a l c o n d itio n s .
Due to th e fa c t th a t p o t a s s i u m c h r o m a t e c o n t a in s a c o n s i d ­
e ra b le

a m o u n t of p o t a s s i u m io n s , th e in f lu e n c e of t h i s io n w as i n ­

v e stig a te d .

It w as a s s u m e d on th e b a s i s of th e e x p e r i m e n t a l d ata

th a t th e p o t a s s i u m io n , in a d d itio n to th e c h r o m i u m , i s a p o te n tia l
in f l u e n c e u p o n th e r e s u l t s

o b ta in e d a t p o t a s s i u m c h r o m a t e c o n c e n t r a ­

t i o n s e q u iv a le n t to 2 0 m g . C r . / l .
A s s u m i n g th e r i s e in h e m a t o c r i t w as an in d e x of c h r o n ic
t o x i c it y , it w a s d e t e r m i n e d t h a t th e h e m a t o c r i t r o s e to i t s g r e a t e s t
d e g r e e a t c o n c e n t r a t i o n s e q u iv a le n t to b e tw e e n 2 m g. to 4 m g. C r . / l .
T h i s f a c t w as of i n t e r e s t b e c a u s e , alth o u g h th e 2 4 - h o u r T i m , a
s t a n d a r d in d e x of t o x i c it y , w a s

100 m g. C r . / l . ;

the ab o v e d a ta i n d i ­

c a t e d p h y s io lo g i c a l c h a n g e s ta k e p la c e a t a f a r l o w e r c o n c e n tr a ti o n .
W ith th e u s e of s p l e n e c t o m i z e d f i s h , it w a s found th a t th e
s p l e e n h a s a te n d e n c y to a c t , in a l i m i t e d c a p a c i t y , a s a b u f f e r
m e c h a n is m a g a in st sudden h em o c o n c e n tra tio n .
by a s ig n if i c a n t r i s e

T h i s w as e v i d e n c e d

in h e m a t o c r i t of s p l e n e c t o m i z e d f is h i n ta p

w a t e r w hen e x p o s e d to t e n m i n u te s of o p e r a t i v e a n d a n e s t h e t i c

stre ss.

AC KNOWLE DG ME NT S

T h e w r i t e r w i s h e s to e x p r e s s h i s g r a t i t u d e to D r . P a u l O.
F r o m m , D e p a r t m e n t of P h y s io lo g y a n d P h a r m a c o l o g y , M ic h ig a n S ta te
U n iv e rsity , for h is g en ero u s

o f f e r in g of l a b o r a t o r y f a c i l i t i e s an d

c o n t in u a l g u id a n c e th r o u g h o u t t h i s

stu d y .

T he w r i t e r w ould a l s o

lik e to e x p r e s s h i s s i n c e r e a p p r e c i a t i o n to th e S ta te of M ic h ig a n
D e p a r t m e n t of C o n s e r v a t i o n f o r i t s c o n s ta n t supply of f is h th r o u g h o u t
a l l th e s e a s o n s of th e y e a r .

v

TABLE

OF CONTENTS

Page
IN TRO D U C TIO N

....................................................................................

. . . .

M A T ER IA LS AND M E T H O D S ..........................................................................
C h e m i c a l M eth o d s
T o tal re sid u e
A lk a l in it y

1
14

.....................................................................................

16

.................................................................................

16

. . .

.................................................................................

16

H a r d n e s s ............................................................................................................

18

C h r o m i u m ...................................................................................................

19

R a d i o a c ti v e Is o to p e M eth o d s
B io l o g ic a l M eth o d s

.............................

.........................

24
25

F i s h b i o a s s a y s ..........................................................................

25

D ra w in g of b lo o d

32

........................................................................................

H e m a t o c r i t s ................................................................................................

33

H e m o g lo b in s

34

.........................................................................................

R e d b lo o d c e l l co u n t

.................................

R e d b lo o d c e l l le n g th

35

.............................................

35

P l a s m a v o lu m e a n d b lo o d v o l u m e .................................

35

S p le n e c to m i e s

40

Body w a t e r

...............................................................................................

.............................................................................................
vi

41

Page
O r g a n w e ig h ts

..............................................................................

41

R E S U L T S AND DISCUSSION O F PHYSIOLOGICAL
V A L U ES O F T H E NORMAL H A T C H E R Y -R A IS E D
RAINBOW T R O U T
....................................................................................................

42

O r g a n W e i g h t s .....................................................................................

42

T o t a l Body W a t e r

49

H e m a to c rits
H e m o g lo b in

......................................................................

.............................

51

............................................

R e d B lo o d C e l l C o u n ts

51

.................................

R e d B lo o d C e l l L e n g th s

. . . .

52

..................................................................

53

P l a s m a V o lu m e an d B lood V o l u m e ....................................................

53

R E S U L T S AND DISCUSSION O F POTASSIUM
C H R O M A T E -IN D U C E D CHANGES IN THE
PH Y SIO LO G IC A L VALUES O F H A T C H E R Y RAISED T R O U T ...........................................................................................................

56

M e d ian T o l e r a n c e L i m i t
T r a c e r E x p erim en ts

..........................................................................

56

.....................................................................................

58

O r g a n W e ig h ts and T o t a l Body W a te r E x p e r i m e n t s
R e s u l t s of B lo od E x p e r i m e n t s
N orm al ch ro m iu m

...........................................................

..........................................................................

S p le n e c to m i z e d c o n t r o l

............................................

S p len e cto m ized c h ro m iu m
H e m a to c rit

. . . .

.............................

.............................................................................................

v ii

60
61
62
62
64
64

Page
H e m o g lo b in

..............................................’ ......................................................

R e d b lo o d c e l l c o u n t .............................................................................
R e d b lo o d c e l l le n g th

64

.

67

.......................................................................

6

P l a s m a v o lu m e an d b lo o d v o l u m e .............................................
D i s c u s s i o n of th e I n c r e a s e i n H e m a t o c r i t

67
.

67

...............................................................................................,

69

D i s c u s s i o n of th e C a u s e s of th e R e d C e l l

Volume Change

D i s c u s s i o n of th e R o le of th e S p le e n in th e I n c r e a s e
in C e ll N u m b er u n d er P o ta s s iu m C h ro m a teS tr e s s . . . .

70

D i s c u s s i o n o f th e P a r t P l a y e d by th e P o t a s s i u m
Io n in P o t a s s i u m C h r o m a t e T o x ic ity « ............. .... ..........................

71

T h e H e m a t o c r i t a s a n Index of T o x ic ity

74

. , . . ,

.........................................................................................................

77

CONC L U S I O N S ........................................................................................................................

80

B I B L I O G R A P H Y ....................................................................................................................

83

S p le e n F u n c ti o n

A P P E N D IX L

R E V IE W O F TO X IC ITY L IT E R A T U R E

. . . .

88

A P P E N D IX I I ...........................................................................................................................
P a r t A.

D a ta f r o m N o r m a l T r o u t

.................................

P a r t B.
D a ta f r o m C h r o m i u m - E x p o s e d and
S p le n e c to m i z e d T r o u t
...............................................................................

v iii

95
96

107

L IS T O F T A B L E S

TABLE

Page

1.

A B r i e f S u m m a r y of th e T o x ic ity L i t e r a t u r e

2.

P h y s i o l o g i c a l R e a c t i o n s of th e
F i s h to th e
S a lt S o lu tio n
.............................................................................................

9

T h e R e l a tio n s h i p of th e T h r e e
T y p e s of
A lk a l in it y to th e T o t a l an d P h e n o lp h th a le in
A lk a l i n i t y
............................................................................................ .

18

R e s u l t s of T e s t s to D e t e r m i n e th e A c c u r a c y
of th e C h r o m i u m M e t h o d ...............................................................

23

M e a n s a n d S t a n d a r d D e v ia tio n s of T e s t s to
D e t e r m i n e th e A c c u r a c y of th e C h r o m i u m
M e th o d ............................................................................................................

23

R e s u l t s of T e s t s to D e t e r m i n e
W h e th e r
F i l t r a t i o n o r A e r a t i o n H a d a n E f f e c t on
th e C h r o m i u m C o n c e n t r a t i o n ....................................................

29

R e s u l t s of T e s t s to D e t e r m i n e
W h e th e r
F i l t r a t i o n o r A e r a t i o n H a d a n E f f e c t on
A lk a l in it y
..............................................................................................

30

A v e r a g e P e r c e n t a g e of T o t a l W eight of S ta b le
T i s s u e s C o n t r i b u t e d by E a c h T i s s u e D i s s e c t e d
f r o m I n d iv id u a l of E ig h t S p e c i e s of F i s h f r o m
W nite O ak L a k e ......................................................................................

48

9.

R e su lts

of T r a c e r E x p e r i m e n t s

..............................................

59

10.

R e su lts

of B lood E x p e r i m e n t s

..................................................

63

11.

R e su lts

of P o ta s s iu m C h lo rid e E x p e rim e n ts

3.

4.

5.

6.

7.

8.

ix

. . . .

. . . .

6

73

TABLE
12.

13.

14.

Page
T h e M e a n H e m a t o c r i t s of F i s h E x p o s e d
to V a r i o u s C o n c e n t r a t i o n s of P o t a s s i u m
C h ro m a te
....................................................................................................

76

R e s u l t s of E x p e r i m e n t s P e r f o r m e d to
I n v e s ti g a te S p le e n F u n c tio n
.................................

78

P h y s i o l o g i c a l V a lu e s of th e N o r m a l
H a t c h e r y - R a i s e d R ain bo w T r o u t ............................................

80

x

L IS T O F F IG U R E S

FIG U R E
1.

Page
E x p e r i m e n t a l a q u a r i a in c o n s ta n t
te m p e ra tu re room
..............................................................................

15

2.

M ic ro -in je c tio n a p p a ra tu s

36

3.

A rtific ia l re s p ira tio n ap p aratu s

4.

N o r m a l k id n e y w e ig h ts

5.

N o r m a l l i v e r w e ig h ts

6.

N o r m a l s p l e e n w e ig h ts

7.

Body w eigh t p lo tte d a g a i n s t body w a t e r ............................

50

8.

E s t i m a t i o n of 2 4 h o u r m e d ia n t o l e r a n c e
l i m i t by s t r a i g h t - l i n e g r a p h i c a l i n t e r p o l a t i o n

. . . .

57

...............................

65

...................................

66

9.

...............................
..............................................

38

.....................................................................

44

* ....................................................................

45

..........................................

C o m p a r i s o n of p l a s m a v o l u m e s

10.

C o m p a r i s o n of t o t a l b lo o d v o lu m e s

11.

I n c r e a s e in h e m a t o c r i t in r e l a t i o n to
p o ta ss iu m c h ro m a te c o n c e n tra tio n
..................

xi

46

75

IN T R O D U C T IO N

T h e p r o b l e m of s t r e a m
one to u c h in g u p o n m a n y f a c e t s

an d la k e p o llu tio n c o n t r o l i s a d i v e r s e
of the b io l o g ic a l and p h y s ic a l s c i e n c e s .

T h e p r e v a i l i n g d i s c i p l i n e i s a i m e d a t the d e t e c ti o n of p o llu tio n a l ­
r e a d y in th e w a t e r s w ith c o n t r o l c o m in g a f t e r l e g a l p r o c e e d i n g s .

As

w a t e r r e s o u r c e s b e c o m e u t i l i z e d to t h e i r f u l l e s t e x te n t by m u n i c i ­
p a l i t i e s a n d i n d u s t r i e s a s w e ll a s f o r t h e i r r e c r e a t i o n a l a n d e s t h e t i c
v a l u e s , th e a t tit u d e i s c h a n g in g r a p i d l y to one of c o n t r o l b e f o r e
p o llu tio n o c c u r s .
q u e stio n s
p a rtic u la r

such a s ,

T h is a s p e c t of c o n t r o l b r i n g s f o r t h m a n y new
''H o w m u c h of a w a s te c a n b e d i s c h a r g e d in to a

s t r e a m b e f o r e it w ill

ad v ersely

a f f e c t th e a q u a tic life ? ''

T h i s i s a q u e s ti o n i n d u s t r y i s a s k in g the r e g u l a t o r y a g e n c y m o r e
and m o re ;

th e a n s w e r to w h ich , if s lig h tly w ro n g , c a n c a u s e e i t h e r

l o s s in v a l u e of a s t r e a m

o r a v e r y r e a l m o n e t a r y l o s s to th e

in d u stry .
C o n te m p la tio n upon t h i s q u e s ti o n w ill r e v e a l t h r e e i n t e r a c t i n g
v a ria b le s.

T h e w a s te , th e s t r e a m , a n d th e f o r m of a q u a tic lif e ,

e a c h of w h ich m u s t be g iv e n

co n sid e ra tio n .

F i r s t th e w a s te :

I g n o r in g th e p u r e ly c h e m i c a l d i l e m m a w hich a r i s e s

from

an a tte m p t

to d e t e r m i n e i t s io n ic a n d m o l e c u l a r c o n s t i t u e n t s a n d f o c u s in g
1

2

a t t e n t i o n on i t s b io l o g ic a l i m p l i c a t i o n s , we fin d th a t v a r i a t i o n in
te rm s

of c o n c e n t r a t i o n i s f u r t h e r c o m p l i c a t e d by th e a n t a g o n is tic

a n d s y n e r g i s t i c a c t io n of v a r i o u s m e t a l l i c

c atio n s.

It h a s long b e e n

know n th a t th e t o x i c it y of s o m e m e t a l c a t io n s to a q u a tic

o rg a n ism s

i s c o u n t e r a c t e d , o r a n t a g o n iz e d , by o t h e r m e t a l c a t io n s in m i x e d
s a l t s o lu ti o n s
R in g e r 's

( H e ilb ru n n ,

1938).

A n e x c e lle n t e x a m p le of t h i s i s

s o lu tio n in w hich th e s o d iu m , c a l c i u m , and p o t a s s i u m io n s

a r e b a l a n c e d in s u c h p r o p o r t i o n s th a t th e s p e c if ic to x ic ity of e a c h i s
n e u tra liz e d .
L o e b a n d W a s te n e y s
stu d ie s
to f is h .

(1911, 1915) r e p o r t e d s o m e of th e e a r l y

of a n t a g o n i s m of th e p r i n c i p l e c a t io n s of s e a w a t e r r e l a t i n g
G arrey

(1916) fo llo w ed i m m e d i a t e l y w ith h i s w o r k on th e

a v e r a g e lo n g e v ity of m in n o w s of th e g en u s N o tr o p is in v a r i o u s m i x ­
tu re s

of s o d iu m , p o t a s s i u m , c a l c i u m , a n d m a g n e s i u m c h l o r i d e s in

d istille d w a te r.

He found th a t m in n o w s in 9,000 m g. N a C l / l . w ould

liv e tw o d a y s , but th e a d d itio n of th e
c a lc iu m ch lo rid e
days.

or m ore,

so d iu m c h l o r i d e to 0.001 M.

p r o lo n g e d th e p e r i o d to eig h t to te n

In t u r n , h e found th e c a l c i u m io n to b e a n t a g o n i s t i c to p o t a s ­

siu m and m a g n esiu m .

A h ig h c o n c e n t r a t i o n of s o d iu m c h l o r i d e

c o u n t e r a c t e d th e to x i c it y of c a l c i u m

ch lo rid e and p o ta ssiu m c h lo rid e ,

a n d m a g n e s i u m c h l o r i d e w as a n t a g o n i s t i c to p o t a s s i u m

ch lo rid e.

3

O th e r w o rk e rs
1924; R a m u l t , 1928a
re la tio n sh ip s

(P o w ers,

1921; K r u g e r ,

1928; G u e y l a r d ,

1923,

1928b; Y oung, 1938) i n v e s t i g a t e d the to x i c it y

of t h e s e io n s upon s t i c k l e b a c k s , m in n o w s , a n d o th e r

s p e c i e s in c lu d in g s a l m o n e g g s a n d found th e s a m e

o v e r-a ll re su lts.

In a d d itio n , E l l i s (1937) sh o w e d th a t s o d iu m n i t r a t e d e c r e a s e d
th e to x i c it y of c u p r i c

s u lf a te

a n d th e c o m b in a tio n of s o d iu m n i t r a t e

a n d c a l c i u m c h l o r i d e w a s e v e n m o r e a n t a g o n is tic .
b e e n done on a n t a g o n is m

Much w o rk h a s

(E llis an d L a d n e r , 1935; J o n e s ,

1938), but

in r e a l i t y it i s not g r e a t l y c o n c e r n e d w ith the p r o b l e m at h a n d a n d
t h u s , it i s c o n s i d e r e d only b r i e f l y h e r e .
S y n e r g i s m b e tw e e n v a r i o u s c a t io n s lik e w is e h a s b e e n show n
to e x i s t .

B an dt (1946) d e m o n s t r a t e d th a t m i x t u r e s of th e s u l f a t e s of

n ic k e l a n d z in c , c o p p e r a n d z in c , an d of c o p p e r a n d c a d m i u m w e r e
up to fiv e t i m e s m o r e to x ic th a n th e y w ould h a v e b e e n if th e to x ic
e f f e c t of th e m e t a l s w e r e

s im p ly a d d itiv e ,

D o u d o ro ff (1952) s u b ­

s t a n t i a t e d the e v id e n c e on t h e s y n e r g i s t i c a c t io n b e tw e e n c o p p e r an d
z in c .
We c a n p r o j e c t th e i d e a s b r o u g h t out in th i s b r i e f d i s c u s s i o n
of th e i n t e r a c t i o n s of th e c o m p o n e n t p a r t s
th e

stre a m

le v e ls.

of th e w a s te a n d s e e th a t

w a t e r i t s e l f i s a v a r i a b l e in any c o n s i d e r a t i o n of to x i c it y

C a l c i u m , f o r e x a m p le , i s an i m p o r t a n t ion in n a t u r a l l y

o c c u rrin g w a te rs; its

p re s e n c e in la rg e and s m a ll d e g re e s depends

4

u p o n th e g e o l o g ic a l f o r m a t i o n of th e w a t e r s h e d .
l a r g e l y to th e h a r d n e s s

It c o n t r i b u t e s

of th e w a t e r , w h ich in t u r n i s

d ire c tly r e ­

l a t e d to th e a l k a l i n i t y , e a c h b e in g i m p o r t a n t to th e p o te n c y of a
w a ste .

In a d d itio n , th e pH, o th e r io n s p e c i e s , s u s p e n d e d s o l i d s ,

d i s s o l v e d g a s e s , a n d , if th e

stre a m

i s e x t e n s i v e ly u t i l i z e d by i n ­

d u s t r y , w a s te p r o d u c t s d i s c h a r g e d u p s t r e a m

c o n t r i b u t e to th e c o m ­

p le x ity of d i s p o s a l p r o b l e m s .
Not th e l e a s t to be c o n s i d e r e d in to x ic ity p r o b l e m s i s the
a m a z i n g l y c o m p le x b io d y n a m ic c y c l e o c c u r r i n g w ith in th e w a t e r
itse lf.

I ts i n t e r r e l a t i o n s h i p s c a u s e p o to m o lo g is ts to c o m p a r e a

stre a m

to a liv in g o r g a n i s m .

W ater,

a

m ost

im p o rta n t

s u s t a i n i n g f a c t o r of l i f e , h o ld s c o n fin e d w ith in i t s n a t u r a l b a s i n s
m a n y f l o r i s t i c a n d f a u n is t ic p hyla.
f a c t o r in s t r e a m

T he s p o r t s m a n , an in f lu e n tia l

c o n t r o l , th in k s in t e r m s

of f is h p r o d u c tio n , w hile

th e a q u a tic b io l o g is t m u s t c o n s i d e r th e v a s t i n t e r digit a tin g food
c h a i n w hich e c o l o g i s t s t e r m

th e

" fo o d w e b . "

E v e n th ough f is h m a y

liv e th r o u g h s o m e c a t a s t r o p h i c c h a n g e s in t h e i r e n v i r o n m e n t , th e
d e l i c a t e l y b a l a n c e d r e l a t i o n s h i p s b e tw e e n tr o p h ic

le v e l s m a y b e u p s e t

s u f f ic ie n tly to m a k e th e h a b i t a t u n s u ita b le f o r th e m an d o th e r f o r m s
lo n g a f t e r th e d i s a s t e r h a s
v ie w e d f r o m
from

passed.

Not only i s th e

p ro b le m

a n e g a tiv e a t tit u d e in w hich i m p o r t a n t s p e c i e s d i s a p p e a r

one o r m o r e t r o p h i c l e v e l s , but it c a n be a p o s iti v e s itu a tio n .

T h e d e g r a d a t i o n of th e s t r e a m
p a rtic u la r

m ay b ecom e a fav o rab le fa c to r fo r a

s p e c i e s w h ich in t u r n i n c r e a s e s i t s n u m b e r s c o n s i d e r a b l y

out of p r o p o r t i o n f o r m i n g a c a r i c a t u r e of th e o r i g i n a l b io d y n a m ic
p ic tu re .
H a v in g b r i e f l y d i s c u s s e d th e p r o b le m

of s t r e a m

p o llu tio n in

g e n e r a l , we s h a l l c o n s i d e r th e p r e s e n t a p p r o a c h to th e q u e s tio n :
1'How m u c h of a w a s te c a n b e d i s c h a r g e d into a p a r t i c u l a r s t r e a m
b e f o r e it w ill a d v e r s e l y a f f e c t the a q u a tic l i f e ? ' 1

T h is q u e s t i o n i s

b e in g a n s w e r e d , f r o m a n i n d u s t r i a l w a s te a s p e c t , by th e to x ic ity
b io a ssa y .

D o u d o ro ff et a l . (1951), c o m m i s s i o n e d by the F e d e r a t i o n

of Sew age an d I n d u s t r i a l W a s te s A s s o c i a t i o n s , h a v e a t t e m p t e d tjo
s t a n d a r d i z e th e p e r f o r m a n c e of t h e s e t e s t s ; h o w e v e r , th e l i t e r a t u r e
a t p r e s e n t i s f i l l e d w ith a s m a n y m e th o d s a s r e s u l t s .
T h e m o s t o b v io u s f a u lt s in th e b i o a s s a y m e th o d s a r e
are

c arried

(1) th e y

out u n d e r c r i t i c a l c o n c e n t r a t i o n s of th e w a s t e s w ithout

c o n s i d e r a t i o n of c h r o n ic le v e l s ;

(2) th e m a j o r i t y of th e w o rk is

done on f i s h , an d th e e f f e c t of th e w a s t e s on lo w e r f o r m s i s i g ­
n o r e d ; an d (3) th e y d is p la y a c o m p le te la c k of c o n c e r n f o r s i t e of
e n t r y and th e m o de of a c t i o n of th e to x ic io n s w ith in th e fis h .
Due to th e v a r i a b l e s d i s c u s s e d , th e l i t e r a t u r e
v a r i a t i o n in th e t o l e r a n c e of f is h to c h r o m i u m .
l i t e r a t u r e i s in T a b l e

1.

sh o w s a wide

A s u m m a r y of t h i s

In o r d e r not to c o n fu s e t h i s d i s c u s s i o n

6

TABLE

1

A B R I E F SUMMARY O F THE TO X IC ITY L IT E R A T U R E

A n im a l
B lu e g ill
S u n fish

Com pound
N a2 C r 0 4

N a2C r 2 ° 7

S tic k le ­
backs
R a in b o w
T rout

Y oung
E els

M innow s

T rout

L argem o u th
B ass

C r 2 ( S 0 4 )3

K2C r ° 4

R e su lts
Z 4 - h r .. T L m - - 93 0 m g.
Na2C r 0 4/ l .

R eferen ce
A b e g g , 1950

2 4 - h r . T L m - - 7Z 8 mg.
N a^ C r^ O ^ /l.
L eth al c o n e . l i m i t - 1 .Z m g. C r . / l .

Jones,

T o x ic ity [100 x ( l / t i m e ) ]
--0.050^+0*66 m in .
Z000
mixi. t e s t , at 50 m g . C r / l .

G rin d le y ,
1946

K X rO ,
Z
4

T o x ic ity - - 0.05 1_+0.044
m i n , - ^; 1946 m in . te x t, a t
50 m g. C r / l .

K C r 2 (S 0 4 )2

S u r v iv a l t i m e at 5.Z m g.
C r / l . a v e r a g e d 18.7 h o u r s

1939

O s h im a , 1931

K2C r ° 4

S u r v iv a l t i m e a t 5Z0 m g.
C r / l . a v e r a g e d 1Z.4 h o u r s

K2C r 2 (' )7

S u r v iv a l t i m e a t 5Z0 m g.
C r / l . a v e r a g e d 5.35 h o u r s

T riv a le n t
C r as
s u lf a te

40 m g . C r / l . i s a m i n i ­
m u m fa ta l c o n c e n tra tio n
in 6 h o u r s

L e C le rc and
D e v la m in c k ,
1950

6 h o u r s ' e x p o s u r e to 100
m g . K ^ C r ^ C ^ / l . f a ta l
w ith in 1Z h o u r s a f t e r
rem oval

R u s h to n ,

48 h r . T L m ; 195 mg.
C r/l.

F r o m m and
S c h iff m a n t
1958

KZC r Z<^>7

K2 C r ° 4

1921

7

TABLE

A n im a l

1 (C o n tin u ed )

R e su lts

Com pound

D a p h n ia

5 0% i m m o b i l i z e d in 16
h o u r s ; < 0 .6 m g . / l .

A nderson,
1944

N a^C rO ^

50% i m m o b i l i z e d in 48
h o u r s ; < 0.32 m g . / l .

A nderson,
1946

N a ^ C r^ O ?

5 0% i m m o b i l i z e d in 48
h o u r s ; < < 0 .3 1 m g . / l .

T riv a le n t
ch ro m iu m

T o x ic a t 6 m g . / l . o r l e s s

K2C l- ° 4

F lo ra

R eferen ce

R u d o lfs et.
a l . , 1950

w ith a d e t a i l e d d e s c r i p t i o n of t h e s e w o r k s , th e r e a d e r w ill find th i s
re v ie w

of l i t e r a t u r e i n A p p en d ix I.
A c o m p a r i s o n of th e d a ta found i n th e l i t e r a t u r e i s

w hat

h a z a r d o u s , s in c e

th e y a r e r e p o r t e d

som e­

in m an y w ay s f r o m

m e d ia n

t o l e r a n c e l i m i t s to to x i c it y p e r m in u te a n d done u n d e r a v a r i e t y
c o n d i tio n s .

of

It i s o b v io u s th a t th e v a r i a n t s , th e a n i m a l s , th e r e ­

c e iv in g w a t e r , an d th e m o l e c u l a r f o r m of th e w a s te a l l c o n t r i b u t e
to th e c o n f u s io n , w ith th e in e v ita b le r e s u l t th a t th e b i o a s s a y d a ta
are
th e n

of p r a c t i c a l u s e only to th e o r i g i n a t o r of th e e x p e r i m e n t , and
only i n a g iv e n s itu a tio n .
In a n a t t e m p t to r e m e d y t h i s , it w as f e lt th a t one m u s t study

t h e in d i v id u a l s w ith in th e p o p u la tio n u n d e r g o in g th e s t r e s s

of a to x ic

8

e n v iro n m e n t.

T h i s a p p r o a c h , of n e c e s s i t y , le d d i r e c t l y to th e f ie l d

of p h y s io lo g y .
T h e l i t e r a t u r e d e a lin g w ith c h a n g e s in th e p h y sio lo g y of f i s h
u nder ch ro m iu m

s t r e s s i s a l m o s t c o m p le te ly la c k in g .

A b e g g (1950),

u n d e r c o n d itio n s d e s c r i b e d in A p p en d ix I, s tu d ie d c h a n g e s in t i s s u e
f lu i d s a n d b lo o d s p e c if ic g r a v i t y of b l u e g i l l s b r o u g h t on by e x p o s u r e
to s o lu ti o n s of s o d iu m c h r o m a t e a n d s o d iu m d ic h r o rn ate .
m o v in g a s t r i p

By r e ­

of d o r s a l m u s c l e t i s s u e , w e ig h in g i t , d e s i c c a t i n g i t

in a n oven a t a t e m p e r a t u r e

of 110° C. f o r 24 h o u r s a n d r e w e ig h in g

t h e d r y t i s s u e , he d e t e r m i n e d th e t i s s u e flu id v o lu m e .
e x p re s s e d a s percen tag e

T h is w as

of w a t e r of th e w e t - t i s s u e w eig h t.

B lo o d s p e c if i c g r a v i t y w a s d e t e r m i n e d by u s e of th e f a llin g drop d e n s io m e te r.
sam p le

siz e ,

T h is m e th o d c o m b in e s th e m e r i t s

of s m a l l

0.01 c m m . , w ith s p e e d a n d a c c u r a c y .

B lo o d f o r th e d e t e r m i n a t i o n w a s o b ta in e d f r o m a p ith e d f i s h ' s
b u lb u s a r t e r i o s u s .

By i n s e r t i n g a 3 / 4 - i n c h n e e d le tu b e b r o k e n f r o m

a 2 3 -g a u g e h y p o d e r m i c n e e d le in th e b u l b u s , it w a s found th a t th e
a c t i o n of th e h e a r t p u m p e d th e b lo o d out th e tu b e so th a t it c o u ld be
c o l l e c t e d on a p a r a f f i n b lo c k .

T h e d a ta a r e r e p o r t e d in T a b le 2.

A b e g g f e lt th a t th e v a r i o u s p h y s io lo g ic a l r e a c t i o n s of th e f is h
to th e s a l t s o lu tio n

w as

d iffic u lt

to

e x p la in

u n le ss

one

assum ed

9

TABLE

2

PH Y SIO LO G IC A L R EA CTIO N S O F T H E FISH
T O THE SA L T SOLUTION

C h e m ic a l

No. of F i s h

H ours
Ex­
posed

X %
T issu e
F lu i d

T issu e

N a ?C r O

N a2C r 2 ° 7

S ta n d a r d
D e v ia tio n

P
V a lu e a

P e t.
Change^3

F lu i d s

10 C o n t r o l
10 T e s t

24
24

81.22
81.63

0.8267
0.6610

N.S.

N.S

10 C o n t r o l
11 T e s t

13
13

80.30
82 .24

0,7457
1.4419

0.01

+2.

B lood S p ecific G r a v ity

N a ,C rO .
c*
4

10 C o n t r o l
10 T e s t

24
24

1.0441
1.0452

0.001451
0,006406

N.S.

N.S.

N a C r~ O

10 C o n t r o l
9 T est

13
13

1 .0399
1.0372

0.011361
0.003501

0„05

-0 .3 7 5

a P r o b a b i l i t y b a s e d on F i s h e r ' s t - t e s t v a l u e s .
no s ig n if i c a n t c h a n g e .
^ C a l c u l a t e d on th e b a s i s

of th e c o n t r o l v a l u e s e q u a lin g 100

t h a t t h e io n s e n t e r t h e f i s h ' s b ody.
p a th w a y s :

N.S. d e n o te s

T h i s i s p o s s i b l e th r o u g h s e v e r a l

th e s k in , g i l l s a n d o r a l m e m b r a n e s , a n d by in g e s t io n .

A b e g g (1949) m a d e a stu d y of th e e f fe c t of s a l t s o lu tio n s on
th e m u c o u s c o v e r i n g of b l u e g i l l s .

T he s a l t c o n c e n t r a t i o n s u s e d w e r e

th e s a m e a s t h o s e r e p o r t e d in h is

195 0 p a p e r .

P h e n o l r e d w as

10

a d d e d to th e t e s t

s o lu ti o n s to a c o n c e n t r a t i o n of 1:1 0 ,0 0 0 .

T h e f is h

w e r e r e m o v e d a f t e r one h o u r ' s e x p o s u r e a n d r i n s e d off u n d e r
r u n n in g t a p w a t e r f o r t h r e e m i n u t e s .

T h e body s u r f a c e s of a l l e x ­

p e rim e n ta lly t r e a t e d fish w ere

s t a i n e d w ith th e dye.

c o n tro ls re m a in e d unchanged.

He fe lt t h i s

The u n tre a te d

sh o w ed a n a l t e r a t i o n of

th e m u c o u s c o v e r w hich m ig h t th e n h a v e p e r m i t t e d th e io n s to p e n e ­
t r a t e th e sk in .

A b e g g found th a t b o th s o d iu m c a r b o n a t e a n d s o d iu m

d i c h r o m a t e c a u s e d a n i n c r e a s e in t i s s u e flu id c o n te n t of th e m u s ­
c u l a t u r e , a n d in b o th c a s e s th e y c a u s e d th e p r e c i p i t a t i o n of th e
m u c o u s c o v e r i n g of th e body*
In th e s a m e

p a p e r , u s i n g H. W. S m i t h 's phenol r e d t e c h n i q u e ,

A b e g g d e m o n s t r a t e d th a t th e f r e s h - w a t e r s te n o h a l in e t e l e o s t d r i n k s
w a t e r u n d e r n o r m a l c o n d itio n s a s w e ll a s in s o lu tio n s of h i g h e r
o sm o tic p r e s s u r e s .
In a d d itio n to th e c o n c l u s i o n s th a t s o d iu m d i c h r o m a t e c a u s e d
a n i n c r e a s e in t i s s u e f lu i d s , A b e g g s t a t e d th e s i z e of f is h u s e d in a
to x i c it y s tu d y i s an i m p o r t a n t f a c t o r in th e i n t e r p r e t a t i o n of th e
d a ta ; f o r h e found t h a t th e l a r g e r f is h (2 0 gm . to 35 g m .) w e r e m o r e
t o l e r a n t th a n th e s m a l l f is h (4 g m . to 10 g m .) ,
F ro m m

a n d S c h if f m a n (1938), i n t h e i r w o r k w ith la r g e m o u t h

b a s s , c o n c lu d e d th a t e x p o s u r e to 94 m g. C r / l .

c a u s e d in itia lly a

s l i g h t , but n o t h ig h ly s ig n if i c a n t i n c r e a s e , in th e ox yg en c o n s u m p t io n

11

of the animal.

This was followed by a gradual decline to 27 percent

below normal after 68 hours' exposure.
A lso , they found, by m icroscopic examination of the intestine,
sev ere pathological changes im m ediately posterior to the pyloric
caeca, which in all probability completely destroyed its digestive
functions.
I n a d d i t i o n , c o a g u l a te d s t r i n g s

of m u c u s f r o m th e a n u s of a l l

e x p e r i m e n t a l f i s h w e r e o b s e r v e d p r i o r to d e a th .
th a t c h ro m iu m

It w a s t h e o r i z e d

e n t e r e d th e g i l l s a n d w a s e x c r e t e d , in p a r t , by th e

liv e r v ia th e b ile .

T h i s c h r o m i u m - l a d e n b ile c a u s e d th e i n t e s t i n a l

dam age.
It i s i n t e r e s t i n g to n o te th a t no d a m a g e to th e g il l t i s s u e w as
r e p o r t e d , a lth o u g h th e c o m m o n c r i t e r i o n i s t h a t th e h e a v y m e t a l s
k i l l b y p r e c i p i t a t i o n of th e m u c u s c o v e r i n g of th e g i l l s .

T h ey did

n o te a co u g h in g r e f l e x in th e f i s h b e f o r e d e a t h , but th e e x a c t s i g ­
n if i c a n c e of t h i s i s n o t known.
B e a r i n g in m in d th e l i t e r a t u r e a s r e v i e w e d h e r e i n , th e i n d i ­
v i d u a l i n t e r e s t e d i n th e e f f e c t s of c h r o m i u m w a s t e s on f i s h , o r o t h e r
a q u a t ic o r g a n i s m s , c a n o b ta in l i t t l e h e lp w ith th e p r o b l e m s a t hand,
s i n c e th e w o r k done a p p lie d to a p a r t i c u l a r s e t of c o n d itio n s a n d n o t
to g e n e r a l i t i e s .

T o r e i t e r a t e , i t w a s f e l t th a t a p h y s io lo g i c a l-

p h a r m a c o l o g i c a l a p p r o a c h to th e p r o b l e m s of s t r e a m

p o llu tio n w ould

12

p r o d u c e a b e t t e r u n d e r s t a n d i n g of th e m a n y in f lu e n c in g f a c t o r s .

For

e x a m p l e , if we u n d e r s t o o d how c h r o m i u m e n t e r s th e f i s h , and if we
knew th e d i s t r i b u t i o n a n d th e m o d e of a c t io n of th e to x ic io n on ce
i t i s w ith in th e o r g a n i s m , we c o u ld b e t t e r u n d e r s t a n d th e l i m i t s of
to x ic

stre ss.
T h i s p r o j e c t w a s a p p r o a c h e d w ith t h e s e i d e a s in mind* n o t a s

a n a n s w e r to to x i c it y
c o m p le x p u z z le .

L ik e th e c o n v e n tio n a l b i o a s s a y , t h i s e x p e r i m e n t

f i x e s th e v a r i a b l e s ;
is used .

p r o b l e m s a s a w h o le , but a s one p ie c e in a

one w a t e r , one w a s t e , a n d one s p e c i e s of a n i m a l

U nlike th e b i o a s s a y , it i s d e s ig n e d to p r o m o t e a n u n d e r ­

s ta n d i n g of th e s t r e s s th e in d iv id u a l f is h i s e x p e r i e n c i n g .

A n o th e r

d i f f e r e n c e - - p e r h a p s th e m o s t im p o r t a n t - - i s th e fa c t t h a t t h i s e x p e r i ­
m e n t d e a l s not w ith c r i t i c a l c o n c e n t r a t i o n s , b u t s u b le t h a l d o s e s of
ch ro m iu m .
T o o r i e n t t h i s w o r k w ith th e l i t e r a t u r e a n d to u s e a s a b a s e ­
lin e f o r th e p r o j e c t a s a w h o le, a 2 4 - h o u r m e d ia n t o l e r a n c e l i m i t
w as d e te rm in e d .

The subsequent e x p e rim e n ts w ere all p e rfo rm e d

at o n e - f if t h of t h i s l i m i t .

O nce th e w o r k in g c o n c e n t r a t i o n l e v e l w as

d e t e r m i n e d , pilot e x p e r i m e n t s u s in g th e r a d i o i s o t o p e C r
th e c i r c u l a t o r y
w ith in th e t r o u t .

sy stem
It i s

51

im p lic a te d

a n d th e l i v e r a s a path of th e c h r o m i u m
s a f e to a s s u m e th a t th e b lo o d i s i n s t r u m e n t a l

in th e t r a n s p o r t of th e to x ic a g e n t f r o m i t s e n t r a n c e in to th e a n i m a l

u n ti l i t s f in a l e x it.

T h e r e f o r e , it w ould be th e f i r s t t i s s u e th a t

m ig h t e x h ib it c h a n g e s due to th e
th is th e s is is

stre ss.

U nder th is a ss u m p tio n ,

p red o m in an tly d ir e c te d at c h ro m iu m -in d u c e d change

i n b lo o d , a n d i n c l u d e s d e t e r m i n a t i o n s

of h e m a t o c r i t s , h e m o g lo b in s

c e l l c o u n t s , p l a s m a v o l u m e s , a n d b lo o d v o l u m e s , a m o n g o t h e r s .

M A TERIA LS AND METHODS

T h e r a in b o w t r o u t u s e d th r o u g h o u t t h i s w o r k w e r e o b ta in e d
f r o m th e

M ic h ig a n D e p a r t m e n t of C o n s e r v a t i o n th r o u g h th e c o o p e r a ­

t i o n of M e s s r s . E d w a r d B aco n a n d R alp h M a r k s .

T h e f is h w e r e

t r a n s p o r t e d in p l a s t i c b a g s in lo ts of one h u n d r e d f r o m th e Wolf
L a k e H a t c h e r y to th e l a b o r a t o r y .

F if ty t r o u t w e r e p la c e d i n a s m a l l

a m o u n t of w a t e r i n e a c h b a g , a f t e r w hich th e b a g w a s i n f l a t e d w ith
oxygen an d s e a le d .

Ice w a s p la c e d a r o u n d th e o u ts id e of th e bags*

w hen th e t e m p e r a t u r e s w a r r a n t e d i t , to k e e p th e f is h at 15° C .
In th e l a b o r a t o r y t h e f i s h w e r e h e ld in f o u r 2 6 - g a llo n g l a s s
a q u a r i a k ep t in a c o n s t a n t t e m p e r a t u r e r o o m

( F i g u r e 1).

T h is r o o m

w a s i l l u m i n a t e d c o n tin u a lly by f l u o r e s c e n t lig h ts o v e r h e a d a n d on
e a c h h o ld in g ta n k .

C o n s ta n t i l l u m i n a t i o n w a s u t i l i z e d b e c a u s e e a c h

t i m e th e li g h ts w e r e t u r n e d on o v e r th e ho ld ing ta n k s th e f is h w ould
b e c o m e e x c e s s i v e l y e x c i ta b l e , th u s i n t r o d u c in g a v a r i a b l e th a t m ig h t
a f f e c t th e f in a l r e s u l t s .
T h e w a t e r u s e d th r o u g h o u t th e e x p e r i m e n t s w a s h e l d at 14° C.
to

15° C . in th e h o ld in g ta n k s and in th e e x p e r i m e n t a l t a n k s , a n d

w a s ta p w a t e r w hich h a d a h a r d n e s s

of 334 m g . of C a C O ^ / l . , a t o t a l

a l k a l i n i t y of 204 m g . of C a C 0 3/ l . a n d a pH of 8.5 to 8.8.
14

Figure

1.

Experimental aquaria in constant temperature

room,

15

16

On each Monday, Wednesday, and Friday the fish were fed
dried trout pellets developed by the Michigan Department of C onser­
vation for use in their hatcheries.

Chem ical Methods

Total residue (Standard Methods> 1955)

Total resid u e, or total solid s, was done by evaporation.
Fifty m illilite r s of sample were placed in a weighed dish and
evaporated to dryness at tem peratures between 95° C. and 105" C.
The dish was reweighed and the difference calculated.

The total

residue in m g ./l. was found by the following equation:
mg. total r e sid u e /l. = (difference x 1000)/(m l. of sample)

Alkalinity (Standard Methods, 1955)

Alkalinity was done by titration.

Fifty m illilite r s of sample

w ere titrated to pH 8.3, using phelolphthalein indicator, with 0.02
N. hydrochloric acid, after which it was further titrated using
F leish er Methyl Purple Indicator to a purple endpoint at a pH of 4.8.
The phenolphthalein alkalinity was calculated as mg. of
C aC 03/ l . by the following formula:
mg. CaCO^/l. = (ml. standard acid x 1000)/(m lo of sample)

17

T h e t o t a l a l k a lin i ty w a s a l s o
a n d w a s c a l c u l a t e d in th e
of s t a n d a r d to b r i n g th e

sam e

e x p r e s s e d in m g.

of C a C O ^ / l .

m a n n e r u s in g th e to ta l m i l l i l i t e r s

s o lu tio n to a pH of 4.8.

T h e t o t a l a l k a lin i ty of a w a t e r i s m a d e up of t h r e e ty p e s;
n a m e l y , th e h y d r o x id e , c a r b o n a t e , and b i c a r b o n a t e a l k a lin i ty found in
th e a p p r o x i m a t e r e l a t i o n s h i p s a s i l l u s t r a t e d in T a b l e
S t a n d a r d M eth o d s

3, ta k e n f r o m

(1955).

De M a r t i n i 's

(1938) e q u a tio n s f o r th e d e t e r m i n a t i o n of the

t h r e e t y p e s of a l k a l i n i t i e s a n d H^CO^ a r e a s follow s:
[Aik. +

(1)

(HCO j

(H+ ) - -2-SL]

=

(H}

3

1 +

2

KZ
(H+ )

K
(H+ ) --------~ ~ ]

[Aik. +
(2)

(c o

^
«

>

1

2

.

2
(H+ )
K

[Aik. +
(3)

(h 2 c o 3 ) =

(H+ )
"k ~ x
1

K
(4)

(OH~) =

(H+ ) (H

)

kI

1 + 2

_ j _
(H+ )

W

(H+ )
" A i k . " a l k a lin i ty i s in e q u iv a le n t s of t i t r a t a b l e b a s e p e r l i t e r
K j a n d Kz a r e th e f i r s t a n d s e c o n d io n i z a ti o n c o n s t a n t s of
c a r b o n i c a c i d a n d Kw of w a t e r .

18

TABLE

3

TH E R E L A T IO N S H IP O F THE T H R E E T Y P E S O F A L K A L IN IT Y TO
TH E T O T A L AND P H E N O L P H T H A L E IN A L K A L IN IT Y

R e s u l t s of
T itra tio n

H y d r o x id e A ik .
a s C aC O ^

C a r b o n a t e A ik.
a s C aC O ^

B i c a r b o n a t e A ik.
a s CaCCh

P =■ 0

0

0

T

P = 1/2 T

0

2P

T -2P

P = l/Z

T

0

2P

0

P -

T

2 (P -T )

2 (T -P )

0

T

0

0

u

1/2

T = t o t a l a l k a lin ity ;

M oore

5

P = p h e n o lp h th a le in a lk a lin ity .

(193 9) t r a n s f o r m e d t h e s e e q u a tio n s in to g r a p h ic f o r m .

T h e s e w e r e u s e d i n t h i s w o rk .

H ardness

(S chw arzenbach T itra tio n )

H a r d n e s s d e t e r m i n a t i o n s w e r e m a d e by t i t r a t i o n u s in g r e ­
a g e n t s m a n u f a c t u r e d by th e H a ll L a b o r a t o r i e s , I n c ., and p u r c h a s e d
f r o m C a l g o n , I n c ., C h ic a g o , I llin o is .

T his is a v e rs e n a te titr a tio n

b a s e d upon th e m e th o d s in v e n te d by D r . G. S c h w a rz e n b a c h and
a s s o c i a t e s i n S w i tz e r l a n d .
H ard n ess

S a m p l e s of 5 0 m l . , w ith 1 m l. of

B u f f e r S o lu tio n a d d e d , w e r e t i t r a t e d w ith H a g a n H a r d n e s s

19

T i t r a t i o n S o lu tio n (l

m l, = ZO m g . C a C O ^ / l . ) u s in g th e h a r d n e s s i n ­

d i c a t o r w h ich c h a n g e s f r o m r e d to b lu e a t th e en d p o in t.

H ardness

w a s c a l c u l a t e d a s fo llo w s:
T o t a l h a r d , a s m g . C a C O ^ / l . = ZO x b u r e t t e r e a d i n g in m l.

C h ro m iu m

( S a ltz m a n ,

195Z)

S a m p l e s f r o m th e a q u a r i a w e r e d ilu te d , w hen n e c e s s a r y , so
th a t th e a m o u n t a n a l y z e d , 1 to 10 m l . , w ould c o n ta in f r o m
m icro g ram s

of c h r o m i u m .

5 to

15

T h is w as a s h e d in a l Z 5 - m l . P h i l l i p s

b e a k e r by a d d in g 0.5 m l, g l a s s d i s t i l l e d r e a g e n t g r a d e c o n c e n t r a t e d
n itric

a c i d , a n d 0.Z5 m l. of 40 p e r c e n t so d iu m b i s u l f a t e , th e n

e v a p o r a t e d to d r y n e s s on a L i n d b e r g ty p e hot p la te .

A t th i s point

S a l t z m a n r e c o m m e n d s r e p e a t i n g th e a d d itio n of a c i d a n d a s h in g
a g a i n , but it w a s found th a t t h i s w as u n n e c e s s a r y u n d e r the c o n d i­
tio n s

e x i s t i n g d u r in g t h e s e t e s t s .

T e n m i l l i l i t e r s of 0.5 N. s u lf u r ic

a c i d , m a d e up w ith double d i s t i l l e d w a t e r , w e r e a d d e d and s w i r l e d
to d i s s o l v e th e a s h .

W hen th e a c i d w as a d d e d to th e hot a s h it went

in to s o lu tio n w ith m u c h l e s s d iffic u lty t h a n did th e c o o le d a s h .
Any w a t e r , in c lu d in g r e a g e n t s m a d e up w ith w a t e r , a d d e d to
th e s a m p l e s a t t h i s
tille d .

point o r a f t e r w a r d s , m u s t b e double g l a s s d i s ­

T h i s p r e v e n t s r e d u c t i o n of the h e x a v a le n t c h r o m i u m w h ich is

f o r m e d by th e a s h in g a n d s u b s e q u e n t s t e p s .

Only th e h e x a v a le n t

20

ch ro m iu m

w ill

d e v e lo p

th e

co lo r

of th e

s-d ip h en y lcarb azid e

re­

agent .
To th e a c i d s o lu tio n of th e a s h i s a d d e d 0.5 m l.
p o ta ssiu m

of 0.1 N„

p e r m a n g a n a t e ; it i s th e n c o v e r e d a n d h e a t e d on th e hot

p la te at a p p r o x i m a t e l y

100° C. f o r 2 0 m i n u te s .

c a l l s f o r th e a d d i tio n of m o r e
p e a r s , but o n ce a g a i n t h i s

S a l t z m a n 's m e th o d

p e r m a n g a n a t e i f th e pink c o l o r d i s a p ­

s te p w as found u n n e c e s s a r y .

A t the end

of t h i s t i m e th e p e r m a n g a n a t e i s

d e c o l o r i z e d by th e a d d itio n of 5

p e r c e n t s o d iu m a z i d e a t th e r a t e

of 1 d ro p e v e r y

ing a f t e r e a c h d r o p .

10 s e c o n d s , s w i r l ­

T h r e e to 5 d r o p s w e r e u s u a ll y enough to

d e s t r o y any b r o w n i s h t i n t , a n d any e x c e s s w as a v o id e d s in c e th i s
w ou ld r e d u c e th e c h r o m i u m .

With th e s a m p l e s once a g a in c o l o r l e s s ,

th e y w e r e r e m o v e d i m m e d i a t e l y f r o m th e h e a t an d p la c e d in a t r a y
of c o ld w a t e r .

T h e s a m p l e s now m a y b e f i l t e r e d , if n e c e s s a r y , o r

t r a n s f e r r e d d i r e c t l y in to 2 5 m l. v o l u m e t r i c f l a s k s u s in g double d i s ­
t i l l e d w a t e r a s a w a s h liq u id .
ric

O nce th e s a m p l e s a r e in th e v o l u m e t ­

f l a s k s , c o l o r d e v e lo p m e n t c a n p r o c e e d .
C o l o r w a s d e v e lo p e d by adding 1.0 m l. of s - d i p h e n y l c a r b a z i d e

reag en t.

T h i s r e a g e n t w a s m a d e up by d is s o lv i n g 10 g r a m s

of

p h ta lic a n h y d r id e in 175 m l. of r e d i s t i l l e d 95 p e r c e n t e th y l a lc o h o l,
w a r m i n g to e f f e c t s o lu tio n .

To th is i s ad d ed 0,625 g r a m s

s-

d y p h e n y l c a r b a z i d e d i s s o l v e d i n 5 0 m l . of r e d i s t i l l e d a lc o h o l,

F in a lly ,

21

t h i s c o m b in a ti o n i s
i s q u ite

m a d e up to 25 0 m l. w ith a lc o h o l.

T h is r e a g e n t

s t a b l e a n d w a s s t o r e d in a b r o w n b o ttle u n d e r r e f r i g e r a t i o n

fo r 8 m o n th s.

Only o c c a s i o n a l r e s t a n d a r d i z a t i o n w a s n e c e s s a r y .

One m in u te a f t e r th e d e v e l o p e r w as a d d e d , 2.5 m l. of 4 M.
s o d iu m d ih y d r o g e n p h o s p h a te w as a d d e d a s a b u f f e r a n d th e
m a d e up to 25 m l.

sam p le

If i r o n w a s p r e s e n t , the b u f f e r c o u ld be a d d e d

b e f o r e th e s - d i p h e n y l c a r b a z i d e an d a llo w e d to s ta n d 15 m i n u t e s b e ­
fo re read in g .

T h i s w ould e l i m i n a t e i n t e r f e r e n c e

due to s m a l l a m o u n ts

of i r o n .
T h e pink c o l o r w a s r e a d at a w a v e le n g th of 540 m g . w ith in
3 0 m i n u t e s on a B a u s c h a n d L o m b S p e c tr o n ic 2 0 c o l o r i m e t e r .
A p o ta ssiu m c h ro m a te

s o lu tio n w as u s e d to s t a n d a r d i z e the

d e t e r m i n a t i o n a n d w as m a d e by d i s s o lv i n g 0.3734 g r a m s of p o t a s s i u m
c h ro m a te in
ch ro m iu m

1 l i t e r of r e d i s t i l l e d w a t e r .

per m illilite r.

ch ro m iu m v e rs u s

S ta n d a r d c u r v e s

T h is c o n ta in e d 100 jjg. of
of m i c r o g r a m s h e x a v a le n t

p e r c e n t t r a n s m i s s i o n w e r e m a d e up a p p r o x im a te l y

e v e r y two w e e k s w hen m a n y a n a l y s e s w e r e b e in g m a d e , but if
c h r o m i u m d e t e r m i n a t i o n s w e r e done p e r i o d i c a l l y , a c u r v e w a s m a d e
w ith e a c h s e r i e s

of d e t e r m i n a t i o n s .

H e x a v a l e n t c h r o m i u m w a s d e t e r m i n e d by p r o c u r i n g a p r o p e r l y
d il u te d s a m p l e in a 25 m l . v o l u m e t r i c f l a s k an d ad d in g th e
d ip h e n y lc a rb a z id e

re a g e n t and buffer a s above.

s-

The sam p les w ere

22

t h e n d il u te d to 2 5 m l. w ith do u b le d i s t i l l e d w a t e r a n d r e a d c o l o r i m e t ric a lly

u s in g th e sa m e
The a c c u ra c y

s ta n d a rd s a s above.

of th e t e s t w as d e t e r m i n e d by m a k in g up 2 ^ug.

-JCr

/m l,

sta n d a rd s

d ic h ro m a te .

T hree

u s in g

b o th

p o ta ss iu m c h ro m a te and p o ta ssiu m

10 jag. s a m p l e s f r o m

e a c h s o lu tio n w e r e a n a l y z e d

f o r t o t a l c h r o m i u m a n d h e x a v a le n t c h r o m i u m , e a c h s a m p l e b e in g
r e a d in th e c o l o r i m e t e r t h r e e t i m e s .
a p p e a r in T a b l e 4.
te sts

are

T h e r e s u l t s of t h e s e a n a l y s e s

T h e m e a n s an d s t a n d a r d d e v i a tio n s of t h e s e

shown i n T a b le 5.

Due to th e f a c t th a t th e s a m p l e s a g r e e w e ll w ith in t h e m s e l v e s ,
it w a s f e lt t h a t th e d e v ia tio n f r o m

the s t a n d a r d of 10 jag. w a s due to

th e d ilu tio n an d m e a s u r e m e n t m e th o d s a n d not th e a n a l y t i c a l p r o ­
ced u re.
th e

T a k in g

s e rie s

10

yxg.

a s th e m e a n of th e p o p u la tio n (p) and u s in g

of s a m p l e s w ith th e g r e a t e s t v a r i a t i o n

(s

2

—

= 0.2 06, X =

10.9) it w a s found th a t th e s t a n d a r d e r r o r of th is g ro u p i s
Jx

+. 0.37.

M a k in g th e a s s u m p t i o n , w h ich i s not s u b s t a n t i a t e d by e x ­

p e r i m e n t a l d a t a , th a t t h i s e r r o r w ould not ch an g e a p p r e c i a t e l y w ith
g r e a t e r d il u tio n s , i t w a s f e lt th a t th i s a c c u r a c y w as s u f f ic ie n t f o r
te stin g a ll w a te r

sam p les.

It w a s a l s o found by u s in g ja = 10 and th e r e s u l t s
p o ta ssiu m c h ro m a te

s a m p le s th a t a F is h e r

" t"

of th e

t e s t sho w ed no

e v i d e n c e of a d i f f e r e n c e b e tw e e n t o t a l a n d h e x a v a le n t c h r o m i u m

23

TABLE

4

R E S U L T S O F T E S T S TO D E T E R M IN E THE A CCUR ACY
O F TH E CHROMIUM M ETHOD

R e a d in g on
C o lo rim e te r

T o ta l C h r o m i u m

H e x a v a le n t C h r o m i u m

S a m p le N u m b e r
2
1
3

S a m p le N u m b e r
2
1
3

K2G r 2

1
2
3

11.6
11.5
11.5

10.8
10.8
10.8

°7

10.6
10,6
10.4

1 0o6
10.6
10.6

11.0
10.8
10.6

11.0
11.0
11.0

10.0
10.2
10.2

10.6
10.6
1 0 o8

10.2
10.2
10.0

K ^C rO ?

1
2
3

10.2
10.6
10.4

10.8
11.8
10.6

10.6
10.6
10,8

TABLE 5
MEANS AND STANDARD DEVIATIONS O F TESTS T O D E T E R M IN E
TH E ACCURACY O F TH E CHROMIUM METHOD
K2C r ° 7

K2C r 2 ° 7

C h ro m iu m

X

s

X

s

T o t a l c h r o m i u m ................... ..................

10.9

0.454

10.6

0.235

...............

10.8

0.200

10.3

0.285

H ex av alen t c h ro m iu m

. .

24

m e th o d s.

Upon t h i s b a s i s , it w a s a s s u m e d th a t th e c h r o m i u m a d d e d

to th e a q u a r i a r e m a i n e d f o r th e m o s t p a r t in th e h e x a v a le n t f o r m ,
and la te r

e x p e r i m e n t s u p h e ld t h i s t h e o r y .

R a d i o a c ti v e Is o to p e M eth o d s

T h e r a d i o i s o t o p e C h r o m i u m 51 w as u s e d in th e t r a c e r
p e rim e n ts.

ex­

T h i s e l e m e n t w a s s u p p lie d by the N a tio n a l L a b o r a t o r y ,

O ak R id g e , T e n n e s s e e , in th e f o r m

of C r C l^ i n HC1 s o lu tio n w ith a

c o n c e n t r a t i o n of 22,85 4, 10 p e r c e n t m g . / m l , a n d a s p e c if ic a c tiv it y
of 1760 m c / g .

T h i s , th e t r i v a l e n t f o r m , h a d to be o x id iz e d to th e

h e x a v a le n t f o r m f o r u s e in t h i s p r o b le m .
c a r r i e d out in th e fo llo w in g m a n n e r .
5 0 p g . of

Cr

51

w as

T h i s t r a n s f o r m a t i o n w as

A s a m p l e of a p p r o x i m a t e l y

m a d e b a s i c w ith 2 m l. of 6 N. S o d iu m h y d r o x ­

i d e a n d 1 m l. of 3 p e r c e n t h y d r o g e n p e r o x id e w a s a d d e d .

T h i s w as

h e a t e d at 110° C . f o r 5 m i n u t e s , a f t e r w h ich it w as b o ile d f o r one
h o u r to d r i v e off th e e x c e s s p e r o x id e .

At th e e n d of th e b o ilin g

p e r i o d , w hile th e s o lu tio n w a s s t i l l w a r m , 0.2 m l . m o r e of th e NaOH
w a s a d d e d an d s w i r l e d .

If b u b b le s w e r e g iv e n off, th e s o lu tio n w as

b o i l e d a g a i n u n t i l no b u b b lin g w as e v id e n t.

When no b u b b lin g

o c c u r r e d , th e s o lu tio n w as n e u t r a l i z e d w ith 2 m l. of 6 N. h y d r o ­
c h l o r i c a c i d , c o o l e d , and u s e d .

25

T he t e s t a n i m a l s w e r e a lw a y s e x p o s e d to the r a d i o a c t i v e
i s o t o p e by m a k in g a d ilu tio n of t h e i r e n v i r o n m e n t a l w a t e r .

W a te r

s a m p l e s of 1 m l. w e r e a n a l y z e d f o r a c tiv it y by e v a p o r a t i n g t h e m in
p o r c e l a i n p la n c h e ts w ith a d i a m e t e r of 3 5 m m .
of t h e s e

G a m m a r a y c o u n ts

p r e p a r a t i o n s w e r e m a d e w ith a N u c l e a r I n s t r u m e n t a n d

C h e m i c a l C o r p o r a t i o n M odel 183 S c a l e r e q u ip p e d with a T r a c e r l a b
tu b e of th e s c i n t i l l a t i o n ty p e .
O r g a n c o u n ts w e r e m a d e by w eig h in g th e f r e s h t i s s u e on a
R o l l e r - S m i t h b a l a n c e a n d d r y a s h in g th e m
in a m u ffle f u r n a c e at 5 00° C .

on th e p o r c e l a i n p la n c h e ts

When th e r e s i d u e t u r n e d w hite o r

lig h t g r a y , th e p la n c h e ts w e r e r e m o v e d f r o m th e oven a n d c o o led .
C o n c e n tra te d n itr ic

a c i d w as a d d e d to d is s o lv e a n d / o r s u s p e n d th e

a s h , th e r e s u l t a n t s o lu tio n b e in g e v a p o r a t e d slo w ly on a hot p la te .
T his

le ft a th in f i l m of th e a s h even ly d i s t r i b u t e d on th e p la n c h e t,

w h ic h w a s c o u n te d a s a b o v e .
B oth w a t e r and o r g a n c o u n ts w e r e m a d e e i t h e r a s c o u n ts p e r
fiv e m i n u t e s , o r a s m i n u t e s p e r 400 c o u n t s , c o r r e c t e d f o r d e c a y and
r e p o r t e d a s c o u n ts p e r m in u te .
B io lo g ic a l M eth o d s

F is h b io a ssa y s

(D o u d o ro ff et a l ., 1951)

T h i s m e th o d s e t up by th e C o m m i t t e e on R e s e a r c h , S u b c o m ­
m itte e

of T o x i c ity , S e c tio n III of th e F e d e r a t i o n of Sew age a n d

26

I n d u s t r i a l W a s t e s A s s o c i a t i o n s , w a s fo llo w e d a s c l o s e l y a s
cab le;

p ra c ti­

d e v i a tio n s w e r e m a d e only w hen th e r e s e a r c h e r f e lt t h e m to

b e of no e f f e c t on th e f in a l r e s u l t s .
S in ce t h i s i s a s t a n d a r d p r o c e d u r e i n th e f ie ld ot to x ic w a s te
c o n t r o l , b u t unknow n in p u r e p h y s io lo g ic a l w o r k , it i s f e lt th a t a
b r i e f o u tlin e of th e m e th o d i s j u s t i f i a b l e

F ish .

in t h i s w o rk .

F i s h b e in g u s e d in b i o a s s a y t e s t s

sh o u ld b e n a tiv e

to

th e w a t e r s h e d r e c e i v i n g th e w a s t e s , and sh o u ld be n e i t h e r a t o l e r a n t
n o r a h ig h ly i n t o l e r a n t s p e c i e s , b u t r a t h e r , one w ith a r e c o r d of
m e d ia n t o l e r a n c e .

T h e fo llo w in g f a m i l i e s a r e r e c o m m e n d e d :

C e n tra rc h id a e

( s u n f is h e s , b a s s e s , c r a p p i e s )

S a lm o n id a e

( tr o u t, c h a r r s , s a lm o n s )

C y p rin id a e

( tr u e m in n o w s) e x c l u s i v e of c a r p a n d g o ld fish

C a to sto m id a e
T e st a n im a ls

(su ck ers)

sh o u ld be o b ta in e d f r o m any s in g le s o u r c e a n d

b r o u g h t to t h e l a b o r a t o r y at ab o u t th e s a m e t i m e .
l a r g e s t f is h
sm a lle st.

T he le n g th of th e

s h o u ld not b e m o r e th a n 1.5 t i m e s th e le n g th of th e

D o u d o ro ff r e c o m m e n d e d f is h a v e r a g i n g l e s s th a n t h r e e

i n c h e s , but t h i s w a s not a d h e r e d to b e c a u s e of th e d iffic u lty th a t
w o u ld be e n c o u n t e r e d i n p e r f o r m i n g o t h e r p h y s io lo g ic a l t e s t s .
u s e d th r o u g h o u t the w o rk a v e r a g e d
of f r o m

10.0 to

F ish

13.6 c m . in le n g th w ith a r a n g e

17.0 c m . a n d a m e a n w eig h t of 19.0 g r a m s .

27

The t e s t a n im a ls

s h o u ld be a c c l i m a t i z e d to l a b o r a t o r y c o n d i ­

t i o n s f o r a t l e a s t one w e e k , p r e f e r a b l y t e n d a y s o r lo n g e r .

D u r in g

t h i s t i m e th e y a r e to b e fe d a t r e g u l a r i n t e r v a l s a t l e a s t t h r e e
ti m e s a w eek.

F ish

s h o u ld not be fe d two d a y s b e f o r e , n o r d u r in g

th e t e s t .
T h e p e r c e n t a g e of f is h dy in g o r s e r i o u s l y d i s e a s e d d u r in g th e
a c c l i m a t i z a t i o n p e r i o d s h o u ld n o t e x c e e d 10 p e r c e n t .

T h e e x p e r i m e n t a l w a t e r ( d ilu e n t) .
r e c e i v i n g th e w a s te o r a r t i f i c i a l s t r e a m

W a te r f r o m th e s t r e a m
w a t e r of s i m i l a r q u a lity

w ith r e s p e c t to th e c a l c i u m , m a g n e s i u m , s u l f a t e , t o t a l d i s s o l v e d
s o l i d s , a n d t o t a l a l k a lin i ty i s r e c o m m e n d e d .
p r o j e c t r e p o r t e d h e r e i n i s not a p r o b l e m

S in ce th e r e s e a r c h

s p e c if ic to a p a r t i c u l a r

s t r e a m , ta p w a t e r w a s d e c id e d upon a s th e d ilu e n t.

O th e r e x p e r i m e n t a l c o n d i t i o n s .

T h e t e s t sh o u ld b e p e r f o r m e d

at u n i f o r m t e m p e r a t u r e s b e tw e e n 68° F . a n d 77°
f o r w a r m w a t e r f is h a n d 54° F . to 64° F .
c o l d w a t e r f is h a r e u s e d .

The ran g e

F.

(2 0° C .- 2 5 ° C .)

(12° C . - 1 8 ° C .) when

s h o u ld n o t e x c e e d 2° F . o r

1° C . ab o v e o r b elo w the c h o s e n t e m p e r a t u r e .
The te s t c o n ta in e r o r a q u aria

sh o u ld be of g l a s s a n d of s u c h

a s i z e t h a t d e p th of w a t e r i s n e v e r l e s s th a n 6 in c h e s .

The d is ­

s o lv e d o x ygen c o n te n t of th e w a t e r s h o u ld n e v e r b e l e s s th a n 4 ppm

28

f o r w a r m w a t e r f is h o r 5 p p m f o r c o ld w a t e r f is h .

A e ra tio n , u n d er

som e

c o n d i tio n s , i s not r e c o m m e n d e d due to th e f a c t t h a t t h i s m a y

d riv e

out g a s e o u s o r v o l a t i l e c o m p o n e n ts of a w a s te .
A t l e a s t t e n a n i m a l s s h o u ld be t e s t e d f o r e a c h d ilu tio n ; th e

t o t a l w e ig h t of a l l f is h in a t e s t c o n t a i n e r s h o u ld not e x c e e d 2 g r a m s
per lite r,

and 1g r a m

per lite r

o r l e s s b e in g p r e f e r r e d .

A e r a t i o n a n d f i l t r a t i o n w e r e b oth u s e d in t h e s e

e x p e rim e n ts

a f t e r due e x a m i n a t i o n of t h e i r e ffe c t on th e d i s s o l v e d c h r o m i u m .
A e r a t i o n w a s a c c o m p l i s h e d by c o m p r e s s e d a i r i n t r o d u c e d in to th e
t a n k s v i a a s to n e a i r b r e a k e r .

A q u a riu m f il te r s

of g l a s s wool and

g r a v e l w e r e u s e d to c l e a r th e w a t e r of s u s p e n d e d s o l i d s .

B efore

u s i n g t h e s e a p p a r a t u s , w hich w e r e c o n t r a r y to r e c o m m e n d e d p r o ­
c e d u r e , t h r e e a q u a r i a w e r e s e t up a s fo llo w s:
A q u a riu m

1

No f i l t e r , no a e r a t i o n

A q u a riu m

2

No f i l t e r , a e r a t i o n

A q u a riu m

3

F iltra tio n , a e ra tio n

T h e w a t e r in e a c h of t h e s e t a n k s c o n ta in e d 10 m g. C r

+6 /
/l.

The

t o t a l a n d h e x a l e n t c h r o m i u m , a n d t o t a l a lk a lin ity m e a s u r e d at
th e s t a r t

of th e

a f t e r th e

a d d i tio n of c h r o m i u m .

s h o w n in T a b l e s

e x p e r i m e n t a n d 24 h o u r s , 48

0 a n d 7.

h o u r s , and 5 d a y s

T h e r e s u l t s of t h e s e t e s t s a r e

29

TABLE

6

R E S U L T S O F T E S T S TO D E T E R M IN E W H ETH ER F IL T R A T IO N
OR A E R A T IO N HAD A N E F F E C T ON THE
CHROM IUM CO N C E N T R A T IO N

T o ta l C h r o m i u m
Tank
No.

S a m p le
A
(m g ./l.)

S a m p le
B
(m g ./l.)

H e x a v a le n t C h r o m i u m
S a m p le
A
(m g ./l.)

X

S a m p le
B
(m g ./l.)

PH
X

S ta rt
1
Z
3

9.6
9,4
9.8

9,8
10.0
10.0

9.7
9.7
9.9

9.6
9.2
9.8

—

—

-----------------

9.6
9.2
9.8

8.03
8.34
8.58

Z4 H o u r s
1
Z
3

10.6
10.6
10.4

10.z
10.7
1 0 .Z

10.4
10.6
10.3

10.4
10.7
10.7

10.6
10.7
10.6

10.5
10.7
10.6

8.34
8.83
8.80

10.6
10.4
10.2

10.6
10.6
10.1

10.6
10.5
10.2

8.33
8.83
8.85

------

-------------

48 H o u r s
1
Z
3

1 0 .Z
10.0
10.0

10.Z
10.0
10.0

1 0.Z
10.0
10.0
5 D ays

1
Z
3

10.6
10.4
10.1

10.6
1 0 .Z
10.6

10.6
10.3
10.3

---------

-------------— — —

8.38
8.91
8.90

30
TABLE

7

R E S U L T S O F T E S T S T O D E T E R M IN E W H ETH ER F IL T R A T IO N
OR A E R A T IO N HAD AN E F F E C T ON A L K A L IN IT Y

Tank
N um ber

T o t a l A lk a lin ity
S a m p le A

S a m p le B

M ean

224
22 0
212

222
220
212

22 4
220
224

224
219
223

- —---------

224
216
222

--------—
—

224
224
224

S ta rt
1
2
3

220
220
212
24 H ours

1
2
3

224
218
222
48 H o u r s

1
2
3

224
216
222
5 D ay s

1
2
3

224
224
224

31

It c a n be c o n c l u d e d r e a d i l y f r o m t h e s e d a t a , ev en w ithout
b e n e f i t of s t a t i s t i c a l a n a l y s i s , th a t n e i t h e r f i l t r a t i o n n o r a e r a t i o n
a l t e r e d th e c h r o m i u m c o n te n t o r t o t a l a l k a lin i ty of th e w a t e r .

As a

r e s u l t it w as f e lt th a t te n f is h c o u ld be kept in e a c h 4 0 - l i t e r a q u a r i u m
w ith o u t a l t e r i n g th e f in a l r e s u l t s

of th e b i o a s s a y , ev en th o ug h th i s

g r e a t l y e x c e e d e d th e r e c o m m e n d e d n u m b e r .

T he d i s s o l v e d oxygen

c o n te n t w a s a l w a y s ab o v e 5 ppm a n d a v e r a g e d 8 ppm .

E x p erim en tal p ro c e d u re .

C o n c e n t r a t i o n s to be t e s t e d w e r e

m a d e up by a d d in g p o t a s s i u m c h r o m a t e c r y s t a l s

(M a llin c k r o d t A n ­

a l y t i c a l R e a g e n t) d i r e c t l y to the f il le d a q u a r i a ; th e r e s u l t a n t s o lu tio n
w a s th e n a n a l y z e d f o r h e x a v a le n t c h r o m i u m .
ti o n s

E x p erim en tal c o n c e n tra ­

s e l e c t e d w e r e b a s e d on p r o g r e s s i v e b is e c t io n of i n t e r v a l s on a

lo g a rith m ic

s c a le .

P i l o t t e s t s w ith few f is h an d a wide r a n g e of

c o n c e n t r a t i o n s w e r e done to e s t a b l i s h th e a p p r o x im a te t o l e r a n c e
l i m i t s b e f o r e th e b i o a s s a y began*

A c o n t r o l ta n k of te n f is h u n d e r

th e e x p e r i m e n t a l c o n d itio n s w as t e s t e d s im u l ta n e o u s l y w ith the b i o ­
assay.

If th e c o n t r o l ta n k h a d a m o r t a l i t y of h i g h e r th a n 10 p e r c e n t ,

th e t e s t r e s u l t s w e r e c o n s i d e r e d u n r e l i a b l e .

T h e f is h w e r e h a n d le d

w ith s m a l l d ip n e ts a n d m e a s u r e d an d w eigh ed p r e v i o u s to th e s t a r t
of th e t e s t .

T h e t e s t s w e r e r u n fo r bo th

T h e d a ta a r e
a n c e l i m i t (T L m ).

24

a n d 48 h o u r s .

re p o r te d a s a 2 4 -h o u r or 4 8 -hour m e d ian t o l e r ­
T h i s i s e s t i m a t e d by s t r a i g h t - l i n e

g ra p h ic a l

i n t e r p o l a t i o n u s i n g p e r c e n t s u r v i v a l on th e o r d in a t e an d log., d o s e
( e x p r e s s e d in m g . C r

/l.)

on th e a b s c i s s a .

In o r d e r to

do t h i s ,

it i s n e c e s s a r y to h a v e

tw o t e s t c o n c e n t r a t i o n s , one in w hich

th e

s u r v i v i n g f r a c t i o n i s ab o v e 0.5 0 but b elo w 0.90, an d th e o th e r with
as u r v i v i n g f r a c t i o n
s e e n th a t th e

b elo w 0.5 0 but ab o v e 0.10.

It c a n r e a d i l y

be

c l o s e r t h e s e f r a c t i o n s a r e to 0.50 th e m o r e a c c u r a t e

th e e s t i m a t e w ill b e .

By e x te n d in g a s t r a i g h t lin e b e tw e e n t h e s e

two p o in t s , th e T L m i s

r e a d in m g. C r

cro sses

su rv iv a l ax is.

th e 50 p e r c e n t

+

6

/ l . at th e point

th e lin e

F o r th e b i o l o g is t i n t e r e s t e d in s t a t i n g a p r e s u m a b l y h a r m l e s s
co n c e n tra tio n

(C) of a w a s t e , H a r t , D o u d o ro ff, a n d G r e e n b a c k (1945)

s u g g e s t e d th e fo llo w in g e q u a tio n with r e s e r v a t i o n s :
C = (48 h r . T L m x 0 .3 ) /( S 2 )
S = (24 h r . T L m ) /( 4 8 h r . T L m )

D r a w i n g of b lo o d

It w a s fou n d e a r l y in th i s w o rk th a t th e p r o c u r i n g of a v a lid
sam p le
ced u re.

of b lo o d f r o m

s m a l l f is h w ould p ro v e to be a d iffic u lt p r o ­

T h e c u ttin g off of th e t a i l a t th e c a u d a l p e d u n c le a n d c o l ­

l e c t i n g th e b lo o d a s it flo w ed f r o m th e c a u d a l v e i n i s a p o p u la r
m e th o d in f is h r e s e a r c h .
m e n ts.

T h e h ig h v a r i a n c e

It w as t r i e d in the e a r l i e r

p ilo t e x p e r i ­

of th e s a m p l e s o b ta in e d by th is m e th o d

33

i n d i c a t e d th e b lo o d w a s b e in g c o n t a m i n a t e d , p r o b a b ly by s p i n a l and
i n t e r s t i t i a l f lu i d s .

A f t e r p r a c t i c e , b lo o d w a s o b ta in e d by h e a r t

p u n c t u r e f r o m f i s h a n e s t h e s i z e d w ith 3 00 m g. M S - 2 2 2 / l.
m e th a n e s u lf o n a te ) .

It

w as

(T ric a in e

v i r t u a l l y i m p o s s i b l e to m a k e a s u c c e s s f u l

h e a r t p u n c t u r e th r o u g h th e s k in in t h e s e

s m a l l fish ; t h u s , a v e n t r a l

i n c i s i o n w a s m a d e to e x p o s e th e h e a r t , an d th e p e r i c a r d i u m w as
s t r i p p e d off to f r e e th e v e n t r i c l e .

Next a 0.25 m i l l i l i t e r

sy rin g e ,

r i n s e d in 1000 U S P / m l . h e p a r i n s o lu tio n , an d f it te d w ith a 27 gauge
n e e d l e , w as i n s e r t e d s t r a i g h t down in to th e v e n t r i c l e a t i t s b a s e n e a r
th e ju n c tio n of th e t r u n c u s a r t e r i o s u s .
ti v e p r e s s u r e
in to i t .

w ith in the

By k e e p in g a s lig h tly n e g a ­

s y r i n g e e a c h h e a r t b e a t would pum p blood

No b lo o d s a m p l e of l e s s th a n 0.1 m l. w as u s e d in t h e s e

d e t e r m i n a t i o n s , s in c e it w a s f e lt th a t th e r e s i d u e of h e p a r i n , a lth o u g h
a s m u c h a s p o s s i b l e h a d b e e n e x p e lle d b e f o r e ta k in g th e
w o u ld c a u s e a s e r i o u s d ilu tio n e r r o r .

sam p le,

The a v e r a g e s a m p l e w as b e ­

tw e e n 0*1 a n d 0.2 m i l l i l i t e r .

H e m a to c rits

T h e b lo o d d r a w n by h e a r t p u n c tu r e w as e x p r e s s e d out of th e
s y r i n g e onto a w ax s u r f a c e ta k in g c a r e to av o id a i r b u b b le s w hich
m ig h t g e t in to th e b lo o d p i p e tte s .
f i l l e d up to th e

A v a n A lle n h e m a t o c r i t tu b e w as

100 m a r k f r o m the pool of b lo o d , a f t e r w hich it w as

34

d il u te d w ith a 0.6 p e r c e n t

s o d iu m c h l o r i d e u n til th e bulb of th e

h e m a t o c r i t tu b e w as t h r e e - f o u r t h s fu ll.

T he tu b e w a s

s e a l e d with

a s p r i n g c lip a n d c e n t r i f u g e d a t 25 00 R P M f o r tw e n ty m i n u t e s .
accu racy
to c rits

T he

of t h i s t e s t w as d e t e r m i n e d by ta k in g fo u r d i f f e r e n t h e m a ­

from

a

sta n d a rd e r r o r

s in g le
w as

b lo o d

sam p le

f r o m one f is h .

T he r e s u l t a n t

0.72 m l./lO O m l.

H e m o g lo b in s

H e m o g lo b in c o n te n t of th e b lo o d w a s d e t e r m i n e d a s a c id
h e m a tin as

o u tlin e d in th e B a u s c h a n d L o m b S p e c t r o n 2 0 C l i n i c a l

T e c h n iq u e M a n u a l.
lite rs

T w en ty cu b ic m i l l i m e t e r s w e r e a d d e d to 5 m i l l i ­

of 1 p e r c e n t HC1 an d a llo w e d to s ta n d one h o u r .

S ince fish

b lo o d i s n u c l e a t e d , th e r e s u l t a n t s o lu tio n w as s lig h tly t u r b i c , w hich
i n t e r f e r e d w ith th e c o l o r i m e t r i c

m e th o d .

T h e n u c le i w e r e r e m o v e d

by c e n t r i f u g i n g a t 2500 R P M f o r 10 m i n u t e s , and th e

s u p e r n a ta n t

w a s r e a d on the c o l o r i m e t e r at 52 5 rnp w av e le n g th .

T he s t a n d a r d s

f o r h u m a n b lo o d p r o v id e d w ith th e m a c h in e w e r e u s e d , a n d th e r e ­
s u l t s w e r e r e p o r t e d in g r a m s of h e m o g lo b in p e r
L ik e
m e th o d

w as

th e

h e m a to c rit

e s ta b lish e d

s in g le b lo o d s a m p l e .

by

d e te rm in a tio n ,
ta k in g

s ix

th e

100 g r a m s of blood.
acc u ra cy

h e m o g lo b in

of th i s

sam p les

from a

T h e s t a n d a r d e r r o r w as 0.32 g r a m s h e m o -

g l o b i n /1 0 0 g r a m s b lood.

35

R e d b lo o d c e l l co u n t

A s t a n d a r d r e d b lo o d p ip e tte w as f il le d f r o m th e pud d led
b lo o d , a n d a 1:200 d ilu tio n w a s m a d e u s in g 0.6 p e r c e n t s o d iu m
c h l o r i d e a s th e d ilu ta n t.

A r e d b lo o d c e l l count w as m a d e in th e

a c c e p t e d c l i n i c a l m a n n e r u s in g a L evy h e m o c y t o m e t e r .

R e d b lo o d c e l l le n g th

C e l l le n g th s w e r e d e t e r m i n e d f r o m a i r - d r i e d b lo o d s m e a r s
w h ich w e r e

s t a i n e d five m i n u te s w ith W r i g h t 's b lo o d s ta in .

Random

m i c r o s c o p i c f i e l d s w e r e b r o u g h t in to fo c u s an d a l l c e l l s th a t would
a l in e p r o p e r l y , a s th e m i c r o m e t e r w as r e v o lv e d th r o u g h a 180° a r c ,
w ere m e a su re d .

T h i s w a s a slow p r o c e s s a s often no c e l l s would

be i n lin e in a g iv e n f ie ld , but s in c e r a n d o m n e s s w a s a d e s i r a b l e
f a c t o r , th i s m e th o d w as fa ith fu lly p u r s u e d .

C e l l le n g th s a r e

re­

p o r t e d in m i c r o n s .

P l a s m a v o lu m e a n d b lo o d v o lu m e

P l a s m a v o lu m e w a s done by th e dye d ilu tio n m e th o d u s in g
E a s tm a n 's

T - 1 8 2 4 (E v a n s B lu e ).

th e e x p o s e d v e n t r i c l e
tu b e rc u la r

T h e dye w as in j e c t e d d i r e c t l y in to

of a n a n e s t h e s i z e d f is h by m e a n s of a 1 m l.

s y r i n g e m a n ip u la t e d by a m i c r o b u r e t m a n u f a c t u r e d by th e

M i c r o - m e t r i c I n s t r u m e n t C o m p a n y ( F i g u r e 2).

A

num ber

30

Figure 2.

Mcro-injection

apparatus.

36

37

g a u g e i n j e c t i o n n e e d l e w a s n e e d e d to in t r o d u c e th e dye with no le a k a g e of b lo o d d u r in g o r a f t e r th e i n j e c ti o n .

U sin g a 6,0 m g . / m l .

s o l u t i o n of d y e , 0.074 m g . w e r e i n j e c t e d in to th e f is h in a t o t a l
v o lu m e of 0.012 m l.

If b le e d in g due to th e i n c i s i o n , o r d u r in g o r

a f t e r th e in j e c t i o n w a s u n u s u a lly p r e v a l e n t , th e f is h w a s d is c a rd e d *
Of c o u r s e , t h e r e w a s a lw a y s a s m a l l a m o u n t of s e e p a g e of b loo d ,
b u t due to th e f a s t c o a g u l a ti o n t i m e

of f is h b l o o d - - a m a t t e r of a

few s e c o n d s - - t h i s w a s a t a m i n im u m .
th is e r r o r
be

It w as f e lt th a t to c o u n t e r a c t

due to b le e d in g , th e v o lu m e of d y e, 0.012 m l . , w ould not

s u b t r a c t e d f r o m th e fin al r e s u l t s .

T h e n e e d le w a s not w ith ­

d r a w n f r o m th e v e n t r i c l e u n til a l l v i s i b l e c o n c e n t r a t i o n of dye w as
r e m o v e d f r o m th e h e a r t .

T h i s p r e v e n t e d le a k a g e of th e d y e , and

a l s o h e l p e d s e a l th e p u n c t u r e q u ic k ly due to th e a c t io n of u n d ilu te d
b lo o d .

It w a s found b e s t to r e m o v e th e n e e d le a t th e p e a k of

s y s t o l e o r b e g in n in g of d i a s t o l e , so th e blood would not be s q u e e z e d
out of th e f r e s h hole*
T he i n j e c t e d f i s h w e r e

p la c e d on t h e i r b a c k s in a V - s h a p e d

t r o u g h lin e d w ith w et g a u z e , a n d a r t i f i c i a l r e s p i r a t i o n w a s in d u c e d
by m e a n s

of w a t e r flo w in g o v e r t h e i r g il ls . ( F ig u r e 3).

W a te r f r o m

a n a q u a r i u m r e s e r v o i r w a s le d to th e f i s h by m e a n s of r u b b e r
tu b in g a n d a g l a s s tu b e w h ich w as p la c e d in th e f i s h ' s m o u th .
W a t e r flo w e d a t th e r a t e

of 15 0 d r o p s p e r minute*

T h e f is h , u n l e s s

Figure

3.

Artificial respiration

apparatus.

38

39

f
it w a s d e e p ly a n e s t h e s i z e d , w ould p u m p the w a t e r o v e r th e g il ls
w ith r e l a t i v e l y n o r m a l o p e r c u l a r m o v e m e n ts .

If o p e r c u l a r m o v e ­

m e n t s w e r e a b s e n t , s lig h t s t i m u l a t i o n on th e v e n t r a d of th e m o u th
w o u ld c a u s e a s p a s m o d i c
re sp ira to ry

m o v e m e n t and h a s t e n th e r e t u r n of th e

re fle x e s.

T h e f is h w ould r e m a i n in t h i s

p o s itio n f o r t e n m i n u t e s ,

a lth o u g h th e y w ould u s u a ll y h av e to b e r e m o v e d to be r e a n e s t h e s i z e d
a t l e a s t once d u r in g th e p e r i o d .

T e n m i n u t e s w as c o n s i d e r e d s u ffi-

c i e n t t i m e to c o m p le te th e e n t i r e m ix in g of dye ev e n th o u g h the
c i r c u l a t i o n t i m e of th e f is h w as unknown.
tim e

of O p s a n u s ta u w a s two m i n u te s

The c a l c u l a t e d c i r c u l a t i o n

(B ro w n , 1957), w h ich , if the

t r o u t w e r e a p p r o x i m a t e l y e q u a l, w ould i n d i c a te five c o m p le te c i r c u l a ­
ti o n s of th e dye w ith in th e p e r i o d allo w ed .
A t th e e n d of th e te n m i n u te s b lo o d w as d ra w n f r o m th e fis h
as

d e s c r ib e d above.

fuge

tu b e

T h e b lo o d w as th e n e x p r e s s e d in to a 1 m l, c e n t r i ­

f r o m w h ich a h e m a t o c r i t s a m p l e w a s r e m o v e d .

The r e ­

m a in in g b lo o d w a s c e n t r i f u g e d a t 25 00 R P M fo r te n m i n u te s .

T he

p l a s m a w a s d il u te d by a d d in g 40 c m m s of it to 0.5 m l. of d i s t i l l e d
w a t e r in a c u v e t t e .

T h i s d ilu tio n w as r e a d in th e S p e c tro n ic 2 0 a t

a w a v e le n g th of 65 0 m q .

The c o n c e n t r a t i o n of th e dye in the u n ­

d il u te d p l a s m a w a s t h e n d e t e r m i n e d f r o m a s t a n d a r d c u r v e m a d e
p r e v io u s ly by r e a d i n g know n d ilu tio n s in the c o l o r i m e t e r .

40

T h e a c c u r a c y of t h i s t e s t is unknow n s in c e two o r m o r e d e ­
t e r m i n a t i o n s c o u ld n o t b e m a d e

on th e

s a m e f i s h , a n d t h e r e a r e no

know n d a t a of t h i s ty p e f o r th i s

s p e c i e s w hich c o u ld be c o m p a r e d .

S p le n e c to m ie s

F i s h to b e s p e l e n e c t o m i z e d w e r e a n e s t h e s i z e d a s p r e v io u s l y
d e s c r i b e d , p la c e d in th e V - s h a p e d tr o u g h a n d a d m i n i s t e r e d a r t i f i c i a l
re sp ira tio n .

A s m a l l i n c i s i o n , about

1.5 c m . lo n g , w a s m a d e by

m e a n s of a p o in te d s c a l p e l in th e v e n t r a l a b d o m e n s lig h tly p o s t e r i o r
to a m id p o in t b e tw e e n th e p e c t o r a l a n d pelvic g i r d l e s .
w ould u s u a ll y lie b e n e a th t h i s lo c a tio n .

T he s p le e n

By m e a n s of a p o in te d

f o r c e p s , th e s p l e e n w a s w ith d r a w n th r o u g h th e i n c i s i o n and cu t off.
It w a s found th a t th e v e s s e l s

s e r v i n g th e s p l e e n w e r e s m a l l , an d

t h e r e w a s no n e e d of ty in g th e m

off.

With th e s p l e e n r e m o v e d , th e

i n c i s i o n w a s c l o s e d w ith one s u t u r e , an d th e a r e a d r i e d w ith a
s p o n g e a n d c o v e r e d w ith c o llo d io n .

By t h i s t i m e th e f is h w ould be

out of th e a n e s t h e t i c en ou g h to r e t u r n i t to an a q u a r i u m .
a t i o n d id n o t a p p e a r to u n duly h a r m th e fis h .

T his o p e r ­

F i f t e e n m i n u te s a f t e r

b e in g r e t u r n e d to th e ta n k th e y sh o w ed no s ig n s of a b n o r m a l b e ­
h a v i o r a n d w ould f e e d on t r o u t p e l l e t s .

41

B ody w a t e r

T o t a l body w a t e r , i n g r a m s

p e r 100 g r a i n s of body w eig h t,

w a s d e t e r m i n e d on f is h w h o se l i v e r , g a ll b l a d d e r , s p l e e n , k id n e y ,
and im m a tu re

gonads w ere rem o v ed .

T h e f is h w e r e w e ig h e d to th e

n e a r e s t te n th of a g r a m an d d r i e d in a n ov en 95° C . to 105° C. f o r
48 h o u rs*

T h e y w e r e c o o le d i n a d e s i c c a t o r a n d a g a in w eig h ed , the

l o s s in w eig h t b e in g c o n s i d e r e d a s t o t a l body w a t e r .
T h e m a i n o b je c tio n to th e m e th o d w ould be th e fa c t th a t th e
o r g a n s w e r e r e m o v e d p r e v i o u s to the d e t e r m i n a t i o n .
e r r o r i n t r o d u c e d by t h i s

The g re a te s t

p r o c e d u r e w ould s t i l l be s m a l l .

m e a n w eig h t of th e f is h w ith o ut o r g a n s a s
a v e r a g e body w a t e r w eig h t a s

U sin g th e

15.6 g r a m s a n d th e

12.2 g r a m s , it c a n b e d e t e r m i n e d th a t

th e w a t e r w o u ld c o n s t i t u t e 7 8 p e r c e n t of th e body w eigh t.

By a d d in g

th e w e ig h t of th e e x c i s e d o r g a n s , th e m e a n w eig h t of th e wet f is h
becom es

16.0 g r a m s ;

an d a s s u m i n g th e o r g a n s to b e 90 to 100 p e r ­

c e n t w a t e r - - a n e x t r e m e e s t i m a t e - - t h e t o t a l body w a t e r w ould b e c o m e
79 p e r c e n t of th e body w e ig h t.
r e m o v a l of t h e s e

T h is m e a n s th e e r r o r

o r g a n s w ould be

in t r o d u c e d by

1.4 p e r c e n t a t th e v e r y

m o s t.

O r g a n w e ig h ts
F re sh ly

r e m o v e d o r g a n s w e r e w e ig h e d on a R o l l e r - S m i t h

b a l a n c e a n d th e r e s u l t s

r e p o r t e d in m i l l i g r a m s .

R E S U L T S AND DISCUSSION O F PHY SIO LOG ICA L V ALUES O F
T H E NORM A L H A T C H E R Y -R A IS E D RAINBOW TR O U T

T h e p a u c ity

of p h y s io lo g i c a l d a ta p e r t a i n i n g to f is h i n g e n e r a l ,

a n d r a in b o w t r o u t in p a r t i c u l a r , m a d e it i m p e r a t i v e to e s t a b l i s h
n o rm al
groups.

p h y s io lo g i c a l

v alu es

T o o b ta in t h i s

th r o u g h

th e

use

of l a r g e

p h y s io lo g ic a l d a ta f r o m

co n tro l

s m a l l f is h , it w as

n e c e s s a r y to d r a s t i c a l l y a d a p t m e th o d s e m p lo y e d in m a m m a l i a n
p h y s io lo g y to th e p r o b l e m s e n c o u n t e r e d .

T h e s e m o d i f ic a t io n s a r e

p r e s e n t e d in th e e x t e n s i v e a n d d e t a i l e d s e c ti o n e n t itl e d

' 'M a te ria ls

a n d M e th o d s .
T h e d a ta a n d t h e i r

s ta tistic a l

e v a lu a tio n p e r t a i n i n g to th i s

s e c t i o n c a n b e fou n d i n A p p en d ix II, P a r t A.

O r g a n W e ig h ts

T h e a n a l y s i s of t h e s e d a ta on th e k id n e y , l i v e r , a n d s p le e n
w a s a p p r o a c h e d in th e follow ing w ay s:

(1) a s a l i n e a r r e g r e s s i o n

of o r g a n w eig h t on body w e ig h t, an d (2) o r g a n w e ig h ts e x p r e s s e d a s
p e r c e n t of body w eig h t.
A ll r e g r e s s i o n a n d c o r r e l a t i o n c o e f f ic ie n ts w e r e
t e s t e d , an d th e y p r o v e d to be h igh ly s ig n if ic a n t.

42

sta tistic a lly

The s tr a ig h t lin e s ,

43

f i t t e d by th e l e a s t

squares

m e th o d , v e r i f y i n g t h i s c o r r e l a t i o n of o r ­

g a n w e ig h t on body w eig h t a r e i l l u s t r a t e d in F i g u r e 4, f o r the kidney;
F i g u r e 5, f o r th e l i v e r ;

and F i g u r e

6, f o r the s p le e n .

T h u s , h a v in g p r o v e d the p r e m i s e th a t o r g a n w e ig h ts a r e
d i r e c t l y r e l a t e d to body w e ig h t, th e p r e d i c t i o n e q u a t io n s , in c lu d in g
th e s t a n d a r d e r r o r

of th e e s t i m a t e , can be s t a t e d a s fo llo w s:

K idney:
Y T, 20,43 m g. = -1 0 .5 0 4- 8.75 x g r a m s
L iv er:
Y +, 40.46 m g, = -38.93 +

12.89 x g r a m s

S p leen:
Y +, 11.53 m g. = -7 .93 + 2.97 x g r a m s
Y i s th e p r e d i c t e d w eig h t of th e o r g a n a n d x i s th e g iven body
w eig h t.
T h e r e i s no e v id e n c e th a t e x t r a p o l a t i o n of th e d ata bey o n d
th e r a n g e d e t e r m i n e d e x p e r i m e n t a l l y w ould be v a lid ; t h e r e f o r e , the
u s e of t h e s e

e q u a tio n s b e y o n d th e r a n g e of th e g r a p h s

sh o u ld be

q u e s ti o n e d .
W h en e x p r e s s e d in t e r m s

of o r g a n w eight a s a p e r c e n t a g e of

body w e ig h t, th e m e a n s of t h e s e d a ta c o l l e c t e d on te n f is h a r e
fo llo w s:

as

44

LO
rv]

OO

CO

a

OJ
tuo

I

X!

tuo

•»—I

cj
m -o
PQ

240

CO

o

o
00

Csj

o

vO

o

Tt<

o

ro

O
O

rv]

K id n e y W eight ( m illig r a m s )

00

o

OO

o

Figure 4, Normal kidney weights,
(Dotted lines indicate standard error of the estim ate.)

oo

45

s

cd
bJO
X!

•OJD
H
<D
£
in

o

PQ

240

CO

o

04

00

o

vO

o

o

CM

L iver

W eight

(m illig ra m s)

o

oo

o

vO

oo
rt*
o

Figure 5.
(Dotted lines indicate

OO to

Normal liver weights.
standard error of the estimate.)

CO
rxj

100

00

o

o

O '*

oo

o

CO
m
S p le e n W eight ( m illig r a m s )

o

o

o

o

o

o

oo

GQ

cd
Sh

s

too

*<u
£

too

O
PQ

Figure 6. Normal spleen weights.
(Dotted lines indicate standard error of the estim ate.)

46

47

Kidney:
°*77 g/lO O g. of body weight £ the standard error
of 0.0034 g . / 100 g.
Liver:
1.02 g./lOO g. of body weight +, the standard error
of 0.08 g./lOO g.
Spleen:
0.24 g./lOO g. of body weight +, the standard error
of 0.023 g . / 100 g.
No information on organ weights pertaining to rainbow trout
or m em bers of the family Salmonidae was found in the literature,
although Krumholz (1956) reported data for eight species of warm
water fish which had been exposed to low level radioactive wastes.
His data can be found in Table 8 of this section.
Krumholz's resu lts on the kidney range from 0.11 to 0.98
percent, and on the liv er from 0.84 to 3.65 percent.

The results

presented herein fall well within the ranges for these organs.

The

data on spleen weight reported by Krumholz ranged from 0.06 to
0.14 percent, which is w ell below that found for the trout, 0.24 per*
cent.
There are two points that should be emphasized in this com ­
parison of data.

F ir st, there are definitely species differences

48

TABLE

8

A V E R A G E P E R C E N T A G E O F T O T A L WEIGHT O F S TA B LE
TISSUES C O N T R IB U T E D BY EACH TISSUE D ISSEC TED
F R O M INDIVIDUAL O F E IG H T S P E C IE S O F FISH
F R O M WHITE OAK LAKE
( K ru m h o lz , 1956)

Fish

Kidney

Liver

Spleen

1.19

0.11

0.11

1.03

0.10

0.16

1.17

0,06

Largemouth B a s s ........................ . . .

0.20

0.84

0.08

Carp <,......................................

0.59

3.65

0.13

............................................, . . .

0.98

2 .25

0.05

Redhorse ............................................ , . . .

0.57

2.00

0.11

Gizzard S h a d ................................... . . .

0.78

1.90

0.14

B l u e g i l l ................. ..............................
Black Crappie

........................... .

White Crappie

............................... . . .

Bullhead

. . .

. . .

i n d i c a t e d by K r u m h o l z 's d a ta on w a r m w a t e r f is h .

The tro u t, a

c o ld w a t e r s p e c i e s , m ig h t e a s i l y d if f e r to a g r e a t e r e x te n t, a s e v i ­
d e n c e d by th e s p l e e n w eig h t.

S ec o n d ly , th e fact th a t the f is h u n d e r

i n v e s t i g a t i o n by K r u m h o lz w e r e e x p o s e d f o r long p e r i o d s of ti m e
to r a d i o a c t i v i t y m u s t b e r e c o g n i z e d 0

49

Data were obtained on gall bladder and gonad weights, a l­
though they were found to be too variable to be of use in this work.
The gall bladder was weighed full of b ile, and its weight reflected
the secretion and expulsion of bile which are variables that may or
may not be logically correlated with body weight.

Gonad weights

were extrem ely variable in these young fish due to their being in
various stages of sexual development.

Both the gall bladder and

gonad resu lts are presented in Appendix II.

T o t a l B ody W a t e r

These data, obtained from ten fish, are handled in the same
manner as the organ weights.

Once again the regression and c o r ­

relation coefficients proved to be highly significant, and the following
r e g ressio n equation was calculated:
Y i . 0,47 g. = -0 .6 2 +

0.82 x

F i g u r e 7 i l l u s t r a t e s t h i s c o r r e l a t i o n b e tw e e n t o t a l body w a t e r on
body w e i g h t .
W hen e x p r e s s e d in t e r m s of p e r c e n t
of t h e

re su lts

of body w eig h t, th e m e a n

d e t e r m i n e d on t e n f is h is:
7 7 .4 g ./lO O g. of body w eig h t
of 1,02 g./lO O g.

±,

th e s t a n d a r d e r r o r

mt

00

CO

o

00

n*

m

o

T o ta l B od y W ater

<— I

I— I

>— I

00

(g ra m s)

o

00

01

u
bJO

scd

•I—
t

<1)

lD TD
O

m

Figure 7,
Body weight plotted against body water.
(Dotted lines indicate standard error of the estimate,)
50

51

Brown (1957), in her recent compilation of published work,
states that the r esu lts of water determinations are difficult to com ­
pare due to the widely differing techniques used by various authors.
After a com plete review of the literature* she came to the con­
clusion that 1'fish in general contain about 80 to 85% water, extreme
values ranging from 53 to 89.3%l"

These data on the trout, although

below 80 percent, still fall w ell within the range.

He mat oc r it s

H em atocrits were found to be 31,8 i, a standard error of
1,39 ml. of red blood c e lls per 100 ml. of blood,

Spector (1956),

in his Handbook of B iological Data, a complete review of all litera­
ture, reported an average hematocrit for rainbow trout of 27.2 ml,
R.B.C./lOO m l. of blood with a range in data between 22 ml. to
36 ml./lOO m l.

Hemoglobin

The mean of the hemoglobin determinations on ten fish were
found to have a value of 6.51 ± the standard error of 1.27 g./lOO ml.
of blood. '“Spector (1956) reports for trout a hemoglobin value of
8,5 g ,/l0 0 m l. with a range of 6.2 g. to 11.5 g./lOO ml.

P ro sser

52

etjal. (1950) gives a range from 8.7 g. to 17.6 g./lOO ml. for fish
in general.
The data reported in this work fall in the lower part of the
range reported by Spector (1956).

This may be due in part to the

general adoption of mammalian techniques without the adaptations
n ecessary for nucleated blood.

The usual methods call for a

colorim etric analysis of hemolyzed blood at some point during the
procedure.

If the interfering nuclei are not removed prior to this

manipulation, the resu lts w ill be high.

The author made a few

determinations of hemoglobin on nucleated sam ples and found these
values agreed quite w ell with the upper range published by Spector.
They also agreed very w ell with Black's (1955) values of 11.0 g ./
100 m l.

Red Blood C ell Counts

The red blood c e ll counts on ten fish averaged 1.11 «±, 0.098
m illion c e lls/c m m .

This agrees very w ell with Spector's (1956)

value for trout of 1.01 m illion c e lls/c m m . with a range of 0,74 to
1.5 m illion c e lls/c m m .

P r o sser et a l. (1950) reports a range for

fish in general of 0.585 to 2.685 m illion/cm m .

53

Red Blood C ell Lengths

By taking an average length of ten c e lls /fis h and using ten
fish, a value of 14.69 p

the standard error of 0.24 was determined.

P ro sser et a l. (195 0) gives a value of 16.7 ju for trout.

It is inter*

esting to note at this point that Brown (1957) published data on the
e e l, 14.6 p. in length and 1.10 m illion c e lls/c m m . in number, which
agree clo sely with the trout data reported here.

This i s , of course,

no more than an interesting observation.

Plasm a Volume and Blood Volume

The regression and correlation coefficients relating plasma
and blood volume to total body weight were found to be highly sig ­
nificant.

The following prediction equations, including the standard

error of the estim ate, were calculated*.
Plasm a volume:
Y ±. 0,07 ml. = 0.094 + 0.0154 x grams
Blood volume:
Y

°. 15
0.08 ml. = frTOS O + 0.023 x grams

When expressed in m illiliters per 100 grams of body weight,
the mean of ten determinations is .

P l a s m a v o lu m e :
2.13 m l./lO O g. i , th e s t a n d a r d e r r o r

of 0.14 m l./lO O

B lo o d v o lu m e :
3.25 m l . / 100 g.

±

th e

sta n d a rd e r r o r

of 0.16 m l./lO O

A lth o u g h no p l a s m a o r b lo o d v o lu m e d a ta s p e c if i c a ll y r e l a t e d
to t r o u t c o u ld b e found in th e l i t e r a t u r e , P r o s s e r et a h

(1950) r e ­

p o r t e d a b lo o d v o lu m e f o r t e l e o s t f i s h e s in g e n e r a l of 1.5 to 3 p e r ­
c e n t of body w e ig h t.

B r o w n (1957), in h e r c o m p ila t io n of d a ta on

A c t i n o p t e r y g i i , r e p o r t e d a r a n g e of 1.4 to 2.8 p e r c e n t of th e body
w e ig h t s .
V a r i o u s w o r k e r s h a v e a l l r e p o r t e d blo o d v o lu m e v a l u e s
l o w e r t h a n t h o s e o b s e r v e d by t h i s a u t h o r .

The m o s t p la u s ib le

ex­

p la n a tio n of t h i s d i s c r e p a n c y i s th a t, u n til th i s r e p o r t , b lo o d v o lu m e
e x p e r i m e n t s h a d b e e n p e r f o r m e d on l a r g e f is h , a l l o v e r
A ls o , sp e c ific

a v e ra g e s w e re ca lc u la te d fro m

f i s h , u s u a l l y o n e , tw o , o r t h r e e .
f i s h , s u c h a s u s e d in t h i s

100 g r a m s .

s m a l l n u m b e r s of

T h e r e i s no e v id e n c e th a t young

s tu d y , h av e a l a r g e r b lo o d v o lu m e th a n

a d u lt f i s h , a lth o u g h t h i s p o s s i b i l i t y m u s t b e c o n s i d e r e d in v ie w in g
th e se

d a ta .

In a d d i t i o n , th e m e th o d s u s e d to o b ta in th e p u b lis h e d

r e s u l t s v a r y f r o m d i r e c t w a s h in g to d ilu tio n t e c h n iq u e s e m p lo y in g a
v a rie ty

of v i t a l d y e s .

F u r t h e r , s in c e s p e c i e s d i f f e r e n c e s h a v e b e e n

i n d i c a t e d i n o t h e r p h y s io lo g i c a l d a ta , t h e r e i s r e a s o n to b e l ie v e th i s

55

m ay

be

p u b li s h e d

a

cause
d a ta .

of th e

d isc re p a n c y

b e tw e e n

th i s

w o rk

and

the

RESULTS AND DISCUSSION OF POTASSIUM CHHOMATEINDUCED CHANGES IN THE PHYSIOLOGICAL VALUES
OF HATCHERY-RAISED TROUT

Median Tolerance Limit

T o e s t a b l i s h a w o r k in g c o n c e n t r a t i o n of p o t a s s i u m c h r o m a t e ,
e x p r e s s e d in t e r m s

of th e h e x a v a le n t c h r o m i u m io n , a tw e n ty - f o u r

h o u r m e d i a n t o l e r a n c e l i m i t (T L m ) w a s d e t e r m i n e d .

F ig u re 8 i l ­

l u s t r a t e s th e l i n e a r i n t e r p o l a t i o n of th e b i o a s s a y d a ta w ith a r e s u l t ­
ing t w e n t y - f o u r h o u r T L m of 100 m g . C r / l .

G r in d le y

(1946) r e p o r t s

a to x i c i t y to r a in b o w t r o u t u s in g p o t a s s i u m c h r o m a t e , but h i s r e ­
su lts a r e

s u c h th a t th e y a r e i m p o s s i b l e to i n t e r p r e t i n lig h t of t h i s

e x p e rim e n t.

F rom m

a n d S c h iffm a n (1958) found a T L m f o r l a r g e -

m o u th b a s s of 195 m g . C r / l . u s in g p o t a s s i u m c h r o m a t e .

O s h im a

(1931) found th a t y o u n g e e l s would s u r v i v e in c o n c e n t r a t i o n s of
p o ta ssiu m
ho urs.

c h ro m a te

e q u a l to 52 0 m g . C r / l . fo r an a v e r a g e of 12,4

We c a n c o n c lu d e f r o m th e above th a t t r o u t , an i n t o l e r a n t

s p e c ie s, a re

m ore

s u s c e p t i b l e to th e to x ic e f f e c ts of p o t a s s i u m

c h r o m i u m t h a n e e l o r l a r g e m o u th b a s s .
The b io a ssa y

p r o d u c e d a l e v e l a t w hich 5 0 p e r c e n t of th e

t r o u t d ie w ith in tw e n t y - f o u r h o u r s ; b u t s in c e th i s w o rk i s p r i m a r i l y

56

o
GO

graphical interpolation.

o
o
r-

o
o

O')
O
o

o
o

rvi

o
o

o
o

.°

vO

o

o

Figure

8.

0O

Q

Estimation

o
o

of 24 hour median tolerance

limit by straight-line

CO

O

o
CT'

o

OO

o

o

o
o
rO
m
[—
vD
P e t . S u r v i v a l of F i s h

o

o

58

a i m e d a t l e v e l s of c h r o n i c t o x i c i t y , a c o n c e n t r a t i o n of 2 0 p e r c e n t of
th e T L m , o r 2 0 m g . C r / l . w a s a r b i t r a r i l y c h o s e n .

Tracer Experiments

O n c e t h e w o r k in g l e v e l w a s e s t a b l i s h e d , t r a c e r e x p e r i m e n t s
w e r e done t o f in d th e t a r g e t t i s s u e s .
b o th th e

s ta b le c h ro m iu m

A q u a r i a w e r e s e t up u s in g

and th e r a d i o a c t i v e C r

51

, so th a t e a c h

m i l l i l i t e r o f w a t e r c o n t a in e d 18.6 jig of c h r o m i u m w ith an a c t iv it y
of tw e lv e c o u n t s p e r m i n u te .

T h r e e f is h w e r e e x p o s e d to t h i s c o n ­

c e n t r a t i o n f o r t w e n t y - f o u r h o u r s , a f t e r w hich th e y w e r e s a c r i f i c e d ,
a n d t h e s p l e e n , l i v e r , g a l l b l a d d e r , an d a s e c ti o n of th e c a u d a l
p e d u n c le w e r e a n a l y z e d .

T h e c a u d a l p ed u n cle w as u s e d a s a c o n t r o l ,

s in c e i t i s p r e d o m i n a n t l y m u s c l e t i s s u e ; t h u s , t h e r e w as no r e a s o n
to s u s p e c t it to c o n c e n t r a t e th e c h r o m i u m .
E x p r e s s e d in t e r m s

of m i c r o g r a m s

of c h r o m i u m / 1 0 0 m g , of

t i s s u e a n d a s a m e a n co u nt p e r m i n u te / 1 0 0 g r a m s of t i s s u e , th e r e ­
su lts a r e

sh o w n in T a b l e 9.

A n a l y s i s of v a r i a n c e m e th o d s sh o w e d th a t th e s p le e n a n d g a ll
b la d d e r w ere

s ig n if i c a n tl y m o r e a c tiv e th a n th e c a u d a l p e d u n c le ,

w h e r e a s th e k id n e y a n d l i v e r did not d if f e r f r o m th e c o n t r o l .
B a s e d u p o n th e ab o v e i n f o r m a t i o n , th e path of th e c h r o m i u m
io n t h r o u g h th e f i s h w a s p o s tu la te d .

T he c h r o m i u m io n , having

59

TABLE

9

RESULTS OF TRACER EXPERIMENTS

Organs

Mean cpm./lOO mg.
of T issue

Spleen ............................

34.92

60

33.24

57

13.88

23

8.28

14

4.83

8

Gall bladder

. . . .

Kidney

............... „ .

Liver

........................ ...

Caudal peduncle

. ,»

.

.

• •

C r /l 00 mg. Tissue

gained entrance to the blood by an unknown m eans, is distributed
throughout the animal and tends to be concentrated in the spleen.
The red blood c e ll of mammals is known to have an intense affinity for hexavalent chromium and Cr
in blood volume determinations.

51

labeled c e lls have been used

This means of transport by the

blood of fish seem ed to be highly probable in light of this experi­
ment.
Although the function of the spleen of fish is unknown, based
upon knowledge of mammalian spleens, it can be reasoned that the
reticulo-endothelial system of the spleen could possibly cause a

60

b r e a k d o w n of th e c h r o m i u m - l a d e n c e l l s w ith a r e l e a s e of c h r o m i u m
p r o d u c t s in to th e e f f e r e n t s p le n ic v e s s e l which l e a d s d i r e c t l y to th e
liv e r.

T h e l i v e r c o u ld th e n e l i m i n a t e t h i s c h r o m i u m p r o d u c t by way

of th e b i l e .

A lth o u g h t h e r e w a s no e v id e n c e th a t th e l i v e r c o n c e n ­

tr a te d c h ro m iu m

ab o v e th e l e v e l found in the n o n s p e c if ic m u s c l e

t i s s u e , th e g a l l b l a d d e r , in c lu d in g th e b i l e , i m p l i c a t e d th i s
a p a th of e l i m i n a t i o n of c h r o m i u m ,

organ as

F r o m m an d S ch iffm an (1958)

t h e o r i z e d th a t i t w a s t h i s c h r o m i u m - l a d e n b ile w hich c a u s e d th e
e x t e n s i v e i n t e s t i n a l d a m a g e th a t w a s o b s e r v e d in th e l a r g e m o u th b a s s .
A lth o u g h th e k id n e y w a s not a s s o c i a t e d with c h r o m i u m r e ­
m o v a l by e v id e n c e g a t h e r e d in t h i s e x p e r i m e n t , n e i t h e r did th e e v i ­
d e n c e o m it it a s a p ath w ay .

P o s s i b l y , lik e th e l i v e r , it i s an

a c t iv e r o u t e , but d o e s not c o n c e n t r a t e th e c h r o m i u m w ith in i t s t i s ­
sues.

A n a l y s i s of u r i n e c o u ld b e done to e n lig h te n t h i s h y p o th e s is .

Or g a n W eig h ts an d T o t a l Body W a te r E x p e r i m e n t s

T h u s , h a v in g d e f in ite ly i m p l i c a t e d th e

s p le e n an d l i v e r an d

p o s s i b ly th e k id n e y , s t u d i e s on t h e s e o r g a n s a n d th e t o t a l body w a te r
w e r e done to d e m o n s t r a t e c h a n g e s in th e w eight of t h e s e

o rgans and

c h a n g e s in w a t e r c o m p o s i t i o n due to e x p o s u r e to a p p r o x im a te l y
2 0 mg, C r / l .
exposed

w ith

It w a s found in c o m p a r i n g t h e d ata f r o m five f is h so
th e

re g re ssio n

lin e s

of th e

no rm al

r a in b o w

tro u t,

61

that no differences

could be detected under the experimental con­

ditions .

R esults of Blood Experiments

H e m a t o c r i t s w e r e d e t e r m i n e d on fo u r of th e ab ove five f i s h ,
a n d th e r e s u l t s

a v e r a g e d 54 m l./lO O m l. a s c o m p a r e d to th e 31.8

m lo/lOO m l . o f t h e n o r m a l t r o u t .

In lig h t of th e f o r e g o in g , it w as

d e c i d e d to s tu d y th e b lo o d in g e n e r a l a n d th e c a u s e s f o r th e r i s e
h e m a t o c r i t in p a r t i c u l a r .

in

S in ce th e m a m a l i a n s p le e n a c t s a s a

r e s e r v o i r f o r r e d b lo o d c e l l s , r e l e a s i n g th o s e c e l l s in t i m e of
s t r e s s , a s tu d y of th e
order.
tio n s

s p le n ic r e l a t i o n s h i p s to th e h e m a t o c r i t w as in

T h e fo llo w in g e x p e r i m e n t w as d e s ig n e d to a n s w e r th e q u e s ­
posed above.
I n ta c t a n d s p l e n e c t o m i z e d f is h w e r e kept in ta p w a t e r f o r

tw e n ty -fo u r h o u rs.
c u p erate.
w ere

T h i s w as done to a llo w o p e r a t e d f is h to r e ­

A f t e r t h i s t w e n t y - f o u r h o u r p e r i o d (1) t e n n o r m a l f is h

e x p o s e d to a p p r o x i m a t e l y 2 0 m g , C r / l .

g ro u p );

(norm al ch ro m iu m

(2) te n s p l e n e c t o m i z e d f is h w e r e kept in ta p w a t e r ( s p l e n e c ­

t o m i z e d c o n t r o l g ro u p ); a n d (3) t e n s p le n e c to m i z e d c o n t r o l s w e r e
e x p o s e d to a p p r o x i m a t e l y 2 0 m g . C r / l B ( s p e le n e c to m i z e d c h r o m i u m
group).

S a m p l e s w e r e o b ta in e d f o r a l l t h r e e g r o u p s a f t e r tw en ty -

f o u r h o u r s f o r th e fo llo w in g d e t e r m i n a t i o n s :

(1) le n g th , (2) w e ig h t,

62

(3) hem atocrit, (4) red blood c e ll count, (5) hemoglobin, (6) plasma
volum e, and (7) blood volum e.

Red blood c e ll length determinations

were not done on splenectom ized fish.

The resu lts of the experi­

ments are shown in Table 10.
Plasm a and blood volum es were expressed as a linear r e ­
gression of volume on body weight.

Once again, all regression and

correlation coefficients proved to be highly significant.

The equa­

tions for the r e g ressio n lin e s, including the standard error of the
estim ate, are as follows:

Normal chromium

Plasm a volume:
Y ±, 0.116 ml. = 0.16 x grams
Blood volume:
Y i, 0.116 ml. = 0.04 + 0.02 8 x grams

S p len e cto m ized c o n tro l

Plasm a volume:
Y i

0.084 ml. = 0.164 -1* 0.017 x grams

B lo o d v o lu m e :

Y i. 0.88 m l. = 0.14 + 0.032 x grams

63

TABLE 10
RESULTS OF BLOOD EXPERIMENTS
D e te rm in a tio n

M ean

S tan d a rd E r r o r

N o r m a l F i s h E x p o s e d to 2 0 m g , C r / l ,
H e m a to c rit

. . . . . .

43.8 m l . / 100 m l.

H e m o g lo b in

...................

6.6 g./lOO m l.

0.25 g.

1.25 m i lli o n c e l l / c m m .

0.032 m illio n

R .B .C . c o u n t
C e l l le n g th

...............
...................

P l a s m a v o lu m e
B lo o d v o lu m e

14,93

yx

1.56 m l.

0.15

)i

. , .

1.61 m l./lO O g.

0,12 m l.

. , . , .

3.01 m l . / 100 g.

0.18 m l.

S p le n e c to m i z e d F i s h in T a p W a te r
H e m a to c rit

. . . . . .

2.85 m l./lO O m l.

1.42 m l.

H e m o g lo b in

. . . . . .

5.61 g . / 100 m l.

1.13 g.

1.04 m i l l i o n c e l l / c m m .

0.18 m i llio n

2 .4 4 m l./lO O g.

0.12 m l.

3.85 m l./lO O g.

0.16 m l,

R .B .C . co u n t

. . . . .

P l a s m a v o lu m e

. .

B lo o d v o l u m e ...............

S p le n e c to m i z e d F i s h E x p o s e d to 2 0 m g . C r / l .
2,92 m l.

H e m a to c rit

. . . . . .

40,6 ml./TOO m l.

H e m o g lo b in

............... ...

7.42 g./lOO m l.

0.51 g.

1.38 m i lli o n c e l l / c m m .

0.11 m illio n

. , .

1.74 m l./lO O g.

0.10 m l.

B lo o d v o l u m e ...............

3.16 m l./lO O g.

0.14 m l.

R . B . C . co u n t

...............

P l a s m a v o lu m e

64

Splenectom ized chromium

P l a s m a v o lu m e :
Y i , 0.069 m l . = 0.05 +

0.015 x g r a m s

B lo o d v o lu m e :
Y

±,

0o077 = 0.18 +

0.022 x g r a m s

T h e r e g r e s s i o n li n e s f o r th e p l a s m a v o lu m e d ata a r e
in F i g u r e

p lo tte d

9* a n d f o r th e b lo o d v o lu m e d a ta in F i g u r e 10.

T h e r e s u l t s of t h e s e e x p e r i m e n t s w e r e c o m p a r e d a m o n g t h e m ­
s e l v e s a n d w ith th e n o r m a l t r o u t d a t a .

A n a l y s i s of v a r i a n c e , c o -

v a r i a n c e , a n d t - t e s t s w e r e u s e d w ith th e follow ing r e s u l t s .

H em a to c rit

T h e s p l e n e c t o m i z e d an d in t a c t a n i m a l s e x p o s e d to c h r o m i u m
had h e m a to c rits

s ig n if i c a n tl y h i g h e r t h a n th o s e f i s h , both s p l e n e c t o ­

m i z e d a n d i n t a c t , not e x p o s e d to c h r o m i u m .

H e m o g lo b in

The

s p l e n e c t o m i z e d a n i m a l s e x p o s e d to c h r o m i u m h ad a s i g ­

n if i c a n tl y h i g h e r h e m o g lo b in c o n te n t th a n d id th o s e s p le n e c to m i z e d
figti t h a t w e r e not e x p o s e d .
b etw een

oth er g ro u p s.

T h e r e w a s no e v id e n c e of d i f f e r e n c e s

65

o

00

on

sO
on

Ed

anj

S
h
tuo
tuo

•rH

CD

^

Figure

00

t3
PQ

Comparison

r-H

9.

o

on

of plasma volumes.

jrH

o

00

o

00

o

vO

lD

^

on

o

o

o

o

P la s m a V o lu m e

(m illilite r s)

o

Figure

10.

Comparison

of total blood volumes.

66

I
ti

-O

O

OO

O
T o ta l B lo o d V o lu m e
,- H

-H

vT>

o
O
(m illilite r s)

fM
O

67

K e d b lo o d c e l l co u n t

A c c o r d i n g to th e a n a l y s i s of v a r i a n c e , th e s p le n e c t o m i z e d
a n i m a l s e x p o s e d to c h r o m i u m h a d s ig n if ic a n tly h i g h e r b lo o d c e l l
c o u n t s t h a n th e i n t a c t a n d s p l e n e c t o m i z e d c o n t r o l s .

By

" t"

te st,

th e n o r m a l a n i m a l e x p o s e d t o c h r o m i u m a l s o h a d a h i g h e r blo o d
c o u n t t h a n t h e n o r m a l co un t.

R e d b lo o d c e l l le n g th

By

" t"

t e s t , th e n o r m a l a n i m a l e x p o s e d to c h r o m i u m h a d

s ig n if i c a n tl y l a r g e r b lo o d c e l l s th a n th e n o r m a l t r o u t i n ta p w a t e r .
No m e a s u r e m e n t s w e r e m a d e on s p l e n e c t o m i z e d f is h .

P l a s m a v o lu m e a n d b lo o d v o lu m e

By c o v a r i a n c e , th e s p l e n e c t o m i z e d f is h in ta p w a te r h a d a
s i g n if i c a n tl y h i g h e r p l a s m a a n d b lo o d v o lu m e th a n th e s p l e n e c t o m i z e d
f i s h e x p o s e d to c h r o m i u m .

T he e x p e r i m e n t a l e v id e n c e d id n o t show

a d i f f e r e n c e in th e o t h e r g r o u p s .

D i s c u s s i o n of th e I n c r e a s e in H e m a t o c r i t

T h e h ig h ly s i g n if i c a n t i n c r e a s e in h e m a t o c r i t s of n o r m a l t r o u t
e x p o s e d t o c h r o m i u m c o u ld b e b r o u g h t ab o u t in t h r e e g e n e r a l w ays:

68

(1) by a n i n c r e a s e i n c e l l s i z e , (2) by an i n c r e a s e i n c e l l n u m b e r ,
a n d (3) by a d e c r e a s e in p l a s m a v o lu m e .

Since t h e r e w as a hig h ly

s i g n if i c a n t i n c r e a s e in c e l l s i z e , t h i s m a t t e r w ill b e d i s c u s s e d f i r s t .
If n o r m a l t r o u t h a v e a r e d blood c e l l co un t of 1.11 x 10
c e l l / c m m . an d a h e m a t o c r i t of 3 1 .8 , th e n (1) t h e r e a r e
c e lls/1 0 0

/L

1.11 x 1011

m l . of b lo o d an d (2) e a c h m i l l i l i t e r of th e p a c k e d r e d

b lo o d c e l l s c o n t a i n s 3.5 0 x 10

9

ce lls.

In th e n o r m a l t r o u t e x p o s e d

t o c h r o m i u m , u s i n g th e s a m e m a t h e m a t i c s , it w as found th a t (1) t h e r e
are

1.25 x 10 * 1 c e l l / 1 0 0 m l . , an d (2) t h a t e a c h m i l l i l i t e r of th e

p a c k e d r e d b lo o d c e l l s c o n t a in s 2.85 x 10

9

c e lls.

A s s u m i n g th a t th e

c h a n g e in n u m b e r s of p a c k e d c e l l s w as due to c h a n g e in v o lu m e only,
th e

1.11 x 1011 c e l l s i n th e n o r m a l t r o u t , if c h a n g e d in v o lu m e lik e

t h o s e i n th e c h r o m i u m - e x p o s e d f i s h , w ould c a u s e a r i s e in h e m a t o c r i t
from

31.8 to 3 8.9.

T h e r e f o r e , th e i n c r e a s e in c e l l v o lu m e a c c o u n ts

f o r 5 9 p e r c e n t of the t o t a l r i s e i n h e m a t o c r i t .

If th e c e l l n u m b e r

i n c r e a s e d by 1.40 x 10*° c e l l / 1 0 0 m l. of b lo o d a n d , a s c a l c u l a t e d
a b o v e , t h e r e a r e 2.85 x 10

9

c e l l p e r m i l l i l i t e r of p a c k e d c e l l s , th e n

th e i n c r e a s e i n n u m b e r w ould a c c o u n t f o r 4.9 m i l l i l i t e r s
c e l l s , o r 41 p e r c e n t of th e i n c r e a s e i n h e m a t o c r i t .

of p a c k e d

S ince t h e s e p e r ­

c e n t a g e s w e r e c a l c u l a t e d f r o m m e a n s , th e s t a n d a r d e r r o r

of t h e s e

m e a n s m u s t b e k ep t in m in e w hen c o n te m p la tin g t h e s e r e s u l t s .

69

W hen th e h e m a t o c r i t of a n a n i m a l i n c r e a s e s

s ig n if ic a n tly , th e

blood, v o lu m e m u s t i n c r e a s e a n d / o r th e p l a s m a v o lu m e m u s t d e ­
crease,

U n f o r t u n a te ly , n e i t h e r of t h e s e e v e n ts co u ld be show n to

o c c u r by s t a t i s t i c a l e x a m in a t io n of th e b lo o d a n d p la s m a v o lu m e r e ­
su lts.

T h e r e f o r e , a lth o u g h c h a n g e s m u s t o c c u r , th e y a r e to o s m a l l

to b e m e a s u r e d by th e m e th o d u s e d h e r e i n .
In lig h t of th e ab o v e e v id e n c e , it c a n be c o n c lu d e d th a t a p ­
p r o x i m a t e l y 59 p e r c e n t of th e i n c r e a s e

in h e m a t o c r i t due to e x p o s u r e

to p o t a s s i u m c h r o m a t e i s due to a n i n c r e a s e in c e l l v o lu m e an d a p ­
p ro x im a te ly
ber

41 p e r c e n t of th e i n c r e a s e i s due to i n c r e a s e in n u m ­

of c e l l s .

No e v id e n c e of a ch an g e in p la s m a o r b lo o d v o lu m e

c o u ld b e d e t e c t e d .

D i s c u s s i o n of th e C a u s e s of th e R e d
C e l l V o lu m e C h an g e

T h e r e a s o n s f o r th e r e d c e l l v o lu m e ch an g e w e r e not c o n ­
s i d e r e d e x p e r i m e n t a l l y in t h i s

stu d y .

T h e o re tic a lly , th is in c re a s e

in v o lu m e c o u ld be c a u s e d by a s t r e s s r e a c t i o n s u c h a s p o s tu la te d
by I r v i n g , B la c k , a n d S a ffo rd (1941).

T h e y found, u s in g tw e n ty - f iv e

r a in b o w t r o u t , e r y t h r o c y t e s would s w e ll 1 to 21 p e r c e n t , w ith an
average

s w e lli n g of 10 p e r c e n t , when s u b je c te d to te n s i o n s

d io x id e a b o v e

10 m m B Hg.

of c a r b o n

T he s w e llin g of th e r e d b lo o d c e l l s w as

70

found to b e m o r e i n t e n s e th a n w ould be e x p e c te d if the CO^ te n s i o n
w a s th e only v a r i a b l e

w h ic h d e t e r m i n e d v o lu m e c h a n g e .

In th e c h r o m i u m
not a f a c t o r .

e x p e r i m e n t s h e r e i n , c a r b o n d io x id e w as

U sin g M o o r e 's

(193 9) g r a p h s in c o n n e c tio n with the

a l k a l i n i t y a n d pH of th e s o l u t i o n s , c a r b o n dio x id e w as n e v e r d e ­
t e c t e d in m e a s u r a b l e a m o u n t s .
A n o t h e r c a u s e of i n c r e a s e in v o lu m e co u ld be due to the
a f f in ity of th e e r y t h r o c y t e s fo r c h r o m i u m .

If th e c h r o m i u m

e n t e r s t h e r e d c e l l , th e o s m o ti c c o n c e n t r a t i o n m ig h t be a l t e r e d
s u f f ic ie n tly to c a u s e w a t e r to b e d r a w n in to th e c e l l .
m o le c u la r fo rm

Since th e

of th e c h r o m i u m c o m p le x in th e b lo o d a n d in the

r e d b lo o d c e l l i s unknow n, c a l c u l a t i o n of th e a m o u n t of c h r o m i u m
n e c e s s a r y to c h a n g e th e o s m o ti c p r e s s u r e

in s u c h a way a s to

c a u s e t h i s i n c r e a s e in v o lu m e w as n o t u n d e r ta k e n .

D i s c u s s i o n of th e R o le of th e S p le e n in th e I n c r e a s e
in C e l l N u m b e r u n d e r P o t a s s i u m C h r o m a t e S t r e s s

T h e m a m m a l i a n s p l e e n i s known to s t o r e e r y t h r o c y t e s f o r
r e l e a s e in to th e c i r c u l a t i n g b lo o d d u r in g t i m e s of s t r e s s .

The

s p l e n e c t o m i z e d f i s h e x p e r i m e n t s w e r e d e s ig n e d to d e m o n s t r a t e
w h e t h e r th e f is h s p le e n a d d e d e r y t h r o c y t e s to the c i r c u l a t i n g blood
u n d e r p o ta ssiu m

ch ro m a te

stre ss.

T h e s p le n e c to m i z e d f is h u n d e r

71

c o n t r o l c o n d i tio n s d id not show a d if f e r e n c e in h e m a t o c r i t , h e m o ­
g lo b in , o r c e l l co u n t w hen c o m p a r e d to n o r m a l c o n t r o l f is h .
s p l e n e c t o m i z e d f is h u n d e r p o t a s s i u m c h r o m a t e

stre ss

The

(20 m g. C r / l . )

d id sh o w a s ig n if i c a n t i n c r e a s e in h e m a t o c r i t s a n d r e d c e l l count
a b o v e th e
creased

sp le n e c to m iz e d and in ta c t c o n tro ls .

T he

h e m o g lo b in

in ­

s i g n i f i c a n t l y a b o v e th e l e v e l of th e s p l e n e c t o m i z e d c o n t r o l

f i s h , b u t not of th a t of th e i n t a c t c o n t r o l s .

None of th e v a l u e s d e ­

t e r m i n e d on c h r o m i u m - e x p o s e d s p le n e c to m i z e d f is h d e v ia te d s t a t i s ­
t i c a l l y f r o m t h o s e of th e c h r o m i u m - e x p o s e d n o r m a l fis h .
It c a n be c o n c lu d e d f r o m th e f o r e g o in g e v id e n c e th a t the
p resen ce

of th e s p le e n i s not n e c e s s a r y to i n c r e a s e th e h e m a t o c r i t

under c h ro m iu m

stre ss,

s in c e t h e r e

w as no d if f e r e n c e in e f f e c ts

w h e th e r th e e x p e r i m e n t s w e r e p e r f o r m e d on s p l e n e c t o m i z e d o r i n ­
t a c t f is h .

D i s c u s s i o n of t h e P a r t P l a y e d by th e P o t a s s i u m Ion
in P o t a s s i u m C h r o m a t e T o x ic ity

The r e s u lts
g r a m s of c h r o m i u m

s o f a r h a v e b e e n e x p r e s s e d in t e r m s

of m i l l i ­

io n p e r l i t e r of w a t e r ev en th ough th e co m p o u n d

u s e d th r o u g h o u t t h i s w o rk w as p o t a s s i u m c h r o m a t e ,

S in ce p o t a s s i u m

c h r o m a t e d o e s c o n t a in c o n s i d e r a b l e a m o u n ts of p o t a s s i u m ion, a
f u r t h e r s tu d y w a s u n d e r t a k e n to c o n s i d e r i t s effect on th e f o r e g o in g
re su lts.

72

T h e g r o u p of f is h u s e d in th is e x p e r i m e n t w a s k ep t in c o n ­
c r e t e t a n k s a n d f e d t r o u t p e l l e t s p r i o r to th e t i m e of i t s a r r i v a l a t
th e l a b o r a t o r y , w h e r e a s a l l o t h e r f is h u s e d th r o u g h o u t t h i s stu dy
w e r e t a k e n f r o m h o ld in g ponds; thus* th e y w e r e d e p e n d e n t on n a t ­
u r a l food.

T h i s f a c t c a l l e d f o r the d e t e r m i n a t i o n of new c o n t r o l and

c h r o m i u m ^ e x p o s e d v a l u e s of th e h e m a t o c r i t .
w e r e e x p o s e d to a p o t a s s i u m c h l o r i d e
e q u a l to 2 9 .8 m g . K / l ,
th e p o t a s s i u m c h l o r i d e
p o ta ssiu m c h ro m a te

In a d d itio n , te n f is h

so lu tio n of a c o n c e n t r a t i o n

T h e c o n c e n t r a t i o n of th e p o t a s s i u m ion in
s o lu tio n w as e q u a l to i t s c o n c e n t r a t i o n in the

s o lu tio n .

The r e s u lts a re

show n in T a b le

11.

f,T fr t e s t s f a i l e d to show an y e v id e n c e of a d if f e r e n c e b e ­
tw e e n th e p o t a s s i u m

c h l o r i d e - e x p o s e d f is h and th e c o n t r o l a n d

c h r o m i u m - e x p o s e d f is h .

The v a r i a n c e

(85.55) of th e p o t a s s i u m

c h l o r i d e - e x p o s e d s a m p l e s w e r e s ig n if ic a n tly h ig h e r th a n th e n o r m a l
s a m p l e s * a n d it w a s a s s u m e d th a t t h i s w as a n in d ic a tio n of a s t a t e
of c h a n g e .

It i s i n t e r e s t i n g to no te th a t a s a m p le of fis h e x p o s e d

to 6.6 m g . C r / l . , th e lo w e s t c o n c e n t r a t i o n of c h r o m i u m m e a s u r e d
th a t w ould i n c r e a s e th e h e m a t o c r i t to 43.8 m l./lO O m l . , a l s o h a d a
h ig h ly s ig n if i c a n t v a r i a n c e

(258.75).

P e r h a p s th i s i n c r e a s e in v a r i ­

a n c e of s a m p l e s c a n be e x p e c te d at c o n c e n t r a t i o n s w hich a r e j u s t
s t r o n g en o u g h to c a u s e an i n c r e a s e in h e m a t o c r i t in only p a r t of th e
fish .

If t h i s a s s u m p t i o n is t r u e , th e n th e p o t a s s i u m io n , in a d d itio n

73

TABLE

11

R E S U L T S O F POTASSIUM CH LO RIDE E X P E R IM E N T S

S o lu tio n

KC1 (29.8 m g . K / l . )
Tap w a te r
KCrO.

N um ber
of F is h

H em ato crit
( m l . / 100 m l . )

10

38.8

85.55

10

34.0

13.77

10

40.3

24,44

, ......................

.................................................

(20 m g . C r / l . ) ...................

T1 r-BTi i— ■»1■ 1 ■ !—

. 1 1i ■
—
■■

to th e c h r o m i u m , i s a p o te n tia l in flu e n c e upon th e r e s u l t s
at p o ta ssiu m

V a ri­
ance

o b ta in e d

c h r o m a t e c o n c e n t r a t i o n s e q u a l to 2 0 m g , C r / l ,

W h e th e r th i s
o r a s y n e rg istic

p o te n tia l in f lu e n c e would be an a d d itiv e e ffect

e f f e c t i s unknow n, but it d o e s add s o m e u n d e r ­

s ta n d in g to th e r e a s o n why p o t a s s i u m c h r o m a t e is c o n s i d e r e d to be
m o r e to x ic th a n s o d iu m c h r o m a t e .
of th in k in g in t e r m s
such com pounds a s

T h i s a l s o q u e s ti o n s th e p r a c t i c e

of c h r o m i u m ion c o n c e n t r a t i o n s w hen t e s t i n g
p o t a s s i u m c h r o m a t e , e s p e c i a l l y at the hig h c o n ­

c e n t r a t i o n s in v o lv e d in a c u te

stu d ie s.

74

T h e H e m a t o c r i t a s a n Index of T o x ic ity

O nce h a v in g d e m o n s t r a t e d th a t th e h e m a t o c r i t i n c r e a s e d in
f is h e x p o s e d to p o t a s s i u m

c h r o m a t e , it w as th e n u n d e r t a k e n to d e ­

t e r m i n e th e lo w e s t c o n c e n t r a t i o n of c h r o m i u m a t w h ich t h i s ch an g e
to o k p la c e .

T he i n f o r m a t i o n in T a b le 12, in c lu d in g the r e s u l t s of th e

n o r m a l c o n t r o l g r o u p a n d th e n o r m a l c h r o m i u m g r o u p , a r e
in F ig u r e

p lo tte d

11.

T h e le n g th of th e l i n e s e x te n d in g above a n d b elo w th e p o in ts
p lo tte d in F i g u r e
th e m e a n .

11 a r e

e q u a l to two t i m e s th e s t a n d a r d e r r o r

of

By c o m p a r i n g t h e s e l i n e s , a ro u g h e s t i m a t e of s t a t i s t i c a l

d iffe re n c e s c an be m ade.
F ro m

th is

g r a p h we c a n c o n c lu d e th a t th e h e m a t o c r i t v a lu e

i s i n c r e a s e d a t 1.4 m g . C r / l . a n d r e a c h e s i t s m a x im u m r i s e in
v a lu e a t a point b e tw e e n 2 a n d 4 m g . C r / l .
T h i s i n f o r m a t i o n i s i n t e r e s t i n g to th e s t r e a m
g is t b e c a u s e , a lth o u g h th e 2 4 - h o u r T L m i s

p o llu tio n b i o l o ­

100 m g , C r / l . , in

r e a l i t y , c o n c e n t r a t i o n s b e tw e e n 2 an d 4 m g. C r / l . c a u s e a c t u a l
p h y s i c a l c h a n g e to o c c u r w ith in th e t r o u t .
S in ce th e d e t e r m i n a t i o n of th e h e m a t o c r i t i s a s i m p l e p r o ­
c e d u r e r e q u i r i n g a m i n im u m of e q u i p m e n t, it would be w e ll w o rth
th e i n v e s t i g a t i o n to d e t e r m i n e w h e th e r t h i s r i s e in h e m a t o c r i t c o u ld

Figure

11.

Increase in hematocrit in relation

to potassium

chromate

concentration.
75

M e a n H e m a t o c r i t (m l./lO O m l.

76

TABLE
THE

12

M EAN H E M A T O C R IT S O F FISH E X P O S E D T O VARIOUS
C O N C E N T R A T IO N S O F POTASSIU M CHROM ATE

N um ber
of F i s h

C h ro m iu m
(m g ./l.)

M ean H e m a to c rit
(m l./lO O m l.)

S ta n d a r d E r r o r

10

18.0

43,8

1.56

9

6 *6

43.8

5.08

10

1.4

39.3

1.47

8

0,5

26.7

2.67

10

0.0

31.8

1.39

be u s e d a s a n in d e x of c h r o n ic to x ic ity in m u c h th e s a m e way a s
th e T L m i s u s e d a s an in d e x of a c u te to x ic ity .

T h is would r e q u i r e

e x p e r i m e n t s w h ich w ould d e t e r m i n e w h e th e r an i n c r e a s e d h e m a t o c r i t
w as a g e n e r a l r e s p o n s e to n o n s p e c if ic
to p o t a s s i u m
As

s t r e s s , o r a s p e c if ic r e s p o n s e

c h ro m a te .

s t a t e d e a r l i e r i n t h i s p a p e r , t h i s p r o j e c t d o e s not a t te m p t

to a n s w e r a l l of th e p r o b l e m s e n c o u n t e r e d in s t r e a m

p o llu tio n co n -

t r o l , b u t r a t h e r , p o in ts th e way to a b e t t e r u n d e r s ta n d i n g of th e m .

77

S p le e n F u n c tio n

D u r in g th e c o u r s e of th e e x p e r i m e n t s c o n c e r n e d w ith blood
s t u d i e s , tw o s e r i e s
(see T a b le

of h e m a t o c r i t s w e r e ta k e n on e a c h g ro u p of fis h

13), one s e r i e s in c o n n e c tio n with th e b lo o d v o lu m e d e ­

t e r m i n a t i o n s a n d one s e r i e s

with th e o th e r b lo o d t e s t s .

e n c e s in th e m e th o d s of r u n n in g t h e s e two s e r i e s w e re :

The d i f f e r ­
(1) th e b lo od

f o r th e h e m a t o c r i t s a n d o t h e r b lo o d t e s t s w as d r a w n i m m e d i a t e l y
a f t e r th e f i s h w a s a n e s t h e s i z e d , a n d (2) th e blood f o r th e h e m a t o c r i t s
a n d b lo o d v o lu m e t e s t s
a n e s th e s iz e d and a f te r

w a s d r a w n te n m i n u te s a f t e r th e f is h w as
0.012 m l. of 6.0 m g. T -1 8 2 4 / m l . w as in je c te d .

D e s ig n a ti n g t h o s e h e m a t o c r i t s t a k e n im m e d i a te l y a s h e m a t o c r i t s at
tim e

q>

an d t h o s e t a k e n a f t e r t e n m i n u te s a s h e m a t o c r i t s at t i m e

th e a v e r a g e r e s u l t s a r e t a b u l a t e d in T a b le

13.

W hen th e hig h ly s ig n if ic a n t d if f e r e n c e b e tw e e n th e two s p l e n e c ­
t o m i z e d c o n t r o l h e m a t o c r i t s w a s n o tic e d , i t w as d e c id e d to e lim i n a te
th e p o s s i b i l i t y of th e T -1 8 2 4 a n d the o p e r a t iv e p r o c e d u r e a s a p o s ­
s ib le c a u s e of t h i s i n c r e a s e

d u r in g th e te n m in u te p e r io d .

Those

s p l e n e c t o m i z e d f is h in w hich no T - 1 8 2 4 w a s in j e c te d d u rin g the te n
m in u te p e r i o d

s t i l l sh o w e d a s ig n if ic a n t i n c r e a s e in h e m a t o c r i t ,

w h ile t h o s e t h a t
in c re a se .

w ere

sham

o p erated

show ed

no

e v id e n c e

of an

78

TABLE

13

R E S U L T S O F E X P E R IM E N T S P E R F O R M E D TO
IN V ESTIG A TE S P L E E N FUNCTION

H e m a to c rits
( tim e Q)

H e m a to c rits
(tim e
)

D if f e r e n c e

S p len e cto m ized c o n t r o l .

28.5

36.6

8.1

S p len e cto m ized c h r o ­
. ......................................
m iu m

40.6

44.9

4.3

S p len e cto m ized c o n tro l
no. T - 1 8 2 4
.................... .

28.5

34,9

6.4

31.8

35.0

3.2

43.8

46.3

2.5

33.6

35.8

2.2

G ro u p

N o rm al co n tro l

...............

N o rm al c h ro m iu m

. . .

S h am o p e r a t e d
c o n t r o l .......................... ...

T h e a b o v e e v id e n c e i n d i c a t e s th a t th e s p le e n of t r o u t a c t s a s
a b u f f e r to s u d d e n h e m o c o n c e n t r a t i o n .

T h is fu n c tio n i s l i m i t e d , a s

e v id e n c e d by th e te n d e n c y of th e h e m a t o c r i t to i n c r e a s e in e v e r y
in sta n c e
m e th o d s.
th e

e v e n th o u g h t h i s i n c r e a s e

c an n o t b e v e r i f i e d by s t a t i s t i c a l

O th e r e v id e n c e of th e l i m i t e d n a t u r e of th i s fu n c tio n i s

s ig n if i c a n t i n c r e a s e in h e m a t o c r i t in both in t a c t a n d s p l e n e c t o ­

m i z e d f i s h e x p o s e d to c h r o m i u m , w hich would in d ic a te th e c o m p le te

79

o v e r w h e l m i n g of th e b u f f e r c a p a c ity
p o ta ssiu m c h ro m a te

of th e

s p le e n due to p r o lo n g e d

stre ss.

M o re ev id en ce, g a th e re d fro m e x p e rim e n ts

s p e c if i c a ll y d e ­

s ig n e d to i n v e s t i g a t e th e te n d e n c y of th e s p le e n to a c t a s a b u f f e r ,
i s n e e d e d b e f o r e d e f in ite c o n c l u s i o n s c a n be m a d e .

S ince th i s

w o r k w a s p r i m a r i l y a i m e d a t a n s w e r s to to x ic ity i n v e s t i g a t i o n s , a nd
s in c e t h i s l i m i t e d b u f f e r a c t io n of th e s p le e n did not in flu e n c e th e
r e s u lts under c h ro m iu m

s t r e s s , f u r t h e r i n v e s t ig a ti o n into s p le n ic

f u n c tio n w a s not u n d e r ta k e n .

CONCLUSIONS

I,

T h e fo llo w in g p h y s io lo g i c a l v a l u e s h av e b e e n e s t a b l i s h e d

f o r th e n o r m a l h a t c h e r y - r a i s e d r a in b o w t r o u t (T ab le

TABLE

14):

14

PH Y S IO L O G IC A L VALUES O F THE NORMAL
H A T C H E R Y -R A IS E D RAINBOW TROUT
D e t e r m i n a t i on

M ean V alu e

H em a to c rit

31,8 m l./lO O m l.

S ta n d a r d E r r o r
1.39 m l.

E r y t h r o c y t e co u n t . . . .

1.11 m i lli o n c e l l / c m m .

0.098 m illio n

H e m o g lo b in

6.51 g ./lO O g.

1.27 g.

..........................

E r y t h r o c y t e le n g th

. .

0.24

14.69

)i

. . . »

2.13 m l./lO O g.

O014 m l.

B lood v o lu m e

...................

3.25 m l . / 100 g.

0 al6 m l.

K idney w eight

. . . . . .

0.77 g./lO O g.

0 o039 g.

L i v e r w eig h t

...................

1.02 g./lO O g.

0.077 g„

S p le e n w eig h t

...................

0.24 g ./lO O g.

0.02 3 g.

P l a s m a v o lu m e

T o t a l body w a t e r

. . . .

7 7 .4 g./lO O g.

80

1.02 g.

81

2.

K id n e y , l i v e r , a n d s p le e n w eight; a n d t o t a l body w a t e r and

p l a s m a a n d b lo o d v o l u m e s w e r e found to c o r r e l a t e highly s ig n if ic a n tly
w ith body w e ig h t,
3.

T h e 2 4 h o u r m e d ia n t o l e r a n c e li m it of t r o u t to p o ta s s iu m

c h r o m a t e i n a c o n c e n t r a t i o n e x p r e s s e d in m g, C r / l . i s
4.

T racer

100 m g, C r / l .

e x p e r i m e n t s in d i c a t e d th a t th e s p le e n and th e g a ll

b l a d d e r , in c lu d in g th e b i l e , a c c u m u l a t e d c h r o m i u m above th e le v e l of
a s e c t i o n of th e c a u d a l p e d u n c le , w h e r e a s t h e r e w as no ev id e n c e th a t
kidney o r l i v e r did.
5.
liv e r,

T h e r e w a s no e v id e n c e th a t th e w eig h ts of th e k id n e y ,

s p l e e n , an d t o t a l body w a t e r of f is h e x p o s e d to p o ta s s iu m

c h r o m a t e w e r e d i f f e r e n t f r o m t h o s e of f i s h in ta p w a t e r .
6.
w ere

T h e h e m a t o c r i t s of b o th s p l e n e c t o m i z e d an d in t a c t fish

s ig n if i c a n tl y h i g h e r th a n c o n t r o l f is h in ta p w a t e r .

T h is r i s e

in h e m a t o c r i t w a s found to b e due to an i n c r e a s e in c e l l n u m b e r and
c e l l v o lu m e .
7.

T h e r e w a s no e v id e n c e th a t th e p la s m a o r b lo o d v o lu m e

i n c r e a s e d o r d e c r e a s e d in f i s h e x p o s e d to p o t a s s i u m c h r o m a t e ,
8.

The p re se n c e

of th e

th e h e m a t o c r i t u n d e r c h r o m i u m

s p le e n i s not n e c e s s a r y to i n c r e a s e
s t r e s s , s in c e t h e r e w a s no d if f e r

e n c e in e f f e c t s w h e th e r th e e x p e r i m e n t s w e r e p e r f o r m e d on s p l e n e c ­
t o m i z e d o r in t a c t f is h .

82

9.

It w a s a s s u m e d , on th e b a s i s

th e p o ta ss iu m

io n , in a d d itio n to

e n c e u po n th e

re su lts

of e x p e r i m e n t a l d a t a , th at

th e c h r o m i u m , i s a p o te n tia l in f lu ­

o b ta in e d a t p o t a s s i u m c h r o m a t e c o n c e n t r a t i o n s

e q u a l to 2 0 m g . C r / l .
10.

It w a s fou n d th a t th e

r i s e in v a l u e

a t a point b e tw e e n

h e m a t o c r i t r e a c h e s i t s m a x im u m
2 a n d 4 m g. C r / l . It i s h o p ed th a t

f u t u r e e x p e r i m e n t a t i o n m ig h t p r o v e th a t th e i n c r e a s e in h e m a t o c r i t
i s an in d e x of c h r o n i c to x ic ity .
11.

D a ta i n d i c a t e d th a t th e s p le e n m a y a c t a s a li m it e d

b u f f e r m e c h a n i s m to s u d d e n h e m o c o n e e n tr a t io n .

BIBLIO G R A PH Y

A b e g g , R . , 1949, S o m e I m p l i c a t i o n s f r o m th e C h a n g e s of th e M ucous
C o v e r i n g of K is h S u b je c te d to S alt S o lu tio n s.
P r o c , L o u is ia n a
A c a d . S c i . , 1 2 :7 -1 0 .
A b e g g , R . , 195 0, S om e E f f e c t s of I n o r g a n ic S a lt s on th e B lood S p e ­
c ific G r a v i t y a n d T i s s u e F lu i d s of th e B lu e g ill, L e p o m is
m a c r o c h i r u s R a f . P h y s i o l . Z o o l ., 2 3 :1 2 4 -1 3 4 .
'
’
A n d e r s o n , B. G ., 1944, T h e T o x ic ity T h r e s h o l d s of V a r io u s S u b s ta n c e s
F o u n d in I n d u s t r i a l W a s te s a s D e t e r m i n e d by th e U se of
D a p h n ia m a g n a . S ew age W o rk s J o u r n a l , 16 (6): 1156- 1163,
A n d e r s o n , B. G.» 1946, T he T o x ic ity T h r e s h o l d of V a r i o u s S odium
S a l t s D e t e r m i n e d by th e U se of D aphnia m a g n a .
Sew age
W o r k s J o u r n . 1 8 ( l) :8 2 - 9 0 .
^ B a n d t , H. J . , 1946, U e b e r r e r s t a r k e S c h a d w ir Kungen au f F i s c h e ,
i n s e b e s o n d e r e lib e r e r h o h te G iftw irk u n g d u r c h K o m b in a tio n
von A b w a sse rg ifte n .
B e itra g e W a s s e r - , A b w a s s e r-u .
F i s c h e r e i c h e m i e , 1 :1 5 -2 3 ,
B la c k , E . C . , 1955, B lood L e v e l s of H e m o g lo b in a n d L a c tic A c id in
Som e F r e s h w a t e r F i s h e s F o llo w in g E x e r c i s e .
J . F ish . R es.
Bd. C a n a d a , 12(6):9 1 7 -9 29 .
B r o w n , M. E . , 1957, T h e P h y s io lo g y of F i s h .
VoL 1, M e ta b o l is m ,
A c a d e m i c P r e s s , I n c ., P u b l i s h e r s , New Y o r k .
De M a r t i n i , F . E . , 1938, C o r r o s i o n a n d th e L a n g e l i e r C a l c i u m C a r ­
b o n a te S a t u r a t i o n In d e x , J o u r n . A .W .W .A . 3 0 :8 5 -9 2 .
D o u d o ro ff, P . , 1952, S o m e R e c e n t D e v e lo p m e n ts in th e Study of T o x ic
In d u s tr ia l W a ste s,
P r o c . F o u r t h P a c if ic N o r th w e s t I n d u s t r i a l
W a s te C o n f ., S tate C o ll. of W a sh in g to n , P u l l m a n , W a s h ., 2 1 25.

83

84

D o u d o r o ff , P . , A n d e r s o n , B, G ., B u r d i c k , G. E . , G a lt s o f f , P . S .,
H a r t , We B 0, P a t r i c k , f t . , S tr o n g , E . R . , S u r b e r , E . W ., and
V a n H o r n , W. M ., 1951, B i o - A s s a y M eth o d s f o r th e E v a l u a ­
t i o n of A c u te T o x ic ity of I n d u s t r i a l W a s te s to F i s h .
Sew age
a n d I n d u s t r i a l W a s t e s , 2 3 (l 1): 1 3 8 0 -1 3 9 7 .
D o u d o ro ff, P . , a n d K a tz , M , , 1953, C r i t i c a l R e v ie w of L i t e r a t u r e on
th e T o x ic ity of I n d u s t r i a l W a s t e s a n d T h e i r C o m p o n e n ts to
F ish .
II, T h e M e ta l s A s S a l t s .
Sew age and I n d u s t r i a l
W a s t e s , 25(7):802 - 839.
E llis,

M. M ., 1937, D e te c tio n an d M e a s u r e m e n t of S t r e a m P o ll u tio n .
B u ll. No. 2 2 , U.S. B u r . of F i s h e r i e s ; B u ll. B u r , F i s h e r i e s ,
4 8 :3 6 5 - 4 3 7 .

E l l i s , M. M . , a n d L a d n e r , G. C . , 1935, A tta c k in g th e N a ti o n 's W a te r
P o ll u tio n M e n a c e .
T r a n s . T w e n t y - f i r s t A m e r . G a m e . C o n f 0,
1 3 5 -1 4 8 ,
F r o m m , P . O . , a n d S c h if f m a n , R . H ., 1958, T o x ic A c tio n of H e x a v a l e n t C h r o m i u m on L a r g e m o u t h B a s s .
J o u r n . W ildlife M a n ­
a g e m e n t.
Jan,
(In P r e s s )
G a r r e y , W. E . , 1916, T h e R e s i s t a n c e of F r e s h W a t e r F i s h to
C h a n g e s of O s m o tic a n d C h e m i c a l C o n d itio n s .
A m er. Jo u r.
P h y s i o l . , 3 9 :3 1 3 -3 2 9 .
G r i n d l e y , J , , 1945, T r e a t m e n t a n d D i s p o s a l of W a ste W a t e r s C o n t a i n ­
ing C h r o m a t e .
J o u r . Soc. C h e m . Inc. (London) 64:33 9 -3 4 4 .
G r in d le y , J . , 1946, T o x ic ity to R ain b o w T r o u t and M innow s of Some
S u b s ta n c e s Known To B e P r e s e n t in W a s te W a te r D is c h a r g e d
to R iv e r s .
A nn. A pp l. B i o l . , 33:10 3 -1 1 2 .
G u e y l a r d , F . , 1923, R e s i s t a n c e d e s E p in o c h e s aux V a r i a t i o n s de
sa lin ite .
C o m p t, R e n d . Soc. B io l . , 8 9 :7 8 -8 0 .
G u e y l a r d , F . , 192 4, De l 'a d a p t a t i o n aux c h a n g e m e n ts de s a li n ite .
R e c h e r c h e s b io lo g iq u e s et p h y s i c o - c h i m i q u e s s u r l'E p i n o c h e .
A r c h . P h y s . B io lo g iq u e , 3 :7 9 -1 9 7 .

85

* B ., D o u d o ro ff, P . , an d G r e e n b a n k , J . r 1945, T he E v a lu a tio n
o f th e T o x ic ity of I n d u s t r i a l W a s t e s , C h e m i c a l s , and O th e r
S u b s t a n c e s t o F r e s h W a te r F i s h e s .
T h e A tla n tic R efin in g
C o ., P h ila d e lp h ia , P a .

H e i l b r u n n , L . V ., 1938, A n O u tlin e of G e n e r a l P h y s io lo g y .
S a u n d e rs C o ., P h ila d e lp h ia , P a.

W. B.

I r v in g , L . , B la c k , E . C . , a n d S a f f o r d , V.» 1941, T e m p e r a t u r e a nd O^
A ffin ity of F i s h B lood.
B io l. B u ll. 8 0 :1 -1 7 .
J o n e s , J . R , E 0, 1938, T h e R e l a tiv e T o x ic ity of S a lts of L e a d , Z i n c ,
a n d C o p p e r to th e S t i c k le b a c k , G a s t e r o s t e u s a c u l e a t u s L ., an d
t h e E f f e c t of C a l c i u m on th e T o x ic ity of L e a d a n d Z in c S a lts .
J o u r n . E x p . B i o l . , 1 5 :3 9 4 -4 0 7 .
J o n e s , J . R . E . , 1939, T h e R e l a t i o n b e tw e e n th e E l e c t r o l y t i c S olu­
ti o n P r e s s u r e s of th e M e ta ls a n d T h e i r T o x ic ity to th e
S t i c k le b a c k , G a s t e r o s t e u s a c u l e a t u s L.
J o u r n . E x p . B io l.,
1 6 :4 2 5 -4 3 7 .
K l a s s e n , C . W ., H a s f u r t h e r , W. A ., a n d Y oung, M. K . , 1949, T he
T o x i c ity of H e x a v a le n t C h r o m i u m to Sunfish a n d B lu e g ill s .
P r o c . F o u r t h I n d u s t r i a l W aste C o n f., P u r d u e Univ. E ng.
B u l l . , E x t . S e r i e s . , 6 8 :2 2 9 -2 3 7 .
K r u g e r , F . , 1928, U n te r s u c h u n g e n iib e r die U r s a c h e n d e s T o d e s d e s
S t i c h lin g s , G a s t e r o s t e u s a c u l e a t u s L . , in r e i n e n C h l o r n a t r i u m
Losungen,
Z e i t s . v e r g l . P h y s i o l . , 7 :6 9 6 -7 3 5 .
K r u m h o lz , L o u i s A . , 1956, O b s e r v a t i o n s on th e F i s h P o p u la tio n of
a L a k e C o n ta m in a t e d by R a d io a c tiv e W a s t e s ,
B u ll. A m .
M u s e u m W at. H i s t . , 110 (4):2 7 7 -3 68 .
* L e C l e r c , E . , a n d D e v la m in c k , F . , 1950, C o n s i d e r a t i o n s p r e l i m i n a i r e s
a la d e t e r m i n a t i o n de la d o se m i n i m a m o r t e l l e , p o u r le
p o i s s o n , de q u e lq u e s s u b s t a n c e s a c a r a c t e r e n o cif.
B ull.
C e n t r e B e ig e E tu d e et D o c u m e n t. E q u x , 8 :4 8 3 -4 8 5 .
L o e b , J . , a n d W a s t e n e y s , H . , 1911, D ie E n tg iftu n g von N a t r i u m c h l o r i d
d u rc h K a liu m c h lo rid .
B io c h e m . Z e i t s . , 3 3 :4 8 0 -4 8 8 .

86

L o e b , J , , a n d W a s t e n e y s , H ., 1915, On th e In flu e n c e of B a la n c e d an d
N o n - B a l a n c e d S alt S o lu tio n s upon th e O s m o tic P r e s s u r e of th e
B ody L iq u id s of F u n d u lu s ,
J o u r n , B io l. C h e m , , 2 1 :2 2 3 -2 3 8 ,
M o o r e , E . W . , 1939, G r a p h ic d e t e r m i n a t i o n of C a r b o n D io x id e and
t h e T h r e e F o r m s of A lk a lin ity .
J o u r n , A m . W a te r W o rk s
A s s o c , 31(1 ):51 -6 5 .
* O s h i m a , S.* 1931, On th e T o x ic A c tio n of D is s o l v e d S a lts a n d T h e i r
I o n s up o n Y oung E e l s , A n g u illa ja p o n ic a .
Jo u rn , Im p erial
F i s h e r i e s E x p , S ta. ( J a p a n e s e ) 2 :1 3 9 -1 9 3 .
P o w e r s , E , B . , 1921, A n ta g o n i s m and i t s P o s s i b l e U tility in P o llu te d
W aters,
T r a n s , A m e r , F i s h e r i e s Soc. 5 0 :2 9 3 -2 9 6 ,
P r o s s e r , C . L . , B ishop* D. W„, B r o w n , F. A ., J r . , J a h n , T , L . , an d
W ulff, Y . J . , 1950, C o m p a r a t i v e A n im a l P h y s io lo g y , W, B,
S au n d er C o ., P h ila d e lp h ia , P a,
* R a m u lt, R , , 192 8 a, In flu e n c e of C e r t a i n S a lts upon th e H a tc h in g a n d
D e v e lo p m e n t of S a lm o n F r y , S a lm o t r u t t a 0 B u ll, I n t e r n a t .
A c a d , P o l o n a i s e S c L , C l a s s e S ci. M ath. N a t., S e r i e B, Sci.
N a t . , 1927, 4 5 - 6 2 .
* R a m u lt, R . , 192 8b, T he In flu e n c e of C e r t a i n S a lts upon th e D e v e lo p ­
m e n t of Y oung S t i c k l e b a c k s , G a s t e r o s t e u s a c u l e a t u s .
B ull,
I n t e r n a t . A c a d , P o l o n a i s e S c i., C l a s s e S ci. M ath. N a t., S e r ie
B. S ci. N a t . , 1927, 6 3 -6 6 ,
R u d o lf s , W ., B a r n e s , G. E„, E d w a r d , G. P H e u k e l e k i a n , H»,
H u r w i t z , E . , R e n n , C . E .* S te i n b e r g , S ., a n d V au g h an , W. E . r
1950, R e v ie w of L i t e r a t u r e on T o x ic M a t e r i a l A ffe c tin g
Sew age T r e a t m e n t P r o c e s s e s , S t r e a m s , a n d B .O .D . D e t e r m i n a ­
tio n s.
S ew ag e a n d In d u s t. W a s t e s , 2 2 (9 ) :1 1 5 7 - 1 191.
* R u s h to n , W ., 1921, B io l o g ic a l N o te s .
2 5 :1 0 1 - 1 1 7 .

S a lm o n a n d T r o u t M a g .,

S a l t z m a n , B. E . , 1952, M i c r o d e t e r m i n a t i o n of C h r o m i u m with
D i p h e n y l c a r b a z i e d by P e r m a n g a n a t e O x id ation , A n a l. C h e m .
2 4 :1 0 1 6 - 1 0 2 4 .

%

87

S p e c t o r , W. S ., 1956, H an d b o o k of B io l o g ic a l D ata.
C o ., P h ila d e lp h ia , P a.

W. B, S a u n d e r s

S t a n d a r d M e th o d s of W a t e r a n d Sew age A n a l y s i s , 1955, U.S. P u b lic
H e a lt h S e r v i c e .
Y o u n g , R . T . , 1938, T h e E f f e c t of B a l a n c e d V e r s u s U n b a la n c e d S o lu ­
t i o n s of H y d r o g e n Ion C o n c e n t r a t i o n s and D i s t i l l e d W a t e r on
F ish .
E c o lo g y , 1 9 :1 3 6 -1 4 0 .

* T h is a r t i c l e w a s not s e e n in i t s o r i g i n a l f o r m .

A P P E N D IX I

R E V IE W O F TO X IC ITY L IT E R A T U R E

A b e g g (1950)* u s in g b l u e g i l l s u n f is h , L e p o m is m a c r o c h i r u s *
w eig h in g 2 0 to 35 g r a m s , t e s t e d b o th N a2CrC>4 an d Na2C r 2 0 ? .
co n d itio n s a s

U nder

s e t f o r t h by H a r t , D o u d o ro ff, an d G r e e n b a n k (1945),

A b e g g u s e d w a t e r w ith a t o t a l a lk a lin i ty a s C a C 0 3 of 60 to 12 0
m g , / l . , t o t a l h a r d n e s s a s C aCO ^ of 75 to 150 m g . / l . , s u lp h a t e s a s
S 0 4 o f 2 0 to 5 0 m g . / l . a n d m a x im u m d i s s o l v e d s o lid s of l e s s th a n
500 m g . / l .

H e r e p o r t e d t h i s w a t e r not wholly s a t i s f a c t o r y a s a

d ilu tio n w a t e r f o r s t u d i e s p e r t a i n i n g to th e e f f e c ts of c h e m i c a l com *
p o u n d s on t h e p h y s io lo g y of f i s h e s .
T h e f i s h w e r e t e s t e d i n 5 g a llo n ro u n d g l a s s b a t t e r y ja r s *
E a c h j a r c o n t a i n e d 12 l i t e r s

of so lu tio n an d w as kept a t 2 2 ° +

C . b y a c o n s t a n t t e m p e r a t u r e b a th .
f i s h in e a c h c o n t a i n e r .
to le ra n c e lim its

0.2

U sin g a e r a t i o n A begg t e s t e d six

H e r e p o r t e d h i s d a ta in 24 h o u r m e d ia n

(T L m ); t h a t is * c o n c e n t r a t i o n s l e t h a l to 5 0 p e r c e n t

of t e s t a n i m a l s in 24 h o u r s , a n d found t h e m to be 93 0 m g . N a ^ C r C ^ /
1. a n d 728 m g . N a2C r 2 C>7/ l .

T h e s e t e s t s o lu tio n s h a d th e follow ing

c h a r a c t e r i s t i c s:

88

89

S p ecific
R e sista n c e
(ohm s a t
22 .2 ° C .)

PH

C h e m ic a l

M o ls ./l.

C a l . O s m o tic
P r e s s u r e in
L i t e r A tm o s .

N a^C rO ^

0.0073

0.19

520

8.1

0 o0044

0 e12

105

5.9

N a2C r 2 ° 7

Jones

(1939) did w o r k on a s e r i e s

w e re added to v e r y

so ft ta p w a t e r

of m e t a l l i c io n s w hich

" o f a hig h d e g r e e of p u rity

T h e c a l c i u m io n c o n c e n t r a t i o n w a s ab o ut 1 m g . / l .

T he f is h w e r e

s t i c k l e b a c k s , G a s t e r o s t e n s a c u l e a t u s , u s u a lly 3 0 to 5 0 m m . in le n g th .
F o u r t o fiv e f i s h w e r e e x p o s e d in 2 l i t e r s
th e s o l u t i o n s r e n e w e d a t d aily i n t e r v a l s .
d iffe re n t c o n c e n tra tio n s

of e a c h t e s t s o lu tio n , and
A s e r i e s of te n to tw en ty

of e a c h s a l t w a s t e s t e d a t 14 C . to 15

C.

J o n e s r e p o r t e d h i s d a ta i n s u r v i v a l t i m e s of 1 day (14-30
h o u r s ) , 2 d a y s (3 8 -6 0 h o u r s ) , 4 d a y s (76-120 h o u r s ) , a n d 1 w eek
(140-2 00 h o u r s ) a n d in l e t h a l c o n c e n t r a t i o n l i m i t s , o r th e c o n c e n t r a ­
tio n of th e m e t a l s a t w h ich th e a v e r a g e
th a t of th e c o n t r o l s , about

s u r v i v a l t i m e n e a r l y e q u a le d

10 d a y s .

F o r t r i v a l e n t c h r o m i u m in th e f o r m of C r z (SC>4 )3 J o n e s found
th e fo llo w in g c o n c e n t r a t i o n s in r e l a t i o n to s u r v iv a l t i m e s :
5.0 m g . / l . i 2 d a y s , 2 .0 m g . / l . ;

1 d ay ,

4 d a y s , 1.4 m g . / l . ; a n d 1 w eek , 1.3

90

m g ./l* w ith a f in a l l e t h a l c o n c e n t r a t i o n l i m i t of 1.2 m g . / l . o r 2.30 x
10

g ram

a to m s/lite r*

G rin d le y

(1945), i n h i s p a p e r on d i s p o s a l of c h r o m a t e w a s t e s ,

r e p o r t e d of t e s t s m a d e on c h r o m i u m u s in g a e r a t e d , d i s t i l l e d w a te r
a s a diluent*

H e found r a in b o w t r o u t , S a lm o g a i r d n e r i v a r .

sh a sta ,

w o u ld t o l e r a t e a p o t a s s i u m c h r o m a t e a n d d i c h r o m a t e s o lu tio n a t
c o n c e n t r a t i o n e q u iv a le n t to 2 0 m g . C r / l . , f o r f r o m 2 to 8 d a y s .

He

c o n c lu d e d th a t th e li m i t i n g c o n c e n t r a t i o n b elow which s u b s t a n ti a ll y
n e u t r a l s o lu ti o n s of c h r o m a t e a r e nontoxic to r a in b o w t r o u t d u rin g
p e r i o d s of 8 d a y s , u n d e r e x p e r i m e n t a l c o n d itio n s , a p p e a r e d to be
s lig h tly l e s s t h a n 2 0 m g . C r / l .
In G r i n d l e y 's

(1946) p a p e r on to x ic ity to f is h he a g a in r e ­

p o r t s on c h r o m a t e to x ic ity to r a in b o w t r o u t .

W h e th e r t h e s e two

p a p e r s c o v e r t h e s a m e e x p e r i m e n t c o u ld not b e d e t e r m i n e d , s in c e
n o r e f e r e n c e w a s m a d e in e i t h e r w o rk to th e o th e r p r o j e c t .

A ssum ­

in g tw o t e s t s w e r e p e r f o r m e d , h e a g a in u s e d w e l l - a e r a t e d d i s t i l l e d
w a t e r to m a k e up th e p o t a s s i u m c h r o m a t e an d d i c h r o m a t e s o lu tio n s
w h ich w e r e m a i n t a i n e d a t 18° C , d u r in g e x p o s u r e to th e f i s h .
T h e to x i c i t y of a s o lu tio n to in d iv id u a l f i s h w a s e x p r e s s e d a s
100 t i m e s th e r e c i p r o c a l of th e t i m e p e r io d , in m i n u t e s , d u r in g w h ich
eac h fish w as im m e r s e d .

The a v e r a g e of t h e s e in d iv id u a l t o x i c i t i e s w as

r e p o r t e d a s t h e m e a n to x ic ity p lu s o r m in u s th e s t a n d a r d d e v ia tio n .

91

R a t h e r th a n r e p o r t i n g h i s c o m p le te d a ta h e r e , w hich c o v e r e d
tw e n t y - t w o d il u tio n s of b o th K2 C r 0 4 and K ^ C ^ C y
w h ich a r e

c l o s e s t to th e 24 h o u r s

only th e r e s u l t s

(1440 m in u te s ) a n d 48 h o u r s

(2 880 m i n u t e s ) l e t h a l c o n c e n t r a t i o n s a r e d i s c u s s e d .

With K C r O
2
4

G r i n d l e y fo u n d 10 t r o u t w ould e n d u r e 2 00 m g . C r / l .

(747 m g .

K ^ C r O ^ / l . ) a t an i n i t i a l pH of 7.3 f o r an a v e r a g e of 374 m i n u te s
b e f o r e o v e r t u r n i n g , w ith a c a l c u l a t e d m e a n to x ic ity of 0.2 7 ± 0.11
m in . 1; 50 m g . C r / l .

(187 K2 C r 0 4 m g . / l . ) a t pH 6.6 c o u ld be e n ­

d u r e d 3580 m i n u t e s w ith a m e a n to x ic ity of 0.02 8

0.010 m in .

With t h e d i c h r o m a t e h i s r e s u l t s w e r e a s fo llow s:
C r/l.

(566 m g . K ^ C r ^ O ^ / l . ) a t pH 5 .4 f o r 188 m i n u te s w ith a m e a n

to x o c ity of 0.53 £
at pH 5 .0 f o r
m in .

2 00 m g .

0.60 m in , \

50 m g . C r / l .

(142 m g . K ^ C r ^ O ^ /l .)

1946 m i n u t e s w ith a m e a n to x ic ity of 0.051 +, 0.044

* and 2 0 m g. C r / l

(57 m g . K ^ C r ^ O ^ / l . ) at pH 5,5 f o r 4348

m i n u t e s w ith a m e a n to x i c it y of 0.02 3

0.008 m in .

It i s i n t e r e s t i n g to n o te th a t th e c h r o m a t e a n d d i c h r o m a te ,
w h e n e x p r e s s e d a s m g , C r / l . , y i e l d a p p r o x im a te l y th e s a m e t o x i c i t i e s .
W nen th e f is h h a d t u r n e d o v e r , th e y w e r e t r a n s f e r r e d to
f r e s h w a t e r , a n d f is h w h ich h a d b e e n i m m e r s e d in s o lu tio n s c o n t a i n ­
in g th e h i g h e s t c o n c e n t r a t i o n o f c h r o m a t e d ie d soo n a f t e r t r a n s f e r .
S o m e of t h e f i s h r w h ich h a d b e e n i m m e r s e d in lo w e r c o n c e n t r a t i o n s ,
d ie d i n a b o u t 3 d a y s a f t e r t r a n s f e r , a n d a few did r e c o v e r .

V ery

92

few f is h w h ich h a d b e e n i m m e r s e d i n s o lu tio n s of th e d i c h r o m a t e
s u r v i v e d w h e n t r a n s f e r r e d to f r e s h w a t e r .

T h o s e th a t did s u r v iv e

w e r e u s u a l l y t h e f is h w h ich h a d b e e n th e f i r s t to o v e rtu rn * s u g g e s t ­
in g th a t t h e s e f is h w e r e a f f e c t e d by th e a c id ity of th e

s o lu tio n

r a t h e r t h a n by a n y s p e c if i c to x ic e ffe c t of th e d ic h ro m a te ,,
F ro m m
e tja l,

a n d S c h iffm a n (1958), u s in g th e m e th o d s of D ou d o ro ff

(1951), fo u n d th e 48 h o u r m e a n t o l e r a n c e l i m i t s of la r g e m o u th

b a s s , le n g th 10,6

0,64 c m . , w eigh t 11.3 ± , 2 . 7

c h r o m i u m , K ^ C r O ^ , w a s 195 m g . C r / l .

g r a m s , fo r h e x a v a le n t

T he d ilu en t h a d a to t a l

h a r d n e s s of 3 34 m g . C a C O ^ / l . , t o t a l a lk a lin ity of 2 06 m g. C a C O ^ /l - ,
a pH o f 8.5 to 8„5, a n d w a s h e ld a t 20° C . to 21° C.
In a d d itio n to th e a b o v e , D o u d o ro ff an d Katz
re v ie w of l i t e r a t u r e

(1953), in t h e i r

p e r t a i n i n g to th e to x ic ity of w a s t e s to f is h , c ite

o t h e r r e f e r e n c e s , w h ich , due to t h e i r u n a v a i la b i lit y , th e a u th o r w as
n o t a b le to c o n s u l t .
th a t th e

T h e y a r e a s fo llow s:

O s h im a (1931) r e p o r t e d

s u r v i v a l t i m e of yo ung e e l s in a s o lu tio n of K C r z (SC>4 )2 at

a c o n c e n t r a t i o n of 5.2 m g . C r / l .
found th a t

a v e r a g e d 18.7 h o u r s .

He a l s o

0.01 M. K ^ C r C ^ a n d 0.005 M. K2C r 2 0 ? s o lu tio n s (520

m g . C r / l . ) w e r e l e t h a l to y o u n g e e l s in an a v e r a g e of 12.4 an d 5.35
hou rs re sp e c tiv e ly , w hereas
K Cr O
Z

2«

s o lu ti o n s
*7

i n 50 h o u r s .

0.002 5 M. K2C r 0 4 an d 0.0005 Mo

(13 0 m g . C r / l . a n d 52 m g. C r / l . ) k i l l e d no e e l s

93

Le C le rc

a n d D e v la m in c k (195 0) found th a t t r i v a l e n t c h r o m i u m ,

a s a s u l f a t e , a t a c o n c e n t r a t i o n of 40 m g , C r / l . w a s a m in im u m fa ta l
c o n c e n t r a t i o n f o r m in n o w s in 6 h o u r s .

R u s h to n (1921) found th a t a

6 h o u r e x p o s u r e to 100 m g . K ^ C r ^ O ^ / l . p ro v e d f a t a l to t r o u t w ithin
12 h o u r s a f t e r r e t u r n to f r e s h w a t e r .
K l a s s e n et a l . (1949) d id w o rk on w a s t e s f r o m a c h r o m i u m
p la tin g i n d u s t r y i n W o o d s to c k , I ll i n o i s .

Since th e a u t h o r i s p e r s o n a ll y

f a m i l i a r w ith t h i s w o r k , h e f e e l s t h a t th e r e s u l t s w e r e so e r r a t i c
th a t th e y do n o t c o n s t i t u t e a v a l i d t e s t .
t h i s w o r k , c i t e d th a t in one t e s t
C r/l.

fo r

D o u d o ro ff, i n h i s r e p o r t on

10 b lu e g ill s u n fis h t o l e r a t e d 83 m g.

10 d a y s o r m o r e , w h e r e a s 6 b lu e g ill s u n fis h d ie d w ith in 4

d ay s in a d il u te d w a s te c o n ta in in g 75 m g . C r / l .

It i s th e a u t h o r 's

o pinion th a t t h e s e c o n f lic tin g d a ta w e r e due to th e fa c t th a t the
w a s te u s e d in th e b i o a s s a y s w a s c o l l e c t e d a t v a r i o u s t i m e p e r i o d s
a n d i t s c o m p o s i tio n w a s in a s t a t e of c o n s ta n t flux.

Knowing th e

s i t u a t i o n , c y a n id e w ould b e s u s p e c t e d a s b e in g a c o n t r i b u tin g f a c t o r
to th e v a r i a t i o n o b s e r v e d .
T h e d a ta r e l a t i n g to th e to x i c it y of c h r o m i u m to i n v e r t e b r a t e s
is even m o re

s c a r c e t h a n th a t of th e f is h .

A n d e r s o n h a s m a d e e x c e lle n t p r o g r e s s in th e f ie ld with h is
w o rk w ith D ap h n ia m a g n a in an a t t e m p t to stu d y th e e ffe c t of v a r i o u s
p o l l u t a n t s on th e food c h a i n of f is h .

In h i s w o rk (1944. 1946) he

94

u s e d t h r e s h o l d c o n c e n t r a t i o n s ; th a t i s , c o n c e n t r a t i o n s w hich would
i m m o b i l i z e 5 0 p e r c e n t of th e t e s t a n i m a l s i n a giv e n p e r i o d of t i m e .
In h i s e a r l i e r w o r k , he e m p lo y e d a t i m e i n t e r v a l of 16 h o u r s , but
found t h i s u n s a t i s f a c t o r y .

T h e n h e c h a n g e d to a 48 h o u r p e r i o d in

1946 w ith a r e s u l t a n t l o w e r in g of t h r e s h o l d s .

In b o th t h e s e p r o j e c t s ,

h e u s e d L a k e E r i e w a t e r a s th e d ilu e n t w hich h a d th e fo llo w in g p e r ­
t i n e n t c h e m i c a l c o m p o s itio n :
m g ./l.;

c a l c i u m , 31 m g . / l . ;

m a g n e s i u m , 7.6

c a r b o n a t e r a d i c a l , 3.1 m g . / l . ; b i c a r b o n a t e r a d i c a l , 114 m g . /

1.; s u lf a t e r a d i c a l , 13 m g . / l . ;
d is s o lv e d so lid ,

c h lo rin e

(C l), 8.7 m g . / l , ;

and to ta l

133 m g . / l .

In 1944 A n d e r s o n found K ^ C rO ^ to i m m o b il iz e d D aphnia
m a g n a in 16 h o u r s a t a c o n c e n t r a t i o n of l e s s th a n 0.6 m g . / l .

In h i s

1946 w o r k w ith s o d iu m s a l t s , he found li t t l e d if f e r e n c e b e tw e e n th e
c h r o m a t e a n d d i c h r o m a t e t h r e s h o l d s w h en c o m p a r e d on th e b a s i s of
th e c h r o m i u m c o n te n t of th e m o l e c u l e , th e c h r o m a t e b e in g to x ic at
< 0 .3 2

m g . / l . a n d th e d i c h r o m a t e at < < 0 .3 1 m g . / l .
R u d o lfs e t a l .

(1950) r e p o r t e d a to x ic e ffe c t to f l o r a a n d

fa u n a due to t r i v a l e n t c h r o m i u m c o n c e n tr a ti o n s of 6 m g . / l . o r l e s s .
H i s a r t i c l e , a c o m p r e h e n s i v e r e v i e w of to x ic ity l i t e r a t u r e p e r ta in in g
to a l l b r a n c h e s of w a s te d i s p o s a l , w as le n g th y , and r e f e r e n c e to th e
p a r t i c u l a r w o r k in w hich t h i s d a tu m w a s found co u ld not be lo c a te d .

A P P E N D IX

T h is

II

s e c t i o n c o n t a in s a l l d a ta and t h e i r s t a t i s t i c a l evaluation*

A ll s t a t i s t i c a l m e t h o d s , e x c e p t th e a n a l y s i s of v a r i a n c e te c h n iq u e
u s e d on t h e r e g r e s s i o n c o e f f i c i e n t s , a r e a c c o r d i n g to S n e d e c o r (1956).
H is c o n v e n ie n t a b b r e v i a t i o n s ,

m e a n in g s ig n if ic a n t (c< = .05) and **

m e a n in g h ig h ly s ig n if i c a n t (c< = .01) a r e u s e d .
T h e a n a l y s i s of v a r i a n c e t e c h n iq u e s u s e d on th e r e g r e s s i o n
c o e f f i c ie n t s a r e th o s e of D r . W. D. B a te n , S t a t i s t i c i a n , M ich ig an S tate
U n i v e r s i t y A g r i c u l t u r e E x p e r i m e n t S tatio n .

95

96

P a rt A.

N orm al T rout

D a ta U s e d to O b ta in th e R e g r e s s i o n C u r v e s of K id n ey , L i v e r , an d
S p le e n W e ig h ts on B ody Weight

T o tal
Wt.
of F i s h
M in u s
O rgans

K id n ey
W eight
(m g .)

K idney
Index
(pet.
body
w t.)

14.6

13.1

131.8

0.90

11.0

11.3

79.8

8.7

11.6

14.8

L iv er
Index
(pet.
body
w t.)

S p leen
W eight
(m g.)

S p leen
Index
(pet.
body
w t.)

66.6

0.46

32.6

0.22

0.72

130.0

t—
>
i—
<
00

T o tal
L e n g th
of
F ish
( c m .)

36.2

0.33

64.6

0.74

74.4

0,86

13.4

0.15

12.9

81.6

0.55

162.0

1.09

46.2

0.31

17.7

13.2

176.0

0.99

181.0

1.02

54.0

0,30

13.7

12.7

120.4

0.88

170.4

1.24

2 7 .4

0.20

24.2

14.6

199.6

0.82

231.8

0.96

79.2

0.33

14.0

12.5

93.6

0.67

153.4

1.10

27.4

0.20

11.1

12.5

82.8

0.74

103.8

0.94

15.6

0.14

26.0

15.6

20 0 .0

0.77

345.4

1.33

51.4

0.20

L iv er
W eight
(mg.)

(*.)

97

D a ta P e r t a i n i n g to O r g a n W e ig h t, G a ll B l a d d e r , an d G onads

T o tal
W eight
of F i s h
M inus
O rgans

T o tal
L e n g th
of F i s h
(c m .)

G a ll
B la d d e r
W eight
(m g.)

G all
B la d d er
Index
(pet.
body
wt.)

14,6

13,1

49.0

0.34

8.6

0,06

11.0

11.3

20.0

0.18

34.2

0.31

8.7

11.6

25.0

0.29

25,6

0,2 9

14.8

12.9

36.4

0.25

44.6

0.30

17.7

13.2

18.0

0.10

379.0

2 .1 4

13.7

12 .7

2 4 .0

0.18

6.4

0,05

24.2

14.6

31.8

0.13

272.0

1.12

14.0

12.5

27.0

0.19

34.8

0,24

11.1

12.5

24.6

0.22

11.6

0.10

2 6,0

15.6

38.6

0.15

28.0

0.11

(g.)

Gonad
W eight
(m g.)

Gonad
Index
(pet.
body
w t.)

98

D a ta U s e d to O b ta in R e g r e s s i o n C u r v e s
W a t e r W eight on B ody Weight

T o tal
W eight
of F i s h
M inus
O rgans

T o tal
L e n g th
of F i s h
( c m .)

D is h
W eight
(g-)

D is h Wt.
P lu s D ry
F i s h Wt.
(g-)

of T o ta l

W a te r
Weight
(go)

(g.)

W a te r
Weight
(pet.
body
w t.)

14.6

13.1

59„3

62.6

11.3

77

11.0

11.3

59.8

62.5

8.3

75

8,7

11.6

60.5

62.5

6.2

71

14.8

12.9

58.2

61,5

11.5

78

17,7

13.2

65.4

69.6

13.5

76

13.7

12.7

57.3

60,2

10.8

79

24,2

14.6

64,4

69.9

18.7

77

14.0

12.5

65.1

68.3

10.8

77

11.1

12.5

58.8

60.8

9.1

82

2 6 .0

15.6

54.8

59.5

21.3

82

99

D a ta P e r t a i n i n g to H e m a t o c r i t , R e d Blood C e l l C o u n t,
H e m o g l o b i n s , a n d R e d B lo o d C e l l L e n g th

T o tal
L e n g th
of F i s h
(c m .)

H em a­
to c rit
(pet.
RBC)

Red
B lood
C ell
C ount
(m illio n s/
c m m ,)

15.5

13.5

35

16.1

12.6

2 1 .4

T o tal
W eight
of
F ish

H em o­
globin
(g*
pet.)

C e ll
L en g th
X
(p.)

1 „08

5.3

14.6

35

1.12

8.3

14.7

14.0

30

1.15

7 .4

15,2

12.2

11.7

39

1.04

8.1

14.6

23.0

14.0

26

0.99

6.3

14.4

2 0.8

15.2

27

1.19

5.3

14.7

11.8

11.3

28

1.2 6

7.2

14.6

11.7

11.4

36

1.08

6.3

15,0

18,7

13.3

29

1.22

6.5

14.7

2 1 „7

14.1

33

0,96

4 .4

14.4

(g.)

100

Data Used to Obtain the R egression Curves of Plasma and
Blood Volumes on Body Weight
T o tal
W eig h t
of
F ish
(g.)

T - 182 4
H em a­
T - 1824
ReP la sm a
to c rit
In je c te d c o v e re d
V o lu m e
(pet.
(m g .)
(m g ./
(m l.)
RBC)
m l.)

P la sm a
P e t . of
T o ta l
W eight

B lood
V o lu m e
(m l.)

Blood
P e t . of
T o ta l
Weight

14.5

0.147

0.377

29

0.39

2.7

0.54

3.8

29.1

0.147

0.273

34

0.54

1.9

0.82

2.8

22.2

0.074

0.198

38

0.37

1.7

0.60

2.7

14.4

0 .074

0.393

47

0.19

1.3

0.36

2.5

22.2

0.074

0.155

37

0.48

2.2

0.76

3.4

14.6

0.074

0.178

27

0.42

2.8

0.57

3.9

10.5

0.074

0.315

34

0.23

2.2

0.36

3.4

2 0.7

0.074

0.178

28

0.42

2.0

0.58

2.8

12.2

0.074

0.2 99

40

0.25

2.0

0.41

3.4

13.5

0.074

0.222

36

0.33

2.5

0.52

3.8

S t a t i s t i c a l V a lu e s of th e D ata on N o r m a l C o n tr o l s

V alue

M ean ( X ) ..................
V a r ia n c e

Z
(s ) . . .

S tan d a rd
D ev iation (s)
S ta n d a rd
E r r o r (s—)
X

„. .

. . . .

Kidney
Weight

L iv er
Weight

Spleen
Weight

T o ta l
Body
W a te r

0.77 pet.

1.0Z pet,

0,24 p et.

77.4 pet.

0.016

0.059

0.005

10.49

0.013

0.24

0.08

3.24

0.039

0.077

0.023

1.02

10Z

S t a t i s t i c a l V a l u e s of th e D a ta on N o r m a l C o n tr o l s (Continued)

H em a­
to c rit

H em o­
g lo b in

RBC
Count

RBC
L en g th

P la sm a
V olum e

Blood
V olum e

Z.13 p c t 0

3.2 5 pet.

31.8 p e t.

6.51 g , /
100 m l .

1.11 x 106
per cm m .

19.33

1.61

0.0096

0.84

0.188

0.26

4.39

1.Z7

0.098

0.9Z

0,42

0.51

1.39

0.40

0.03

0.09Z

0.14

0.16

14,67

p .

103

S t a t i s t i c a l V a lu e s R e l a t e d t o th e R e g r e s s i o n of th e A bove
on Body W eight

T o tal
Body
W a te r

P la sm a
V o lu m e

B lood
V o l­
um e

8.57

0.82

0.015

0,023

-3 8 .9 5

- 1 0 .5 0

-0.62

0.094

0.05

0 a84

0.88

0.93

0.996

0.80

0.89

11.53

40.46

20.43

0.47

0.073

0.077

K idney
W eight

L iv er
W eight

S p le e n
W eight

2.97

12.89

In te rse c t
(a)

-7 .9 3

C o rre l.
C o e f. (r)
S ta n d a r d
E r r o r of
E stim a te

V a lu e

Reg.
C o ef.

(b)

104

R e g r e ssio n

a n a ly sis

K id n e y w e i g h t .
A n a l y s i s of V a r i a n c e of R e g r e s s i o n C o e f f ic ie n t

S ource

D e g r e e s of
F reedom

Sum of
S quares

T o t a l ...............................................................................

9

24,160.92

R e g r e s s i o n ...............................................................

1

2 0 ,8 2 2 .8 7

E r r o r ...........................................................................

8

3,338.05

M ean
S quare

2 0 ,8 2 2 .8 7 * *
417.26

A n a l y s i s of t h e c o r r e l a t i o n c o e f f ic ie n t:
t = r / ( m - 2 ) / 1 - r ^ , d.f. = n - 2 .
t = 1 9.3 9 * *

L i v e r w e ig h t.
A n a l y s i s of V a r i a n c e
^

of R e g r e s s i o n C o e f f ic ie n t
D e g r e e s of
F reed o m

T o t a l ..............................................................................

9

R e g r e s s i o n ......................................................
E r r o r ...........................................................................

Sum of
S quares

M ean
S quare

6 0 ,199.14
1 47,103.41

8

A n a l y s i s of t h e c o r r e l a t i o n c o e ffic ie n t:
t = 11.00**

13,0 9 5 .7 3

47,103.4.1**
1 ,6 3 6.96

105

Spleen weight.
Analysis of Variance of Regression Coefficient
Source

D e g r e e s of
Freedom

Sum of
Squares

T o t a l ...............................................................................

9

3563.12

R e g r e s s i o n ...............................................................

1

2501.07

E r r o r ...........................................................................

8

1062.05

Mean
Square

2501.07**
132.76

A n a l y s i s of t h e c o r r e l a t i o n c o e ff i c ie n t :
t = 8.05**

T o t a l b od y w a t e r .
A n a l y s i s of V a r i a n c e of R e g r e s s i o n C o e f f i c i e n t

S O IU T C G

D e g r e e s of
Freedom

T o t a l ..............................................................................
R e g r e s s i o n .................................................... .
E r r o r ..........................................................................

9
1

Sum of
Squares
192.77
190.99

8

A n a l y s i s of th e c o r r e l a t i o n c o e f f i c i e n t :
t = 140.2 9**

Mean
Square

1.78

190.99**
0.22

10 6

Plasm a volume.
Analysis of Variance of Regression Coefficient
D e g r e e s of
Freedom

Sum of
Squares

Mean
Square

T o t a l ..............................................................................

9

0.1138

R e g r e s s i o n ...............................................................

1

0.0715

0.0715**

E r r o r ...........................................................................

8

0.0423

0.0053

A n a l y s i s of t h e c o r r e l a t i o n c o e f fi c ie n t :
t = 6.27 **

B lo od v o l u m e .
A n a l y s i s of V a r i a n c e of R e g r e s s i o n C o e f f i c i e n t

ource

D e g r e e s of
Freedom

T o t a l ..........................................................................
R e g r e s s i o n ...............................................................
E rror

..............................................

Sum of
Squares

9

M ean
Square

0.2136

1
8

A n a l y s i s of t h e c o r r e l a t i o n c o e f f i c ie n t :
t = 12.07**

0.165 7

0.165 7**

0.0479

0.0060

107

Part

B.

C h ro m iu m -in d u c e d Changes

D a t a U s e d f o r t h e S t r a i g h t - L i n e I n t e r p o l a t i o n of th e
24- H o u r M e dia n T o l e r a n c e L i m i t
24-Hour
Survival
(pet.)

C hrom ium
(m g./l.)

Number
of F i s h

71.0

10

100

97.0

10

70

127.0

10

0

135.0

10

10

144.0

10

0

154.0

10

0

D a t a U s e d t o C a l c u l a t e t h e A c t iv i t y of t h e
A quaria W a te r in T r a c e r E x p e rim e n ts

Background
(count/
5 min.)

Background
(cpm)

W ater
(count/
5 m i n .)

W ater
(cpm)

C orrected
W ater
(cp m /m l.)

190

38

263

53

15

195

39

252

50

11

1 84

37

217

46

9

Mean

12

1 08

D a ta U s e d t o D e t e r m i n e A m o u n t of C h r o m i u m in
A q u a r i a W a t e r in T r a c e r E x p e r i m e n t
Sample
No.

P et.
T rans.

Chrom ium
(H-g*/sample)

Chromium
(m g./l.)

1

44.0

9.3

18.6

2

43.5

9.3

18.6

3

43.5

9.3

18.6

Weight s of t h e O r g a n s of F i s h

U s e d in T r a c e r E x p e r i m e n t

(g.)

Spleen
Wt.
(mg.)

Liver
Wt.
(mg*)

Gall
B ladder
Wt. (mg.)

K i dney
Wt,
(mg.)

Caudal
Peduncle
Wt. (mg.)

8.1

1 7.0

131 .8

16.2

78.0

235.4

14.7

21 .6

158.0

24.8

94.4

1 70.4

15.8

63.0

1 74.2

41 .0

1 28.2

218.0

Fish
Wt.

109

Activity of Organ of F ish Exposed to Radioactive Chromium

Spleen
Count
(5 m i n . )

Liver
Count
(5 m i n . )

G a ll
Bladder
Count
(5 m i n . )

K id ne y
Count
(5 m i n .)

Caudal
Peduncle
Count
(5 m i n .)

182

190

1 84

204

206

193

2 34

186

220

179

186

204

183

213

203

O r g a n C o u n t s C o r r e c t e d t o C o u n t s p e r Minute
p e r 100 m g . of O r g a n

S p le e n
Count
C orrected
(cpm /
100 m g .)

Liver
Count
C orrected
(cpm /
100 m g .)

G a ll
Bladder
Count
C orrected
(cpm /
100 m g.)

K idn ey
Count
C orrected
(cpm /
100 m g.)

Caudle
Peduncle
Count
C orrected
(cpm /
100 m g .)

43.53

6.83

48.14

15.30

5.18

44,44

11.26

33.06

15.89

3.99

13.10

6.77

18.54

10.61

5.32

110

A nalysis

of V a r i a n c e

of T r a c e r

Source

S u m s of
Squares

f

T o t a l .........................................

Mean
Square

3386.13

B e t w e e n ........................................................
E rror

Data

. ....................................................

. . . .

4

2273,83

568.46**

10

1112.30

111 .23

s — = 6.09
2

Q T e s t {Studentized Range)

Q
Qs—
X
S plee n
G all b l a d d e r
K i d n ey
L iver
Caudal peduncle

2

3

4

3.01

3.67

4.08

22,35

24.84

26.61

34.92 33.24 13.88
8.28
4.83

26.61 = 8.31
24.84 = 8.40

Ill

O r g a n W e i g ht, T o t a l Body W a t e r , and H e m a t o c r i t Data
Which Was C o m p a r e d with N o r m a l C o n t r o l Data
F ish
Wt.

W ater
Wt.

<g-)

(g->

S plee n
Wt.
(mg.)

Liver
Wt.
(mg.)

Kidney
Wt.
(mg.)

H em atocrits
(m l./
100 m l .)

7.2

5.6

10.0

94.0

62.8

10.2

8.4.

17.2

153.2

68.4

15.6

11.7

44 .0

1 78.0

92.4

53

13.8

10.1

18.8

186.0

129.4

53

12.3

9*6

17.4

166.0

99.6

51

56

11 z

Data from Normal Fish Exposed to IB mg. C r/l.
(normal chromium group)
Hematocrit
(m l./
1 0 0 ml.)

Red Blood Hemoglobin
Cells (mil(g*/
1ions/cm m .) 1 0 0 ml.)

Cell
Length
(p)

Fish
Length
(cm.)

Fish
Weight
(g.)

36

1 .21

5.5

14.2

14.6

23.2

45

1.19

6.1

15.6

10.0

7.8

43

1 .30

7.6

15.2

1 2.9

12.9

43

1.33

6.9

14.7

14.0

1 4.6

46

1.12

6.7

14.7

10.9

9.4

42

1 .37

6.1

14.1

12.6

15.3

38

1.05

5.6

16.5

16.9

33.9

44

1.26

6.5

12.1

12.0

54

1 .28

7.2

15.0

1 0.7

8.5

47

1 .39

7.8

14.4

13.2

1 7.4

113

P lasm a

P las­
ma
Vol.
(m l./
100

Wt.
(g->

and B loo d V o l u m e D a ta f r o m N o r m a l F i s h E x p o s e d
t o 18 m g . C r / l . ( n o r m a l c h r o m i u m g roup)

T - 1 824
Length
Injected
(cm .)
(mg-)

T - 1824
Recov­
ered
(m g./
m l. )

Hema­
tocrit
(m l-/
100
ml.)

P las­
ma
Vol.
(m l.)

Blood
Vol.
(ml.)

g*>

Blood
Vol.
(m l./
100 g.
body
wt.)

1 .3

26.2

14.9

0,074

0.221

58

.33

.80

3.0

2.3

12.7

12.4

0.074

0.257

45

.29

.52

4.1

2.2

38.6

17.0

0.074

0.086

35

.86

1 .32

3,4

1 .1

33.0

15.9

0.074

0.198

60

.37

.94

2.8

1 .8

14.7

13.0

0.074

0.280

51

.26

.54

3.7

1 .5

20.9

14,9

0.074

0.236

48

.31

.60

2.9

1 .4

31 .4

16.3

0.074

0.164

49

.45

.88

2.8

1 .5

34.0

16.4

0.074

0,147

40

.50

.84

2.5

1 .5

14.0

13.0

0.074

0.343

34

.22

.33

2.3

1 .5

21.2

14.6

0.074

0,239

43

.31

.55

2.6

1 14

Data fro m

S p l e n e c t o m i z e d F i s h E x p o s e d to T a p W a t e r
(splenecto m ized control)
R ed Blood
C el l Count
(m illions/
cm m .)

H e m o g l o b in
(g./lOO m l.)

Length
(cm .)

Weight
(g->

H em atocrit
( ml./lO O m l . )

11 -8

12.9

28

1 .08

6.3

12.3

11.9

23

.99

5.6

1 0.6

7.5

27

1 .04

4.7

1 3.9

15.3

24

.68

4.0

1 3.7

16.7

26

1 .18

5 .4

14.4

16.5

30

.85

5.1

13.1

17.8

34

1 .22

7.2,

13.3

1 7.3

25

.92

4.4

14.1

18.3

37

1 .34

7.8

13.4

19.0

31

1.10

5.6

1 15

Plasm a and Blood Volume Data from Splenectomized Fish
Exposed to Tap Water (splenectomized control group)

T - 1824
T - 1824 Recov­
length Weight
Injected
ered
(cm.)
(g.)
(mg.)
(m g ./
ml.)

P la s­
ma
Vol.
(ml.)

P la s­
ma
Vol.
(m l./
100

g.)

Hema­
tocrit Blood
(m l./
Vol.
100
(ml.)
ml.)

Bloo
Vol
(ml.,
100

g-)

15.8

34.6

0.074

0.093

.80

2.3

47

1.50

4.3

16.0

31.6

0.074

0.103

.72

2.3

33

1 .07

3.4

15.2

22,4

0.074

0.105

.70

3.1

32

1.04

4.6

16.0

34.0

0.074

0.110

.67

2.0

40

1.12

3.3

15.2

26.5

0.074

0.147

.50

1 .9

44

.90

3.4

14.0

19.5

0.074

0.134

.55

2.8

27

.76

3.9

12.4

14.6

0.074

0.215

.34

2.4

36

.54

3.7

12.7

13.5

0.074

0.192

.38

2.8

37

.61

4.5

14.2

21.4

0.074

0.147

.50

2.4

44

.90

4.2

15.9

26.3

0.074

0.118

.63

2.4

26

.85

3.2

1 16

D a ta f r o m

S p l e n e c t o m i z e d F i s h E x p o s e d to 24.8 mg.
( s p l e n e c t o m i z e d c h r o m i u m g ro up )

C r/l.

(g->

H em atocrit
( ml./lO O m l . )

R e d Blood
Cell Count
(m illions/
c m m .)

Hemoglobin
(g./lOO m l . )

13.7

20.6

53

1 .57

9.4

13.8

15.6

40

1 .31

8.1

12.9

11.9

52

1 .97

8.1

1 3.8

16.2

42

1 .50

8.1

13.2

15.2

25

0.98

4.9

15.1

21.3

32

0.92

5 .4

16.7

35.1

47

1 .74

8.3

14.8

18.6

41

1 .35

8.3

15.3

21.3

30

0.99

5.1

13.0

13.9

44

1 .43

8.5

Length
(cm .)

Weight

117

P l a s m a a n d Blood V o l u m e D a ta f r o m S p l e n e c t o m i z e d F i s h
E x p o s e d t o 24.8 mg. C r / l .

T - 1824
^ength Weight
Injected
(cm .)
(g.)
(mg.)

T - 1824
Recov­
ered
(m g ./
m l.)

P las­
ma
Vol.
(m l.)

P las­
ma
Vol.
(m l./
100

Hema­
tocrit
(m l./
100
ml .)

Blood
Vol.
(m l.)

Bloo
Vol
(ml.,
100
g->

g>)

15.8

33.3

0.074

0.116

.64

1 .9

36

1 .00

3.0

13.9

17.4

0.074

0.247

.30

1 .7

53

.64

3.6

1 5.6

29.5

0.074

0.196

.38

1 .3

48

.73

2.5

13.9

18.2

0.074

0.230

.32

1 .8

50

.64

3.5

12.9

15.3

0.074

0.350

.21

1 .4

52

.44

2.9

14.0

18.8

0.074

0.264

.28

1 .5

46

.52

2,8

13.8

15.7

0.074

0.221

.33

2.1

35

.51

3.3

1 3.9

14.8

0.074

0.204

.36

2.4

39

.59

4.0

14.6

21.4

0.074

0.210

.35

1 .6

47

.67

3.1

14.7

19.8

0.074

0.221

.33

1 .7

43

.58

2.9

118

R e g r e s s i o n of p l a s m a v o l u m e on body weight

R e g r e s s i o n of P l a s m a V o l u m e on Body Weight
Norm al
Chromium

Splenectom ized
Control

Splenectom ized
Chromium

0.016

0.01 7

0.015

( a ) ...................

0.00

0.016

0.05

C orrelation coef­
f i c i e n t (r)
.......................

0.81

0.86

0.81

0.116

0.084

0.069

6.64**

9.31**

6.67**

R eg ressio n coef­
f i c i e n t (b) .......................
Intersect

S t a n d a r d e r r o r of
e s t . (S—) ...........................
e
1' t M v a l u e of c o r ­
rela tio n coefficient

.

119

A n a ly s is of v a ria n c e ta b le s — te stin g r e g r e s s io n c o e ffic ie n ts .
Normal Chromium

f

Sums of
Squares

T o t a l ..............

9

0.3092

R egression

1

0.2016

0 . 2 0 1 6 **

Error

8

0.1076

0,0135

Source

....

Mean
Square

Splenectomiz ed Control

f
I

Sums of
Squares

T o t a l ..............

9

0.2042

Regression ,

1

0.1483

0.1483**

Error

8

0.0559

0.0070

Source

....

Mean
Square

Splenectomized Chromium
Mean
Square

f

Sums of
Squares

T o t a l ..............

9.

0.1138

R egression .

1

0.0763

0.0763**

8

0.0375

0.0047

Source

120

R e g r e s s i o n of blood v o lu m e on body weight

R e g r e s s i o n of Blood V o l u m e on Body Weight
Norm al
Chromium

Splenectomized
Control

Splenectomized
C hrom ium

R e g re ssio n coef­
f i c i e n t (b) .......................

0.028

0.032

0.022

I n t e r s e c t (a)

...................

0.04

0.14

0.18

C o r r e la tio n Coef­
f i c i e n t (r) .......................

0.92

0.88

0.88

0.116

0.14

0.077

S t a n d a r d e r r o r of
e s t . (S—) ..........................
e
Mt M v a l u e of c o r ­
rela tio n coefficient

.

16.89**

11.00**

11 .00**

121

Analysis of variance tab le--testin g regression coefficients.
Normal Chromium
Source

f

Sums of
Squares

Mean
Square

T o t a l ........................................................................

9

0.2136

R e g r e s s io n

1

0.1657

0.1657**

E rror

8

0.0479

0.0060

Splenectomized Control

Source

Sums of
Squares

f

Mean
Square

T o t a l ..............................................................................

9

0.2042

. . .. .......................

1

0.1483

0.1483**

E r r o r ........................................... '..............................

8

0.0559

0.0070

R e g r e s s io n

Splenectomized Chromium
Mean
Square

Source

f

Sums of
Squares

T o t a l ............................................. ........................

9

0.2154

R e g r e s s io n ..................................

........................................................

1

0.1679

0 . 1 679**

.................

8

0.0475

0.0059

.

122

S ta tis tic a l v a lu e s fo r h e m a to c r its

S ta tistic a l V a lu es fo r H em a to crits
Normal
Chromium

Splenectomized
Control

Splenectomiz ed
Chromium

Mean (X) ........................

43.8

28.5

44.9

Variance (s2) ..............

24.44

20.33

41.44

Standard deviation
(s) ...................................,

4.94

4.50

6.44

Standard error ( s r - ) .

1.56

1.42

1.36

Statistical values for red blood cell counts

Statistical Values for Red Blood Cell Counts
Normal
Chromium

Splenectomized
Control

Splenectomiz ed
Chromium

Mean (X) ........................

1.25

1.04

1.38

Variance ( s ^ ) ..............

0.01

0.33

0.12

Standard deviation
(s) ......................................

0.10

0.57

0.34

Standard error (s—) .

0.032

0.18

0.11

123

S ta tis tic a l v a lu e s for' h em oglobin

S ta tistic a l V alu es fo r H em oglobin
Normal
Chromium

Splenectomized
Control

Splenectomized
Chromium

Mean (X) ........................

6.60

5.16

7.42

Variance (s 2) ..............

0.62

12.99

2.65

Standard deviation
(s) ......................................

0.79

3.60

1.62

0.25

1.14

0.51

Standard error { s ~ ) •
X

Statistical values for plasma volume
as percent of total body weight

Statistical Values for Plasma Volume
as Percent of Total Body Weight
Normal
Chromium

Splenectomized
Control

Splenectomized
Chromium

Mean ( X ) ........................

1.61

2.44

1.74

Variance (s ) ..............

0.15

0.14

0.108

Standard deviation
(s) ......................................

0.38

0.37

0.33

Standard error (s^.) •

0.12

0.12

0.10

124

S t a t i s t i c a l v a l u e s f o r bloo d v olum e
a s p e r c e n t of t o t a l body weight

S t a t i s t i c a l V a l u e s f o r Blood V o l u m e
as P e r c e n t of T o t a l Body Weight

M e a n (X)
V ariance

2
(S ) ...............

Normal
Chromium

Splenectomized
Control

3.01

3.85

3.1 6

0.32

0.27

0.20

0.56

0.52

0.44

0.18

0,16

0.14

Splenectomized
Chromium

Standard deviation
( s)

. . • •

S t a n d a r d e r r o r ( s —) .

125

C o v a r i a n c e a n a l y s i s o f p la s m a v o lu m e d a ta

Covariance Analysis of Plasma Volume Data
Mean
Square

Source

f

2 d2

Normal c o n t r o l.............................................................

.8

0.040

0.005

.........................................

8

0.054

0.007

Normal c h r o m iu m .......................................................

8

0.106

0.013

..................................

8

0.039

0.005

W it h in ............................................................................... .

32

0.238

0.007

R egression c o e f f i c i e n t ............................................

3

0.003

0.001

Common ( e r r o r ) ...................................................... ...

35

0.241

0.007

3

0.218

0.073**

38

0.459

Splenectomized control

Splenectomized chromium

Adjusted means

..........................................................

T o t a l ............................................................. ...

S— = 0.026
yA
''Q''-Test .
2

3

4

Q

2 .8 7

3 .4 6

3.81

QSyA

0.075

0.090

0.099

Splenecto m ized chrom ium
N orm al chrom ium
N o rm a l control
Splenectom ized control

.579 ~ .099 = .480
.390 - . 0 9 0 = .300
. 3 6 2 - .075 = .287
.350

126

C o v a r i a n c e a n a l y s i s o f b lo o d v o lu m e d a ta

C o v a ria n ce A n a ly sis of B lood V olum e Data
Source

f

2
2d

Mean
Square

Normal c o n t r o l............................

8

0.046

0.006

Splenectomized control

8

0.136

0.017

Normal c h r o m iu m ....................

8

0.107

0.013

Splenectomized chromium

8

0.140

0.018

W it h in ................................................

32

0.429

0.013

Regression coefficient . . . ,

3

0.35

0.012

Common ( e r r o r ) ........................

35

0.464

0.013

........................

3

0.115

0.038*

Total . ................................................

38

0.579

Adjusted means

. . .

S— = 0.079
yA
1'QM-T est

Q
QSyA

2

3

4

2.87

3.46

3.81

.227

Sple nectom ized control
N orm al chrom ium
Splenecto m ized chrom ium
N o rm a l control

.273

.301

.924 - .301 = .628
.732 - .273 = .459
.632 - .227 = .405
.552

127

A n a l a y s i s of v a r i a n c e of h e m a t o c r i t data

A n a l y s i s of V a r i a n c e of H e m a t o c r i t Data

Source

f

S u m s of
Squares

Mean
Square

T o t a l ...............

......................

39

2902

Between . . .

......................

3

1557

519**

E rror

......................

6

1345

37.3

. , . .

sx

=

1.93

MQM-Test

Q

^ X

N orm
S p len
N orm
S p len

a
e
a
e

2

3

4

2.87

3.46

3.82

5.64

6.68

7.37

l ch rom ium
ctom ized ch rom ium
l control
cto m ized control

43.8 - 7.37 = 36.4
40.6 - 6.68 = 33.9
31.8 - 5.64 = 26.2
28.5

128

A naly sis

of v a r i a n c e of h e m o g l o b i n d at a

A n a l y s i s of V a r i a n c e

of H e m o g lo b in D ata
S u m s of
Squares

Mean
Square

Source

f

T o t a l ..................................................................................

39

73.35

3

16.43

5.4 8*

36

56,92

1.55

Between
E rror

....................................................

. . . ...................................................................

sx

=

0.39

1’Q11- T e s t
2

3

4

Q

2.87

3.46

3.82

OS-

1.12

1.35

1.49

S p len
N orm
N orm
S p len

e
a
a
e

ctom ized ch rom ium
l chrom ium
l control
ctom ized control

7,42
6.60
6.51
5.61

-

1.49
1.35
1.12

= 5.93
= 5.25
= 5.39

129

A nalysis

of v a r i a n c e of r e d blo o d c e l l count data

A nalysis

of V a r i a n c e of Red Blood Cell Count Data

Source

f

T o t a l ...............

......................

39

Between . . .
E rror

. ..

s5

.

=

......................

36

S u m s of
Squares

Mean
Square

2.26
0.67

0.22**

1 .59

0.044

0.066

" Q11- T e s t
Z

3

4

Q

2,87

3.46

3.82

OS-

0.19

0.Z1

0.25

Splenectom ized chrom ium
N orm al chromium
N o rm al control
S p l e n e c t o m i z e d cont rol

1 .38 - 0.25 = 1.13
1.25 - 0.21 = 1.04
1.11 - 0.19 = 0.92
1 .04

130

111 1T- T e s t s

B e t w e e n n o r m a l c h r o m i u m an d n o r m a l control:
Red blood c e l l counts

t = 3.18**

Red blood c e ll l e n g t h s

t = 4.7 7**

D a ta u s e d in p o t a s s i u m c h l o r i d e
experim ents- - hem atocrits

Data

U s e d in P o t a s s i u m

C h l o r i d e Expe r i m e nt s - - H e m a t o c r i t s

Tap W ater
S a m p l e s ,..............................

29.8 mg. K / l

20 mg, C r / l .

39

38

47

32

44

44

33

37

33

30

38

44

34

45

40

36

34

35

28

35

43

32

39

34

37

35

38

39

43

44

Mean . . .

...............

34.0

38.8

40.2

Ya r i a n c e

...............

13.77

85.55

24.44

Standard deviation . .

3.71

9.25

4.9

Standard e r r o r

1.17

2.92

1.56

. . . .

131

Mt T' - T e s t s .
B e t w e e n h e m a t o c r i t s f r o m KC1 s o l u t i o n and t a p w a te r:
t = 1.53
B e t w e e n h e m a t o c r i t s f r o m KCl an d C r so lu ti on s:
t = 0.42

D a ta u s e d in d e t e r m i n a t i o n of s p l e e n function

R e s u l t s of H e m a t o c r i t s D e t e r m i n e d on Sham
O p e r a t e d and S p l e n e c t o m i z e d F i s h
Splenecto m ized Control
No T - 1824

Sham
O perated

Sham
O perated

Timeio

Time0

Time10

26

36

29

30

32

29

38

35

38

32

33

40

36

50

32

36

44

26

36

38