Acute Toxicity of Hydrogen Cyanide to Freshwater Fishes

7/30/2019 Acute Toxicity of Hydrogen Cyanide to Freshwater Fishes

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Arch. Environm. Contam. Toxicol. 7, 325-337 Archives of EnvironmentalContaminationand Toxicology

A cut e T ox i c i t y o f H y drogen C yan i de t o F reshw a t er F i shes 1'2Lloyd L. Smith, Jr., Steven J. Broderius, Donavon M. Oseid, Gary L. Kimball,and Walter M. Koens tDepartment of Entomology,Fisheries, and Wildlife,Universityof Minnesota, 1980 FolweUAvenue,St. Paul, Minnesota55108

Abstract. Acute toxicity of hydrogen cyanide was determined at varioustemperatures from 4~ to 30~ and oxygen concentrations of 3.36 to 9.26 mg/Lon different life history stages of five species of fish: fathead minnow,P i m e p h a l e s p r o m e l a s Refinesque; bluegill, L e p o m i s m a c r o c h i r u s Rafin-esque; yellow perch, P e r c a f l a v e s c e n s (MitchiU); brook trout, S a l v e l i n u sf o n t i n a l i s (Mitchill); and rainbow trou t, S a l m o g a i rd n e r i Richardson. Medianlethal threshold concentrations and 96-hr LC50's were established by flow-through type bioassays. Acute toxicity varied from 57 /zg/L for juvenilerainbow trout to 191 /zg/L for field stocks of juvenile fathead minnows.Juvenile fish were more sensitive at lower temperatures and at oxygen levelsbelow 5 mg/L. For most species juvenile s were most sensitive and eggs moreresistant.

Compounds containing the cyanide group are present in many industrial andmunicipal effluents, including those from iron and steel mills, oil refineries, andplating plants, and constitute a significant source of toxicants introduced intoaquatic ecosystems. In aqueous solution the cyanide radical from simple alkalicyanides such as NaCN hydrolyzes to form free cyanide (CN- ion and molecularHCN). The molecular (un-ionized) component predominates at pH values (6.0-8.0) found in most natural waters, w ith less than 6% free cyanide occurring in theionic form below pH 8 at 25~ As the pH of aqueous simple cyanide solutions isincreased the percentage of free cyanide present as the CN- ion is increased tosatisfy the equilibrium reaction of HCN ~ H + CN-.The world literature on the toxici ty to fish of various cyanides was reviewedby Doudoroff (1976). Wuhrmann and Woker (1948), Bridges (1958), and Doudo-roff e t a l . (1966) have concluded tha t HCN is the principal toxic cyanide form.However, Broderius e t a l . (1977) have demonstra ted that even though molecularHCN is more toxic than the CN- ion, the anion does contribute to the totaltoxicity and to a greater degree as the test pH increases. Since HCN is highlytoxic to fish and most invertebrates, and further since some relatively nontoxic

1 Paper No. 9954, Scientific Journal S e r i e s , Minn esota Agricultural Experiment Station, St. Paul,Minnesota.Research supported by the U.S. Environmental Protection Agency, Environmental ResearchLaboratory, Duluth, Minnesota, under Grant No. R802914.0090-4341/78/0007-0325 $02.60@ 1978 Springer-Verlag New York Inc.


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3 26 L . L . S m i t h , J r . e t a l .

i ro n -c y ar ti de c o m po u n d s ph o t o d e c o m po s e , r e le a si n g H C N , c y a n id e c o n s t i t u te s ahazard in certain waste receiving water.The present paper reports on 337 acute tox ic i ty te st s des igned to de termine

the 96-hr med ian l e tha l tox icant concentrat ion (LC50) o f HCN and the med ianle tha l threshold concentrat ion (LTC) (Sprague 1969) a t var ious temperatures andd i sso lv ed ox yge n concentrat ions for three l if e h i story s tages of four spec ie s o ff i sh and for juven i l e s o f one other . F i sh u sed in the te sts were the fatheadm i n n o w , P im epha l e s p r o r n e l a s Raf inesque ; b luegil l , Lepor n i s mac r o c h i r u s Ra f -inesque ; ye l l ow perch , Pe r c a f l a v e sc e n s (Mitchi l l ) ; brook trout, Sa l v e l i n u sf o n t i n a l i s (MitchiU); and rainbow trout, Sa lmo g a i r d n er i Richardson .A wide range of reported l etha l concentrat ions to freshwater fi shes sup po s-edly attr ibutable to difference in sp ecies, condit ion of test f i sh , and test cond i-t ions l ed to the present ser i e s o f te st s conducted wi th a s ing l e water source andusing un i form exper imenta l procedures .

Mater ia l s and MethodsD i f f e r e n t s p e c i e s u s e d f o r a c u t e t e s t s w e r e s e c u r e d a s e g g s , f r y, a n d j u ve n i l e s , o r w e r e r e a r e d i n t h el a b or a t o r y . B r o o k a n d r a i n b o w t r o u t w e r e o b t a i n e d a s n e w l y h a r d e n e d e g g s o r a s 2 4 - h r fr y f r o m s t a teh a t c he r i e s . F a t h e a d m i n n o w s w e r e c u l tu r e d i n o u r l a b o r a to r y f r o m b r o o d s t o c k o r i gi n al ly o b ta i n e df r o m t h e U . S . E n v i r o n m e n t a l P r o t e c t i o n A g e n c y ' s E n v i r o n m e n t a l R e s e a r c h L a b o r a t o r y i n D u l u t h ,M i n n e s o t a . J u v e n i l e w i l d - s t o c k f a t h e a d s w e r e c o l l e c t e d f r o m C o m o L a k e i n S t . P a u l , M i n n e s o t a .B l u e g i ll s w e r e o b t a i n e d f r o m w i l d s t o c k w i t h e g g s s p a w n e d a n d f r y h a t c h e d i n t h e l a b o r a t o r y . J u v e n i l eb l u e g i ll s w e r e c o l l e c t e d f r o m lo c a l w a t e r s . Y e l l o w p e r c h e g g s w e r e o b t a i n e d f r o m w i l d a d u lt s t o c kw h i c h s p a w n e d i n t h e l a b o r a t o r y . Y e l l ow p e r c h e g g s w e r e a l s o c o ll e c t e d i n t h e f i e l d a n d h a t c h e d i n th el a b o r a t o r y f o r f r y te s t s ; j u v e n i l e p e r c h w e r e c o l l e c t e d i n th e f i e l d. D u r i n g r e a r i n g a n d m a i n t e n a n c e ,f i s h w e r e f e d d i f f e r e n t f o o d s i n c l u d i n g ( 1) m a s h e d h a r d - b o i l e d e g g y o l k , ( 2) a m i x t u r e o f g r o u n d h a r d -b o i l e d e g g a n d l e t t u c e , ( 3 ) l i v e , n e w l y h a t c h e d b r i n e s h r i m p (Ar t emia sa l i na ) ( " S a n F r a n c i s co B a yB r a n d , " M e t a f r a m e , I n c . 3 ) , ( 4 ) f r o z e n m a t u r e b r i n e s h r i m p , a n d ( 5 ) " G l e n c o e " d r y p e l l e t s .

W a t e r s u p p l y f o r t h e l a bo r a t o r y t e s t s w a s f r o m a d e e p w e l l a n d w a s t r a n s m i t t e d t o e x p e r i m e n t st h r o u g h p o l y v i n y l c h l o r i d e p i p e . T h e e x p e r i m e n t a l w a t e r h a d a t o t a l h a r d n e s s a n d a l k a l i n i t y o fa p p r o x i m a t e l y 2 20 a n d 23 5 m g / L a s C a C O 3 , r e s p e c t i v e l y . A c o m p r e h e n s i v e a n a l ys i s o f t h e w a t e r w a sr e p o r t e d b y S m i t h et a l . (1976).

T e m p e r a t u r e a c c l i m a t i o n f o r e g g s a n d f r y w a s a t t h e r a t e o f l l Y C / h r a n d f o r j u v e n i l e s 2 * C /h r . E g g sa n d f r y w e r e r a n d o m l y p la c e d i n t e s t c h a m b e r s a s s o o n a s t e s t c o n d it i o ns w e r e r e a c h e d . J u v e n i l e sw e r e h e l d a t t e s t c o n d i ti o n s f o r s e v e n d a y s b e f o r e b e i n g p la c e d i n t e s t c h a m b e r s . J u v e n i l e f i s h b r o u g h tf r o m t h e f i e l d w e r e g i v e n p r o p h y l a c ti c t r e a t m e n t w i t h n e o m y c i n a n d t e t r a c y c l i n e a t 20 m g / L f o r 4 - h rp e r i o d s o n t h r e e c o n s e c u t iv e d a y s .

T e s t w a t e r a d j u s t e d to a p p r o p r i a te t e m p e r a t u r e a n d o x y g e n w a s c o n v e y e d b y g r a v i t y f r o me l e v a t e d h e a d t a n k s t o t h e d i l u t e r a p p a r a t u s . T h e t o x i c a n t w a s d e l i v e r e d t o t e s t c h a m b e r s f r o mi n t e r m i t t e n t - f l o w d i l u te r s m o d i f i e d f r o m t h o s e o f M o u n t a n d B r u n g s ( 1 96 7) . S o d i u m c y a n i d e s t o c ks o l u ti o n s w e r e a d j us t e d t o p H 1 1 w i t h N a O H . W a t e r f lo w to e a c h t e s t c h a m b e r w a s 1 .3 L a t e a c h 3 -m i n c y c l e a s s u r i n g 9 9 % d i s p l a c e m e n t i n 3 . 5 h r .

T e s t c h a m b e r s f o r j u v e n i l e s a n d t r o u t s w i m - u p f r y w e r e g l a s s a q u a r i a 50 x 2 4 x 2 0 c m h i g h f il le dt o 2 0 L o f t e s t s o l u t i o n . E gg s a n d f r y w e r e t e s t e d i n s c r e e n b o t t o m e d a c r y l i c c y l i n d e r s e a c h c o v e r e dw i t h a b a k e l i t e l i d a n d h e l d i n a 20 - L c h a m b e r s o t h a t a p o r t i o n o f th e w a t e r f r o m e a c h c y c l e f l o w e du p w a r d t h r o u g h t h e s c r e e n t o t h e o u t le t . A l l t e s t s w e r e c o n d u c t e d u n d e r t w o f l uo r e s c e n t l a m p s ( L u x o r

3 U s e o f p r o d u c t o r tr a d e n a m e s m e n t i o n e d t h r o u g h o u t t h e t e x t d o e s n o t c o ns t i t u te e n d o r s e m e n t .


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T o x i c i t y o f H C N t o F i s h 32 7

I n c . " V i t a - L i t e " ) p r o v i d i n g a n i n t e n s i t y o f 5 5 t o 7 8 f o o t c a n d l e s a t t h e w a t e r s u r f a c e a n d a p h o t o p e r i o do f 1 2 h r li g h t.

T h e f r e s h w a t e r f i s h u s e d f o r t e s t i n g a nd t h e i r l i f e h i s t o r y s t a g e , s i z e , a n d d e n s i t y i n t h e b i o a s s a yc h a m b e r s a r e p r e s e n t e d i n T a b l e 1 . O n l y i d e n t if i e d l i v e e g g s w e r e t e s t e d . E g g s a n d f r y w e r e t e s t e di m m e d i a t e l y o n i n t r o d u c t i o n t o th e t e s t c h a m b e r s , b u t f o l lo w i n g t e m p e r a t u r e a c c l i m a t i o n j u v e n i l e sw e r e h e l d i n te s t c h a m b e r s f o r t hr e e d a y s p r i o r t o t o x ic a n t e x p o s u r e . J u v e n i le s w e r e f a s t e d d u r i n g th el a s t 2 4 h r o f a cc l i m a t i o n to t h e b i o a s s a y s y s t e m s a n d d u r i n g t h e f i r s t 96 h r o f t o x i ca n t e x p o s u r e . A l lr e p o r t e d c o n c e n t r a t io n s o f t ox i c a nt , p H , 0 3 , a n d t e m p e r a t u r e w e r e b a s e d o n a n a ly s e s m a d e o n w a t e rf r o m t h e t e s t c h a m b e r s . F r e e c y a ni d e c o n c e n t r a ti o n s i n e a c h c h a m b e r w e r e d e t e r m i n e d d a i l y b y t h eE p s t e i n c o l or i m e t r i c m e t h o d ( A m e r i c a n P u b l i c H e a l t h A s s o c i a t i o n e t a l . 1 9 71 ) w i t h c a l c u l a t e d H C Nc o n c e n t r a t i o n s b a s e d o n c o r r e s p o n d i n g p H a n d t e m p e r a t u r e m e a s u r e m e n t s a n d u s i n g d i s s o c i a t i o nc o n s t a n t s o f m o l e c u l a r H C N a s d e f i n e d b y I z a t t e t a l . (1962).

O b s e r v a t i o n s o n m o r t a l i ty o f t e s t o r g a n i s m s e x p o s e d t o k n o w n c y a n i d e c o n c e n t r a t i o n s w e r em a d e d a i l y . T h e c o n c e n t r a t i o n - p e r c a n t m o r t a l i t y d a t a w e r e a n a l y z e d w i t h a l o g a r i t h m i c - p r o b a b i l i t y( l og - p r o b i 0 p r o g r a m ( D i x o n 1 9 7 3) . U p p e r a n d l o w e r 9 5 % c o n f i d e n c e l i m i t s f o r t h e 9 6 - h r m e d i a n l e t h a lt o l e r a n c e ( L C 5 0 ) a n d m e d i a n l e t h a l t h r e s h o l d c o n c e n t r a t i o n s ( L T C ) w e r e c a l c u l a t e d f r o m t h ee q u a t i o n s l o g L C S 0 1 . 96 ( 1/ b )( N '/ 2 ) -1 ~ f o r h o m o g e n e o u s d a t a o r

log L C5 0 t .0~K_z>(1/b)(x2/K-2) l/~(N'/2) -~j~ for h et e r o ge ne ou s data .T h e s y m b o l , N ' , r e f e r s t o t h e n u m b e r o f t e s t o r g a n i s m s e x p e c t e d t o d i e w i t h i n t h e m o r t a l i ty i n t e r v a l o f1 6 t o 8 4 % . T h e r e c i p r o c a l o f th e l o g - p r o b i t l i n e ' s s l o p e ( l/ b) i s e q u i v a l e n t t o t h e s t a n d a r d d e v i a t i o n o ft h e l o g a r i t hm o f t h e p o p u l a t i o n ' s t o l e r a n c e f r e q u e n c y d i s t r i b u t i o n ( & ) o r t h e l o g a r it h m o f L i t c h f i e l da n d W i l c o x o n ' s ( 19 4 9) s l o p e fu n c t i o n , S . T h e s y m b o l , K , r e f e r s t o t h e n u m b e r o f t r e a t m e n t l e v e l se x c l u d i n g c o n t r o l s , a n d t o t h e ( C h i ) z v a lu e c a l c u l a t e d f o r g o o d n e s s o f f i t o f t h e r e g r e s s i o n l i n e t o t h ed a t a p o i n t s . I f t h i s v a l u e i s g r e a t e r t h a n t h e t a b u l a t e d ( C h i) z w i t h K - 2 d r , t h e d a t a a r e s i g n i f i c a n t l yh e t e r o g e n e o u s . T h e a b o v e f o r m u l a s w e r e d e r i v e d f r o m L i t c h f i e l d a n d W i l c o x o n ( 1 9 4 9 ) a n d F i r m e y( 19 7 1) . T h e m e d i a n l e t h a l t h r e s h o l d c o n c e n t r a t i o n s w e r e d e t e r m i n e d a t t h e e n d o f a t i m e i n t e r v a l w h e nn o m o r t a l i t y in a n y t e s t t r e a t m e n t h a d o c c u r r e d f o r a t l e a s t 4 8 h r . W h e n t h e d a t a w e r e s u c h t h a t p r o b i ta n a l y s i s b y c o m p u t e r w a s n o t p o s s i b l e , c a l c u l a t i o n s w e r e m a d e g r a p h i c a l l y .

R e s u l t sA c u t e t o x i c i t y o f H C N v a r i e d f o r d i f f e r e n t f i s h l i f e h i s t o r y s t a g e s w i t h e g g s b e i n gm o s t r e s i s t a n t a n d n e w l y h a t c h e d f r y a n d j u v e n i l e s t h e m o s t s e n s i t i v e . M e d i a nl e t h a l c o n c e n t r a t i o n s f o r 9 6 - h r a n d l e t h a l t h r e s h o l d p e r i o d s , t h e i r 9 5 % c o n f i d e n c e

Table 1 . N u m b e r o f o r g a ni s m s p e r t e s t c h a m b e r a n d r a n g e i n t o ta l le n g t h a t d i ff e r e n t l i fe h i s t or ys t a g e s

S a c f r y S w i m - u p f r y J u v e n i l e sN o . L e n g t h L e n g t h L e n g t h

S p e c i e s e g g s N o . ( m m ) N o . ( r am ) N o . ( r am )F a t h e a d m i n n o w 2 5 - 5 0 2 5 5 - 6 2 5 5 - 6 1 0 2 6 - 4 5B l u e g i l l 2 5 1 0 - 5 0 4 1 0 - 5 0 4 1 0 - 2 0 1 3 - 2 8Y e l l o w p e r c h 2 0 - 7 0 2 5 4 2 5 4 1 0 - 2 0 4 8 - 6 2B r o o k t r o u t 2 5 1 0 9 - 1 0 1 0 1 4 - 1 6 1 0 4 0 - 6 8R a i n b o w t r o u t . . . . . 10 4 0 - 6 8


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3 28 L.L. Smith, Jr. et al.limits, and log-probit regression analysis of the concentration-percent mortalitycurves for each exposure period are detailed in Tables 2 through 13.

F a t h e a d M i n n o wTests with different life history stages of the fathead minnow were conducted atabout 15~ to 25~ and 4 to 7 mg/L dissolved oxygen . Delayed embryologicaldevelopment at low temperature and low DO resulted in no apparent differencein resistance with decreasing DO but an increase in resistance with decreasingtemperature for 96-hr egg tests (Table 2). Median lethal HCN concentrations athatching were markedly reduced for tests at about 25~ and when the DO wasless than 5.52 mg/L. At comparable Do levels it appears that a reduction intemperature from 25.0 ~ to 15.2*C resulted in some increased sensitivity athatching. The time required to hatching for eggs at about 25~ 20~ and 15~ was 6,10, and 17 days, respectively.For fry and juvenile tests there was little difference in the 96-hr and medianlethal threshold concentrations. There was no clear trend associated with tem-perature for fry tests, but for tests at about 25~ and below 5.14 mg/L DO anincrease in sensitivity was apparent (Table 3). No change in sensitivity related totest temperature was observed for juvenile s (Table 4).

B l u e g i l lBluegill eggs incubated at about 25~ and DO ranging from 6.90 to 3.39 mg/Lshowed no correlation in sensitivity at hatching to high concentrations of HCN(Table 5). At the hatching LC50 values essentially all the newly hatched sac frywere premature and deformed. Fry were considerably more sensitive to cyanidethen eggs, showing at 25~ a substantial decrease in 7-day LTC values withdecrease in DO.In juvenile tests a t 8.4~ to 24.9~ and comparable DO levels the LTC valuesincrease progressively from 61.7 to 120 txg/L HCN (Table 6). However, only aslight reduction in sensitivity was observed for uvenil es tested at 3.48 to 6.90 mg/L DO and 25~

Y e l lo w P e r c hResults from yellow perch egg and fry experiments conducted at about 10~ to18~ and 4 to 7 mg/L dissolved oxyge n are so variable that specified medianlethal concentr ations cann ot be cons idered definitive (Table 7). For juvenile testsat 15.0~ to 21.0~ and at comparable DO levels the LTC values were determinedto increase progressively from 87.6 to 101 tzg/L HCN (Table 8). An increase insensitivity was obse rved for juveniles t ested a t 7.10 to 3.56 mg/L DO and 21ocwith LTC values decreasing from 107 to 75.0 /zg/L HCN. There was littledifference between the 96-hr and LTC values for juvenile perch.


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-

-

1

-5

11

-

-

1

1

20

35

77

4

I

-85

64

1

1

-

-

I

-99

71

-

-

1

1

18

46

7.80

2

4

-24,538

14

82.4

76.4-88,9

82.4

76.4-88.9

20

52

77

4

8

-0

14

1

1

~

-

9

-12

I16

-

-

1

1

20

60

79

4

1

-4

19

1

1

1

1

20.0

7.13

7.90

4

9

-28.818

19

1

124-138

1

124-138

28

35

77

2

7

-14

15

1

89

1

89

20

50

78

2

7

-5

17

1

I1

1

I1

21

61

79

8

2

-0

18

1

1

1

1

22

70

79

2

6

-0

14

1

1

1

1

Te5AceocyoHCNobu

easw/m-uye

a9hL

ameaehh

do

hcnc

o

Me

c

o

L

oay

9L

LohL

9%

9%

D

No

Cd

Cd

~

mgL

pH

t

Tme

a

b

~gL

lm

~L

lm

E 22

33

77

2

6

-13

22

-

-

6

4

20

49

77

2

6

-49

36

-

-

.5

2

21

60

79

2

8

-4

61

-

-

6

5

20

69

79

2

6

-38

31

-

-

5

3

F 20

59

78

6

t

-26

29

3

1

~

-

I

-83

57

-

-

2

1

29

35

77

3

4

-3

22

-

-

1

99

29

50

78

4

1

-27

32

2

1

-

-

7

-6

18

-

-

1

1

29

60

79

8

2

-5

76

2

2

~

-

2

-1

98

-

-

2

1

2

68

79

4

9

-82

54

2

2

-

-

.5

-g

73

-

-

1

i1

c .a.


7/13

Te6AeocyoH

tobu

u

ee

a9hL

ameaehh

dc

o

ID

~Meec

o

Lpo

oay

9hL

L

9%

9%

D

No

Cd

Cd

mL

pH

te

Tm

a

b

#L

lm

pgL

lm

,< (3 ~ 8

84

60

78

2

-

-

-

80

-

-

-

5

-42

22

-

-

67

5362

97

83

79

1

-

-

-

-

1

10

60

78

2

6

-50

48

>

-

1

11

72

79

2

8

-00

23

>

-

1

10

61

79

2

-

-

-

>

-

-

-

-

-

1

602

10

36

77

2

-

-

-

>

-

-

1

942

11

81

78

3

-

-

-

>

-

~

-

10

59

77

1

4

-36

43

>

-

90

492

Sf~

10

35

76

2

4

-21

35

1

711

Acute Toxicity of Hydrogen Cyanide to Freshwater Fishes

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