Effects of prolonged exposure to ammonia on rainbow trout (Salmo gairdneri) eggs and sac fry
by Dalton Earl Burkhalter
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Zoology
Montana State University
© Copyright by Dalton Earl Burkhalter (1975)
Abstract:
Laboratory experiments were conducted to determine the effects of ammonia on the eggs and sac fry of
rainbow trout (Salmo gairdneri). Two experimental runs were made. Each run exposed eggs and the
resulting sac fry to five concentrations of ammonia ranging up to 0.37 mg/l un-ionized ammonia as N
(NH3-E). Exposure was continuous throughout the incubation period and for 42 days thereafter.
Controls were maintained in essentially ammonia-free water.
A concentration of 0.1 mg/l NH3-N caused retardation of growth and development of sac fry
throughout most of the test period. Evidence existed that 0.05 mg/l NH3-N caused retardation of
growth and development early in the growth period.
A concentration of 0.25 mg/l NH3-N was suggested as the incipient LC50 (lethal threshold
concentration) for rainbow trout sac fry. There was no differential egg mortality or effect on incubation
period.
Hypertrophy of the epithelium of the secondary lamellae of gill tissue occurred at 0.19 mg/l NH3-N.
Karyolysis and karyorrhexis occurred in the same tissue at 0.28 mg/l NH3-N.
Pale coloration occurred in sac fry at concentrations of 0.19 mg/l HN3-N and higher. This effect was
attributed to a lack of blood coloration through possible reduction in erythrocyte numbers or reduced
hemoglobin content in the erythrocytes. S T A a m E M 1 OF EERMISSION TO COPY
I n p r e s e n tin g t h i s t h e s i s in p a r t i a l f u l f i l l m e n t o f th e r e q u ir e ­
m ents f o r an advanced degree a t Montana S ta te U n iv e rs ity , I ag ree
t h a t th e L ib ra r y s h a l l make i t f r e e l y a v a ila b le f o r i n s p e c t i o n . ' I
f u r t h e r a g re e t h a t p e rm is sio n f o r e x te n s iv e copying o f t h i s t h e s i s .
f o r s c h o la r ly p u rp o se s may be g ra n te d by my m ajor p r o f e s s o r , o r, in
h i s a b se n c e, by th e D ire c to r o f L i b r a r i e s .
I t i s u n d e rsto o d t h a t any
copying o r p u b lic a tio n o f t h i s t h e s i s f o r f i n a n c i a l g a in s h a l l n o t be
a llo w ed w ith o u t my w r i t t e n p e rm is sio n .
S ig n atu re
pate
3/
/
975-
EFFECTS OF PROLONGED EXPOSURE TO AMMONIA ON RAINBOW TROUT
(SALMO GAIRDNEBI) EGGS AND SAC FRY
by
DALTON EARL BUREHALTER
A t h e s i s s u b m itte d •i n p a r t i a l f u l f i l l m e n t
o f th e re q u ire m e n ts f o r th e degree
of
MASTER OF SCIENCE
in
Zoology-
Approved:
^K ead, M ajor D epartm ent
MONTANA STATE UNIVERSITY
Bozeman, Montana
March, 1975
iii
'
.
ACK EdW iEDGM EH1
The -w riter e x p re ss e s h is a p p r e c ia tio n to th o s e who a s s i s t e d in
th e s tu d y .' - D r. C alv in Kaya d ir e c te d th e stu d y and a s s i s t e d in p re ­
p a r a tio n o f th e m a n u s c rip t.
D rs. John W right and R ich ard Gregory
c r i t i c a l l y review ed th e m a n u sc rip t.
•
D rs. R obert T h u rsto n .a n d
Rosem arie Russo p ro v id e d in fo rm a tio n and review ed th e m a n u sc rip t.
E rn e s t Vyse p ro v id e d photom icrograph equipm ent.
Dr
The Montana Co­
o p e ra tiv e F is h e ry U n it p ro v id e d s p a c e . and f a c i l i t i e s .
Thanks a.re .due Mr. R obert P ip e r f o r use o f f a c i l i t i e s a t th e
Bozeman, Montana, F is h C u ltu r a l Development C enter (FCDC).
S p e c ia l •
th a n k s go t o Mr. C h a rlie Smith o f th e FCDC f o r h i s ■t e c h n i c a l ad v ice
in f i s h c u ltu r e and h i s .v e r y generous a s s is ta n c e in th e h i s t o l o g i c a l
s tu d y .
Mr. Wes O rr o f th e E nnis N a tio n a l F is h .H atchery p r o v id e d •
rainbow t r o u t eggs.,
Mr. C hris. C a lv e rt a s s i s t e d i n th e s t a t i s t i c a l
a n a ly s is .
■ "
S p e c ia l a p p r e c ia tio n goes t o my w ife and fa m ily f o r t h e i r
encouragem ent and s u p p o r t..
The p ro je c t" w a s su p p o rte d in p a r t by a U .S. E n v iro n m en tal P ro - ■
t e c t i o n Agency T ra in in g G rant No. 7-6009-716.
' . " ■■■
iv
TABLE OF CONTENTS
VITA . ....................................................
'
Page
H
ACKNOWLEDGMENT . . . ................................... ......................... .... . . .
iii
LIST
OF
TABLES ......................................................................
LIST
OFFIGURES
vi
. . . . . . . . . . . . . . . . . . . . .
v iii
ABSTRACT .................................................................
ix
INTRODUCTION .....................
'I
CHEMISTRY OF AMMONIA H AQUEOUS SOLUTION .
. . . . '.
VARIABLES AFFECTING T O X IC IT Y .................................... ... .
.
pH and Tem perature ...................... '.....................................
F ree Carbon D i o x i d e ...............................'.........................
D isso lv e d Oxygen . . . . . . . . . . . . . . . .
A lk a lin ity and H ardness . . . . . . . . . . . .
AMMONIA TOXICITY TO FISHES
•
• •. . . .
'k
.4
5
5
5'
' A cute T o x ic ity S tu d ie s . . . . . . . . . . . . .
. Long-term T o x ic ity S tu d ie s . . . . . . . .
. . .
5
7
MATERIALS AND METHODS
• DILUTION WATER
'
3
. . .
MECHANISM OF TOXICITY......................
. . .. . . .
. , .
DILUTION APPARATUS. . .
TROUT EGGS
.:
SAC FRY''. . . .....
. .
................... 8
.. . . .
.....................
. . .
. . .
2
. . .
. . .. .
. .. . .
. ... ... . .
. . . .
H
11.
. . .
11
............................... . . .. .
17
. . . . .. . . ...
■WATER CHEMISTRY MEASUREMENTS
. . .
. .
. .. '
. . . . . . . . . . . .
BLOODSMEARS AND HISTOLOGICAL SECTION'S . . .
. . . . . . .'
18
■1820
''
RESULTS
. . .
. . . ............................ ............................... .
GROWTH AHD DEVELOPMENT . . . . .
.
..
.
. . . . . .
MORTALITY ........................................ . . ; . . . . . .
HISTOLOGICAL- CHANGES
..................'. . . . - .
BLOOD ANOMALIES . ■............................ ......................... .
DISCUSSION . . . . . . . .
APPENDIX . . . . . . . .
. . . . . .
. . . . . .
. . .
. .
. ' .............. ......
34
■35
...................... 48
..
..
.
. . . . . .
.
.. . .
55’
56
. . . . .
OPERATION . . . . . . . . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . .
21
. ■. .-. ' 44
DESIGN AND OPERATION OF THE DILUTION APPARATUS
DESIGN OBJECTIVES. .
Page
21
...........................................
56
56
63
VX
L IS T OF TABLES
■Table
1.
Page
P e rc e n ta g e o f u n -io n iz e d ammonia in aqueous s o lu ­
t i o n a t d i f f e r e n t pH 's and te m p e ra tu re s ..........................
3
2.
Chem ical and p h y s ic a l p r o p e r tie s o f d i l u t i o n
w a t e r .....................................................................................................12
3.
U n -io n ized ammonia c o n c e n tra tio n s a t each t e s t
s t a t i o n .....................
.1 5
4.
Run I . Mean t o t a l le n g th s (TL) in cm a t g iv e n
days a f t e r h a t c h i n g ........................................................................... 21
5.
Run 2 . Mean t o t a l len g th 's (TL) i n cm a t g iv en
days a f t e r h a t c h i n g ...............................'......................................22
6.
Runs I and 2 combined. Mean t o t a l le n g th s (TL)
in cm a.t g iv e n days a f t e r h a tc h in g '......................
23
7.
Run I . • Comparisons among mean t o t a l le n g th s
. . . .
8.
Run 2 .
. . . .
9.
Runs I and 2 combined. Comparisons among mean
t o t a l l e n g t h s .....................
33
10.
Egg m o r ta lity d u rin g in c u b a tio n ..............................................
34
11.
Run I . C h i-sq u are m o r ta lity com parisons f o r eggs
and sac f r y ............................... ............................................... . .
36
Run 2 . C h i-sq u are m o r ta lity com parisons f o r eggs
and sac f r y . .............................................................
38
12.
Comparisons' among mean t o t a l le n g th s
13.
C um ulative m o r ta lity e x p re sse d a s n e a r e s t p e rc e n t
a t g iv e n exposure' t i m e s .......................... ■................................. 40
14.
Mean LC50 v a lu e s from Runs I and 2, m g/l
• • •
30■■
31
44
v ii
T able
Page
15.
T o ta l ammonia c o n c e n tra tio n s (m g /l
r e q u ir e d t o produce a g iv e n l e v e l o f un­
io n iz e d ammonia (m g /l HH3-N) a t d i f f e r e n t
pH 's and t e m p e r a t u r e s ....................................................................... 53
16.
Run I .
A n a ly sis o f v a ria n c e o f t o t a l le n g th
. . . .
58
IT .
Run 2 .
A n a ly sis o f v a ria n c e o f t o t a l le n g th
. . . .
59
18 .
Runs I and 2 combined. A n a ly sis o f v a ria n c e
o f t o t a l le n g th . . . ....................................................................... 60
19.
Run I . M o rta lity f ig u r e s showing numbers o f sac
f r y a l i v e o r dead and p o t e n t i a l m o r t a lit y ( p o t.
mort
................................... ■........................ - .......................6l
20.
Run 2 . M o r ta lity f ig u r e s showing numbers o f sac
f r y a l i v e o r dead and p o t e n t i a l m o r ta lity ( p o t.
m o rt.)
......................................................
62
v iii
L I S T OF FIGURES
F ig u re
1.
2.
Page
Schem atic diagram o f c o n sta n t flo w to x ic a n t
d i l u t i o n a p p a r a t u s .....................................................
13
C onstant flow to x ic a n t d i l u t i o n a p p a ra tu s ( to p ) .
In c u b a tio n tu b e s and r e a r in g pa,ns w ith sac
f r y (bottom )
.......................................................
3.
Growth r e l a t i o n s h i p s o f sac f r y a t g iv e n s t a t i o n s
4.
Growth r e l a t i o n s h i p s o f sac f r y a t g iv e n tim es
a f t e r h a tc h in g ......................................................
l4
. .
25
26
5.
Sac f r y developm ent a t 28 d a y s ,a f t e r h a tc h in g . . . . . 2J
6.
Sac f r y developm ent a t 7-day i n t e r v a l s a t
s e le c te d s t a t i o n s ..........................................................................
28
7.
Cum ulative m o r ta lity o f sac f r y . .............................................4 l
8.
T o x ic ity cu rv es . . . . . . . . .
9.
LC50 v a lu e s from t o x i c i t y cu rv es i n F ig . 8
.......................................
42
................ 43
10.
G i l l fila m e n ts showing norm al t i s s u e (X 325)
. . . .
45
11.
G i l l fila m e n ts showing h y p e rtro p h y o f e p ith e liu m
o f secondary la m e lla e (arro w s) (X325) . . . . . . . .
46
ix
ABSTRACT
L a b o ra to ry ex p erim en ts were conducted t o d eterm in e th e e f f e c t s
o f ammonia on th e eggs and sac f r y o f rainbow t r o u t (Salmo g a ir d n e r i) .
Two e x p e rim e n ta l ru n s were made. Each ru n exposed eggs and th e r e - .
s u i t i n g sac f r y t o f iv e c o n c e n tra tio n s o f ammonia ra n g in g up t o
0 .3 7 m g /l u n -io n iz e d ammonia a s N (NH3- E ) . Exposure was co n tin u o u s
th ro u g h o u t th e in c u b a tio n p e rio d and f o r 42 days t h e r e a f t e r . C o n tro ls
were m a in ta in e d i n e s s e n t i a l l y am m onia-free w a te r.
A c o n c e n tra tio n o f 0 .1 m g /l HH3-N caused r e t a r d a t i o n o f growth
and developm ent o f sac f r y th ro u g h o u t most o f th e t e s t p e r io d . ' E v i­
dence e x is te d t h a t 0 .0 5 m g /l HH3-E caused r e t a r d a t i o n o f grow th and
developm ent e a r ly in th e grow th p e rio d .
A c o n c e n tra tio n o f 0 .2 5 m g /l EH3-E was su g g ested a s th e in c ip ie n t
LC50 ( l e t h a l th r e s h o ld c o n c e n tra tio n ) f o r rainbow t r o u t sac f r y . There
was no d i f f e r e n t i a l egg m o r t a lit y o r e f f e c t on in c u b a tio n p e rio d .
H ypertrophy o f th e e p ith e liu m o f th e secondary la m e lla e o f g i l l
t i s s u e o c c u rre d a t 0 .1 9 m g /l M 3-R. K a ry o ly sis and k a ry o rrh e x is
o c c u rre d i n th e same t i s s u e a t 0 .2 8 m g /l HH3-N.
P a le c o lo r a tio n o c c u rre d i n sac f r y a t c o n c e n tra tio n s o f 0 .1 9
m g /l HHo-K and h ig h e r . T his e f f e c t was a t t r i b u t e d t o a la c k o f blood
c o lo r a tio n th ro u g h p o s s ib le r e d u c tio n i n e ry th ro c y te numbers or r e ­
duced hem oglobin c o n te n t i n th e e r y th r o c y te s .
INTRODUCTION
The p re se n c e o f ammonia in a q u a tic ecosystem s has re c e iv e d
in c re a s e d a t t e n t i o n i n r e c e n t y e a r s .
A grow ing aw areness o f th e
t o x i c i t y o f ammonia t o a q u a tic an im als has f o s te r e d much o f t h i s
a tte n tio n .
Ammonia f in d s i t s way in to th e a q u a tic ecosystem th ro u g h th e
m u n ic ip a l sewage tre a tm e n t p l a n t, a g r i c u l t u r a l and f e e d lo t ru n o f f,
c o a l coking and g a s i f i c a t i o n p l a n t s , and f e r t i l i z e r m an u factu rin g
p la n ts ( EIFAC 1 9 7 3 ).
Organisms r e s id in g w ith in a w a te rc o u rse a re a
f u r t h e r so u rce o f ammonia b u t g e n e r a lly i n such nom inal q u a n titie s
t h a t n a t u r a l mechanisms o f d e g ra d a tio n can m a in ta in c o n c e n tra tio n s
w e ll below to x ic l e v e l s .
In tr o d u c tio n s o f ammonia by th e a c t i v i t i e s
o f man can be so lo c a l iz e d and c o n c e n tra te d t h a t n a t u r a l means o f
d e g ra d a tio n a re overwhelmed and ammonia c o n c e n tra tio n s can re a ch
to x ic l e v e l s .
Many s tu d ie s o f ammonia t o x i c i t y t o warm and c o ld w a ter f is h e s
have been made.
Most c o ld w ater s tu d ie s have used salm onids as th e
t e s t a n im al w ith th e rainbow t r o u t b e in g u sed most f r e q u e n tly in t h i s
c o u n try .
Much a c u te t o x i c i t y d a ta e x i s t b u t lo n g e r te rm t o x i c i t y d a ta
a r e l e s s numerous.
T his stu d y was u n d e rta k e n i n an e f f o r t t o o b ta in more t o x i c i t y
d a ta on v e ry young c o ld w a ter f i s h .
T h is p a p er d is c u s s e s a stu d y o f
th e t o x i c i t y o f aqueous, ammonia t o th e . eggs and sac f r y o f rainbow
t r o u t (Salmo g a iv d n e r i.) .
-2 CHEMISTRY OF AMMONIA H
AQUEOUS SOLUTION
The c h e m istry o f ammonia i s r e l a t i v e l y sim ple in most n a tu r a l
w a te r s .
Ammonia gas i s v e ry s o lu b le in w ater and may d is s o lv e by
sim ple d i s s o l u t i o n .
Ammonia may a ls o e n te r w ater a s th e ammonium io n ,
NH^+, from th e d i s s o lu t io n o f such compounds a s NHijlC l, NH^HCO^, and
(NHili) 2SO^.
Ammonia behaves i n w ater a s a B r^n sted a c id o r b ase a s shown in
th e fo llo w in g e q u a tio n s ( Stumm and Morgan 1970):
NHil+ + HgO = NH3 + H3O+ (a c id )
NH3 + HgO
— NHili + SH- (b ase)
The pK v a lu e s a t 20 C f o r th e s e r e a c tio n s a re g iv e n below ( Hodgman
1958):
PKa = 9 AOO '
PK-J3- = 4.767
I n c o n tr a s t t o e a r l i e r co n cep ts th e NHiliOH m olecule p ro b a b ly does .
not e x is t.
I n s te a d , th e NH3 m olecule i s most l i k e l y lo o s e ly bound t o
many w a ter m o lecu les th ro u g h hydrogen bonding ( Stumm and Morgan 1970).
T h is ammonia h y d ra te (NH3 -H2O).- i s most f r e q u e n tly c a ll e d " u n -io n iz e d
ammonia" w h ile th e NHifi+. io n i s r e f e r r e d t o a s " io n iz e d ammonia".
sum o f th e two f r a c t i o n s i s th e n term ed " t o t a l ammonia".
The
In th is
p a p e r th e term s 11NH3" and " u n -io n iz e d ammonia" w i l l be u sed i n t e r ­
changeably a s w i l l a ls o "NHiji+" and " io n iz e d ammonia".'
The u n -io n iz e d f r a c t i o n o f ammonia has long been re c o g n iz e d a s
-3 -
•the a g e n t which i s to x ic t o f i s h (Wuhrmann and Woker 1948, c ite d hy
EIFAC 1973; Downing and Merkens 19 5 5 ).
T h is f r a c t i o n i s n o t c o n sta n t
f o r a g iv e n t o t a l ammonia c o n c e n tra tio n b u t v a r ie s . s i g n i f i c a n t l y w ith
.pH and te m p e ra tu re and s l i g h t l y w ith io n ic s tr e n g th .
e q u a tio n s above show an in c re a s e ' i n th e
pH.
The chem ical
f r a c t i o n w ith in c re a s in g
S im ila r ly an in c re a s e i n te m p e ra tu re r e s u l t s in an in c re a s e in
th e
f r a c t i o n (Hodgman 195.8).
These v a r i a t i o n s w ith pH and tem p er­
a tu r e a re i l l u s t r a t e d by th e fo llo w in g r e p r e s e n ta tiv e f ig u r e s (T able l )
e x tr a c te d from T r u s s e ll (1 9 7 2 ).
T able I .
P e rc e n ta g e o f u n -io n iz e d ammonia in aqueous s o lu tio n a t
d i f f e r e n t p H 's and te m p e ra tu re s .
T em perature, C
8
10
7 .0
0 .1 6
0 .1 9
7 .5
0 .5 0
0
PH
CO
.
8 .5
12
; 14
16
' 0.21
; 0.25
0.29
0.59
0.68
0.80
0.92
1.58
1.8 3
2 .1 2
2.48 •
2.86
4 .8 2
5.55
6.4 o
7.4 5
8.52
VARIABLES AFFECTING TOXICITY
The t o x i c i t y o f ammonia t o f is h e s depends upon s e v e r a l f a c to r s
in c lu d in g d is s o lv e d oxygen, f r e e carbon d io x id e , pH, te m p e ra tu re , p r i o r
exposure t o a.mmonia, p h y s ic a l s t r e s s , g e n e r a l p h y s io lo g ic a l s t a t u s , and
I
-li­
th e p re se n c e o f o th e r to x ic su b sta n c e s o r m itig a tin g p a ra m e te rs
(W illingham 1973).
I n t h i s stu d y th e s e f a c t o r s were e i t h e r e lim in a te d
o r c lo s e ly c o n tr o lle d .
The more s a l i e n t o f th e s e f a c t o r s w i l l be
d is c u s s e d in th e fo llo w in g p a ra g ra p h s .
pH and T em perature
An in c re a s e i n pH a n d /o r te m p e ra tu re in c re a s e s th e t o x i c i t y o f a
g iv e n t o t a l ammonia c o n c e n tra tio n t o f i s h due t o th e p re v io u s ly
d is c u s s e d pH and te m p e ra tu re dependence o f th e
fra c tio n .
T his
c o r r e l a t i o n w ith te m p e ra tu re may be o f f s e t , however, by a g r e a te r
s u s c e p t i b i l i t y o f f i s h t o ammonia p o is o n in g a t low te m p e ra tu re s .
Burrows (1964) found re d u c ed re c o v e ry i n chinook salmon f in g e r li n g s
exposed t o ammonia a t ab o u t 6 C compared w ith th o s e exposed a t about
l k C.
Brown ( 1968) in d ic a te d a l i n e a r r e l a t i o n s h i p showing a d e­
c re a s in g 48- h r LC50 f o r HHg w ith d e c re a s in g te m p e ra tu re .
For rainbow
t r o u t th e 48- h r LC50 a t 3 C was l e s s th a n o n e - th ir d th e v a lu e a t 20 C.
F ree Carbon D ioxide
L loyd' and H e rb e rt ( i 960) found t h a t ammonia t o x i c i t y d e crea se d
w ith d e c re a s in g carbon d io x id e c o n c e n tr a tio n .
They re a so n e d t h a t th e
r e s p i r a t o r y w a te r in c re a s e d i n carbon d io x id e c o n te n t and hence . .
d e c re a se d i n pH a s i t p a ss e d over th e g i l l s u r f a c e s .
T h is caused a
lo w e rin g o f th e HH^ c o n c e n tra tio n a t th e g i l l s u r f a c e s .
When i n i t i a l
carbon d io x id e l e v e l s were h ig h , th e lo w erin g o f pH would be l e s s th a n
when i n i t i a l carbon d io x id e le v e l s were low (when added carbon d io x id e
would be r e l a t i v e l y g r e a t e r ) .
D isso lv e d Oxygen
Downing and Merkens (1955) found t h a t s u r v iv a l p e rio d s o f rainbow
t r o u t exposed t o ammonia in c re a s e d w ith in c r e a s in g d is s o lv e d oxygen
l e v e l s ra n g in g fro m -1.5 t o 8 .5 m g /l.
The in c re a s e in s u r v iv a l tim e
a s a f u n c tio n o f d is s o lv e d oxygen was g r e a t e s t a t th e low er concen­
t r a t i o n s o f ammonia.
Oxygen e f f e c t s were o b serv ed th ro u g h o u t th e
ran g e b u t were m ost pronounced i n th e low er e x tre m es.
Lloyd ( l9 6 l a , 1961b) a ls o found an in c re a s e d t o x i c i t y o f ammonia
a t low d is s o lv e d oxygen l e v e l s .
For example he found t h a t th e
th r e s h o ld LC 50 a t kO'jo d is s o lv e d oxygen s a t u r a t i o n was h a l f t h a t a t
100/o s a t u r a t i o n (L loyd 1961b) .
He re a so n e d t h a t low er oxygen l e v e l s '
r e s u l t e d i n low er o u tp u t o f carb o n d io x id e a t th e g i l l s u rfa c e which
le s s e n e d th e downward change i n pH.
A l k a l i n i t y and H ardness
No. d i r e c t in flu e n c e on th e t o x i c i t y o f ammonia t o f i s h has been
o b serv ed f o r a l k a l i n i t y and i t s e f f e c t i s lim ite d t o th e e x te n t t h a t
i t h e lp s d eterm in e pH ( EIFAC 1973).
H e rb e rt ( 1962) found no e f f e c t s on ammonia t o x i c i t y t o rainbow
t r o u t .f r o m v a r i a t i o n s in calciu m w a ter h a rd n e ss .
AMMONIA- TOXICITY TO FISHES.
A cute T o x ic ity S tu d ie s .
Lloyd and H e rb e rt ( i 960) showed t h a t th e 500-m in u te LC 50 f o r
-
6-
rainbow t r o u t was a p p ro x im a te ly 0 .4 m g /l u n -io n iz e d ammonia a s N
(IE 3-N) a t th e g i l l s u r f a c e .
Merkens and' Downing (1957) found a
th r e s h o ld LC'50 o f a p p ro x im a te ly 1 . 7 m g /l HHg-It in th e p re se n c e o f
v e ry lo w .c arb o n d io x id e ( l e s s th a n 4 m g / l ) .
Lloyd ( 1961b) g r a p h ic a lly r e l a t e s th e th r e s h o ld LC50 t o pH,
a l k a l i n i t y , d is s o lv e d oxygen, te m p e ra tu re , and carbon d io x id e .
When
a p p lie d t o a t y p i c a l w a ter o f pH rJ.6 , a l k a l i n i t y 200 m g /l a s CaCOg,
te m p e ra tu re 18 C, d is s o lv e d oxygen 60/0 o f a i r s a t u r a t i o n , and carbon
d io x id e 11 m g /l, th e th r e s h o ld LC50 f o r rainbow t r o u t i s 0 .4 m g/l .
HH3-H.
B a ll ( 1967) found th e th r e s h o ld LC 50 f o r rainbow t r o u t t o be
0 . 4 l m g /l HH3-H.
Lloyd and O rr ( 1969) found a s im ila r th r e s h o ld
LC50 o f 0 .3 9 m g /l HH3-H f o r rainbow t r o u t .
R ice (1971) found rainbow t r o u t eggs and e a r ly sac f r y u n a ffe c te d
by c o n c e n tra tio n s o f 3 .0 m g /l HH3-H.
D uring a b s o r p tio n o f th e y o lk
sac th e r e s i s t a n c e t o HHg d e c re a se d and n e a r co m p letio n o f a b s o rp tio n
th e 24- h r l e t h a l c o n c e n tra tio n (LC50?) had dropped t o a p p ro x im a te ly
0 .0 8 m g /l HH3-H1
H e rb e rt and Shurben ( 1963) found a 24- h r LC50 o f 0 .3 - 0 .4 m g/l
HH3-H f o r rainbow tr o u t..
A l a t e r s tu d y (H e rb e rt and Shurben 1965,
c i t e d by EIPAC 1973) showed 24- h r LC50. v a lu e s o f 0 .4 0 -0 .5 8 m g /l HH3-H 1
Penaz ( 1965) showed t h a t brown t r o u t eggs were r e s i s t a n t t o tw oh our ex p o su res t o c o n c e n tra tio n s o f 4 l m g /l HH3-H, how ever, some ■
-7 -
evidence existed that hatching success was reduced if;exposure occurred
during later stages of development. Other work hy Penaz ( 1965)
suggested that the threshold LC50 for brown trout fry was 0 .3 3 mg/l
HH3-N.
'
.
Wuhrmann and Woker (1948, c i t e d by EIFAC 1973) found a th re s h o ld
f o r t r o u t spawn ( f r y ? ) o f 0 .2 5 -0 .3 3 m g /l HH^-N.
Liebmann ( i 960, c i t e d
by EIFAC 1973) found a th r e s h o ld v a lu e o f 0 .2 m g /l NH3-N f o r rainbow
tro u t fry .
Long-term Toxicity Studies
Burrows (1964) found no m o r t a lit y among Chinook salmon f in g e r lin g s
a f t e r 6 -week ex p o su res t o ammonia c o n c e n tra tio n s o f 0 .005- 0 .0 0 7 m g/l
NH3-N.
P a th o lo g ic a l symptoms w ere, how ever, o b serv ed .
S evere h y p e r-
.p la s i a o f th e seco n d ary la m e lla e r e s u l t e d a t th e low er c o n c e n tra tio n
w h ile h y p e r p la s ia and f u s io n o f th e seco n d ary la m e lla e o c c u rre d a t
th e h ig h e r c o n c e n tr a tio n .
F l i s ( 1968) found t i s s u e damage i n g i l l s ,
s k in , i n t e s t i n e , l i v e r , and k id n ey s o f c a rp r e s u l t i n g from exposure t o
s u b le t h a l l e v e l s ' o f ammonia.
Larmoyeux and P ip e r (1973) found t h a t an 8 -month exp o su re o f
■rainbow t r o u t t o 0 .0 1 m g /l NH^.-N caused h y p e rp la s ia and f u s io n o f th e
secondary la m e lla e o f th e g i l l s .
E vidence o f t i s s u e damage was a ls o
n o te d i n th e l i v e r , s p le e n , and th y r o i d .
These r e s u l t s were o b ta in e d
. a t d is s o lv e d oxygen l e v e l s r a n g in g .down t o 3 .3 m g /l.
Reichenbach-KLinke (1967) found that concentrations up to
.
.
■8-
0 .4 m g / l c a u s e d h y p e rp la sia ,- s w e llin g , and in flam m a tio n o f th e
g i l l s i n rainbow t r o u t .
■
I n a d d itio n , he found t h a t rainbow, t r o u t
s u f f e r e d a d i s t i n c t and i r r e v e r s i b l e d e c re a se i n th e number o f
e ry th ro c y te s ' i n th e b lo o d .
MECHANISM OF TOXICITY
The e x a c t m echanism -of t o x i c i t y o f HHb t o f i s h e s . i s - n o t known a t
'
U
1
t h i s tim e .
S e v e ra l s t u d i e s . have been, done i n an a tte m p t t o id e n tif y
t h i s mechanism.
Brockway (1950) c o r r e la te d ammonia i n th e su rro u n d in g w ater w ith
red u ced oxygen c a r ry in g c a p a c ity o f t r o u t b lo o d .
He found t h a t ammonia
c o n c e n tra tio n s o f I m g /l (assumed a s to tal-am m o n ia) red u ced th e oxygen
c o n te n t ..of th e b lo o d t o abou t o n e -se v e n th th e -n o rm a l v a lu e and i n ­
c re a s e d th e carbon d io x id e c o n te n t o f th e b lo o d by 15$.
He concluded
t h a t ammonia red u ced th e a b i l i t y o f f i s h hem oglobin t o combine w ith
oxygen o r t o l i b e r a t e carbon d io x id e .
S u ffo c a tio n o c c u rre d a s a
re s u lt.
Fromm and G i l l e t t e - ( 1968) c o r r e la te d blo o d ammonia and n itro g e n
e x c r e tio n w ith am bient ammonia.
They found t h a t b lo o d ammonia i n ­
c re a s e d w ith in c r e a s in g am bient ammonia w h ile a t th e same tim e t o t a l
n itr o g e n e x c r e tio n d e c re a se d .
Ammonia e x c r e tio n d e c re a se d by a
g r e a t e r p e rc e n ta g e th a n d id t o t a l n itr o g e n e x c r e tio n .
They a ls o found
t h a t th e a b i l i t y o f hem oglobin t o combine w ith oxygen i n v i t r o was ■
n o t a l t e r e d by. ammonia c o n c e n tra tio n s , up t o 0..05 m g /l NH^--N.
-
-9 -
Lloyd and O rr ( 1969) found t h a t u r in e e x c r e tio n i n rainbow t r o u t
in c re a s e d w ith in c r e a s in g am bient ammonia l e v e l s .
They su g g est t h a t
t h i s d i u r e s i s r e s u l t s from an in c re a s e i n p e rm e a b ility o f th e f i s h
t o w a te r in th e p re se n c e o f ammonia.
They su g g est t h a t ammonia con- ■ ■
c e n tr a ti o n s may in c r e a s e u n t i l u r in e e x c r e tio n re a c h e s a p la te a u a t
w hich tim e p e rm e a b ility exceeds e x c r e tio n and d e a th r e s u l t s .
O lson and Fromm (1971) c o r r e la te d a d e c re a se i n t o t a l n itro g e n
e x c r e tio n by rainbow t r o u t w ith an in c r e a s e in am bient ammonia.
They
su g g e ste d t h a t th e d e c re a se in ammonia e x c r e tio n was due t o a d e c re a se
i n g r a d ie n t betw een blo o d and w a te r.
Mo com pensatory e x c r e tio n o f
u re a o r p r o t e i n n itr o g e n was n o ted t o o f f s e t th e in c re a s e d blood
ammonia.
R eichenbach-K linke ( 1967) m a in ta in s t h a t ammonia a c t s on th e
r e s p i r a t o r y organs and th e b lo o d o f th e f i s h .
H is fin d in g s t h a t
rainbow t r o u t s u f f e r e d an i r r e v e r s i b l e d e c lin e i n e ry th ro c y te numbers
le d him t o h y p o th e siz e t h a t e ry th ro c y te numbers co u ld become so low
t h a t th e f i s h co u ld no lo n g e r m a in ta in l i f e .
Some w orkers c u r r e n tly f e e l t h a t am bient.am m onia p ro d u ces to x ­
i c i t y i n f i s h e s due t o th e in c re a s e i n b lo o d ammonia which r e s u l t s .
S ince ammonia i s to x ic th e f i s h must e i t h e r d e to x ify o r e x c r e te i t .
F o r s te r and G o ld s te in ( 1969) s t a t e t h a t t e l e o s t f is h e s la c k two enzymes,
which mammals u t i l i z e i n th e o r n ith in e u re a c y cle f o r u re a s y n th e s is .
They b e lie v e th e s e f i s h e s produce u re a i n sm all q u a n t i t i e s from p u rin e s
-
'and amino a c id s .
10-
Urea s y n th e s is in th e t e l e o s t cannot keep pace
w ith r i s i n g blo o d ammonia and th e f i s h becomes p o iso n e d .
Fromm and
G i l l e t t e ( 1968) b e lie v e in c re a s e d le v e l s o f blood ammonia s tim u la te
g ly c o ly s is in n e u ra l t i s s u e .
T h e ir work d id n o t show t h a t ammonia
i n h i b i t e d exchange o f r e s p i r a t o r y g a se s o r d e crea se d th e a f f i n i t y , o f
hem oglobin f o r oxygen.
MATERIALS AED METHODS
Two t e s t ru n s were perform ed d u rin g th e tim e p e rio d December,
1973, th ro u g h March, 197^•
T e s tin g was perform ed in a la b o r a to r y on
th e campus o f Montana S ta te U n iv e rs ity , Bozeman, M ontana.
Rainbow
t r o u t eggs and sac f r y were exposed t o c o n tr o lle d c o n c e n tra tio n s o f
re a g e n t g rad e
C l.
DILUTION WATER
The t e s t w a ter was from th e d om estic supply t o th e c i t y o f Bozeman,
M ontana. ■ T reatm ent o f t h i s w ater b e fo re d i s t r i b u t i o n i s lim ite d t o '
s e t t l i n g and c h lo r in a tio n .
th ro u g h a c t i v a t e d c h a r c o a l.
The w ater was d ec h lo r in a te d by f i l t e r i n g
T e st w a ter was h e a te d and m a in ta in e d a t
c o n s ta n t te m p e ra tu re by an e l e c t r i c flo w -th ro u g h h e a te r c o n tr o lle d by
a v o lta g e r e g u l a t o r .
The chem ical and p h y s ic a l c h a r a c t e r i s t i c s o f th e t e s t w ater r e ­
mained q u ite c o n s ta n t th ro u g h o u t th e t e s t p e rio d s ’.
c h a r a c t e r i s t i c s m o n ito red a re p re s e n te d i n T able 2 .
The v a lu e s f o r th e
The io n s o f c a l ­
cium, magnesium, and b ic a rb o n a te a cc o u n te d f o r a p p ro x im a te ly 85/0 o f
th e c o n d u c tiv ity m easured.
Chem ical m easurem ents a re d e s c rib e d l a t e r
i n t h i s s e c tio n .
DILUTION APPARATUS
The d i l u t i o n a p p a ra tu s shown i n F ig u re s I and 2 was u sed t o .
in tro d u c e th e t e s t w ater c o n ta in in g to x ic a n t t o th e t r o u t eggs a n d /o r
sac f r y .
T h is a p p a ra tu s combined some o f th e co n cep ts o f Brungs and
Mount ( 1967) , G re n ie r ( i 960) , an d M c A llis te r e t a l . (1 9 7 2 ).• I added
-1 2 -
T able 2 .
Chem ical and p h y s ic a l p r o p e r tie s o f d i l u t i o n w a te r .
A lk a lin ity , m g /l a s CaCOg
100-110
H ardness, m g /l a s CaCOg
106-123
Ca++, m g /l a s CaCOg
7k -82
Mg++, m g /l a s CaCOg
32- 4 l
D isso lv e d oxygen, m g /l
pH
>8
.
7 . 4 - 7 .6
C o n tro lle d te m p e ra tu re , C
10-12
C o n d u c tiv ity , micromhos/cm
202-262
NOg-, m g/l
<1
NO2 " , m g /l
0 .0 7 max
T o ta l ammonia, NH^+-N
0 .1 5 max
R e s id u a l c h lo rin e
0 .0
-1 3 -
Eeater
Chlorine-free tap
Constant head reservoir
water
Voltage control
Waste
■120 VAC
10-ml
syringe
tube.7
20-1
Mariotte
bottle
D-2
I
500-ml filter flask
"Y" venturi—
Waste
Dilution water
Concentrated toxicant
Diluted toxicant
Mixing
Chamber
Incubation tube
with eggs----Rearing pan
Stainless steel
wool
y Waste
Figure I.
Schematic diagram of constant flow toxicant dilution
apparatus.
-1 4 -
F ig u re 2 .
C onstant flow to x ic a n t d i l u t i o n a p p a ra tu s ( t o p ) . In c u b a tio n
tu b e s and r e a r in g pans w ith sac f r y (b o tto m ).
-1 5 -
o th e r in n o v a tio n s t o a d a p t th e d i l u t i o n a p p a ra tu s t o th e s p e c if ic
needs o f th e t e s t .
D esign o b je c tiv e s and o p e ra tio n o f th e a p p a ra tu s
a r e d is c u s s e d i n th e a p p e n d ix .
The eggs were in c u b a te d in tu b e s p a tte r n e d a f t e r th o s e d e sc rib e d
by H urley (1 9 7 2 )(F ig u re s I and 2 ) .
Because t r o u t eggs a re s e n s itiv e
t o p h y s ic a l shock (D avis 1953)* th e w a ter e n te r e d a s id e .a rm and
e x ite d th ro u g h a bottom tu b e t o p re v e n t egg a g i t a t i o n .
Each o f th e s ix in c u b a tio n tu b e s and r e a r in g pans c o n s ti tu te d a
te s t s ta tio n .
S ta tio n numbers were a s s ig n e d a c c o rd in g t o th e nom inal
u n -io n iz e d ammonia c o n c e n tra tio n a t. t h a t s t a t i o n .
The c o n tr o l s t a t i o n
was i d e n t i c a l i n a l l r e s p e c ts t o th e o th e r f iv e s t a t i o n s ex cep t no
c o n n e c tio n e x is te d on t h e ,t o x i c a n t s id e o f G- (F ig u re l ) .
The nom inal
and a c t u a l mean u n -io n iz e d ammonia c o n c e n tra tio n s a t each s t a t i o n a re
g iv e n i n T able 3.
T able 3-
U n -io n iz e d ammonia c o n c e n tra tio n s a t each t e s t s t a t i o n .
S ta tio n
■■0. 4'
0 .3
0 .2 ■
0 .1
0 .0 5
c o n tro l■
Nominal
c o n c e n tra tio n
m g /l NH^-N
o .4 o
0 .3 0
0 .2 0
0 .1 0
0 .0 5
0 .0 0
A c tu a l mean
c o n c e n tra tio n
m g/l NH3-N
■ Run I
0 .3 7
0 .2 7
0 .1 9
0 .0 9
0 .0 5
0 .0 0
Run 2
.
0 .3 7 ■
0 .2 8
0 .1 9
0 .1 0
0 .0 5
0 .0 0
-
16 -
A ll tu b in g u sed was g la s s , l a t e x ru b b e r, o r Tygon®.
t a i n e r s were g la s s o r p o ly e th y le n e .
A ll con­
S to p p e rs were made o f ru b b e r.
Nominal flow t o each s t a t i o n was 400 m l/m in.
Large d iam eter
c a p i l l a r y tu b in g i n th e d i s t r i b u t i o n m a n ifo ld c o n tr o lle d th e flow s t o
th e s i x s t a t i o n s t o w ith in 5$ betw een e x tre m e s.
■
Flow t o each s t a t i o n
rem ained w ith in ' 10$ o f th e 400 m l/m in nom inal v a lu e .
Each s t a t i o n was se rv e d by a s e p a ra te 2 0 -1 M a rio tte b o t t l e .
The
to x ic a n t c o n c e n tra tio n a t each s t a t i o n was p r im a r ily c o n tr o lle d by th e
make-up c o n c e n tra tio n in each M a rio tte b o t t l e and s e c o n d a rily by manual
a d ju stm e n t o f th e h e i g h t ■o f K (F ig u re l ) .
The in c u b a tio n tu b e s were c o n s tr u c te d from 20 cm x 2 .5 cm t e s t
tu b e s .
The p o ly e th y le n e r e a r in g pans were 32 cm x 28 cm x 13 cm deep.
W ater d ep th was f cm r e s u l t i n g i n a p p ro x im a te ly 6 I w a te r volum e.
T e st s o lu tio n flo w was s u f f i c i e n t t o e q u a l th e volume o f th e
in c u b a tio n tu b e s i n ab o u t 6 s e c .
The q u a n tity in tro d u c e d in to th e
pans e q u ale d th e volume o f th e pans i n ab o u t 15 m in u te s „
These flow
r a t e s were much h ig h e r th a n th e minimum flo w o f th e c o n ta in e r volume
e v ery s ix h o u rs recommended by AFHA e t a l . (1971)•
A flo w r a t e o f a t
l e a s t 10 I p e r gram o f f i s h p e r day e x is t e d a t th e c o n c lu s io n o f th e
te s t.
T his flow r a t e exceeded th e 2 - 3 - l» g “!* d ay “l recommended by
Sprague ( 1969) .
The f i s h mass was b ased on d a ta from Bowen and
S tu d d ard (1970) u s in g th e g r e a t e s t f i s h le n g th and assum ing 100$
s u rv iv a l.
.
-IT -
I u s e d 'th e s e h ig h flo w r a t e s t o m inim ize in flu e n c e on w ater
q u a lity by th e t e s t f i s h ; o f . s p e c i a l i n t e r e s t was th e m aintenance o f
h ig h d is s o lv e d oxygen l e v e l s and th e av o id an ce o f s i g n i f i c a n t ammonia
a d d itio n from n itro g e n o u s w a ste s .
TROUT EGGS
I o b ta in e d t r o u t eggs from th e E nnis N a tio n a l F is h H atchery
(U .S . D ept, o f I n t . , F is h and W ild lif e S e rv ic e ) a t E n n is, M ontana.
Eggs f o r b o th t e s t ru n s were from th e E nnis s t r a i n o f rainbow t r o u t .
The eggs were s tr ip p e d and f e r t i l i z e d u s in g th e s ta n d a rd d ry method
(D avis 1953).
They were th e n w a ter h ard en ed and tr a n s p o r te d t o th e
t e s t la b o r a to r y . • •
A f te r g r a d u a l e q u a liz a tio n t o th e t e s t w ater te m p e ra tu re ,' 310
eggs vWere counted in to each in c u b a tio n tu b e u sin g a 'p o ly e th y le n e
s t r a i n e r c o n ta in in g a known number o f h o le s .
and any o b v io u sly deform ed.
I removed any dead eggs
Exposure t o th e ammonia c o n c e n tra tio n s
commenced w i t h i n .24- h o u rs a f t e r egg f e r t i l i z a t i o n . ■
D uring in c u b a tio n th e eggs were t r e a t e d every second d a y ,w ith .f o r
maldebyde s o lu tio n d i l u t e d 1 : 300- f o r 15- 20. min -t o c o n tr o l fungus
(S m ith, C.' E ., F is h C u ltu r a l Development ’C en ter, Bozeman, M ont.;
p e r s o n a l com m unication, Septem ber, 1 973)..
I c o n tr o lle d te m p e ra tu re w ith in ± 0 . 5 C o f th e nom inal v a lu e .
The. nom inal te m p e ra tu re f o r Run I eggs w as'1 2 C and f o r Run 2 eggs
was 10 C.
The nom inal te m p e ra tu re f o r sac f r y o f b o th ru n s was 10 C. '■
- l 8Eggs were l e f t u n d is tu rb e d ..in th e in c u b a tio n tu b e s u n t i l f i r s t
in d ic a tio n s o f h a tc h in g o c c u rre d .
I
I th e n d i s t r i b u t e d th e eggs over
th e bottom o f th e r e a r in g pans where h a tc h in g was completed-.
Dead eggs
and sac f r y were removed and re c o rd e d a t t h i s tim e .
SAC FRY
Sac f r y were in s p e c te d r e g u la r ly and m o r t a l i t i e s re c o rd e d .
I con­
s id e r e d a f i s h dead when r e s p i r a t o r y and o th e r o v e rt movements ceased
(AREA e t a l . 1 9 7 1 ) I removed sam ples o f l i v e f i s h p e r i o d i c a l l y f o r
le n g th m easurem ents, p h o to g ra p h s, and h i s t o l o g i c a l e x am in atio n .
These
were im m ediately f ix e d i n aqueous B ouin1s s o lu tio n and p re s e rv e d i n .
rJO0Io e th a n o l.
A lthough some sac f r y d eveloped t o th e p o in t where fe e d in g n o r­
m a lly commences, no fe e d in g was done d u rin g th e t e s t r u n s .
I m easured sac f r y t o t a l le n g th w ith a s t e e l r u l e u n d er a w ideI
f i e l d m icroscope (Hubbs and L a g le r 1958)•
WATER CHEMISTRY MEASUREMENTS
W ater te m p e ra tu re was m o n ito red w ith a c a li b r a t e d m e rc u ry -in ­
g la s s therm om eter immersed i n one m ixing chamber (F ig u re l ) .
As a
check I immersed a re c o rd in g therm om eter i n th e o v e rflo w w aste from
th e c o n s ta n t head r e s e r v o i r .
a Beckman model rJGk pH m e te r.
I m easured th e d i l u t i o n w a te r pH with- •
A g ita tio n o f th e t e s t s o lu tio n th ro u g h
th e d i l u t i o n a p p a ra tu s caused an in c r e a s e i n pH, a p p a r e n tly due t o lo s s
o f carbon d io x id e .
The a d d itio n o f N%C1 should have caused a s l i g h t
-1 9 -
d e c re a s e i n pH "but none was n o te d .
The n e t change i n pH o f th e
d i l u t i o n w a ter was c o n s is te n tly 0 .0 5 -0 .1 0 pH u n i t upw ard. . C onsequently,
th e pH v a lu e u sed i n c a lc u la tin g u n -io n iz e d ammonia was 0 .1 u n it
h ig h e r th a n t h a t m easured i n th e d i l u t i o n w a te r.
I m easured .c o n d u c tiv ity w ith a YSI model 31 c o n d u c tiv ity b r id g e .
I c o n s tr u c te d a c o n d u c tiv ity v s . te m p e ra tu re curve f o r th e d i l u t i o n
w a te r t o c o r r e c t re a d in g s t o 25 C.
I m easured a l k a l i n i t y u s in g th e
p o te n tio m e tr ic method (AEHA e t a l . 1971) •
A Beckman model rJGk pH
m eter was u sed t o d e term in e end p o in t.
I m easured h a rd n e ss by t i t r a t i o n w ith th e d i sodium s a l t o f
ey elo h e x a n ed ia m in e te t r a a c e t i c a c id ( CDTA; Hexa Ver®, Hach Chemical
C o., Ames, Io w a ).
I m easured calciu m w ith th e same t i t r a n t and a
s u i t a b l e c alc iu m i n d i c a t o r .
:
Magnesium and calciu m were c o n sid e re d as
th e o n ly s i g n i f i c a n t components o f h a rd n e s s .
Magnesium w as, t h e r e ­
f o r , c a lc u la te d by d if f e r e n c e betw een h a rd n e ss and calciu m (AEHA e t a l .
1 9 7 1 ).
I m easured d is s o lv e d oxygen by th e io d o m e tric method (a z id e
m o d if ic a tio n ) a c c o rd in g t o APHA e t a l . (1 9 7 1 ).
(PAO) was s u b s t i t u t e d f o r sodium t h i o s u l f a t e .
c o lo r com parison w ith a I m g /l s ta n d a rd .
P h e n y la rs in e oxide
I m easured HO^*" by
The cadmium r e d u c tio n method
was u sed w ith N itr a V er TV®(Hach Chem ical C o .).
I m easured NOg"" w ith
N i t r i V e r® ( Hach Chem ical Co.) and a Bausch and Lomb S p e c tro n ic 20
c o lo r im e te r .
R e s id u a l c h lo rin e was m o n ito red u sin g O -T o li Ver®
-2 0 -
s o l u t io n (Each Chem ical Co.) and a c o lo r w heel.
I m easured ammonia n itr o g e n u s in g d i r e c t n e s s l e r i z a t i o n (AEHA
e t a l . 19T 1)•
c ip ita te .
R o ch e lle s a l t was u sed t o p re v e n t fo rm a tio n o f p r e ­
Ammonia was q u a n tita te d w ith a K le tt model 800-3 c o lo r ­
im e te r w ith 4 cm l i g h t p a th and $20-600 nm f i l t e r .
A s ta n d a rd curve
was p re p a re d u sin g re a g e n t g rad e RHijiCl i n am m onia-free, d i s t i l l e d ,
d e io n iz e d w a te r.
I c a lc u la te d u n -io n iz e d ammonia u s in g T r u s s e ll
(1 9 7 2 ).
BLOOD SMEARS AHD HISTOLOGICAL SECTIONS
I o b ta in e d b lo o d sm ears from sac f r y by s e v e rin g th e c a u d a l f i n •.
and sp re a d in g a t h i n f ilm on m icroscope s l i d e s .
F ix in g ' was done in
m ethanol and s ta i n in g i n Leishm an1s and Giemsa.
The sac f r y were vacuum i n f i l t r a t e d and embedded whole a f t e r th e
p ro c e d u re s o f Luna ( 1968) .
and e o s in .
The s e c tio n s were s ta in e d w ith hem ato x y lin
RESULTS,
GROWTH AND DEVELOPMENT
E v a lu a tio n o f grow th was b ased on t o t a l le n g th a t t a i n e d by th e
sac f r y .
The mean t o t a l le n g th s from Runs I and 2 a re p re s e n te d in
T ables 4 and 5»
T able 4 .
Sam pling began 21 days a f t e r h a tc h in g in Run I , and
Run I . Mean t o t a l le n g th s (TL) in cm a t g iv e n days a f t e r
h a tc h in g . ( S ta n d a rd e r r o r o f th e mean = 0 .0 2 cm f o r each
m e a n ;.number o f o b s e rv a tio n s = 20 f o r each m ean.)
Days a f t e r h a tc h in g
S ta tio n
21
28
35
42
C o n tro l
2 .3 8
2 .5 9
2 .6 8
2.67
0 .0 5
2 .2 9
2 .5 3
2 .6 6
2.6 8
0 .1
2.15
2 .3 7
2 .5 1
2 .6 3
0 .2
1 .7 2
I ..81
1.90
. I . 97
0 .3
1 .4 3
1 .4 4
1.55
1.64
0 .4
•••
••.
• *"
7 days a f t e r h a tc h in g in Run 2 .
•
D ata from b o th ru n s were combined
where a p p lic a b le and th e ru n s were c o n sid e re d as r e p l i c a t i o n s .
The
mean t o t a l le n g th s from th e ru n s combined a re p re s e n te d in Table 6 .
A bsences o f d a ta occur where no sam ples were ta k e n b ecau se h ig h mor­
t a l i t i e s l e f t few a v a il a b le s u rv iv o rs '.
No samples were a v a ila b le
from S ta tio n 0 .4 i n Run I due t o h ig h e a r ly m o r t a lit y .
-2 2 -
T atile 5 .
Run 2 . Mean t o t a l le n g th s (TL) i n cm a t g iv e n days a f t e r
h a tc h in g . (SE = s ta n d a rd e r r o r o f th e mean; N = number o f
_______ o b s e r v a tio n s .)
___________________ _______________
j
Days a f t e r h a tc h in g
S ta tio n
C o n tro l
TL
SE
E
0.05
TL
SE
E
7
■ 1.74
0.03
9
14
28
35
2.03 ■ 2.25
o.o4
. 0.03
ll
10
2.39
0.02
20
2.42
0.03
19
2.35
0.02
20
'
1.93'
0.04
10
2.20
0.03
11
2.38
0.02
20
2.36
0.03
20
2.36
0.02
20
'1.53
0.03
10
1.78
o.o4
10
2.01
0.03
10
2.22
0.02
20
2.26
0.03
20
2.28
0.02
20
1.31
0.03
10
1.42
o.o4
10
1.57
0.03
10
1.67
0.02
20
I.67
0,03
20
1.18
o.o4
5
#• •
• 0•
...
. 1.36
0.03
10
1.17
o.o4
5
1.24
0.05
5
1.35
o.o4
5
1.39
0.02
18
0.2
TL
SE
E
0.3
TL
SE
E
0.4
TL
SE
E
42 '
1.67
0.03
9
0.1
TL
SE
E
21
•
1.71
0.02
20 .
•••
■ 1.42
o.o4
9
e ®•
1.41
0.03
10
-2 3 -
T able 6 .
Runs I and 2 combined. Mean t o t a l le n g th s (TL) i n cm a t
g iv e n days a f t e r h a tc h in g . (SE = s ta n d a rd e r r o r o f th e mean;
E = number o f o b s e r v a tio n s .)
Days a f t e r h a tc h in g
S ta tio n
C o n tro l
TL
SE
E
0.05
TL
SE
E
0.1
TL
SE
E
0.2
TL
SE
E
0 .3
TL
SE
E
21
28
35
2.31
2.49
2.55
0 .0 2
0 .0 2
0 .0 2
31
39
40.
2.25
2.46
0 .0 2
0 .0 2
31
40
2 .0 8
0.02
30 ■
.
40
•
4o
1.65
1.74
1.78
0 .0 2
0 .0 2
40 .
i.4o
0.02
30 .
0 .0 2
4o
2 .5 2
0.02
40
2.46
2.39
0 .0 2
0 .0 2
30
2.51
2 .5 1
0.02
40
2 .2 9
0 .0 2
'4 2
4o
.
0 .0 2
4o
1.84
0 .0 2
' 4o
1.53
0 .0 2
29
-24-
The mean t o t a l le n g th s (T ab les 4 and 5) a re p l o t t e d in F ig u re s
3 and 4 .
F ig u re 3 shows le n g th v a r i a t i o n w ith age (d ay s a f t e r h a tc h ­
in g ) a t e a c h / s t a t i o n and F ig u re 4 shows le n g th v a r i a t i o n among
s t a t i o n s a t f ix e d age's.
The p h y s ic a l appearance o f th e f i s h a t 28 d a y s.a g e i s i l l u s t r a t e d
by F ig u re 5*
The s u b je c ts i n th e p h o to g rap h s Were s e le c te d t o r e p r e ­
s e n t th e mean le n g th from each sam ple.
A specim en from S ta tio n 0 .4
i n Run I was n o t in c lu d e d due t o h ig h m o r t a lit y .
The specim en from
S ta tio n 0 .4 i n Run 2 i s r e p r e s e n ta tiv e o f b o th S ta tio n s 0 .3 and 0 .4 o f
t h i s ru n .
Comparison o f f i s h developm ent a s' a f u n c tio n o f tim e i s
i l l u s t r a t e d by F ig u re 6 . . These p h o to g rap h s show developm ent a t 7-
'
day i n t e r v a l s and compare c o n tr o l f i s h w ith th o s e from s e le c te d
s ta tio n s .
The p h o to g rap h s d em o n strate t h a t r e t a r d a t i o n o f growth
a t th e h ig h e r ammonia c o n c e n tra tio n s i s accom panied by a' g e n e r a l in h i­
b i t i o n o f developm ent and f a i l u r e t o a b so rb th e y o lk .s a c .
L e a st sq u ares a n a ly s is o f v a ria n c e was perform ed on th e le n g th
d a ta , and v a r i a t i o n s among mean le n g th s Were h ig h ly s i g n i f i c a n t (P < .0 l)
a t a l l a g e s . ■A n a ly sis o f v a ria n c e t a b l e s a re p re s e n te d in T ables 16,
17, and 18 (A ppendix). . The ru n s combined showed s i g n i f i c a n t d if f e r - - ,
ences (P < .0 l) betw een r e p l i c a t i o n s -at e a c h .a g e and (w ith one ex ce p tio n )
no s i g n i f i c a n t i n t e r a c t i o n betw een tre a tm e n t and r e p l i c a t i o n a t' th e
5$ I e v e l i
T his e x c e p tio n o c c u rre d a t 42 days (.0 1 < P < .0 5 )..............
-25-
Run I
Run 2
DAYS
AFTER
HATCHING
O
CONTROL
•
0.2
#
0.05
D
0.3
O
0.1
#
0.4
STATION
F ig u re 3.
Growth r e la tio n s h ip s o f sac f r y a t g iv en s t a t i o n s .
-2 6 -
RUN I
STAT I ON
CONTROL
TL,c m
0.03
RUN 2
DAYS
F ig u re 4 .
AFTER
HATCHIN G
Growth r e l a t i o n s h i p s o f sac f r y a t g iv e n tim e s a f t e r
h a tc h in g .
PO
-<
I
Run 2
Run I
A C o n tro l
B 0 .0 5
C 0 .1
D 0 .2
E 0 .3
A C o n tro l
B 0 .0 5
C 0 .1
S ta tio n
Figure 5
Sac fry development at 28 days after hatching
S ta tio n
- 2 8 -
m
m
M
S ta tio n 0 .2
EM
-
S ta tio n 0 .4
A T
B 14
C 21
D 28
Days a f t e r h a tc h in g
F ig u re 6 .
Sac f r y developm ent a t 7-d-ay i n t e r v a l s a t s e le c te d s t a t i o n s .
A ll f i s h a re from Run 2 .
-29-
Comparisons among mean t o t a l le n g th s a re p re s e n te d i n T ables
8, and 9.
These t a b l e s a llo w a b s o lu te and p e rc e n ta g e com parisons
betw een th e c o n tr o l and each tre a tm e n t and a ls o betw een each tre a tm e n t
and e v ery o th e r tr e a tm e n t.
For exam ple, from Run I a t 21 days
( T able j ) th e mean le n g th d if f e r e n c e betw een S ta tio n 0 .1 and th e
c o n tr o l was 0 .2 3 cm; th e mean le n g th o f S ta tio n 0 .1 f i s h was ^Ofo t h a t
o f th e c o n tr o l; t h i s d if f e r e n c e i s s i g n i f i c a n t (P < .0 5 ).
o f d if f e r e n c e s among means were e s ta b lis h e d
P ro b a b ilitie s
by r e f e r e n c e t o a
S tu d e n tiz e d Range T able ( Snedecor and Cochran 196%).
D iffe re n c e s i n g ro s s b e h a v io r among t e s t f i s h were n o te d in
r e l a t i o n t o s t a t i o n lo c a t i o n .
A c tiv ity among th e c o n tr o l f i s h fo llo w ­
in g h a tc h in g in v o lv e d weak w rig g lin g m otions a s th e f i s h la y on th e
bottom o f th e r e a r in g p a n .
W ith tim e th e w rig g lin g m otions in c r e a s ­
in g ly resem b led swimming m otions and s h o r t p e rio d s o f swimming began
t o o c c u r.
E v e n tu a lly th e y were c o n s ta n tly suspended and swimming
f r e e l y i n th e su rro u n d in g w a te r.
days a f t e r h a tc h in g .
T h is s ta g e o c c u rre d a t ab o u t 15-20
A c tiv ity a p p ea re d t o in c re a s e th ro u g h o u t th e
4 2 -day p e rio d fo llo w in g h a tc h in g .
B eh av io r and a c t i v i t y o f f i s h in
S ta tio n 0 .0 5 co u ld n o t be d is tin g u is h e d from t h a t o f th e c o n tr o l.
B ehavior o f f i s h i n S ta tio n s 0 .1 and h ig h e r was r e a d i l y d i s ­
tin g u is h a b le from t h a t o f th e c o n tr o l.
F is h i n S ta tio n 0 .1 reach ed
a f u l l y suspended swimming c o n d itio n a b o u t 6 -7 days l a t e r th a n th e
c o n tr o l.
As i n th e c o n tr o l, a c t i v i t y ap p eared t o in c r e a s e th ro u g h o u t
-30Table J .
Run I. Comparisons among mean tptal lengths. Enter table at
left in desired row and read absolute difference in cm and
relative length in percent ( ) in desired column. Differences
are significant (P<.0 5 ) unless otherwise indicated.
0 .1
S ta tio n
C o n tro l •
0 .0 5
21 days
0 .3
0 .2
0 .1
.0.05
0 .9 5 (6 0 )
0 . 66( 72)
0 .2 3 (9 0 )
0 .0 9 (9 6 )
0 . 86( 62)
0 .5 7 (7 5 )
0 .1 4 (9 4 )
0 .7 2 (6 7 )
0 .4 3 (8 0 )
28 days
0 .3
0 .2
■ 0 .1
0 .0 5
1 .1 5 (5 6 )
0 .7 8 (7 0 )
0 . 22( 92)
0 .0 6 (9 8 )*
1 .0 9 (5 7 )
0 .7 2 (7 2 )
0 . 16( 94)
0 .9 3 (6 1 )
0 .5 6 (7 6 )
0 .3 7 (8 0 )
35 days
0 .3
0 .2
0 .1
0 .0 5
1 .1 3 (5 8 )
0 .7 8 (7 1 )
0 .1 7 (9 4 )
0 .0 2 (9 9 )*
1 . 11( 58)
0 . 76( 71)
0 .1 5 (9 4 )
0 .9 6 (6 2 )
0 .6 1 (7 6 )
0 .3 5 (8 2 )
42 days
.0 .3
'
0 .2
0 .1
0 .0 5
1 . 03( 61)
0 .7 0 (7 4 )
0 .0 4 (9 9 )*
0 .0 1 (1 0 0 )*
l.o 4 ( 6 l)
0 .7 1 (7 4 )
0 .0 5 (9 8 )*
0 .9 9 (6 2 )
0 .6 6 (7 5 )
0 ,3 3 (8 3 )
.
* D iffe re n c e n o t s i g n i f i c a n t a t th e 5$> l e v e l .
■ 0 .2
0 .2 9 (8 3 )
-31Table 8.
Run 2. Comparisons among mean total lengths. Enter-table at
left in desired row and read absolute difference in cm and
relative length in percent ( ) in desired column. Differences
are significant (P<.0 5 ) unless otherwise indicated.
S ta tio n
C o n tro l
7 days
0.40 .3
0 .2 '
0 .1
0 .0 5
l 4 days
. 0 .4 0 .3 .
0 .2
0 .1 ■
0 .0 5
21 days
0 .4 .
0 .3
0 .2
' 0 .1 '
0.05,
28 days
0 .4
0 .3 .
0 .2
0 .1
0 .0 5 '
35 days
0 .4 .
0 .3 .
0 .2 .
■ 0 .1
0 .0 5
0 .1.
0 ,2
0 . 50( 70)
0 .5 7 ( 6 7 ) '
0 . 56( 68) : 0 . 49( 71)
0 .4 3 (7 5 ) - 0 . 36( 78)
0 .2 1 (8 8 )
0 .1 4 (9 2 ) ,
0 . 07( 96)*
0 . 36( 76)
0 .3 5 (7 7 )
0 . 22( 86)
0 .1 4 (8 9 )
0 .1 3 (9 0 )
0 .7 9 (6 1 )
. 0 ,6 9 (6 4 ) .
0 .5 4 (7 0 )
0 . 18( 87)
©©*
0 . 61( 70)
0 . 25( 88).
0 .1 0 (9 5 )*
0 .5 1 (7 4 )
0 . 15( 92)
0 . 90( 60)
0 . 85( 61)
0 .2 2 (8 6 )
©©©
" 0 . 68( 70)
0 .2 4 (8 9 )
0 .0 5 (9 8 )*
0 .6 3 (7 1 )
0 .1 9 (9 1 )
0 . 66( 67)
a-.# ©
0 .4 4 (7 8 )
0 .9 9 (5 8 )
1 .02 ( 57)
0 .7 1 (7 0 )
0 .1 6 (9 3 )
0 . 83( 63)
0 . 86( 61)
0 .5 5 (7 5 )
0 .2 8 (8 3 ) :
0 .3 l(8 l) •
•• • •
.• • •
0 . 69( 71)
0 . 10( 96)
0 .5 9 (7 4 )
0 .0 5
1 . 00( 58)
1 .0 3 (5 7 )
0 . 72( 70)
0 .1 7 (9 3 )
0 .0 1 ( 100)*
...
’■
0 .7 5 (6 9 )
q/U % 93)
0 . 06( 98)*
0 .3
0 .0 1 (9 9 )*
0 . 36( 80)
■
* Difference not significant at the. 5$- level.
•••
0 . 03( 102)*
-3 2 -
■Table 8 .
C ontinued.
S ta tio n -
C o n tro l
42 days
0.4,
. o .3
0V2.
0 .1
0 .0 5 .
0 .9 4 (6 0 )
0 .9 3 (6 0 )
0 .6 4 (7 3 )
0 .0 7 (9 7 )*
0 .0 1 (1 0 0 )*
0 .0 5
0 . 95( 60)
0 .9 4 (6 0 )
0 .6 5 (7 2 )
0 .0 8 (9 7 )* .
* Difference not significant ■at the
,
0 .1
0 . 87( 62)
0 .8 6 (6 2 )
0 . 57( 75)
0 .2
.
0 . 30( 82)
0 .2 9 (8 3 )
0 .3
0 . 01( 99)*
level.'
,1
■•-v
' -33Table 9-
Runs I and 2 combined. Comparisons among mean total lengths.
Enter table at- left in.desired row and read absolute differ­
ence ■in cm and relative length in percent ( ) in desired
column. Differences are significant (P<.0$) unless other­
wise indicated.
S ta tio n
C o n tro l
21 days
0 .3
0 .2
' 0*1
. 0 .0 5
0 .6 6 (7 1 )
0 .2 3 (9 0 )
0 .0 6 (9 7 )
0 . 60( 73)
0 . 17( 92)
28 days
0 .3
0 .2
■ 0 .1
0 .0 5
■ 1 .0 9 (5 6 )
0 .7 5 (7 0 )
0 . 20( 92)
0 .0 3 (9 9 )*
1 .0 6 ( 57)
0 . 72( 71)
0 . 17( 93)
35 days . ■,
0 .3
0 .2
' 0 .1
. 0 .0 5 ■
0 .7 7 (7 0 )
0 .1 6 (9 4 )
- 0 .0 4 (9 8 )*
0 . 7 3 ( 71)
0 .1 2 (9 5 )
42 days
0 .3
0 .2
o .i ■
0*05
• • e
0 . 98( 61)
0 .6 7 (7 3 )
0 .0 5 (9 8 )*
0 .0 1 (1 0 0 )*
'
' 0 .1
0 .0 5
. . .
0 ;2
0 • 0
0 . 43( 79)
.
0 . 89( 61)
0 . 55( 76)
0 .3 4 (8 0 )
0 .6 1 (7 4 )
-
0 .9 9 (6 1 )
0 .6 8 (7 3 )
0 .0 6 (9 6 ) .
* D iffe re n c e n o t s i g n i f i c a n t a t th e 5^ l e v e l .
0 .9 3 (6 2 )
0 .6 2 (7 5 )
.
0 .3 1 (8 3 ).
-3 4 -;.
the 42-day period.
Behavior of fish in Station 0.2; was very different■
from that of the control.
The activity of these fish was greatly
inhibited and at the end of 42 days only an estimated IOf0 of the sur­
viving fish were swimming freely.
Activity of fish in Stations 0.3
and 0.4 was limited to weak and infrequent Wriggling on the bottoms of
the rearing pans.
MORTALITY
y
Eggs were exposed to ammonia concentrations for all but the first
few hours of incubation.
Incubation periods were normal (Run I - 25: '
days at 12 0; Run 2 - 33
days
at 10 C) (Embody 1934).
Egg mortality
during incubation is shown in Table 10.
Table 10.
Egg mortality during incubation.. 310 eggs per station.
Station
Run
•I
2 ■
’
0 .1
Control
0 .0 5
, 7 .
14
9
103
88
■94
-
0 .2 '
; . 's
i n .
0 .4
0 .3 . .
' .21
.
• 100. ’ •
17
93
.
■The greater mortality among eggs in Run 2 was probably due to
differences in egg quality. Run 2 eggs were obtained very near the
end of the spawning period. .Inferiority of these eggs may be due in
part to frequent handling of the brood fish and their close confinement
-35-
f o r s e v e r a l weeks (D avis 1953).
C h i-sq u are p r o b a b i l i t i e s f o r egg and sac f r y m o r ta lity ap p ear in
T ab les 11 and '12.
i n T able 13.
Cum ulative p e rc e n t m o r ta lity o f sac f r y a re shown
V alues were c o rre c te d f o r c o n tr o l m o r ta lity by d ed u ctin g
th e p e rc e n ta g e dead i n th e c o n tr o l from b o th th e p e rc e n ta g e dead in
th e tre a tm e n t and from 100 p e r c e n t.
The form er f ig u r e o b ta in e d was
d iv id e d by th e l a t t e r and m u ltip lie d by 100 ( Sprague 1969) .
M o rta lity
p e rc e n ta g e s were, b ased on th e number o f in d iv id u a ls rem ain in g and
e x clu d e s th o s e removed f o r p r e s e r v a tio n .
i n T ables I 9 and 20' (A p p en d ix ).
The m o r ta lity d a ta a re l i s t e d
The cu m u lativ e u n c o rre c te d m o r ta lity '
p l o t s (F ig u re 7) te n d t o .be skewed sigm oid curves t a i l i n g o f f more
g ra d u a lly a t th e low er c o n c e n tra tio n s a s d e s c rib e d by Sprague ( 1969)..
B e s t - f i t lo g - p r o b a b il ity p l o t s o f th e c o rre c te d m o r ta lity p e r ­
c en ta g e s ly in g on e i t h e r s id e o f $0^ a re p re s e n te d in F ig u re 8 (APHA
e t a l . 1971)•
E s tim a te s o f LC50 a t w eekly i n t e r v a l s from th e s e p lo ts
a r e p re s e n te d a s a fu n c tio n o f tim e in F ig u re 9*
The mean' LC50 v a lu e s
from th e s e p l o t s (F ig u re 9) a re shown i n Table l 4 . I n b o th ru n s , where s iz a b le m o r t a lit y o c c u rre d , a la r g e p e rc e n ta g e
o c c u rre d a t h a tc h in g a s sac f r y were a tte m p tin g t o emerge from th e egg ■
s h e ll.
M o r ta lity a t t h i s tim e l e f t th e egg s h e l l r u p tu r e d b u t c lin g in g
t o th e f r y body w ith o nly th e head exposed. .
HISTOLOGICAL CHANGES
'
H is to lo g ic a l changes were n o t a s pronounced a s th o s e re p o rte d
-3 6 -
T ab le 11.
S ta tio n
Run I , . C h i-sq u are m o r t a lit y com parisons f o r eggs and sac
f r y . E n te r t a b l e a t l e f t i n d e s ir e d row and re a d s i g n i f i ■ cance in d e s ir e d column.
.
C o n tro l
0 .0 5
0 .1
'
0 .2 •
0 .3
0
X
0
0
X ■■
■ 0-
■
Eggs
0 .4
0 .3
0 .2
0 .1
■0 .0 5
0
0
. 0
0
\ o
X
0
0
0
'
Sac f r y
7 days
■ 0 .4
0 .3
0 .2
0 .1
0 .0 5
X
X
0 0
0
l 4 days
0 .4
■ 0 .3
.^ 0 .2
0 .1
0 .0 5
X
X ■
X .
o .
0
21 days
0 .4
0 .3
0 .2
0 .1
. . 0 .0 5
X ■
X
X
0
0
28 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
■X
X
.X
■ O 1
.0 '
X -P C .0 5 ;
0 - P > .0 5
■X
X
0
X .
X
0
■ 0
X
X
X
■
X
X
0
X
X
0
0
X
x
0
■
V
•
X •
X
0
0 .
•X
• X
0
. 0
<.
■X
■X
X '.
X
X
•X
X
X
X
x. ■
x
• X
.
-3 7 -
Table 11.
Continued.
S ta tio n
C o n tro l
35 days
0 .4
0 .3
0.2
0 .1
0 .0 5
X
X
.X
0
X
42 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
X
X
X
0
X
X -P < .0 5 ;
0 - E >.05
0 .0 5
X
X
X
X -
X
X
X
X
0 .1
x ■
X
X
X
X
X
0 .2
0 .3
X
X
X
X
X
X
-3 8 -
Table 12.
Run 2. Chi-square mortality comparisons for eggs and. sac
fry. Enter table at left in desired row and read signifi­
cance in desired column.
Eggs - Eo m o r t a lit y com parisons were s i g n i f i c a n t a t th e 5% l e v e l .
Sac f r y
S ta tio n
C o n tro l
7 days
0 .4
0 .3
■ 0 .2 '
0 .1
0 .0 5 '
X
X
0
0
X
X
X
X
X
X
X
0■
l 4 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
X
X
0
0
X
X
X
0
X
X
X
0
21 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
X
X
0
0
0
X
X
. 0
X
28 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
X
X
0
0
0
X - PC.05; 0 - P>.05
0 .0 5
.
X
X
0
X
0 .1
0 .2
0 .3
X
X
X
X
X
X
X
X
0
X
X
X
X•
X '
0
X
X
X
■
-39-
Table 12.
S ta tio n
Continued.
C o n tro l
0 .0 5
0 .1
0 .2
0 .3
35 days
0 .4
0 .3
.
0 .2
0 .1
0 .0 5
X
X
'X
0
0
X
X
0
X
X
.X
X
X
• X
-X
42 days
0 .4
0 .3
0 .2
0 .1
0 .0 5
X
' X
X
0
0
X
X
0
X
X
X
X
X
X
X
X - PC.05; 0 - PX 05
-4o-
T able 13.
C um ulative m o r t a lit y e x p re ss e d a s n e a r e s t p e rc e n t a t g iv en
exposure tim e s . A c tu a l m o r t a lit y i s fo llo w e d by c o rre c te d
f ig u r e i n ( ) .
Days exposure
S ta tio n
7
14
21
28
35
42
Run I
C o n tro l
3 (0 )
3 (0 )
4 (0 )
5 (0 )
6 (0)
6 ( 0)
0 .0 $
6 (4)
6 (4)
7 (3)
10 (5)
I l (6)
12 ( 6 )
0.1
V(I)
4 (2)
4 (0 )
5 ( 0)
5 ( 0)
6 ( 0)
0.2
5 (3)
8 ( 6)
11 (7)
1 3 (8 )
20 (15)
39 (35)
0.3
32 (30)
^1(50)
72 (71)
76 (75)
77 (76)
91 (90)
0.4
4 l (4o)
58 (57)
99 (99)
99 (99)
99 (99)
99 (99)
Run 2
22(o)
24 (0)
25 (0)
26 (0 )
32 (0)
33 (16)
34 (15)
36 ( 16)
36 (15).
37 (15)
4 l (13)
0.1
19(0)
20
( 0)
21 ( 0 )
23 (0 )
25 (0)
27 ( 0 )
0.2
22 ( 3 )
2 4 (3 )
25- ( I )
28 (4)
38 ( 16)
50 ( 27)
0 .3
81 (76)
8 7 ( 83)
88 (84)
89 ( 85)
91 (88)
94 ( 91)
0.4
52 (4o)
6 1 ( 50)
66 (55)
67 (56)
78 ( 70)
86 ( 80)
C o n tro l
20
0 .0 5
(0)
RUN
I
RUN
2
DAYS
O
CONTROL
•
■
0.05
□ 0.3
0.2
O
0.1
#
0.4
STATION
F ig u re 7 .
C um ulative m o r ta lity o f sac f r y .
U n co rrected f o r c o n tr o l m o r ta lity
-42-
0.20
O
■
g/1
NH
DAYS
EXPOSURE
E
0.30
RUN
2
P
0.20
30
SO
7. MORTALITY
Figure 8 .
T o x ic ity c u rv e s .
-43-
0.30
HUN
#
RUN
LC 5 0 , m g / I NH
O
DAYS
F ig u re 9»
EXP OSU RE
LC50 v a lu e s from t o x i c i t y cu rv es in F ig . 8 .
-44-
T able 14.
Mean LCjpO v a lu e s from Runs. I and 2, m g /l KH3-K. .
Days e x p o s u re ’
14 ■
7
:o .26
LC50
21
28
, 35 ' .
0 .2 5
'0.24
0 .2 3
42 '
0 .2 1
-
"by some w orkers (Larmoyeux and P ip e r 1973J Burrows 1964; R eichenbachK lin k e 1967) .
These w orkers used f i n g e r l i n g f i s h o r l a r g e r in w h ich '
developm ent was s u f f i c i e n t l y advanced t o p ro v id e good h i s t o l o g i c a l
s e c tio n s .
The e a r ly developm ent s ta g e p re s e n t i n sac f r y p re s e n ts
d i f f i c u l t i e s i n e v a lu a tin g h i s t o l o g i c a l s e c tio n s a n d , some anom alies ,■
: may have been o b scu red .
'
-
' A n a ly sis o f h i s t o l o g i c a l s e c tio n s was lim ite d t o g i l l t i s s u e , th e . .
on ly s tr u c t u r e which p ro v id e d ad eq u ate d e t a i l .
There were som e.occur­
re n c e s o f h y p e rtro p h y o f th e e p ith e liu m o f th e secondary la m e lla e in
f i s h from S ta tio n s 0 .2 , 0 .3 , and 0 . 4 ^(F ig u re s 10 and 1 1 ). . K a ry o ly sis ,
■I
and k a ry o rrh e x is were sometimes found i n ; g i l l e p i t h e l i a l c e l l s in
f i s h from S ta tio n s 0 . 3 .and 0 .4 a f t e r 28 days ex p o su re.
BLOOD ANOMALIES
In both runs the coloration of the sac.fry distinctly varied
among stations. .This variation was due partly (if not entirely) to
blood coloration.
Blood color in' fish from the control and from•■
Stations 0.05 and 0.1 was- dark pink.
Blood color in fish from.
-4 5 -
C o n tro l - 28 days a f t e r h a tc h in g
F ig u re 10.
G i l l f ila m e n ts showing norm al t i s s u e (X 325)•
-4 6 -
S ta tio n 0 .3 - 42 days exposure
S ta tio n 0 .4 - 28 days exposure
F ig u re 11.
G i l l fila m e n ts showing h y p e rtro p h y o f e p ith e liu m o f
secondary la m e lla e (arro w s) (X 325)«
-4 7 -
S ta tio n s 0 .2 , 0 .3 , and 0 .4 was p a le s tra w c o lo re d .
n o t d e m o n strate any blo o d c e l l a n o m a lie s.
Blood smears d id
E x tr a c ta b le b lo o d volume
was n o t g r e a t enough t o a llo w d e te rm in a tio n , o f h e m a to c rit o r hemo­
g lo b in c o n te n t o f th e e r y th r o c y te s .
DISCUSSION
T his stu d y d e m o n s tra te s -th a t ammonia .causes r e t a r d a t i o n in grow th
o f rainbow t r o u t sac fr y ..
I n h i b i t i o n o f grow th was accom panied by.
g e n e r a l i n h i b i t i o n o f developm ent and o f - y o lk sac a b s o r p t i o n .' The
grow th curves and p h o to g rap h s (F ig u re s 3,- k and 5) i l l u s t r a t e two
d i s t i n c t c a te g o r ie s o f e f f e c t s .
S ta tio n s 0 .0 5 and 0 . 1 . com prise th e
f i r s t w ith m oderate r e t a r d a t i o n in grow th and d e v e lo p m e n t;■ S ta tio n s
0 .2 , 0 .3 , and 0 .4 com prise th e second w ith g ro ss r e t a r d a t i o n o f grow th,
and developm ent.
■Maximum d if f e r e n c e s in le n g th betw een c o n tr o l and t e s t ,fis h ■
o c c u rre d a t about 1 4 -2 1 •days in S ta tio n s 6 .0 5 and 0 .1 and a t about
28-35 days in S ta tio n s 0 .2 - 0 .4 .
Why d if f e r e n c e s d id n o t c o n tin u e t o
in c r e a s e u n t i l 42 days age i s e x p la in e d .by th e grow th cu rv es in F ig u re 3.
At about 28-35 days age th e s lo p e .o f th e c o n tr o l grow th curves-
d e c re a se s , m arkedly i n b o th ru n s .
A bsorption- o f th e y o lk sac in th e .
c o n tr o l i s e s s e n t i a l l y com plete a t 28 days (F ig u re 5 )•
T h is su g g ests
t h a t , w ith o u t fe e d in g , th e c o n tr o l f i s h e x p e rie n c e d a' n u t r i t i o n a l
d e f i c i t a s a b s o r p tio n o f th e y o lk sac n e ared co m p letio n r e s u l t i n g in
red u ced grow th ra te '..
;
Mean le n g th d i f f e r e n c e s ■betw een th e ■c o n tr o l and .S ta tio n s .6 .4 , . 0 .3 , and 0 .2 were s i g n i f i c a n t (PC.0 5 ) a t ' a l l ages i n b o th ru n s .
L ength d if f e r e n c e s betw een th e c o n tr o l a n d .S ta tio n 0 .1 were s i g n i f i ­
c a n t (P < .05')' a t a l l ages ex cep t 42 days i n each ru n a n d -in th e ru n s •
com bined.
The le n g th d if f e r e n c e betw een th e c o n tr o l and S t a t i o n . 0.0 5
■was s i g n i f i c a n t (P <.05) a t 21 days in Run I a.nd in th e ru n s combined. '
A lthough th e d if f e r e n c e s were no t s i g n i f i c a n t a t th e 5$ l e v e l th e
c o n tr o l mean le n g th s were g r e a t e r th a n S ta tio n 0 .0 5 mean le n g th s
th ro u g h about 28 days a g e .
T his " c lo s in g " o f d if f e r e n c e s may be
,
I
a t t r i b u t e d t o th e d e c re a s in g s lo p e s o f th e c o n tr o l grow th curves w ith
advancing y o lk sac a b s o r p tio n .
F u r th e r ex am in atio n o f th e growth
c u rv es (F ig u re 3) shows t h a t S ta tio n 0 .0 5 and 0 .1 grow th r a t e s d e c lin e
l a t e r in tim e th a n th e c o n tr o l grow th r a t e .
The s t a t i s t i c a l evidence
s u p p o rts an i n h i b i t i o n o f grow th and y o lk sac a b s o r p tio n in th e s e two ■
s ta tio n s .
T his i n h i b i t i o n v e ry l i k e l y d e lay e d th e c o n d itio n o f
i
n u t r i t i o n a l d e f i c i t a llo w in g grow th i n th e s e s ta t i o n s t o g a in on th e
c o n tr o l.
I t i s l i k e l y t h a t , w ith fe e d in g , th e le n g th d if f e r e n c e s
betw een th e c o n tr o l and S ta tio n s 0 .0 5 and 0 .1 would have c o n tin u e d .
The e f f e c t s o f ammonia on th e sac f r y b e h a v io r were s t r a i g h t ­
fo rw a rd .
The b e h a v io r o f S ta tio n 0 .0 5 f i s h could no t be d is tin g u is h e d
from t h a t o f th e c o n tr o l.
B e h a v io ra l d if f e r e n c e s o f f i s h from a l l
:
o th e r s t a t i o n s were s t r i k i n g . ■ I n h i b i t i o n o f p h y s ic a l a c t i v i t y and
d e la y in a c h ie v in g a s t a t e o f swimming c o r r e la te d w ith r e ta r d e d grow th
and developm ent.
The im p lic a tio n s o f grow th r e t a r d a t i o n and b e h a v io r m o d ific a tio n
d e se rv e a t t e n t i o n .
One obvious consequence im p lied i s lo n g e r .tim e
! ■
'
t o a c h ie v e a. g iv e n le n g th which co u ld c e r t a i n l y e q u ate w ith d e c lin e
i n q u a lity o f a f i s h e r y .' A more s u b tle r e s u l t co u ld be i n d i r e c t
-5 0 -
m o r t a lit y .
Sac f r y o f salm onids a re v u ln e ra b le t o many f a c to r s and
th e lo n g e r th e y rem ain i n t h i s l i f e phase th e h ig h e r w i l l be th e
cu m u lativ e m o r t a l i t y .
R ic k er ( 194l) documents th e p r e d a tio n o f sock-
eye salmon by o th e r salm onids and squaw fish from th e tim e th e salmon
le a v e th e re d d s a s l a t e sac f r y .
d w e llin g s c u lp in a l s o .
He in d ic a te s p r e d a tio n by th e bottom '
H e rtin g and W itt ( 1.967) showed p r e f e r e n t i a l
p r e d a tio n by th e bow fin tow ard c e n tr a r c h id s w ith im p aried p h y s ic a l
c o n d itio n .
C la ire and P h i l l i p s ( 1968) observ ed p r e d a tio n o f rainbow
t r o u t sac f r y by s to n e f ly (P le c o p te ra ) nymphs.
There i s l i t t l e eviden ce from t h i s stu d y t h a t ammonia a f f e c t s th e
h a tc h in g su cc e ss o f rainbow t r o u t e g g s.
At th e c o n c e n tra tio n s t e s t e d
on ly S ta tio n 0 .3 i n Run I showed s i g n i f i c a n t d i f f e r e n t i a l egg mor­
t a l i t y (P<.05) when compared w ith th e c o n tr o l.
A ll egg m o r t a l i t i e s
were sm all i n t h i s ru n ra n g in g from 2/0 t o 7%.
Under h a tc h e ry c o n d i­
i
t i o n s , h a tc h in g su cc e ss o f rainbow t r o u t i s ab o u t 90$ ( Sm ith, C. E ., ■
F is h C u ltu r a l Development C en ter, Bozeman, M ont.; p e rs o n a l communica­
t i o n , December, 197^-) •
T h is lo n e ev id en ce o f d i f f e r e n t i a l egg m ortalf-
i t y i s weak s in c e no su p p o rt i s g iv e n by o th e r s t a t i o n s i n e i t h e r
ru n .
Ho e f f e c t s o f ammonia on in c u b a tio n tim e s were o b se rv e d .
In c u b a ­
t i o n p e rio d s were w ith in a 3-^- day v a r i a t i o n c o n sid e re d norm al (D avis
1953).
A f te r h a tc h in g , m o r ta lity d if f e r e n c e s among s t a t i o n s q u ic k ly
became e v id e n t.
S ta tio n s 0 .4 and 0 .3 were s i g n i f i c a n t l y d i f f e r e n t
1
-5 1 - ■
(P<.05) from th e c o n tr o l at. a l l ages i n b o th r u n s .
S ta tio n .0 .2 in
b o th ru n s was s i g n i f i c a n t l y d i f f e r e n t (P<.05) from th e c o n tr o l d u rin g
th e l a t t e r p a r t o f th e r u n s .
S ta tio n 0 .1 was n o t s i g n i f i c a n t l y d i f - '
f e r e n t from th e c o n tr o l a t any time-.
S ta tio n 0 .0 5 in b o th ru n s was .
s i g n i f i c a n t l y d i f f e r e n t (P<.05) from th e c o n tr o l o n ly i n some in s ta n c e s
and m o r t a lit y was low th ro u g h o u t b o th r u n s .
Cause f o r th e s i g n i f i c a n t .
■d i f f e r e n t i a l m o r t a lit y betw een S ta tio n 0 .0 5 and th e c o n tr o l i s l i k e l y
sam pling e r r o r s in c e no su p p o rt i s g iv e n by S ta tio n 0 .1 .
E vidence i s s tro n g t h a t ammonia c o n c e n tra tio n s above 0 .2 m g /l■
b e g in tfe evoke m ajor m o r ta lity i n rainbow t r o u t sac f r y .
The -
t o x i c i t y c u rv es (F ig u re 8) i l l u s t r a t e t h i s in showing LC^O v a lu e s o f
0 .2 - 0 .3 m g /l
Run I p ro v id e d a b e t t e r d e fin e d p ro g r e s s io n o f
t o x i c i t y c u rv es w ith tim e .
T h is was l i k e l y due t o much low er c o n tr o l
m o r t a lit y i n t h i s r u n . ' C lose agreem ent o f LC50 v a lu e s from b o th ru n s
e x i s t s from 21 days and on (F ig u re 9 )•
. The q u e s tio n a r i s e s : a t which exposure tim e sh o u ld th e i n c ip ie n t
LC50 be e stim a te d ?
E xam ination o f th e -cum ulative m o r t a lit y p lo ts
(F ig u re 7) shows an upward i n f l e c t i o n i n s e v e r a l cu rv es a f t e r 28 or.
35 d a y s.
T his may be due s t r i c t l y t o ammonia o r from o th e r e f f e c t s
a s s o c ia te d w ith ag in g and y o lk sac a b s o r p tio n .
Age 21 days g e n e r a lly
r e p r e s e n ts th e p o in t where th e sigm oid curves' p la te a u .
T h is su g g ests
u s in g th e 21 d a y .LC^O a s th e e s tim a te o f th e in c i p i e n t LC50.
The
LC^O a t t h i s tim e i s 0 .2 5 m g /l EHy-E (a v e rag e o f r u n s ) . ' T h is v a lu e
■
(
-5 2 -
i s low er th a n th e th r e s h o ld v a lu e s o f 0 . 3 - 0 . 4 m g /l EHg-Iii g e n e r a lly
found f o r rain b o w s.
The v a lu e i s in te rm e d ia te t o th r e s h o ld v a lu e s
o f 0 .2 m g /l EHg-E found by Liebmann ( i 960 , c i t e d by EIFAC 1973) f o r
rainbow t r o u t f r y and 0 .3 3 m g /l EHg-E found by Penaz ( 1965 ) f o r brown
tro u t fry .
A lthough h i s t o l o g i c a l changes were n o t pronounced, some anom alies
were o b serv ed which co u ld be d e tr im e n ta l.
The o b serv ed h y p e rtro p h y
o f th e g i l l secondary la m e lla e e p ith e liu m co u ld i n t e r f e r e w ith oxygen
and carbon d io x id e exchange a c ro s s t h i s membrane.
K a ry o ly s is and
1
1
k a ry o rrh e x is in t i s s u e could c e r t a i n l y i n t e r f e r e w ith f u n c tio n o f t h e ,
tis s u e .
The la c k o f re d c o lo r a tio n in th e blo o d in f i s h from S ta tio n s
0 . 2 , 0 .3 , and 0 .4 co u ld have r e s u l t e d from a re d u c tio n i n o r s u p p re s -'
s io n o f e ry th ro c y te numbers a s o b serv ed by R eichenbach-K linke ( 1967 ) .
H is to lo g ic a l changes coupled w ith a p o s s ib le r e d u c tio n in
e ry th ro c y te numbers v e ry l i k e l y co u ld have low ered th e oxygen tr a n s p o r t
c a p a b i l i t y o f th e b lo o d t o a l e v e l where grow th and developm ent were
in h ib ite d .
T h is h y p o th e s is i s su p p o rte d by e x p e rim e n ta l evidence t h a t
low le v e l s o f d is s o lv e d oxygen cause r e t a r d a t i o n o f grow th and d ev elo p ­
ment o f young salm onids (W arren 1971 ) and young n o rth e rn p ik e ( S i e f e r t
e t a l . 1 9 7 3 ) . . The c o n d itio n o f sac f r y dying as th e y were a tte m p tin g
t o emerge from th e egg s h e l l was n o te d by S i e f e r t e t a l . ( 1973) in th e
n o rth e rn p ik e embryos exposed t o d e p re sse d le v e ls o f d is s o lv e d oxygen.
-5 3 -
The co rresp o n d en ce betw een a g iv e n u n -io n iz e d ammonia l e v e l and
th e t o t a l ammonia c o n c e n tra tio n re q u ir e d t o produce i t is. i l l u s t r a t e d
by T able 15 ( T r u s s e ll 1972).
T able 1 5 .
T o ta l ammonia c o n c e n tra tio n s (m g /l KHj^+-W) r e q u ire d to p r o ­
duce a. g iv e n l e v e l o f u n -io n iz e d ammonia, (m g /l HHg-U) a t
d i f f e r e n t pH 's and te m p e ra tu re s .
T em perature, C '
U n -io n iz e d ammonia
0 .0 5
8
PH
7.0
7.5
8 .0
8 .5
o .i
7.0
7.5
■
8 .0
8.5
0.2
0.3
0.4' •
10
12
l4
31.2
10.0
3.2
1.0
2 6 .3
62.5
20.0
52.6
47.6
17.0 ' . 14.7
4.7
5-5
1.8
1.6'
6 .3
'
2.1
2 3 .8
2 0 .0
8.5
2.7
7.4
2.4
6.3
2 .0
' 17.2
5.4
1.8
0 ;9
0 .8
0.7
0 .6
4o.o
12.5
4.0
1.3
34.5 .
1 0 .9
8 0 .0
2 5 .0
8 .1
69.0
105.3
8 .0
125.0
4o.o
12.7
8.5 .
. 9 .2
3 .6
187.5
• 60.0
7.0
7.5
7.0
7.5
8.0
8.5
7.0
7.5 '
8 .0
8.5
19.0
6 .2
250.0
8 0 .0
2 5 .3
8 .3
16
95.2
29.4
3 3 .9
10.9
■ 9 .4
.
3.5
1.2
21.7
7.0'
2.4
3.1
2.7
1 5 7 .9
5 0 .9
142.9.
120.0
37.5
12.1
4.0
103.5
210.5 ■
190.5
58.8
1 8 .9
6 .3
160.0
137.9
.
l6.4
5.4
6 7 .8
. 2 1 .9
7.2
. 44.1
14.2
4.7
50 .0
16.1
5.4
32.6
10.5
' 3.5
43.5
i4.o
4.7 •
At a. t y p i c a l summertime w ater te m p e ra tu re o f l4 C and pH 8 an ammonia
-54-:
c o n c e n tra tio n o f about 4 m g /l
grow th in rainbow t r o u t f r y .
( 0 .1 m g /l KH^-N) can i n h i b i t
At th e same te m p e ra tu re and pH a
c o n c e n tra tio n o f about 10 m g /l HHj4.*1"-N (0 .2 5 m g /l N H yN )' can produce
50^ m o r t a lit y a f t e r p ro lo n g e d e x p o su re .
Recommended a llo w a b le le v e l s f o r long te rm exposure a re le s s th a n
o n e -te n th th e i n c ip ie n t LC50 su g g este d by t h i s s tu d y .
An u n -io n iz e d
ammonia c o n c e n tra tio n .of 0 .0 2 mg/l- HHyN i s recommended as maximum
by EIFAC (19.73) and. W illingham .(1973) f o r w a ters c l a s s i f i e d a s f i s h - .
e rie s .
T his v a lu e c o rresp o n d s t o a t o t a l ammonia c o n c e n tr a tio n .o f
0 .8 m g /l NHj4*1"-H a t l 4 C .and pH 8 .
Ammonia c o n c e n tra tio n s in n a t u r a l w a te rs can exceed th e maximum
recommended v a lu e s and, depending on pH and te m p e ra tu re , can re a ch
l e v e l s which can i n h i b i t grow th o r e f f e c t m o r t a lit y i n rainbow t r o u t
sac f r y .
For exam ple, .the Bozeman, M ontana, sewage tre a tm e n t p la n t
■p ro v id e d o nly p rim ary tre a tm e n t p r i o r t o 1970.
The t o t a l ammonia
c o n c e n tra tio n ' in th e E a st G a lla t in R iver, below t h i s p la n t averaged
2 .8 6 m g /l ( S tu a r t 'e t a l . 19 7 ^).
A f te r 1970,. a new sewage tre a tm e n t ■
p la n t upgraded tre a tm e n t t o p rim ary p lu s ' p a r t i a l .seco n d ary .
During
th e summer o f 1973, th e t o t a l ammonia c o n c e n tra tio n i n th e E a s t.
G a lla t in below th e new. p la n t av erag ed 0 .85 m g/l ( S tu a r t e t a l . ,1 9 7 4 ).',
Ammonia can be a s e r io u s problem t o rainbow t r o u t sac f r y and t o
a fis h e ry .
The t o x i c i t y o f ammonia may be in flu e n c e d by many f a c t o r s .
The r e s u l t s o b ta in e d i n t h is , stu d y c o u ld b e - a l t e r e d s i g n i f i c a n t l y .by
even m oderate changes in su rro u n d in g c o n d itio n s,. .
APPENDIX
DESIGN AHD OPERATION OF THE DILUTION APPARATUS
DESIGN OBJECTIVES
1)
A sm all q u a n tity o f c o n c e n tra te d to x ic a n t was t o be i n t r o ­
duced in to a la r g e q u a n tity o f d i l u t i o n w a te r.
2)
The q u a n tity o f to x ic a n t in tro d u c e d was t o be dependent upon
th e flow o f d i l u t i o n w a te r;
any i n t e r r u p t io n o f d ilu tio n
w a ter flow would th u s i n t e r r u p t to x ic a n t in tr o d u c tio n .
3)
Tubing and o r i f i c e s were t o be a s la rg e in d ia m e ter as,
p o s s ib le t o a v o id p lu g g in g .
4)
A c o n s ta n t, uniform , b a la n c e d flo w was d e s ir e d th ro u g h th e
in c u b a tio n tu b e s and re a rin g , p a n s.
OPERATION
C h lo rin e -fr e e w a ter e n te re d th e c o n s ta n t head r e s e r v o i r , A,
(F ig u re I ) i n q u a n tity s u f f i c i e n t t o c o n s ta n tly o v erflo w th ro u g h a
w aste tu b e , B.
W ater th e n p a sse d th ro u g h .a d i s t r i b u t i o n m an ifo ld , C,
t o s ix , 500-ml f i l t e r f l a s k s , D, a c tin g a s c y c lic sip h o n r e s e r v o i r s .
Upon f i l l i n g , th e w a ter l e v e l in D ro s e in to a v e n t tu b e , D -l, u n t i l
flow th ro u g h a sip h o n tu b e , E, a t p o in t F i n i t i a t e d a s ip h o n .
The
f l a s k c o n te n ts d is c h a rg e d th ro u g h th e p o ly e th y le n e "Y", G, and in to
th e m ixing chamber, H.
A 5 -ml g la s s b e a k e r, D-2, a c te d a s a. v a lv e by
f l o a t i n g upward, when em ptied, a g a in s t th e bottom o f th e siphon tu b e .
T his a c ti o n r a is e d th e rim o f D-2 above th e w ater l e v e l in D r e s u l t i n g
in a c le a n b re a k in g o f th e sip h o n .
G a c te d as a v e n t u r i .
- ■
The flo w o f w ater th ro u g h G allo w ed a.
-57-
m etered q u a n tity o f c o n c e n tra te d to x ic a n t t o be fo rc e d by atm o sp h eric
p r e s s u r e from th e 10-m l g la s s s y rin g e tu b e , K ,■th ro u g h a "U" tu b e , L,
and in to t h e •flow o f d i l u t i o n -water.
The nom inal d i l u t i o n a t t h i s
p o in t was 1 0 0 :1 . , C o n cen trated to x ic a n t and d i l u t i o n w a ter mixed
th ro u g h tu rb u le n c e in th e m ixing chamber, H.
A "U" tu b e , M, a ss u re d
b e t t e r m ixing by a llo w in g o n ly bottom draw rem oval o f t e s t s o lu tio n
from th e m ixing chamber, H.
The t e s t s o lu tio n was th e n conveyed t o
s ix in c u b a tio n tu b e s , N, a n d /o r r e a r in g p an s, P. . C a p illa r y tu b in g was
i n s e r te d betw een M and N o r P t o r e s t r i c t flo w so t h a t th e mixing
chamber n ev er em p tied .
The 10-m l sy rin g e tu b e , K, was s u p p lie d w ith to x ic a n t by th e 20-1
p o ly e th y le n e M a rio tte b o t t l e , J .
The l e v e l o f l i q u i d i n th e sy rin g e ,
tu b e was c o n tr o lle d by r a i s i n g and lo w erin g th e tu b e w ith in a h o ld in g
clam p.
-58-
T able 16.
Run I .
A n a ly sis o f v a ria n c e o f t o t a l le n g th .
Source o f
v a ria tio n
Age
Mean
sq u are
F
2.138501
•••
•••
4
2.023370
0.505842
510.639*
95 '
0.094108
0.000991
100
3.450499
•♦•
4
3.153941
0.788485
836. 615*
e rro r
95
0.089535
0.000942
""
to ta l
100
■4.174488
. •«
4
3.242215
0.810554
95
0.127669
0.001344
to ta l . .
100
4.292690
*. .
tre a tm e n t
4
2.899856
0.724964
. 95
0.100074
0.001053
to ta l
21 days
tre a tm e n t
e rro r
to ta l
28 days
tre a tm e n t
\
35 days
tre a tm e n t
e rro r
b2 days
e rro r
D egrees o f
freedom
Sum o f
sq u ares
100
.
-
..
603. l4 i*
•••'
6 8 8 .20&* .
■
-59-
T able 17.
Age
7 days
14 days
21 days
28 days
35 days
42 days
* PC.or
Run 2 .
A n a ly sis o f v a ria n c e o f t o t a l le n g th .
Source of.
v a ria tio n
D egrees o f
freedom
Sum o f
sq u ares
Mean
sq u are
to ta l
tre a tm e n t
e rro r
48'
5
42
2.676600
0.336813
0.042914
0.067363
0.001022
to ta l
tre a tm e n t
e rro r
45
4
1.255797
0.541081
0.081038
0.135270'
0.002026
6 6 . 768*
•••
to ta l
tre a tm e n t
e rro r
47
0.191826
0.001090
175.958*
•42
0.862900
0.767306
0.045788
to ta l
tre a tm e n t
e rro r
108
5
102
3.332598
3.083708
0.162189
0.616742:
0.001590
387. 867*
to ta l
tre a tm e n t
e rro r
79
3
75
1.501500
1.105762
■ 0.142817
0.368587
0.001904
193.563*
-
to ta l
tre a tm e n t
e rro r
99
5
93
2.447501
2.283239
0.164262
0.456648 ■
0.001766
258.540*
4o
4
F
65.927*
*#•
. . .
. . .
• • .
. . .
• • •
.
.
e
-6o-
T able 18.
Age
21 days
28 days
35 days
42 days
Runs I and 2 combined.
Source o f
v a ria tio n
A n a ly sis o f v a ria n c e o f t o t a l le n g t h .
D egrees oii" Sum o f ■
freedom
. sq u ares
Mean
sq u are
F
to ta l
tre a tm e n t
re p lic a tio n
t r e t , x re p .
e rro r
122
3
I
3
ll4
1.508797
1 .142661
0.067351
0.002339
0.120334
0.380887
0.067351
0.000780
O.OOIO56
to ta l
tre a tm e n t
re p lic a tio n
t r e t , x re p .
e rro r
190
4
I
4
180
5.590898
4 . 819726.
0.146647
0.009017
0.224879
1.204931
0.146647
0.002254 •
0.001249
964.462*
117. 380*
to ta l
tre a tm e n t
re p lic a tio n
t r e t , x re p .
e rro r
159
3
I
3
151
4.902091
. 2.367061
0.415039
0.230030
...
0.789020
0.415039
0.001380
0.001523
. *.
517.941*
272. 447*
0 . 906**
•••
to ta l
tre a tm e n t
re p lic a tio n
t r e t , x re p .
e rro r
189
4
6.125891
4.155007
0.596425
0.014349
0.250286
...
1.038752
0.596425
0.003587
0.001398
742. 896*
426.552*
2 . 565***
•••
*
PC.Ol
** p >.05
*** . o k p <.05
I
4
179
o.oo4i4i
•••
360.839*
6 3 . 806* .
0.739**
•••
l.8o4**
•••
-61 -
T able 1 9 .
Run I . M o r ta lity f ig u r e s showing numbers o f sac f r y a liv e
o r dead and p o t e n t i a l m o r t a lit y ( p o t . m o rt.)
■
Days- after hatching
Station
Pot.
mort.
control
dead
alive
238
0 .0 5
dead
alive
233
0.1
dead
alive
236
0 .2
dead
alive
24o
0 .3
dead
alive
■224
0 .4
dead
alive
290
7
14
21
28
35
42
6
232
6
232
9
229
12
226
13
225
14
224
13
220
' 13
220
16
217
23
210
27
206
27
206
8
228
10
226
10
226
12
224
12
224
13
223
20
220
26
214
32
208
51
189
93
147
72
152
114
HO
161
63
171
53
176
48
204
20
119
171
167
123
288
2
288
2
288
2
288
2'
12
228
'
- 62-
T able 20.
Run 2 . M o r ta lity f ig u r e s showing numbers o f sac f r y a liv e
o r dead and p o t e n t i a l m o r t a lit y ( p o t. m o rt. ) .
Days a f t e r h a tc h in g
S ta tio n
P o t.
m o rt.
c o n tr o l
dead
a liv e
137
0 .0 5
dead
a liv e
129
0 .1 '
dead
a liv e
- 150 '
0 .2
dead
a liv e
127
0 .3
dead
a liv e
195
0 .4
dead
; a liv e
176
7.
14
21
28
35 .
42
27
IlO ’
31
106
33 ■
104
34
103^
■ 35
102 ■
44,
- 93
42
87
44
85
46
83
47
82
48
81.
53
76
29.
.121 .
32
118
32
118
. 35 .
115
38
112
' 41
109
28
99
30
97
32
95
■36 .
91
48
79
■ 64
63
■
■
158 .
' 37
173
22
173
22
91
■ 85
107
69
116
60
■ 174
21
.
.
118
58
178
17
-
1
■ 183
12
137 ' ' ' 152
39 ■ . 24
'
,
LITER A TU RE C IT E D
AFHA, AWWA, WPCF. 1971. S ta n d a rd methods f o r th e e x am in atio n o f "water
and w a ste w a te r. American P u b lic .H ealth A s s o c ia tio n , W ashington,
D.C.
B a ll, I . R. 1967. The r e l a t i v e s u s c e p t i b i l i t i e s o f some s p e c ie s o f
fre s h w a te r f i s h t o p o iso n s - I . Ammonia. • W ater R e s e a rc h '1 :7 6 7 775.
Bowen, J . T .,. and N. S tu d d a rd . 1970. E n g lish a n d 'm e tric le n g th w eight r e l a t i o n s h i p s f o r rainbow , brown, and brook t r o u t . ■
Appendix A .I . t o Manual o f f i s h . c u l t u r e . U .S. D ep ti o f I n t . , ■
■ Bureau, o f S p o rt F is h e r ie s and W ild lif e , W ashington, D.C;.
Brockway, D. R. 1950. M etab o lic p ro d u c ts and t h e i r e f f e c t s .
P ro g re s s iv e F i s h - C u l t u r i s t 1 2 :127-129.
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D A T E
B u r k h a lte r , D alto n E
E f f e c ts o f p ro lo n g e d
ex p o su re to ammonia on
rainbow t r o u t . . .
!A S .U E D
T O
A'ZIX