Arch. Environm.Contam.Toxicol.7, 325-337
Archives of
Environmental Contamination and Toxicology
Acute Toxicity of Hydrogen Cyanide to Freshwater Fishes 1'2 Lloyd L. Smith, Jr., Steven J. Broderius, Donavon M. Oseid, Gary L. Kimball, and Walter M. Koenst Departmentof Entomology,Fisheries,and Wildlife,Universityof Minnesota,1980FolweUAvenue, St. Paul, Minnesota55108
Abstract. Acute toxicity of hydrogen cyanide was determined at various temperatures from 4~ to 30~ and oxygen concentrations of 3.36 to 9.26 mg/L on different life history stages of five species of fish: fathead minnow, Pimephales promelas Refinesque; bluegill, Lepomis macrochirus Rafinesque; yellow perch, Perca flavescens (MitchiU); brook trout, Salvelinus fontinalis (Mitchill); and rainbow trout, Salmo gairdneri Richardson. Median lethal threshold concentrations and 96-hr LC50's were established by flowthrough type bioassays. Acute toxicity varied from 57 /zg/L for juvenile rainbow trout to 191 /zg/L for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg/L. For most species juveniles were most sensitive and eggs more resistant. Compounds containing the cyanide group are present in many industrial and municipal effluents, including those from iron and steel mills, oil refineries, and plating plants, and constitute a significant source of toxicants introduced into aquatic ecosystems. In aqueous solution the cyanide radical from simple alkali cyanides such as NaCN hydrolyzes to form free cyanide (CN- ion and molecular HCN). The molecular (un-ionized) component predominates at pH values (6.08.0) found in most natural waters, with less than 6% free cyanide occurring in the ionic form below pH 8 at 25~ As the pH of aqueous simple cyanide solutions is increased the percentage of free cyanide present as the CN- ion is increased to satisfy the equilibrium reaction of HCN ~ H § + CN-. The world literature on the toxicity to fish of various cyanides was reviewed by Doudoroff (1976). Wuhrmann and Woker (1948), Bridges (1958), and Doudoroff et al. (1966) have concluded that HCN is the principal toxic cyanide form. However, Broderius et al. (1977) have demonstrated that even though molecular HCN is more toxic than the CN- ion, the anion does contribute to the total toxicity and to a greater degree as the test pH increases. Since HCN is highly toxic to fish and most invertebrates, and further since some relatively nontoxic
1 Paper No. 9954, ScientificJournal Series, Minnesota Agricultural Experiment Station, St. Paul, Minnesota. Research supported by the U.S. Environmental Protection Agency, EnvironmentalResearch Laboratory, Duluth,Minnesota,under Grant No. R802914. 0090-4341/78/0007-0325 $02.60 @ 1978 Springer-Verlag New York Inc.
326
L . L . Smith, Jr. et al.
iron-cyartide compounds photodecompose, releasing HCN, cyanide constitutes a hazard in certain waste receiving water. The present paper reports on 337 acute toxicity tests designed to determine the 96-hr median lethal toxicant concentration (LC50) of HCN and the median lethal threshold concentration (LTC) (Sprague 1969) at various temperatures and dissolved oxygen concentrations for three life history stages of four species of fish and for juveniles of one other. Fish used in the tests were the fathead minnow, Pimephales prornelas Rafinesque; bluegill, Lepornis macrochirus Rafinesque; yellow perch, Perca flavescens (Mitchill); brook trout, Salvelinus fontinalis (MitchiU); and rainbow trout, Salmo gairdneri Richardson. A wide range of reported lethal concentrations to freshwater fishes supposedly attributable to difference in species, condition of test fish, and test conditions led to the present series of tests conducted with a single water source and using uniform experimental procedures.
Materials and Methods Different species used for acute tests were secured as eggs, fry, and juveniles, or were reared in the laboratory. Brook and rainbow trout were obtained as newly hardened eggs or as 24-hr fry from state hatcheries. Fathead minnows were cultured in our laboratory from brood stock originally obtained from the U.S. Environmental Protection Agency's Environmental Research Laboratory in Duluth, Minnesota. Juvenile wild-stock fatheads were collected from Como Lake in St. Paul, Minnesota. Bluegills were obtained from wild stock with eggs spawned and fry hatched in the laboratory. Juvenile bluegills were collected from local waters. Yellow perch eggs were obtained from wild adult stock which spawned in the laboratory. Yellow perch eggs were also collected in the field and hatched in the laboratory for fry tests; juvenile perch were collected in the field. During rearing and maintenance, fish were fed different foods including (1) mashed hard-boiled egg yolk, (2) a mixture of ground hardboiled egg and lettuce, (3) live, newly hatched brine shrimp (Artemia salina) ("San Francisco Bay Brand," Metaframe, Inc.3), (4) frozen mature brine shrimp, and (5) "Glencoe" dry pellets. Water supply for the laboratory tests was from a deep well and was transmitted to experiments through polyvinyl chloride pipe. The experimental water had a total hardness and alkalinity of approximately 220 and 235 mg/L as CaCO3, respectively. A comprehensive analysis of the water was reported by Smith et al. (1976). Temperature acclimation for eggs and fry was at the rate of llYC/hr and for juveniles 2*C/hr. Eggs and fry were randomly placed in test chambers as soon as test conditions were reached. Juveniles were held at test conditions for seven days before being placed in test chambers. Juvenile fish brought from the field were given prophylactic treatment with neomycin and tetracycline at 20 mg/L for 4-hr periods on three consecutive days. Test water adjusted to appropriate temperature and oxygen was conveyed by gravity from elevated head tanks to the diluter apparatus. The toxicant was delivered to test chambers from intermittent-flow diluters modified from those of Mount and Brungs (1967). Sodium cyanide stock solutions were adjusted to pH 11 with NaOH. Water flow to each test chamber was 1.3 L at each 3min cycle assuring 99% displacement in 3.5 hr. Test chambers for juveniles and trout swim-up fry were glass aquaria 50 x 24 x 20 cm high filled to 20 L of test solution. Eggs and fry were tested in screen bottomed acrylic cylinders each covered with a bakelite lid and held in a 20-L chamber so that a portion of the water from each cycle flowed upward through the screen to the outlet. All tests were conducted under two fluorescent lamps (Luxor
3 Use of product or trade names mentioned throughout the text does not constitute endorsement.
327
Toxicity of HCN to Fish
Inc. "Vita-Lite") providing an intensity of 55 to 78 footcandles at the water surface and a photoperiod of12 hr light. The freshwater fish used for testing and their life history stage, size, and density in the bioassay chambers are presented in Table 1. Only identified live eggs were tested. Eggs and fry were tested immediately on introduction to the test chambers, but following temperature acclimation juveniles were held in test chambers for three days prior to toxicant exposure. Juveniles were fasted during the last 24 hr of acclimation to the bioassay systems and during the first 96 hr of toxicant exposure. All reported concentrations of toxicant, pH, 03, and temperature were based on analyses made on water from the test chambers. Free cyanide concentrations in each chamber were determined daily by the Epstein colorimetric method (American Public Health Association et al. 1971) with calculated HCN concentrations based on corresponding pH and temperature measurements and using dissociation constants of molecular HCN as defined by Izatt et al. (1962). Observations on mortality of test organisms exposed to known cyanide concentrations were made daily. The concentration-percant mortality data were analyzed with a logarithmic-probability (log-probi0 program (Dixon 1973). Upper and lower 95% confidence limits for the 96-hr median lethal tolerance (LC50) and median lethal threshold concentrations (LTC) were calculated from the equations log LCS0 • 1.96(1/b)(N'/2) -1~ for homogeneous data or log LC50 • t.0~K_z>(1/b)(x2/K-2)l/~(N'/2) -~j~for heterogeneous data. The symbol, N', refers to the number of test organisms expected to die within the mortality interval of 16 to 84%. The reciprocal of the log-probit line's slope (l/b) is equivalent to the standard deviation of the logarithm of the population's tolerance frequency distribution (&) or the logarithm of Litchfield and Wilcoxon's (1949) slope function, S. The symbol, K, refers to the number of treatment levels excluding controls, and • to the (Chi) z value calculated for goodness of fit of the regression line to the data points. If this value is greater than the tabulated (Chi) z with K-2 dr, the data are significantly heterogeneous. The above formulas were derived from Litchfield and Wilcoxon (1949) and Firmey (1971). The median lethal threshold concentrations were determined at the end of a time interval when no mortality in any test treatment had occurred for at least 48 hr. When the data were such that probit analysis by computer was not possible, calculations were made graphically.
Results A 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 fish life 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 g most resistant and newly hatched fry and juveniles the most sensitive. Median lethal concentrations for 96-hr and lethal threshold periods, their 95% confidence
Table 1. Number of organisms per test chamber and range in total length at different life history stages Sac fry Species
No. eggs
No.
Length (mm)
Fathead minnow Bluegill Yellow perch Brook trout Rainbow trout
25-50 25 20-70 25 . .
25 10-50 25 10 .
5-6 4 4 9-10 .
Swim-up fry
.
No.
Length (ram)
25 10-50 25 10
5-6 4 4 14-16
Juveniles No.
Length (ram)
10 10-20 10-20 10 10
26-45 13-28 48-62 40-68 40-68
328
L.L. Smith, Jr. et al.
limits, and log-probit regression analysis of the concentration-percent mortality curves for each exposure period are detailed in Tables 2 through 13.
Fathead Minnow
Tests with different life history stages of the fathead minnow were conducted at about 15~ to 25~ and 4 to 7 mg/L dissolved oxygen. Delayed embryological development at low temperature and low DO resulted in no apparent difference in resistance with decreasing DO but an increase in resistance with decreasing temperature for 96-hr egg tests (Table 2). Median lethal HCN concentrations at hatching were markedly reduced for tests at about 25~ and when the DO was less than 5.52 mg/L. At comparable Do levels it appears that a reduction in temperature from 25.0 ~ to 15.2*C resulted in some increased sensitivity at hatching. 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 median lethal threshold concentrations. There was no clear trend associated with temperature for fry tests, but for tests at about 25~ and below 5.14 mg/L DO an increase in sensitivity was apparent (Table 3). No change in sensitivity related to test temperature was observed for juveniles (Table 4).
Bluegill
Bluegill eggs incubated at about 25~ and DO ranging from 6.90 to 3.39 mg/L showed no correlation in sensitivity at hatching to high concentrations of HCN (Table 5). At the hatching LC50 values essentially all the newly hatched sac fry were premature and deformed. Fry were considerably more sensitive to cyanide then eggs, showing at 25~ a substantial decrease in 7-day LTC values with decrease in DO. In juvenile tests at 8.4~ to 24.9~ and comparable DO levels the LTC values increase progressively from 61.7 to 120 txg/L HCN (Table 6). However, only a slight reduction in sensitivity was observed for juveniles tested at 3.48 to 6.90 mg/ L DO and 25~
Yellow Perch
Results from yellow perch egg and fry experiments conducted at about 10~ to 18~ and 4 to 7 mg/L dissolved oxygen are so variable that specified median lethal concentrations cannot be considered definitive (Table 7). For juvenile tests at 15.0~ to 21.0~ and at comparable DO levels the LTC values were determined to increase progressively from 87.6 to 101 tzg/L HCN (Table 8). An increase in sensitivity was observed for juveniles tested at 7.10 to 3.56 mg/L DO and 21oc with LTC values decreasing from 107 to 75.0 /zg/L HCN. There was little difference between the 96-hr and LTC values for juvenile perch.
6.36 6.13 3.51 4.46 5.52 6.34 7.25
~
15.2
20.0
24.9
24.8
25.0
25.0
24.9
7.99
8.00
7.90
7.95
7.72
7.88
7.86
pH
1
5
3
2
2
11
2
No. of tests
eggs expressed
8 5 40 36 5 8 6 6 9 I0 12 10 --
Treatments b - 5.033 -15.508 2.175 - 2.063 - 5.358 - 8.411 - 8.488 -13.291 - 6.262 - 5.288 - 4.612 .-14.863 --
a 3.940 9.765 1.159 3.411 4.494 6.491 6.475 8.909 4.973 4.563 4.194 8.992 --
b 352 -273 -202 -121 -184 -196 -202
p.g/L 274-453 -162-463 -130-314 -77.3-190 -115-293 -140-274 ---
95% Confidence limits
96-hr L C 5 0
-126 -118 -116 -113 -180 -162 187
-90.9-174 -97.3-142 -86.6-157 -83.0--154 -122-266 -135-193 --
95% Confidence limits
Hatch LC50
at hatching
p,g/L
as 96-hr LC50 and median lethal concentrations
L o g probit r e g r e s s i o n analysis a
to f a t h e a d m i n n o w
DO mg/L 6.38 6.14 3.77 5.14 6.17
~
15.0
20.0
24.6 24.7 24.9
7.84 7.96 8.02
7.89
7.86
pH
M e a n test conditions
3 2 9
6
6
Treatments 13 15 21 13 lO 8 28
swim-up fry expressed
-12.743 -13.376 - 6.419 - 6.145 - 9.989 - 8.170 - 7.971
a
122 -99.1 -81.6 108 I13
p,g/L
104-143 -88.9-111 -71.2-93.6 90.3-130 96.5-133
95% Confidence limits
96-hr L C 5 0
-102 -96.1 81.6 108 113
p.g/L
-92.8--113 -83.6-I lO 71.2-93.6 90.3-130 96.5-133
95% Confidence limits
LTC
as 96-hr LC50 and median lethal threshold concentrations
8.507 9.143 5.721 5.621 7.840 6.471 6.314
b
L o g - p r o b i t regression analysis
to fathead minnow
No. of tests
T a b l e 3. A c u t e t o x i c i t y o f H C N
a A probit o f 4.0, 5.0, a n d 6.0 c o r r e s p o n d s to 16, 50, and 84% mortality, r e s p e c t i v e l y , w h e n ~ l is the m a x i m u m likelihood probit v a l u e o f p e r c e n t mortality and Xt is log H C N c o n c e n t r a t i o n in p.g/L for the r e g r e s s i o n equation r = a + b(Iog Xl) b N u m b e r o f H C N c o n c e n t r a t i o n s in the r e g r e s s i o n analysis
DO mg/L
M e a n test conditions
T a b l e 2. A c u t e t o x i c i t y o f H C N
~D
~r
o
6.07 3.58 4.68 5.20 6.07
7.13 3.58 5.08 6.13 7.04
*C 15.0 20.0
19.8
20.0
20.0 24.8 25.0 25.1 25.2
7.75 7.83 7.98 7.96
7.90
7,91
7.78
7.80
7.70
7.86
pH
2 2 8 2
4
4
4
2
4
4
NO. of tests
7 7 28 6
9
8 9 13
4
15 15 II II
Treatments
- 18.494 -23.502 -20.032 -27.069
-28.818
-21.086 - 19.292 -20,485
-24,538
-27.167 -28.565 - 8.512 - 9.924
a
11.592 13.722 11.868 15.415
15.978
12,446 I 1.616 11.933
15,415
15.455 16.176 6.417 7.146
b
Log-probit regression analysis
106 119 129 120
131
125 -137
82.4
121 -128 --
IcJL
87.9-129 I 11-129 124-133 113-128
124-138
117-133 -122-153
76.4-88,9
116-125 -109-149 --
95% Confidence limits
96-hr L C 5 0
106 119 129 120
131
~ 123 137
82.4
-119 -123
pg/L
87.9-129 I I I - 129 124-133 113-128
124-138
-116-132 122-153
76.4-88.9
-115-123 -105-143
95% Confidence limits
LTC
7,89
5.99 3.59 5.08 6,01 6.81
24.9 24,9 24.9 24~8
7.90
7,93
7.72 7.80
7.70 7,79 7.92 7,90
3,39 4.99 6.09 6.90
pH
25.2 25,0 25,1 25.0 Fry 20.0
Egg__2s
~
DO mg/L
Mean test conditions
4
8
3 4
6
2 2 2 2
No. o f tests
tI II 4 10 7 22 28 9 .5
6 6 8 6
Treatments
- 2.618 - 8.388 -40.381 - 2.756 -18.630 -13.580 -18.117 - 8.222 - I l.g20
- 1.349 - 4,902 -12.494 - 3.802
a
2.973 5.791 22.256 3,279 10.872 7.611 9.883 5.433 7,355
2.236 3.630 6.157 3.185
b
Log-probit regression analysis
365 --232 -276 -271 --
-----
~.g/L
188-709 --147-366 -241-316 -200-368 --
-----
95% Confidence limits
96-hr L C 5 0
~ 205 109 -149 ~ 218 -194
690 .535 693 580
~g/L
-156-270 99.9-120 -117-189 -193-247 -i 10-340
461-1033 240-1192 572-841 343-980
95% Confidence limits
L T C o r hatch L C 5 0
Table 5. Acute toxicity of HCN to bluegill eggs and sw/m-up fry expressed as 96-hr LCS0 and median lethal threshold or hatching concentrations
20.0
DO mg/L
Mean test conditions
Table 4. Acute toxicity of HCN to fathead minnow juveniles expressed as 96-hr LC50 and median lethal threshold concentrations.
.a.
c~
8.35 6.07 7.03 7.97 6.06 3.48 5.05 6.17 6.90
9.7 15.0 15.1 17.8 20.0
25.1 25.0 24.9 24.9
7.71 7.78 7.92 7.86
7.94 7.83 7.92 8.12 7.86
7.80
pH
6 6 5 6
1 3 2 1 6
2
7.90 7.70 7.72 7.87 7.94
7.04 3.84 5.32 7.44 7.00
7.66 7.76 7.87 7.90 7.90
Eggs 14.0
pH
4.96 6.04 7.20 6.17
3.36
~
14.1 14.0 14.1 18,0 Fry 10.0 14.0 14.0 14.0 18.1
DO mg/L
Mean test conditions
hatching concentrations
No. of tests
2 2 2 4 2
2 2 2 2
2
--40.261 --44.847 - 15.982 --31.160 -32.839 -16.173 -21.671 -19.485 -30.955
a -25.283 -25.692 I 1.190 -17.797 18.609 10.593 13.003 11.766 17.160
b
-8 8 ---
8 3 6 6 8 --
Treatments
--10.431 - 1,910 ---
- 11.968 -14.050 -14.491 - 5,099 - 0.072 --
a
-6.249 2,734 ---
6.900 9.006 8.849 4.813 2.340 --
b
Log-probit regression analysis
---81.0-93.8 66.0-85.2 -103-112 -86.0-116 55.8-227 109-133 115-135
95% Confidence limits
96-hr LC50
-61.7 -87.1 -m m 108 99.7 113 120 125
p.g/L
-104-112 86.0-116 55.8-227 109-133 115-135
-59.3-64.2 -81.0-93.8 --
95% Confidence limRs
LTC
>357 295 337 >395 >338
288 ->329 >317 >389 >276
/zg/L
-256-339 202-561 ---
270-307 ------
95% Confidence limits
96-hr LC50
< 170 --
Archives of
Environmental Contamination and Toxicology
Acute Toxicity of Hydrogen Cyanide to Freshwater Fishes 1'2 Lloyd L. Smith, Jr., Steven J. Broderius, Donavon M. Oseid, Gary L. Kimball, and Walter M. Koenst Departmentof Entomology,Fisheries,and Wildlife,Universityof Minnesota,1980FolweUAvenue, St. Paul, Minnesota55108
Abstract. Acute toxicity of hydrogen cyanide was determined at various temperatures from 4~ to 30~ and oxygen concentrations of 3.36 to 9.26 mg/L on different life history stages of five species of fish: fathead minnow, Pimephales promelas Refinesque; bluegill, Lepomis macrochirus Rafinesque; yellow perch, Perca flavescens (MitchiU); brook trout, Salvelinus fontinalis (Mitchill); and rainbow trout, Salmo gairdneri Richardson. Median lethal threshold concentrations and 96-hr LC50's were established by flowthrough type bioassays. Acute toxicity varied from 57 /zg/L for juvenile rainbow trout to 191 /zg/L for field stocks of juvenile fathead minnows. Juvenile fish were more sensitive at lower temperatures and at oxygen levels below 5 mg/L. For most species juveniles were most sensitive and eggs more resistant. Compounds containing the cyanide group are present in many industrial and municipal effluents, including those from iron and steel mills, oil refineries, and plating plants, and constitute a significant source of toxicants introduced into aquatic ecosystems. In aqueous solution the cyanide radical from simple alkali cyanides such as NaCN hydrolyzes to form free cyanide (CN- ion and molecular HCN). The molecular (un-ionized) component predominates at pH values (6.08.0) found in most natural waters, with less than 6% free cyanide occurring in the ionic form below pH 8 at 25~ As the pH of aqueous simple cyanide solutions is increased the percentage of free cyanide present as the CN- ion is increased to satisfy the equilibrium reaction of HCN ~ H § + CN-. The world literature on the toxicity to fish of various cyanides was reviewed by Doudoroff (1976). Wuhrmann and Woker (1948), Bridges (1958), and Doudoroff et al. (1966) have concluded that HCN is the principal toxic cyanide form. However, Broderius et al. (1977) have demonstrated that even though molecular HCN is more toxic than the CN- ion, the anion does contribute to the total toxicity and to a greater degree as the test pH increases. Since HCN is highly toxic to fish and most invertebrates, and further since some relatively nontoxic
1 Paper No. 9954, ScientificJournal Series, Minnesota Agricultural Experiment Station, St. Paul, Minnesota. Research supported by the U.S. Environmental Protection Agency, EnvironmentalResearch Laboratory, Duluth,Minnesota,under Grant No. R802914. 0090-4341/78/0007-0325 $02.60 @ 1978 Springer-Verlag New York Inc.
326
L . L . Smith, Jr. et al.
iron-cyartide compounds photodecompose, releasing HCN, cyanide constitutes a hazard in certain waste receiving water. The present paper reports on 337 acute toxicity tests designed to determine the 96-hr median lethal toxicant concentration (LC50) of HCN and the median lethal threshold concentration (LTC) (Sprague 1969) at various temperatures and dissolved oxygen concentrations for three life history stages of four species of fish and for juveniles of one other. Fish used in the tests were the fathead minnow, Pimephales prornelas Rafinesque; bluegill, Lepornis macrochirus Rafinesque; yellow perch, Perca flavescens (Mitchill); brook trout, Salvelinus fontinalis (MitchiU); and rainbow trout, Salmo gairdneri Richardson. A wide range of reported lethal concentrations to freshwater fishes supposedly attributable to difference in species, condition of test fish, and test conditions led to the present series of tests conducted with a single water source and using uniform experimental procedures.
Materials and Methods Different species used for acute tests were secured as eggs, fry, and juveniles, or were reared in the laboratory. Brook and rainbow trout were obtained as newly hardened eggs or as 24-hr fry from state hatcheries. Fathead minnows were cultured in our laboratory from brood stock originally obtained from the U.S. Environmental Protection Agency's Environmental Research Laboratory in Duluth, Minnesota. Juvenile wild-stock fatheads were collected from Como Lake in St. Paul, Minnesota. Bluegills were obtained from wild stock with eggs spawned and fry hatched in the laboratory. Juvenile bluegills were collected from local waters. Yellow perch eggs were obtained from wild adult stock which spawned in the laboratory. Yellow perch eggs were also collected in the field and hatched in the laboratory for fry tests; juvenile perch were collected in the field. During rearing and maintenance, fish were fed different foods including (1) mashed hard-boiled egg yolk, (2) a mixture of ground hardboiled egg and lettuce, (3) live, newly hatched brine shrimp (Artemia salina) ("San Francisco Bay Brand," Metaframe, Inc.3), (4) frozen mature brine shrimp, and (5) "Glencoe" dry pellets. Water supply for the laboratory tests was from a deep well and was transmitted to experiments through polyvinyl chloride pipe. The experimental water had a total hardness and alkalinity of approximately 220 and 235 mg/L as CaCO3, respectively. A comprehensive analysis of the water was reported by Smith et al. (1976). Temperature acclimation for eggs and fry was at the rate of llYC/hr and for juveniles 2*C/hr. Eggs and fry were randomly placed in test chambers as soon as test conditions were reached. Juveniles were held at test conditions for seven days before being placed in test chambers. Juvenile fish brought from the field were given prophylactic treatment with neomycin and tetracycline at 20 mg/L for 4-hr periods on three consecutive days. Test water adjusted to appropriate temperature and oxygen was conveyed by gravity from elevated head tanks to the diluter apparatus. The toxicant was delivered to test chambers from intermittent-flow diluters modified from those of Mount and Brungs (1967). Sodium cyanide stock solutions were adjusted to pH 11 with NaOH. Water flow to each test chamber was 1.3 L at each 3min cycle assuring 99% displacement in 3.5 hr. Test chambers for juveniles and trout swim-up fry were glass aquaria 50 x 24 x 20 cm high filled to 20 L of test solution. Eggs and fry were tested in screen bottomed acrylic cylinders each covered with a bakelite lid and held in a 20-L chamber so that a portion of the water from each cycle flowed upward through the screen to the outlet. All tests were conducted under two fluorescent lamps (Luxor
3 Use of product or trade names mentioned throughout the text does not constitute endorsement.
327
Toxicity of HCN to Fish
Inc. "Vita-Lite") providing an intensity of 55 to 78 footcandles at the water surface and a photoperiod of12 hr light. The freshwater fish used for testing and their life history stage, size, and density in the bioassay chambers are presented in Table 1. Only identified live eggs were tested. Eggs and fry were tested immediately on introduction to the test chambers, but following temperature acclimation juveniles were held in test chambers for three days prior to toxicant exposure. Juveniles were fasted during the last 24 hr of acclimation to the bioassay systems and during the first 96 hr of toxicant exposure. All reported concentrations of toxicant, pH, 03, and temperature were based on analyses made on water from the test chambers. Free cyanide concentrations in each chamber were determined daily by the Epstein colorimetric method (American Public Health Association et al. 1971) with calculated HCN concentrations based on corresponding pH and temperature measurements and using dissociation constants of molecular HCN as defined by Izatt et al. (1962). Observations on mortality of test organisms exposed to known cyanide concentrations were made daily. The concentration-percant mortality data were analyzed with a logarithmic-probability (log-probi0 program (Dixon 1973). Upper and lower 95% confidence limits for the 96-hr median lethal tolerance (LC50) and median lethal threshold concentrations (LTC) were calculated from the equations log LCS0 • 1.96(1/b)(N'/2) -1~ for homogeneous data or log LC50 • t.0~K_z>(1/b)(x2/K-2)l/~(N'/2) -~j~for heterogeneous data. The symbol, N', refers to the number of test organisms expected to die within the mortality interval of 16 to 84%. The reciprocal of the log-probit line's slope (l/b) is equivalent to the standard deviation of the logarithm of the population's tolerance frequency distribution (&) or the logarithm of Litchfield and Wilcoxon's (1949) slope function, S. The symbol, K, refers to the number of treatment levels excluding controls, and • to the (Chi) z value calculated for goodness of fit of the regression line to the data points. If this value is greater than the tabulated (Chi) z with K-2 dr, the data are significantly heterogeneous. The above formulas were derived from Litchfield and Wilcoxon (1949) and Firmey (1971). The median lethal threshold concentrations were determined at the end of a time interval when no mortality in any test treatment had occurred for at least 48 hr. When the data were such that probit analysis by computer was not possible, calculations were made graphically.
Results A 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 fish life 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 g most resistant and newly hatched fry and juveniles the most sensitive. Median lethal concentrations for 96-hr and lethal threshold periods, their 95% confidence
Table 1. Number of organisms per test chamber and range in total length at different life history stages Sac fry Species
No. eggs
No.
Length (mm)
Fathead minnow Bluegill Yellow perch Brook trout Rainbow trout
25-50 25 20-70 25 . .
25 10-50 25 10 .
5-6 4 4 9-10 .
Swim-up fry
.
No.
Length (ram)
25 10-50 25 10
5-6 4 4 14-16
Juveniles No.
Length (ram)
10 10-20 10-20 10 10
26-45 13-28 48-62 40-68 40-68
328
L.L. Smith, Jr. et al.
limits, and log-probit regression analysis of the concentration-percent mortality curves for each exposure period are detailed in Tables 2 through 13.
Fathead Minnow
Tests with different life history stages of the fathead minnow were conducted at about 15~ to 25~ and 4 to 7 mg/L dissolved oxygen. Delayed embryological development at low temperature and low DO resulted in no apparent difference in resistance with decreasing DO but an increase in resistance with decreasing temperature for 96-hr egg tests (Table 2). Median lethal HCN concentrations at hatching were markedly reduced for tests at about 25~ and when the DO was less than 5.52 mg/L. At comparable Do levels it appears that a reduction in temperature from 25.0 ~ to 15.2*C resulted in some increased sensitivity at hatching. 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 median lethal threshold concentrations. There was no clear trend associated with temperature for fry tests, but for tests at about 25~ and below 5.14 mg/L DO an increase in sensitivity was apparent (Table 3). No change in sensitivity related to test temperature was observed for juveniles (Table 4).
Bluegill
Bluegill eggs incubated at about 25~ and DO ranging from 6.90 to 3.39 mg/L showed no correlation in sensitivity at hatching to high concentrations of HCN (Table 5). At the hatching LC50 values essentially all the newly hatched sac fry were premature and deformed. Fry were considerably more sensitive to cyanide then eggs, showing at 25~ a substantial decrease in 7-day LTC values with decrease in DO. In juvenile tests at 8.4~ to 24.9~ and comparable DO levels the LTC values increase progressively from 61.7 to 120 txg/L HCN (Table 6). However, only a slight reduction in sensitivity was observed for juveniles tested at 3.48 to 6.90 mg/ L DO and 25~
Yellow Perch
Results from yellow perch egg and fry experiments conducted at about 10~ to 18~ and 4 to 7 mg/L dissolved oxygen are so variable that specified median lethal concentrations cannot be considered definitive (Table 7). For juvenile tests at 15.0~ to 21.0~ and at comparable DO levels the LTC values were determined to increase progressively from 87.6 to 101 tzg/L HCN (Table 8). An increase in sensitivity was observed for juveniles tested at 7.10 to 3.56 mg/L DO and 21oc with LTC values decreasing from 107 to 75.0 /zg/L HCN. There was little difference between the 96-hr and LTC values for juvenile perch.
6.36 6.13 3.51 4.46 5.52 6.34 7.25
~
15.2
20.0
24.9
24.8
25.0
25.0
24.9
7.99
8.00
7.90
7.95
7.72
7.88
7.86
pH
1
5
3
2
2
11
2
No. of tests
eggs expressed
8 5 40 36 5 8 6 6 9 I0 12 10 --
Treatments b - 5.033 -15.508 2.175 - 2.063 - 5.358 - 8.411 - 8.488 -13.291 - 6.262 - 5.288 - 4.612 .-14.863 --
a 3.940 9.765 1.159 3.411 4.494 6.491 6.475 8.909 4.973 4.563 4.194 8.992 --
b 352 -273 -202 -121 -184 -196 -202
p.g/L 274-453 -162-463 -130-314 -77.3-190 -115-293 -140-274 ---
95% Confidence limits
96-hr L C 5 0
-126 -118 -116 -113 -180 -162 187
-90.9-174 -97.3-142 -86.6-157 -83.0--154 -122-266 -135-193 --
95% Confidence limits
Hatch LC50
at hatching
p,g/L
as 96-hr LC50 and median lethal concentrations
L o g probit r e g r e s s i o n analysis a
to f a t h e a d m i n n o w
DO mg/L 6.38 6.14 3.77 5.14 6.17
~
15.0
20.0
24.6 24.7 24.9
7.84 7.96 8.02
7.89
7.86
pH
M e a n test conditions
3 2 9
6
6
Treatments 13 15 21 13 lO 8 28
swim-up fry expressed
-12.743 -13.376 - 6.419 - 6.145 - 9.989 - 8.170 - 7.971
a
122 -99.1 -81.6 108 I13
p,g/L
104-143 -88.9-111 -71.2-93.6 90.3-130 96.5-133
95% Confidence limits
96-hr L C 5 0
-102 -96.1 81.6 108 113
p.g/L
-92.8--113 -83.6-I lO 71.2-93.6 90.3-130 96.5-133
95% Confidence limits
LTC
as 96-hr LC50 and median lethal threshold concentrations
8.507 9.143 5.721 5.621 7.840 6.471 6.314
b
L o g - p r o b i t regression analysis
to fathead minnow
No. of tests
T a b l e 3. A c u t e t o x i c i t y o f H C N
a A probit o f 4.0, 5.0, a n d 6.0 c o r r e s p o n d s to 16, 50, and 84% mortality, r e s p e c t i v e l y , w h e n ~ l is the m a x i m u m likelihood probit v a l u e o f p e r c e n t mortality and Xt is log H C N c o n c e n t r a t i o n in p.g/L for the r e g r e s s i o n equation r = a + b(Iog Xl) b N u m b e r o f H C N c o n c e n t r a t i o n s in the r e g r e s s i o n analysis
DO mg/L
M e a n test conditions
T a b l e 2. A c u t e t o x i c i t y o f H C N
~D
~r
o
6.07 3.58 4.68 5.20 6.07
7.13 3.58 5.08 6.13 7.04
*C 15.0 20.0
19.8
20.0
20.0 24.8 25.0 25.1 25.2
7.75 7.83 7.98 7.96
7.90
7,91
7.78
7.80
7.70
7.86
pH
2 2 8 2
4
4
4
2
4
4
NO. of tests
7 7 28 6
9
8 9 13
4
15 15 II II
Treatments
- 18.494 -23.502 -20.032 -27.069
-28.818
-21.086 - 19.292 -20,485
-24,538
-27.167 -28.565 - 8.512 - 9.924
a
11.592 13.722 11.868 15.415
15.978
12,446 I 1.616 11.933
15,415
15.455 16.176 6.417 7.146
b
Log-probit regression analysis
106 119 129 120
131
125 -137
82.4
121 -128 --
IcJL
87.9-129 I 11-129 124-133 113-128
124-138
117-133 -122-153
76.4-88,9
116-125 -109-149 --
95% Confidence limits
96-hr L C 5 0
106 119 129 120
131
~ 123 137
82.4
-119 -123
pg/L
87.9-129 I I I - 129 124-133 113-128
124-138
-116-132 122-153
76.4-88.9
-115-123 -105-143
95% Confidence limits
LTC
7,89
5.99 3.59 5.08 6,01 6.81
24.9 24,9 24.9 24~8
7.90
7,93
7.72 7.80
7.70 7,79 7.92 7,90
3,39 4.99 6.09 6.90
pH
25.2 25,0 25,1 25.0 Fry 20.0
Egg__2s
~
DO mg/L
Mean test conditions
4
8
3 4
6
2 2 2 2
No. o f tests
tI II 4 10 7 22 28 9 .5
6 6 8 6
Treatments
- 2.618 - 8.388 -40.381 - 2.756 -18.630 -13.580 -18.117 - 8.222 - I l.g20
- 1.349 - 4,902 -12.494 - 3.802
a
2.973 5.791 22.256 3,279 10.872 7.611 9.883 5.433 7,355
2.236 3.630 6.157 3.185
b
Log-probit regression analysis
365 --232 -276 -271 --
-----
~.g/L
188-709 --147-366 -241-316 -200-368 --
-----
95% Confidence limits
96-hr L C 5 0
~ 205 109 -149 ~ 218 -194
690 .535 693 580
~g/L
-156-270 99.9-120 -117-189 -193-247 -i 10-340
461-1033 240-1192 572-841 343-980
95% Confidence limits
L T C o r hatch L C 5 0
Table 5. Acute toxicity of HCN to bluegill eggs and sw/m-up fry expressed as 96-hr LCS0 and median lethal threshold or hatching concentrations
20.0
DO mg/L
Mean test conditions
Table 4. Acute toxicity of HCN to fathead minnow juveniles expressed as 96-hr LC50 and median lethal threshold concentrations.
.a.
c~
8.35 6.07 7.03 7.97 6.06 3.48 5.05 6.17 6.90
9.7 15.0 15.1 17.8 20.0
25.1 25.0 24.9 24.9
7.71 7.78 7.92 7.86
7.94 7.83 7.92 8.12 7.86
7.80
pH
6 6 5 6
1 3 2 1 6
2
7.90 7.70 7.72 7.87 7.94
7.04 3.84 5.32 7.44 7.00
7.66 7.76 7.87 7.90 7.90
Eggs 14.0
pH
4.96 6.04 7.20 6.17
3.36
~
14.1 14.0 14.1 18,0 Fry 10.0 14.0 14.0 14.0 18.1
DO mg/L
Mean test conditions
hatching concentrations
No. of tests
2 2 2 4 2
2 2 2 2
2
--40.261 --44.847 - 15.982 --31.160 -32.839 -16.173 -21.671 -19.485 -30.955
a -25.283 -25.692 I 1.190 -17.797 18.609 10.593 13.003 11.766 17.160
b
-8 8 ---
8 3 6 6 8 --
Treatments
--10.431 - 1,910 ---
- 11.968 -14.050 -14.491 - 5,099 - 0.072 --
a
-6.249 2,734 ---
6.900 9.006 8.849 4.813 2.340 --
b
Log-probit regression analysis
---81.0-93.8 66.0-85.2 -103-112 -86.0-116 55.8-227 109-133 115-135
95% Confidence limits
96-hr LC50
-61.7 -87.1 -m m 108 99.7 113 120 125
p.g/L
-104-112 86.0-116 55.8-227 109-133 115-135
-59.3-64.2 -81.0-93.8 --
95% Confidence limRs
LTC
>357 295 337 >395 >338
288 ->329 >317 >389 >276
/zg/L
-256-339 202-561 ---
270-307 ------
95% Confidence limits
96-hr LC50
< 170 --
