EPA-600/3-76-062a
July 1976
Ecological Research Series
EFFECT OF HYDROGEN SULFIDE ON
FISH AND INVERTEBRATES
Parti- Acute and
Chronic Toxicity Studies
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
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'I; ft, ••••- -'
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into five series. These five broad
categories were established to facilitate further development and application of
environmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The five series are:
]. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal
species, and materials. Problems are assessed for their long- and short-term
influences. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides the technical
basis for setting standards to minimize undesirable changes in living organisms
in the aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-062a
July 1976
EFFECT OF HYDROGEN SULFIDE ON FISH AND INVERTEBRATES
Part I - Acute and Chronic Toxicity Studies
by
Lloyd L. Smith, Jr.
Donavon M. Oseid
Ira R. Adelman
Steven J. Broderius
Department of Entomology, Fisheries, and Wildlife
University of Minnesota
St. Paul, Minnesota 55108
Grant No. R800992
Project Officer
Kenneth E. F. Hokanson
Environmental Research Laboratory-Duluth
Monticello, Minnesota 55362
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
DULUTH, MINNESOTA 55804
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory -
Duluth, U.S. Environmental Protection Agency, and approved for publication.
Approval does not signify that the contents necessarily reflect the views
and policies of the U.S. Environmental Protection Agency, nor does mention
of trade names or commercial products constitute endorsement or recommendation
for use.
11
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ABSTRACT
Acute and chronic toxicity of hydrogen sulfide to seven fish species
and eight invertebrates were determined in continuous-flow bioassays.
Fish species were fathead minnows, goldfish, bluegill, walleye, white
sucker, brook trout, and rainbow trout. Invertebrates were Asellus,
Crangonyx, Gammarus, Baetis, Hexagenia, Ephemera, Procambarus, and Cam-
barus. In 159 acute tests lethal threshold concentration for juvenile
fish varied from 0.0087 mg/liter in rainbow trout to 0.0840 mg/liter in
goldfish. Except in goldfish, fry stage was up to three times more
sensitive than the juvenile. In 96 tests on invertebrates the 96-hr
LC50 ranged from 0.020 mg/liter in Baetis to 1.070 mg/liter in Asellus.
Acute toxicity of H_S to fathead minnows varied 24-fold between 6.5 and
24.0 C. Temperature effects were not as marked on invertebrates. In
chronic exposure to H^S in 29 tests running up to 825 days, maximum no-
effect concentration to fish ranged from 0.0004 mg/liter in bluegills
to 0.0100 mg/liter in goldfish. No-effect level was determined from
growth, survival, reproduction, or swimming performance. In nine chronic
tests running up to 138 days, maximum safe levels ranged from 0.0012
mg/liter in Gammarus to 0.0152 mg/liter in Hexagenia. Application
factors relating acute toxic (96-hr LC50 for juveniles) to no-effect
levels varied from .231 in rainbow trout to .013 in bluegills and from
.091 in Gammarus to .048 in Procambarus.
This report was submitted in fulfillment of Project 18050 DGG, HLW,
Grant No. R800992 by the Department of Entomology, Fisheries, and Wild-
life, University of Minnesota, under the sponsorship of the Environ-
mental Protection Agency. Work was completed in May, 1974.
ill
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CONTENTS
Page
Abstract ill
List of Figures vi
List of Tables vii
Acknowledgments xvi
Sections
I Conclusions 1
II Recommendations 3
III Introduction 4
IV Materials and Methods 8
V Fathead Minnow 17
VI Goldfish 72
VII Bluegill 102
VIII Walleye 138
IX Brook Trout 150
X Rainbow Trout 176
XI White Sucker 198
XII Crayfish 200
XIII Benthic Invertebrates 218
XIV Effect of pH on Toxicity 252
XV Discussion 254
XVI References 277
XVII Publications 281
XVIII Glossary 283
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FIGURES
No. Page
1 Schematic Diagram of the Continuous-Flow Bioassay 11
Apparatus
2 The Effect of Temperature on the 96-hr LC50 (TL50) 80
of H2S to Goldfish with 95% Confidence Limits
3 The Effect of Temperature on the 96-hr LC50 (TL50) 81
of H S to Three Groups of Fish
4 Changes in LC50 (TL50) Values During 11 Days of 83
Exposure to H~S at Three Temperatures with 95%
Confidence Limits
5 The Effect of Oxygen on the 96-hr LC50 (TL50) of 86
H2S to Goldfish
6 Toxicity Curves for Brook Trout Juveniles at 2.5 C 158
Intervals from 8.5 to 21.0 C
7 Mean 96-hr LC50's (Quadratic) and Lethal Threshold Con- 160
centrations (Linear) for Brook Trout at 8.5 and 21.0 C
8 Length-Frequency Distribution of Gammarus After 65 251
Days of Exposure to Varied Concentrations of H»S
VI
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TABLES
No. page
1 Analysis of Well Water 9
2 Source of Fathead Minnows and Stage at Start of Acute 18
Tests with H S
3 Acute Toxicity of H S (LC50 and LTC) to Fathead Minnow 21
Eggs and Fry
4 Acute Toxicity of H2S (LC50 and LTC) to Wild Stock 22
Juvenile Fish at Various Temperatures
5 Acute Toxicity of H S (LC50 and LTC) to Field-reared 24
and Laboratory-reared Juvenile Fish at 20 C
6 Acute Toxicity of H S (LC50 and LTC) to Fathead 25
Minnows of Wild and Duluth Stock
7 Source of Fathead Minnows and Stage at Start of 26
Chronic Tests with H?S
8 Test Conditions in Chronic Fathead Minnow Tests 2 28
and 3 Started with Wild Stock Juveniles
9 Test Conditions in Chronic Fathead Minnow Test 5 30
Started with Wild Stock Juveniles
10 Test Conditions in Fathead Minnow Chronic Test 4 32
Started with Duluth Stock Sac Fry
11 Test Conditions in Fathead Minnow Chronic Test 6, 33
Started with Duluth Stock Sac Fry
12 Test Conditions in Fathead Minnow Chronic Test &„ 34
Started with Fish from Chronic 6, Spawnings
13 Survival of Wild Stock Fathead Minnows with Long-term 36
Exposure to H?S in Chronic Tests 2 and 3
14 Survival of Duluth Stock Fathead Minnows Started as 38
Sac Fry with Long-term Exposure to H2S in Chronic Test 4
15 Survival of Duluth Stock Fathead Minnows in Chronic Test 61 40
Started as Sac Fry with Long-term Exposure to H S
vii
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No. Page
16 Survival of Fathead Minnows Started as Juveniles from 42
Chronic Test 6, Eggs with Long-term Exposure to t^S
17 Survival of Wild Stock Juvenile Fathead Minnows with 44
Different Concentrations of Fish and H2S in Chronic
Test 5
18 Growth of Wild Stock Fathead Minnows at Various Concen- 46
trations of H-S in Chronic Tests 2 and 3
19 Growth of Duluth Stock Fathead Minnows Started as Eggs 47
in Chronic Test 4
20 Growth of Juvenile Fathead Minnows in Chronic Test 5 48
with Varied Concentrations of HLS and Different Numbers
of Fish in Test Chambers
21 Growth of Duluth Stock Fathead Minnows Started as Sac Fry 50
in an Experiment Covering Two Generations
22 Spawning Success, Eggs per Female and Number of Spawnings 52
per Female in Duluth Stock Fathead Minnows with Con-
tinued Exposure to Low Levels of H_S in Chronic Test 6,
23 Spawning Success, Eggs per Female and Number of Spawnings 53
per Female in Duluth Stock Fathead Minnows with Con-
tinued Exposure to Low Levels of H2S in Chronic Test 62
24 Number of Days to Hatch of Fathead Minnow Eggs Deposited 54
in Various Concentrations of H~S and Hatched in the
Same Concentration or in Control Water
25 Survival to Hatch of Fathead Minnow Eggs Laid in Various 56
Concentrations of H-S and Incubated in the Same Concen-
trations or in Control Water
26 Length of Fathead Minnow Fry at Hatch from Eggs Laid in 57
Various Concentrations of H~S and Incubated in the Same
Concentration or in Control Water
27 Length of Fathead Minnow Adults at Termination of Chronic 58
Tests 6., and &„
28 Weight of Fathead Minnow Adults at Termination of Chronic 60
Test 6,
Vlll
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No. Page
29 Source of Fathead Minnows Used for Population Com- 62
parison Tests
30 Lake Water Chemistry 64
31 Acute Test Conditions and LC50 Values for Fathead Minnows 65
in Population Comparison (Wakefield Lake)
32 Acute Test Conditions and LC50 Values for Fathead Minnows 66
in Population Comparison (Hay Lake)
33 Acute Test Conditions and LC50 Values for Fathead Minnows 67
in Population Comparison (Harrier Lake)
34 Acute Test Conditions and LC50 Values for Fathead Minnows 68
in Population Comparison (Porter Lake)
35 Acute Test Conditions and LC50 Values for Fathead Minnows 70
in Population Comparison (Duluth-Newtown Stock)
36 LTC Values of Fathead Minnows from Seven Populations 71
37 Percentage Survival of Goldfish Eggs and Percentage 77
Malformed Fry in Various H2S Concentrations
38 Percentage Survival of Goldfish Fry and Mean Length of 77
Fry at End of Exposure to Various H?S Concentrations
39 LC50 of H2S to Goldfish at Different Temperatures 79
40 Effect of Oxygen on H2S Toxicity to Goldfish in 84
Paired Bioassays
41 Effect of Temperature, Acclimation Time, and Goldfish 88
Stock on the 96-hr and 11-day LC50
42 Acute Mortality of Goldfish at Three Temperatures 89
Expressed as 96-hr and 11-day LC50
43 Test Conditions in Goldfish Chronic I 94
44 Test Conditions in Goldfish Chronic II 96
45 Weights of Goldfish in Three Chronic Bioassays 98
46 H~S Concentrations not Affecting Goldfish Adversely 99
Determined from Three Chronic Bioassays
47 Reproduction of Goldfish in Chronic 1-0 99
IX
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No. Page
48 Time to Death of Goldfish from Various Chronic H2S 101
Concentrations Exposed to an Acutely Lethal H^S
Concentration of 0.322 mg/liter
49 Source of Bluegills and Stage of Fish Used for Acute 103
Tests with H S
50 Acute Test Conditions and LC50 Values for Bluegills 106
Tested in H S
51 Mean Daily H2S Exposure and Percentage Survival of 110
Fingerling Bluegills on Successive Days in 10
Treatment Chambers
52 H2S Concentrations at Which 100%, 50%, and 0% of Bluegills 112
Survived in 2 to 11 Days after Acclimation to H_S
53 Source of Bluegills and Stage of Fish at Start of Chronic 113
Test with H S
54 Physical and Chemical Conditions of Chronic Bluegill Tests 114
55 Survival of Bluegills, with Long-term Exposure to H_S 118
56 Growth of Bluegills with Long-term Exposure to H_S 120
57 Minnow Consumption of Bluegills with Long-term 122
Exposure to H S
58 Food Conversion Efficiency of Bluegills with Long-term 124
Exposure to H9S
59 Spawning Success of Bluegills with Long-term Exposure 125
to H2S
60 Time to Anesthesia of Bluegills Subjected to Long- 128
term Exposure to H S
61 Test Conditions of Series A and B During Chronic 132
Bluegill Tests
62 Effect of Chronic Exposure of Bluegills to Various 133
Levels of H2S for 126 Days (Series A) on Growth,
Gill Irrigation Rate and Swimming Endurance
63 Effect of Chronic Exposure of Bluegills to Various 134
Levels of H?S for 148 Days (Series B) on Growth,
Gill Irrigation Rate and Swimming Endurance
x
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No. Page
64 Resistance of Bluegills with Chronic Treatment of H S 135
to Subsequent Exposure of Copper Sulfate (as Cu)
(Series A) and Malathion (Series B)
65 Acute Test Conditions and LC50 Values for Walleye 140
Juveniles Tested in H~S
66 Physical and Chemical Conditions in Chronic Tests on 142
Walleyes
67 Survival of Walleye During Chronic Exposure to H2S 143
68 Mean Weight of Juvenile Walleye after Exposure to H^S 144
69 Mean Length of Juvenile Walleye after Exposure to H_S 145
70 Minnow Consumption of Juvenile Walleyes Exposed to H~S 146
71 Efficiency of Food Conversion by Walleye Exposed to H~S 147
72 Time to Immobility in MS:222 of Walleye Juveniles 149
Exposed to H_S
73 Threshold Toxicity (LTC) of H S to Brook Trout Eggs 152
74 96-hr LC50 and LTC Values of H S to Brook Trout Sac Fry 153
75 96-hr LC50 and LTC Values of H2S to Brook Trout 154
Feeding Fry
76 96-hr LC50 and LTC Values of H S to Brook Trout 155
Juveniles
77 A Comparison of 96-hr LC50 and LTC Values of H S for 159
2.5 C Increments from 8.5 to 21.0 C in Brook Trout
Juveniles
78 Percentage Differences in Mean LTC Values of H2S 162
Between 8.5 and 13.5 C for Each Life History
Stage of Brook Trout
79 Percentage Differences in Mean LTC Values of H S 153
Between Each Successive Life History Stage of
Brook Trout at 8.5 and 13.5 C
80 Weight of Juvenile Brook Trout at Succeeding Intervals in 165
Various H?S Concentrations at 9 C in 72 Days
81 Weight of Juvenile Brook Trout in Various H~S Concen- 166
trations at 13 C in 120 Days
XI
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No. Page
82 Brook Trout Reproduction Data in Various H S 168
Concentrations
83 Spawning Dates of Brook Trout During the Month 169
of October
84 Percentage Hatch of Brook Trout Eggs, Days to Hatch, 172
and Length of Fry at Hatch
85 Swimming Endurance of Brook Trout Juveniles after 45 174
and 120 Days of Exposure to H-S at 13 C
86 Source of Rainbow Trout and Stage of Fish at Start 177
of Tests with H2S
87 Acute Test Conditions and LC50 Values for Rainbow 179
Trout Tested in H-S
88 Duration of Rainbow Trout Sperm Viability in H~S 181
in Acute Test 1
89 Fertility of Rainbow Trout Eggs Inseminated in H2S 182
90 Length of Rainbow Trout Fry at Hatch and Percentage 183
Survival of Eggs Incubated in H S at 12.6 C
91 LC50 Values for Rainbow Trout Fry and Length of Fry 184
at Various H?S Concentrations in 20 Days of
Exposure (Acute Test 6)
92 LC50 Values for Rainbow Trout Juveniles at Various Days 186
and Length and Weight After 17 Days Exposure to
Different Concentrations of H^S (Acute Test 5)
93 Test Conditions in Chronic Test 1 with Rainbow Trout 187
94 Test Conditions in Chronic Tests 2, 3, and 4 with 188
Rainbow Trout
95 Test Conditions in Chronic Test 5 with Rainbow Trout 190
96 Survival of Rainbow Trout During Chronic Exposure to 191
Various Concentrations of H9S
97 Mean Weight of Rainbow Trout After Various Periods 193
of Exposure to Different Concentrations of H?S
98 Mean Length of Rainbow Trout Juveniles After Various 194
Periods of Exposure to Different Concentrations of H_S
xn
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No. Page
99 Survival, Weight, and Length in Chronic Test 2 of 195
Rainbow Trout Started as Eyed Eggs and Exposed to
Various Concentrations of Phenol
100 Survival, Length, and Weight of Rainbow Trout in a 197
Mixture of H«S and Phenol After Various Periods
of Exposure in Chronic Test 5
101 Test Conditions and LC50 Values of H S in Acute Tests 199
with White Sucker
102 Stage of Crayfish at Collection and at Start of 201
Acute Tests
103 Acute Test Conditions and LC50 Values for Crayfish 204
in H£S
104 Hatching Success of Procambarus clarkii Exposed to 206
H~S at Various Temperatures
105 Test Conditions in Chronic Tests of Crayfish 210
106 Survival of Crayfish (Procambarus clarkii) with 213
Long-term Exposure to H-S
107 Reproduction of Crayfish (Procambarus clarkii) in 214
Chronic Test 1
108 Weight of Crayfish (Procambarus clarkii) with Long- 216
term Exposure to H_S
109 96-hour LC50 Values of Subsamples of Juvenile 217
Crayfish (Procambarus clarkii) after Long-term
Exposure to H-S
110 Summary of Acute Bioassays Conducted with H?S on Six 220
Species of Invertebrates
111 Survival Time of Hexagenia in Different Type Chambers 222
with H?S in Three Paired Tests
112 Effect of Chamber Type on Hexagenia Survival without 222
Toxicant Present
113 LC50 Values of H2S to Crangonyx in Cylinders and 224
Box and Mud Chambers
Xlll
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No. Page
114 Effect of Substrate Type on Survival of Gammarus 224
at Similar H S Levels and Varied Times
115 Effect of Chamber Area and Volume on 96-hr LC50 225
of H«S with Gammarus
116 LC50 Values of tLS to Gammarus after Various Intervals 227
at 4 and 6 mg/liter <}„ and 10 and 15 C
117 Survival Time of Hexagenia Held on Mud in Fresh 229
Water for Varying Periods and Then Transferred to
Nitex Baskets Without Food or Toxicant
118 LC50 Values of H S for Hexagenia Collected in 231
Different Months
119 Emergence of Ephemera with Constant H~S and Varied 233
Oxygen Concentration and with Constant Oxygen and
Varied H?S Concentration in Succeeding Days
120 Emergence of Hexagenia with 2.0 mg/liter 0_ and 234
Varied Concentrations of H^S
121 Feeding of Gammarus on Populus alba pyramidalis 235
Leaves at Various Levels of H«S
122 96-hour LC50 Values of H2S for Six Invertebrates 236
Collected at Different Seasons
123 Characteristics of Test During Exposure of Hexagenia 239
in the Acute Test
124 Survival of Nymphs and Calculated LC50's of H~S to 241
Hexagenia on Succeeding Days During the Acute Test
125 Test Conditions During Chronic Exposure of Hexagenia 242
126 Summary of Survival of Hexagenia Nymphs and Emergence 243
of Subimagos During the Chronic Test
127 Relationships Between LC50, Chronic Safe, and Appli- 244
cation Factor Values for Hexagenia and Gammarus
128 Acute Toxicity of H2S to Gammarus Used in Chronic 247
Studies
129 Conditions During Chronic Tests of Gammarus with H2S 248
xiv
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No. Page
130 Acute Toxicity of H S to Seven Species of Fish 255
131 Median Tolerance Limits (LC50) to H2S of Eggs, Fry 260
and Juveniles of Various Species
132 Acute Toxicity of H-S to Eight Species of Invertebrates 264
133 Chronic Toxicity of H2S to Seven Species of Fish and 266
Three Invertebrate Species
134 No-effect Concentrations of H_S for Various Stages and 272
LTC Concentrations for Juveniles in Seven Species
of Fish
135 LC50 (96-hour) and LTC Values of Juveniles and 273
Application Factors Based on Chronic No-effect Level
136 Comparison of Application Factor for H«S with Fathead 275
Minnows and Goldfish Tested for Acute Response at
Different Temperatures
XV
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ACKNOWLEDGMENTS
The authors wish to acknowledge the financial assistance of the Minne-
sota Agricultural Experiment Station in completion of these studies.
They also wish to thank the Minnesota and Wisconsin Departments of
Natural Resources for assistance in obtaining trout, walleye, and
sunfish for experimental purposes.
Various graduate students, junior and assistant scientists and labora-
tory technicians assisted or were largely responsible for portions of
the study. Melvin Matson had primary responsibility for the brook
trout studies, Gary Kimball and Sayed El-Kandelgy for the bluegill,
Larry Olson for sections of the fathead minnow study, David Lind for
the geographical comparisons of fathead sensitivity, and Gary Kimball
for work on crayfish.
Dennis Swanson assisted in fathead minnow and walleye experiments. The
entire staff assisted in most experiments for which special responsi-
bility was not assigned to one person.
xvi
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SECTION I
CONCLUSIONS
The results described in the following report permit certain basic
conclusions to be made concerning the toxicity of dissolved undis-
sociated hydrogen sulfide to aquatic organisms. These general con-
clusions are:
1) H»S is highly toxic to fish and other aquatic organisms at
concentrations frequently found in natural and polluted
situations.
2) Toxic concentrations because of their low level or location in
the ecosystem are frequently overlooked and their importance
not evaluated.
3) The most sensitive life history stage is not the same in all
species.
4) Acute toxicity estimates will vary with fish species, source of
fish, and temperature of test.
5) The ratio of the no-effect level of lUS to the 96-hr LC50
varies from 1/3-5 in trout to 1/15-20 in warmwater fish.
6) Recommended ELS levels for fish will protect macro-invertebrate
species.
7) Since threshold LC50 (LTC) concentrations of the most sensitive
life history stage in some species approaches the no-effect
level, safe level specifications should be 0.05 of the juvenile
(LTC) at 20 C in most warmwater species and 0.10 in trout at
15-18 C.
8) To insure no-effect (as defined) on all stages of all warmwater
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aquatic species (except some spawning adults) a level of 0.002
mg/liter H2S at 20 C should be applied.
9) Where fish mortality is associated with oxygen reduction or
water movements, hydrogen sulfide levels should be examined.
10) When reproductive behavior brings fish, eggs, or fry into the
proximity of areas in which anaerobic decomposition can be
expected, such as the soil-water interface, poor reproduction
or survival may be caused by continuous evolution of low con-
centrations of H?S although dissolved oxygen is adequate.
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SECTION II
RECOMMENDATIONS
1. It is recommended that 0.002 mg/liter H~S be considered a safe
limit for protection of all fish species and hottorn-inhabiting
invertebrates in areas of lakes and streams not used for spawning
by nesting fish.
2. It is recommended that in areas used for reproduction by nesting
fish that concentrations of 0.001 mg/liter H«S not be exceeded.
3. It is recommended that appropriate application factors be applied
to lethal threshold concentration where particular species are
concerned rather than 96-hr LC50.
4. It is recommended that ample oxygen concentration for sustaining
fish life not be considered as an index of safe levels of tLS
without analysis for H?S, particularly in strata of water near
the soil-water interface.
>. It is recommended that field studies be conducted to determine the
occurrence of H9S in non-polluted waters to determine natural back-
ground levels in potential fish habitats.
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SECTION III
INTRODUCTION
A study of the effects of hydrogen sulfide on fish and invertebrates
has been conducted over a period of 5 years to determine lethal and
sublethal concentrations which have adverse effect on fish and inverte-
brates. Available literature at the start of the study indicated that
there was a wide discrepancy in assumed acute lethal values and that
the greater part of the experimental results did not include the con-
ditions under which experimental work had been done. Field work carried
out by the principal investigator and his colleagues indicated that
very low levels of hydrogen sulfide occurred in nature and in polluted
situations and that these levels had detrimental effects on fish
1 2
reproduction (Colby and Smith ; Adelman and Smith ). Special gear
developed during previous studies enabled the sampling of water near
the mud-water interfaces in areas where spawning and egg incubation
3
occurred (Colby and Smith )-
Hydrogen sulfide was demonstrated to be constantly evolved from organic
sludge in streams and to be a normal product found in hypolimnetic
situations during various seasons of the year. In the State of Minne-
sota hatcheries have been abandoned because of the occurrence of
hydrogen sulfide at toxic levels in their water supplies. A variety
of industrial effluents contain hydrogen sulfide or their break-down
products result in hydrogen sulfide generation. Although hydrogen
sulfide oxidizes rapidly, its continued evolution from bottom muds and
formation from break-down products may result in a substantial concen-
tration occurring at all times in some fish habitats. The foregoing
considerations led to the development of the research program reported
herein.
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TOXIC PROPERTIES OF HYDROGEN SULFIDE
The toxic properties of hydrogen sulfide are exerted in the undis-
sociated form. The extent of dissociation is strongly dependent on
pH with approximately 50% dissociation in the vicinity of 7.0 pH.
Hydrogen sulfide affects oxygen relationships in the organism and also
may affect mitochondrial systems. The toxicology of hydrogen sulfide
in freshwater aquatic organisms has been little studied and there are
few definitive references in the literature. A poorly investigated
area with reference to effect on organisms has been the actual concen-
tration of undissociated hydrogen sulfide at the point of oxygen trans-
fer on the gills. Discharge of carbon dioxide through gill structures
may result in pH levels which alter the apparent hydrogen sulfide level
in the ambient solution at the point of bodily entry. Since no direct
evaluation of this has been possible, it has been assumed that the
ambient levels as determined in the test chambers were the toxic levels.
Indirect tests described herein suggest that at high ambient pH, hydro-
gen sulfide appears more toxic than expected.
BASIS OF INVESTIGATION
The investigations reported here were all conducted in the fishery labor-
atory of the Department of Entomology, Fisheries, and Wildlife, Univer-
sity of Minnesota, with deep well laboratory water. The organisms used
for tests were of wild stock with the exception of laboratory-reared
fathead minnows and hatchery-reared goldfish and trout. Both wild and
laboratory-reared fathead minnows were used in an extensive series of
comparative experiments.
COVERAGE OF PRESENT REPORT
The present report embodies results of chronic and acute tests which
established 96-hr LC50 concentrations of hydrogen sulfide and lethal
threshold concentration (LTC), defined as the concentration at which no
deaths occur for 48 hours, and no-effect levels of the toxicant based
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on chronic test running up to 826 days. Six species of freshwater fish
and eight invertebrates were tested at all life history stages. The
problem of application factor to relate acute to chronic responses is
discussed in connection with the various species and appropriate factors
described for the organisms used.
The seven species of fish used in the investigations were selected to
cover a wide spectrum of presumed sensitivity and environmental prefe-
rence. They included the fathead minnow, Pimephales promelas Rafinesque,
goldfish, Carassius auratus (Linnaeus), white sucker, Catostomus com-
mersoni (Lacepede), bluegill, Lepomis macrochirus Rafinesque, walleye,
Stizostedion vitreum vitreum (Mitchill), rainbow trout, Salmo gairdneri
Richardson, and brook trout, Salvelinus fontinalis (Mitchill).
Invertebrates used included two crayfish, Procambarus clarkii (Girard)
and Cambarus diogenes Girard, one isopod, Asellus militaris Hay, two
amphipods, Crangonyx richmondensis laurentianus Bousfield and Gammarus
pseudolimnaeus Bousfield, and three Ephemeroptera, Baetis vagans^
McDonough, Ephemera simulans Walker, and Hexagenia limbata (Serville).
OBJECTIVES OF RESEARCH
The specific objectives of the research program were (1) to determine
acutely toxic concentrations of hydrogen sulfide (96-hr LC50 and lethal
threshold concentration (LTC)); (2) to determine the effect of exposure
to chronic low concentrations of hydrogen sulfide on growth, mortality,
reproduction, and other responses; (3) to explore factors affecting
toxicity of hydrogen sulfide to freshwater organisms; (4) to establish
application factors for use with acute tests; and (5) to develop a
method for determination of molecular hydrogen sulfide and its first
dissociation constants.
PLAN OF PRESENTATION
Presentation of the report has been planned to include most of the
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pertinent data on materials, methods and results in separate discussions
of each species. General aspects of methods applying to all work is
included in the Materials and Methods Section. A brief discussion and
summary is included at the end of the results section applying to each
species. General discussion and comparisons are treated in the section
entitled Discussion.
Part I of this report contains all the data on bioassay results,
methodology, and evaluation of results and recommendations of safe
levels of H_S for fish and invertebrates. Part II (presented under
separate cover) contains discussion of analytical methods for H^S
and a revised set of ionizatiosi constants for sulfides dissolved in
water;
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SECTION IV
MATERIALS AND METHODS
GENERAL LABORATORY CONDITIONS
All water for flow-through tests is transmitted from the well to the
laboratory through PVC pipe after catalytic iron removal has reduced
Fe to less than 0.1 mg/liter. The water is hard and comes from the
Jordon sandstone stratum underlying the Minneapolis-St. Paul metropoli-
tan area (Table 1). Water is cooled when necessary with a Dunham-Bush
chiller incorporating stainless steel cooling coils. Heating, aeration,
and pH adjustment is done in the head tank above each diluter. Light
was controlled automatically to a predetermined schedule according to
the needs of individual experiments. Unless otherwise noted, light was
from daylight-fluorescent tubes which varied in intensity with various
experiments. Ambient room temperature in the laboratory was maintained
at 20 C.
TEST ORGANISMS
Fish for experiments were secured from the field or from state and
commercial hatcheries. Walleye eggs came from Cutfoot Sioux Lake and
fingerlings from various Minnesota State rearing ponds. Fathead minnows
came from various lakes and from Duluth-Newtown laboratory stock? Gold-
fish were all secured from Ozark Fisheries, Inc., Missouri, or were
raised in our laboratory from the same stock. Bluegills came from lakes
in the Twin City metropolitan area. White suckers were reared from eggs
taken at Bemidji Fishery Station of the Minnesota Department of Natural
Resources. Brook trout eggs juveniles, and adults were from the Wis-
-------
Table 1. ANALYSIS OF WELL WATER
(milligrams/liter)
Item
Concentration
Total hardness as CaCO,
Calcium as CaCO.,
Iron
Chloride
Sulfate
Sulfide
Fluoride
Total phosphates
Sodium
Potassium
Copper
Manganese
Zinc
Cobalt, nickel
Cadmium, mercury
Ammonia nitrogen
Organic nitrogen
220
140
0.02
-------
consin State Hatchery at Osceola, Wisconsin. Rainbow trout eggs were
purchased from White Trout Farm, Paradise, Utah and from Ennis National
Hatchery, Ennis, Montana. Fish were reared in the laboratory. Inverte-
brates were secured from various sources as described in the discussion
of particular experiments.
Prophylactic Treatment
All fish stock was given prophylactic treatment for disease control on
entrance to the laboratory. They were subsequently treated in chronic
tests when evidence of disease was noted. The protocol for all treat-
ments is described in each species section.
APPARATUS
Acute Tests
Acute test apparatus was of two basic designs, one using H~S gas and
one using sodium sulfide, adjusted pH, and proportional diluters with
chemical-metering apparatus ("dipping-bird"-type toxicant dispensers).
The gas dispenser is described by Colby and Smith. It consists of two
tanks, one containing aerated water and the other oxygen-free water,
supplying a constant head of water. Well water prior to entering these
tanks is heated in a constant temperature water bath and then aerated
with compressed air or stripped of oxygen and carbon dioxide in a second
column with nitrogen. Figure 1 is a schematic diagram showing the essen-
tial features of the apparatus and the path of the water and H_S.
The hydrogen sulfide gas passes from a Number 3 cylinder (T) through an
H2S corrosive-free regulator (R), through a needle valve (V), and into
the H-S mixing chamber (X^) . Deoxygenated water (W..) from one head tank
passes through a Roger Gilmont Instruments flowmeter (F) (flow range 10-
850 mg/min) and into the mixing chamber (X..) where the H^S gas is
bubbled through. The mixing chamber (X..) was made from a sealed 6-liter
plastic egg-hatching jar set on a magnetic stirrer (S) with a 1-inch
teflon-covered magnet included in the jar. The H2S content of the water
10
-------
Figure 1. Schematic diagram of the continuous-flow bioassay apparatus
showing essential features and path of H-S and water through the appa-
ratus. H^S mixing chamber (X,); manifold for water with dissolved H9S
(X2) ; manifold with deoxygenated water (X.,) ; manifold with aerated water
(X^); environmental chambers (X5); deoxygenated water (W,); deoxygenated
water (W2); aerated water (W,,); minerators (M and M2); tlowmeter (F);
H2S gas tank (T); regulator (R); needle valve (V); exhaust fan (E);
magnetic stirrer (S); water bath (WB); water bath drain (D ); final drain
(D,) . Insert of individual environmental chamber (X,-) ; confinement area
of organisms (A); gravel (B); Jarrell-Ash oxygen probe receptacle (C);
water with dissolved H^S (I-); water adjusted for oxygen concentration (I )
11
-------
was controlled by variation of the H_S bubble rate with a needle valve
(V) and the water flow rate with the flowmeter (F). Hydrogen sulfide
gas that is not dissolved in the water in the mixing chamber (X.^ is
pulled through a hood and duct to an exhaust fan (E). The H2S-water
mixture then passes through a stainless steel tempering coil in a con-
stant temperature water bath (WB) and into a manifold (X2), immersed in
the water bath (WB), where it is metered with Fischer and Porter Min-
erators (M..) .
Deoxygenated water (W9) also passes from the same head tank as water
going to the mixing chamber (X.,), through stainless steel tempering
coils and into manifold (X3), immersed in the water bath (WB). Oxygen-
saturated water (W,,) passes from the other head tank through stainless
steel tempering coils and into the manifold (X,), immersed in water
bath (WB). Water from manifolds (X. and X,) is fed into a common tube
and is metered with Fischer and Porter Minerators (M9). Water from
minerators (M.. and M9) flows into a common tube and passes into environ-
mental chambers (X,-), past the test organisms in an area confined by
Nitex screens, and to the drain (D.,) . The desired oxygen content is
obtained by controlling the ratio of aerated water from manifold (X,)
to deoxygenated water from manifold (X.,) using stopcocks. The desired
sulf ide concentrations are controlled by minerators (M..) , and the flow
rate is controlled by minerators (M_). Tygon tubing was used exclusively
in the continuous flow system except for the stainless steel tempering
coils. The manifolds, head tanks and environmental chambers were made
from acrylic plastic. Each bioassay apparatus contained six environ-
mental chambers (X,). Water with the dissolved H9S (I,) is mixed with
•3 Z. _L
water adjusted for dissolved oxygen concentration (I9) at a Y junction
and enters the chamber through an acrylic plastic tube. Test chambers
are modified in accordance with needs of particular organisms or stages.
Details are given in the discussion of each species.
The interval between mixing and flow over the test organisms and out of
12
-------
the chamber did not exceed 1-1/2 min. Desired concentrations of dis-
solved H-S were attained by adjusting the water flow on the basis of
analysed water from the bioassay test chambers. Flow rates, test cham-
ber size, design and other details peculiar to the tests on each organism
are described in the experimental design section relating to each
species.
The second type of apparatus used was modified from that described by
4
Brungs and Mount. It was a continuous flow-through type with chemical-
metering apparatus (Mount and Brungs ) delivering sodium sulfide to each
test chamber. Each "dipping bird" in the apparatus was calibrated to
deliver the volume required for a particular concentration. Ranges of
test concentrations were adjusted by changing concentration of the stock
solution. Water flow was controlled by the multiple diluter apparatus.
Hydrogen ion concentration was adjusted with H_SO, dispensed with a
"dipping bird" apparatus in the head tank above each diluter. Chilled
water was introduced to the head tank where needed and final adjustment
of temperature made by hot water coils of polyethylene or stainless
steel controlled by a thermostat and solenoid in the hot water lines.
Flow rates, test chamber size, and variations peculiar to each experiment
are described in the design section of the report on that experiment.
Desired pH levels were attained by manually adjusting the chemical-
metering apparatus on the basis of analysis in the test chambers with
a Beckman line-operated meter.
Test chambers in both types of apparatus were of glass or acrylic plastic.
Glass chambers were constructed with silicone cement and all connections
below the head tank were of glass or tygon tubing.
Chronic Tests
All tests discussed in this report which run longer than 3 weeks, in-
cluding egg to egg tests, are referred to as "chronic tests1.1 Chronic
test apparatus was the same as that described above for the sodium sul-
13
-------
fide acute tests except that test chambers were varied in size to accomo-
date the size and numbers of fish used in the particular test. Details
of tank size, flow rates, and water retention time are presented in the
design section of experiments on each species.
Chemical Analyses
The analytical method used for determination of H^S was a modification
of Method C for sulfide described in Standard Methods for the Examination
of Water and Wastewater (American Public Health Association ). Samples
were taken from the center of the open test chamber and from the outlet
in small sealed chambers. They were immediately fixed with zinc acetate;
the standard procedures were then followed and absorbance measured with
a spectrophotometer. Comparison was made against water from the control
test chamber taken at the same time as the sample. Precise repeatability
to 0.01 mg/liter total sulfide was attained. At the pH where most bio-
assays were run (7.7), undissociated H_S was approximately 0.1 of total
sulfide or 0.001 mg/liter with an estimated accuracy of +0.0005 mg/liter.
Calculation of H.S was made from Pomeroy tables for pH and temperature.
All concentrations of ELS reported for various tests are from analyses
in test chambers and are not nominal or target concencrations based on
calculated dilution from stock solutions. Samples were taken three
times daily in acute tests and at 1- to 3-day intervals in chronic tests.
After the completion of the bioassay phases of the project, a revised set
of ionization constants were experimentally determined. They are dis-
cussed in Part II of the present report. A comparison of results deter-
mined from the Pomeroy tables with the new determination is made in the
discussion section of Part 1 of this report. Molecular H?S was deter-
mined in laboratory well water and deionized water after addition of a
known quantity of sulfide. Results were the same at the same pH and
temperature. It is therefore concluded that no materials occur in the
well water which interfere with determination of H S.
14
-------
METHOD OF REPORTING CALCULATION OF LC50 AND LTC
Acute Tests
Median lethal concentrations (LC50) values have been computed by graphic
interpolation on semilog paper (American Public Health Association ).
The median lethal concentrations in most cases were calculated for 48-
72 and 96 hours and where possible for lethal threshold concentrations
(LTC). Lethal threshold concentration was considered to be reached
when no deaths in treatments occurred for 48 hours.
Chronic Tests
The results of chronic tests with levels of H_S which did not cause
death during the first 3 weeks were assessed in terms of survival, rate
of growth, efficiency of food utilization, success of reproduction, and
response to other stresses (swimming endurance, response to other toxi-
cants) . When a no-effect concentration of H_S is specified it refers to
a level where no demonstrable adverse effect is noted in the parameters
measured. In no species were all criteria of adverse effects of toxi-
cant used. Details of criteria used are included with discussion of
each species. In some cases (trout) when response was less than 10%
from the control, the results of treatment were considered to be no
different than the controls because response of individual fish appeared
to account for variability of less than this amount.
EXPERIMENTAL DESIGN
In the majority of experiments one control and four to five treatments
were employed in both acute and chronic studies. In some studies two
controls and 10 treatments were used. Treatment levels were set up on
a logarithmic or arithmatic scale, depending on the range of sensitivity
of the organism. Final tests were run and test levels of H~S determined
on the basis of preliminary tests which defined the general acute range
of toxicity. Test concentrations for chronic tests were usually selected
with the highest acute test concentration which showed no deaths being
used as the highest concentration in the chronic series.
15
-------
All acute tests were done on fish stages not previously subjected to
H2S except as specifically noted. Chronic tests were started with eggs,
fry, fingerlings, and adults and results compared. In some species of
fish more than one generation was subjected to low concentrations of
H?S. Where possible,' chronic tests were run through spawning. Some
short term tests for growth rate analysis were also run.
Invertebrate tests were started with eggs, nymphs, or adults in accor-
dance with availability and objective of the test.
Details of design of each experiment are included with the discussion
of each particular species.
16
-------
SECTION V
FATHEAD MINNOW
(Pimephales promelas Rafinesque)
The response of the fathead minnow to H-S was tested by (1) a series of
acute bioassays at various temperatures with two stocks of fish, (2)
long-term tests at low concentrations, (3) a chronic test through two
complete generations, and (4) acute tests on fish from different
locations and habitat conditions.
ACUTE BIOASSAY
Experimental Design
Fish for tests described in this section were collected in the St. Paul
vicinity (Table 2) and from laboratory stock originating in the National
Water Quality Laboratory at Duluth, Minnesota. Fish were treated when
brought in and as needed with 20 mg/liter neomycin or tetracycline for
3 days and then with methylene blue at 0.2 mg/liter of 1% solution for
each of 3 days. During pretest holding, fish were fed Glencoe dry fish
food pellets, Oregon moist pellets, a ground egg and lettuce mixture, and
brine shrimp once per day until 24 hr before test. Fish were not fed
during first 96 hr of test but daily thereafter. Fish were acclimated to
test tanks for 5 days before introduction of toxicant. Eggs and fry
were tested in chambers 10 x 16 x 20 cm with three glass sides and
bottom and one side of #351 Nitex screen. These test cells were immersed
in 50 x 25 x 20 cm glass chambers which were also used for juvenile
tests. Eggs rested on the bottom of the small chamber until hatched.
17
-------
Table 2. SOURCE OF FATHEAD MINNOWS AND STAGE AT START
OF ACUTE TESTS WITH HS
Tempera-
Stage at Date of ture at
Stage at collec- collec- collec- Method of
Test start tion Sources tion tion, C collection
30 Egg
34
36
40
41
42
43 Fry
44
45
22 Juvenile
24
25 "
26
4
7
8
9
11
12
15
16
17 "
18
19
20
21
Egg Duluth stock 9/12/72
" 12/11/72
" 1/15/73
„ 2/6/73
„ 2/19/73
11 3/4/73
„ 3/14/73
i. 3/21/73
" 3/30/73
" 11/12/70
n 3/24/71
" 10/18/71
11 10/18/71
Juvenile Cleveland Pond 10/29/68
10/29/68
" 10/29/68
" " " 12/23/68
12/23/68
" 8/20/69
" Lake Como 3/4/70
" 7/21/70
" 9/21/70
" " 10/12/70
" 11/2/70
" 1/4/71
„ 1/12/71
24
n
n
n
M
II
II
It
II
20
22
n
n
9
n
M
4
M
22
4
23
19
10
8
4
"
Spawning tiles
n n
M n
ii n
n ii
n M
ti n
M II
II II
II II
II II
II M
M II
Seine
n
n
Trap
"
Seine
Trap
Seine
n
"
"
Trap
n
18
-------
Table 2 (continued). SOURCE OF FATHEAD MINNOWS AMD STAGE AT START
OF ACUTE TESTS WITH HS
Stage at
Stage at collec-
Test start tion
Sources
Te.rnp era-
Date of ture at
collec- collec-
tion tion, C
23 Juvenile Juvenile Lake Como 2/3/71
28
29
31
32
33
" Cleveland Pond 8/2/72
Egg " " 5/22/72
" 5/22/72
Juvenile " " 9/27/72
" " " 9/27/72
Method of
collection
4 Trap
24 Seine
18 Natural spawn-
ing sites
ti i?
15 Seine
19
-------
Day length was 12 hr. The water flow through the test chambers was
300 ml/min.
All tests consisted of one control and five H2S concentrations supplied
by diluters with "dipping-bird" dispensers. Eggs were taken off spawn-
ing tiles within 24 hr of deposition. Fry were hatched in the labora-
tory and juveniles were seined from ponds or reared in the laboratory.
Thirty-two acute tests were run in six series. One series was done on
eggs, one on fry, one to determine effect of temperature, one to deter-
mine difference in response of wild and laboratory-reared fish, and two
to compare acute lethal concentrations with chronic concentrations.
Acute Toxicity
Eggs—Six bioassays were run on fathead eggs (Table 3) at 23.8-24.2 C
and oxygen of 5.6-6.0 mg/liter. The 96-hr LC50 varied between tests
from 0.0190 to 0.0610 mg/liter, with a mean of 0.0350 mg/liter H2S. LTC
or time to hatch was attained in 5 to 8 days at 0.0190 to 0.0595 mg/
liter with a mean of 0.0345 mg/liter H S.
Fry—Three tests on fry started within 24 hr of hatching were run at
24 C and 5.4-6.2 mg/liter 02- The mean LC50 varied from 0.0136 at 24 hr
to 0.0070 mg/liter H2S at 96 hr. Mean LTC at 6 days was 0.0061 mg/liter
H2S. LC50 at 96 hr varied from 0.0066 to 0.0075 mg/liter H2S and LTC
from 0.0057 to 0.0066 mg/liter HLS.
Juveniles at Various Temperatures—Bioassays were run at six temperatures:
6.5, 7.6, 10.0, 15.0, 20.2, and 25.0 C. LC50 at 96 hr ranged from 0.5800
mg/liter at 6.5 C to 0.0280 mg/liter H2S at 25.0 C (Table 4). Fish used
for test at 6.5 C were taken in January when lake water was 4 C. Other
tests were made on fish caught when lake temperature approximated test
temperatures.
20
-------
Table 3. ACUTE TOXICITY OF H0S (LC50 AND LTC)
a/
TO FATHEAD MINNOW EGGS AND FRY~
Test
Days
to
b/
test-
Mean Fish or
length, eggs/
mm chamber
Tempera-
ture, 0~
C mg
»
/I 24 hr 48 hr
LC50,
me/1 H2S
72 hr 96 hr
LTC-^davs)
Eggs - Duluth stock
30
34
36
40
41
42
0
0
0
0
0
0
25
50
25
20
20
30
24
23
24
24
24
24
.0
.8
.1
.2
.0
.1
5.
5.
5.
6.
5.
5.
6
8
7
0
5 _ _ _ _
9
x = — —
43
44
45
0
0
0
5.6 20
5.9 20
5.6 20
24
24
24
.1
.0
.0
Fry
5.
6.
6.
X
- Duluth stock
4 0.0190 0.0130
2 0.0087 0.0072
2 — 0.0096
= 0.0136 0.0099
0.
0.
0.
0.
0.
0.
0.
0.0115 0.
0.0063 0.
0.0081 0.
0.0086 0.
0205
0440
0190
0285
0610
0370
0350
0069
0066
0075
0070
0
0
0
0
0
0
0
0
0
0
0
.0205
.0445
.0190
.0285
.0595
.0350
.0345
.0061
.0066
.0057
.0061
(5)
(5)
(4)
(6)
(8)
(8)
(6)
(6)
(6)
f/pH: 7.9.
—.Days elapsed between collection of specimen and start of test,
—Total hatch.
-------
Table 4. ACUTE TOXICITY OF H2S (LC50 AND LTC) TO WILD STOCK
JUVENILE FISH AT VARIOUS TEMPERATURES3
Test
21
20
19
18
17
16
Mean
Days fish
to length,
b
test mm
6
7
7
7
7
6
47
50
30
34
28
28
.0
.0
.0
.0
.5
.0
Fish Temp-
per era-
chain- ture, 0-
ber C mg/1
40
40
40
40
40
40
6
7
10
15
20
25
.5
.6
.0
.0
.2
.0
5.2
6.3
6.0
6.0
5.7
6.1
LC50,
mg/1 H0S
24 hr 48 hr
-
0.7100 0.6500
-
0.0600
0.0420
0.0340 0.0310
0
0
0
0
0
72 hr
-
.6000
.2400
.0570
.0370
.0300
96 hr
0.5800
0.5200
0.1500
0.0570
0.0320
0.0280
*PH 7.9.
Days elapsed between collection of specimen and start of test.
22
-------
Acute Tests with Field and Laboratory-reared Fish—Two tests with field-
reared juvenile stock and two with fish of the same stock reared from
eggs in the laboratory were run at 24.1-23.8 C (Table 5) to determine
whether laboratory conditions during pretest life had a significant
influence on sensitivity to H_S. The mean 96-hr LC50 for field-reared
fish was 0.0208 mg/liter and the LTC was 0.0204 mg/liter and for labor-
atory-reared fish was 0.0212 and 0.0198 mg/liter H-S, respectively.
Juvenile Acute Tests for Chronic Test Comparison—Nine tests were run
on juvenile fish of wild stock and four on Duluth laboratory stock to
determine LCSO's (Table 6) for comparison with chronic tests at low
concentrations of H~S. The acute tests were run at the time chronic
tests were started. The mean 96-hr LC50 of six tests on 7 to 10 wild
fish at 19.9 to 20.0 C was 0.0367 mg/liter H-S.
Four tests run on Duluth stock were somewhat less than half as tolerant
as the wild stock at 96 hr. The tests were run at 19.8 to 22.2 C
(Table 6). The mean 96-hr LC50 was 0.0162 mg/liter and the mean LTC
was 0.0120 mg/liter H-S. Variation in LCSO's between tests did not
exceed 0.006 mg/liter at 96 hr and did not exceed 0.003 mg/liter at LTC.
CHRONIC BIOASSAY
Experimental Design
Five chronic tests were conducted on fathead minnows and were started
as eggs or juveniles (Table 7). Three tests (chronic 2, 3, and 5) were
with wild juvenile fish. Test 4 was started with sac fry of Duluth
stock eggs. The chronic tests, designated as chronic 6, and &„, were
continuous for two generations and initially employed sac fry of Duluth
stock eggs. Tests 2, 3, 4, 6,, and 6« were run to determine no-effect
concentration and 5 to check effects of fish density.
Diluters were those described for use with sodium sulfide and toxicant
23
-------
Table 5. ACUTE TOXICITY OF H2S (LC50 AND LTC) TO FIELD-REARED
AND LABORATORY-REARED JUVENILE FISH AT 20 C3
to
Days Mean fish Number of Tempera-
to length, fish/ ture, 0_}
Test test mm chamber C mg/1
LC50,
mg/1 Hn
24 hr
48 hr
72 hr
S
96 hr
LTC (days)
Laboratory-reared
29 79 20.0 20
31 133 34.0 20
24.0
24.0
5.2
5.4
x =
0.0270
0.0300
0.0285
0.0260
0.0280
0.0270
0.
0.
0.
0192
0245
0218
0
0
0
.0179
.0245
.0212
0.
0.
0.
0175
0220
0198
(8)
(7)
Field-reared
32 34 26.0 20
33 46 32.0 20
23.8
24.1
5.4
5.3
x =
0.0287
0.0310
0.0298
0.0226
0.0280
0.0253
0.
0.
0.
0220
0203
0212
0
0
0
.0215
.0200
.0208
0.
0.
0.
0210
0197
0204
(9)
(7)
bpH: 7.9.
Days elapsed between collection of specimen and start of test.
-------
Table 6. ACUTE TOXICITY OF H2S (LC50 AND LTC) TO FATHEAD MINNOWS
OF WILD AND DULUTH STOCK3
(Tests Run to Provide Base for Application Factor)
Mean
Days fish
to length,
Test test mm
Fish Temp-
per era-
chain- ture, 0«,
her C mg/1
LC50,
mg/1 H^S
48 hr
72 hr
96 hr
LTC (days)
Wild stock
4 39
7 67
8 74
9 35
11 70
12 19
35
34
36
38
34
—
.1
.9
.2
.3
.0
7
8
8
10
10
10
20.0
20.0
19.9
19.9
19.9
20.0
6
6
6
6
6
6
.5
.1
.1
.0
.0
.0
x =
Duluth
22
24
25
26
34
36
39
32
.0
.0
.0
.0
15
18
16
11
19.8
22.2
22.0
22.0
5
5
6
6
.5
.8
.1
.0
x =
0
0.0420 0
0
0.0430 0
-
-
0.0425 0
stock
0.2000 0
0
-
-
0.2000 0
.0390
.0390
.0400
.0390
-
-
.0392
.0170
.0210
-
-
.0190
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0350
0380
0390
0340
0350
0390
0367
0160
0180
0180
0127
0162
-
-
-
-
-
-
-
-
0.0130(8)
0.0120(10)
0.0109(9)
0.0120
*PH 7.9.
Days elapsed between collection of specimen and start of test.
25
-------
Table 7. SOURCE OF FATHEAD MINNOWS AND STAGE
AT START OF CHRONIC TESTS WITH H^S
Test
2
3
4
5
61
62
Stage at
start
Juvenile
Juvenile
Sac fry
Juvenile
Sac fry
Juvenile
Source
Cleveland Pond
Cleveland Pond
Duluth stock
Lake Como
Duluth stock
Duluth stock
Date of
collec-
tion
12/23/68
8/20/69
11/12/70
12/16/71
3/24/71
10/18/71
Water
temp, at
collec-
tion, C
4
22
22
4
22
23
Method of
collection
Trap
Seine
Spawning tiles
Trap
Spawning tiles
Spawning tiles
26
-------
dispensers described in the previous section. In chronic tests 2 and 3,
five glass-silicone tanks were divided into two sections (a and b)
so that water flowed from diluter through section "a" into "b". Each
section measured 15 x 40 x 31 cm with a water depth of 21 cm and a
total volume of 12.6 liters. Concentrations of H«S reported were based
on analyses of water from each section (Table 8). The 20-liter tanks
described above for the acute tests were used for chronic tests 4, 5,
6,, and 6». In tests 4, 6,, and 6? there were three replications (a,
b, c) each with a separate diluter. In chronic test 5 there were five
replications on the control and each of the three concentrations of
H2S (Table 9). In chronic test 4 there were three replications of the
control and the four HLS concentrations (Table 10)-
In chronic tests 6, and 69, run consecutively in the same diluter, the
three replications (Rep a, b, c) were run in parallel on separate levels
of the diluter bank. On a fourth level another series designated as
Fry Bank was operated for 80 days after spawning started in test 6.. to
hatch eggs and rear fry (Table 11). After 80 days these juveniles were
placed in the three replications of chronic test 62- When spawning
started in test 6? eggs were placed in the Fry Bank o-f test 6_ to hatch
and the fry were held for the 80-day period (Table 12)-
Chronic tests 2 and 6? were started with 20 fish per chamber and chronic
4 with 15 fish per chamber. Fish were randomly removed from all tanks
in each test just prior to spawning so all tanks contained 10 fish per
chamber unless prior mortality reduced the number to less than 10.
Chronic 3 and 6, were started with 10 fish per tank and were not thinned.
Chronic test 5 was started with various numbers of fish from 4 to 19
to determine the effect of density on survival and growth. Controls and
two series of H«S concentrations were employed (target concentrations 1
and 2).
The day length was varied with seasonal changes in all tests. Day
27
-------
Table 8. TEST CONDITIONS IN CHRONIC FATHEAD MINNOW TESTS 2 AND 3
STARTED WITH WILD STOCK JUVENILES3
Chronic 2: Tank
x H~S concentration(mg/l)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0~ (mg/1)
x total alkalinity (mg/1)
Tank
x H_S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0? (mg/1)
x total alkalinity (mg/1)
Chronic 3: Tank
x H_S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (c)
x dissolved 0« (mg/1)
x total alkalinity (mg/1)
Tank
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0? (mg/1)
x total alkalinity (mg/1)
5a
—
—
7.85
20.1
6.60
—
5b
—
—
7.85
20.1
6.60
—
la
—
—
7.67
21.2
7.52
194
Ib
—
—
7.67
21.3
7.99
194
2a
0.0019
0.0010
7.89
20.0
6.60
—
2b
0.0015
0.0010
7.86
19.8
6.60
—
5a
0.0005
0.0006
7.69
21.3
8.15
194
5b
0.0004
0.0004
7.67
21.3
7.61
194
4a
0.0070
0.0038
7.87
20.1
6.60
—
4b
0.0048
0.0028
7.85
20.1
6.60
—
4a
0.0024
0.0012
7.69
21.3
8.25
194
4b
0.0010
0.0008
7.69
21.3
8.10
194
3a
0.0093
0.0046
7.90
20.1
6.60
—
3b
0.0060
0.0029
7.87
20.0
6.60
—
3a
0.0068
0.0027
7.73
21.3
8.20
194
3b
0.0022
0.001?
7.71
21.3
7.96
194
la
0.0126
0.0054
7.94
19.9
6.60
—
Ib
0.0080
0.0033
7.94
20.0
6.60
—
2a
0.0198
0.0078
7.84
21.2
8.23
194
2b
0.0078
0.0034
7.85
21.2
8.23
194
20 fish started in each chamber and thinned to 10 before first spawning.
28
-------
length in chronic tests 2 and 5 was synchronized with that of St. Paul,
Minnesota. The day length for Evansville, Indiana was used in tests
3, 4, 6.. , and 6~ as recommended by the National Water Quality Laboratory
at Duluth. The temperature was varied seasonally in tests 2 and 3 and
was held constant in tests 4, 5, 6-. , and 6«.
A substrate for spawning was provided by half cylinders cut from 3-inch
lengths of cement-asbestos pipe and placed in the test chambers with
the convex side up.
Eggs were incubated in Nitex screen-bottomed glass cylinders which were
continuously moved up and down 1.5 cm in a 10-sec cycle. The oscil-
lation was provided by attachment of the cylinder to a variable speed
gear box and an off-set cam wheel.
All spawned eggs from each chamber were counted. From at least one of
each three spawnings in each test chamber of test 6, and 6~ a random sub-
sample of 50 eggs was incubated to hatch to determine percentage sur-
vival to hatch. Eggs from each treatment were hatched in that treatment
and in control water.
If fish appeared stressed by diagnosed bacterial disease they were
treated with tetracycline or neomycin at the rate of 20 mg/liter for
3 days with continuous inflow of freshwater without the H~S used for
treatment. Combinations of formalin and methylene blue or copper sul-
fate were used for protozoan infections.
After reaching 20 mm total length, fish were weighed in water at 4-week
intervals until sexual maturity. All the fish of a single test chamber
were weighed in water as a group.
Newly hatched fry were fed a suspension of mashed hard-boiled egg yolk
at least three times per day. When a size of about 8 mm total length
29
-------
Table 9. TEST CONDITIONS IN CHRONIC FATHEAD MINNOW TEST 5
STARTED WITH WILD STOCK JUVENILES
Control Tank
No. fish/ tank
x H2S concentration (ing/1)
H2S std, dev. (mg/1)
x pH
x temperature (C)
x dissolved 0« (mg/1)
x total alkalinity (mg/1)
Target concentration _! Tank
No. fish/ tank
x H^S concentration (mg/1)
H2S std. dev, (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
Target concentration 2_ Tank
No. fish/ tank
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0-
x total alkalinity (mg/1)
3
4
-
-
7.76
19.6
9.54
203
5
4
0.0014
0.0008
7.78
19.9
8.97
203
4
4
0.0029
0.0025
7.77
21.9
8.80
203
2
6
_
-
7.75
19,6
8.86
203
4
6
0.0018
0.0010
7.76
19.8
8.77
203
2
6
0.0052
0.0028
7.75
21.5
8.52
203
4
10
-
-
7.75
19.6
9.52
203
3
10
0.0016
0.0008
7.74
19.9
8.31
203
5
10
0.0038
0.0024
7.75
21.6
8.44
203
5
14
-
-
7.74
19.5
8.81
203
1
14
0.0019
0.0011
7.75
20.0
7.87
203
3
14
0.0046
0.0024
7.75
21.5
8.29
203
1
19
-
-
7.72
19.8
8.92
203
2
19
0.0017
0.0009
7.72
19.8
7.71
203
1
19
0.0038
0.0021
7.72
21.5
6.79
203
30
-------
Table 9 continued. TEST CONDITIONS IN CHRONIC FATHEAD MINNOW TEST 5
STARTED WITH WILD STOCK JUVENILES
Target concentration 3 Tank 1
No. fish/ tank
x
t
x
X
X
X
H^S concentration (mg/1)
?S std. dev.
pH
temperature
dissolved 0,
i
total alkald
(mg/1)
(C)
, (mg/1)
.nity (mg/1)
4
0.0094
0.0083
7.76
20.7
8.57
203
4
6
0.0111
0.0062
7.78
20.9
8.09
203
2
10
0.0070
0.0044
1 .11
21.1
7.97
203
3
14
0.0099
0,0077
7.74
20.8
7.47
203
5
19
0.0088
0.0045
7.77
20.7
6.83
203
31
-------
Table 10. TEST CONDITIONS IN FATHEAD MINNOW CHRONIC TEST 4
STARTED WITH DULUTH STOCK SAC FRY&
Replicate a Tank
x H.S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0~ (mg/1)
x total alkalinity (mg/1)
Replicate b Tank
x H_S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved Q~ (mg/1)
x total alkalinity (mg/1)
Replicate c Tank
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0^ (mg/1)
x total alkalinity (mg/1)
5a
—
—
7.68
23.8
8.66
208
5b
—
—
7.76
23.0
8.99
208
5c
—
—
7.69
23.9
8.56
208
4a
0.0010
0.0007
7.64
23.9
8.73
208
4b
0.0006
0.0004
7.74
22.9
8.33
208
4c
0.0010
0.0007
7.65
24.1
8.16
208
3a
0.0022
0.0028
7.65
23.9
8.31
208
3b
0.0016
0.0019
7.72
22.8
7.92
208
3c
0.0021
0.0024
7.63
24.2
8.06
208
la
0.0052
0.0038
7.67
23.5
7.99
208
Ib
0.0045
0.0037
7.70
22.5
7.77
208
Ic
0.0046
0.0048
7.67
23.8
7.45
208
2a
—
—
—
—
—
— —
2b
0.0078
0.0063
7.78
22.7
7.82
208
2c
0.0102
0.0076
7.67
24.0
7.46
208
a
Fifteen sac fry per chamber at start and thinned to 10 fish prior to
first spawning.
32
-------
Table 11. TEST CONDITIONS IN FATHEAD MINNOW CHRONIC TEST 6]
STARTED WITH DULUTH STOCK SAC FRY&
Replicate j. Tank
x H-S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0™ (mg/1)
x total alkalinity (mg/1)
Replicate b Tank
x H^S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0- (mg/1)
x total alkalinity (mg/1)
Replicate c Tank
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0_ (mg/1)
x total alkalinity (mg/1)
Fry bank Tank
x H~S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
5a
7.75
23.1
7.14
214
5b
—
7.76
22.7
7.39
214
5c
—
7.75
23.1
6.98
214
5d
7.81
22.5
7.07
214
4a
0.0004
0.0003
7.73
23.1
6.81
214
4b
0.0004
0.0003
7.77
22.7
7.27
214
4c
0.0005
0.0003
7.74
23.2
6.74
214
4d
0.0004
0.0002
7.83
22.4
7.18
214
3a
0.0012
0.0004
7.72
23.1
6.48
214
3b
0.0011
0.0004
7.73
22.7
7.07
214
3c
0.0012
0.0004
7.73
23.2
6.62
214
3d
0.0007
0.0003
7.84
22.4
7.03
214
la
0.0033
0.0008
7.72
22.9
6.39
214
Ib
0.0026
0.0010
7.77
22.5
7.18
214
Ic
0.0033
0.0011
1 .Ik
22.9
6.49
214
Id
0.0026
0.0011
7.85
22.2
6.68
214
2a
0.0063
0.0019
7.77
23.0
6.31
214
2bb
—
^^
—
^^
2c
0,0068
0.0020
7.76
23.1
6.48
214
2d
0.0041
0.0011
7.89
22.3
6.95
214
Started with 10 fry.
3Fish all killed by low
33
-------
Table 12. TEST CONDITIONS IN FATHEAD MINNOW CHRONIC TEST 6,
STARTED WITH FISH FROM CHRONIC 6^ SPAWNINGS3
Replicate a Tank
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0? (mg/1)
x total alkalinity (mg/1)
Replicate b Tank
x H»S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0,., (mg/1)
x total alkalinity (mg/1)
Replicate c Tank
x H_S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
Fry bank Tank
x H-S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
5a
—
—
7.68
24.1
6.78
201
5b
—
—
7.71
23.6
6.83
201
5c
—
—
7.69
24.3
6.76
201
5d
—
—
7-79
24.2
7.41
201
4a
0.0008
0.0005
7.65
24.2
6.55
201
4b
0.0006
0.0005
7.71
23.6
6.79
201
4c
0.0007
0.0005
7.66
24.3
6.56
201
4d
0.0007
0.0004
7.74
24.1
7.45
201
3a
0.0014
0.0007
7.67
24.2
6.44
201
3b
0.0012
0.0005
7.71
23.7
6.83
201
3c
0.0014
0.0005
7.66
24.3
6.37
201
3d
0.0009
0.0005
7.80
24.1
7.26
201
la
0.0035
0.0016
7.67
24.0
6.31
201
Ib
0.0032
0.0016
7.69
23.5
6.46
201
Ic
0 . 0044
0.0028
7.68
24.0
6.52
201
Id
0.0018
O.OOL0
7.81
24.0
7.14
201
2a
0.0080
0.0035
7.67
24.1
6.17
201
2b
0.0054
0.0025
7.73
23.6
6.58
201
2c
0.0074
0.0030
7.68
24.3
6.21
201
2d
0.0050
0.0023
7.83
24.9
6.88
201
STest started with 20 80-day juveniles from spawning in test
34
-------
was attained, finely pulverized dry Glencoe //I fry granules were added
to the diet, at least twice per day. At a size of about 10 mm, newly
hatched brine shrimp were added twice per day. At 15 mm, feeding of
egg-yolk suspension was stopped and a mixture of finely blended hard-
boiled eggs and lettuce was substituted. At a size of about 30 mm the
#1 fry granules were fed without pulverizing.
All tanks were illuminated with incandescent lights located about 30 cm
above the water surface. Bulbs varied from 40 to 100 watts depending
on the life history stage. For egg incubation and the early fry stages
40 watt bulbs were used, and by the time of sexual maturity the wattage
was gradually increased to 100.
Survival in Chronic Exposure
Chronic tests 2 and 3 were started with wild stock juveniles (Table 13).
Test 2 ran for 191 and test 3 for 345 days. After 51 days in both series
of test 2, survival declined in control and all test concentrations of
H2S. At 0.0093 mg/liter H S in series a., survival was 61% with 93% in
control at 79 days and at 191 days was 6% and 65%, respectively.
In series b_, survival through 79 days was essentially the same as con-
trols in all H9S treatments except at 0.0080 mg/liter where it was sub-
stantially lower. Excessive loss in controls made comparisons doubtful
for the remainder of the test.
Chronic 3, series a_, had reduced survival at 0.0198 mg/liter HLS by the
37th day and thereafter but at lower concentrations survival was similar
or better than controls. Survival in series b with a maximum H0S con-
£.
centration of 0.0078 mg/liter was not lower than control in any concen-
tration.
Chronic 4, with three replications (a,b,c) was started with Duluth stock
sac fry and continued for 84 days (Table 14). Maximum concentration
35
-------
Table 13. SURVIVAL OF WILD STOCK FATHEAD MINNOWS WITH LONG-TERM
EXPOSURE TO H2S IN CHRONIC TESTS 2 AND 3
(expressed as percentage)
Exposure
Test days
Chronic 2
series &_ 24
51
79
107
135
163
191
series b
24
51
79
107
135
163
191
Chronic 3
series ji 9
37
66
177
345
, H«S concentration,
me/1
Control
100
100
93
93
93
86
65
Control
100
100
95
85
77
68
34
Control
100
80
60
60
60
0.0019
90
84
84
84
84
84
76
0.0015
95
90
84
84
76
67
42
0.0005
100
100
100
90
90
0.0070
100
75
75
75
75
75
68
0.0048
100
100
90
86
86
86
69
0.0024
100
100
90
90
90
0.0093
100
61
61
50
50
16
6
0.0060
100
96
96
91
82
82
73
0.0068
100
89
67
67
67
0.0126
91
0
0
0
0
0
0
0.0080
94
59
59
59
53
35
24
0.0198
70
20
20
20
10
36
-------
Table 13 continued. SURVIVAL OF WILD STOCK FATHEAD MINNOWS WITH
LONG-TERM EXPOSURE TO HZS IN CHRONIC TESTS 2 AND 3
(expressed as percentage)
Exposure, H^S concentration,
Test days mg/1
Chronic 3 Control 0.0004 0.0010 0.0022 0.0078
series Jj. 9 89 100 100 100 100
37 67 89 89 80 60
66 67 89 78 80 60
177 56 89 78 80 50
345 56 89 67 70 50
37
-------
Table 14. SURVIVAL OF DULUTH STOCK FATHEAD MINNOWS STARTED
AS SAC FRY WITH LONG-TERM EXPOSURE TO H2S IN CHRONIC TEST 4
(expressed as percentage)
Exposure
Test days
Series a
28
56
84
Series b
28
56
84
Series c
28
56
84
, H S concentration,
me/1
Control
100
100
100
Control
100
100
100
Control
100
100
100
0.0010
100
100
100
0.0006
100
87
87
0.0010
100
93
93
0.0022
100
93
93
0.0016
100
100
100
0.0021
100
100
100
0.0052
100
100
100
0.0045
100
80
80
0.0046
100
100
100
a
—
—
0.0078
100
67
67
0.0102
100
20
20
3Test fish accidentally killed by low pH.
38
-------
in series a. was 0.0052 mg/liter H_S and no difference from controls was
noted in any H~S concentration. Survival in series b_ after 56 days was
lower than controls in 0.0045 and 0.0078 mg/liter H S, but no further
loss occurred up to 84 days. In series c^ survival was substantially
less than controls only at a concentration of 0.0102 mg/liter H-S.
Chronic test 6, with three replications was started with Duluth stock
sac fry and run for 297 days (Table 15). In the three replications
survival was similar to that in controls except at 0.0063 and 0.0068
mg/liter H~S where survival was reduced to 60% and 40%, respectively,
by the end of the test.
Mortality of eggs and fry from adults in 6, held in series <1 (Fry Bank)
for 80 days was not consistently different from controls in any H_S
concentration up to 0.0041 mg/liter.
Chronic test 6« was started with fish derived from spawning in test 6, ,
series d_, and held for 80 days at the same nominal concentrations. In
test 6« run with the same nominal concentrations, significant mortality
occurred above 0.0032 mg/liter H2S (Table 16). At 0.0080 mg/liter in
series a_, survival was 71% of control after 196 days; in series b^, 76%
of control at 0.0054 mg/liter after 274 days; and in series £, 52% of
control at 0.0074 mg/liter after 274 days. Maximum mortality at the
highest levels was reached in 56 to 140 days and did not increase there-
after. Mortality of eggs and fry generated in test 6~ and held for 80
days at same nominal concentrations (series d) varied from controls
substantially at 0.0050 mg/liter and to a lesser degree at 0.0018
mg/liter H2S.
Chronic 5 was conducted with three concentrations of H_S plus one con-
trol with 4 to 19 juveniles fish per tank (Table 17) for 112 days.
Fish were distributed on the basis of a random numbers table. The num-
ber of fish in the tanks did not affect the survival. At 0.0092 mg/liter
39
-------
Table 15. SURVIVAL OF DULUTH STOCK FATHEAD MINNOWS IN CHRONIC TEST
STARTED AS SAC FRY WITH LONG-TERM EXPOSURE TO H2S
(expressed as percentage)
Exposure
Test days
Series a
28
56
84
112
140
168
196
224
252
280
297
Series b
28
56
84
112
140
168
196
224
252
280
297
5
Control
100
100
100
100
100
100
100
100
100
100
100
Control
100
100
100
100
100
100
100
100
100
90
90
H~S concentration,
mg/1
0.0004
100
100
100
100
90
90
90
90
90
90
90
0.0004
90
90
90
90
90
90
90
90
80
80
80
0.0012
100
100
100
100
100
100
100
100
100
100
100
0.0011
80
80
80
80
80
80
80
80
80
80
80
0.0033
90
90
90
90
90
90
90
90
90
90
90
0.0026
100
100
100
100
100
100
100
100
100
100
100
0.0063
70
70
70
70
70
70
70
60
60
60
60
a
r>—
1—
—
__
40
-------
Table 15 continued. SURVIVAL OF DULUTH STOCK FATHEAD WINNOWS IN CHRONIC
£ SAC FRY WITH LONG-TERM ]
(expressed as percentage)
TEST 61 STARTED AS SAC FRY WITH LONG-TERM EXPOSURE TO
Exposure,
Test days
Series c
28
56
84
112
140
168
196
224
252
280
297
S eries d
80
Control
100
100
100
100
100
100
100
100
100
100
100
Control
55
H_S concentration,
mg/1
0.0005
100
100
100
100
100
100
100
100
100
100
100
0.0004
62
0.0012
100
100
100
100
100
100
100
100
100
100
100
0.0007
56
0.0033
100
100
100
100
100
100
100
100
100
90
90
0.0026
61
0.0068
70
70
70
70
70
70
70
70
60
50
40
0.0041
78
fTest fish, accidentally killed by low 0,,.
Series d. started with eggs from adults reared in 61 .
41
-------
Table 16. SURVIVAL OF FATHEAD MINNOWS STARTED AS JUVENILES FROM
CHRONIC TEST 6^^ EGGS WITH LONG-TERM EXPOSURE TO H2S
(expressed as percentage)
Exposure
Test days
Chronic 60
z
series a 28
56
84
112
140
168
196
224
274
series b
28
56
84
112
140
168
196
224
274
a
>
Control
100
100
100
100
100
100
100
100
100
Control
100
100
100
100
100
100
100
100
100
H_S concentration,
rng/1
0.0008
100
100
100
100
100
100
100
100
100
0.0006
100
100
100
100
100
100
100
100
90
0.0014
100
100
100
100
100
100
100
100
100
0.0012
100
100
100
100
100
100
100
100
90
0.0035
100
100
100
100
100
100
100
100
100
0.0032
100
100
90
90
90
90
90
90
80
0.0080
100
71
71
71
71
71
71
ob
0
0.0054
100
95
95
95
76
76
76
76
76
42
-------
Table 16 continued. SURVIVAL OF FATHEAD MINNOWS STARTED AS JUVENILES
FROM CHRONIC TEST 6.^ EGGS WITH LONG-TERM EXPOSURE TO H_S
(expressed as percentage)
Exposure ,
Test days
Chronic 60
series _c 28
56
84
112
140
168
196
224
274
Control
100
100
100
100
100
100
100
100
90
H S concentration,
mg/1
0.0007
100
100
100
100
90
90
90
90
90
0.0014
100
100
100
100
100
100
100
100
80
0.0044
100
100
100
100
100
c
—
—
0.0074
86
86
52
52
52
52
52
52
52
series d
@80
Control 0.0007 0.0009 Q.0018 0.0050
100 79 100 85 64
Exposure times do not include @80-day period of treatment in series d
bf V
Fish lost to disease.
°Fish lost to low 02.
43
-------
Table 17- SURVIVAL OF WILD STOCK JUVENILE FATHEAD MINNOWS WITH
DIFFERENT CONCENTRATIONS OF FISH AND H2S IN CHRONIC TEST 5
(expressed as percentage)
Test
Control
0.0017 mg/1 H2S
0.0041 mg/1 H2S
0.0092 mg/1 H2S
Exposure,
days
28
56
84
112
28
56
84
112
28
56
84
112
28
56
84
112
4
100
100
100
100
100
100
100
100
100
100
100
100
100
100
50
50
Number
6
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
83
of fish/ tank
10
100
100
100
100
100
100
100
100
100
100
100
100
100
90
90
90
14
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
93
19
100
100
100
100
100
100
100
100
100
100
100
100
95
95
95
95
44
-------
H S some loss occurred with the highest percentage loss in the tank with
the smallest number of fish.
Growth
Growth of wild stock juvenile minnows in various concentrations of H_S
was observed in chronic 2 and 3 (Table 18). After 107 days in chronic
2, series £i, growth was adversely affected at 0.0093 mg/liter; and in
series b_, at 0.0080 mg/liter H0S. In series a of chronic 3, fish grew
2.
approximately 33% less at 0.0198 mg/liter H-S than in control in 121
days but no reduction occurred at 0.0068 mg/liter. In series b_ of
chronic 3, percentage increment was not appreciably different from con-
trol at 0.0078 mg/liter H~S. At lower levels growth was greater than
in the control.
Chronic 4 was started with Duluth stock sac fry and carried for 84 days
(Table 19) . At termination in series _a_, growth was greater than con-
trols in all H_S concentrations. In series b_, growth was the same or
greater than control with concentrations of H»S up to 0.0045 mg/liter
but at 0.0078 mg/liter was lower. In series ^, growth was retarded at
0.0102 mg/liter H-S.
Chronic 5 was conducted to determine the effect of fish density in test
chambers on the growth rate in H^S (Table 20). Three concentrations of
H_S and one control with five densities of fish (4 to 19) were tested.
With 6 to 19 fish there were no consistent differences associated with
fish density in the various treatments. It was concluded that within
the fish density range tested, reaction to H«S was not affected by
number of fish per chamber.
In chronic 6, growth in the three series (a,b,c) was not closely related
j.
to ELS concentration except during the first part of the exposure (Table
21). After 112 days at 0.0063 mg/liter tLS in series a_, growth was less
than control but in series _c_ at 0.0068 mg/liter H2S, there was little
45
-------
Table 18. GROWTH OF WILD STOCK FATHEAD MINNOWS AT VARIOUS
CONCENTRATIONS OF H2S IN CHRONIC TESTS 2 AND 3
(expressed as mean weight in grams)
Exposure
Test days
Chronic 2
series a_ 0
51
79
107
series b
0
51
79
107
Chronic 3
series a
0
9
37
66
93
121
series b
0
9
37
66
93
121
, H?S concentration,
mg/1
Control
0.8933
1.347
2.162
2.227
Control
0.893a
1.100
1.784
1.943
Control
0.362
0.525
0.985
1.970
2.010
2.110
Control
0.417
0.635
1.098
1.560
1.620
1.770
0.0019
0,893
1.256
1.875
1.989
0.0015
0.893
1.200
1.800
1.826
0.0005
0.343
0.664
0.877
1.640
1.670
1.740
0.0004
0.477
0.830
1.121
2.290
2.470
2.480
0.0070
0.893
1.225
1.981
2.242
0.0048
0.893
1.165
1,669
1.866
0.0024
0.441
0.757
1.186
2.050
2.150
2.210
0.0010
0.488
0.980
1.439
2.400
2.600
2.630
0.0093
0.893
1.096
1.493
1.866
0.0060
0.893
1.342
1.825
1.945
0.0068
0.419
0.657
1.160
1.990
2.120
2.240
0.0022
0.454
0.849
1.164
1.980
2.190
2.260
0.0126
0.893
1.023
-
-
0.0080
0.893
1.138
1.729
1.786
0.0198
0.351
0.564
1.718
1.090
1.160
1.390
0.0078
0.384
0.538
0.971
1.620
1.690
1.820
Based on mean weight (g) of a random
test.
sample of the stock used to start
46
-------
Table 19. GROWTH OF DULUTH STOCK FATHEAD MINNOWS STARTED AS EGGS
IN CHRONIC TEST 4
(expressed as mean weight in grams)
Test
Series a
Series b
Series c
Exposure,
days
Control
56 0.416
84 0.652
Control
56 0.375
84 0.710
Control
56 0.421
84 0.747
H~S concentration,
mg/1
0.0010
0.392
0.710
0.0006
0.329
0.708
0.0010
0.379
0.678
0.0022
0.474
0.686
0.0016
0.368
0.690
0.0021
0.381
0.788
0.0052
0.386
0.674
0.0045
0.354
0.818
0.0046
0.337
0.664
a
—
—
0.0078
0.269
0.519
0.0102
0.165
0.438
Accidentally killed by low pH.
47
-------
Table 20. GROWTH OF JUVENILE FATHEAD MINNOWS IN CHRONIC TEST 5
WITH VARIED CONCENTRATIONS OF H-S AND DIFFERENT NUMBERS OF FISH IN
TEST CHAMBERS
(expressed as mean weight in grams)
Test
Control
0.0017 mg/1 H2S
0.0041 mg/1 H2S
0.0092 mg/1 H2S
Exposure,
days
28
56
84
112
28
56
84
112
28
56
84
112
28
56
84
112
4
0,76
1.31
1.80
2.52
0.80
1.45
1.95
2.65
0.96
1.78
2.16
2.70
1.12
1.64
2.80
3.65
Number
6
0.88
1.40
2.03
2.31
0.94
1.64
2.20
2.76
0.85
1.68
2.50
2.86
0.97
1.41
2.10
2.48
of fish/ tank
10
0.80
1.51
2.17
2.73
0.89
1.71
2.24
2.86
0.91
1.62
1.98
2.20
1.05
1.71
2.14
2.83
14
0.88
1.55
2.14
2.79
0.94
1.98
2.66
3.27
0.83
1.60
2.03
2.43
0.91
1.66
1.77
2.18
19
0.91
1.69
2.38
2.77
0.93
1.71
2.24
2.69
0.87
1.85
2.32
2.86
0.94
2.02
2.09
2.47
48
-------
difference from the control. In the second cycle of this experiment
described, as chronic 6~ growth after 56 days was not uniformly related
to tUS concentration up to 0.0032 mg/liter. Again, early effects were
overcome later.
Reproductive Success
Spawning success in chronic 6.. started with Duluth sac fry was not
changed consistently with H_S concentrations as measured by number of
eggs deposited per female or total number of spawnings (Table 22).
During the reproductive period the total number of eggs per female
varied from 180 to 2614 in 4 to 33 spawnings in different H S treat-
ments. Mean number of spawnings per female varied from 1.25 to 13.50.
Total eggs spawned per female fell off at 0.0068 mg/liter H_S in series
c^ because there was mortality during the progress of the experiment.
Some treatments had larger numbers of eggs per female than the controls.
In the second cycle, chronic 6« (Table 23), the number of eggs laid per
female was smaller in most cases than in the first cycle but there was
no consistent relationship between number of eggs spawned and H?S con-
centration. At treatments of E^S up to 0.0074 mg/liter in both chronic
6, and 6?, no trends in spawning and egg deposition were related to in-
creased concentrations of H~S. During test 6, there was a delay of
about 20 days in the start of spawning for all treatments as compared
with the controls. For test 6? there was a delay of about 40 days for
the highest H-S levels but the other treatments were equal to the
controls.
Time _tp_ Hatch
Eggs deposited in the three series (a_, b_, c) of chronic 6- and 62 were
hatched in the same H9S concentrations where they were deposited and
some were removed from treatments and hatched in control water (Table
24). No variation in days to hatch attributable to H_S concentration
during hatch was noted.
49
-------
Table 21. GROWTH OF DULUTH STOCK FATHEAD MINNOWS STARTED AS SAC FRY
IN AN EXPERIMENT COVERING TWO GENERATIONS
(expressed as mean weight in grams)
Test
Exposure,
days
H»S concentration,
mg/1
Chronic 6,
series a
Control 0.0004 0.0012 0.0033 Q.0063
28 0.102 0.120 0.118 0.110 0.105
56 0.445 0.411 0.398 0.339 0.337
84 0.704 0.711 0.584 0.782 0.549
112 1.335 1.237 1.094 1.281 0.985
series b
Control 0.0004 0.0011 0.0026
28 0.104 0.098 0.119 0.087
56 0.429 0.400 0.386 0.368
84 0.661 0.716 0.634 0.557
112 1.114 1.130 1.229 1.207
series c
Chronic 6,
i
series a
Control 0.0005 0.0012 Q.Q033 0.0068
28 0.111 0.114 0.110 0.110 0.112
56 0.497 0.452 0.364 0.482 0.394
84 0.690 0.641 0.575 0.809 0.671
112 1.274 0.978 0.908 1.067 1.230
Control 0.0008 0.0014 0.0035 0.0080
0 0.614 0.376C 0.630 0.54? 0.436
28 0.959 0.797 0.960 0.828 0.700
56 1.188 1.165 1.233 1.054 0.653
50
-------
Table 21 continued. GROWTH OF DULUTH STOCK FATHEAD MINNOWS STARTED
AS SAC FRY IN AN EXPERIMENT COVERING TWO GENERATIONS
(expressed as mean weight in grams)
Exposure,
Test days
Chronic 6.
series b
0
28
56
series c
0
28
56
Control
0.631
1.007
1.376
Control
0.602
0.928
1.183
^S concentration,
tng/1
0.0006
0.462C
0.916
1.354
0.0007
0.324°
0.685
0.983
0.0012
0.688
1.025
1.267
0.0014
0.628
0.895
1.166
0.0032
0.536
0.817
1.105
0.0044
0.519
0.790
1.078
0.0054
0.386
0.664
0.962
0.0074
0.352
0.600
0.875
.Fish accidentally killed by low O-.
Exposure times do not include @ 80-day period of treatment in d^ series
of
n .
I
The smaller mean size at day 0 for the lowest concentration was
probably due to overcrowding during exposure in the jl series of
(about 50% more fry were accidentally placed in this chamber) .
51
-------
Table 22. SPAWNING SUCCESS, EGGS PER FEMALE AND NUMBER OF SPAWNINGS
PER FEMALE IN DULUTH STOCK FATHEAD MINNOWS WITH CONTINUED EXPOSURE
TO LOW LEVELS OF H-S IN CHRONIC TEST 6^
Test
Series a
No . females
No. spawnings
Total eggs/tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
Series b
No . females
No. spawnings
Total eggs/tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
Series c
No . females
No. spawnings
Total eggs/tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
H~S concentration,
mg/1
Control
2
27
2591
96
13.50
1296
Control
5
44
8503
193
8.80
1701
Control
4
21
1900
90
5.25
475
0.0004
4
22
2638
120
5.50
660
0.0004
4
33
10456
317
8.25
2614
0.0005
5
25
4221
169
5.00
844
0.0012
2
9
853
95
4.50
426
0.0011
3
16
2417
151
5.33
806
0.0012
4
5
722
144
1.25
180
0.0033
2
19
3023
159
9.50
1512
0.0026
4
33
7215
219
8.25
1804
0.0033
3
8
1591
199
2.67
530
0.0063
3
20
4037
202
10.50
1346
a
—
—
—
—
—
—
0.0068
1
4
399
100
4.00
399
Fish all killed by low 0«,
52
-------
Table 23. SPAWNING SUCCESS, EGGS PER FEMALE AND NUMBER OF SPAWNINGS
PER FEMALE IN DULUTH STOCK FATHEAD MINNOWS WITH CONTINUED EXPOSURE
TO LOW LEVELS OF H2S IN CHRONIC TEST 62
Test
Series a
No. females
No. spawnings
Total eggs /tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
Series b
No . females
No . spawnings
Total eggs /tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
Series c
No. females
No . spawnings
Total eggs /tank
Eggs /spawning
Mean spawnings /female
Mean eggs /female
H2S concentration,
mg/1
Control
7
27
3164
117
3.86
452
Control
6
27
4301
159
4.50
717
Control
8
62
11691
188
7.75
1461
0.0008
4
28
3625
129
7.00
906
0.0006
3
11
603
55
3.67
201
0.0007
7
62
11588
186
8.86
1655
0.0014
6
21
2675
127
3.50
445
0.0012
4
3
482
160
0.75
120
0.0014
6
40
3446
86
6.67
574
0.0035
4
13
1336
102
3.25
334
0.0032
5
23
3480
151
4.60
696
__b
~—
—
—
—
~
a
~
—
—
—
—
—
0.0054
2
2
189
94
1.00
94
0.0074
3
28
3774
134
9.33
1258
all killed by low 02.
All fish died of disease at start of spawning.
53
-------
Table 24. NUMBER OF DAYS TO HATCH OF FATHEAD MINNOW EGGS DEPOSITED
IN VARIOUS CONCENTRATIONS OF H2S AND HATCHED IN THE
SAME CONCENTRATION OR IN CONTROL WATER
Test
Test
conditions'
~S concentration,
me/1
Chronic 6-
series a
series b
series c
Chronic 6~
series a
series b
series c
T— C
T--T
T— C
T— T
T--C
T— T
T— C
T--T
T— C
T — T
T— C
T— T
T— C
Control
6.75
6.75
Control
6.75
6.75
Control
6.00
6.00
Control
5.25
5.25
Control
5.75
5.75
Control
5.75
5.75
0.0004
6.75
6.50
0.0004
6.25
6.25
0.0005
6.35
6.25
0.0008
5.75
5.25
0.0006
5.50
5.00
0.0007
5.25
5.25
0.0012
6.50
6.50
0.0011
6.75
—
0.0012
7.00
—
0.0014
6.00
5.50
0.0012
5.00
5.00
0.0014
6.00
5.50
0.0033
6.35
6.35
0.0026
6.75
6.75
0.0033
6.00
6.00
0.0035
5.50
5.75
0.0032
6.25
5.25
0.0044
—
—
0.0063
8.00
6.50
__b
—
—
0.0068
6.00
6.00
0.0080
6.00
—
0.0054
6.50
6.00
0.0074
7.50
5.50
T—T designates eggs laid in H2S and incubated in the same concentra-
tion; T—C designates eggs laid in treatment and hatched in control
water.
Fish killed by low Q~.
54
-------
Survival to Hatch and Length of Fry at Hatch
Percentage survival of eggs to hatch in controls and H S treatments
varied from 80 to 92 in chronic tests 6- and 6~ (Table 25)„ Eggs laid
and hatched in the same H9S concentration did not survive at different
rates in different treatments nor when hatched in control water during
chronic 6,. In chronic 6~ eggs laid in H2S and incubated in control
water in most cases had lower survival than those laid and incubated
in the same H~S treatment. Survival rate was not directly related to
H«S concentration during incubation.
The length of fry at hatch did not vary significantly with H?S con-
centration during incubation in either chronic 6.. or &„ (Table 26) .
Lengths and weights of adults at termination are given in Tables 27
and 28. No differences attributable to H2S concentrations were noted.
COMPARISON OF FATHEAD POPULATIONS
Experimental Design
The possibility that different populations of fathead minnows might
react differently to H~S was tested by determining acute toxicity of
H-S to fish from four wild populations and two artificially reared
fathead minnow populations (Table 29). The four wild populations of
the same year class were collected at three different seasons (fall,
winter and spring) from upper midwest lakes which varied widely in
chemical characteristics (Table 30). The wild stocks were taken in
the fall near the end of the first growing season, in January during
the winter period of low dissolved oxygen concentration, cold tempera-
ture and maximum concentration of dissolved solids, and in June when
the fish with least resistance to overwintering stresses had been elimi-
nated and before spawning activity began to interfere with bioassay
results. The wild populations were all considered to be natural with
independent genetic characteristics.
55
-------
Table 25. SURVIVAL TO HATCH OF FATHEAD MINNOW EGGS LAID IN VARIOUS
CONCENTRATIONS OF H2S AND INCUBATED IN THE
SAME CONCENTRATIONS OR IN CONTROL WATER
(expressed as percentage)
Test
Test
conditions'
H»S concentration,
mg/1
Chronic 6,
series a
series b
series c
T—T
T--C
T—T
T—C
T—T
T___P
\j
Control 0.0004 0.0012 0.0033 0.0063
67
80 47 88
80 47 88 43
Control 0.0004 0.0011 0.0026
81
54
72
73
54
66 43
66 43
Control 0.0005 0.0012 0.0033 0.0068
59 58 91 73 90
59 51 62 78 92
Chronic 6,
series a
series b
series c
T—T
T—C
T—T
T—C
T—T
T—C
Control 0.0008 0.0014 0.0035 0.0080
70 74 57 82 69
70 66 45 62
Control 0.0006 0.0012 0.0032 0.0054
68 76 27 68 82
68 65 58 53 57
Control 0.0007 0.0014 0.0044 0.0074
73 68 62 — 66
73 52 46 — 44
T--T designates eggs laid in H~S and incubated in same concentration;
T — C designates eggs laid in treatment and hatched in control water.
Fish killed by low 0 .
56
-------
Table 26. LENGTH OF FATHEAD MINNOW FRY AT HATCH FROM EGGS LAID IN
VARIOUS CONCENTRATIONS OF H2S AND INCUBATED IN THE
SAME CONCENTRATION OR IN CONTROL WATER
(expressed in millimeters)
Test
Test
conditions'
~S concentration,
me/1
Chronic 6,
series a
series b
series c
Chronic 6~
series a
series b
series c
T— T
T— C
T— T
T— C
T— T
T — C
T — T
T— C
T— T
T— C
T— T
T— C
Control
5.5
5.5
Control
5.2
5.2
Control
5.4
5.4
Control
5.4
5.4
Control
5.6
5.6
Control
5.4
5.4
0.0004
5.4
5.4
0.0004
5.3
5.3
0.0005
5.2
5.3
0.0008
5.5
5.3
0.0006
5.5
5.5
0.0007
5.6
5.6
0.0012
5.5
5.3
0.0011
5.3
—
0.0012
5.2
—
0.0014
5.5
5.5
0.0012
5.3
5.4
0.0014
5.5
5.6
0.0033
5.2
5.4
0.0026
5.4
5.4
0.0033
5.4
5.1
0.0035
5.6
5.5
0.0032
5.5
5.4
0.0044
—
""•
0.0063
5.4
5.4
b
—
—
0.0068
5.4
5.6
0.0080
5.4
—
0.0054
5.9
5.9
0.0074
5.5
5.4
aT—T designates fry from eggs laid and incubated in same treatment;
T—C, fry from eggs laid in H2S and incubated in control water.
Fish killed by low 0.
57
-------
Table 27. LENGTH OF FATHEAD MINNOW ADULTS AT TERMINATION
OF CHRONIC TESTS f>^ AND 62
(millimeters total length)
Test
Chronic
series
series
series
Chronic
series
series
H^S concentration,
me/1
61
a
x
X
X
b
x
X
X
c
X
X
X
Control
length
length
length
-. a
males
females
M & F
70
59
65
.8
.8
.2
Control
length
length
length
males
females
M & F
78
57
63
.2
.0
.1
Control
length
length
length
males
females
M & F
76
54
66
.0
.8
.6
0.0004
72
54
63
.9
.6
.8
0.0004
72
57
63
.7
.2
.9
0.0005
69
55
61
.8
.2
.7
0.0012
73
56
70
.2
.8
.0
0.0011
73
58
67
.5
.2
.8
0.0012
73
55
66
.3
.2
.1
0.0033
73.6
55.5
68.4
0.0026
71.5
54.9
58.2
0.0033
78.0
55.5
66.8
0.0063
74.2
56.5
63.6
b
—
—
0.0068
70.5
—
70.5
62
a
x
x
x
b
Control
length
length
length
males
females
M & F
72
.2
59.9
63.6
Control
0.0008
71
53
.7
.5
64.4
0.0006
0.0014
70
56
62
.8
.5
.2
0.0012
0.0035
66.4
54.8
61.8
0.0032
0.0080
— —
—
—
0.0054
x length males
x length females
x length M & F
72.2 71.8 70.0 67.0 72.6
57.3 51.8 54.8 54.9 52.8
63.3 67.3 64.9 59.4 67.6
58
-------
Table 27 continued. LENGTH OF FATHEAD MINNOW ADULTS AT
TERMINATION OF CHRONIC TESTS 6^ AND &2
(millimeters total length)
Test
Chronic 62
series c
H-S concentration.
mg/1
Control 0.0007 0.0014 0.0044
0.0074
x length males
x length females
x length M & F
71.5 71.0 72.8 — 69.8
56.9 55.4 54.2 — 54.7
58.5 58.8 61.2 — 62,2
,"-- fish lengths reported as total length.
Fish killed by low 00.
59
-------
Table 28. WEIGHT OF FATHEAD MINNOW ADULTS AT TERMINATION
OF CHRONIC TEST 6^
(grams wet weight)
H?S concentration,
Test
Series
Mean weight males
Mean weight females
Mean weight M & F
Control 0.0004 0.0012 0.0033 0.0063
4.054 4.145 4.058 4.221 4.552
2.155 1.370 1.612 1.522 1.529
3.105 2.757 3.569 3.450 2.738
Series
Mean weight males
Mean weight females
Mean weight M & F
Control 0.0004 0.0011 0.0026
5.184 5.201 4.293 4.257
1.574 1.655 1.788 1.388
2.605 3.175 3.353 1.962
Series _c
Mean weight males
Mean weight females
Mean weight M & F
Control 0.0005 0.0012 0.0033 0.0068
4.760 3.758 4.479 5.774 3.980
1.495 1.466 1.452 1.181
3.303 2.485 3.268 3.478 3.980
HFish killed by low 09.
60
-------
The artificially reared stocks came from fish reared in the National
Water Quality Laboratory at Duluth, Minnesota and from fish reared in
the fishery laboratories at the University of Minnesota. Both stocks
originated from ponds at the Newtown Laboratory and presumably from the
same gene pool. Prior to acute toxicity tests, minnows collected in the
fall and spring were acclimated to 20 C and from winter collections to
14 C, the test temperatures, for at least 10 days prior to the start of
tests. Prophylactic treatments with 20 mg/liter neomycin were carried
out in the acclimation tanks immediately after collection of each group.
Acute bioassays were conducted in the diluter systems previously des-
cribed for acute fathead minnow tests with juveniles. Four treatments
and one control were used in each test. The pH was held at 7.7 and
dissolved oxygen at 5-6 mg/liter. Sulfide concentrations were measured
in the center of each test chamber twice each day and other determina-
tions were made once daily. Bioassays were continued until 48 hr elapsed
without additional mortality. All tests were replicated three times.
Acute Toxicity
Fall collections from the four lakes had mean threshold LCSO's at 20 C
from 0.023 mg/liter in Wakefield Lake (Table 31) to 0.029 mg/liter H2S
in Hay Lake (Table 32). The fall Wakefield Lake sample had an abnormally
high mortality rate during acclimation, which may account for its rela-
tively low resistance to H~S (Table 34). Winter collections could be made
only from Harrier Lake (Table 33) and Hay Lake. These fish showed
greater resistance than fish collected in the fall with LTC at 14 C of
0.038 mg/liter in Harrier Lake and 0.032 mg/liter H2S in Hay Lake (Table
34). This discrepancy is consistent with the effect of test temperature
on H-S toxicity shown in Table 4. There were no winter collections from
Porter Lake. Samples of wild fish collected in the spring of 1973 were
all in healthy condition. Mean LTC's at 20 C varied between lakes from
0.028 to 0.030 mg/liter H2S.
The two groups of cultured fish varied little between themselves with
61
-------
Table 29. SOURCE OF FATHEAD MINNOWS USED FOR
POPULATION COMPARISON TESTS
Test No.
A1F
A2F
A3F
A4W
A5W
A6W
A7S
ASS
A9S
B1F
B2F
B3F
B4S
B5S
B6S
GIF
C2F
C3F
C4W
C5W
C6W
Source
Harrier
T137N R71W
Kidder Cty, ND
Harrier
ii
it
Harrier
it
ii
Wakefield
T29N R22W
Ramsey Cty, MN
Wakefield
ti
it
Hay
T32N R20W
Washington Cty, MN
Hay
ii
u
Date
9/21/72
u
u
1/7/73
IT
fl
5/30/73
ii
ii
10/16/72
u
u
5/23/73
it
ii
10/25/72
u
ii
2/2/73
u
it
Temperature, Collection
C method
11.5 Seine
M it
u it
Ice cover Trap
u it
it it
16.0 Seine
M II
It II
9.5 Seine
tt it
it tt
18 . 5 Seine
ii u
u M
6.0 Trap
u ii
ii ii
Ice cover Trap
u ii
ii M
62
-------
Table 29 continued. SOURCE OF FATHEAD MINNOWS USED FOR
POPULATION COMPARISON TESTS
Test No.
C7S
CSS
C9S
C10S
D1F
D2F
D3F
DAS
D5S
D6S
E1S
E2S
E3S
F1S
F2S
CIS
G2S
G3S
Source Date
Hay 5/31/73
M M
ii ii
ii ii
Porter 10/26/72
T118N R30W "
Meeker Cty, MN "
Porter 5/22/73
tl 11
11 It
U of M 6/27/73
ii
ii it
Newtown, Ohio 8/15/73
ii M
Duluth NWQL 8/31/73
ii ii
ii ii
Temperature, Collection
C method
22.0 Seine
it it
it (i
ii ii
6.5 Seine
ti it
ti U
20.0 Seine
n u
if ft
25.0
•'
ii _
26. Oa
it _
24.0
»
tt _
On receipt by air freight.
63
-------
Table 30. LAKE WATER CHEMISTRY'
(concentrations in mg/1)
Compound
or element
Total hardness as CaCCL
Total alkalinity as CaCO
Total phosphorus
Ammonia nitrogen
Organic nitrogen
Sulfate
Chloride
Manganese
Iron
Calcium as CaCOn
Sodium
Potassium
Magnesium as CaCO«
Fluoride
Specific conductivity
Hmho/cm @ 25 C
Hay
9
14
0.10
0.05
2.4
5.1
3
0.05
0.27
7
3
2
2
0.1
27
Wakefield
40
46
0.08
0.20
1.4
6.4
48b
0.01
0.06
32
48b
27b
8
0.1
200
Porter
190
200
0.11
0.17
1.9
8.5
8
0.01
0.06
93
8
6
97
0.1
280
Harrier
250
450
0.24
0.05
1.1
430
22
0.07
0.35
30
310
46
220
0.1
1500
Water samples taken with fall collection of fish; analyses by Minn.
Dept. of Health.
High concentrations probably due to wintertime salting of residential
streets near the lake.
64
-------
Table 31. ACUTE TEST CONDITIONS AND LC50 VALUES FOR FATHEAD MINNOWS
IN POPULATION COMPARISON (WAKEFIELD LAKE)
cn
Test No.
BlFa
B2F
B3F
B4Sb
B5S
B6S
Days from
collection
to start
of test
16
29
38
30
37
37
Mean
length,
mm
28.5
28.5
33.0
43.5
47.0
49.5
Mean test
conditions
Temp . ,
C
20.1
20.1
20.2
20.1
19.9
20.0
0
mg/1
5.5
5.8
5.6
5.5
5.5
5.6
24 hr 48 hr
0.033
-
0.032
0.037 0.028
0.036 0.035
0.032
LC50,
mg/1 H0S
72 hr
0.025
-
0.023
0.026
0.031
0.030
96 hr
0.031
0.024
0.022
x =
0.026
0.029
0.030
x =
LTC (days)
0.024 (9)
0.024 (6)
0.022 (7)
0.023
0.026 (6)
0.029 (6)
0.030 (6)
0.028
Fall collections.
Spring collections.
-------
Table 32. ACUTE TEST CONDITIONS AND LC50 VALUES FOR FATHEAD MINNOWS
IN POPULATION COMPARISON (HAY LAKE)
Si
Test No.
GIF3
C2F
C3F
C4Wb
C5W
C6W
C7S°
C8S
C9S
Days from
collection Mean
to start length,
of test mm
39
64
80
57
75
75
46
55
55
37.0
38.5
41.5
50.0
51.5
50.5
46.5
50.5
51.5
Mean test
conditions
Temp . , 02 ,
C me/1
19.9
20.0
20.0
13.9
14.0
13.8
20.0
20.0
19.9
5.7
5.5
5.6
5.9
5.8
5.9
5.7
5.5
5.7
LC50,
mg/1 H.,8
24 hr 48 hr 72 hr
0.037 0.032
0.033
0.040 0.035
0.065 0.052 0.043
_
0.045
0.038 0.035
0.041 0.036
0.042 0.036
96 hr
0.032
0.030
0.028
X
0.039
0.041
0.040
X
0.032
0.033
0.031
X
LTC (davs)
0.032 (5)
0.029 (7)
0.027 (7)
= 0.029
0.032 (11)
0.032 (11)
= 0.032
0.032 (6)
0.029 (7)
0.029 (7)
= 0.030
?Fall collection (CF) .
Winter collection (CW).
°Spring collection (CS).
-------
Table 33. ACUTE TEST CONDITIONS AND LC50 VALUES FOR FATHEAD MINNOWS
IN POPULATION COMPARISON (HARRIER LAKE)
05
-g
Test No.
AlFa
A2F
A3F
A4Wb
A5W
A6W
A7S°
A8S
A9S
Days from
collection
to start
of test
41
54
63
44
44
82
40
40
47
Mean
length ,
tnni
33.0
32.0
36.0
45.0
43.5
47.5
49.0
45.5
50.0
Mean test
conditions
Temp . ,
C
19.9
20.1
20.0
14.0
13.9
13.9
20.1
20,0
20.0
mg/1
5.6
5.6
5.4
6.1
6.4
6.3
5.4
5.6
5.7
24 hr 48
-
0.
0.
0.
0.
0.039 0.
0.040 0.
0.039 0.
hr
-
056
031
071
045
033
032
035
LC50,
mg/1 H«S
72 hr
0.033
0.027
0.029
0.061
0.066
0.043
0.030
0.032
0.031
96
0.
0.
0.
0.
0.
0.
0.
0.
0.
hr
028
027
026
x =
059
059
043
x =
029
030
031
x =
LTC (days)
0.026
0.027
0.026
0.026
0.037
0.042
OJ336
0.038
0.029
0.030
0.031
0.030
(9)
(6)
(7)
(13)
(13)
(12)
(6)
(6)
(6)
Fall collection (AF).
Winter collection (AW).
Spring collection (AS).
-------
Table 34. ACUTE TEST CONDITIONS AND LC50 VALUES FOR FATHEAD MINNOWS
IN POPULATION COMPARISON (PORTER LAKE)
00
Test No.
DlFa
D2F
D3F
D4Sb
D5S
D6S
Days from
collection
to start
of test
37
62
78
15
15
30
Mean
length ,
mm
35.0
39.0
42.5
46.0
47.5
43.5
Mean test
conditions
Temp . ,
C
20.1
20.0
20.1
20.1
19.9
20.0
0
2'
mg/1
5
5
5
5
5
5
.7
.3
.7
.6
.7
.6
24 hr 48
0.
-
0.
0.041 0.
0.
0.040 0.
hr
047
-
040
036
038
034
LC50,
me/1 H0S
£-
72 hr
0.032
0.033
0.030
0.032
0.035
0.032
96 hr
0.028
0.028
0.028
X
0.031
0.031
0.031
X
LTC (days)
0.027
0.028
0.026
= 0.027
0.030
0.029
0.031
= 0.030
(6)
(6)
(8)
(7)
(7)
(6)
Fall collections.
Spring collections.
-------
mean LTC's ranging from 0.018 to 0.019 tag/liter H2S (Table 35). These
acutely toxic levels were substantially lower, however, than the LTC
levels of the wild populations (Table 36). Reasons for this discrepancy
in resistance appear to be related either to continued laboratory cul-
ture in which less resistant fish were held in the gene pool or genetic
differences in the original Newtown stock. In work reported above where
wild eggs were brought into the laboratory and hatched and reared to
juveniles under laboratory conditions, the fish had essentially the same
resistance as wild juveniles from the same stock. If disease in the fall
Wakefield sample is taken into account, the results described here indi-
cate that various wild stocks, in the limited geographic area covered by
these experiments, do not have significantly different resistance to H_S.
Temperature variations and the season when fish are collected have more
influence than difference in genetic stock.
SUMMARY
Acute tests of H_S with fathead minnows show that 96-hr LC50 ranged from
0.0280 mg/liter at 25 C to 0.5800 mg/liter at 4 C. Wild stock from
various lakes in Minnesota and North Dakota varied in response with season
and temperature but not with geographic location. Laboratory-reared fish
from Minnesota stock did not vary from wild stock but laboratory-reared
fish from Duluth (Newtown) culture stock were more than twice as sensitive
at 20 C as local wild stock (96-hr LC50 of 0.0162 and 0.0367 mg/liter
HLS, respectively).
Chronic tests (time periods greater than that required for LTC) with wild
stock showed survival was affected adversely at 0.0078 mg/liter H?S and
higher. Growth of wild stock was inhibited at 0.0080-0.0093 mg/liter and
higher and Duluth stock at 0.0052 mg/liter H2S and higher when tests were
started with juveniles. When all life history stages were exposed,
growth of Duluth stock was reduced at 0.0033 mg/liter H-S and higher.
Fecundity in chronic exposure from egg to spawning adult did not appear
to be related to H S concentration up to 0.0070-0.0080 mg/liter although
delay in first spawning occurred at the higher test levels.
69
-------
Table 35. ACUTE TEST CONDITIONS AND LC50 VALUES FOR FATHEAD MINNOWS
IN POPULATION COMPARISON (DULUTHr-NEWTOWN STOCK)
Days from
collection Mean
to start length,
Test No?
E1S
E2S
E3S
F1S
F2S
CIS
G2S
G3S
of test
51
51
58
32
32
10
10
21
nsni
40.5
41.0
42.0
39.5
37.5
41.0
41.5
46.5
Mean test
conditions
Temp . ,
C
20.0
19.9
20.0
20.0
20.0
20.1
20.0
20.0
0 ,
ma/1
5.4
5.8
5.5
5.4
5.7
5.5
5.8
5.6
24 hr 48 hr
0.028 0
0.030 0
-
0.032 0
-
0
0
0
.020
.026
-
.020
-
.024
.025
.029
LC50,
ma/1 EnS
72 hr
0.018
0.022
0.022
0.018
-
0.019
0.023
0.021
96 hr
0
0
0
0
0
0
0
0
.017
.019
.021
x =
.017
.022
x =
.018
.022
.020
x =
LTC (days)
0.017
0.018
0^.020
0.018
0.017
0.021
0.019
0.017
0.020
£.019
0.019
(6)
(6)
(8)
(6)
(6)
(7)
(7)
(7)
a
Eseries- University of Minnesota stock; F series - Newtown stock; G series - Duluth stock.
-------
Table 36. LTC VALUES OF FATHEAD MINNOWS FROM SEVEN POPULATIONS
Population
Harrier Lake
Fall
Winter
Spring
Wakefield Lake
Fall
Spring
Hay Lake
Fall
Winter
Spring
Porter Lake
Fall
Spring
University of
Minnesota stock
Newtown stock
Duluth stock
Test
temperature ,
C
20
14
20
20
20
20
14
20
20
20
20
20
20
LTC (days),
mg/liter H0S
t-
0.026 (6-9)
0.038 (12-13)
0.030 (6)
0.023 (6-9)
0.028 (6)
0.029 (57)
0.032 (11)
0.030 (6-7)
0.027 (6-8)
0.030 (6-7)
0.018 (6-8)
0.019 (7)
0.018 (7)
71
-------
SECTION VI
GOLDFISH
(Carassius auratus (Linnaeus))
The response of goldfish to H S was tested by (1) a series of acute
bioassays at various temperatures on a strain of goldfish from a
commercial hatchery and a second strain from a federal hatchery, (2)
a series of chronic bioassays, and (3) a series of acute bioassays to
determine factors affecting bioassay variability.
ACUTE TESTS
Experimental Design
Acute bioassays of H-S were conducted on the various life history stages
of the goldfish. A total of one test with eggs, one with newly hatched
fry, and 112 with juveniles were run. Of the juvenile tests, 29 were
to determine temperature effects, 20 oxygen effects, and 63 bioassay
method effects.
The egg bioassays were conducted using the modified proportional diluter
previously described. A control and four toxicant concentrations were
dispensed from the diluter into a two-chambered glass container. The
incoming water and sodium sulfide stock solution flowed into a 20 x 20
x 10 cm deep mixing chamber and then through a perforated glass tube
into a 10 x 10 x 5 cm deep test chamber. The eggs were not moved by
the flow of water. Eggs were artificially spawned from a stock of
adult goldfish obtained from Ozark Fisheries, Inc., Stoutland, Missouri.
72
-------
Adults were raised from 11 to 20 C in 2 days to initiate spawning and
eggs from four females were hand stripped. Eggs were fertilized with
milt from excised testes of four males. They were exposed to H«S from
4 hr after fertilization through hatching.
One 96-hr bioassay with fry was conducted using the modified propor-
tional diluter. Fry test chambers were 7.5 x 7.5 x 10 cm deep with
three glass sides and bottom and one Nitex (nylon) screened side. The
water flowed from the mixing chamber into the fry chamber, then out
through the screen into an aquarium in which the chamber was immersed.
Fry were not fed during the bioassay and were not ready to accept food
until the last day of the test.
Acute bioassays with juveniles for determination of the effect of tem-
perature on H~S toxicity were conducted in two identical continuous-flow
units in which gaseous H9S was mixed with well water in an apparatus
1 2
described by Colby and Smith with modifications by Adelman and Smith.
Each unit included six 25-liter glass test chambers (one control and
five treatments), measuring 40 x 25 x 28 cm deep. With a flow rate of
280 ml/min, 90% replacement of water occurred in approximately 1-1/4 hr.
Two stocks of goldfish from Ozark Fisheries, Inc. and three stocks from
the Federal Hatchery at Lake Mills, Wisconsin were tested for tempera-
ture effects during a 16-month period. Variation in fish age in dif-
ferent bioassays was from approximately 4 months to 2-1/2 years, and
the range in mean weights was from 1.40 to 14.63 g. Within any bioassay
the age, weight, and stock of fish was the same.
Fish from both sources were treated with 0.86 mg/liter methylene blue
for 2 days with an additional 0.86 mg/liter added on the third day to
control skin fluke (Gyrodactylus sp.) infections. The fish were held
in the solution for 5 days. Fish held in the laboratory were retreated
periodically when the incidence of flukes increased. Fish were not
used for bioassay until at least 2 weeks after treatment.
73
-------
One week prior to a bioassay 65 fish from each stock to be tested were
removed from holding tanks where temperatures ranged from 10 to 13 C
and placed in a 38-liter acclimating aquarium and then transferred to
test chambers. Oxygen concentration was maintained near saturation and
water flow was continuous. Temperatures were raised or lowered from that
of the holding room to the desired bioassay temperature at the rate of
4 C per day, and held at test temperature at least 3 days prior to the
start of the test. Fish were fed Oregon moist pelleted trout food until 1
day prior to the bioassay but not during test. In each bioassay 10
fish were placed in each chamber after random stratified assortment.
Hydrogen sulfide was then raised to the desired concentration within a
period of 3 hr. Mortality was recorded at 24-hr intervals.
The two units used for temperature series were also used for bioassays
with different oxygen levels. The test chambers for oxygen tests
were acrylic plastic egg-hatching jars (depth 45 cm, diameter 14.5 cm,
volume of water 6.1 liters). Water replacement was 90% in approximately
50 min.
Five stocks of goldfish from Ozark Fisheries, Inc., all hatched during
May and June, 1970, were used for bioassays to determine effects of
oxygen. Age of fish ranged from 6 to 17 months and mean weights from
3.95 to 6.06 g. During the holding period skin flukes were treated
for 1 hr on 2 consecutive days with 0.25 mg/liter formalin. Bioassays
were conducted not less than 10 days after treatment.
Tests were conducted in pairs with a different oxygen concent*ation in
each of the two. One week before the start of a test, 100 fish were
divided between two 38-liter aquaria for acclimation. Oxygen was held
at saturation when temperature acclimation only was required. Accli-
mation to specified oxygen concentrations was achieved by continuous
withdrawal of water from a reaeration system described by Brungs.
Oxygen concentrations were adjusted to the desired level at intervals
74
-------
over 24 hr and temperature was raised from 10 to 11 C to 17.5 C in 2
days for both groups. After desired levels were attained, fish were
held for 1 week before start of test.
At the start of a pair of bioassays fish were assigned in a stratified
random manner, eight per chamber, to five treatments and one control
in which the desired oxygen and H^S concentrations had previously been
set. Transfer was done as rapidly as manipulation permitted.
Sixty-three bioassays were conducted to determine factors affecting
bioassay variability. In this series three proportional diluters were
used to deliver water to three series of four treatments and one control.
Test chambers measured 50 x 25 x 20 cm and contained 20 liters. With
a flow rate of 445 ml/min, 90% replacement of water occurred in approxi-
mately 1-3/4 hr.
This experiment was conducted in a 3 x 3 factorial design with three
temperatures (14, 20, and 26 C), three acclimation times (1, 3, and
5 weeks), and seven blocks (all fish were from the same source but
seven different batches). Approximately every 8 weeks a new shipment
of goldfish was obtained from Ozark Fisheries. Six of these stocks
were hatched in May and June, 1970 and one in May, 1971. Their age
at time of bioassay ranged from 6 to 15 months, and mean weights
ranged from 3.17 to 5.70 g. On arrival in the laboratory the tempera-
ture was lowered from that of the shipment water (15-23 C) to the
holding tank temperature (10-13 C) within 24 hr, and on the second and
third day after arrival the formalin treatment described above was
applied. Three days later 500 of the fish were divided into three
groups, and each group was placed in a 400-liter acclimation tank. The
temperature was raised to the desired levels of 14, 20, and 26 C at the
rate of 5 C per day.
After 1, 3, and 5 weeks from the start of acclimation approximately
one-third of the stock in each tank was removed for an 11-day bioassay
75
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of H9S at the acclimation temperature. The fish from each group were
placed in the five chambers of the appropriate diluter in a stratified
random manner. The desired ELS concentrations had been attained in
these chambers and fish were not fed during the entire bioassay. Hydro-
gen sulfide, pH, and temperature were measured once per day and dis-
solved oxygen once per week.
Acute Toxicity
Eggs and Fry—In the egg bioassay the mean pH reading was 7.62 (range
7.55-7.73); mean temperature 22.1 C (range 22.0-22.2); and mean dis-
solved oxygen 8.72 mg/liter (range 8.25-9.25).
The percent survival at hatch decreased in the two highest H2S concen-
trations (Table 37) with 90% loss at 0.0291 mg/liter H2S. The LC50 at
hatching computed from percentage of all survivals corrected for
Q
mortality in controls (Abbott ) was 0.022 mg/liter HLS and for normal
fry only 0.020 mg/liter. In the two highest concentrations where egg
mortality occurred, a greater percentage of malformed fry were observed
(Table 37), with 62% at 0.0291 mg/liter H2S.
In the fry bioassay the mean pH was 7.66 (range 7.52-8.08); mean tem-
perature 21.6 C (range 19.8-22.2); and mean oxygen was 8.29 mg/liter
(range 8.15-8.50).
Fry survival was considerably less in the two highest H-S concentrations
(Table 38) with complete kill at 0.0485 mg/liter. The 96-hr LC50 was
0.025 mg/liter. The length of fry at the termination of the bioassay
was less in all H S treatments and decreased with increasing tLS. A
t-test indicated that the length of fry in the lowest H7S concentration
was significantly different than the control (t = 2.3448).
Juveniles—Temperature effects—In the 29 bioassays to determine the
effects of temperature on H2S toxicity the mean pH reading was 7.80
76
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Table 37. PERCENTAGE SURVIVAL OF GOLDFISH EGGS AND PERCENTAGE
MALFORMED FRY IN VARIOUS HS CONCENTRATIONS
H2S (mg/1) - mean
range
No. surviving fry
Survival (%)
Malformed fry (%)c
Control 0.0052 0.0097 0.0169 0.0291
(0.0044- (0.0075- (0.0124- (0.0241-
0.0066) 0.0112) 0.0197) 0.0357)
68 75 76 58 8
85 94 95 72 10
0 1 0 14 62
Percentage of total fry.
Table 38. PERCENTAGE SURVIVAL OF GOLDFISH FRY AND MEAN LENGTH OF FRY
AT END OF EXPOSURE TO VARIOUS H^S CONCENTRATIONS
(mg/1) - mean
range
Control 0.0140 0.0207 0.0271 0.0485
(0.0056- (0.0092- (0.0131- (0.0213-
0.0187) 0.0282) 0.0350) 0.0593)
No. surviving fry
Survival (%)
Mean length (mm)
50
100
6.83
47
94
6.54
47
94
6.53
13
26
5.53
0
0
—
50 fry at start of test in each treatment,
77
-------
(range 7.63-7.99; standard deviation, 0.097), the mean dissolved oxygen
was 5.8 mg/liter (range 4.8-7.1; standard deviation 0.68), and mean
total alkalinity was 237 mg/liter CaC03 (range 225-262; standard devi-
ation, 7.3).
The 96-hr LC50 ranged from 0.556 mg/liter at 6.7 C to 0.044 mg/liter at
25 C (Table 39). The log of the LC50 increases proportionally to a
decrease in the log of temperature (Figure 2). The linear regression
is described by the equation:
log y = 4.2325 - 1.8527 log x (1)
where y = 96-hr LC50
x = temperature.
The regression is highly significant (F 25 = 254.86, p<.01), and 91%
of the variation in LC50 is attributable to temperature (Steel and
Torrie ). The regression line for the goldfish is compared with that
for fathead minnows and with that for the uniform stocks of goldfish in
the tests of bioassay variability reported below (Figure 3). The in-
crease in toxicity of H~S with increasing temperatures is similar for
the three groups with all goldfish more tolerant than the fathead
minnows and the uniform stock of goldfish more tolerant than the mixed
stocks. Since the three points defining the regression line for the
uniform group are the means of 21 bioassays (described in the following
section), they are more precise than any single point defining the
regression for the mixed stock where a maximum of six bioassays was
conducted at an individual temperature. The regression line for the
uniform stock indicates that at least with this group the effect of
temperature is slightly curvilinear on a logarithmic plot.
78
-------
Table 39. LC50 OF H2S TO GOLDFISH AT DIFFERENT TEMPERATURES
Test3
LM-10
LM-17
0-9
0-16
0-7
LM-8
0-4
0-2
LM-3
0-5
LM-6
0-18
0-1
0-20
LM-19
LM-14
0-15
LM-23
LM-27
LM-24
0-12
LM-13
0-11
0-25
LM-26
LM-28
0-21
LM-22
Temperature,
C
25.0
24.9
24.8
24.6
24.2
24.0
23.1
23.0
23.0
23.0
23.0
22.9
20.3
20.0
19.8
17.0
17.0
15.1
15.0
13.9
12.4
12.3
12.2
10.2
10.2
8.8
6.7
6.7
Mean length
of fish,
mm
37.5
60.3
28.8
40.9
25.7
39.1
1.77
14.0
27.8
22.8
27.6
51.6
17.5
57.8
66.0
47.2
33.0
88.7
144.0
109.8
35.1
50.5
34.9
116.1
134.4
146.3
69.9
75.2
Mean 96-hr
LC50,
mg/1 H,,S
£.
0.044
0.065
0.037
0.049
0.034
0.050
0.051
0.057
0.063
0.050
0.054
0.050
0.048
0.061
0.057
0.094
0.053
0.118
0.110
0.193
0.175
0.276
0.133
0.271
0.286
0.241
0.556
0.375
0 - Ozark stock; LM - Lake Mills stock
79
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7 8 9 10 12 14 16 18 20 30
TEMPERATURE C
Figure 2. The effect of temperature on the 96-hr LC50 (TL50) of H S
to goldfish with 95% confidence limits.
80
-------
800
600
CO
OJ
X
^ 200
o>
fo 100
1-
oc
o
I
CD
80
60
40
20
i i i i I_L
i i i
6 8 10 15 20 30 40
TEMPERATURE C
Figure 3. The effect of temperature on the 96-hr LC50 (TL50) of H S to
three groups of fish: (A) fathead minnow, (B) goldfish from
bioassays on temperature effects, (C) goldfish from bioassays
for determination of variability in LC50 caused by various
factors.
81
-------
Since the relationship of the 96-hr LC50 to temperature is logarithmic,
increases in tolerance at the colder temperatures are larger than at
higher temperatures. A 10 C change from 7 to 17 C increases toxicity
about 5.5 times, but a 10 C increase from 15 to 25 C increases toxicity
about 2.6 times. Sprague reviewed the literature concerning the
effect of temperature on acute fish mortality from various toxicants
and noted that increased mortality with increased temperatures was
quite common. Increases in toxicity with higher temperatures are
related to exposure time. Sprague points out that at lower tempera-
tures a slower mortality rate early in a bioassay will not always
result in an overall decrease in toxicity. The relationship of H2S
toxicity to temperature in goldfish expressed in Figure 2 is for 96-hr
tests. In the 11-day bioassays reported below the differences in LC50
values between tests conducted at 14, 20, and 26 C decreased with time,
but there was a significant difference between each temperature at
every time interval (Figure 4). At 11 days the toxicity curves at each
temperature appear to have reached an asymptote implying little sub-
sequent change.
Oxygen effects—In the 21 bioassays with varied oxygen concentrations
the mean pH reading was 7.69 (range 7.64-7.80), the mean temperature
was 17.46 C (range 17.0-17.8), and the mean total alkalinity was 229
mg/liter CaC03 (range 205-245).
In bioassays without prior oxygen acclimation of fish the mean 96-hr
LC50 was 0.071 mg/liter H2S at 6.0 mg/liter 02 and 0.053 mg/liter H_S
at 1.5 mg/liter 0_. In tests with acclimation to test oxygen concen-
tration, the mean 96-hr LCSO's were 0.062 and 0.048 mg/liter H?S at the
same oxygen concentrations. In eight of ten pairs, the bioassay con-
ducted at the lower 0 concentration resulted in a lower 96-hr LC50
12 1 9
(Table 40). Shelford , Colby and Smith , and Adelman and Smith found
this same effect in other species although the latter authors found no
effect of oxygen differences on toxicity of H2S to northern pike eggs.
82
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oo
eo
300 -
C/3
(M200
X
^ ISO
o 100
LO
h-
80
60
14 C
20 C
26 C
I
I
J I
i i i
3
D
4 5
Y S
6
8
10 12
Figure 4. Changes in LC50 (TL50) values during 11 days of exposure to tLS at three temperatures
with 95% confidence limits.
-------
Table 40, EFFECT OF OXYGEN ON H2S TOXICITY TO QOLDFISR
IN PAIRED BIOASSAYS
Bioassay
No.
1A
IB
2A
2B
3A
3B
4A
4B
10A
10B
12A
12B
5A
5B
6A
6Bb
7A
7B
8A
8B
9A
9B
Mean acclima- Mean oxygen
tion oxygen, in bioassay, 96-hr LC50,
me/1 ms/1 me/1 H^S
With prior oxygen acclimation
2.88 3.41
4.83 4.97
2.13 1.96
6.07 6.13
1.25 1.05
4.16 4.36
3.08 3.07
6.55 6.13
1.39 1.00
4.06 4.16
1.50 1.40
2.84 2.29
Without prior oxygen acclimation
Sat.a 1.18
" 4.28
1.95
- -
1.81
5.72
2.81
6.31
" 1.08
4.83
0.051
0.058
0.058
0.063
0.070
0.054
0.055
0.066
0.095
0.049
0.046
0.050
0.044
0.053
0.055
_
0.049
0.080
0.066
0.069
0.060
0.065
bSaturation. Dissolved oxygen varied from approximately 9-11 mg/1
Bioassay not completed due to apparatus failure.
84
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In two bioassays (10A, 3A, Table 40) where the fish were acclimated to
the 0- concentration of the bioassay, the 96-hr LCSO's were higher than
all but one in the unacclimated series. These two values were elimi-
nated because they deviated considerably from the other values and since
they occur at very low 09 concentrations they may be indicative of a
separate phenomenon. With these two values eliminated, the relationship
of the 96-hr LC50 to the 0- concentration forms the significant linear
regression F = 9.78, p<.05 (Steel and Torrie ):
J_, o
y = .0443 + 2.83 x (2)
where y = 96-hr LC50
x = oxygen concentration
(Figure 5) . The linear regression from bioassays without oxygen accli-
mation is also significant (F 7 = 8.09, p<.05):
-Lj '
y = .0465 + 4.08 x (3)
where y and x are the same as above (Figure 5) . There is a considerable
variation among points around both regression lines since in the former
only 55% and in the latter 54% of the variation in the 96-hr LC50 is
attributable to 0~ concentration. The rate of increase in H2S toxicity
with decreasing oxygen is the same with or without prior acclimation as
was shown by an analysis of covariance (F ^ = .52, n.s. at .05) (Snede-
13 '
cor and Cochran ) .
The analysis also indicated that the elevation of the regression line
for bioassays without acclimation was significantly higher than that
for bioassays with acclimation (F ,, = 5.23, p<.05). Acclimation to
lower oxygen concentration prior to testing does not increase the resis
tance of goldfish to acute H?S toxicity but makes them more sensitive.
85
-------
CO
O
m
oo
O5
cr
ID
O
X
Figure 5.
100
80
60
40
80
60
CD
CD 40
<£>
A
0
•T B
1234567
DISSOLVED OXYGEN (mg/l)
The effect of oxygen on the 96-hr LC50 (TL50) of H,S to goldfish: (A) with oxygen
acclimation prior to the bioassay and (B) without prior oxygen acclimation. The
circled points are not included in the regression calculation, and 95% confidence
limits are spaced at uniform intervals.
-------
However, the actual difference in elevation of the regression lines is
small and since there is a great variation in individual points, accli-
mation to oxygen does not affect H2S toxicity appreciably, except at
very low oxygen concentrations.
Tests of Bioassay Variability
In the series of tests of bioassay variability the means and standard
deviations for temperature, pH meter readings, oxygen, and total alka-
linity for 21 bioassays at each temperature were:
Diluter 1 Diluter 2 Diluter 3
Temperature (C) 14.1 + 0.12 20.1 + 0.12 25.9 + 0.10
pH meter reading 7.65+ 0.02 7.64+ 0.04 7.65+ 0.05
Oxygen (mg/liter) 8.04+0.43 7.96+0.40 7.56+0.31
Total alkalinity (mg/liter) 216.0 +17 210.0+9 158.0+9
A summary of the LC50 values for these juvenile goldfish is presented
in Table 41. Mean 4-day LCSO's at 14, 20, and 26 C were 0.145, 0.083,
and 0.063 mg/liter, respectively, and mean 11-day LCSO's were 0.084,
0.071, and 0.060 mg/liter H.S, respectively.
The 3x3 factorial design of the experiment in variability permitted
analysis for differences due to acclimation time, temperature, and fish
stocks. Since the pH and fish size may affect the toxicity of H-S and
could not be held precisely constant in different bioassays, an analysis
13
of covariance (Snedecor and Cochran ) with pH and weight of the fish
as covariates was used for both the 4-day and 11-day LC50 values (Table
42).
There was a significant difference in 4-day and 11-day LC50 values in
tests conducted at different temperatures: F2 ^ = 167.27 (p^.Ol) for
the former and F ., = 39.23 (p 4.01) for the latter (Figure 4). No
z, 44
significant difference in the 4-day or 11-day LC50 values was noted in
87
-------
Table 41. EFFECT OF TEMPERATURE, ACCLIMATION TIME, AND GOLDFISH STOCK
ON THE 96-HR AND ll-DA^ LC50
(mg/liter HS)
Temp-
era-
ture,
c
Accli-
mation
time.
weeks 1
Fish stock
234567
14
0.120 0.081 0.080 0.082 0.094 0.080 0.073
(0.162) (0.125) (0.156) (0.142) (0.124) (0.118) (0.129)
0.085 0.078 0.079 0.079 0.103 0.087 0.075
(0.126) (0.150) (0.132) (0.163) (0.195) (0.152) (0.180)
0.082 0.068 0.066 0.080 0.096 0.099 0.076
(0.163) (0.140) (0.132) (0.134) (0.155) (0.150) (0.112)
20
26
0.076 0.065 0.077 0.078 0.075 0.057 0.066
(0.081) (0.076) (0.080) (0.083) (0.082) (0.086) (0.068)
0.079 0.074 0.073 0.084 0.067 0.055 0.052
(0.115) (0.098) (0.089) (0.092) (0.081) (0.068) (0.071)
0.081 0.076 0.072 0.079 0.084 0.061 0.069
(0.087) (0.082) (0.077) (0.088) (0.094) (0.076) (0.076)
0.054 0.052 0.056 0.055 0.062 0.064 0.050
(0.054) (0.057) (0.058) (0.064) (0.076) (0.071) (0.050)
0.065 0.052 0.051 0.061 0.060 0.084 0.046
(0.071) (0.052) (0.053) (0.063) (0.064) (0.084) (0.055)
0.063 0.063 0.065 0.056 0.066 0.072 0.055
(0.066) (0.064) (0.068) (0.057) (0.066) (0.072) (0.059)
96-hr LC50 in parentheses.
-------
Table 42. ACUTE MORTALITY OF GOLDFISH AT THREE
TEMPERATURES EXPRESSED AS 96-HR AND 11-DAY LC5Q£
(rag/liter HS)
96-hr LC50
Mean
Range
Coefficient of variability
11-day LC50b
Mean
Range
Coefficient of variability
14
0.145
0.112-0.195
14%
0.084
0.066-0.120
15%
Temperature , C
21
0.083
0.068-0.115
13%
0.071
0.052-0.084
13%
26
0.063
0.050-0.084
14%
0.060
0.046-0.084
14%
^Results from 21 bioassays at each temperature.
The 11-day LC50 is equivalent to the lethal threshold concentration at
20 and 26 C and approaches the LTC at 14 C.
89
-------
bioassays conducted after 1, 3, or 5 weeks of temperature acclimation
with the 4-day LC50's, F£ ^ = 2.56 (n.s. at .05) and with the 11-day
LC50's, F9 = .76 (n.s.'at .05). Brett showed that acclimation of
6, ) H-'t
goldfish to increased temperature as measured by their thermal tolerance
proceeded at different rates depending on the range in which the in-
crease occurred. An increase from 4 to 12 C required 20 days for accli-
mation to the higher temperature whereas the same increase from 20 to
28 C required 3 days. An increase from 12 to 20 C required 7 days of
acclimation. This rise is similar to that in one group of fish of the
present study where temperature was increased from approximately 12 to
20 C. Since these goldfish should require 7 days to acclimate in accor-
dance with Brett's data, it might be expected that those fish raised
from 12 to 26 C would take longer with a resulting increase in LCSO's
after the third or fifth week of acclimation. Fish stocks 1, 2, and
3 tested at 26 C became more tolerant to EJ5 after 3 or 5 weeks of accli-
mation, but stocks 4-7 did not show this trend (Table 41). Stocks 4-7,
obtained April-September, experienced seasonal mean temperatures ranging
from approximately 12 to 26 C in the supplier's holding pond, and stocks
2 and 3S obtained in January and February, were exposed to pond tem-
peratures of about 4-7 C. This exposure to cold temperatures may have
resulted in a longer acclimation time in the experiments. Stock ls
obtained in November, was exposed to pond temperatures of about 13 C.
This stock would be expected to respond as stocks 4-7 with no increased
acclimation time, but increased time was required.
The analysis of covariance indicated that there was no significant dif-
ference between the 4-day LC50 values for the seven stocks of fish
(Fg ^ = 1-57; n.s. at .05), but there was a significant difference
between the 11-day LC50 values (Fg ^ = 4.98, p^.Ol). Stocks that are
different in their overall resistance to a toxicant after 11 days may
have similar rates of mortality during the earlier period of bioassay.
Handling and sudden exposure to the toxicant may have increased the
initial rates of mortality sufficiently in these bioassays to mask dif-
90
-------
ferences between stocks, and thus longer exposure was requried to show
real differences.
To test the effect of temperature on the variability of the results,
the coefficient of variability was computed from the 4-day and 11-day
LC50's for each temperature. At 14, 20, and 26 C the coefficient of
variability of the 4-day LC50 was 14, 13, and 14%, respectively, and of
the 11-day LC50 was 15, 13, and 14%, respectively, indicating no essen-
tial difference in the variability due to temperature (Table 42).
Summary of Acute Tests
The increased resistance of the goldfish to H_S at lower temperatures is
probably of adaptive advantage. In the summer naturally occurring H»S
is generally restricted to the hypolimnion of lakes, so that fish may
escape to the upper water strata. However, in shallow ice-covered lakes
H~S may be found at most all depths and may contribute to winter fish
kills (Scidmore ). Under these conditions the goldfish could survive
higher H~S concentrations than in the summer when escape would be
possible. If H~S persists for a long period during the winter, dele-
terious sublethal effects may occur, but present data does not permit
any predictions of effects of temperature.
Since H?S usually occurs only at relatively low oxygen concentrations,
its toxicity at these levels may have important environmental signifi-
cance. However, the rate of increased toxicity with lowered oxygen is
not very great, with the 96-hr LC50 reduced only about 26% between oxygen
concentrations of 6 and 1 mg/liter. Furthermore, the difference in H-S
concentrations between bioassay chambers where all or none of the fish
died was small. A mean H9S concentration of 0.087 mg/liter caused 100%
mortality and a mean of 0.036 mg/liter caused 0% mortality. Since com-
plete mortality or survival occurs within a narrow range of H»S and since
the rate of decreased resistance with lowered oxygen is small, the oxygen
concentration will not have an important effect on H~S toxicity in the
91
-------
environment. When H2S concentrations are greater than about 0.036
mg/liter at 17.5 C some goldfish mortality will probably occur regard-
less of the oxygen concentration.
The analysis of factors that could affect bioassay variability shows
that prior acclimation of goldfish to the oxygen concentration of the
bioassay had little effect on acute H2S toxicity. One week of accli-
mation to temperatures of 14, 20, and 26 C appeared adequate. However,
goldfish that experience different thermal histories than the stocks
tested may require a longer time to acclimate. Fish held in winter
conditions and tested at high temperatures and fish held in summer
conditions and tested at low concentrations should be acclimated for a
longer period of time.
The toxicity of H?S to different stocks of fish was not the same although
early mortality rates were similar. It is believed that this difference
may relate to the season of year when fish were tested rather than
genetic differences.
Although the range in 4-day and 11-day LCSO's was greater at colder
temperatures, the coefficient of variability was essentially the same
at 12, 20, and 26 C. Therefore, variability of bioassay results is not
affected by temperature in that range.
CHRONIC TESTS
Experimental Design
Three chronic bioassays with goldfish were conducted, two at the same
time and in the same apparatus but with different stocks of fish and a
third separate bioassay. The bioassays were designated Chronic 1-0,
Chronic I-LM, and Chronic II. The three bioassays were conducted in
the modified proportional diluter. In Chronic II the control and four
treatment chambers were galvanized metal stock watering tanks painted
with an asphalt-aluminum roof coating. The tanks were 122 x 45.7 x 30.5
92
-------
cm deep and fitted with a drain to keep the water level at 21.6 cm con-
taining 123 liters. The same tanks were used in Chronic I but were
divided by a longitudinal glass partition so that two stocks of fish
were kept separately in one tank. A flow-splitter divided the incoming
water equally between each side.
The goldfish tested in Chronic 1-0 were obtained from Ozark Fisheries,
Inc. The fish were 18 months old at the start of the bioassay, and
their mean weight was 2.96 g (range 1.56-5.30). Fish in Chronic I-LM
were obtained from the federal hatchery at Lake Mills, Wisconsin. They
were 6 months old and weighed 3.56 g (range 1.80-6.36) at the start.
The Lake Mills and Ozark fish were assigned in a stratified random
manner to the appropriate section of each experimental chamber, 20 fish
per chamber. After approximately 4 months fish were randomly thinned,
Ozarks to 13 and Lake Mills to 14 fish per tank. The fish obtained from
thinning were tested for resistance to acute malathion and acute ELS
toxicity. When fish began active spawning, artificial tropical fish
spawning grass made of plastic strands entwined in a stainless steel
wire was provided to each tank. All fish were fed Oregon moist trout
pellets ad libitum three times each day.
The Chronic I bioassay lasted 294 days and a cyclic temperature regime
was used. The initial temperature of approximately 14 C was held for
1 month, then raised to 19 C for 3 months, raised to about 22 C for 2
months, lowered to about 13 C for 1-1/2 months, and finally raised to
19 C for 2 months. 'The H9S and dissolved oxygen concentrations varied
slightly between the Lake Mills and Ozark sections of the tank, probably
due to differences in the fish's metabolism or flow characteristics
(Table 43) . The extreme range of Or. resulted from conditions occurring
only occasionally.
Chronic II was conducted for 430 days starting with eggs. Four male and
four female 2-year-old Ozark goldfish were artificially spawned. Five
93
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Table 43. TEST CONDITIONS IN GOLDFISH CHRONIC I
Lake Mills Goldfish
H2S (mg/1) - mean Control 0.002 0.005 0.010 0.025
std. dev. - 0.0009 0.0022 0.0050 0.0105
pH - mean 7.61 7.51 7.52 7.56 7.57
range 7.10-8.01 7.20-7.96 7.01-7.92 7.10-8.03 7.12-8.01
Temperature (C)-mean 18.5 18.4 18.9 18.5 18.7
range 12.8-24.0 12.8-23.7 13.2-24.1 12.8-23.8 13.0-23.9
Dissolved 0~ (mg/1)
mean 6.33 5.31 5.24 5.23 5.54
range 2.60-10.55 2.20-9.90 2.00-9.45 2.30-9.90 2.75-10.95
Total alkalinity
(mg/1 CaC03) -mean 184 184 184 184 184
range 162-230 162-230 162-230 162-230 162-230
Ozark Goldfish
E~S (mg/1) - mean
std. dev.
pH - mean
Control
-
7.61
0.002
0.0010
7.51
0.005
0.0023
7.52
0.010
0.0047
7.56
0.028
0.0125
7.57
range 7.10-8.01 7.02-7.96 7.01-7.92 7.10-8.03 7.12-8.01
Temperature (C) -mean 18.5 18.4 18.9 18.5 18.7
range 12.8-24.0 12.8-23.7 13.2-24.1 12.8-23.8 13.0-23.9
Dissolved 02 (mg/1)
mean 6.65 4.96 4.84 5.31 5.39
range 2.65-10.40 2.20-9.85 1.35-9.35 2.35-9.75 2.75-10.80
Total alkalinity
(mg/1 CaC03) -mean 184 184 184 184 184
range 162-230 162-230 162-230 162-230 162-230
94
-------
hours after fertilization 20 fertile eggs from each spawning were
placed in each experimental chamber for a total of 80 eggs. The egg
chambers are described in the section on acute bioassays of eggs. On
the third day after hatching the fry were thinned to 50 per tank. Since
complete mortality of eggs occurred in the highest H2S concentration,
25 fry from each of the two lowest H_S concentrations were placed in
this chamber. Young fry were fed egg yolk, brine shrimp, and ground
Glencoe trout pellets. Fry chambers at this time were glass tanks
30 x 15 x 30 cm deep, containing 10 liters. On the 33rd day after
hatching the fish were again thinned and transferred, 30 per chamber,
to a 38-liter glass aquarium. After 125 days all fish were again trans-
ferred to the stock watering tanks. From this time on the fish were fed
Oregon moist trout pellets of various sizes, ad libitum. After 304 days
a final thinning left 15 fish per chamber.
A constant temperature of approximately 22 C occurred for all but 15
days late in the bioassay when the temperature was lowered to approxi-
mately 13 C, then raised again in an attempt to induce spawning. The
chemical conditions in Chronic II are summarized in Table 44.
Eggs and Fry
Data on eggs and fry were obtained in Chronic II. The effect of H S on
eggs was reported in the acute bioassay section (Table 37). No increased
mortality over controls occurred at 0.0052 and 0.0097 mg/liter H~S, but
at 0.0169 mg/liter mortality was 13% greater and at 0.0291 mg/liter, 75%
greater than controls. These latter two H-S concentrations also caused
malformed fry at hatching, 14% at 0.0169 mg/liter and 62% at 0.0291
mg/liter (Table 37).
Since nearly all eggs had died at the highest H S concentration, the
fry tested at this concentration were hatched from eggs in the two
lowest concentrations. No mortality of fry occurred at any H~S concen-
tration tested (Table 44).
95
-------
Table 44. TEST CONDITIONS IN GOLDFISH CHRONIC II
H2S (mg/1) - mean Control 0.007 0.009 0.015 0.024
std. dev. - 0.0029 0.0072 0.0056 0.0186
pH - mean 7.62 7.59 7.57 7.63 7.63
range 7.38-8.02 7.40-8.02 7.33-8.18 7.39-8.10 7.30-7.99
Temperature (C)-mean 21.4 21.4 21.5 21.6 21.4
range 12.4-25.7 12.4-26.0 12.3-25.7 12.3-26.0 12.4-25.7
Dissolved 02 (mg/1)
mean 5.93 5.95 5.47 6.13 6.62
range 2.05-9.00 2.00-9.25 2.85-9.65 2.50-9.05 2.00-8.25
Total alkalinity
(mg/1 CaC03)-mean 194 194 194 194 194
range 178-225 178-225 178-225 178-225 178-225
96
-------
Growth Rate
The goldfish were weighed at monthly intervals in the three chronic
bioassays. At termination of Chronic I-LM, only the fish in the highest
concentration weighed less than the controls and intermediate concen-
trations stimulated growth (Table 45). The no-effect level of H?S on
growth was between 0.010 and 0.025 mg/liter. In Chronic 1-0 inter-
mediate levels also stimulated growth with only the highest H_S con-
centration weighing less than the control at termination (Table 45).
The level of no effect with the Ozark fish was between 0.010 and 0.028
mg/liter H S.
In Chronic II the mean weights decreased at all increasing H_S concen-
trations before thinning at 308 days and at termination, 430 days
(Table 45). The analysis of variance indicated a significant difference
at both times, F. n00 = 8.34, p <.01, at the earlier time and F. ,0 =
H , J-JO *t , DO
5.28, p<..01, at the final weighing. The test of least significant
difference showed that the level of effect occurred at one lower H«S
concentration by the end of the test than at 308 days. The lowest con-
centration significantly different than the control was 0.0616 mg/liter
H2S at 308 days, but after 430 days it was 0.009 mg/liter. The no-
effect level was between 0.007 and 0.009 mg/liter H2S (Table 46).
Reproduction
Spawning occurred only in the Chronic 1-0 bioassay. The largest number
of spawnings and spawnings per female occurred at 0.005 mg/liter H-S
(Table 47). No spawning occurred at 0.002 mg/liter, but there was only
one immature female in that tank. One spawning per female occurred in
the control, but there were only two females in this tank compared to
four in the three highest treatments. Fish in the two highest treat-
ments spawned at a reduced rate compared to spawning at 0.005 mg/liter
even though the tanks contained the same number of females. It appears,
therefore, that the safe level for optimum frequency of spawning is
between 0.005 and 0.010 mg/liter HS (Table 47).
97
-------
Table 45. WEIGHTS OF GOLDFISH IN THREE CHRONIC BIOASSAYS
(mean weight in grams)
Time of Days from
weighing start
Chronic I-LM
Start
Before thinning
End
Chronic 1-0
Start
Before thinning
End
Chronic II
Before thinning
Before thinning
End
0
120
294
0
120
294
30
308
430
0
3.36
13.41
38.09
0
2.75
11.99
32.28
0
0.12
42.03
78.68
H~S concentration,
mg/liter
0.002
3.72
15.74
48.34
0.002
2.93
14.98
36.68
0.007
0.10
40.64
67.05
0.005
3.47
14.77
46.60
0.005
2.91
13.84
33.14
0.009
0.13
34.76
56.81
0.010
3.51
14.36
46.70
0.010
3.10
14.10
38.97
0.015
0.10
30.94
54.76
0.025
3.74
11.39
31.40
0.028
3.09
10.91
26.77
0.024
0.09
20.32
34.85
98
-------
Table 46. H2S CONCENTRATIONS NOT AFFECTING GOLDFISH ADVERSELY
DETERMINED FROM THREE CHRONIC BIQASSAYS
(mg/liter)
Bioassay
Basis of
determination
Highest Lowest level
level of of
no effect measured effect
Chronic I-LM
Final weight
0.010
0.025
Chronic 1-0
Final weight
Spawnings per female
a
0.005
a
0.010
Chronic II
Estimate from
Egg survival
Weight after 308 days
Final weight
all effects
0.010
0.009
0.007
0.005
0.017
0.016
0.009
0.009
^o significant difference between highest level of no effect and lowest
level of measured effect.
Table 47. REPRODUCTION OF GOLDFISH IN CHRONIC 1-0
H0S concentration, mg/liter
Number of
Number of
Number of
males
females
spawnings
0
9
2
2
0.002
12
1
0
0.005
9
4
12
0.010
8
4
2
0.025
7
4
3
99
-------
Since the goldfish scattered their eggs among the artificial grass
strands and since non-spawning fish ate many eggs, it was not possible
to make an accurate estimate of fecundity. Attempts to determine sur-
vival of spawned eggs and fry were unsuccessful due to technique and
apparatus malfunction.
Other Chronic Effects
The fish from Chronic 1-0 that were removed after thinning at 120 days
were subjected to two acute treatments. Five fish from each H-S con-
centration were subjected to 1.0 mg/liter malathion for 96 hr to deter-
mine if chronic exposure to H.S affected their tolerance to another
toxicant. The level of malathion was ineffective since no mortality
occurred among any goldfish.
After this treatment the same fish remained in pure well water for 3
days and then were subjected to 0.322 mg/liter H-S until all fish died
by 135 hr of exposure. The time of death of each fish was recorded.
It appeared that those fish exposed to the highest H?S concentration
were the least resistant to an acutely toxic H_S concentration (Table
48), however an analysis of variance indicated no significant difference
SUMMARY
In acute tests egg survival decreased at 0.017 mg/liter H~S and above.
The LC50 at hatching was 0.022 mg/liter. LC50 at 96 hr for fry was
0.025 mg/liter. Ninety-six-hr LC50 for juveniles varied from 0.556
mg/liter at 6.7 C to 0.044 mg/liter at 25 C. At different oxygen levels
96-hr LC50 varied from 0.071 at 6.0 mg/liter 0 to 0.053 mg/liter H2S
at 1.5 mg/liter 0« at 17.5 C. In chronic tests the level of H_S not
adverse to growth is between 0.007 and 0.009 mg/liter H S. The number
of spawnings per female was not affected between 0.005 and 0.010 mg/liter
H2S°
100
-------
Table 48. TIME TO DEATH OF GOLDFISH FROM VARIOUS CHRONIC
CONCENTRATIONS EXPOSED TO AN ACUTELY LETHAL
H2S CONCENTRATION OF 0.322 MG/LITER
(hours)
Order of
mortality
1
2
3
4
5
0
76
83
103
122
124
508
Chronic H~
0.002
100
106
127
127
127
587
S concentration,
0.005
79
102
106
116
135
538
mg/liter
0.010
88
89
114
115
126
532
0.025
84
90
94
101
102
471
101
-------
SECTION VII
BLUEGILL
(Lepomis macrochirus Rafinesque)
Toxicity of H2S to bluegills was determined by (1) a series of acute
tests on eggs, fry, juveniles and adults, (2) an acute test on juve-
niles acclimated to H2S, and (3) a series of chronic and partial
chronic tests.
ACUTE BIO AS SAY
Experimental Design
Acute toxicity tests were run on bluegill eggs, sac fry, juveniles,
and adults. A total of two tests on green eggs, one on newly hatched
sac fry, one on 35-day-old fry, eight on young-of-the-year (yy), and seven
on adult bluegills were conducted (Table 49). Green eggs were stripped
in the laboratory from adults taken in the field. Eggs were fertilized
and tests were started on the same day. Sac fry were incubated at 22 C
from eggs stripped and fertilized in the laboratory and were placed in
test chambers within 24 hr after hatching. Thirty-five-day-old fry
were reared in the laboratory from natural spawning in laboratory tanks.
Fry were fed mashed hard boiled egg yolk, brine shrimp, ground beef
liver, Glencoe dry granules, and ground minnows. Juvenile fish were
seined in the field, brought to the laboratory, and treated for 3 con-
secutive days with 20 rag/liter neomycin, then with 2.65 mg/liter methy-
lene blue for the following 3 days. All fish were held at 21 C and
fed Oregon moist pellets, Glencoe dry fry granules, brine shrimp, and
ground pork liver prior to tests. Adults were secured in the field,
102
-------
Table 49. SOURCE OF BLUEGILLS AND STAGE OF FISH USED
FOR ACUTE TESTS WITH H^S
Test Stage
BE-1 Green egg
BE-3 Green egg
BF-2 Sac fry
BF-4 35-day fry
Lmyy-1 yy
-2
-3
-4
-5
-7
-8
-9
Lma-1 Adults
-3
Lma-4
-5
-6
-7
-10
Date
col-
lected
1/7/71
13/7/71
13/7/71
26/9/68
ii
"
7/10/69
"
"
3/10/69
8/11/68
16/10/68
11
22/1/69
"
10/2/73
30/9/70
Water temp-
erature at
collection, Method of
C capture
24
23
23
20
"
"
17
"
n
18
6
17
"
4
"
"
18
Adults by
hook and
Adults by
hook and
Adults by
hook and
Seine
"
n
ti
n
n
n
Trap net
n n
n M
Source
Carnelian Lake
line
Pleasant Lake
line
Pleasant Lake
line
Laboratory
reared
Medicine Lake
ii ••
n n
II M
II II
M II
II II
Laboratory
reared
Crystal Lake
n n
n n
Hook and line " "
n ti n
M M n
Trap net
n n
n n
n n
103
-------
brought to the laboratory, and treated prophylactically as described
above for fingerlings, held at 13 C, fed Oregon moist pellets, Glencoe
dry pellets, live minnows, fresh and frozen minnows, and ground pork
liver. Adult fish were held 7 days at test temperature prior to test.
On the basis of preliminary acute tests, H2S concentrations for acute
tests were set to bracket the probable LC50 value. Green egg tests had
two controls and ten H9S concentrations. One adult test, designated as
Lma-7 (Table 49), also had two controls and ten H2S concentrations. All
other tests were conducted with one control and five H_S concentrations.
The light regime was 12 hr of light and 12 hr of darkness through the
period of the test.
All acute tests were of the flow-through type with flushing water and
toxicant provided for one control and five treatment chambers from
apparatus described by Colby and Smith and modified by Adelman and
2
Smith. This system used deoxygenated water and H~S gas to introduce the
toxicant to the test chambers. Acrylic test chambers for eggs and fry
were similar to those described by Colby and Smith. Young-of-the-year
fish were tested in 36-liter slate-bottomed glass-walled aquaria, 6-
liter round-bottomed acrylic hatchery jars (15 x 44 cm), or in 25-liter
glass aquaria constructed with silicone glue. Adult fish were tested
in the 36-liter aquaria described above. Tests designated as BE-1, BE-3,
and Lma-7 had two controls and ten H^S concentrations. During the acute
test period all test chambers were subjected to 12 hr of light and 12 hr
of darkness. Fish were not fed during the first 96 hours of test but were
fed thereafter when the test duration was longer.
Acute Toxicity
Green Eggs—The two green egg tests (BE-1 and -3) were run at 21.9 C
and 6.1 and 5.9 mg/liter 02> respectively (Table 50). The eggs in the
test run at 6.1 mg/liter 02 hatched in 66 hr with an LC50 of 0.0162
mg/liter H2S. The second test run at 5.9 mg/liter 02 hatched in 77 hr
with an LC50 of 0.0125 mg/liter H2S. A mean LC50 (66-72 hr) for the two
104
-------
tests was 0.0144 mg/liter H^S.
Sac Fry—Sac fry were tested at 21.7 C and 5.8 mg/liter 0~ 4 days after
egg fertilization and within 24 hr after hatch (BF-2). The 96-hr LC50
was greater than 0.0292 mg/liter H2S (Table 50). After 9 days the LC50
was 0.0169 mg/liter H2S. Advanced feeding fry were tested at 21.8 C
and 6.0 mg/liter 02 39 days after egg fertilization (BF-4). The 96-hr
LC50 was 0.(
(Table 50).
LC50 was 0.0086 mg/liter and after 8 days was 0.0084 mg/liter H S
Juveniles—Seven tests were run on juvenile bluegills ranging in size
from 3.2 to 5.3 cm (Table 50). Temperatures at which the tests (Lmyy
4-9) were conducted varied from 20.1 to 19.9 C, with 0~ ranging from 5.7
to 6.6 mg/liter. The 96-hr LC50 values varied from 0.0290 to 0.0325
mg/liter H_S with a mean value for these tests of 0.0316 mg/liter. One
test (Lmyy-7) run for 8 days to threshold LTC was 0.0325 mg/liter H S
and a second run to 10 days (Lmyy-9) was 0.0310 mg/liter. Fish in tests
Lmyy-1 and -2 with a mean 96-hr LC50 of 0.0222 mg/liter lUS may have a
greater sensitivity than would be normally expected since fish from the
same stock held in fresh water developed an infection of "ich" several
days after the completion of the test.
Adult Fish—Seven acute tests were run on adult bluegills held in fresh
laboratory water for 6 to 174 days prior to bioassay. Fish in the various
tests had mean lengths varying from 11.6 to 13.0 cm. Temperatures in
various tests were 19.6 to 20.3 C and 0» levels were 4.6 mg/liter in one
test (Lma-10) and in others ranged from 5.8 to 6.4 mg/liter. The 96-hr
LC50 values ranged from 0.0198 to 0.0375 mg/liter H2S with a mean for
all tests of 0.0297 mg/liter (Table 50).
Effect of Acclimation to H^S on Acute Response—It will be noted from
Table 50 that LC50 values did not decline significantly after 48 hr
exposure in most tests. To determine to what extent acclimation to H~S
105
-------
Table 50. ACUTE TEST CONDITIONS AND LC50 VALUES FOR BLUEGILLS
TESTED IN H2S
Days
from col- Mean Mean test
lection fish conditions'1
to start length, Temp., Oj,
Test of test cm C mg/1
BE-1
BE-3
BF-2
BF-4
d
Lmyy-1
Lmyy-2
Lmyy-4
Lmyy-5
Lmyy-7
Lmyy-8
Lmyy-9
Lma-1
Lma-3
Lma-4
Lma-5
Lma-6
0
0
4b
39°
60
67
28
42
99
164
262°
31
45
89
33
103
0
0
3
3
3
3
3
4
5
12
12
12
12
12
-
.3
.8
.7
.8
.2
.2
.2
.9
.3
.0
.0
.0
.0
.0
21.
21.
21.
21.
19.
19.
20.
20.
20.
19.
20.
19.
19.
19.
20.
20.
9
9
7
8
8
8
1
1
0
9
0
8
8
6
0
3
6.1
5.9
5.8
6.0
6.3
6.5
6.6
6.3
5.9
5.9
5.7
6.3
6.4
6.0
6.1
5.8
i
. 48 hr
-
LC50 values,
mg/1 H^S
72 hr
(66 hr)
0.0162
(77 hr)
0.0125
- >0
-
r-
_
0.0345
0.0345
0.0340
0.0290
_
0.0237
-
0.0317
0.0280
0.0270
-
-
_
*-
0.0345
0.0320
0.0325
0.0290
_
x =
0.0195
-
0.0317
0.0280
0.0270
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
96 hr
-
.0292
.0086
.0173
.0270
.0222
.0325
.0320
.0325
.0290
.0320
.0316
.019$
.0375
.0280
.0280
.0270
LTC(davs)
-
0.0169(9)
0.0084(8)
-
_
_
-
0.0325(8)
-
0.0310(10)
_
-
-
-
_
106
-------
Table 50 (continued). ACUTE TEST CONDITIONS AND LC50 VALUES
FOR BLUEGILLS TESTED IN HS
Days
from col- Mean
Mean test
lection
to start
Test of test
Lma-7 174
Lma-10 6
fish conditions LC50 values ,
length, Temp., 0^, mR/liter H_S
L ~ "
cm C mg/1 48 hr 72 hr
13.0 20.2 5.8 0.0328 0.0328
11.6 20.0 4.6 0.0353 0.0353
x =
£.
96 hr LTCfH*v.^
0.0328
0.0353
0.0297
pH range 7.8-8.0.
Days since spawned artificially in the laboratory from parents col-
lected in the field.
Days since spawned naturally in the laboratory from parents which
were controls in chronic tests "Bluegill-small".
Fish infected with Ichthyophthirius multifiliis and LC50 values not
included with other yy tests.
107
-------
might be a factor in these results, 10 test chambers were set up with
serial concentrations of H S varying from 0.0144 to 0.0308 mg/liter
(Table 51). On each succeeding day of the 11-day test, except days 6 and
7, the H_S concentration was raised in each chamber. At the lowest
starting concentration (0.0144 mg/liter), 100% of the fish survived 8
days or until the concentration had been raised to 0.0322 mg/liter.
On the succeeding day with the concentration raised to 0.0502 mg/liter,
50% of the fish died and 100% died on the llth day at a concentration
of 0.0873 mg/liter. At the highest initial concentration of 0.0308
mg/liter, 25% of the fish survived at the end of the first day and with
the increase in H?S concentration on the second day to 0.0349 mg/liter,
100% mortality occurred. At intermediate starting concentrations the
ultimate concentration at which 50% and 100% of the fish died increased
with the decrease in starting concentration (Table 52). These data
indicate that acclimation to H_S occurred during the progress of the
experiment provided that initial levels were not acutely toxic within
24 to 48 hr. This experiment also suggests the reason why the 48-hr
and longer LC50 values in tests reported above did not vary significantly
(Table 52).
The acute tests of different life history stages of bluegill indicate
that feeding swim-up fry with a 96-hr LC50 of 0.0086 mg/liter H S are
the most sensitive and that the most resistant is the juvenile stage.
The apparent difference between juvenile and adult resistance was not
shown to be significant.
CHRONIC BIDASSAY
Experimental Design
Eight chronic tests were conducted and varied in duration from 93 to 826
days. Fish were started in the various tests as green eggs, young-of-
the-year, or adults (Table 53). H2S concentrations ranged from means of
0.0007 to 0.0105 mg/liter and temperatures from 24.4 to 14.7 C, 07 from
108
-------
6.2 to 9.0 mg/liter, and pH from 7.58 to 8.08 in the various tests
(Table 54). In one test a seasonal temperature variation was developed.
The criteria for judging effect levels were growth rate, long-term
mortality, food consumption, food conversion efficiency, and repro-
ductive success. At the very low levels variation in concentration was
considerable in some tests but the mean condition was sustained the
greater part of the time. One additional chronic test was made to
determine the effect of sublethal exposure to HLS on swimming endurance
and resistance to copper.
All chronic tests were of flow-through type with flushing water provided
by diluters modified from that described by Mount and Brungs and Brungs
4
and Mount. Rate of flow was 0.5 liter per minute. When fish were less
than 2 cm long they were fed yeast, finely mashed hard boiled egg yolk,
and brine shrimp. After fish exceeded 2 cm in total length, yeast and
egg yolk were discontinued and the diet changed to ground beef liver,
ground fresh minnows, Glencoe dry fry granules, and brine shrimp. The
test chambers varied in size depending on the size of the fish. BG-1,
-2, and -3, and BG-small were tested in 503-liter insulated fiber glass
tanks (208 x 55 x 52 cm) with water depth carried at 44 cm. Bluegill
fry (BG-sp 1 & 2) were started in a 20-liter glass-silicone glue aquarium
and after 145 days of exposure, fish were transferred to the 503-liter
tanks described above. Bluegill in tests BG-sp 3 & 4 and BG-sp 5 & 6
were tested in 20-liter glass aquaria. Light regime was regulated in
accordance with seasonal changes in the St. Paul, Minnesota area.
Survival in Chronic Exposure
Three chronic tests ranging in duration from 93 to 316 days were started
with green eggs hatched in H?S concentrations which varied from 0.0010
to 0.0092 mg/liter in the various tests (Table 54). Percentage sur-
vival in the controls at temperatures from 22 to 24 C was 39% and 32%
after 316 and 130 days, respectively, in tests BG-sp 1 & 2 and 3 & 4
(Table 55). After initial egg and fry mortality, survival decreased
109
-------
Table 51. MEAN DAILY H2S EXPOSURE AND PERCENTAGE SURVIVAL OF FINGERLING BLUEGILLS ON SUCCESSIVE DAYS
IN 10 TREATMENT CHAMBERS
(H^S concentration expressed as mg/liter)
H2S
% s urvival
H2S
% survival
H2S
% survival
H2S
% survival
H2S
% survival
H2S
% survival
H2S
% survival
H2S
% survival
1
0.0144
100
0.0171
100
0.0179
100
0.0205
100
0.0219
100
0.0231
100,
0.0247
50
0.0276
100
2
0.0201
100
0.0234
100
0.0235
100
0.0234
100
0.0295
100
0.0395
25
0,0333
25
0.0264
100
3
0.0206
100
0.0253
100
0.0225
100
0.0316
100
0.0304
100
0.0401
0
0.0461
25
0.0314
100
4
0.0288
100
0.0361
100
0.0409
100
0.0467
100
0.0400
100
-
-
0.0628
25
0.0448
100
Days
5
0.0325
100
0.0460
100
0.0455
100
0.0361
100
0.0336
100
-
-
0.0361
25
0.0403
100
of exposure
6
0.0325
100
0.0460
75
0.0455
75
0.0361
100
0.0336
100
-
-
0.0361
25
0.0403
100
7
0.0325
100
0.0460
75
0.0455
75
0.0361
100
0.0336
100
-
-
0.0361
25
0.0403
100
8
0.0332
100
0.0438
75
0.0446
75
0.0551
100
0.0435
100
-
-
0.0568
0
0.0541
100
9
0.0502
50
0.0612
25
0.0800
0
0.0582
50
0.0560
100
-
-
-
-
0.0898
0
10
0.0671
50
0.0877
0
-
-
0.0658
0
0.0560
50
-
-
-
-
-
-
11
0.0873
0
-
-
-
-
-
-
0.0774
0
-
-
-
-
-
—
-------
Table 51 (continued). MEAN DAILY H2S EXPOSURE AND PERCENTAGE SURVIVAL OF FINGERLING BLUEGILLS ON
SUCCESSIVE DAYS IN 10 TREATMENT CHAMBERS
(H-S concentration expressed as mg/llter)
H2S
% survival
V
1
0.0284
75
0.0308
2
0.0336
50
0.0349
3
0.0589
25
-
Days
4 5
0.0533
0
-
of exposure
6 7 8 9 10 11
„_-.---
______
------
% survival
-------
Table 52. H-S CONCENTRATIONS AT WHICH 100%, 50%, and 0% OF
BLUEGILLS SURVIVED IN 2 TO 11 DAYS AFTER ACCLIMATION TO H2S
(H S concentration expressed as mg/liter, days exposure in parentheses)
Initial
concen-
tration
0
0
0
0
0
0
0
0
0
0
.0144
.0171
.0179
.0205
.0219
.0231
.0247
.0276
.0284
.0308
% Highest concen- Highest concen-
Survival tration with tration with
(24 hr) 100% survival 50% survival
100
100
100
100
100
100
50
100
75
25
0
0
0
0
0
0
<0
0
.0332
.0460
.0455
.0551
.0560
.0231
(8)
(5)
(5)
(8)
(9)
(1)
.0247(0)
.0541
(8)
<1 0.0284 (41)
-------
Table 53. SOURCE OF BLUEGILLS AND STAGE OF FISH
AT START OF CHRONIC TEST WITH HS
Test
Stage
Date
col-
lected
Water tem-
perature at
collection,
C
Method of
capture
Source
BG—sp Green
1 & 2 egg
BG-sp Green
3 & 4 egg
BG-sp Green
5 & 6 egg
BG-small yy
BG-1 Adult
BG-2 Adult
BG-4 Adult
27/5/71
10/6/71
22/6/71
27/8/69
30/1/69
10/2/70
10/5/72
17
4
4
15
Laboratory
spawned
Laboratory
spawned
Laboratory
spawned
Seine
Hook & line
Hook & line
Trap net
Laboratory
Laboratory
Laboratory
Medicine Lake
Crystal Lake
Crystal Lake
Marion Lake
113
-------
Table 54. PHYSICAL AND CHEMICAL CONDITIONS OF CHRONIC BLUEGILL TESTS
BG-sp
x H2S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
BG-sp
l&2a Egg (316 days)
0.0021 0.0042
0.0012 0.0029
7.70 7.70 7.70
22.6 22.4 22.3
7.6 7.6 7.6
195 195 195
3&4a Egg (120 days)
x H-S concentration (mg/1)
H2S std. dev.
x pH
x temperature
(mg/1)
(C)
BG-sp
0.0012 0.0018
0.0016 0.0010
7.71
24.1
5&6a
x H2S concentration (mg/1)
H2S std. dev.
x pH
x temperature
x dissolved 0«
(mg/1)
(C)
(mg/1)
-
7.79
22.5
9.0
7.72
24.0
Egg (93
0.0010
0.0006
7.86
22.2
9.0
BG-small YY (826
x H?S concentration (mg/1)
H2S std. dev.
x pH
x temperature
x dissolved 02
(mg/1)
(Ob
(mg/1)
x total alkalinity (mg/1)
-
7.98
18.1
8.0
219
0.0015
0.0012
7.98
17.9
8.0
219
7.73
23.9
days)
0.0013
0.0007
7.88
22.2
9.0
days)
0.0031
0.0023
8.03
17.8
8.0
219
0.0075
0.0041
7.73
22.3
7.6
195
0.0034
0.0015
7.71
24.4
0.0040
0.0015
7.90
22.1
9.0
0.0061
0.0043
8.03
17.8
8.0
219
0.0092
0.0042
7.72
22.4
7.6
195
0.0087
0.0034
7.74
23.8
0.0073
0.0029
7.90
22.2
9.0
0.0064
0.0063
7.90
18.5
8.0
219
BG-1 Adult (288 days)
x H2S concentration (mg/1)
H2S std. dev.
x pH
x temperature
(mg/1)
(C)
-
8.04
20.7
0.0014
0.0012
8.05
20.2
0.0023
0.0016
8.04
20.3
0.0071
0.0047
8.08
20.2
0.0098
0.0066
8.03
20.3
114
-------
Table 54 (continued). PHYSICAL AND CHEMICAL CONDITIONS
OF CHRONIC BLUEGILL TESTS
BG-1
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
BG-2
x H«S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
BG-4
x H»S concentration (mg/1)
H2S std. dev.
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
Adult
6.3
232
Adult
-
-
7.58
14.7
7.9
Adult
-
-
7.82
23.9
6.4
181
(288 days)
6.3 6.3
232 232
(200 days)
0.0010 0.0025
0.0007 0.0014
7.60 7.61
14.9 15.2
7.9 7.9
(97 days)
0.0007 0.0014
0.0007 0.0020
7.81 7.82
23.8 23.6
6.3 6.2
181 181
6.3
232
0.0062
0.0046
7.63
14.9
7.9
0.0027
0.0048
7.87
23.6
6.4
181
6.3
232
0.0105
0.0103
7.67
15.2
7.9
0.0078
0.0121
7.85
23.5
6.5
181
.Eggs composited from two spawnings of same parents.
Temperatures varied seasonally from 7.4-25.5 C.
Temperatures varied seasonally from 6.8-24.3 C.
Temperatures varied seasonally from 20.0-25.8 C.
115
-------
little with time. Survival decreased progressively with increased con-
centration of H2S to 14% and 11%, respectively, in 0.0092 and 0.0087
ing/liter H2S at the end of the test (Table 55). A third test started
with eggs and run for 93 days was not conclusive since excessive mor-
tality occurred in the controls. However, survival at 0.0073 mg/liter
H S was approximately half that in the controls. The no-effect concen-
tration for maximum long-term survival when fish are exposed to E^S
starting with the egg stage is less than 0.0012 mg/liter I^S.
The chronic test designated as BG-small was run for 826 days starting
with juveniles having a mean weight of 4.02-5.18 g in various treatments
after 56 days of exposure. H2S concentrations were 0.0015 to 0.0064
mg/liter. Little mortality occurred at any level before 362 days (Table
55). At the highest level (0.0064 mg/liter), survival dropped to 60%
after 392 days and to 0 after 420 days. At 0.0061 mg/liter, survival
was 100% at 717 days and 70% by the 826th day. It is apparent that the
safe range for fish exposed to H~S first as juveniles is approximately
0.0061 mg/liter and that lethal ranges are somewhat higher. The dif-
ference in the means of the two highest concentrations are not considered
to be definitive since the standard deviation of both was high.
Three tests of bluegills started as adults (BG-1, -2, and -4) were con-
ducted with H S concentrations varying from 0.0007 to 0.0105 mg/liter.
After 288 days there was 88% survival at 0.0098 mg/liter in one test, or
approximately the same as the control. In a second test, survival de-
creased after 56 days to 83% at 0.0062 mg/liter and fell to about half
of the controls (56%) at 0.0105 mg/liter. The loss at the higher con-
centration occurred at the end of 28 days and did not increase through
the remainder of the 200-day test period. The third test with a range
of treatments from 0.0007 to 0.0078 mg/liter H S ran for 97 days with no
mortality. The low survival in the tests started with eggs appeared to
be attributable to mortality occurring during the very early stages
since once fish reached a mean weight of 4-5 g, mortality was low at
116
-------
levels as high as 0.0061 mg/liter H S.
Growth Rate
Growth rate of fish started as eggs in BG-sp 1 & 2 and BG-sp 3 & 4 which
ran for 316 and 113 days, respectively, decreased at the higher levels
but some apparent stimulation occurred at 0.0012 and 0.0018 mg/liter H2S
(Table 56). After 316 days of exposure to 0.0092 mg/liter, mean weight
was 40.8% of control and after 113 days of exposure in a second test at
0.0087 mg/liter was 85% of control.
When fish were started as juveniles, growth was retarded at all H~S
levels tested above 0.0015 mg/liter. At 0.0061 mg/liter, weight was
63% of the control after 826 days but the reduced mean size at the end
of the test was due primarily to mortality among the larger fish.
In the three tests with adult bluegills (BG-1, -2, and -4), significant
decrease in growth did not occur at concentrations less than 0.0071
mg/liter H~S after 288 days of exposure. The mean weight of fish held
for 288 days at 0.0098 mg/liter was 61% of control. A test (BG-2),
running a shorter period of time (200 days), showed marked decline only
at 0.0105 mg/liter where mean weight was 73% of control. It appears
that growth inhibition will occur after long exposure to all levels of
H^S when fish are started as eggs, although there may be some apparent
stimulation after shorter exposure at low levels. In fish started as
adults, growth inhibition occurred at 0.0061 to 0.0071 mg/liter H2S.
Food Consumption and Conversion
Consumption of live minnows by subadult bluegills at 28 days (BG-2)
varied from 51.46 mg/g fish/day in the controls to 14.22 mg/g fish/day
at 0.0105 mg/liter H?S (Table 57). There was a substantial decrease in
food intake only above 0.0062 mg/liter in 114 days. In a second test
running for 97 days with prespawning adults, no difference was noted at
levels up to 0.0078 mg/liter. Food conversion efficiency was decreased
117
-------
Table 55. SURVIVAL OF BLUEGILLS WITH LONG-TERM EXPOSURE TO
•a
(expressed as percentage)
Exposure t
Test days
BG-sp
1&2
BG-sp
3&4
BG-sp
5&6
BG-small
28
57
116
200
316
28
56
86
113
130
28
93
28
362
392
420
717
826
H^S, me/liter
Control
42
39
39
39
39
Control
43
34
34
32
32
Control
21
2
Control
100
100
100
100
100
100
0.0021
32
31
27
27
27
0.0012
25
14
12
12
12
0.0010
17
2
0.0015
100
100
100
100
100
100
0.0042
54
30
29
27
27
0.0018
25
20
18
18
18
0.0013
13
3
0.0031
90
90
90
90
90
90
0.0075
25
23
23
16
16
0.0034
25
25
25
20
20
0.0040
18
2
0.0061
100
100
100
100
100
70
0.0092
18
14
14
14
14
0.0087
17
12
11
11
11
0.0073
10
1
0.0064
90
90
60
0
0
0
118
-------
Table 55 (continued). SURVIVAL OF BLUEGILLS WITH
LONG-TERM EXPOSURE TO H2S
(expressed as percentage)
Test
BG-1
BG-2
BG-4
Exposure ,
days
28
224
288
28
56
200
97
Control
100
100
90
Control
100
100
100
Control
100
0.0014
100
100
100
0.0010
100
100
100
0.0007
100
H«S, mg/liter
£.
0.0023 0
100
100
100
0.0025 0
100
100
100
0.0014 0
100
.0071
100
100
88
.0062
100
83
83
.0027
100
0.0098
88
88
88
0.0105
56
56
56
0.0078
100
Initial numbers for test BG-sp 1 & 2 were 200; BG-sp 3 & 4, 900; BG-
sp 5 & 6, 800; BG-small, 10; BG-1, 10; BG-2, 6; BG-4, 10.
119
-------
Table 56. GROWTH OF BLUEGILLS WITH LONG-TERM EXPOSURE TO
(expressed as mean weight in grams)
Test
BG-sp
1&2
BG-sp
3&4
BG-small
BG-1
Exposure,
days
78
200
316
86
113
56
112
252
392
493
603
717
826
196
257
288
H,,S, mg/liter
Control
0.29
5.37
10.70
Control
0.17
0.45
Control
4.02
13,62
45.65
50.81
81.73
84.09
97.36
99.91
Control
182.43
183.57
187.04
0.0021
0.16
3.70
7.12
0.0012
0.21
0.56
0.0015
4.50
10.50
39.59
46.34
74.12
76.59
92.36
98.71
0.0014
155.83
155.36
175.18
Z-
0.0042
0.09
3.22
8.70
0.0018
0.20
0.54
0.0031
5.00
12.03
30.65
34.37
62.14
68.05
81.89
90.35
0.0023
163.03
161.28
189.50
0.0075
0.13
1.88
5.29
0.0034
0.15
0.31
0.0061
5.18
11.98
38.57
43.35
72.36
75.41
86.96
63.05a
0.0071
132.38
115.70
120.10
0.0092
0.17
2.37
4.36
0.0087
0.11
0.39
0.0064
4.44
14.04
29.83
31.76
-
-
-
-
0.0098
157.58
150.30
110.05
120
-------
Table 56 (continued). GROWTH OF BLUEGILLS WITH LONG-TERM
EXPOSURE TO H2S
(expressed as mean weight in grams)
Test
BG-2
BG-4
Exposure,
days
0
28
56
84
114
200
0
97
Control
46.85
50.06
51.51
56.23
71.05
124.66
Control
75.90
116.90
0.0010
41.46
43.40
42.06
43.50
49.50
99.20
0.0007
84.00
132.60
H^S, mg/liter
0.0025
43.90
46.11
48.18
48.35
60.23
99.36
0.0014
78.20
132.80
0.0062
55.88
55.26
55.40
47.72
58.90
113.36
0.0027
76.10
120.90
0.0105
48.53
50.16
45.66
44.76
50.28
91.49
0.0078
76.70
118.00
for concentration of 0.0061 dropped due to mortality of a
large fish.
121
-------
Table 57. MINNOW CONSUMPTION OF BLUEGILLS WITH LONG-TERM
EXPOSURE TO H2S
(expressed as milligrams food/gram fish/day)
a
Exposure,
Test
BG-2
BG-4
days
28
56
84
114
97
Control
51.46
73.65
111.32
149.47
x = 96.48
Control
35.89
H«S concentration,
mg/liter
0.0010
25.69
24.01
76.13
104.47
57.58
0.0007
31.95
0.0025
64.85
58.58
116 . 14
166.99
101.64
0.0014
34.31
0.0062
15.35
13.30
72.03
150.40
62.77
0.0027
32.39
0.0105
14.22
15.82
43.02
94.91
41.99
0.0078
36.26
value expressed for each designation of exposure day is the
sumption for the preceding 28-day period.
mean con-
122
-------
at all levels from 0.0010 to 0.0105 mg/liter H S in the first test but
in the second, conversion efficiency did not appear to drop until a
concentration of 0.0078 mg/liter was reached (Table 58). These con-
version factors are consistent with the observed decrease in growth rate
noted previously.
Reproduction
One chronic test (BG-small) running for 826 days failed to produce any
spawning in treatments of 0.0015 to 0.0064 mg/liter H S (Table 59).
Fish in the controls spawned with an average of 9,928 eggs/female and
130 eggs/g female. A second test (BG-4), started with adults and carried
for 97 days in H2S concentrations of 0.0007 to 0.0078 mg/liter, pro-
duced 155.5 eggs/g female in controls, 100.8 eggs/g female at 0.0007
mg/liter, and 51.1 eggs/g female at 0.0014 mg/liter H S. At 0.0027 and
0.0078 mg/liter H S there were no eggs deposited.
Failure to deposit eggs appeared directly related to behavior. In the
two highest treatments there was no significant activity by the male
over the nesting gravel and females did not lay eggs. In the lower
concentrations activity was more restricted at 0.0014 than at 0.0007
mg/liter. Since no lower concentrations were attempted, the exact lower
level of inhibition was not determined. The one third lower deposition
of eggs/g female in this treatment than in the controls strongly
suggests that any measurable level of H~S will have some inhibitory
effect.
Effect of H?S Acclimation on Response to Anesthesia
During the progress of chronic experiments BG-sp 1 & 2 and 3 & 4, BG-
small, and BG-1 and -2, fish were anesthetized periodically with MS:222
to permit weighing. In tests started with eggs and exposed for 288 days,
the mean number of seconds required for anesthesia with increased H S
concentration decreased from 89 in the control to 79 at 0.0092 mg/liter
123
-------
Table 58. FOOD CONVERSION EFFICIENCY OF BLUEGILLS WITH
LONG-TERM EXPOSURE TO H2S
(expressed as percentage)
Test
BG-2
Exposure,
days
28
56
84
114
H0S, mg/liter
Control
4.57
1.36
2.80
5.56
0.0010
3.59
0.00
1.57
4.41
0.0025
2.67
2.64
0.11
4.72
0.0062
0.00
0.68
0.00
4.98
0.0105
0.70
0.00
0.00
4.37
x = 3.57
2.39
2.54
1.42
1.27
BG-4
97
Control 0.0007 0.0014 0.0027 0.0078
12.2 13.2 15.5 14.4 9.0
Calculated from increment in grams divided by grams of food consumed.
124
-------
Table 59. SPAWNING SUCCESS OF BLUEGILLS WITH LONG-TERM
EXPOSURE TO H S
concentration,
ing/liter
Test
BG- small
(826
days)
BG-4
(97
days)
Item
Sex ratio (M:F)
Total eggs
Eggs/female
Eggs/g female
Sex ratio (M:F)
Total eggs
Eggs /female
Eggs/g female
Control
3:3
29,784
9,928
130
Control
4:6
104,967
17,562
155.5
0.0015
2:3
0
0
0
0.0007
3:7
89,315
12,795
100.8
0.0031
5:2
0
0
0
0.0014
4:6
36,935
6,157
51.1
0.0061
4:3
0
0
0
0.0027
5:5
0
0
0
0.0064
-
0
0
0
0.0078
3:5
0
0
0
125
-------
(Table 60). In a second test started with eggs (BG-sp 3 & 4) after 113
days of exposure, mean time to anesthesia decreased more markedly with 102
seconds in the control and 70 seconds at 0.0087 mg/liter H2S. When fish
were started as young-of-the-year and exposed for 826 days, the time for
anesthesia was much shorter in all treatments through 578 days than in
the controls but for the remainder of the test period there appeared to
be no great difference. In tests started with bluegill adults (BG-1 and
-2), exposures varied from 200 to 252 days and fish showed a substantial
decrease in time to anesthesia in all ELS treatments. The greatest dif-
ference was the reduction from 304 seconds in the control of one experi-
ment to 188 seconds at a concentration of 0.0098 mg/liter KLS.
Resistance _tp_ Other Stresses after Chronic Exposure
Experimental Design— Young-of-the-year bluegills (mean total length
3.2 cm) exposed 126 and 148 days to concentrations of ELS. ranging from
0.0004 to 0.0146 mg/liter were subjected to swimming endurance tests
and to 5.5-9.2 mg/liter copper and to 0.075 mg/liter malathion. When
fish were brought into the laboratory they were given prophylactic
treatment with neomycin sulfate. After 3 days they were given a 3-day
treatment with methylene blue. Fish were held at 21+1 C and fed brine
shrimp and ground pork liver prior to chronic tests.
A series of 96-hr LC50 tests were conducted 28, 42, and 99 days after
o
collection of fish in apparatus described by Adelman and Smith. Test
chambers were 6-liter acrylic cylinders 14.5 cm in diameter. Gaseous
ILS was employed and test concentrations were determined by analysis of
water in centers of test chambers. Four levels of ELS and on,e control
were used and water flowed through chambers at a rate of 300 ml/min.
Long-term exposure tests were started on 100 fish 14 days after collec-
tion. They were placed in five tanks, each divided into three sections.
Four test levels and one control were maintained and checked by analysis
in each section. Fish in long-term tests were fed six times per day
126
-------
with various combinations of brine shrimp, ground beef liver, fresh
ground minnows, and 1/32-inch Oregon moist pellets. A constant level
of 7.8 pH was maintained with HUSO, dispensed from a diluter similar
to that used for the toxicant.
The swimming endurance was tested in a stainless steel oval raceway with
a channel width of 8.5 cm, a depth of 15 cm, and a length of 218 cm.
The channel included two straight sections each 50 cm long. Untreated
laboratory water was maintained at a depth of 9 cm in the channel and
the desired current was created with a stainless steel paddle wheel.
The apparatus was similar to that described by Lemke and Mount. Water
velocity was regulated by a rheostat on the 1/8 h.p. motor drive of the
paddle wheel. Fish were kept in position until they fell back by an
electrical shocker placed downstream from their swimming location. One
hundred twenty-volt AC current was used with the minimum amperage required
to keep the fish away from the poles. One fiber glass screen was placed
ahead of the poles to prevent fish from being injured by drifting between
the electrodes and a second screen 22 cm upstream to form a swimmming
area 8.5 x 22 cm.
Test procedure was designed to minimize bias. A single fish was selected
at random from an exposure tank, placed in a 400 ml beaker without
removal from water, and transferred to the raceway swimming area. It
rested in this position without electrical stimulation or water current
for 30 min. Water temperature was held the same as that in the long-
term exposure. In low speed tests after the 30-min rest period, current
velocity was set at 8 cm/sec. After 5 min, current was increased to
11.2 cm/sec, and after 7 min to 15 cm/sec. At this time the electrical
shocker was activated to prevent the fish from lying against the screen
or holding its position by resting the caudal fin against the screen.
At 9, 11, and 13 min the velocity was increased to 17.0, 19.5, and 22.5
cm/sec, respectively. The maximum velocity was maintained until the
fish fell back against the screen. Electrical current was shut off, and
127
-------
Table 60. TIME TO ANESTHESIA OF BLUEGILLS SUBJECTED
TO LONG-TEEM EXPOSURE TO H-S
(seconds)
Test
BG-sp
1&2
BG-sp
3&4
BG-
small
MS: 222
mg/1
80
80
121
130
137
137
137
137
80
80
137
137
137
137
137
137
137
137
Exposui
days
86
116
145
170
200
230
260
288
86
113
56
112
252
392
493
578
111
826
•e,
Control
73
79
104
86
75
106
92
98
x = 89
Control
110
95
x = 102
Control
357
443
138
101
155
196
87
90
x = 196
H,,S, mg/liter
0.0021
79
77
81
93
73
74
86
89
82
0.0012
112
94
103
0.0015
156
124
162
89
123
192
81
106
129
0.0042
82
70
82
99
74
74
85
81
81
0.0018
98
94
96
0.0031
148
122
166
85
115
159
67
121
123
0.0075
73
79
80
105
86
107
82
74
86
0.0034
93
60
76
0.0061
101
128
175
82
99
141
54
104
110
0.0092
72
80
67
100
73
68
91
78
79
0.0087
87
53
70
0.0064
105
122
172
69
-
-
-
-
117
128
-------
Table 60 (continued). TIME TO ANESTHESIA OF BLUEGILLS
SUBJECTED TO LONG-TERM EXPOSURE TO H S
(seconds)
Test
BG-1
BG-2
MS:222
me/1
137
137
137
137
137
Exposure,
days
196
252
X
84
114
200
X
Control
283
326
= 304
Control
194
97
124
= 138
H.S,
Z
0.0014
169
219
194
0.0010
170
94
96
120
rag/liter
0.0023
149
186
168
0.0025
159
101
113
124
0.0071
139
180
160
0.0062
128
86
123
112
0.0098
147
228
188
0.0105
102
89
128
106
Time notation based on complete anesthesia of all survivors in each
concentration after specified days of exposure.
129
-------
water movement stopped. After 5 min fish were anesthetized in MS:222,
weighed, measured, and returned to an unoccupied section of their
original test tank. One fish was taken from each tank successively
until all fish from one section were tested. They were then returned to
their original test section.
In high speed tests, fish were taken from section B of each long-term
test and placed in the raceway. After a 30-min rest, water velocity
was started at 8 cm/sec. After 5 min velocity was increased to 10.7
cm/sec, at 7 min to 17.5 cm/sec, and the electrical shocker activated.
Subsequently at 9, 11, 13, 30, and 45 min, velocity was increased to
19.7, 22.5, 25.0, 27.0, and 28.0 cm/sec, respectively. After swimming
failure of the fish, the procedure was the same as in low speed tests.
Chemical stress tests were carried out with apparatus similar to the
proportional diluter used in long-term tests except that it was designed
to dispense the same toxicant levels to all test chambers. The toxicant
was introduced in a mixing box and then to a distribution box which
split the volume into five equal portions for distribution to test
chambers each of which was 50 x 26 x 30 cm. Water depth was 22 cm and
cycle time was 2 min.
Reaction to Long-term and Short-term Exposure—The 96-hr LC50 tests were
conducted on untreated fish 28, 42, and 99 days after bluegills were
collected. Mean water conditions during the tests were 20 + 0.1 C, 6.2
+ 0.4 mg/liter dissolved oxygen, 7.90 + 0.05 pH meter reading, and 235
+ 0 mg/liter CaC03 total alkalinity. LC50 was calculated from five test
levels and one control. Ninety-six-hour LC50 values for the three tests
were 0.0325, 0.0320, and 0.0325 mg/liter H2S. As noted above, each
test tank in long-term tests was divided into three sections with fish
placed in the first two. Since there was a reduction of H?S levels be-
tween the first (A) and second (B) sections, analyses were run on each
section. In series "A", H0S concentrations extended from 0.0004 to 0.0146
130
-------
mg/liter; in series "B", from 0.0004 to 0.0067 mg/liter (Table 61).
After 126 days of exposure in series "A", the concentrations had no
effect on survival (Table 62) and growth in length appeared to be
affected adversely only at the highest concentration (0.0146 mg/liter)
where weight was less than two-thirds that attained in the control.
Gill irrigation rate of treated fish was significantly increased over
that in controls for all treatments and was 139% greater in the
highest concentration.
After 148-day exposure in series "B", survival was not affected and
growth was not adversely affected (Table 63). Gill irrigation rate
determined visually increased in fish from all treatments and those
from the highest (0.0067 mg/liter) had 42.5% greater irrigation rate
than the controls.
Swimming endurance was adversely affected by chronic exposure to H_S.
In series "A", the low speed tests indicated that those from the lowest
concentration (0.0004 mg/liter) of the series had slightly increased
capability to endure swimming stress. Controls swam for 201 min before
failure and fish from the highest treatment (0.0146 mg/liter) for 31
min or 84% shorter time than the controls (Table 62). Fish from series
"B" in the lowest concentration had the least resistance (36% less than
controls) (Table 63)-
Resistance to Copper and Malathion—After fish finished swimming tests,
they were returned to the treatment tanks and allowed to remain for 18
days before being subjected to tests with copper or malathion. Fish of
the "A" series were used in copper tests. Copper in filtered samples
was 3.8 mg/liter from all chambers except the one containing fish treated
at 0.0015 mg/liter H-S where concentration was 4.0 mg/liter (Table 64).
Survival time varied from 13.5 hr in controls and lowest EJB group to
52.5 hr in highest H,,S group (+288% of control survival time).
131
-------
Table 61. TEST CONDITIONS OF SERIES A AND B DURING
CHRONIC BLUEGILL TESTS3
Series A
x H^S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0- (mg/1)
x total alkalinity (mg/1)
-
-
7.72
24.1
6.20
191
0.0004
0.0006
7.74
24.0
6.49
191
0.0015
0.0012
7,77
24.0
6.58
191
0.0048
0.0020
7.80
24.1
6.66
191
0.0146
0.0050
7.90
24.1
6.49
191
Series B
x H-S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 02 (mg/1)
x total alkalinity (mg/1)
-
-
7.72
23.7
5.87
191
0.0004
0.0007
7.73
23.5
6.21
191
0.0007
0.0006
7.77
23.6
6.18
191
0.0022
0.0014
7.79
23.7
6.13
191
0.0067
0.0032
7.91
23.7
6.01
191
All values given are means of weekly or biweekly tests.
132
-------
Table 62. EFFECT OF CHRONIC EXPOSURE OF BLUEGILLS TO VARIOUS LEVELS
OF H S FOR 126 DAYS (SERIES A) ON GROWTH, GILL IRRIGATION
RATE3 AND SWIMMING ENDURANCE
Item
H~S concentration (mg/1)
Fraction of 96-hr LC50
Survival (%)
Q
Mean total length (cm)
Mean weight (g)
Mean gill irrigation
j i f i. Si
Series A (velocity 8.
-
90
5.68
3.69
46
0.0004
1/81
100
5.21
2.88
57
0.0015
1/22
90
4.78
2.03
65
0 cm/ sec)
0.0048
1/7
90
5.51
3.58
62
0.0146
1/2
90
4.54
1.91
110
% of control
Swimming endurance
Time to failure (min)
% of control
201
+24
241
+20
+41
95
-53
+35
119
-41
+139
31
-84
, Gill irrigation check after 106 days exposure.
Text explanation for speed change.
Fish in all chambers had a mean length at start of chronic tests of
3.23 cm (range 2.70-4.80).
133
-------
Table 63. EFFECT OF CHRONIC EXPOSURE OF BLUEGILLS TO VARIOUS LEVELS
OF H2S FOR 148 DAYS (SERIES B) ON GROWTH, GILL IRRIGATION
RATE3 AND SWIMMING ENDURANCE
Item
H_S concentration (mg/1)
Fraction of 96-hr LC50
Survival (%)
Q
Mean total length (cm)
Mean weight (g)
Mean gill irrigation
•a
rate (no . /min)
% of control
Swimming endurance
Time to failure (min)
% of control
Series B (velocity 8-28 cm/sec)
0 0.0004
1/81
100 100
5.42 5.00
2.86 2.16
52 71
+36.5
28 18
-36
0.0007
1/46
90
5.22
2.59
73
+40.5
21
-25
0.0022
1/15
100
5.40
3.18
73
+40.5
19
-32
0.0067
1/5
100
5.14
3.42
74
+42.5
22
-21
, Gill irrigation check after 106 days exposure.
Text explanation for speed change.
Fish in all chambers had a mean length at start of chronic tests of
3.23 cm (range 2.70-4,80).
134
-------
Table 64, RESISTANCE OF BLUEGILLS WITH CHRONIC TREATMENT OF
TO SUBSEQUENT EXPOSURE OF COPPER SULFATE (AS CU) (SERIES A)
AND MALATHION (SERIES B)a
Item
Q
H_S concentration (mg/1)
Copper (mg/1) - unfiltered
filtered
Temperature (C)
Dissolved 02 (mg/1)
Survival time (hr)
% control
Series
0
5
3
24
6
13
.5
.8
.3
.65
.5
-
0
7
3
24
6
13
0
.0004
.5
.8
.1
.61
.5
0
9
4
24
6
12
-7
.0015
,2
.0
.3
.63
.5
Series
H_S concentration (mg/1)
Malathion (mg/1)
Temperature (C)
Dissolved 02 (mg/1)
Survival time (hr)
0
0
24
7
72
.075
.0
.79
.5
0
0
24
8
94
.0004
.075
.0
.05
.0
0
0
24
8
75
.0007
.075
.1
.05
.5
A
0
8
3
24
6
24
+78
B
0
0
23
7
72
.0048
.2
.8
.5
.63
.0
0
7
3
24
6
52
.0146
.0
.8
.0
.58
.5
+288
.0022
.075
.9
.87
.5
0
0
23
8
72
.0067
.075
.9
.02
.7
aTotal alkalinity in Series A was 212 mg/1 and in Series B, 216 mg/1;
pH in Series A was 7.5 and in Series B, 7.6.
135
-------
The "B" series fish were tested with malathion at a concentration of
0.075 mg/liter. Controls and highest treatment survived for 72.5 hr.
Fish conditioned at the lowest H2S treatment (0.0004 mg/liter) had a
longer survival time than controls or highest treatment (94.0 hr)
(Table 64).
SUMMARY
The most sensitive stage of bluegill development as measured by LC50
is the swim-up fry with the median lethal threshold concentration of
0.0084 mg/liter H2S at 39 days after hatching. The most resistant
stages are juvenile and adult with mean 96-hr LC50 of 0.0316 and 0.0297
mg/liter H~S, respectively. In chronic exposures the gross response
most sensitive to sublethal levels of H-S is spawning which was inhibited
at 0.0007 mg/liter and eliminated at 0.0027 mg/liter H2S. Growth rate
gave no consistent response to the toxicant in most tests until more
than 100 days of exposure. In tests started with fry measurable response
was obtained in 78 days.
The gross effects of long-term exposure to H«S were reduced growth in
the highest concentration and progressively increased gill irrigation
rate with increased concentrations of H-S. This increase in irrigation
rate suggests decreased efficiency in the oxygen uptake or transport.
In the lower speed swimming stress tests given the series "A" fish, pre-
treatment at the lowest concentration appeared to increase endurance;
but at treatment levels of 0.0015 mg/liter H2S and higher, fish had
progressively less endurance. Fish treated at the highest level (0.0146
mg/liter) were much more easily stunned by electrical shock at the time
of failure than those treated at lower levels. In the high speed tests
all fish showed much less resistance to the swimming stress but dif-
ferences among treatments and controls was less marked than in low
speed tests at comparable treatment levels.
Resistance to copper was increased by exposure to H-S but resistance to
136
-------
malathion was not affected except in the lowest concentration. Since
copper affects oxygen uptake by gills and H^S-oxygen relationships in
the blood, the increased irrigation rate induced by exposure to higher
levels of H^S may account for the higher tolerance to copper by treated
fish. At the cellular level, H?S combines with metallic elements
17
(Goodman and Gillman ). Whether this reaction influenced resistance
to copper in the present experiments was not determined.
From the data developed in this study, it is apparent that slow speed
swimming tests will reveal adverse effects of long-term exposure to
H?S on bluegills except at the highest concentrations better than the
other gross indicators of changes except where spawning behavior is
involved. It is also apparent that extended exposure to H~S levels of
0.0015 mg/liter and greater reduces the physical capability of the fish.
137
-------
SECTION VIII
WALLEYE
(Stizostedion vitreum vitreum (Mitchill))
Toxicity of H?S to walleye was determined by (1) four acute tests on
juveniles and (2) two chronic tests started as juveniles. Egg and
fry data were taken from a previous project reported by Smith and
Oseid18.
ACUTE TESTS
Experimental Design
Acute tests to determine LC50 values for H-S were made on juvenile
walleyes collected from Sand Shore Lake near Bethel, Minnesota. Fish
were planted in the lake as fry and later removed by seine as juveniles.
Fish were held in the laboratory prior to testing at 12 C and were fed
live fathead minnows in excess of consumption once a day. Immediately
after arrival at the laboratory fish were treated for 3 days with 20
mg/liter neomycin. They were subjected to a routine of 12 hr of light
and 12 hr of darkness. Fish were raised gradually to test temperature
and held at test temperature for 7 days prior to start of acute tests.
2
Flow-through apparatus described by Adelman and Smith was used in all
tests. The test chambers were of glass-silicone construction with
Acute tests 1-3 having 38-liter capacity and test 4, 25-liter capacity.
Flow rate through each chamber was 30 ml/min. Each test consisted of
one control and five H-S concentrations. The same light regime used for
holding was maintained during the test. Water samples for H2S analyses
138
-------
were made three times daily during each test and for temperature,
oxygen, and total alkalinity once per day. Fish were not fed during
the 96-hr test.
Acute Toxicity
The four acute tests (Table 65) were run at 14.8-16.1 C and at 0 levels
of 5.9-6.8 mg/liter. The 96-hr LC50 varied from 0.0166 to 0.0214 with
a mean of 0.0193 mg/liter lUS.
Acute toxicity of H?S to eggs and fry was determined prior to the present
18
project and reported by Smith and Oseid. In three tests on eggs at
15 C they found that the 96-hr LC50 varied from 0.074 to 0.087 mg/liter
H2S. In four tests at 12 C, 96-hr LC50 ranged from 0.052 to 0.066
mg/liter H~S. LC50 at hatch in periods from 7 to 19 days ranged from
0.022 to 0.066 mg/liter H S with temperature varying from 12-15 C and
0- from 3 to 6 mg/liter. The higher values were obtained with 0? levels
at 4 and 3 mg/liter. Tests at longer periods were from start of incu-
bation and short tests from partial incubation until hatch was complete.
Fry survival at all H S levels tested was substantially below the control
and at concentrations of 0.040 mg/liter and higher was greatly reduced.
At the higher levels the percentage of fry deformity ran from 50% to 84%
with the probability of survival of deformed fry negligible.
CHRONIC TESTS
Experimental Design
Two chronic tests started with walleye juveniles collected from Sand
Shore Lake were conducted for 225 and 231 days. Two additional tests
were carried on but abandoned after disease problems destroyed their
4
validity. Test apparatus was that described by Brungs and Mount which
provided a flow-through of 500 ml/min. Test chambers were insulated
fiber glass tanks 200 x 53 x 53 cm filled with water to contain 340
liters. Each test included one control and four H~S concentrations
139
-------
Table 65. ACUTE TEST CONDITIONS AND LC5Q VALUES FOR
WALLEYE JUVENILES TESTED IN HS
Days from
collection Mean
Test
1
2
3
4
to start
of test
76
89
76
143
Mean test
o
conditions
length, Temp., 00 ,
mm
90
89
92
100
C
15.9
16.1
14.8
15.0
z.
mg/1
5.9
6.0
6.8
6.2
LC50,
me/1 EnS
24 hr 48 hr 72 hr 96 hr
0.0413 0,0314 0.0196 0.0183
0.0274 0.0229 0.0214
0.0178 0.0174 0.0166
- 0.0210
x = 0.0193
,pH 7.8-8.0 in separate tests.
5 fish per chamber.
140
-------
(Table 66). Tests 1 and 2 started with 10 hr of light and 14 hr of
darkness and were gradually adjusted to 16 hr of light and 8 hr of
darkness by the time the test was terminated.
Fish were fed during the test with live minnows in excess of consump-
tion. The number of dead minnows removed from the tank were subtracted
from the number of live minnows introduced to give daily consumption
of minnows. Daily minnow consumption was converted to grams on the
basis of mean weekly weight of minnows fed. Fish were held prior to
start of test and acclimated in the same manner as those used in acute
tests. Chronic test 1 concentrations of H_S ranged from 0.0013 to
0.0051 mg/liter and in test 2 from 0.0031 to 0.118 mg/liter (Table 66).
Survival
Survival in test 1 appeared to be affected adversely in 225 days at
0.0051 mg/liter H2S and in test 2 at 0.0118 mg/liter H2S in 231 days
(Table 67). The large variation in concentration maintained during the
test as indicated by standard deviation throws question on the exact
concentration which could be assumed to affect survival. Misfunction
of dispensing apparatus accounted for the variability.
Growth Rate
Growth as indicated by weight (g) and length (mm) was not significantly
affected at any of the concentrations of H~S tested (Tables 68 and 69).
Consumption of minnows (mg/g fish/day) by juveniles which were exposed
to H_S is shown in Table 70. Efficiency of food utilization based on
increased fish weight compared to food intake in grams was higher in
H2S concentration than in controls and especially at the lower concen-
trations (Table 71). This relationship may have resulted from reduced
general activity noted in the tanks treated with H S.
Fish were measured and weighed after immobilization with MS:222. The
time in seconds to immobility for fish in both tests was substantially
141
-------
Table 66. PHYSICAL AND CHEMICAL CONDITIONS
IN CHRONIC TESTS ON WALLEYES
Test 1
x H?S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0., (mg/1)
x total alkalinity (mg/1)
x H^S concentration (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0- (mg/1)
x total alkalinity (mg/1)
Control
7.59
17.6
8.1
235
Test
Control
7.51
19.6
8.3
182
0.0013
0.0008
7.62
17.8
8.1
235
2
0.0031
0.0027
7.62
19.6
8.3
182
0.0020
0.0020
7.65
17.8
8.1
235
0.0053
0.0081
7.60
19.7
8.3
182
0.0031
0.0025
7.67
17.8
8.1
235
0.0057
0.0070
7.60
19.7
8.3
182
0.0051
0.0042
7.71
18.0
8.1
235
0.0118
0.0105
7.61
19.6
8.3
182
142
-------
Table 67- SURVIVAL OF WALLEYE DURING CHRONIC EXPOSURE TO
(expressed as percentage)
Exposure,
Test days
1 28
56
112
140
225
2 28
197
231
H^S, me/1
Control
100
100
100
100
100
Control
100
100
100
0.0013
100
100
100
100
100
0.0031
100
100
100
0.0020
100
100
100
100
100
0.0053
100
100
100
0.0031
100
100
100
100
100
0.0057
100
100
100
0.0051
90
82
82
73
73
0.0118
93
93
87
10 fish used per tank. Some fish lost from causes unrelated to treat-
ment.
143
-------
Table 68. MEAN WEIGHT OF JUVENILE WALLEYE AFTER EXPOSURE TO
(grams)
Exposure,
Test days
1 28
56
84
112
140
168
196
225
2 0
28
57
85
113
141
169
197
231
Control
7.49
14.28
22.72
34.62
47.57
56.80
59.76
61.60
Control
5.11
9.59
15.51
21.39
24.31
26.77
29.95
41.02
60.81
0.0013
8.35
14.83
24.53
40.03
53.70
61.37
72.46
88.36
0.0031
5.11
11.09
17.11
22.70
25.34
25.94
28.75
33.73
66.17
H^S, rns/1
£~
0.0020
8.07
15.34
25.06
34.50
50.01
55.58
64.12
74.93
0.0053
5.56
11.14
16.18
19.53
21.78
23.53
26.19
35,23
59.23
0.0031
7.67
14.55
23.44
35.27
45.68
54.81
61.89
70.74
0.0057
4.76
10.44
15.83
21.01
25.16
27.41
30.53
50.13
71.68
0.0051
7.52
13.64
20.94
21.81
43.18
55.61
65.85
72.79
0.0118
5.14
10.39
15.01
19.03
22.69
24.24
25.38
29.38
59.41
10 fish per tank.
144
-------
Table 69. MEAN LENGTH OF JUVENILE WALLEYE AFTER EXPOSURE TO
(millimeters)
Exposure,
Test days
1 28
56
84
112
140
168
196
225
2 0
28
57
85
113
141
169
197
231
Control
9.64
12.19
14.59
16.41
18.40
19.20
19.83
20.27
Control
9.54
11.27
12.97
14.67
15.48
15.96
16.62
17.04
20.24
H
0.0013
9.71
11.12
14.53
16.81
18.48
19.79
20.68
22.50
0.0031
9.47
11.59
13.31
14.55
14.42
15.63
15.78
17.00
20.50
«S, mg/1
0.0020
9.69
12.40
14.88
16.71
18.24
19.20
20.03
21.37
0.0053
9.68
11.54
13.24
14.36
14.96
15.40
15.88
17.37
19.78
0.0031
9.36
12.17
14.22
16.30
17.90
19.05
19.94
20.80
0.0057
9.34
11.23
13.13
14.60
15.40
16.00
16.51
19.38
21.36
0.0051
9.51
11.90
13.65
16.30
17.57
19.10
20.32
21.23
0.0118
9.38
11.26
12.99
14.32
15.00
15.60
16.12
17.32
19.85
10 fish per tank.
145
-------
Table 70. MINNOW CONSUMPTION OF JUVENILE WALLEYES EXPOSED TO H Sc
(milligrams/grams fish/day)
Exposure,
Exposure,
Test days
1 29-56
57-84
85-112
113-140
141-168
169-196
197-225
2 0-28
29-57
58-85
86-113
114-141
142-169
170-197
198-231
H~S concentration,
rag/liter
Control
88.24
100.54
69.59
58.64
29.32
25.74
23.73
x = 56.54
Control
179.59
36.65
66.12
48.14
31.32
42.67
54.11
78.55
x - 67.14
0.0013
85.42
106.20
69.08
53.99
31.46
37.36
42.28
60.83
0.0031
182.72
33.33
53.27
42.05
30.81
39.14
87.39
103.90
71.58
0.0020
82.91
100 . 00
64.47
59.87
29.55
34.92
42.58
59.19
0.0053
156.89
30.75
50.95
53.73
36.63
44.65
77.82
85.12
67.07
0.0031
89.11
96.32
73.91
61.76
35.03
34.96
39.81
61.56
0.0057
177.63
33.49
59.17
52.86
31.58
34.86
50.83
63.88
63.04
0.0051
75.61
97.17
84.66
75.38
48.18
42.15
43.42
66.65
0.0118
135.31
32.28
62.28
55.13
33.67
46.35
61.36
97.07
65.43
10 fish per tank.
146
-------
Table 71. EFFICIENCY OF FOOD CONVERSION BY WALLEYE EXPOSED TO
(expressed as percentage)
Exposure,
Test days
1 28- 56
57- 84
85-112
113-140
141-168
169-196
197-225
2 0- 28
29- 57
57- 85
86-113
114-141
142-169
170-197
198-231
Control
25.1
16.2
21.3
19.2
21.5
7.0
4.6
Control
12.1
44.4
17.2
9.5
11.0
9.4
20.6
17.7
0.0013
23.4
16.6
24.8
19.3
15.1
15.8
16.7
0.0031
14.4
44.2
18.8
9.3
2.7
9.4
6.5
22.3
EUS, mg/1
0.0020
26.7
17.2
23.2
17.7
12.7
14.6
13.0
0.0053
15.2
41,4
17.0
12.1
9.7
11.0
34.1
19.1
0.0031
24.7
17.3
19.5
14.8
18.5
12.4
12.0
0.0057
15.0
42.2
13.1
7.2
7.5
8.6
13.5
22.0
0.0051
27.2
15.6
21.5
16.6
18.6
14.3
8.2
0.0118
17.8
38.8
14.4
10,5
7.0
3.5
8.5
24.9
10 fish per tank; efficiency calculated from increment in grams
divided by food intake in grams.
147
-------
shortened at all levels of H2S treatment (Table 72).
SUMMARY
Acute toxicity as measured by 96-hr LC50 had a mean among four tests
on juveniles of 0.0193 mg/liter H-S. In the chronic tests a potentially
adverse effect at all treatment levels was observed through the response
of fish to anesthesia. On the basis of survival, a no-effect level on
juveniles and older fish is 0.0030 mg/liter H~S. High variation in test
concentrations make effect data less accurate.
148
-------
Table 72. TIME TO IMMOBILITY IN MS:222 OF WALLEYE JUVENILES£
EXPOSED TO H2S
(seconds)
Exposure,
Test days
1 28
56
84
112
140
168
196
225
2 28
57
85
113
141
169
197
Control
145
100
100
130
87
94
142
144
Control
60
73
66
82
79
61
73
0.0013
71
91
145
190
92
126
117
83
0.0031
65
65
63
66
64
57
57
H0S, ma/1
0.0020
84
90
207
157
89
124
99
83
0.0053
51
62
55
59
61
54
66
0.0031
93
140
349
172
95
123
109
87
0.0057
55
52
49
46
56
48
53
0.0051
114
183
101
192
87
107
88
77
0.0118
55
46
44
42
46
43
49
10 fish per tank; time based on immobility of all fish from each
treatment.
149
-------
SECTION IX
BROOK TROUT
(Salvelinus fontinalis (Mitchill))
Toxic effects of H?S to brook trout were determined by (1) 96-hr LC50
and LTC tests on eggs, sac fry, feeding fry, and juveniles, (2) chronic
exposure of adults and juveniles, and (3) swimming endurance tests on
fish previously exposed to low levels of H_S.
ACUTE TESTS
Experimental Design
Six bioassays were conducted on eggs with a proportional diluter similar
5 4
to that designed by Mount and Brungs and Brungs and Mount. Sodium
sulfide was used as a source of H~S. The egg chamber consisted of
acrylic plastic cylinders 18 cm long with an inner diameter of 4.5 cm.
Eggs were supported on a Nitex screen attached to the bottom of the
cylinder and were shielded from light. Water flowed through a 25-liter
tank with the cylinders attached to the outlet. Flow was 600 ml/min.
Eggs used in the tests were stripped from trout held in the laboratory
and from a trout hatchery near Osceola, Wisconsin. Four tests were
made on sac fry from eggs incubated in fresh water. Apparatus was the
same as that used for eggs. Four bioassays were run on feeding fry
ranging in length from 2.1-2.8 cm. Fry were fed five times daily on
ground Glencoe trout food and Oregon moist and acclimated to test tem-
perature for 7 days prior to test. Test chambers were glass (19.5 x 20.5
x 21 cm) . Ninety-five percent replacement of water in the test chambers
was made in 30 min. Sixteen tests on juvenile trout were run in glass
150
-------
chambers (50.8 x 25.4 x 25.4 cm) with the outlet adjusted to maintain a
volume of 25 liters. The same diluters used for eggs and fry were used
for juveniles. Ninety-five percent replacement of water was made in
3 hr 20 min. Fish were not fed during first 96 hr of test but received
food thereafter.
Tests for H«S were done from water at outlet five times per day. Fluo-
rescent light was applied to all tests for 12 of each 24 hr. In each
test of eggs, fry, and juveniles five levels of toxicant and one control
were used.
Acute Toxicity
Eyed Eggs—Eyed eggs were used in acute tests. H.S concentrations
ranged from 0.0189 to 0.1026 mg/liter. No 96-hr LC50 was calculated.
Mean lethal threshold concentrations (LTC) were 0.0761 mg/liter H2S
at 8.5 C in 240 and 312 hr and 0.0501 mg/liter H2S at 13.5 C in 192
and 216 hr (Table 73). At 9.4 C and 8.9 C, 50% mortality had not
occurred after 460 and 108 hr, respectively, because treatment levels
were not sufficiently high.
Sac Fry—Sac fry 48 hr after hatch were tested over a range of H~S
concentrations of 0.0077-0.0370 mg/liter. Mean 96-hr LC50 and lethal
threshold concentration in 240 hr at 8.5 C were 0.0210 and 0.0160
mg/liter lUS, respectively (Table 74). Mean 96-hr LC50 and lethal
threshold con<
respectively.
threshold concentrations at 13.5 C were 0.0148 and 0.0120 mg/liter H S,
Feeding Fry—Feeding fry were tested over a range of fLS concentrations
of 0.0140-0.0255 mg/liter. Mean LTC's for 8.5 and 13.5 C were 0.022
and 0.186 mg/liter H S, respectively (Table 75).
Juveniles—Prior to testing juveniles were acclimated at the test tem-
151
-------
Table 73. THRESHOLD TOXICITY (LTC) OF H2S TO BROOK TROUT EGGSa
Cn
to
Mean
tempera- Mean
Test ture, C
1 9.4
2 8.9
3 8.5
4 8.5
5 13.5
6 13.5
pH
7.71 0
(0
7.76 0
(0
7.67 0
(0
7.71 0
(0
7.70 0
(0
7.72 0
(0
H^S concentrations, mg/liter LTC3
me/liter, H^S Hours
.0325
.0087)
.0586
.0149)
.0290
.0044)
.0277
.0049)
.0189
.0102)
.0199
.0076)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0407
0084)
0728
0306)
0391
0067)
0364
0032)
0351
0146)
0349
0132)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0461
0167)
0968
0171)
0551
0097)
0488
0030)
0450
0155)
0416
0143)
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
.0538
.0121)
.1062
.0383)
.0725 0.0915 0.0783
.0122) (0.0170)
.0632 0.0805 0.0738
.0047) (0.0101)
.0583 - 0.0516
.0213)
.0541 - 0.0485
.0157)
460
108
312
240
192
216
Standard deviations in parentheses; eyed eggs used for tests.
-------
Table 74. 96-HOUR LC50 AND LTC VALUES OF
TO BROOK TROUT SAC FRY
Ul
to
Mean
tempera- Mean
Test ture, C pH
1 8.5 7.69 0
(0
2 13.5 7.67 0
(0
3 8.5 7.69 0
(0
4 13.5 7.67 0
(0
H^S concentrations ,
.0126
.0012)
.0077
.0016)
.0121
.0013)
.0084
.0012)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0160
0030)
0098
0012)
0162
0019)
0120
0022)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0216
0036)
0128
0022)
0214
0028)
0149
0013)
0
(0
0
(0
0
(0
0
(0
mg/liter
.0291
.0036)
.0151
.0026)
.0279
.0019)
.0176
.0013)
0
(0
0
(0
0
(0
0
(0
96-hour
LC50,
mg/liter
H.S
.0370 0.0206
.0064)
.0225 0.0138
.0047)
.0363 0.0214
.0024)
.0245 0.0158
.0009)
LTC
mg/liter Hours
BUS
0.0161 240
0.0117 240
0.0160 240
0.0124 240
Standard deviations in parentheses.
-------
Table 75. 96-HOUR LC50 AND LTC VALUES OF
TO BROOK TROUT FEEDING FRY'
01
Mean
tempera-
ture , Mean
mg/liter
9 6 -hour
LC50, LTC,
Test C
1 8.5
2 13.5
3 13.5
4 8.5
pH
7.69 0
(0
7.74 0
(0
7.71 0
(0
7.73 0
(0
.0180
.0020)
.0150
.0025)
.0140
.0014)
.0185
.0018)
0
(0
0
(0
0
(0
0
(0
.0210
.0020)
.0171
.0029)
.0171
.0019)
.0218
.0019)
0
(0
0
(0
0
(0
0
(0
7
.0220
.0022)
.0200
.0029)
.0189
.0016)
.0230
.0022)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0235
0027)
0220
0035)
0206
0020)
0242
0027)
0
(0
0
(0
0
(0
HC TJ O
m^J n.,-,0
.0255 0.0234 0.0220
.0029)
.0250 0.0215 0.0186
.0037)
.0240 0.0217 0.0187
.0022)
0.0232 0.0226
—
Hours
240
216
216
240
Standard deviations in parentheses; fry 95 days after fertilization of eggs.
-------
peratures for 7 days except at 18.5 and 21 C when 14-day periods were
used. Values were obtained for 96-hr LC50 and threshold (LTC)
after 144 to 244 hr at 8.5, 11, 13.5, 16.0, 18.5, and 21.0 C. Sixteen
tests were conducted over a period from April to October and mean size
increased from 5 to 16 cm in successive bioassays.
Fish were tested over a range of H-S concentrations of 0.0053 to 0.0346
mg/liter. Mean 96-hr LC50's showed a range of 0.0266 mg/liter H?S at
8.5 C to 0.0178 mg/liter H2S at 21 C. Mean lethal threshold concentra-
tions had a range of 0.0197 mg/liter at 8.5 C to 0.0078 mg/liter H2S
at 21 C (Table 76, Figure 6).
The percentage changes in mean 96-hr LC50 and threshold values for each
2.5 C increment from 8 to 21 C were determined (Table 77). Mean 96-hr
LC50 values decreased 33.1% with increasing temperature and mean
threshold values decreased 60.4% from 8 to 21 C (Figure 7).
Mean times in hours to threshold LC50 decreased with increasing tempera-
ture from 8.5 to 13.5 C, but began to increase with increasing tempera-
ture between 13.5 and 16 C (Table 77). At the lowest temperature
tested (8.5 C) deaths occurred slowly and evenly throughout the test.
Intermediate temperatures (11, 13.5, and 16 C) produced death less slowly
but evenly over a shorter time period. At higher temperatures (18.5 and
21 C) most deaths occurred quickly, but some occurred unevenly over a
long time period. In the bioassays at higher temperatures most mor-
tality occurred quickly, but some continued sporadically over extended
time periods (144 and 288 hr; Table 76) similar in duration to the low
temperature bioassays.
Influence of Temperature on Various Stages—To determine the effects of
temperature on acute toxicity to all life history stages except adults
(eyed egg, sac fry, feeding fry, and juvenile) and the relative resis-
tance of various stages, a comparison was made of the H«S threshold
155
-------
Table 76. 96-HOUR LC50 AND LTC VALUES OF H2S TO BROOK TROUT JUVENILES
Mean
tempera-
ture , Mean
Test C
1 8.1
2 8.1
3 8.5
4 11.0
5 11.1
6 13.5
7 13.6
8 13.4
PH
7.72 0
(0
7.71 0
(0
7.69 0
(0
7.69 0
(0
7.70 0
(0
7.68 0
(0
7.78 0
(0
7.85 0
(0
H^S concentrations , mR/liter
.0165
.0034)
.0166
.0030)
.0148
.0020)
.0107
.0016)
.0089
.0014)
.0109
.0019)
.0116
.0142)
.0107
.0050)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0178
0039)
0189
0033)
0181
0027)
0140
0033)
0141
0019)
0136
0017)
0196
0217)
0143
0056)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0200
0041)
0208
0048)
0213
0049)
0175
0034)
0170
0023)
0172
0018)
0224
0244)
0156
0081)
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
.0226
.0042)
.0235
.0043)
.0254
.0044)
.0214
.0025)
.0210
.0046)
.0211
.0016)
.0233
.0301)
.0183
.0086)
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
0
(0
9 6 -hour
LC50, LTC,
mg/liter mg/liter
H^S H^S
•£• /-
.0251 0.0245 0.0187
.0049)
.0245 0.0256 0.0191
.0058)
.0346 0.0297 0.0212
.0033)
.0264 0.0228 0.0171
.0038)
.0258 0.0233 0.0184
.0041)
.0252 0.0219 0.0153
.0027)
.0271 0.0174
.0302)
0.0156 0.0156
-
Hours
264
288
288
216
216
264
144
144
-------
Table 76 (continued). 96-HOUR LC50 AND LTC VALUES OF H2S TO BROOK TROUT JUVENILES
en
-q
Mean
tempera-
ture , Mean
Test C pH
9 15.9 7.73 0
(0
10 15.9 7.72 0
(0
11 16.0 7.67 0
(0
12 16.0 7.67 0
(0
13 18.5 7.67 0
(0
14 18.5 7.67 0
(0
15 21.0 7.68 0
(0
16 21.0 7.70 0
(0
o
H^S concentrations
£.
.0125
.0028)
.0124
.0021)
.0097
.0024)
.0094
.0018)
.0116
.0020)
.0110
.0015)
.0063
.0016)
.0053
.0013)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0141
0020)
0149
0028)
0120
0016)
0117
0024)
0123
0023)
0119
0023)
0083
0013)
0074
0015)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0149
0021)
0168
0030)
0133
0021)
0129
0020)
0138
0022)
0140
0018)
0111
0021)
0103
0015)
, mg/liter
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0171
0028)
0183
0027)
0155
0022)
0153
0022)
0156
0018)
0154
0019)
0146
0025)
0155
0018)
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
0.
(0.
96-hour
LC50, LTC,
mg/liter rag/liter
HnS H^S
0188 0.0163 0.0141
0046)
0189 0.0173 0.0146
0022)
0171 - 0.0129
0029)
0178 - 0.0137
0021)
0173 0.0168
0025)
0173 0.0168
0022)
0183 0.0177 0.0078
0034)
0207 0.0179 0.0078
0018)
Hours
192
192
240
240
-
-
240
288
Standard deviations in parentheses.
-------
21 C
8.5 C
288-!
264-*
240-
216-
192
QC
r>
O
x
168'
5 144-
UJ
H 120-
96-
72'
.008 .010 .012 .014 .016 .018 .020 .022 .024 .026 .028
LC50 CONCENTRATION
Figure 6. Toxicity curves for brook trout juveniles at 2.5 C intervals
from 8.5 to 21.0 C (expressed as mg/liter H?S).
158
-------
Table 77. A COMPARISON OF 96-HOUR LC50 AND LTC VALUES OF H S FOR 2.5 C
INCREMENTS FROM 8.5 TO 21.0 C IN BROOK TROUT JUVENILES3
Number
of
tests
3
2
3
4
2
1
15
Tempera-
ture,
C
8.5
11.0
13.5
16.0
18.5
21.0
8.5-
21.0
Mean,
mg/1
0.0266
0.0230
0.0183
0.0168
0.0168
0.0178
96-hr LC50
Mean difference,
mg/1 %
-0.0036 -13.6
-0.0047 -20.5
-0.0015 -8.2
0 0
+0.0010 +6.0
-0.0088 -33.1
LTC Mean
Mean, Mean difference, time,
mg/1 mg/1 % hr
0.0197 280
-0.0019 -9.6
0.0178 216
-0.0023 -12.9
0.0155 204
-0.0017 -11.0
0.0138 216
-
-
-
0.0078 252
-0.0119 -60.4 234
Mean differences are between LC50 or LTC at successive temperatures.
159
-------
.027-
.025-
.023-
.021-
.019-
cc
o
z
o
u
o
m
O
.015-
.013-
.011-
.009-
.007-
LETHAL THRESHOLD
10 12 14 16
TEMPERATURE C
18
20
22
Figure 7. Mean 96-hr LC50's (quadratic) and lethal threshold concen-
trations (linear) for brook trout at 8.5 to 21.0 C (expressed
as ing/liter H2S; 96-hr LC50 fitted by eye).
160
-------
values (Table 78). At 8.5 and 13.5 C the most resistant stage to H2S
was the egg, followed by feeding fry, juvenile, and sac fry in that
order (Table 79). Eggs were more than three times as tolerant as juve-
niles. The influence of temperature on the various stages was greatest
in eggs where a 34.2% decrease in resistance occurred with an increase
in temperature from 8.5 to 13.5 C. The least difference was in feeding
fry with a decrease of 16.1%. The resistance of eyed eggs to H_S
toxicity is probably due to the low respiration rates of incubating
trout eggs. Sac fry susceptibility to H-S toxicity is due to a variety
19
of reasons (Olson and Marking ): large surface area capable of absorp-
tion of H_S, no well developed detoxifying system, and a high metabolic
rate.
CHRONIC TESTS
Three chronic bioassays were run on juveniles and adult brook trout to
determine the effects of low concentrations of H_S on growth of juve-
niles, reproduction by adults, and swimming endurance of juveniles.
Experimental Design
Two chronic tests running for 72 and 120 days were conducted on brook
trout to determine effects of H?S on growth. Fish in the 72-day test
were started as 23-day-old fry with a mean weight of 0.09 g. The 120-
day test was started with 166-day-old juveniles with a mean weight of
5 g. Proportional diluters previously described were used to supply
water and toxicant to the test chambers. In the 72-day experiment," 25
fry were placed in each of 10 aquaria (51 x 24 x 20 cm). After 30 days
fish from the aquaria were placed in 10 fiber glass tanks (164 x 56 x 52
cm), each divided into two equal parts containing 28 liters. The experi-
ment consisted of eight H?S levels ranging from 0.0015 to 0.0142 mg/
liter and two controls (Table 80). Mean pH was 7.72 (7.59-7.95), tem-
perature 9.0 C (9.0-9.1), and oxygen 7.9 mg/liter (7.8-8.0). Fish were
fed ad libitum Oregon moist and Glencoe pellets two times per day.
Periodic weighings of fish were made en masse from each tank. They were
161
-------
Table 78. PERCENTAGE DIFFERENCES IN MEAN LTC VALUES OF H2S BETWEEN
8.5 AND 13.5 C FOR EACH LIFE HISTORY STAGE OF BROOK TROUT
Stage
Temperature,
C
Mean LTC, Mean LTC time,
mg/1 H0S % change hr
Eyed egg
8.5
13.5
0.0760
0.0500
-34.2
276
204
Sac fry
8.5
13.5
0.0160
0.0120
-25.0
240
240
Feeding fry 8.5
13.5
0.0223
0.0187
-16.1
244
216
Juvenile
8.5
13.5
0.0197
0.0154
-21.8
280
204
162
-------
Table 79. PERCENTAGE DIFFERENCES IN MEAN LTC VALUES OF H2S BETWEEN
EACH SUCCESSIVE LIFE HISTORY STAGE OF BROOK TROUT AT 8.5 AND 13.5 C
Stage
8.5 C
13.5 C
Mean LTC, Mean LTC,
mg/liter H^S % change mg/liter H^S % change
Eyed egg
Sac fry
Feeding fry
Juvenile
0.0760
0.0160
0.0223
0.0197
-78.9
+30.9
-11.7
0.0500
0.0120
0.0187
0.0154
-76.0
+55.8
-17.6
163
-------
transferred to water in a beaker and differential weight between water
and water containing fish was determined.
In the 120-day test 20 juvenile fish were placed in each of the 10 fiber
glass tanks described above with eight levels of toxicant ranging from
0.0015 to 0.0130 ing/liter and two controls (Table 81). Fish were fed
ad libitum Oregon moist and Glencoe pellets two times per day. Mean pH
was 7.69 (7.30-8.15), temperature 13.2 C (13.0-13.3), and oxygen 7.8
mg/liter (7.6-8.3). Weighing was done en masse as in the 72-day test.
Flow-through rate in both tests was 600 ml/min. H?S was tested from
water taken out of the central portion of the tank five times per week.
Growth Rate
In the 72-day test changes in growth rate were not progressive with
increased concentrations of H-S but at 0.0032 mg/liter and higher some
reduction occurred (Table 80). After 60 days there was a marked decrease
in growth rate at 0.0090 mg/liter and higher levels. At the conclusion
of the test fish kept in 0.0140 mg/liter H2S were 45% smaller than the
mean of the controls. Since no statistical treatment on the basis of
individual fish was made, exact significance of the effect of fish varia-
bility on apparent growth difference cannot be made. On the assumption
that difference less than 10% may not have been significant, it is
believed that no adverse effect was shown at concentrations less than
0.0066 mg/liter H S.
The 120-day test started with 166-day-old juveniles showed depressed
growth rate at all treatment levels (Table 81). After 60 dgys, treat-
ments with H2S concentration of 0.0090 mg/liter and higher had mean
weights 16 to 24% lower than the controls. After 90 days, growth was
6% below controls at 0.0067 mg/liter and 22% below at 0.0090 mg/liter.
After 120 days, growth was 14% below control at 0.0067 mg/liter and 53%
below at 0.0125 mg/liter. The major growth reduction appeared to occur
between 0.0067 and 0.0090 mg/liter H S.
164
-------
Oi
en
Table 80. WEIGHT OF JUVENILE BROOK TROUT AT SUCCEEDING INTERVALS
IN VARIOUS H2S CONCENTRATIONS AT 9 C IN 72 DAYS3
(grains)
H0S concentration, me/liter
Day Control.,
j.
0 0.09
30 0.25
60 1.01
72 1.28
Total0 1.19
0.0015
Control,, Mean (0.0005)
£-
0.09 0.09 0.09
0.32 0.28 0.28
[0]
0.91 0.96 0.92
[-4]
1.22 1.25 1.24
[-1]
1.13 1.16 1.15
[0]
0.0032
(0.0011)
0.09
0.20
[-29]
0.92
[-4]
1.18
[-6]
1.09
[-6]
0.0051
(0.0014)
0.09
0.21
[-25]
1.02
[+6]
1.15
[-8]
1.06
[-8]
0.0066
(0.0018)
0.09
0.29
[+4]
0.96
[0]
1.21
[-3]
1.12
[-3]
0.0090
(0.0027)
0.09
0.23
[-18]
0.86
[-10]
1.07
[-14]
0.98
[-15]
0.0119
(0.0018)
0.09
0.24
[-14]
0.71
[-26]
0.86
[-31]
0.77
[-33]
0.0140
(0.0015)
0.09
0.16
[-43]
0.55
[-33]
0.72
[-42]
0.63
[-45]
0.0142
(0.0032)
0.09
0.21
[-25]
0.61
[-36]
0.74
[-41]
0.65
[-43]
Q
Percentage comparison of weight in various H^S concentrations with the mean weight of control groups
in brackets.
Standard deviations in parentheses.
Increment in 72 days.
-------
Ol
Oi
Table 81. WEIGHT OF JUVENILE BROOK TROUT IN VARIOUS H2S CONCENTRATIONS AT 13 C IN 120 DAYS3
(grams)
H^S concentration, ing/liter
Day Control., Control,-, Mean
-L /
0 5.0 5.0 5.0
30 7.7 7.5 7.6
60 12.3 12.4 12.4
90 19.2 18.9 19.0
120 33.2 33.3 33.2
Totald28.2 28.3 28.2
0.0015
(0.0015)
5.0
7.2
[-5]
11.5
[-7]
18.8
[-1]
30.2
[-9]
25.2
[-11]
0.0034
(0.0015)
5.0
7.4
[-3]
11.6
[-6]
17.8
[-6]
26.4
[-20]
21.4
[-24]
£.
0.0050
(0.0013)
5.0
7.6
[0]
11.5
[-7]
17.0
[-11]
30.3
[-9]
25.3
[-10]
0.0067
(0.0017)
5.0
7.2
E-5]
12.0
[-3]
17.8
[-6]
29.2
[-12]
24.2
[-14]
0.0090
(0.0030)
5.0
6.4
[-16]
9.4
[-24]
14.8
[-22]
23.2
[-30]
18.2
[-35]
0.0097
(0.0024)
5.0
7.0
[-8]
11.0
[-24]
16.6
[-13]
26.5
[-20]
21.5
[-24]
0.0125
(0.0033)
5.0
6.3
[-17]
10.4
[-16]
13.5
[-29]
18.2
[-45]
13.2
[-53]
0.0130
(0.0029)
5.0
6.5
[-14]
10.4
[-16]
15.0
[-21]
c
-
-
-
percentage comparison! of weight in various H^S concentrations with the mean weight of control groups
in brackets.
Standard deviations in parentheses.
This tank not weighed due to poor survival (35%) of smaller fish.
Increment in 120 days.
-------
Reproduction
A third chronic test was started with adult brook trout to determine
the influence of prespawning exposure to spawning success. The experi-
mental design was the same used for the previous chronic tests except
that spawning boxes were provided shortly prior to anticipated first
spawning. The spawning boxes consisted of marine plywood with two
fiber glass screen windows. Box size was 51 x 58 x 16 cm. Temperatures
were gradually reduced from 13.8 C in August to 9 C in October. A
nominal temperature of 9 C was then maintained throughout the spawning
and subsequent incubation period. One hundred trout, each weighing
approximately 500 g, were distributed between 10 tanks on August 8 with
five males and five females in each tank. Size grading at the hatchery
where fish were secured provided an extremely uniform stock. To insure
sex identification during bioassay, adipose fins on male trout were
clipped. Test concentrations varied from 0.0055 to 0.0128 mg/liter H2S
(Table 82). Equipment malfunction eliminated three treatments at 0.0010,
0.0030, and 0.0050 mg/liter. Mean pH was maintained at 7.78 (7.40-8.23),
temperature at 9.1 C (6.2-13.8), and mean oxygen at 7.5 mg/liter. H-S
concentrations were checked three times per week.
Fifty-five days after start of treatment on October 5 trout in the
control first spawned and continued thereafter until October 18 (Table
82). In most cases spawning took place in the early morning hours and
eggs were removed from spawning boxes within 12 hr of deposition. A
spawning was defined as deposition of 50 or more eggs in the spawning
box. Two days after spawning started in the control, spawning started
in a concentration of 0.0055 mg/liter H2S, the lowest treatment. Seven
spawnings were made over the following 27-day period until October 30.
Four females spawned between October 4 and 10 but there was no further
spawning until October 27 and 30, when the fifth female spawned twice
(Table 83). At a concentration of 0.0079 mg/liter H2S spawning started
on October 5. By October 15 five of the six total spawnings had taken
place. On October 26 the final spawning occurred. With a treatment of
167
-------
Table-82. BROOK TROUT REPRODUCTION DATA IN VARIOUS H S CONCENTRATIONS
00
H-S Number
Tank
10
3
9
6
7
8
concentration ,
a/
mg/liter—
control
0.0055(0.0021)
0.0079(0.0024)
0.0109(0.0026)
0.0121(0.0030)
0.0128(0.0043)
Spawnings
10
7
6
1
3
3
Dates
10/2-10/18
10/4-10/30
10/5-10/26
10/31
10/20-10/30
10/6-10/10
Number
of eggs
3,797
2,222
1,731
328
1,254
1.396
all°veb/
76.8
73.6
74.1
70.4
52.4
72.7
of
M
4
5
5
5
4
4
fish
F
5
5
5
5
4
5
Mean eggs/
spawning
380
317
288
328
418
465
Mean eggs/
female
759
444
346
66
314
279
a/
—.Standard deviations in parentheses.
— Percentage of eggs alive immediately after spawning (within 12 hours).
-------
Table 83. SPAWNING DATES OF BROOK TROUT DURING THE MONTH OF OCTOBER
H?S concentration, mg/liter
Control 0.0055 0.0079 0.0109 0.0121 0.0128
2 4 5 31 20 6
2 47 27 8
4 68 30 10
4 7 14
6 9 15
9 27 26
10 30
12
16
18
169
-------
0.0109 mg/liter H?S a single spawning took place on October 31, 29 days
after initial spawning in the control. Throughout much of the period
preceding egg deposition in this concentration spawning behavior was
greatly depressed. Occasionally a female lingered in the spawning box
but males showed no interest. Not until October 24 was any significant
activity noted, when a female fanned out the spawning box. The initial
spawning at 0.0121 mg/liter H~S occurred on October 20. Two more
spawnings followed on October 27 and 30. In this concentration spawning
activity was also greatly depressed throughout the greater part of the
month. On October 19 the first fanning of the spawning box was noted.
In each case spawning in this tank followed a 24-hr period of accidentally
decreased H_S concentration. It will be noted from the standard deviation
+0.0030 mg/liter that variability of H2S concentration was quite large.
In the highest concentration (0.0128 mg/liter H-S) trout were being held
at near the lethal concentration. Any increase over that target level
caused the trout great stress. There were large variations in the
standard deviation of concentration in this tank and the three spawnings
which did occur on October 6, 8, and 10 followed a 3-day period of de-
creased H-S levels. The concentration was reduced to prevent mortality
following a slug of higher concentration. It was evident that fish at
all H^S concentrations tested were capable of spawning throughout Oc-
tober if the stress were temporarily relieved. However, at 0.0109
mg/liter H-S and higher fish were prevented from spawning (that is, they
showed little or no interest in spawning activity) unless short periods
of reduced H-S concentration occurred. All spawning activity was stopped
at 0.0109, 0.0121, and 0.0128 mg/liter H2S when target levels were re-
stored. It is believed that consistently maintained levels of 0.0109
mg/liter and higher would totally prevent spawning. The tes\ was con-
tinued until January but no spawnings occurred after October 30.
In the control there was an average of two spawnings per female with
the first spawning usually producing the largest number of eggs. Al-
though the number of spawnings by individual females is not known, in
no case was the average of two spawnings per female obtained in the H S
170
-------
concentrations. The percentage of eggs alive immediately after spawning
in the treatments differed little from that in the control (Table 82).
The number of eggs deposited per female decreased with increased con-
centration of H_S (Table 82). The total number of spawnings, the total
number of eggs spawned per tank, and the number of eggs per female de-
creased rapidly with increased concentration. The control had 759 eggs
per female and the H^S treatments varied from 444 in the lowest concen-
tration to 279 in the highest concentration. At the lower levels where
spawning activity was not greatly depressed the effective spawning period
was much extended.
Viability of Eggs—After each spawning the eggs were removed from the
spawning box and a random sample placed in an incubation basket in the
treatment where eggs were spawned and also in the control tank. Eggs
from successive spawnings were randomly distributed to the same baskets
as the first spawning. Samples of eggs deposited in the control tank
were placed in baskets in the control and also in each of the treatment
levels (Table 84). Baskets were attached to oscillating arms which
maintained water movement over the eggs. Hatching success of eggs
spawned and incubated in the control tank was 77%. The best hatch of
eggs spawned in any H?S treatment and incubated in the control tank was
30% (Table 84). Eggs spawned in the control tank and incubated in
various H_S treatments had much better hatching success than eggs spawned
and incubated in the same H S treatments. This difference may be caused
by water absorption immediately following egg deposition for a 30-hr
period. Eggs spawned and incubated in individual treatments may absorb
a lethal or near-lethal dosage of H2S. Eggs spawned in a treatment tank
but incubated in a control tank had decreased mortality. Eggs spawned
in the control tank and incubated in the various treatment tanks had
survival levels only slightly less than those spawned and incubated in
controls.
171
-------
Table 84. PERCENTAGE HATCH OF BROOK TROUT EGGS, DAYS
TO HATCH, AND LENGTH OF FRY AT HATCH
Eggs spawned in control and incubated in H2S treatments (Group A); eggs
spawned in each treatment and incubated in same treatment (Group B); and
eggs spawned in each treatment and incubated in control tank (Group C).
H2S
concentration,
Q
Group mg/ liter
A 0
0
0
0
0
B 0
0
0
0
0
.0055
.0079
.0109
.0121
.0128
.0055
.0079
.0109
.0121
.0128
(0
(0
(0
(0
(0
(0
(0
(0
(0
(0
.0021)
.0024)
.0026)
.0030)
.0043)
.0021)
.0024)
.0026)
.0030)
.0043)
C Control
0
0
0
0
0
.0055
.0079
.0109
.0121
.0128
(0
(0
(0
(0
(0
.0021)
.0024)
.0026)
.0030)
.0043)
Number
of eggs
150
200
150
200
70
225
250
100
250
150
100
192
225
100
150
150
Number
hatched
59
104
79
123
49
87
13
ob
ob
37
77
54
67
0
28
42
7
/o
hatch
39
52
52
61
70
38
5
0
0
24
77
28
29
0
18
28
.3
.0
.7
.5
.0
.7
.2
.7
.0
.1
.8
.7
.0
Mean fry
length, Days to
cm hatch
2.
2.
2.
2.
2.
2.
1.
_
-
2.
2.
2.
2.
-
2.
2.
0
0
0
0
0
0
9
1
0
0
1
0
1
54.
56.
55.
59.
56.
49.
51.
_
-
61.
57.
57.
57.
-
55.
56.
5
2
8
2
4
8
8
4
8
6
2
4
4
cl w
Standard deviation in parentheses. Treatment concentrations are those
continued through prespawning, spawning, and incubation periods.
Loss due to temporary malfunction of apparatus.
172
-------
Examination of hatching data indicates that eggs spawned in a treatment
and incubated in a treatment have poor resistance down to 0.0055 mg/
liter H«S (39% survival). Survival of control spawned and incubated
eggs (77%) at 9 C appears to be a good measure of spawning and hatching
success under experimental conditions and is consistent with the observed
production hatchery practice. The length of fry at hatch (Table 84)
does not appear to be affected by exposure of eggs or adults prior to
spawning. Days to mean hatch were not significantly affected by H_S
concentrations in the two lowest concentrations except that eggs
hatched slightly faster than controls. In the higher levels eggs
hatched somewhat slower than controls.
Swimming Endurance
After 45 and 120 days of exposure in the 120-day chronic growth bioassay
previously described, 10 fish from each control and six treatment levels
(70 fish) were tested for swimming endurance in a raceway filled with
untreated laboratory water at 13 C. The raceway consisted of a revol-
ving acrylic hood, 128 cm in diameter, covering a circular channel.
The revolving hood which had alternate strips of black markings gave
the test fish an illusion of water movement in the raceway and caused
it to swim around the circular channel. An electrical barrier of probes
attached to the hood forced the fish to swim at the revolving hood
velocity. When the fish could no longer keep pace with the hood, it
fell against or through the probes and was stunned. Two simulated water
velocities, 50.3 cm/sec and 66.4 cm/sec, were used after 45 days and
120 days of exposure, respectively. The time in seconds to failure was
noted for each H S concentration. After the 45-day tests the two highest
H_S concentrations were increased from mean values of 0.0114 and 0.0119
mg/liter to 0.0125 and 0.0130 mg/liter (Table 85). A consistent decrease
in endurance times with an increase in ELS concentration was observed.
A multiple range test at 99% confidence level showed significant dif-
ferences between performance of control fish and fish from treatments
of 0.0090 mg/liter H-S and greater after 45 and 120 days of chronic
173
-------
Table 85. SWIMMING ENDURANCE OF BROOK TROUT JUVENILES
AFTER 45 AND 120 DAYS OF EXPOSURE TO H2S AT 13 C&
(time in seconds)
Exposure,
45 days Control 0.0037
H0S concentration, mg/liter
0.0070 0.0090 0.0097 0.0114 0.0119
Mean 315 304 303 228 232 182 194
Range 221-456 168-616 134-610 168-288 188-302 100-261 94-415
120 days0 Control 0.0034 0.0067 0.0090 0.0097 0.0125 0.013Qd
Mean 376 358 345 214 182 182
Range 280-545 240-435 225-530 125-230 135-230 75-300
Raceway hood velocity set at 50.3 cm/sec (45 days) and 66.4 cm/sec
/120 days); 10 fish per treatment level.
Approximately 35% of the fish in this tank died and most were smaller
than the average size fish in the tank.
°After 45 days of exposure to lower levels (0.0037-0.0119 mg/liter)
concentrations were raised above starting levels in the two highest
treatments.
After 120 days poor fish survival (35%) and poor fish condition pre-
vented testing.
174
-------
exposure (Table 85). Direct examination of the fish during endurance
tests often showed that fish larger and smaller than average swam for
shorter times than the average within any given treatment, particularly
at the higher concentrations. Endurance time, however, did not differ
greatly with size of fish within a given concentration.
SUMMARY
Acute test were run on brook trout eggs, sac fry, feeding fry, and
juveniles to determine 96-hr LC50's and LTC values up to 460 hr. These
tests showed that at 13.5 C the most resistant stage was the eyed egg
(LTC 0.0501 mg/liter H2S), followed by feeding fry (LTC 0.0186 mg/liter),
juvenile (LTC 0.0155 mg/liter), and sac fry (LTC 0.0120 mg/liter) in
that order. The greatest influence of temperature on resistance to H«S
was noted in the egg stage and the least difference in the feeding fry.
With increase in temperature from 8.5 to 21.0 C there was a steady
decrease in resistance of juveniles to tLS. A slightly greater than
twofold decrease occurred in LTC and about 35% decrease in 96-hr LC50.
Chronic tests running up to 120 days indicated that growth rate at all
H~S concentrations was lower than in the control. Reproduction was
reduced in all H^S concentrations tested. The number of spawnings per
female was less and the number of eggs per spawning was reduced from
that in controls. At all levels of H«S there was a definite inhibition
of spawning success and at 0.0079 mg/liter and higher reduction was to
less than half the controls. The effect of H«S appeared to be primarily
suppression in spawning behavior. Swimming endurance was significantly
reduced in all fish exposed for periods of 45 to 120 days at 0.0090
mg/liter H«S and higher concentrations tested. At the highest level
(0.0125 mg/liter) after 120 days of exposure, swimming endurance
expressed in seconds to exhaustion was approximately half that of the
controls. The data show that levels for protecting all life history
stages and functions will be less than 0.0055 mg/liter HS.
175
-------
SECTION X
RAINBOW TROUT
(Salmo gairdneri Richardson)
Toxicity of H?S to rainbow trout was tested by (1) acute tests on sperm
viability, success of fertilization, eggs, fry, and juveniles and (2)
chronic tests to determine survival, growth rate in H2S and in a com-
bination of H«S and phenol.
ACUTE TESTS
Experimental Design
The acute tests of H~S toxicity on rainbow trout include one on sperm
viability, one on success of fertilization, one on eggs, one on fry,
and two on juveniles (Table 86). The test on sperm viability (Acute 1)
was conducted by stripping males taken from 13 C water and held for 2
days at 10 C. One male was used for each series with no pooling of
sperm. Sperm were distributed at random in each series. One drop of
sperm was placed in control or HLS concentration water contained in a
Syracuse watch glass. A drop of mixture was immediately placed on a
slide and duration of motility determined under a microscope (200x)
equipped with heat-absorbing filter over the light source. The fer-
tilization experiment (Acute 2) was done by stripping male %nd female
fish treated as in the previous test. Ten cc of unfertilized eggs were
placed in water from control or H?S treatment and 2 drops of milt were
added and mixture stirred gently. The mixture was held for 3 min and
eggs were then transferred to control water and incubated for 10 days
176
-------
Table 86. SOURCE OF RAINBOW TROUT AND STAGE OF FISH
AT START OF TESTS WITH HS
Test
Acute
1
2
3
4
5
6
Chronic
1
2
3
4
5
6
7
Stage
at start
Sperm
Fertilization
Egg
Fry
Juvenile
Juvenile
Fry and
juvenile
Eyed egg
Newly ferti-
lized eggs
Eyed egg
Eyed egg
Eyed egg
Juvenile
Date
obtained Source
2/11/71 State Hatchery, Lanesboro, Minn.
2/11/71 " " " "
2/11/71 " "
2/11/71
23/3/70 White's Trout Farm, Paradise, Utah
2/11/71 State Hatchery, Lanesboro, Minn.
23/3/70 White's Trout Farm, Paradise, Utah
5/10/71 Ennis National Fish Hatchery, Ennis,
Montana
3/11/71 State Hatchery, Lanesboro, Minn.
3/11/71 " " "
16/12/71 White's Trout Farm, Paradise, Utah
16/12/71
16/12/71
177
-------
in a Heath-Techna hatching battery with laboratory water at 12.2 C
(Table 87). At 10 days the percentage of fertile eggs was determined.
The acute egg test (Acute 3) was started with eggs stripped from adults
as described for the previous test- Eggs were fertilized in control
water and after water hardening for 2 hr were placed in test tanks at
various H.S concentrations. Twelve H2S concentrations and three controls
were maintained through egg hatch at 12.6 C. Test chambers were glass-
silicone (20 x 20 x 20 cm) with a water volume of 6 liters. Eggs rested
on the bottom of the chambers. The apparatus for dispensing toxicant
4
was a modified Mount and Brungs diluter which provided a flow-through
rate of 200 ml/min. Test chambers were illuminated at 12 hr per day
with 40-watt incandescent bulbs.
The fry test (Acute 4) was started with fry hatched from eggs incubated
in control water as in previous tests. At hatching the fry were placed
in twelve ELS concentrations and three controls. Light was controlled
as in the egg test.
Two juvenile tests (Acute 5 and 6) were started with fish 45 and 54 mm
long, respectively. Flow-through apparatus as described by Adelman and
2
Smith was used in both tests. Test chambers were glass-silicone type
(50 x 25 x 20 cm) containing 20 liters of water. Flow-through rate was
300 ml/min. Fish were reared from eggs at 12 C and were fed Glencoe
fry granules up to the time of testing. No feeding was done during the
96 hr of the tests. Feeding with Glencoe granules was resumed in test
5 which ran more than 96 hr. Illumination was by fluorescent tubes.
In all acute tests samples of water were taken daily from the center of
each test chamber and tested for temperature, pH, dissolved 09, and
total alkalinity. Water samples for ILS determinations were taken
twice daily from the center of each test chamber. Day length in acute
tests 5 and 6 was 12 hr.
178
-------
Table 87. ACUTE TEST CONDITIONS AND LC50 VALUES FOR RAINBOW TROUT TESTED IN
Test
1
2
3
4
5
6
Stage
Sperm
Fertili-
zation
Egg
Fry
Juvenile
Juvenile
Days from
collection
to start
of test
2
3
1
31
91
113
Mean test
Number
fish per
chamber
—
—
150
30
10
10
Mean conditions
length, Temp.,
mm C
12.2
12 . 2
12.6
22 13.1
45 12.3
54 15.1
0
mg/1
9.4
9.4
9.4
7.8
8.4
6.4
LC50,
me/1 H.S
48 hr 72 hr 96 hr LTC (days)
_
_
- 0.0154 (29)
- 0.0056 (20)
0.0130 0.0087 (17)
0.0150 0.0130 0.0125 -
pH 7.7 (average of meter readings).
-------
Sperm Motility
Newly ejected sperm were placed in ELS concentrations ranging from
0.0022 to 0.0589 mg/liter in two replications each from a single male
(Table 88). In two replications of three control water tests (six
determinations), the mean survival time in seconds as judged by dis-
cernible motion under magnification varied from 68.5 to 97.5. In the
various ELS concentrations survival time varied between 82.0 and 92.5
sec. In all but one of the ELS treatments survival was slightly longer
than the mean of all controls.
Egg Fertility
Fertility of eggs inseminated in various ELS concentrations from 0.0096
to 0.0570 mg/liter and incubated for 10 days in control water at 12.2 C
showed no trends with increasing concentrations (Table 89).
Egg Survival
Eggs fertilized in control water and incubated in various ELS concen-
trations from 0.0045 to 0.0598 mg/liter had decreasing survival to
hatch with increase in ELS concentration (Table 90). From 0.0045 to
0.0236 mg/liter ELS survival of eggs was better than in controls. Above
that level survival was very low, with none hatching at 0.0598 mg/liter.
Length of fry at hatch did not vary significantly from the controls
except at 0.0469 mg/liter H S. The calculated LC50 of eggs at 29 days
when hatch was completed was 0.0154 mg/liter ELS.
Fry Survival
Fry were hatched from eggs incubated in control water and subjected to
various concentrations of ELS from 0.0027 to 0.0068 mg/liter for 20 days
(Table 91). In controls with three replications length of fry after 20
days was 26.0 mm. At all ELS concentrations growth was slightly slower
than in controls and markedly so at 0.0068 mg/liter ELS. Fry did not
survive in concentrations greater than 0.0068 mg/liter.
180
-------
Table 88. DURATION OF RAINBOW TROUT SPERM
VIABILITY IN H2S IN ACUTE TEST 1
H2S
concentration ,
mg/1
Control
Control
Control
0.0022
0.0031
0.0083
0.0086
0.0105
0.0164
0.0214
0.0246
0.0420
0.0436
0.0484
0.0589
Duration of motility,
seconds
1
69.0
84.0
73.0
103.0
88.0
84.0
88.0
90.0
98.0
79.0
95.0
76.0
89.0
77.0
79.0
2
68.0
111.0
74.0
83.0
67.0
78.0
97.0
89.0
94.0
95.0
79.0
92.0
92.0
87.0
100.0
Mean
68.5
97.5
73.5
93.0
77.5
81.0
92.5
89.5
96.0
87.0
87.0
84.0
90.5
82.0
89.5
181
-------
Table 89. FERTILITY OF RAINBOW TROUT EGGS INSEMINATED IN
H«S concentration,
rng/1
Control
0.0096
0.0113
0.0144
0.0158
0.0451
0.0570
Fertilization,3
%
84
75
78
90
85
87
94
Determined after 10 days by presence of an embryo.
182
-------
Table 90. LENGTH OF RAINBOW TROUT FRY AT HATCH AND PERCENTAGE
SURVIVAL OF EGGS INCUBATED IN H2S AT 12.6 C
H2S
concentration ,
mg/1
Control
Control
Control
0.0045
0.0067
0.0105
0.0116
0.0117
0.0236
0.0279
0.0293
0.0469
0.0598
Mean
length,
nun
13.8
13.8
13.5
14.1
13.7
14.0
13.6
13.4
12.9
13.1
13.2
11.5
—
Egg
survival
to hatch,
%
46b
27b
17b
66
87
16
87
70
55
7
8
4
0
Time to
first
hatch,
days
26
26
26
26
26
26
26
26
26
26
27
26
—" —
,A11 eggs dead or hatched in 29 days.
Survival in controls was low from excessive turbulence; therefore sur-
vival in other treatments was not corrected to controls.
183
-------
Table 91. LC50 VALUES FOR RAINBOW TROUT FRY AND LENGTH OF FRY
AT VARIOUS H S CONCENTRATIONS IN 20 DAYS OF EXPOSURE (ACUTE TEST 6)
LC50
Days
mg/1 H0S
Fry length (20 days)
H£S,
mg/1
Length,
TTTm
5
7
10
12
13
17
20
0.0127
0.0097
0.0079
0.0075
0.0076
0.0057
0.0056
Control
Control
Control
0.0027
0.0032
0.0037
0.0050
0.0054
0.0068
26.0
26.0
26.0
25.5
25.5
25.5
25.0
24.0
23.0
184
-------
Juvenile Survival
Two acute tests were run on juveniles: Acute 5 was run for 17 days in
H2S levels from 0.0014 to 0.0095 mg/liter and Acute 6 was run for 96 hr
at levels from 0.0075 to 0.0292 mg/liter. In Acute 6 the LC50 was
0.0125 mg/liter H2S at 96 hr. In Acute 5 the LC50 was 0.0130 mg/liter
at 96 hr and 0.0086 mg/liter at 10 days. The LC50 did not change
thereafter through 17 days of exposure (Table 92). At lower exposure
levels in 17 days juveniles grew faster in weight than average of con-
trols up to exposures of 0.0062 mg/liter. At 0.0095 mg/liter H-S mean
weight of fish was approximately half the mean of the controls.
CHRONIC TESTS
Experimental Design
Five chronic tests were run with rainbow trout; three with H?S (Chronic
1, 3, and 4), one with phenol (Chronic 2), and one with combined H~S
and phenol (Chronic 5) .
Chronic 1 was divided into three segments, a_, ^b_, and c^. Diluter appa-
ratus was the same as described for acute tests. In each segment of
the tests four H9S concentrations and one control were maintained (Table
93). Test chambers were 50 x 25 x 20 cm with 16-liter volume. Flow
rates were 134 ml/min. Test 1-a was started with newly hatched fry,
l-b_ with 10-day-old fry, and l-c_ with 50-day-old juveniles. The test
was started with 30 fish per tank. They were fed Glencoe fry granules.
Test chambers were illuminated with 40-watt incandescent lights on a
12-hr light cycle.
Chronic 3 was started with eggs immediately after fertilization in the
laboratory. One hundred and fifty eggs were placed in each of one con-
trol and four H2S treatments (Table 94). For the first 33 days eggs
and fry were held in cylinders 6 cm in diameter with Nitex screen
bottoms. Fry were then transferred to 20-liter tanks described for
185
-------
Table 92. LC50 VALUES FOR RAINBOW TROUT JUVENILES AT VARIOUS
DAYS AND LENGTH AND WEIGHT AFTER 17 DAYS EXPOSURE TO DIFFERENT
CONCENTRATIONS OF HS (ACUTE TEST 5)
H2S
concentration,
mg/1
Control
Control
Control
0.0014
0.0018
0.0025
0.0031
0.0062
0.0075
0.0095
Mean length
weight (17
TTgP
45.0
47.6
46.0
47.8
46.9
46.1
48.0
46.9
42.8
38.5
and
days),
g
1.248
1.502
1.314
1.458
1.435
1.386
1.499
1.367
0.945
0.674
LC50,
mg/1 HnS
0.0130
0.0113
0.0104
0.0103
0.0102
0.0091
0.0086
0.0088
0.0087
0.0087
(days)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(17)
186
-------
Table 93. TEST CONDITIONS IN CHRONIC TEST 1 WITH RAINBOW TROUT'
Item
Test
x H-S cone, (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0 (mg/1)
Test
x H_S cone, (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0,., (mg/1)
Test
x H2S cone, (mg/1)
H S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0~ (mg/1)
1 2
Chamber
3
4
5
1-a (100 days duration)
Control 0.0018
0.0020
7-72 7.74
14.4 14.6
10 . 1 9.8
0.0032
0.0024
7.77
14.7
9.7
0.0075
0.0037
7.79
15.0
9.9
0.0131
0.0063
7.80
14.7
9,7
1-b (90 days duration)
Control 0.0011
0.0011
7.72 7.73
14.7 14.8
9.6 9.8
0.0033
0.0046
7.76
14.8
9.7
0.0048
0.0027
7.78
15.0
9.7
0.0106
0.0098
7.79
14.8
9.6
1-c (50 days duration)
Control 0.0011
0.0008
7.82 7.80
14 . 6 15 . 0
9.6 9.4
0.0037
0.0062
7.81
14.9
9.4
0.0059
0.0033
7.82
15.0
9.4
0.0102
0.0115
7.82
15.0
9.4
30 fish per chamber,
187
-------
Table 94. TEST CONDITIONS IN CHRONIC TESTS 2, 3, AND 4
WITH RAINBOW TROUT
Item
x phenol cone, (mg/1)
Phenol std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0~ (mg/1)
x total alkalinity (mg/1)
x H-S cone- (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0,, (mg/1)
x total alkalinity (mg/1)
x H~S cone, (mg/1)
H2S std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0,., (mg/1)
x total alkalinity (mg/1)
1
Test
Control
-
7.78
12.4
10.5
220
Test
Control
7.78
13.5
9.8
216
Test
Control
1 .Ik
1.3.3
9.6
218
2
2a
1.50
0.16
7.79
12.5
10.5
220
3b
0.0009
0.0006
7.75
14.0
9.8
216
4C
0.0012
0.0012
7.72
13.7
9.6
218
Chamber
3
3.40
0.17
7.81
12.5
10.5
220
0.0020
0.0015
7.75
13.9
9.8
216
0.0040
0.0019
7.71
13.4
9.6
218
4
7.70
0.25
7.79
12.5
10.5
220
0.0025
0.0024
7.75
13.5
9.8
216
0.0062
0.0017
7.71
13.2
9*6
218
5
26.20
1.77
7.79
12.5
10.5
220
0.0052
0.0059
7.78
13.6
9.8
216
0.0123
0.0042
7.72
13.4
9.6
218
, 40 days duration; 50 fish per chamber.
145 days duration; 150 fish per chamber.
Ill days duration; 150 fish per chamber.
188
-------
acute tests where they were held to day 56. At that time the finger-
lings were transferred to 208 x 55 x 52 cm fiber glass tanks filled to
contain 503 liters of water. Fish were fed as in chronic test 1 and
remained in these tanks to completion of test. Flow-through rate was
500 ml/min. A 12-hr light cycle was maintained with 200-watt incan-
descent lights.
Chronic 4 was started with 150 eyed eggs in one control and four H?S
treatments (Table 94). For the first 13 days eggs were held in cylinders
as described for chronic 3. Fry were then transferred to 20-liter tanks
as described for previous tests and fed as in previous tests and held to
day 71 when fingerlings were placed in the fiber glass tanks previously
described.
Chronic 2 was started with 50 eyed eggs placed in each of four concen-
trations of phenol and one control (Table 94). Eggs were held in cylin-
ders as described above for 6 days and then transferred to 20-liter
tanks where they were held to the end of the test (40 days). Flow rate
was 500 ml/min. Tanks were illuminated as in chronic 3 and fish feeding
was done as in chronic 1.
Chronic 5 was started with 50 juvenile fish in 20-liter tanks and
exposed to H S for 74 days. One treatment with H?S, one control, and
four treatments with one concentration of H^S combined with four con-
centrations of phenol constituted the test (Table 95).
Survival
Rainbow trout survival in 100 days when tests were started with newly
hatched fry (1-a) was significantly affected at 0.0075 mg/liter but not
at 0.0032 mg/liter H2S (Table 96). At a concentration of 0.0132 mg/liter
no fish survived to 28 days. When fish were started as 10-day-old fry
(1-b), a marked reduction in survival was noted at 46 days in a concen-
189
-------
Table 95. TEST CONDITIONS IN CHRONIC TEST 5 WITH RAINBOW TROUT'
Chamber
Item
x H2S (mg/1)
x H2S std. dev. (mg/1)
x phenol (mg/1)
Phenol std. dev. (mg/1)
x pH
x temperature (C)
x dissolved 0? (mg/1)
x total alkalinity (mg/1
123
Control 0.0052 0.0054
0.0018 0.0017
0.13
0.06
7.78 7.84 7.82
13.1 12.8 12.7
6.4 6.4 6.4
223 223 223
4
0.0056
0.0016
0.34
0.17
7.80
12.7
6.4
223
5
0.0061
0.0024
0.60
0.30
7.81
12.6
6.4
223
6
0.0048
0.0020
1.40
0.24
7.84
12.8
6.4
223
74 days duration; 50 fish per chamber.
190
-------
Table 96. SURVIVAL OF RAINBOW TROUT DURING CHRONIC EXPOSURE
TO VARIOUS CONCENTRATIONS OF H2S
(percentage)
Stage at Days of
Test start exposure
la Newly 28
hatched 56
fry 84
100
Ib 10-day-old 18
fry 46
74
90
Ic 50-day- old 6
juveniles 34
50
3 Fertilized 28
eggs 56
86
114
145
b
4 Eyed eggs 27
54
82
111
Control
100
90
90
90
Control
100
100
97
97
Control
100
93
93
Control
56a
34
31
31
30
Control
58
80
52
40
H,
£
0.0018
100
90
87
87
0.0011
100
100
97
97
0.0011
97
97
97
0.0009
80
75
73
73
64
0.0010
49
66
42
42
,S (mg/1)
0.0032
100
97
97
93
0.0033
97
97
87
87
0.0037
100
100
100
0.0020
89
71
65
62
56
0.0039
60
85
54
42
0.0075
97
57
57
57
0.0048
100
77
74
74
0.0059
93
93
90
0.0025
83
80
79
78
62
0.0063
56
48
46
30
0.0132
0
0
0
0
0.0106
83
60
33
33
0.0102
97
43
43
0.0052
80
15
13
9
7
0.0124
55
33
4
4
f High mortality caused by excessive water agitation in chamber.
Terminal stage - feeding larvae.
191
-------
tration of 0.0048 mg/liter H2S. In 90 days, survival was 33% at 0.0106
mg/liter and 87% at 0.0033 mg/liter H S. When fish were started as
50-day-old juveniles, survival was 43% in 50 days at 0.0102 mg/liter IU
with no appreciable mortality at the lower levels.
In chronic 3 started with fertilized eggs, some reduction in survival
after 28 days occurred at all concentrations from 0.0009 to 0.0052
mg/liter H?S. After 56 days, survival was 15% of eggs at start with a
concentration of 0.0052 mg/liter. Appreciable mortality occurred in
control during egg stage and in HLS treatments most mortality occurred
during fry stage.
These data indicate that when rainbow trout are subjected to H_S from
fertilization onward any concentration above 0.0025 mg/liter (chronic
3) will cause some mortality. When fish are started as eyed eggs
(chronic 4) they are more resistant and show no effect on survival at
0.0039 mg/liter but some effect at 0.0063 mg/liter HS.
Growth
Growth of fry was retarded at 0.0075 mg/liter H«S and higher (chronic
1-a) (Tables 97 and 98). In the test started with fertilized eggs
growth was retarded at all test levels. The apparent better growth
at 0.0052 mg/liter H-S was caused by survival of only a few large fish.
When a test was started with eyed eggs (chronic 4) , there appeared to
be some increased growth at 0.0010 mg/liter H-S but retardation at
0.0063 mg/liter and higher.
Tests with Combined Phenol and H S
As a preliminary to testing the chronic effects of combined H S and
phenol, a 40-day test on eyed eggs was run on phenol (chronic 2) at
four concentrations from 1.50 to 26.20 mg/liter (Table 99). At all
concentrations tested survival and growth were affected. At 26.20 mg/
liter no fry survived at 40 days .
192
-------
Table 97. MEAN WEIGHT OF RAINBOW TROUT AFTER VARIOUS PERIODS
OF EXPOSURE TO DIFFERENT CONCENTRATIONS OF H S
(grams)
Stage at Days of
Test start exposure
la Newly
hatched
fry
57
85
100
H.S
Control
0
1
2
.408
.279
.410
Control
Ib 10-day-old
fry
47
75
90
0
1
2
.576
.304
.053
Control
Ic 50-day-old
j uveniles
6
34
50
0
1
1
.435
.286
.952
Control
3 Fertilized
eggs
145
7
.900
Control
4 Eyed eggs
111
4
.920
0
0
1
2
0
0
1
1
0
0
1
1
0
6
0
5
.0018
.491
.608
.609
.0011
.627
.477
.918
.0011
.403
.160
.763
.0009
.180
.0010
.110
0
0
1
2
0
0
1
2
0
0
1
1
0
6
0
4
. mg/1
.0032
.491
.499
.339
.0033
.555
.524
.134
.0037
.441
.203
.854
.0020
.290
.0039
.320
0
0
1
1
0
0
1
1
0
0
1
1
0
5
0
2
.0075
.327
.229
.905
.0048
.506
.442
.548
.0059
.354
.505
.887
.0025
.960
.0063
.460
0
0
0
1
2
0
0
1
1
0
8
0
1
.0132
-
-
-
.0106
.562
.550
.472
.0102
.382
.252
.724
.0052
.680
.0124
.670
193
-------
Table 98. MEAN LENGTH OF RAINBOW TROUT JUVENILES AFTER VARIOUS
PERIODS OF EXPOSURE TO DIFFERENT CONCENTRATIONS OF H2S
(millimeters)
Test
la
Ib
Ic
3
4
Stage at
start
Newly
hatched
fry
10-day-old
fry
50-day-old
juveniles
Fertilized
eggs
Eyed eggs
Exposure,
days
57
85
100
47
75
90
6
34
50
b
145
lllb
Control
3.35
4.80
5.69
Control
3.76
4.86
5.52
Control
3.46
4.79
5.35
Control
8.90
Control
7.50
I
0.0018
3.57
5.19
5.87
0.0011
3.91
5.18
5.67
0.0011
3.47
4.72
5.34
0.0009
8.10
0.0010
7.60
US, mg/1
0.0032
3.57
5.10
5.65
0.0033
3.80
5.20
5.68
0.0037
3.45
4.79
5.38
0.0020
8.30
0.0039
7.10
0.0075
3.25
4.62
5.65
0.0048
3.62
5.00
5.33
0.0059
3.25
4.80
5.36
0.0025
8.00
0.0063
6.40
0.0132
_a
-
-
0.0106
3.76
5.26
6.11
0.0102
3.35
4.75
5.37
0.0052
9.30
0.0124
5.20
fry dead at 57 days.
Terminal stage - juveniles.
194
-------
Table 99. SURVIVAL, WEIGHT, AND LENGTH IN CHRONIC TEST 2 OF RAINBOW
TROUT STARTED AS EYED EGGS AND EXPOSED TO VARIOUS
CONCENTRATIONS OF PHENOL
Item
Exposure,
days
Phenol, mg/1
Control 1.50 3.40 7.70 26.20
Survival (%)
At hatch
40
94
90
94
66
94
32
94
20
64
0
Weight (g)'
40
0.338 0.152 0.099 0.092
Length (mm)
Wet weight.
40
29.9 24.2 24.4 22.6
195
-------
Chronic 5 with 0.0048-0.0061 mg/liter HLS combined with four levels of
phenol from 0.13 to 1.40 mg/liter (Table 100) showed no additive or
synergistic effect on survival when fish were exposed to the two
materials at the same time. Growth rate was slowed by H?S alone at
0.0052 mg/liter and with 1.40 mg/liter phenol added a further retar-
dation occurred. Although the two materials acting together at this
concentration of phenol resulted in a greater retardation than either
alone, the retardation was not as much as would have been expected from
results of H.S and phenol tested separately.
SUMMARY
Sperm motility and egg fertilization were not adversely affected up to
concentrations of 0.0589 mg/liter H2S. LTC of fry at 20 days was 0.0056
mg/liter. Mean LC50 of juveniles at 96 hr was 0.0130 mg/liter and at
17 days was 0.0087 mg/liter HLS. When trout are subjected to H?S from
fertilization onward, any concentration above 0.0025 mg/liter H-S will
have adverse effects on growth and survival. When eggs or fish are
started in H^S at the eyed egg stage or later they are more tolerant.
Phenol and H~S in combination increase mortality but not as much as
anticipated from separate tests.
196
-------
Table 100. SURVIVAL, LENGTH, AND WEIGHT OF RAINBOW TROUT IN A
MIXTURE OF H2S AND PHENOL AFTER VARIOUS PERIODS OF
EXPOSURE IN CHRONIC TEST 5
Item
Exposure,
days
Chamber
6
H£S (mg/1)
Phenol (mg/1)
Control 0.0052 0.0054 0.0056 0.0061 0.0048
Control Control 0.13 0.34 0.60 1.40
Survival (%)
Weight
Length
(g)b
(mm)
28
74
18
74
74
98
98
2.89
5.66
7.9
100
100
2.50
4.92
7.5
100
100
2.67
5.07
7.5
100
100
2.51
5.08
7.7
100
92
2.42
4.17
7.1
98
98
2.29
4.35
7.3
Test started with 49-day-old juveniles.
Wet weight.
197
-------
SECTION XI
WHITE SUCKER
(Catostomus commersoni (Lacepede))
A series of four acute tests were conducted on sucker juveniles and a
series of three chronic tests were started from sucker fry. Excessive
mortality in the three successive chronic attempts necessitated their
termination. The experimental design for the acute tests was the same
as that described for walleye juveniles. The length of test fish
varied between experiments from 32 - 121 mm (Table 101). Test tem-
perature was 20-20.1 C, oxygen levels 5.9-6.2 mg/liter and pH 7.8-7.9.
The 96-hr LC50 values varied from 0.0185 to 0.0290, with a mean of
0.0219 mg/liter H-S. Tests on eggs and fry were conducted as part of
20
another study prior to the start of this project (Smith and Oseid ).
They are reported in the summary tables in the subsequent discussion
section.
198
-------
Table 101. TEST CONDITIONS AND LC50 VALUES OF H2S IN ACUTE TESTS WITH WHITE SUCKER
Test
1
2
3
4
Source
Reared from
eggs
Bait dealer
it ii
it it
Number
fish per
chamber
10
3
3
3
Mean
length,
mm
32
123
124
121
Mean test
conditions
Temp . ,
C
20.0
20.0
20.1
20.1
0 ,
mg/1
6.2
6.1
5.9
6.0
PH
7.8
7.9
7.9
7.9
LC50,
mg/1 H0S
24 hr 48 hr 72 hr
0.0185 0.0185
0.0247
0.0290 0.0230 0.0208
— — —
96 hr
0.0185
0.0193
0.0208
0.0290
-------
SECTION XII
CRAYFISH
(Procambarus clarkii (Girard) and Cambarus diogenes Girard)
Acute tests were run on Procambarus clarkii (Girard) to determine 96-hr
LC50 and LTC of H~S. Chronic tests at low levels determined the in-
fluence of H~S on survival, growth, and reproduction. During chronic
tests the sensitivity of various life history stages was determined.
Cambarus diogenes Girard was subjected to acute tests only to deter-
mine the differences between northern and southern species.
ACUTE TESTS
Collection, Treatment, Holding, and Acclimation
Procambarus clarkii was trapped and shipped from a commercial crayfish
farm near Fairbanks, Louisiana (Table 102). They were shipped as adults,
subadults, and berried females by air freight in styrofoam coolers with
wetted and frozen potato sacks. Transportation time from farm to holding
tanks in the laboratory was 12-18 hr. All crayfish were treated on
arrival with 0.2% formalin to remove external oligochaetes before accli-
mation and long-term holding. Berried females provided material for
egg, larval, and juvenile tests. Juveniles were reared to a weight of
1-3 g at 15-18 C and fed Glencoe pellets before testing. Crsfyfish were
reared to 5-7 mm for use in one test. Adults were held in fiber glass
tanks and transferred to acclimation tanks as needed. Acclimation of
juveniles and adults (Tests 6-21) was done in a stock tank coated with
aluminum asphaltum. Cinder blocks were placed in the tank for cover
and all size groups of crayfish were fed Glencoe pellets and lettuce £id
200
-------
Table 102. STAGE OF CRAYFISH AT COLLECTION AND AT START OF ACUTE TESTS
Test
1,2,3
4,5
6
7-11
12
13-15
16-19
20,21
22-25
Stage at
a
start
Procambarus clarkii
Egg
Larvae
Juvenile
Juvenile
Subadult
Subadult
Subadult
Subadult
Adult
Stage at Shipment or
collection collection date
trapped in Fairbanks, LA
Egg
Egg
Egg
Egg
Subadult
Subadult
Subadult
Subadult
Adult
9-8-72
9-8-72
9-29-71
9-8-72
9-29-71
12-7-71
3-9-72
5-31-72
3-11-74
Cambarus diogenes seined in Dakota County, MN
26
27,28
Juvenile & subadult
Juvenile & subadult
Juvenile & subadult
Juvenile & subadult
6-4-74
5-11-74
o
,See glossary.
A permanent pond.
201
-------
libitum. All crayfish were acclimated for 8 to 16 days to test tempera-
tures. Juveniles (tests 6-11) were held in 25-liter glass aquaria and
fed Glencoe pellets twice per day. Eggs and larvae (tests 1-5) were
derived from berried females held in fiber glass tanks. These tanks
received the control water from egg test diluters. Eggs were accli-
mated 1 day at temperature and larvae were collected and used after eggs
were hatched. Cambarus diogenes (tests 26-28) were collected from a
Dakota County, Minnesota pond by hand seine and transported to the labor-
atory in an aerated stock tank. They were given 10 min treatment with
0.2% formalin solution and transferred immediately to stock tanks for
acclimation. Acclimation time varied from 10 to 18 days at test tem-
peratures and crayfish were fed Glencoe pellets and lettuce ad libitum.
Egg Tests
Eggs of 7_. clarkii were taken from berried females, acclimated for 1 day
at test temperature and then tested at three temperatures (Table 103).
Twenty-five eggs each were placed in 1-3/4-inch diameter acrylic cylin-
ders with 1/16-inch mesh Nitex screen bottoms and outlets. The cylin-
ders were attached to the outlets of the glass aquaria receiving the flow
from diluters described for previous tests using sodium sulfide. The
entire water volume from each cycle passed through the test chambers. A
total of eight H?S treatments and two controls were used for the egg
tests. Ninety-five percent turnover time in the tanks at the head of
each cylinder was 4 hr. H-S concentration was determined from samples
taken at the inlet to the cylinders. Tests were conducted for 25, 35,
and 54 days at 14.2, 18.0, and 21.9 C, respectively. Photoperiod was
12 hr of light and 12 hr of darkness. Hatching was considered to have
occurred with the breaking of the egg shell.
Ninety-six hr LC50 values of H2S were >0.408, >0.433, and 0.370 mg/liter
H2S at 14.2, 18.0, and 21.9 C, respectively. LTC's were 0.282, 0.208,
and 0.151 mg/liter H-S at the same test temperatures. Percentage hatch
in 8-15 days at 21.9 C was 32-37% of controls at concentrations of HS
202
-------
up to 0.266 mg/liter (Table 104). Corrected hatch at 18 C in 19 days
was 89% with 0.188 mg/liter H2S and in 26 days was 20% with 0.270 mg/
liter. Corrected hatch was 76% at 0.208 mg/liter H2S and 14.4 C in 29
days and 33% at 0.395 mg/liter H2S and 14.0 C in 26 days.
Larval Tests
Two tests (acute tests 4 and 5) were run on first two instar larvae for
96 hr at 22 and 18 C. The same equipment and conditions were used as
described for egg tests. Ninety-six-hr LC50 was 0.058 mg/liter H2S at
22 C and 0.125 mg/liter at 17.9 C (Table 103).
Juvenile Tests
Juvenile test 6 was done on the H_S gas apparatus previously described
using one control and five treatments. Crayfish were acclimated for 2
days at 25 C and oxygen saturation. Mean test conditions were 24.6 C
and 4.08 mg/liter 0« with eight animals per chamber. Test chambers were
20-liter tanks with a 95% water replacement in 32 hr. Juvenile tests
7-11 were run in 20-liter tanks served by proportional diluters with
sodium sulfide. Ninety-five percent replacement time was 2 hr. Ten
crayfish were placed in each of one control and five treatments. Tests
were conducted at temperatures of 22.1, 18.0, 14.1, and 13.9 C with
acclimation for 10 days at 22, 18, and 14 C. Photoperiods were 12 hr
of light and 12 hr of darkness. Cover was provided in the tanks by
cement-asbestos tiles and fish were fed with Glencoe pellets after the
first 96 hr. Ninety-six-hr LC50 at 22.1 C was 0.034 mg/liter HZS, at
18 C was 0.083 mg/liter, and at 14.1 C was 0.147 mg/liter (Table 103).
LTC at 18 C in 14 days was 0.053 mg/liter and at 13.9 C in 11 days was
0.126 mg/liter H?S. Small crayfish (5-7 mm total length) in test 6 had
96-hr LC50 of 0.051 mg/liter H2S at 24.6 C.
Subadult Tests
Ten acute tests were run (tests 12-21) in the same equipment as for
203
-------
Table 103. ACUTE TEST CONDITIONS AND LC50 VALUES FOR CRAYFISH IN
to
o
Test
Days from
collection Number/
Stage to start chamber
Mean Mean
carapace Mean temper-
length, weight, ature,
mm g C
Mean
PH
Mean 0^
mg/1
LC50 ,
96 hr
rng/1 H^S
LTC (days)
Procambarus clarkii
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Egg 3
Egg 3
Egg 3
Larvae Reared from
eggs
Larvae "
Small "
juvenile
Juvenile "
Juvenile "
Juvenile "
Juvenile "
Juvenile "
Sub-adult 12
Sub-adult 12
Sub-adult 12
25
25
25
25
25
8
10
10
10
10
10
6
5
5
-
-
-
-
-
5-7
17
21
21
21
17
39
48
49
.2
.0
.5
.9
.3
.4
.4
.9
21
18
14
22
17
24
1.07 22
1.96 18
1.98 18
2.25 14
1.08 13
15.3 25
30.1 18
35.0 18
.9
.0
.2
.0
.9
.6
.1
.0
.0
.1
.9
.9
.2
.0
7.67
7.68
7.69
7.69
7.62
7.76
7.71
7.69
7.70
7.67
7.69
7.82
7.81
7.83
5.74
6.40
6.95
6.71
7.20
4.08
6.49
6.84
6.79
6.41
7.95
4.15
5.61
5.48
0
>o
7°
0
0
0
0
0
0
0
0
0
.370
.433
.408
.058
.125
.051
.034
.083
-
.147
-
.075
.080
.095
0.151 (25)
0.208 (35)
0.282 (54)
-
-
-
0.034 (4)
-
0.053 (14)
-
0.126 (11)
-
-
_
-------
Table 103 (continued). ACUTE TEST CONDITIONS AND LC50 VALUES FOR CRAYFISH IN
to
o
en
Test
15
16
17
18
19
20
21
22
23
24
25
Days from
collection
Stage to start
Sub -adult
Sub-adult
Sub-adult
Sub-adult
Sub-adult
Sub-adult
Sub-adult
Adult
Adult
Adult
Adult
23
23
23
27
40
19
48
14
50
25
59
Number
per
chamber
6
8
8
8
8
7
10
10
10
10
10
Mean Mean
carapace Mean temper-
length, weight, ature,
mm g C
49.
44.
46.
40.
43.
41.
41.
58.
56.
58.
57.
4
7
7
9
7
4
5
5
3
1
8
Cambarus
26
27
28
Sub-adult
Sub-adult
Sub-adult
9
14
18
10
10
10
26.
25.
24.
4
2
7
33.8
35.5
32.6
36.8
40.0
37.6
39.1
40.4
42.7
45.3
44.0
diogenes
5.2
4.1
4.4
18.2
18.0
17.9
17.9
18.1
17.9
18.1
21.7
18.1
18.1
14.0
22.0
18.1
13.9
Mean
PH
7.83
7.82
7.78
7.76
7.83
7.81
7.83
7.64
7.67
7.67
7.65
7.70
7.69
7.68
Mean 0»
mg/1
5.20
5.42
5.80
5.06
4.96
6.51
4.96
6.11
6.47
6.88
7.15
5.95
7.45
7.60
LC50, me/1 H.S
96 hr
0.090
0.100
-
0.115
0.093
0.091
0.081
0.121
0.215
-
0.271
0.070
0.108
0.150
LTC (days)
0.052 (10)
-
0.060 (11)
-
-
-
0.058 (15)
0.121 (4)
-
0.130 (11)
0.202 (10)
-
-
-
o
i Acclimation ranged from 8 to 16 days; 1 day at temperature for eggs.
Wet weight.
-------
Test 104. HATCHING SUCCESS OF Procambarus clarkii EXPOSED TO H0S
£
AT VARIOUS TEMPERATURES
H2S,
Test mg/1
1 Control
Control
0.156
0.176
0.208
0.221
0.266
0.324
0.377
0.389
2 Control
Control
0.170
0.188
0.214
0.270
0.279
0.305
0.402
0.433
3 Control
Control
0.159
0.208
0.237
0.246
Temper-
ature,
C
21.9
21.8
21.6
21.9
21.6
21.8
21.9
22.0
22.1
21.9
18.0
18.1
18.1
18.2
18.0
18.0
18.1
18.0
17.9
18.0
14.0
14.3
14.4
14.4
14.4
14.0
Number
of eggs
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
25
Hatch, a
%
76
72
24
28
0
0
26
0
0
0
64
63
55
56
0
5
0
1
0
0
87
83
92
63
26
68
Corrected
hatch,
%
100
100
32
37
0
0
34
0
0
0
100
100
87
89
0
20
0
4
0
0
100
100
100
76
31
78
Time to
50% hatch,
days
8
9
12
15
-
-
12
-
-
-
14
13
20
19
-
26
-
31
-
-
25
23
23
29
43
25
206
-------
Table 104 (continued). HATCHING SUCCESS OF Procambarus clarkii
EXPOSED TO H2S AT VARIOUS TEMPERATURES
Test
3
H2S,
mg/1
0.263
0 . 336
0.395
0.408
Temper-
ature,
C
14.4
14.0
14.0
14.0
Number
of eggs
25
25
25
25
Hatch, a
%
44
52
29
0
Corrected
hatch,
%
53
60
33
0
Time to
50% hatch,
days
31
44
26
—
kissing eggs due to diluter malfunction were removed from considera-
tion in calculation of % hatch.
Hatch was corrected to 100% of controls.
207
-------
juvenile test 6 except that 25-liter tanks with a 4-hr 95% water turn-
over time were used. One test (test 12) was run at 25.9 C and the others
at 17.9 to 18.2 C. In the various tests five to ten individuals were
placed in each of one control and five treatments. Acclimation at test
temperature prior to testing was for 8-16 days. Glencoe pellets were
fed after 96 hr of treatment. Photoperiod was 12 hr of light and 12 hr
of darkness. Ninety-six-hr LC50 was 0.075 mg/liter H2S at 25.9 C and
at 17.9-18.2 C was 0.080-0.115 mg/liter, with a mean of 0.093 mg/liter
H2S (Table 103). LTC was 0.052-0.060 mg/liter H2S in 10-15 days at
17.9-18.1 C.
Adult Tests
Four tests (tests 22-25) were run on adults at temperatures from 14.0 to
21.7 C. Ten individuals were placed in each of one control and six
treatments in 70-liter stock tanks served by proportional diluters and
sodium sulfide. Cement-asbestos tile shelters were provided in the
tanks. Acclimation was at 14, 18, and 22 C for 10-16 days. Photoperiod
was 12 hr of light and 12 hr of darkness. Crayfish were fed Glencoe
pellets after 96 hr. Ninety-six-hr LC50 was 0.121 mg/liter H2S at 21.7 C,
0.215 mg/liter at 18.1 C, and 0.271 mg/liter at 14.0 C (Table 103).
LTC at 18.1 C was 0.130 mg/liter H2S at 11 days and 0.202 mg/liter at
14.0 C in 10 days. LTC at 21.7 C was the same as 96-hr LC50.
Subadult Tests with Cambarus diogenes
Three tests were conducted on subadult Cambarus diogenes with ten indi-
viduals in each of one control and six test chambers. The 96-hr LC50
was determined at 13.9, 18.1, and 22.0 C (Table 103). Equipment was
identical to that described for adult tests with Procambarus. The 96-hr
LC50 at 13.9 C was 0.150 mg/liter H S, at 18.1 C was 0.108 mg/liter, and
at 22.0 C was 0.070 mg/liter.
Summary of Acute Tests
In Procambarus clarkii, eggs are the most resistant life history stage
208
-------
with LTC vaues varying from 0.151 mg/liter H-S at 21.9 C to 0.282 mg/
liter at 14.2 C. The juvenile stage is the least resistant with LTC's
varying from 0.034 mg/liter H2S at 22.1 C to 0.126 mg/liter at 13.9 C.
Yolk-sac larvae are intermediate in sensitivity between eggs and juve-
niles with 96-hr LC50 of 0.058 mg/liter H2S at 22.0 C and 0.125 mg/liter
at 17.9 C. Subadults have the same resistance as juveniles with LTC
values varying from 0.052 to 0.060 mg/liter H2S at 17.9-18.2 C.
Adults were more resistant than all stages except eggs with LTC's of
0.121 mg/liter at 21.7 C and 0.202 mg/liter H S at 14 C. The Cambarus
diogenes subadults tested at 18.1 C had similar sensitivity to P_. clarkii
subadults at 96 hr (0.108 and 0.093 mg/liter H-S, respectively).
CHRONIC TESTS
Experimental Design
Three chronic tests were conducted on Procambarus clarkii. Chronic test
1 (1-a and 1-b) was run on two generations for 447 days. Test 1-a was
run for the entire period and test 1-b was started after the 335th day
in the same tanks using offspring of females of the original test. Tem-
perature and daylight were carried in accordance with seasonal changes
in Louisiana where the original stock was collected. The temperature
varied from 9 to 24 C and light periods from 14 to 10.5 hr of light
(Table 105). The intensity varied from 62 to 153 ft candles. The five
2
test tanks were of fiber glass with a surface of 1.144 m and a volume
of 172 liters. Two berried females were placed in each tank. The num-
ber of young produced was 244, 148, 272, 54, and 161 for tanks 1-5,
respectively. After 138 days the total number in each tank was reduced
to 50 and at 168 days to 20 individuals. Cover was provided by cement-
asbestos tiles and Glencoe pellets and lettuce were available continuously
jid libitum. The four treatments ranged from 0.0041 to 0.0183 mg/liter
H2S for the first 364 days and from 0.0048 to 0.0158 mg/liter for the
last 112 days. In 1-b, which ran for 112 days, the two treatments were
0.0048 and 0.0097 mg/liter H?S. There was no reproduction in the two
highest treatments.
209
-------
Table 105. TEST CONDITIONS IN CHRONIC TESTS OF CRAYFISH
(Procambarus clarkii)
Test
conditions
Diluter
45123
x H2S cone, (mg/1)
Std. Dev.
x temperature (C)
Range
x pH
Range
x dissolved 0~ (mg/1)
Range
x total alkalinity
Range
Test 1-a (364 days)
Control 0.0041 0.0086 0.0135 0.0183
0.0018 0.0040 0.0060 0.0081
18.1 18.2 18.0 18.1 18.2
9.1-24.3 10.0-24.1 9.8-23.9 9.8-24.1 9.8-24.1
7.71 7.70 7.69 7.69 7.73
7.39-8.17 7.40-8.17 7.42-8.02 7.40-8.15 7.39-8.19
7.46 6.80 6.58 6.41 6.35
5.7-9.9 4.2-9.7 3.7-9.6 3.5-9.6 2.7-9.5
201.2 201.2 201.2 201.2 201.2
175-235 175-235 175-235 175-235 175-235
x H2S cone, (mg/1)
Std. Dev.
x temperature (C)
Range
x pH
Range
x dissolved 0« (mg/1)
Range
x total alkalinity
Range
Test 1-b (112 days)
Control 0.0048 0.0097 0.0127a 0.0158a
0.0023 0.0035 0.0062 0.0076
16.0 16.0 15.8 21.6 21.6
8.8-24.3 9.0-24.1 8.8-23.9 20.0-24.1 20.1-24.1
7.71 7.69 7.69 7.69 7.76
7.54-7.84 7.54-7.81 7.55-7.82 7.54-7.82 7.66-7.89
8.05 6.96 6.78 4.37 4.78
6.3-10.9 4.7-10.2 4.6-9.9 3.6-4.6 2.7-6.0
203.2 203.2 203.2 203.2 203.2
170-225 170-225 170-225 170-22*5 170-225
210
-------
Table 105 (continued). TEST CONDITIONS IN CHRONIC TESTS
OF CRAYFISH (Procambarus clarkii)
Test
conditions
Diluter
14235
x H2S cone, (mg/1)
Std. Dev.
x temperature (C)
Range
x pH
Range
x dissolved 02 (mg/1)
Range
x total alkalinity
Range
Test 2 (196 days)
Control 0.0044 0.0078 0.0140 0.0199
0.0033 0.0047 0.0082 0.0093
14.6 14.2 14.2 14.1 14.2
9.8-23.9 9.1-24.2 9.8-24.0 9.8-24.0 10.0-23.8
7.74 7.72 7.73 7.72 7.76
7.62-8.05 7.60-8.11 7.60-8.09 7.60-8.10 7.60-8.10
8.40 6.44 7.02 6.78 7.63
4.7-9.6 5.8-9.9 4.6-9.6 3.8-9.5 4.4-9.7
216.5 216.5 216.5 216.5 216.5
200-235 200-235 200-235 200-235 200-235
x H_S cone, (mg/1)
Std. Dev.
x temperature (C)
Range
x pH
Range
x dissolved 02 (mg/1)
Range
x total alkalinity
Range
Test 3 (196 days)
Control 0.0044 0.0088 0.0115 0.0172
0.0040 0.0069 0.0087 0.0146
20.76 20.63 20.67 20.61 20.59
19.3-25.7 19.2-25.9 19.2-25.8 19.1-25.0 18.8-25.1
7.73 7.75 7.73 7.73 7.73
7.50-7.98 7.58-8.09 7.54-8.05 7.58-8.08 7.56-8.00
6.86 6.02 5.97 5.36 6.31
4.00-8.65 5.15-7.70 4.10-8.05 3.00-7.95 4.10-7.90
210.6 210.6 210.6 210.6 210.6
198-231 198-231 198-231 198-231 198-231
Data from first 28 days only.
211
-------
Chronic test 2 ran for 196 days under the same seasonal conditions as
described for test 1. Light intensity was 34-40 ft candles and test
tanks were 40-gal stock tanks containing 140 liters of water. The test
was started with 20 juveniles 5-10 mm total length in each tank. Cover
was provided by cement-asbestos tiles and Glencoe pellets and lettuce
were fed ad libitum. H2S treatments ranged from 0.0044 to 0.0199
mg/liter.
Chronic test 3 was conducted for 196 days at a temperature of 25 C for
the first 34 days and at 20 C for the remainder of the run. Photoperiod
was 12 hr of light and 12 hr of darkness. The light intensity was 34-
37 ft candles. R S treatments ranged from 0.0044 to 0.0172 mg/liter.
Survival
Survival in chronic test 1-a which was started with larvae from berried
females varied from 28% for 447 days in the control to 1% after 332
days at 0.0183 mg/liter H2S (Table 106). Percentages were derived from
the product at the end of each culling period. At 232 days there was
reduction at the three highest levels. The first marked loss was at the
juvenile stage after 232 days in all treatments and control. The
second generation (test 1-b) survival was 40% in the controls and 67.6%
at 0.0097 mg/liter H-S. In chronic test 2 after 196 days survival was
100% at 0.0078 mg/liter H2S and 54% at 0.0199 mg/liter. In chronic test
3 after 196 days survival in the control was 86% and at 0.0172 mg/liter
ELS was 11%. The no-effect level for survival in test 1-a after 447
days was 0.0041 mg/liter ILS and in test 1-b after 112 days was 0.0097
mg/liter since higher levels were not tested. In test 2 the no-effect
*
level was 0.0078 mg/liter and in test 3, 0.0088 mg/liter H2S at 196 days.
Reproduction
Reproduction in chronic test 1-a was derived from 1 to 3 surviving
berried females at 235 days (Table 107). One female surviving at a con-
centration of 0.0183 mg/liter H2S had no eggs attached. Of three females
212
-------
Table 106. SURVIVAL OF CRAYFISH (Procambarus clarkii)
WITH LONG-TERM EXPOSURE TO H2S
(expressed as percentage )
Exposure,
Test days
1-a (first
b
generation)
1-b (second
generation)
2
3
55
139
232
322
447
112
28
84
140
196
28
84
140
196
H^S , mg/liter
Control
98
94
70
37
28
Control
40
Control
94
77
73
70
Control
100
91
89
86
0.0041
92
89
71
36
27
0.0048
42.3
0.0044
100
100
100
100
0.0044
100
73
60
60
0.0086
96
91
46
27
14
0.0097
67.6
0.0078
100
100
100
100
0.0088
100
100
90
83
0.0135
100
82
53
16
12
0.0127
-
0.0140
95
79
69
64
0.0115
100
63
31
31
0.0183
26
13
3
1
1
0.0158
-
0.0199
94
73
69
54
0.0172
100
50
11
11
Corrected for loss from culling and sampling.
First generation derived from hatched larvae.
213
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Table 107. REPRODUCTION OF CRAYFISH (Procambarus clarkii)
IN CHRONIC TEST 1
Item
H~S cone, (mg/liter)
Number berried females
Q
Number of larvae
Survival -447 days (%)
Total number of females
alive at end of test
Total number berried
females
Total weight berried
females (g)
Total number eggs
Number eggs/berried
female
Number eggs/g berried
female
H2S cone, (mg/liter) -
second generation
Survival (%) - second
generation (112 days)
4
Control
2
54
28
2
2
38.7
120
60
3.10
Control
40.0
5
0.0041
2
161
27
4
3
48.3
201
67
4.17
0.0048
42.3
Diluter
1
0.0086
2
249
14
3
2
30.2
102
51
3.38
0.0097
67.6
2
0.0135
2
148
12
3
lb
27.8
35
35
1.26
0.0127
0
3
0.0183
2
272
1
1
0
0
0
0
0
0.0158
-
Numbers culled to 50 at day 138 and to 20 at day 168; percentage ad-
justed to numbers before culling. •
Eggs produced were infertile.
214
-------
at 0.0135 mg/liter H~S, only one produced eggs and they were infertile.
At 0.0086 mg/liter, two of the three surviving females and at 0.0041
mg/liter, three of four surviving females were berried. The two sur-
viving females in the control were berried. The number of eggs per gram
of berried female varied from 3.10 in the controls to 1.26 at 0.0135
mg/liter tUS. As noted above, the eggs at this latter concentration
were infertile. Survival of eggs and survival of larvae from eggs
through 112 days in the second generation were 40.3 to 67.6%. No detri-
ment to survival was noted in the three test levels examined. The safe
concentration of H~S for reproduction appears from the foregoing data to
be 0.0086 mg/liter. At higher concentrations eggs are either infertile
or none are produced.
Growth
In chronic test 2 growth was inhibited at 0.0078 mg/liter H S after 196
days (Table 108). In chronic test 3 growth was inhibited after 196 days
at 0.0088 mg/liter. The no-effect concentration of H~S on growth was
0.0044 mg/liter in chronic tests 2 and 3.
Acute 96-hr tests were run on subsamples of chronic 1-a and 1-b organisms
(Table 109). All tests were conducted at 18 C. The 96-hr LC50 for
second generation controls is 0.0800 mg/liter H2S. In the first genera-
tion LC50 values were higher in individuals taken from the higher con-
centration, indicating some degree of acclimation. At concentrations of
0.0041 mg/liter H~S, the LC50 was approximately the same as found in the
more extensive acute tests reported above. In the second generation (1-b)
LCSO's were lower at comparable concentrations than in the first genera-
tion (1-a).
jummary of Chronic Tests
The no-effect concentration among the levels tested was 0.0041 mg/liter
ELS and was based on both survival or gain in mean weight in tests la and
2. Among survivors and without reference to growth rate, the no-effect
level for reproduction was 0.0086 mg/liter HS.
215
-------
Table 108. WEIGHT OF CRAYFISH (Procambarus clarkii)
WITH LONG-TERM EXPOSURE TO H2S
(grams)
Exposure, H0S,
Test
1-a
First genera-
tion
1-b
Second
generation
2
3
days
125
139
169
204
232
447
112
28
84
140
196
28
84
140
196
Control
0.47
0.69
1.99
6.91
11.26
28.74
Control
0.50
Control
0.14
0.39
1.56
11.22
Control
0.39
3.63
12.53
16.19
0.0041
0.25
0.36
1.30
4.49
6.91
18.11
0.0048
0.47
0.0044
0.26
0.31
1.59
10.76
0.0044
0.34
4.39
14.09
17.43
mg/liter
0.0086
0.12
0.18
0.70
2.99
5.47
20.89
0.0097
0.11
0.0078
0.21
0.43
1.37
7.57
0.0088
0.32
3.53
7.20
11.49
0.0135
0.18
0.26
0.80
3.38
5.92
27.65
0.0127
-
0.0140
0.11
0.27
0.89
7.57
0.0113
0.25
2.58
12.30
15.11
0.0183
0.24
0.32
1.07
2.37
7.33
14.63
0.0158
-
0.0199
0.11
0.24
0.61
5.22
0.0171
0.22
2.38
12.84
14.93
216
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Table 109. 96-HOUR LC50 VALUES OF SUBSAMPLES OF JUVENILE CRAYFISH
(Procambarus clarkii) AFTER LONG-TERM EXPOSURE TO HS
Test
Chronic exposure,
mg/liter H0S
96-hour LC50,
mg/liter H0S
1-a First generation
1-b Second generation
Control
0.0041
0.0086
0.0135
0.0183
Control
0.0048
0.0097
0.1080
0.1140
0.1270
0.0800
0.0950
0.1000
217
-------
SECTION XIII
BENTHIC INVERTEBRATES
Laboratory studies on one isopod, Asellus militaris Hay, two amphipods,
Crangonyx richmondensis laurentianus Bousfield and Gammarus pseudo-
limnaeus Bousfield, three Ephemeroptera, Baetis vagans McDonough,
Ephemera simulans Walker, and Hexagenia limbata (Serville), were con-
ducted. Field studies and surveys have indicated that H-S is often
present in both polluted and some natural ecosystems at levels that are
detrimental to fish and invertebrates. Limited references in the liter-
ature to the effect of H2S on various invertebrates suggested that a
rigorous evaluation of acute toxicities in a representative series of
aquatic organisms associated with fish populations would permit an
overall evaluation of the importance of H-S in the aquatic systems.
The species used in these tests were selected to include organisms from
a wide range of habitat conditions varying from clear, cool water and
firm substrate to warm, turbid waters with substrates where insects
might burrow in the mud and where tUS levels might normally be rela-
tively high. In addition to the two crayfish discussed in Section XII,
a series of acute tests was run on six other species and chronic ex-
posure tests were run on Gammarus and Hexagenia.
ACUTE TOXICITY TESTS
Experimental Design
The isopods, Asellus militaris, used in the experiments were collected
from Jackfish Bay, Rainy Lake, Minnesota and had a mean length of 8 mm
218
-------
(range 5-13 mm) . Crangonyx was also taken from Jackfish Bay and ranged
from 6-15 mm in length, with a mean of 10 mm. Gammarus was collected
in Valley Creek, Washington County, Minnesota and varied in size from
8-16 mm, with a mean of 11 mm. Baetis was also taken from Valley Creek
and ranged in size from 4-6 mm, with a mean of 5 mm. Nymphs of Hexa-
genia varied from 14-35 mm, with a mean of 23 mm, and were taken from
Jackfish Bay and Crystal Beach, Rainy Lake, Minnesota. Ephemera
ranged from 13-21 mm, with a mean of 17 mm, and were taken from Crystal
Beach. The organisms were collected with a Peterson dredge, hardware
cloth scoop, or drift net. Organisms were held in the laboratory at
the same temperature as tests and were fed detritus from the collection
site.
All tests were conducted in flow-through apparatus as described by
1 2
Colby and Smith and Adelman and Smith. The former apparatus consisted
of a cylindrical chamber 3.8 cm in diameter by 9.5 cm deep with a Nitex
screen at the bottom. The second type was an acrylic box 7.6 x 7.6 x
5 cm with various substrates into which the organisms could burrow. H^S
levels were maintained by dissolving tLS gas in oxygen-free water and
mixing this water at the entrance to the test chambers with a propor-
tioned amount of oxygen-saturated water to achieve the desired oxygen
and H_S concentrations. After mixing, the solution passed through the
test chamber in not more than 90 sec. Analyses of the HLS content were
routinely made at least once each day.
Tests set up to determine median tolerance limits consisted of five H2S
concentrations and one control (Table 110). Temperature and oxygen
were adjusted to meet the requirements of the various tests as subse-
quently outlined. In some cases substrate, oxygen, and pH were varied
while a constant level of H_S was maintained. Experiments on the effect
of feeding and non-feeding of Gammarus during bioassay and on the effect
of H S on feeding characteristics were conducted.
219
-------
Table 110. SUMMARY OF ACUTE BIOASSAYS CONDUCTED WITH
ON SIX SPECIES OF INVERTEBRATES
Species
Asellus
Crangpnyx
Gamma r us
Baetis
Ephemera
Hexagenia
Number
of
tests
4
8
10
2
5
39
Duration,
days
4
4
4-10
2-4
4-11
2-11
Temper-
ature,
C
15.1
15.1
12.4
14.8
15.0
15.0
Range H^S
concentration ,
mg/1
0.044-2.196
0.029-2.671
0.008-0.112
0.008-0.064
0.106-0.617
0.005-0.702
LC50,a
rng/1 H.S
1.070-1.700
0.310-0.840
0.030-0.059
0.020-0.026
0.135-0.380
0.026-0.680
LC50 at conclusion of test period. Ranges represent all tests and
all exposure times.
220
-------
Effect of Test Chamber on Results
First tests with Hexagenia were done in cylinders (Colby and Smith ).
21 22
Consideration of work by Eriksen ' suggested that chamber design and
substrate could have a significant effect on the sensitivity of an
organism to a toxicant. A box was designed, therefore, which permitted
inclusion of a substrate into which the insects could burrow (Adelman
2 21
and Smith ). Eriksen showed that oxygen consumption was 0.65 cc/g
dry body weight/hr when nymphs were in burrows and 1.1 cc when on bare
substrate. During the present study comparative tests with cylindrical
chambers and boxes with substrate were made. With an oxygen level of
2 mg/liter and temperature of 15 C, the 48-hr LC50 for Hexagenia in
cylinders was 0.460 mg/liter H_S and in the box with substrate was
0.520 mg/liter H2S. Oxygen at 2.0 mg/liter was selected for species
collected in Rainy Lake because significant H~S concentrations were
usually not found where oxygen was higher (Colby and Smith ). The num-
bers of days to 50% and 25% survival in three pairs of box and cylinder
tests under the same conditions were more than twice as great in boxes
with mud substrate (Table 111). After 2 days of exposure, controls from
nine tests were transferred to Nitex baskets with and without mud sub-
strates for 6 days. When both experimental period and post-treatment
periods were with mud substrate, the survival was greater (Table 112).
In another test, mud substrate was provided in two series of H_S concen-
trations with box chambers. In one set burrowing was prevented by an
overlay of Nitex screen. Where burrowing was permitted, the 96-hr LC50
was 0.120 mg/liter H S and where prevented, 0.060 mg/liter H S. A 12-
day test without H_S was conducted in boxes with mud, with mud and screen
to prevent burrowing, and with no mud. Where burrowing was permitted,
survival was 62-75%. When burrowing was not permitted with mud present,
survival was 0-12%. In tests without mud present, survival was 12-38%.
Type ^f_ Substrate—To determine whether type of mud substrate was a
factor in resistance, two tests were run with sludge from below a paper
mill and mud from an unpolluted habitat of Hexagenia. The test chamber
221
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Table 111. SURVIVAL TIME OF Hexagenia IN DIFFERENT TYPE
CHAMBERS WITH H2S IN THREE PAIRED TESTSa
(days)
Chamber
Item
Cylinder Box Cylinder Box Cylinder Box
H2S (mg/liter)
Survival time
50% or more
25% or more
0.143 0.178 0.266 0.276 0.355 0.36
3.5 8 3 6 2.5 4.5
4.5 9.5 3.5 8.5 3.5 5.5
2 mg/liter 0,; 15 C.
Box contained mud for burrowing.
Table 112. EFFECT OF CHAMBER TYPE ON Hexagenia SURVIVAL
WITHOUT TOXICANT PRESENT3
Experimental
chamber
(2 days)
Cylinder without mud
c
Box with mud
Box with mud
Number
of
tests
2
2
5
Post-treatment
Nitex basket
chamber
(6 days)
Without mud
Without mud
With mud
Survival
Mean,
%
22.5
40.0
80.0
Range,
%
0-50
30-50
60-100
All tests at 2.0 mg/liter 02 and 15 C except as noted.
2 days of treatment, 6 days in post-treatment.
One test at 4.0 mg/liter 09.
222
-------
was a trough, half of which contained sludge and half mud so that water
flowed over both and nymphs had access to both. In the first test, after
3 days 78% of nymphs had selected the mud and in the second, after 4 days
87% selected the mud in preference to the sludge. In a subsequent 96-hr
LC50 bioassay conducted over mud and sludge there was little difference
with 0.320 mg/liter H2S on mud and 0.310 mg/liter H2S on sludge. Ap-
parently the type of material into which burrows were made did not alter
the reaction to ELS. When Crangonyx, a species found where natural ELS
may be abundant, was tested in boxes with mud and in cylinders without
mud, resistance to ELS was greater when animals could burrow (Table 113).
Gammarus were tested to determine the effect of various substrates on
resistance to ELS. Box chambers with mud, fine sand, pebbles (diameter
1 cm), and no substrate except the box floor were used in a 96-hr test
at 0.049 and 0.052 mg/liter ELS with one control. Oxygen was 3.84
mg/liter and temperature 15.1 C. Percentage survival varied from 4%
on mud to 36% on pebbles (Table 114). The course substrate had the
highest survival rate and the finest (mud), the lowest. The observed
reaction may have been related to reduced activity where suitable cover
was available.
Area and Volume—In one test with Gammarus at 5.92 mg/liter 02 and 14.8
C, designed to determine the effect of size of chamber on 96-hr LC50, an
increase of tenfold in bottom area and threefold in volume decreased
the 96-hr LC50 of ELS approximately 25%. Other tests conducted by Sie-
23
sennop and Smith (unpublished data) where chamber sizes were 6 and
20 liters, respectively, the LC50 was depressed still further (Table
115). It is believed that increased area decreased activity and hence
the LC50.
Effect of Water Quality
Oxygen—To determine the influence of ambient oxygen concentration on
resistance to ELS, Gammarus, Ephemera, and Hexagenia were subjected to
223
-------
Table 113. LC50 VALUES OF H2S TO Crangonyx IN CYLINDERS
AND BOX AND MUD CHAMBERS3
(mg/liter H9S)
Chamber
Cylinder
Box
48 hr
0.540
0.770
LC50
72 hr
0.425
0.590
96 hr
0.310
0.510
2 mg/liter
substrate.
, 15 C, pH 7.4; cylinders without mud; boxes with mud
Table 114. EFFECT OF SUBSTRATE TYPE ON SURVIVAL OF Gammarus
AT SIMILAR H2S LEVELS AND VARIED TIMES
(percentage survival)
H2S cone, (mg/1)
Substrate
24 hr
48 hr
72 hr
96 hr
Control
None
100
100
96
96
0.049
Mud
96
20
4
4
Survival
0.049
Fine sand
96
24
16
8
0.051
None
96
48
35
26
0.052
Pebble3
100
100
100
36
Diameter 1 cm.
224
-------
Table 115. EFFECT OF CHAMBER AREA AND VOLUME ON 96-HR LC50
OF HnS WITH Gammarus
/.
Bottom area,
Number of
individuals
25
25
20
40
Total
11
116
400
1,288
2
cm
Per
0
4
20
32
ind.
.44
.6
.0
.2
Volume,
Total
107
325
6,600
20,000
cc
Per ind.
4.3
13.0
330
500
96-hr LC50,a
mg/liter H0S
0.059
0.044
b
0.035
0.022°
Oxygen and temperature were approximately 6 mg/liter and 15.0 C for
all tests.
b_ . 2 j
Siesennop.
L. L. Smith, Jr. (unpublished data).
225
-------
various levels of oxygen and H_S. In the first series of tests Gammarus
and Ephemera were treated with constant levels of H_S and varied concen-
trations of oxygen. Gammarus was subjected to 0.094 mg/liter H^S and
5.77, 4.86, 3.18, and 2.30 mg/liter 02 at 11 C for 10 days or until all
organisms were dead. At the highest oxygen concentration 80% survived
for 10 days and at the lowest, 4% survived through 6 days. Ephemera
were exposed to 0.20 mg/liter H S and 7.9, 6.1, 3.9, and 1.7 mg/liter Q~
at 15 C. With 7.9 mg/liter ()„ no mortality occurred in 10 days but at
1.7 mg/liter 02 all died in 3 days. At intermediate oxygen levels 40%
or more survived 10 days. Hexagenia were exposed to 2 and 4 mg/liter
0 and five levels of H9S from 0.076 to 0.880 mg/liter at 15 C in
^ £•
cylindrical chambers without mud (two replications). At 2 mg/liter 0-,
LC50 was 0.620 and 0.460 mg/liter H2S at 24 and 48 hr, respectively.
In 4 mg/liter 0 , LC50 was 0.640 and 0.535 mg/liter H2S at 24 and 48 hr.
A series of four tests on Gammarus at 6 and 4 mg/liter 0_ and at 10 and
15 C was run to .determine the combined effect of the two variables on
LC50 of H-S. The tests were run for 10 days at five EJS levels (0.011-
0.075 mg/liter) in the cylindrical chambers without mud. After 10 days
the LC50 varied only 0.007 mg/liter (Table 116). The lowest was 0.042
mg/liter H2S at 4 mg/liter 0 and 15 C.
At 10 C and 6 mg/liter 0 early resistance was high (0.095 mg/liter lUS)
but decreased rapidly up to 6 days and little thereafter. At 15 C early
resistance was less and after 6 days decreased slowly. With 4 mg/liter
02 resistance to H-S was less at the higher temperature. These data
indicate that after initial difference in resistance, oxygen«and tem-
perature within the ranges tested do not have much influence on long-
term response.
Effect of pH—The effect of pH on H?S toxicity aside from its relation-
ship to dissociation was considered to be substantial by Bonn and
24
Follis on the basis of 3-hr tests with fish. During the present study
226
-------
Table 116. LC50 VALUES OF H2S TO Gammarus AFTER VARIOUS INTERVALS
AT 4 AND 6 MG/LITER 02 AND 10 AND 15 C
(mg/liter H0S)
6 mg/1 00 4 gm/1 0.
Days
2
4
6
8
10
10 C
0.095
0.059
0.053
0.050
0.049
^.
15 C
0.071
0.059
0.056
0.054
0.045
10 C
-
0.054
0.051
0.050
0.049
15 C
0.062
0.058
0.052
0.045
0.042
227
-------
with Hexagenia which were acclimated at test conditions, three 96-hr
tests at 7.4 pH and five 96-hr tests at 7.7 pH run in winter had a mean
LC50 of 0.365 and 0.151 mg/liter H S, respectively. Seven summer tests
run at 7.7 pH and the same temperature had a mean 96-hr LC50 of 0.111
mg/liter H~S. Oxygen concentration and temperature during all tests
were 2.0 mg/liter 02 and 15 C.
Effect of Laboratory Acclimation
A series of experiments were run to determine the effect of holding
test organisms in the laboratory prior to bioassay tests. Asellus,
Ephemera, and Hexagenia were held for varied lengths of time prior to
the start of each test. Results indicated a marked influence of labora-
tory acclimation time on resistance when pH, temperature, and food
availability were the same in comparative tests. Asellus were held in
fresh water in the laboratory for 9, 16, 30, and 44 days prior to testing
and then subjected to 96-hr LC50 tests. The LCSO's were 1.07, 1.21, 1.52,
and 1.70 mg/liter H~S for succeeding acclimation periods. Temperature
was 15 C, 02 was 2 mg/liter and pH, 7.3-7.5. Ephemera were held for 2
and 17 days in fresh water at 15 C and 2 mg/liter 0« before testing.
After 5 days LCSO's were 0.21 and 0.20 mg/liter lUS. At 7 days LCSO's
were 0.20 and 0.14 mg/liter H_S, respectively. Hexagenia were held in
fresh water on mud substrate with plant and animal detritus for 3, 6,
10, 14, and 18 days and then transferred to Nitex baskets without food
to determine subsequent survival rates in the absence of toxicants.
Holding up to 6 days resulted in reduced survival after transfer to
baskets but longer holding periods did not significantly increase mor-
tality beyond initial losses (Table 117). These results suggest the
advisability of a 6-day acclimation period prior to toxicity tests
because reactions do not vary with longer holding time.
Effect of_ H2S Acclimation
Because bottom-living organisms, which burrow in the substrate, may be
exposed continuously to low levels of H S, two tests were run on Hexa-
228
-------
Table 117. SURVIVAL TIME OF Hexagenia HELD ON MUD IN FRESH WATER
FOR VARYING PERIODS AND THEN TRANSFERRED TO NITEX BASKETS
WITHOUT FOOD OR TOXICANT
(days)
Holding period Survival rate in baskets
(days) 50% 10% 0%
3 10.2 10.8 11
6 5.5 8.5 9
10 7.2 7.8 8
14 4.5 7.5 10
18 5.6 11.5 13
229
-------
genia to determine the effect of H2S acclimation on acute response to
H9S. Test organisms were exposed in duplicate to 0.016 mg/liter ^S,
2 mg/liter 02, and 15 C at 7.7 pH for 13 days. The 96-hr LC50 was
0.108 mg/liter and 0.140 mg/liter H S. The 96-hr LC50 of organisms held
in freshwater for the same period and tested simultaneously were 0.103
and 0.098 mg/liter H2S. After 10 days of exposure to 0.036 mg/liter H2S
under the same conditions as the 13-day acclimation, the 96-hr LC50 was
0.108 mg/liter H-S. Simultaneous tests with the organisms held in
fresh water was 0.135 mg/liter lUS. The data suggest that pretreatment
with very low levels may increase resistance to acute levels of H~S
but that higher pretreatment levels increase sensitivity. The results
are inconclusive but indicate the need for more careful evaluation of
effects of low level exposure of bottom-living invertebrates on sub-
sequent acute lethal tests.
Effect of Season, Sex and Size
It is usually more desirable to catch wild invertebrates shortly before
testing rather than to maintain cultures. Because seasonal differences
in resistance may occur, acute tests were run at different seasons from
1967-1969 on Hexagenia (Table 118). It is evident from the data that
organisms taken in summer are more sensitive than those taken in fall
and winter and also that wild organisms taken in different years may
vary in sensitivity.
In seven lUS bioassays with Gammarus ranging in size from 8.0 to 16.0
mm mortality rates of males and females were compared and size of mor-
talities and survivors noted. No significant difference in sensitivity
to H2S of males and females or different sizes was apparent. *There was
considerable variability between tests but no trends.
Effect of H S on Behavior
Behavioral effects of ILS on Ephemera and Hexagenia were noted by obser-
vation of emergence of nymphs from burrows during 10-day tests in
230
-------
Table 118. LC50 VALUES OF H2S FOR Hexagenia COLLECTED
IN DIFFERENT MONTHS3
(mg/liter H0S)
Month
1967-68
August
October
December
Feb ruary
July
1968-69
November
January
March
May
July
48 hr
0.23
0.40
0.46
0.39
0.25
0.16
-
-
-
0.13
LC50
72 hr
0.16
0.18
0.26
0.24
0.17
0.16
0.14
0.17
0.11
0.06
96 hr
0.12
0.15
0.16
0.10
0.12
-
0.09
0.12
0.07
0.03
Tests at 15 C, 2 mg/liter O^j and 7.7 pH; acclimation 10 days at test
•mrl T i~ 1 rvr> c
conditions.
231
-------
chambers with mud substrate at different concentrations of H^S and 0^.
Percentage of emergence was based on the total number of individuals in
the test period. At an exposure of 0.20 mg/liter H2S and four levels of
0 from 7.9 to 1.7 mg/liter, emergence of Ephemera varied from 100% in
3 days at 1.7 mg/liter 0- to no emergence in 9 days at 7.9 mg/liter 0^
(Table 119). At 2.00 mg/liter 02 and five levels of H2S from 0.16 to
0.30 mg/liter, emergence was earlier at the higher H2S concentrations.
With Hexagenia subjected to 2.0 mg/liter 0^ and five H2S concentrations
from 0.18 to 0.54 mg/liter, emergence of nymphs was sooner at higher con-
centrations (Table 120). In all test series using mud substrates only
two nymphs died in burrows rather than emerging before death. The
difference in days between 50% emergence of nymphs and 50% mortality of
Ephemera varied from less than 1 day at 0.5 mg/liter tUS to 4 days at
0.15 mg/liter. With Hexagenia the difference was 3 days throughout the
same range of sulfide concentrations.
Effect of H.S on Feeding of Gammarus
When Gammarus was fed Populus alba pyramidalis leaves and treated with
0.010-0.050 mg/liter H2S at 10 C and 5.8-6.0 mg/liter 02> food consump-
tion varied from 0.431 mg/individual/day in controls to 0.135 mg/indi-
vidual/day at 0.050 mg/liter H2S (Table 121).
Summary of Acute Tests
From the foregoing data it is apparent that conditions under which a
bioassay is run and the time of year when specimens are collected alter
the response to H_S. It is also important that an unnatural degree of
activity in test organisms not be induced by test conditions. *To deter-
mine a useful LC50 for purposes of habitat evaluation, the data from
tests done under conditions in our judgment most similar to the natural
habitat were selected (Table 122).
A comparison of relative sensitivity of the invertebrates tested showed
Asellus to be the most resistant, followed by Crangonyx and the burrowing
232
-------
Table 119. EMERGENCE OF EphemeraaWITH CONSTANT H S
AND VARIED OXYGEN CONCENTRATION AND WITH CONSTANT OXYGEN
AND VARIED H2S CONCENTRATION IN SUCCEEDING DAYS
(expressed as percentage)
. b
0^, me/1 H,
Days
1
2
3
4
5
6
7
8
9
10
1.7 3.9
0 10
100 10
100 10
d
10
20
20
40
50
60
60
6.1
10
10
10
20
20
20
20
20
20
30
7.9
0
0
0
0
0
0
0
0
0
0
0.0
10
0
0
10
10
10
10
10
10
10
A
0.16
20
20
70
30
50
50
100
100
80
100
,S, mg/lc
0.19
40
70
100
100
80
100
100
100
100
100
0.26
80
100
100
100
100
100
100
100
100
100
0.30
100
100
100
d
-
-
-
-
-
—
Emergence of nymphs from burrows in two tests.
With 0.20 mg/liter H S.
2.0 mg/liter
Dash indicates no data - all nymphs emerged and died.
233
-------
Table 120. EMERGENCE3 OF Hexagenia WITH 2.0 MG/LITER C>2 AND
VARIED CONCENTRATIONS OF H2S
(expressed as percentage)
H0S concentration, mg/liter
Day
1
2
3
4
5
7
8
9
10
0.00
0
0
10
20
0
30
40
20
60
0.18
0
11
11
22
33
78
100
100
100
0.25
0
22
78
78
89
100
-
-
—
0.35
0
20
80
100
100
-
-
-
—
0.43
30
70
100
100
100
-
-
-
—
0.54
90
100
100
_b
-
-
-
-
-
o
, Emergence of nymphs from burrows.
Dash after 100 indicates all dead.
234
-------
Table 121. FEEDING OF Gammarus ON Populus alba, py.ramidalis LEAVES
AT VARIOUS LEVELS OF H2S
(intake expressed as mg/individual/day)
H«S concentration,
mg/liter Test 1E
0.0 0.677
0.010
0.013 0.550
0.016
0.020
0.028 0.489
0.031
0.033
0.039
0.047
0.050
Test 2a Test 3b
0.604 0.431
0 . 381
-
0.680
0.367
-
0 . 340
0.428
0.244
0.196
0.135
* 6.0 mg/liter 02, 15 C, and pH 7.51.
5.8 mg/liter 09, 10 C, and pH 7.52.
235
-------
Table 122. 96-HOUR LC50 VALUES OF H2S FOR SIX INVERTEBRATES
COLLECTED AT DIFFERENT SEASONS
Species
As ell us
Crangonyx
Gamma r us
Baetis
Ephemera
Hexagenia
PH
7.5
7.4
7.5
7.6
7.4
7.7
7.7
7.4
mg/1
2.0
2.0
5.9
6.2
1.9
2.0
2.0
2.0
Temper-
ature,
C
15.2
14.9
15.0
14.8
15.0
15.0
15.0
15.0
Days
4
4
4
4
4
4
4
4
96-hr
LC50,
mg/1 HnS
1.07
0.84
0.059
0.0203
0.316
o.mb
0.151
0.365
Season
Winter
Winter
Winter
Summer
Winter
Summer
Winter
Winter
Mean of two tests.
Mean of seven tests - July collection.
236
-------
mayflies. The most sensitive were the flowing stream forms with a 96-
hr LC50 that was 2-6% that of the most resistant forms. In general, as
might be expected from flowing stream forms, the degree of resistance
was closely related to the probable occurrence of low oxygen or high
H S in their normal habitat.
CHRONIC TESTS
Hexagenia limbata (Serville)
Hexagenia nymphs inhabit U-shaped burrows dug in fine bottom sediment.
25
Eriksen demonstrated that the stratum of water available to 20-35 mm
nymphs was the 6-7 mm layer above the mud-water interface. Earlier
nymphal stages (<1 mm at hatch) have a correspondingly reduced water
stratum available. The work reported here was designed to test the
effects of chronic exposure to H~S under general habitat conditions as
close as possible to optimum for the species. Acute tests were run
for comparison.
Experimental Design—Nymphs for the study were collected from the
Crystal Beach area of Rainy Lake near Ranier, Minnesota on May 19, 1973
at 2 to 4 ft. Mud with nymphs still in the burrows was placed in a
screen-bottomed box, suspended in lake water, and the mud gently washed
out. Nymphs were transferred to the laboratory in lake water. The mean
length of the nymphs from the tip of the rostrum to the tip of the
abdomen was 1.77 cm. Prior to testing nymphs were held in glass aquaria
(50 x 25 x 20 cm) with a water depth of 16 cm. Three cm of mud from
the same area in which the nymphs were collected was placed on the
bottom. The 200 nymphs held in each aquarium formed burrows within a
few hours after placement. Water at 18 C, pH 7.8, and saturated with
0? was passed through each aquarium at the rate of 500 ml/min during
pretest holding period. The nymphs were fed a finely ground suspension
of lettuce twice daily and recently hatched brine shrimp once each day.
The acute test was conducted in flow-through apparatus with five toxicant
237
-------
concentrations and one control as described by Colby and Smith and
2
Adelman and Smith. Toxicant was added as sodium sulfide from stock
solution by the "dipping bird" dispenser. Test chambers for acute
studies were identical with those used for laboratory maintenance of
the nymphs. Water flow through each chamber was 300 ml/min and a light
cycle of 12 hr of light and 12 hr of darkness was maintained. Nymphs
were not fed during the first 96 hr of each test but thereafter were
fed on the same schedule as the laboratory stock. Analyses were made
three times each day for HLS and once each day for pH, temperature,
dissolved 0 , and total alkalinity- Water samples for the determina-
tion of H~S, pH, temperature, and dissolved CL were siphoned from near
the mud-water interface of each chamber. H-S samples were fixed im-
mediately to insure that the exact concentration assumed to be bathing
the nymphs in the burrows was determined.
Chronic tests employed two diluters modified from that of Brungs and
4
Mount and the same test chambers described for nymph maintenance. Each
diluter served four concentrations and one control. H~S concentrations
from one diluter were alternated randomly with those of the other to
form a continuous series. Temperature and pH were controlled and water
was saturated with 0 in the head tanks. Chemical analyses were made
in the same manner as in the acute tests. H S, pH, and temperature were
determined three times per week and dissolved 0 once weekly. The
feeding schedule was that used for laboratory maintenance. Chronic
treatment of nymphs was started on July 9, 1973 and terminated 138 days
later. Mortality of nymphs and emergence of subimages to adults were
determined daily.
Acute Toxicity—Acute tests were run in connection with chronic tests
for critical comparison. H S concentrations in these tests ranged from
0.0251-0.4723 mg/liter with one control, temperature from 17.8-18.3 C,
pH from 7.67-7.99, and 0 from 4.53-6.63 mg/liter in the various cham-
bers (Table 123). Total alkalinity as CaCO was constant at 235 mg/liter.
238
-------
Table 123. CHARACTERISTICS OF TEST DURING EXPOSURE OF
Hexagenia IN THE ACUTE TEST
Test chamber
Item 1
x H2S (mg/1) 0
Std. Dev. 0
x pH 7.99
x temperature (C) 18.3
x dissolved 00 6.63
5
0.0251
0.0123
7.90
18.2
6.03
4
0.0466
0.0159
7.90
18.2
6.22
3
0.1078
0.0227
7.84
18.1
5.81
2
0.2890
0.0230
7.73
17.8
4.94
6
0.4723
0.0151
7.67
17.9
4.53
(mg/1)
x total alkalinity 235
(mg/1)
235
235
235
235
235
239
-------
A total of 10 individuals was placed in each test chamber. The LC50
concentration of H_S dropped from 0.312 mg/liter at 48 hr to 0.165
mg/liter at 96 hr and at 12 days was 0.060 mg/liter (Table 124). Per-
centage survival dropped to 80% in the lowest treatment after 6 days
but in all others after the first or second day- Survival in control
over the entire period was 70%.
Chronic tests—The test conditions in the two chronic bioassays (Table
125) included one control and four H S concentrations ranging from 0.0-
0.0762 mg/liter. Temperature, pH, and dissolved 0_ were similar to those
in acute tests. A total of 10-13 nymphs were placed in each tank (Table
126). Mortality was low (0-9%) in all concentrations lower than 0.0290
mg/liter ELS. At this concentration mortality was 37% and at 0.0762
mg/liter H.S none survived. Emergence of subimagos varied from 30-75%
at levels below 0.0348
no emergence occurred.
at levels below 0.0348 mg/liter BUS. At this concentration and higher
There was a small but non-significant reduction in length of subimagos
as the H_S concentration increased in each experiment. Nymphs exposed
to 0.0152 mg/liter H2S were 6% shorter and at 0.0129 and 0.0290 mg/liter
H_S were 3% shorter than the controls.
Summary—On the basis of nymphal survival and the percentage which
emerged as subimagos in the chronic tests, it is apparent that concen-
trations up to 0.0152 mg/liter H2S in the Diluter 1 test and 0.0129
mg/liter in the Diluter 2 test are not different from the controls
(Table 126).
With a 96-hr LC50 of 0.165 mg/liter H2S and the highest safe level of
0.0152 mg/liter, the application factor (.0152/.165) would be .09.
A ratio based on the 12-day LC50, highest safe concentration, and appli-
cation factors is made between the Hexagenia and data from the amphipod,
Gammarus (Table 127). It is apparent that although there are wide
differences in the 96-hr LC50, 12-day LC50, and highest chronic safe
240
-------
Table 124. SURVIVAL OF NYMPHS AND CALCULATED LC50'S OF lU
TO Hexagenia ON SUCCEEDING DAYS DURING THE ACUTE TEST3
(expressed as percentage)
Day
1
2
3
4
5
6
7
8
9
10
11
12
LC50,
me/1 EnS
4.
0.312
0.185
0.165
0.135
0.134
0.120
0.110
0.090
0.072
0.072
0.060
H0S concentration, mg/1
0.0
100
100
100
100
100
100
100
100
100
100
100
100
0.0251
100
100
100
100
100
100
90
90
80
80
80
80
0.0466
100
90
90
80
80
60
50
50
50
50
50
50
0.1078
100
100
90
80
80
80
70
60
40
20
20
0
0.2890 0.4723
100 100
60 0
30
20
0
-
-
_
-
-
-
— ~
a Survival values have been corrected on the basis of survival in the
control tank (Abbott ).
241
-------
Table 125. TEST CONDITIONS DURING CHRONIC EXPOSURE OF Hexagenia
Test chamber - Diluter 1
Item 3
x H-S concentration (mg/1)
Std. Dev.
x pH 7.9
x temperature (C) 17.7
x dissolved 0,, (mg/1) 7.53
2
0.0011
0.0009
7.9
17.8
7.65
1
0.0060
0.0022
8.0
18.0
7.37
5
0.0152
0.0050
8.0
17.7
6.86
4
0.0348
0.0105
8.0
17.8
5.52
Test chamber - Diluter 2
8
x H«S concentration (mg/1) -
Std. Dev.
x pH 7.8
x temperature (C) 17.5
x dissolved 00 (mg/1) 7.34
10
0.0042
0.0017
7.9
17.8
7.11
9
0.0129
0.0060
7.9
17.6
6.84
7
0.0290
0.0096
7.9
17.7
6.06
6
0.0762
0.0153
8.2
17.9
4.75
242
-------
Table 126. SUMMARY OF SURVIVAL OF Hexagenia NYMPHS AND
EMERGENCE OF SUBIMAGOS DURING THE CHRONIC TEST
Test chamber
Diluter 1 Diluter 2
Item 3 2 1 5 4 8 10 9 7 6_
H2S (mg/1) 0 0.0011 0.0060 0.0152 0.0348 0 0.0042 0.0129 0.0290 0.0762
Number of 12 13 10 11 11 13 13 12 11 11
nymphs
Percentage 88 0 9 36 80 037 100
deaths
Percentage 50 38 30 36 0 54 62 75 36 0
subimagos
emerged
Percentage 92 92 100 91 64 92 100 100 73 0
emerged
or sur-
vived in
138 days
243
-------
Table 127. RELATIONSHIPS BETWEEN LC50, CHRONIC SAFE, AND
APPLICATION FACTOR VALUES FOR Hexagenia AND Gammarus
Item Hexagenia Gammarus
96-hr LC50 (mg/1 H2S) 0.165 0.022
12-day LC50 (mg/1 H2S) 0.060 0.011
Highest safe level (mg/1 H2S) 0.015 0.002
Application factor based on .09 .09
96-hr LC50
Application factor based on .25 .20
12-day LC50
244
-------
level, the differences in application factors are quite small.
Gammarus pseudolimnaeus Bousfield
To determine the long-term effect of exposure to sublethal levels of
H-S, four tests were run on Gammarus from 65 to 105 days. This period
permitted a minimum of one reproductive cycle.
Experimental Design—Gammarus for the experiments were secured from two
spring-fed streams; Valley Creek flowing into the St. Croix River near
Afton, Minnesota and an unnamed stream flowing into the St. Croix River
near Marine on St. Croix, Minnesota. Collections were made by placing
a screen on the bottom and gently stirring bottom vegetation to dis-
lodge the organisms. Collections were made on November 20, February 16,
July 27, and December 29 when stream temperatures were 7, 6, 12, and
3 C, respectively.
Acute tests were made prior to the start of each chronic test to deter-
mine the 96-hr LC50 of H?S to the test organisms. Chronic tests were
then started with individuals from the same stock and continued for 65
to 105 days. Specimens were raised to a temperature of 18 C over a
period of 5 to 7 days and then held for 10 days prior to the start of
acute and chronic tests. During the acclimation and chronic test
periods they were fed leaves of Populis alba pyramidalis. Leaves were
presoaked in flowing laboratory water for 2 wk before they were used as
food.
2
Acute tests were made in the apparatus described by Adelman and Smith
employing glass test chambers 50 x 25 x 20 cm. Water depth was 16 cm
and total volume 20 liters. Flow rate through the chambers was 300
ml/min. H?S concentrations at the center of each chamber were deter-
mined three times a day during the test. Acute tests were run with five
levels of H«S and one control.
245
-------
Chronic exposures on new individuals were made in the same chambers as
were used for acute tests with a diluter modified as described by Brungs
and Mount. Four H9S concentrations and one control were employed with
a flow-through rate of 125 ml/min. The concentration was analysed
three times per week. Gammarus were fed leaves as described above with
a sufficient number placed in each tank to provide both food and cover.
Tanks were illuminated with 61-cm Duratest Vitalite fluorescent lamps
providing 50 ft-candles at the water surface. Photoperiod was 16 hr
of light and 8 hr of darkness. The pH and temperature were maintained
18
by methods described by Oseid and Smith. At the start of each chronic
experiment, except test 3, 20 coupled pairs were placed in each chamber.
In test 3, 40 individuals were used without reference to sex. At the
completion of each test after 65, 96, and 105 days, formalin was added
to each tank to kill and preserve the organisms. They were counted and
measured within 1 month and the entire group was weighed after centri-
fuging to remove adherent water.
Effect of H^,S—Acute tests were run with H«S concentrations ranging
from 0.008 to 0.093 mg/liter (Table 128). Temperature varied from
17.8-18.1 C, oxygen from 5.8-7.4 mg/liter,, and pH from 7.7-7.9. Total
alkalinity ranged from 222-232 mg/liter CaCO . The total length of
organisms exclusive of antennae was 0.7-1.2 cm. For the successive
tests the 96-hr LCSO's were 0.022, 0.022, 0.024, and 0.021 mg/liter
H^S. A single threshold test run through 18 days gave an LTC value of
0.011 mg/liter H2S.
H_S concentration in the four chronic tests varied from 0.0007 to 0.0192
mg/liter (Table 129). Temperature in three tests varied from 17.1-17.8
C and in one test was 18.0-18.2 C. Dissolved 0 ranged from 7.4-8.9
mg/liter. Survival at the higher levels was as low as 4% of the control
and as high as 57% in another test. The duration of the exposure did
not seem to have a direct relationship to the survival totals but the
maximum number was assumed to be controlled by chamber size which was
constant. Length-frequency distribution of the organisms surviving at
246
-------
Table 128. ACUTE TOXICITY OF H2S TO Gammarus USED IN CHRONIC STUDIES
Test
1
2
3
4
Range of
u c a
tlyd cone. ,
me/1
0.012-0.093
0.012-0.061
0.009-0.044
0.008-0.054
Temper-
ature,
C
17.9
17.8
18.0
18.1
PH
7.7
7.9
7.8
7.9
Threshold
96-hr LC50, LTC,b
mg/1 H^S mg/1 H0S
0.022
0.022
0.024
0.021 0.011
, Set up in logarithmic series.
18-day duration.
247
-------
Table 129. CONDITIONS DURING CHRONIC TESTS OF Gammarus WITH H0SC
x H2S cone, (mg/1)
Std. Dev.
x Temperature (C)
x pH
No. survivors
ft
Weight survivors (g)
x H«S cone, (mg/1)
Std. Dev.
x Temperature (C)
x pH
No. survivors
Weight survivors (g)
x H~S cone, (mg/1)
Std. Dev.
x Temperature (C)
x pH
No. survivors
Weight survivors (g)
Test 1
Control
-
17.2
7.80
446
1.961
Test 2
Control
17.4
7.66
477
3.320
Test 3
Control
-
17.7
7.83
229
0.932
(65 days)
0.0007
0.0007
17.1
7.81
548
2.349
(105 days)
0.0007
0.0012
17.3
7.65
380
1.628
(95 days)
0.0012
0.0013
17.6
7.82
432
1.361
0.0019
0.0013
17.1
7.83
504
2.648
0.0013
0.0017
17.4
7.67
793
3.402
0.0032
0.0024
17.7
7.85
374
1.179
0.0031
0.0021
17.3
7.76
117
1.083
0.0029
0.0027
17.6
7.60
458
2.254
0.0064
0.0032
17.8
7.80
241
0.914
0.0128
0.0075
17.2
7.86
19
0.091
0.0071
0.0044
17.5
7.69
363
2.390
0.0153
0.0051
17.7
7.85
19
0.302
248
-------
Table 129 (continued). CONDITIONS DURING CHRONIC TESTS OF
Gammarus WITH H0Sa
.. _. _,_
Test 4 (65 days)
x H2S cone, (mg/1)
Std. Dev.
x Temperature (C)
x pH
No. survivors
Weight survivors (g)
Control
-
18.1
7.68
581
2.478
0.0012
0.0006
18.1
7.70
421
2.150
0.0026
0.0008
18.2
7.75
53
0.367
0.0076
0.0026
18.0
7.78
607
2.778
0.0192
0.0062
18.0
7.71
336
1.174
, Dissolved 0? range 7.4-8.9 rag/liter.
Standard Methods of Water Analysis - colorimeter used instead of
tube comparison.
£
Total weight of all survivors.
249
-------
the end of the experiment was roughly the same for the four tests
(Figure 8). The distribution within a single test was essentially the
same except that total numbers in each size class varied with the H-S
level.
Summary—The results presented above suggest that at concentrations of
H_S below 0.002 mg/liter there was increased reproduction or survival
in some tests but there was no consistent effect on mean weight of
individuals. At levels in excess of 0.002 mg/liter there was a reduc-
tion in numbers and a consequent reduction in total weight of the test
group. Treatments between 0.0013 and 0.019 mg/liter showed a mean
reduction of 71% in numbers and 76% in total weight when compared to
the controls. On the assumption that 0.002 mg/liter ILS is the maximum
safe level for Gammarus and the mean 96-hr LC50 of 0.022 mg/liter H2S
is employed, an application factor of .10 would apply. It is apparent
from these data that the sensitivity of Gammarus is at approximately
the same level as that of the brook trout. Although the expectancy of
any significant level of H2S in a flowing stream which is normally in-
habited by Gammarus remains to be demonstrated, the extremely low level
which is toxic might well be attained in bottom soil or in decaying
detritus. Field studies on this point would be useful.
250
-------
80
60
40
20
0
80
0 20
Z
0
IOO
U.8O
O
60
4O
(T
LJ
0)20
40
20
0
20
CONTROL
TOTAL NUMBER=446
0.0007 MG/L H2S
TOTAL NUMBER=548
0.0019 MG/L H2S
TOTAL NUMBER = 504
0.0031 MG/L H2S
TOTAL NUMBER* 117
O.OI28 MG/L H2S
TOTAL NUMBER = I 9
3.0 9.0 fO.O T2.0
LENGTH (MM)
14.0
16.0
18.0
Figure 8. Length-frequency distribution of Gammarus after 65 days of
exposure to varied concentrations of H S.
251
-------
SECTION XIV
EFFECT OF pH ON TOXICITY
The relationship between pH of various sulfide test solutions and
apparent toxicity of the ambient H S levels for a series of tests is
summarized here with details presented in Part II of this report.
It was observed that over the pH range of 6.5 to 8.7 the apparent
toxicity of molecular H S to the fathead minnow increases by approximately
fourfold with the greatest increase in toxicity occurring above pH
values of about 7.1. An attempt was made to explain this change in
toxicity by assuming it resulted from a greater depression in the pH
value of the respiratory water as it passes over the gill surface when
the carbon dioxide content of the original test solution is low com-
pared with when it is relatively high. This would result in the actual
pH at the gill surfaces of fish being substantially lower than the
measured ambient levels. Consequently, the concentration of H_S would be
increased at the gill surface as a result of the HS ions forming H?S
to a greater degree in test solutions containing a relatively low con-
centration of free carbon dioxide. Theoretically such an explanation
may account for the apparent increase in the toxicity of H-S with a
reduction in the concentration of free carbon dioxide and accompanying
increase of pH in the test solution. However, it appears that the
above explanation may not be appropriate and it has been demonstrated
in Part II that the acute toxicity of sulfide solutions to the fathead
minnow is not entirely related to the ambient concentration of molecular
HS but is linearly related to the ambient concentration of dissolved
252
-------
sulfide (i.e., H-S plus HS ). It is proposed that the acute toxicity
of sulfide solutions of relatively low pH values results mainly from
the penetration of the gills by molecular HLS, But the increased
apparent toxicity of molecular H.S with increasing pH may result from
a greater contribution in the penetration of the gills by the HS ion
and because of the effect which the internal pH, as related to ambient
C0~ tensions, would have on the blood and intracellular sulfide equi-
librium.
253
-------
SECTION XV
DISCUSSION
DIFFERENCE IN ACUTE SENSITIVITY BETWEEN SPECIES
A wide range of acute sensitivity to H S exists between various fish
species (Table 130). At comparable temperatures, LTC values for juve-
nile fish ranged from 0.0087 mg/liter H2S at 12 C in 17 days with
rainbow trout to 0.0840 mg/liter H S at 14 C for goldfish in 11 days.
Other fish species varied between these extremes. The maximum diffe-
rence between species was approximately fourfold for comparable temper-
atures and life history stage. However, temperature and life history
stage have a large influence on response of individual species as will
be discussed below.
DIFFERENCES IN ACUTE TOXICITY OF VARIOUS LIFE HISTORY STAGES
The acute sensitivity of various life history stages in the same species
varies with fry being the most sensitive, except in the goldfish where
eggs are the most sensitive (Table 131). The difference in sensitivity
between the most and least sensitive stage was approximately three- to
elevenfold. As fish increase in age beyond the fry stage, they become
more resistant to the acute effects of H-S. Presumably this increase is
related to differences in metabolic rates.
These findings suggest that no prediction of the relative sensitivity
of various stages can be projected for untested species. While tempera-
tures of tests varied between stages in some cases, they were close to
conditions usually encountered by the fish in nature.
254
-------
Table 130. ACUTE TOXICITY OF H2S TO SEVEN SPECIES OF FISH
(expressed as rag/liter)
to
in
en
Species
Fathead
b
minnow
Duluth
stock
Field
stock
Number
of
Stage tests
Egg 6
Fry 3
Juve- 1
nile
3
Juve- 4
nile
1
1
7
6
Original values
Temp . ,
C
24
24
20
22
6
10
15
20
24
96-hr
Mean
.0350 .
•
.0070 .
.
.0160
.0162 .
•
.5150 .
.
.1500
.0570
.0360 .
.
.0212 .
.
LC50
Range
0190-
0610
0066-
0075
-
0127-
0180
4600-
5800
-
-
0320-
0390
0152-
0280
LTC
Mean Range
.0345 .0190-
.0595
.0061 .0057-
.0066
-
.0120 .0109-
.0130
_ _
_
-
-
.0189 .0143-
.0220
96-hr
Mean
.0536 .
•
.0107 .
.
.0238
.0246 .
•
.7089 .
.
.2041
.0814
.0545 .
•
.0324 .
o
Corrected values
LC50
Range
0291-
0935
0101-
0115
-
0193-
0273
6332-
7983
-
-
0484-
0590
0232-
0428
LTC
Mean Range
.0529 .0291-
.0912
.0093 .0087-
.0101
-
.0182 .0166-
.0198
_ _
-
-
-
.0289 .0219-
.0336
Days
4-8
6
-
8-10
_
-
-
-
7-9
-------
Table 130 (continued). ACUTE TOXICITY OF H2S TO SEVEN SPECIES OF FISH
(expressed as mg/liter)
to
01
Oi
Species Stage
Goldfish Egg
Fry
Juve-
nile
"
M
Sucker Egg
ii ^
Fry°
Juve-
nile
Number
of
tests
1
1
21
21
21
1
1
3
4
Original values
Temp. ,
C
22
22
14
20
26
15
13
15
20
96-hr
Mean
.0220
.0250
.1450 .
.
.0830 .
.
.0630 .
•
.0280
-
.0210 .
•
.0219 .
m
LC50
Range
-
-
1120-
1950
0680-
1150
0500-
0840
—
-
0130-
0260
0185-
0290
LTG 96-hr
Mean Range Mean
.0321
.0370
.0840 .0660- .2002
.1200
.0710 .0520- .1201
.0840
.0600 .0460- .0935
.0840
.0383
.0190
.0287
.0330
*a
Corrected values
LC50
Range
-
-
.1546-
.2692
.0984-
.1664
.0742-
.1247
_
-
.0178-
.0356
.0279-
.0437
LTC
Mean Range
-
-
.1160 .0911-
.1656
.1027 .0752-
.1215
.0891 .0683-
.1247
_ _
.0268
-
-
Days
-
-
11
11
11
_
12
-
-
-------
Table 130 (continued). ACUTE TOXICITY OF H^S TO SEVEN SPECIES OF FISH
(expressed as mg/liter)
to
Species Stage
Bluegill Egg
Sac fry
Swim- up
fry
Juve-
nile
Adult
c
Walleye Egg
c
c
Fry
Juve-
Number
of
tests
2
1
1
5
7
2
2
1
4
Temp. ,
C
22
22
22
20
20
12
15
15
15
96-hr
Mean
.0144d
-
.0086
.0316
.0297
.0520
.0805
.0070
.0193
nile
Original values
LC50 LTC
Range Mean Range
.0125-
.0162
.0169
.0084
.0290- .0318 .0310-
.0325 .0325
.0198-
.0375
.0290 .0220-
.0360
^
.0740-
.0870
- - -
.0166-
.0214
96-hr
Mean
.0219 .
.
-
.0131
.0478 .
.
.0448 .
t
.0720
.1103 .
t
.0096
.0282 .
o
Corrected values
LC50 LTC
Range Mean Range
0190-
0246
.0253
.0128
0438- .0481 .0468-
0491 .0491
0298-
0565
.0402 .0305-
.0499
1014-
1192
- - -
0242-
0312
Days
-
9
8
8-10
-
10-19
-
-
-
-------
Table 130 (continues). ACUTE TOXICITY OF H^S TO SEVEN SPECIES OF FISH
(expressed as mg/liter)
to
01
00
Species
Rainbow
trout
Brook
trout
Stage
Egg
Fry
Juve-
nile
it
Egg
it
Sac fry
ii ii
Swim- up
Number
of
tests
1
1
1
1
2
2
2
2
2
Original values
Temp . ,
C
13
13
12
15
9
14
9
14
9
96-hr LC50
Mean Range
-
-
.0130
.0125
-
-
.0210 .0206-
.0214
.0148 .0138-
.0158
.0233 .0232-
.0234
Mean
.0154
.0056
.0087
-
.0760
.0500
.0160
.0120
.0223
LTC
Range
-
-
-
-
.0738-
.0783
.0485-
.0516
.0160-
.0161
.0117-
.0124
.0220-
.0226
o
Corrected values
96-hr LC50
Mean Range Mean
.0215
.0077
.0181 - .0121
.0179
.1035
.0697
.0286 .0280- .0218
.0291
.0204 .0190- .0165
.0218
.0318 .0316- .0304
.0319
LTC
Range
-
-
-
-
.1005-
.1066
.0676-
.0719
.0218-
.0219
.0161-
.0171
.0300
.0308
Days
29
20
17
-
10-13
8-9
10
10
10
-------
Table 130 (continued). ACUTE TOXICITY OF H2S TO SEVEN SPECIES OF FISH
(expressed as rag/liter)
to
en
Number
Original values
of Temp. , 96-hr LC50
Species Stage tests C Mean
Brook Swim-up 2 14 .0216
trout fry
Juve- 3 8 .0266
nile
" 2 11 .0230
3 14 .0183
4 16 .0168
2 19 .0168
2 21 .0178
Range
.0215-
.0217
.0245-
.0297
.0228-
.0233
.0156-
.0219
.0163-
.0173
.0168
.0177-
.0179
LTC
Mean Range
.0186 .0186-
.0187
.0197 .0187-
.0212
.0178 .0171-
.0184
.0155 .0153-
.0156
.0138 .0129-
.0146
-
.0078 .0078
o
Corrected values
96-hr LC50
Mean
.0302
.0358
.0313
.0263
.0240
.0244
.0264
Range
.0301-
.0304
.0330-
.0400
.0310-
.0317
.0224-
.0315
.0233-
.0247
.0244
.0262-
.0265
LTC
Mean Range
.0260 .0260-
.0262
.0265 .0252-
.0286
.0242 .0232-
.0250
.0223 .0220-
.0224
.0197 .0184-
.0209
-
.0116 .0116
Days
9
11-12
9
6-11
8-10
-
10-12
Values derived from Pomeroy constants corrected to new values from ionization constants worked
determine seasonal and geographical
put in this study.
Does not include 40 sets of acute tests on fathead minnows to
variation.
Data from Smith and Oseid.
LC50 at hatch time of 66 and 77 hr.
-------
Table 131. MEDIAN TOLERANCE LIMITS (LC50) TO H2S OF EGGS, FRY, AND JUVENILES OF VARIOUS SPECIES
(mg/liter H?S - duration days in parentheses)
Temp . ,
Species C Egg
Brook trout 9 0.0760
(10-13)
14 0.0500
(8-9)
to
o
Rainbow trout 13 0.0154
(29)
Sucker 15 0.0280°
(4)
Northern piked 13 0.037
(4)
Fathead 24 0.0345
(4-8)
Original
Sac fry
0.0160
(10)
0.0120
(10)
0.0056
(20)
0.0210C
(4)
0.026
(4)
0.0061
(6)
values Corrected values
Swim-up Swim-up
fry Juvenile Egg Sac fry fry Juvenile
0.0223 0.0197 0.1035 0.0218 0.0304 0.0265
(10) (11-12)
0.0186 0.0155 0.0697 0.0165 0.0260 0.0223
(9) (6-11)
0.0087 0.0215 0.0077 - 0.0121
(17)
0.0219 0.0383C 0.02876 - 0.0330
(4)
0.052 0.036
0.0120e 0.0529 0.0093 - 0.01826
(8-10)
-------
Table 131 (continued). MEDIAN TOLERANCE LIMITS (LC50) TO H2S OF EGGS, FRY, AND JUVENILES
OF VARIOUS SPECIES
(mg/liter H9S - duration days in parentheses)3
to
CT5
Original values
Species
Goldfish
Walleye
Blue gill
Temp . ,
C Egg
22 0.0220
(4)
15 0.0805C
(4)
22 0.0144
(3)
Sac fry
0.0250
(4)
0.0070°
(4)
0.0169
(9)
Swim- up
fry Juvenile
0.0830f
(4)
0.0193
(4)
0.0084 0.0318f
(8) (8-10)
Corrected values
Swim- up
Egg Sac fry fry Juvenile
0.0321 0.0370 - 0.1201f
0.1103° 0.0096° - 0.0282
0.0219 0.0253 0.0128 0.0481f
Values in excess of 4 days are LTC values.
Corrected values represent change from Pomeroy ionization constants to new constants worked out in
this study. -. ~
Smith and Oseid. „
Adelman and Smith.
'TAt 22 C.
At 20 C.
-------
FACTORS INFLUENCING ACUTE SENSITIVITY OF FISH
Temperature and acclimation influence the acute sensitivity of fish to
H S. The most influential factor is temperature. Sensitivity in fat-
head minnows varied in 96-hr LC50 values from 0.5150 mg/liter H2S at
6.1 C to 0.0212 mg/liter H2S at 24 C, approximately a twenty-fivefold
difference. In goldfish tested at 14.1 and 26 C, the difference was
approximately 2.5 times. While the differences are not as marked as in
the fathead, extrapolation of the temperature values approximates the
same slope. Brook trout tested between 8.2 and 21 C did not show as
marked a difference as fathead minnows or goldfish. When threshold
LC50 concentrations are compared, the differences are not as marked. In
a range of 12 C in goldfish the difference is approximately 1.5 times.
In brook trout, however, the threshold value with a 13 C difference in
temperature changes sensitivity approximately threefold. The implica-
tions of these data for natural distribution of the various organisms
suggest that the cyprinids can stand for short periods at least extreme
concentrations at low temperatures while the other species have a much
lower degree of increased tolerance at the low temperatures. The im-
plications for year round standard setting are evident.
The season of the year at which wild fish are subjected to H9S appears
to have some influence on their resistance. Fathead minnows taken late
in the winter appear to have somewhat less resistance at comparable
temperatures than those taken during the open water season.
Another factor influencing sensitivity is acclimation. It is apparent
from data collected on both fathead minnows and bluegills that a certain
degree of acclimation to ELS can take place provided that initial ex-
posure is not in the acutely lethal range. This finding suggests that a
continuous low exposure, although it may have effects on long-term sur-
vival, growth, reproduction etc., does make the organisms more tolerant
(Tables 51, 52).
262
-------
ACUTE TOXICITY TO INVERTEBRATES
The sensitivity of invertebrate organisms varied from an LC50 of 1.070
mg/liter H2S in Asellus to 0.020 mg/liter H2S in Baetis (Table 132).
The two species (Gammarus and Baetis) which inhabit cold and well-aerated
flowing waters were the most sensitive. Conversely, the two species
(Asellus and Crangonyx) which are typically found where anoxic conditions
are common showed the most resistance. The coldwater species were in
the same general area of sensitivity as the coldwater fish. The tests
on invertebrates were run under conditions assumed to be preferential
both as to habitat and to temperature for the particular period of the
year. The burrowing forms, as would be expected, are more tolerant than
those which live on the surface.
RESPONSE OF FISH AND INVERTEBRATES TO LONG-TERM EXPOSURE TO H S
The tLS concentrations at which growth, reproduction, survival, and per-
formance were tested indicated that in most species the no-effect level
was much lower than the LTC and in some species any measurable level of
H?S had some adverse effect (Table 133). The length of exposure in the
chronic tests extended to 825 days in one case but in most tests for a
shorter period of time. Growth rate in most species of fish was found
to be as good or better an index to adverse effect as survival or repro-
duction. Exceptions were found in bluegills and brook trout in which
reproductive success was the most sensitive indicator of adverse effect.
In bluegills and brook trout the adverse effect was related to behavior
which limited sexual activity. When fish which had poor spawning per-
formance or no spawning performance at all were placed in fresh water,
they immediately proceeded to go through the spawning act and deposit
eggs. Non-spawning fish in high levels of H2S which were examined
showed no apparent reduction in eggs contained or in other anatomical
features. Brook trout were inhibited completely from spawning activity
at higher levels but like the bluegill spawned effectively when trans-
ferred into non-toxic medium. However, brook trout at all levels of
exposure showed a reduced production of eggs. The relative influence of
263
-------
Table 132. ACUTE TOXICITY OF
TO EIGHT SPECIES OF INVERTEBRATES
Temp-
Number era-
of ture,
Species tests C
Asellus '
Crangonyx
a
Gammarus
a
Baetis
a
Ephemera
TT . a , c
Hexagenia
Procambarus
Egg
Larvae
Juvenile
4
8
10
4
2
5
39
1
1
1
1
1
1
1
1
1
15.
14.
15.
18.
14.
15.
15.
18.
14.
18.
21.
17.
22.
14.
18.
22.
,2
9
0
0
8
0
0
0
2
0
9
9
0
1
0
1
Preferredd
96-hr LC50,
me/1 HnS
LC50
range,
mg/1 HnS
1.010-
1.700
0.310-
0.840
0.030-
0.059
0.021-
0.024
0.020-
0.026
0.135-
0.380
0.026-
0.680
0.165
-
-
-
-
-
-
-
-
Dura-
tion,
days
4
4
4-10
4
2-4
4-11
2-11
4
4
4
4
4
4
4
4
4
Orig-
inal
Season value
Winter 1.
Winter 0.
Winter 0.
Summer & 0.
winter
Summer 0 .
Winter 0.
Summer 0.
Winter 0.
Summer 0.
- >0.
70.
0.
0.
0.
0.
0.
0.
07
84
059
022
020
316
111
151
165
408
433
370
125
058
147
083
034
Cor-
rected
value
1.
1.
0.
0.
0.
0.
0.
0.
0.
>o.
70.
0.
0.
0.
0.
0.
0.
46
14
081
032
028
429
155
211
244
568
626
55.7
177
087
204
121
051
pH
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
7.
5
4
5
8
6
4
7
7
8
7
7
7
6
7
7
7
7
264
-------
Table 132 (continued). ACUTE TOXIC1TY OF
OF INVERTEBRATES
TO EIGHT SPECIES
Preferred
96-hr LC50,
Species
Procambarus
Sub adult
Adult
Cambarus
Sub adult
Number
of
tests
8
1
1
1
1
1
1
1
Temp-
era-
ture,
C
18.0
25.9
14.0
18.1
21.7
13.9
18.1
22.0
LC50 Dura-
range, tion,
mg/1 H^S days
Z.
4
4
4
4
4
4
4
4
niR/1
Orig-
inal
Season value
0.093
0.075
0.271
0.215
0.121
0.150
0.108
0.070
HnS
Cor-
rected
value
0.137
0.114
0.375
0.308
0.176
0.205
0.157
0.105
PH
7.8
7.8
7.7
7.7
7.6
7-7
7.7
7.7
o ,-
^ Data from Oseid and Smith
Tests conducted after varying periods of laboratory acclimation.
C Tests made at various seasons and test conditions account for extreme
variation of range.
"Preferred LC50" is level determined under conditions judged to be
most appropriate to the species.
e Value corrected from Pomeroy constants to ionization constants worked
out in this study.
265
-------
Table 133. CHRONIC TOXICITY OF
to
,S TO SEVEN SPECIES OF FISH AND THREE INVERTEBRATE SPECIES
(expressed as mg/liter -H9S)
Species
Rainbow
trout
Brook
trout
Fathead
minnow
Stage
at
start
Sac fry
10- day fry
50-day ju-
veniles
Green eggs
Eyed eggs
Adult
0.09 g
finger-
lings
0.5 g
fing.
Juvenile
Juvenile
Dura-
tion,
days
100
90
50
145
111
45-75
72
120
191
345
Temp-
era-
ture,
C
14.7
14.8
15.0
13.7
13.4
12.9
13.0
13.0
20.0
21.3
Original values
Test range
.0018-. 0131
.0011-. 0106
.0011-. 0102
.0009-. 0052
.0012-. 0123
.0055-. 0128
.0015-. 0142
.0015-. 0130
.0015-. 0126
.0004-. 0198
No
effect
.0032
.0033
.0059
.0025
.0039
<.0055
. 0066
.0067
.0070
.0078
Lowest
effect
cone.
.0075
.0048
.0102
.0052
.0063
.0055
.0090
.0090
.0080
.0198
o
Corrected values
Test range
.0026-
.0016-
.0016-
.0013-
.0017-
.0079-
.0021-
.0020-
.0023-
.0006-
.0192
.0154
.0148
.0073
.0172
.0183
.0197
.0178
.0190
.0295
No
effect
.0047
.0048
.0085
.0035
.0055
<.0079
.0091
.0092
.0106
.0116
Lowest
effect
cone.
.0110
.0070
.0148
.0073
.0088
.0079
.0125
.0124
.0120
.0295
Factor
of
effect
S+G
S+G
S+G
S
S+G
R
G
G
S
S+G
-------
Table 133 (continued). CHRONIC TOXICITY OF t^S TO SEVEN SPECIES OF FISH AND THREE
INVERTEBRATE SPECIES
(expressed as mg/liter HS)
to
Oi
Species
Fathead
minnow
Goldfish
Bluegill
Stage
at
start
Sac fry
Juvenile
Sac fry
Juvenile
Juvenile
Adult
Eggs
Adult
Juvenile
Juvenile
Adult
Eggs
Eggs
Dura-
tion,
days
84
112
297
354
294
294
430
288
826
126
& 148
200
316
120
Temp-
era-
ture,
C
23,0
20.0
23.0
24.0
18.6°
18.6°
21.5
c
20.2
11. 8°
24.0
15.0
22.4
24.0
Original values
Test range
.0006-. 0102
.0014-. 0111
.0004-. 0068
.0006-. 0080
.0020-. 0250
.0020-. 0280
.0070-. 0240
.0014-. 0098
.0015-. 0064
.0004-. 0146
.0010-. 0105
.0021-. 0092
.0012-. 0087
No
effect
.0052
.0041
.0033
.0035
.0100
.0050
.0070
.0023
.0015d
.0004
.0062
-
-
Lowest
effect
cone.
.0078
.0092
.0068
.0074
.0250
.0100
.0090
.0071
.0031
.0015
.0105
.0021
.0012
Corrected values3
Test range
.0009-
.0021-
.0006-
.0009-
.0028-
.0028-
.0101-
.0021-
.0022-
.0006-
.0014-
.0031-
.0018-
.0152
.0165
.0101
.0120
.0350
.0392
.0346
.0147
.0094
.0220
.0144
.0136
.0127
No
effect
.0077
.0061
.0049
.0052
.0140
.0070
.0101
.0034
.0022
.0006
.0085
-
—
Lowest
effect
cone.
.0116
.0136
.0101
.0111
.0350
.0140
.0130
.0106
.0046
.0023
.0144
.0031
.0018
Factor
of
effectb
S+G
S
S+G+R
S+G+R
G
R
G
G
G+S+R
SE
G
G+S
S
-------
Table 133 (continued). CHRONIC TOXICITY OF lUS TO SEVEN SPECIES OF FISH AND THREE
INVERTEBRATE SPECIES
(expressed as mg/liter HS)
Stage
at
Species start
Bluegill Eggs
Adult
o> Walleye Juvenile
CD
Juvenile
Gammarus Adult
Adult
Adult
Adult
Dura-
tion,
days
93
97
225
234
65
105
95
65
Temp-
era-
ture,
C
22.2
23.6
17.8
19.6
17.2
17.4
17.7
18.1
Original values
Test range
.0010-
.0007-
.0013-
.0031-
.0007-
.0007-
.0012-
.0012-
.0073
.0078
.0051
.0118
.0128
.0071
.0153
.0192
No
effect
-
-
.0031
.0057
.0019
.0013
.0012
_
Lowest
effect
cone.
-
.0007
.0051
.0118
.0031
.0029
.0032
.0012
Corrected valuesa
Test range
.0015-
.0010-
.0018-
.0043-
.0010-
.0010-
.0018-
.0018-
.0110
.0118
.0071
.0165
.0189
.0100
.0223
.0284
No
effect
-
-
.0043
.0080
.0028
.0018
.0018
_
Lowest
effect
cone.
-
.0010
.0071
.0165
.0046
.0041
.0047
.0018
Factor
of
effect
-
R
S
S
R+S
R+S
R+S
R+S
Hexagenia Nymphs
138 17.!
.0011-.0762 .0152
.0290
.0016-.1128 .0225
.0429
Procam- Eggs
barus Eggs
447 18.1 .0041-.0183 .0041 .0086
112 15.9 .0048-.0158 .0097 .0127
.0060-.0267 .0060
.0068-.0222 .0136
.0126 S
.0179 S
-------
Table 133 (continued). CHRONIC TOXICITY OF H2S TO SEVEN SPECIES OF FISH AND THREE
INVERTEBRATE SPECIES
(expressed as rag/liter HS)
to
05
co
Temp-
Species
Procam-
barus
Stage
at
start
Juvenile
Juvenile
Dura-
tion,
days
196
196
Original values
era-
ture,
C
14.
20.
Test range
3
6
.0044-
.0044-
.0199
.0172
No
effect
.0044
.0044
Lowest
effect
cone.
.0078
.0088
0
Corrected values
Test range
.0062-
.0066-
.0281
.0256
No
effect
.0062
.0066
Lowest
effect
cone.
.0110
.0131
Factor
of
b
effect
G
G
Values corrected from Pomeroy constants to constants worked out in this study.
S = Survival, G = Growth, R = Reproduction based on survival of offspring, SE = Swimming
Endurance.
, Mean of seasonal variation.
Reproduction inhibited at 0.0015 mg/liter H S.
-------
H9S in reducing egg potential and the effect on behavior which reduced
actual deposition was not determined definitively. Fathead minnows
running through two generations showed no highly significant difference
in reproductive capacity at the various KLS levels tested, but growth
was a sensitive index.
The chronic toxicity of H-S to invertebrates was determined from Gam-
mar us , Hexagenia, and Procambarus. In Gammarus the no-effect level was
0.0013 mg/liter at 17.4 C in 105 days and in Hexagenia was 0.0152
mg/liter at 17.8 C in 138 days. Procambarus at 18.1 C showed an adverse
effect at 0.0086 mg/liter in 447 days. Reproduction was not affected
at 0.0090 mg/liter.
USE OF APPLICATION FACTORS
The present study has afforded an opportunity to compare the use of
application factors derived from no-effect levels of a single toxicant
to predicted probable long-term adverse effects on fish from acute
27
tests. The concept was proposed by Mount and Stephan as a way to
determine safe levels of toxicants for species which could not be
tested on a long-term basis. Initially many workers in the field hoped
that a common, if not exact, factor might be proposed which would
permit extension to many toxicants and species. Their work did not
support this assumption. Failing this result, a more restricted
objective was sought, namely, use of a single factor for a single toxi-
cant that would be applicable across a wide spectrum of species. The
O Q
National Academy of Science Panel on Water Quality Standards accepted
this general approach and assigned an application factor for use with
*
all toxicants on which acute toxicity data but not chronic toxicity
data were available. In general, the most sensitive species was used
as a basis for final recommendation.
Subsequent research has shown that a single factor cannot be used for
all toxicants and that with a single toxicant, its application to many
270
-------
species may not be tenable.
In the study reported here several species of fish and invertebrates
were tested under the same conditions in flow-through apparatus under
one principal investigator and, for the most part, by the same staff.
In the seven species of fish tested, LTC values were used where possible
or 96-hr LC50 where threshold values were not available (Tables 134, 135)
Chronic test temperatures and acute test temperatures as close as
possible were used for comparison. Estimates of no-effect levels were
based on survival, growth, or success of reproduction, whichever showed
the greatest sensitivity. Application factors were calculated by
dividing no-effect concentrations established for the most sensitive
criterion by the acute toxic value for various life history stages.
The no-effect values may not be precise since the true no-effect level
is usually between the observed no-effect test concentration and the
lowest concentration showing adverse effects. Various methods have been
proposed for surmounting this difficulty. In the present report the
highest test concentration which showed no effect has been used as the
"no-effect" level.
Calculation of a meaningful application factor is complicated by several
factors. The first is related to the life history stage used for the
acute base level and whether a 96-hr or LTC test is employed. A second
is the criterion of no-effect concentration. This criterion may be
growth, long-term survival, normal reproduction when entire life cycle
from egg to egg is covered, or various performance and behavioral obser-
vations. Meaningful assessment is further complicated by relating
observed chronic responses to their true ecological significance. A
third limitation is the selection of appropriate test temperatures for
establishing the base values. As data on H S show, response may vary
drastically with changes in temperature. Tests on which application
factors are based in this study were done at varied temperatures but are
those which in general would be found in nature during the corresponding
stage of life.
271
-------
Table 134. NO-EFFECT CONCENTRATIONS OF H S FOR VARIOUS STAGES AND LTC CONCENTRATIONS FOR JUVENILES
IN SEVEN SPECIES OF FISH
(mg/liter HS)
Spawn-
Species ing
Brook .0055
trout
Rainbow
trout
Northern
pike
Fathead0 .0035
minnow
Goldfish .0050
Walleye
Bluegill .0007
Original values
Juve- Fry
nile survi- Hatch Juvenile
growth val success LTC (days)
.0067 - - .0155(6-11)
.0059 - .0025 .0087 (17)
.004a .014a
.0033 .0035 .0035 .0120(8-10)
.0070 .0207 .0097 .0710 (11)
.007b .012b .0193 (4)
.0015 - - .0318(8-10)
Corrected values
Juve- Fry
Spawn- nile survi- Hatch Juvenile
ing growth val success LTC (days)
.0079 .0092 - - .0223(6-11)
.0085 - .0035 .0121 (17)
.006a .020a
.0052 .0049 .0061 .0061 .0182(8-10)
.0070 .0101 .0306 .0142 .1027 (11)
.010b .017b .0282 (4)
.0010 .0022 - - .0481(8-10)
a 2
Adelman and Smith.
Smith and Oseid.18
£
Duluth stock.
-------
Table 135. LC50 (96-HOUR) AND LTC VALUES OF JUVENILES AND APPLICATION
FACTORS BASED ON CHRONIC NO-EFFECT LEVEL3
Juvenile
Species
Brook trout
Rainbow trout
Q
Fathead minnow (D)
Fathead minnow (F)
Goldfishb
Walleye
Bluegillb
Gammarus
Hexagenia
Procambarus
96-hr
LC50,
mg/1 H2S
0.0266
0.0130
0.0162
0.0367
0.0830
0.0193
0.0316
0.0220
0.1650
0.0830
LTC,
mg/1 H2S
0.0155
0.0087
0.0120
-
0.0710
-
0.0318
0.0110
0.0600
0.0530
Chronic
no-effect
level,
mg/1 H2S
0.0055
0.0025
0.0033
0.0041
0.0050
0.0031
0.0004e
0.0020
0.0150
0.0040
Application
96-hr
LC50
0.207
0.192
0.203
0.112
0.060
0.161
0.013
0.091
0.091
0.048
Factor
LTC
0.355
0.287
0.275
-
0.070
-
0.013
0.182
0.250
0.076
aThese comparisons made on basis of values uncorrected for new ionization
constants and criterion showing lowest no-effect level; acute values based
on temperature most similar to mean chronic temperatures.
Fish subjected to partial chronic tests and not through entire life cycle
(egg-to-egg).
£
Duluth stock-
Field stock.
Q
Based on swimming performance ,
273
-------
Most acute testing is based on response of juvenile fish and usually
for a 96-hr period or, in some cases, on LTC. Long-term chronic tests
usually do not include an egg-to-egg cycle, the most desirable no-effect
base.
As pointed out above, the application factor will vary in a single
species depending on the life history stage used for comparison. When
juveniles at a selected temperature (trout, 15 C and bluegill, 20 C)
are compared to the most sensitive stage, the factor will be about 3-4
times higher if figures for the most sensitive stage are used. When
application factors for various species based on acute tests with
juveniles are calculated at temperatures presumed to be preferred by
this stage, a large difference is found (0.231 for rainbow trout, 0.013
for bluegill).
Depending on the criterion used for no-effect, application factors may
vary considerably. In the case of bluegills, if growth of juveniles
is used to determine the no-effect level, the factor will be 0.13, but
if the reproductive success of adults is compared to acute response of
juveniles, the factor will be 0.013. Even this factor may not give
true protection since at the lowest concentration tested (0.0007 mg/
liter H-S) some inhibition of egg deposition occurred.
Another factor complicating assessment of appropriate application
factors is the temperature at which acute tests are made. As was dem-
onstrated in fathead minnows and goldfish, the differences may be
extreme. When application factors were calculated for the various
*
temperatures, a wide range of results was obtained (Table 136). In
the fathead minnows, the difference in application factors may be
twentyfold (0.005 to 0.100) depending on the temperature of acute
tests. With goldfish it can be fifteenfold. Tests at the same tem-
peratures were not conducted on all species. It was, therefore, not
demonstrated that the relative difference in application factors
between species would be the same at all temperatures. Application
274
-------
Table 136. COMPARISON OF APPLICATION FACTOR FOR
WITH FATHEAD
MINNOWS AND GOLDFISH TESTED FOR ACUTE RESPONSE AT
DIFFERENT TEMPERATURES3
Fathead minnow
Tempera-
ture,
C
6.5
7.6
10.0
15.0
20.3
25.0
96-hr
LC50
mg/1 H.,8
0.580
0.520
0.150
0.057
0.032b
0.028
Appli-
cation
factor
.005
.006
.020
.050
.930
.100
Tempera-
ture,
C
6.7
10.2
12.4
17.0
20.3
24.8
Goldfish
96-hr
LC50,
mg/1 H0S
0.556
0.271
0.175
0.053
0.048
0.037
Appli-
cation
factor
.009
.019
.029
.094
.104
.135
No-effect level for fathead minnow 0.003 mg/liter and for goldfish,
P. 005 mg/liter.
Single test in temperature series (Table 4).
275
-------
factors applied to invertebrates are within the range exhibited by fish
species.
In view of the foregoing considerations, it is apparent that determina-
tion of safe levels or no-effect levels should be based on a test of
the full life cycle. Reproduction may not be more sensitive than other
responses with some toxicants and species; but, until this is demon-
strated, assessment of safe levels for growth, survival, or physical
performance must remain an approximation. In the setting of water
quality standards and appraisal of effect on ecosystems a "no-effect"
level may not be required to maintain an acceptable or even normal fish
population structure and production.
REGIONAL DIFFERENCE IN FISH TOLERANCE
Acute tests on fathead minnows from different populations and limnologi-
cal sites showed that the nature of the habitat made little difference
but that populations from widely separated areas, such as Minnesota and
southern Ohio, may have significantly different responses to H_S. This
fact makes the use of application factors based on acute tests from one
population and chronic tests from the same species from a widely sepa-
rated population of doubtful value.
NEW IONIZATION CONSTANTS
In Part II of this report (published separately) an analytical tech-
nique for molecular H~S is described, and a new calculation of ioniza-
tion constants has been prepared. A factor of 1.35-1.52 must be
applied to the Pomeroy constants to obtain revised values in thg
range of pH 7.5-8.0 and temperatures of 10-25 C. The summary tables
in the discussion section show corrected values taken from the com-
plete table as well as those originally calculated from the Pomeroy
tables and reported in the body of the report. It is believed that
the new constants will not affect the basic recommendations which have
been made for standard-setting purposes.
276
-------
SECTION XVI
REFERENCES
1. Colby, P. J., and L. L. Smith, Jr. Survival of Walleye Eggs and
Fry on Paper Fiber Sludge Deposits in Rainy River, Minnesota.
Trans. Amer. Fish. Soc. 96/3):278-296, July 1967.
2. Adelman, I. R., and L. L. Smith, Jr. Effect of Hydrogen Sulfide
on Northern Pike Eggs and Sac Fry. Trans. Amer. Fish. Soc.
_9_9 (3): 501-509, July 1970.
3. Colby, P- J., and L. L. Smith, Jr. A Microstrata Water Sampler
for Stream Study. Prog. Fish-Cult. 30(2):116-117, April 1968.
4. Brungs, W. A., and D. I. Mount. A Water Delivery System for Small
Fish-holding Tanks. Trans. Amer. Fish. Soc. 99(4);799-802,
October 1970.
5. Mount, D. I., and W. A. Brungs. A Simplified Dosing Apparatus for
Fish Toxicology Studies. Water Res. 1^:21-29, January 1967.
6. American Public Health Association, American Water Works Associa-
tion, Water Pollution Control Federation. Standard Methods for
the Examination of Water and Wastewater. 13th ed. New York, Ameri-
can Public Health Association, Inc., 1971. 874 p.
7. Pomeroy, R. D. Hydrogen Sulfide in Sewage. Sewage Works J. 13:
498-505, May 1941.
277
-------
8. Brungs, W. A. Chronic Effects of Low Dissolved Oxygen Concentra-
tions on the Fathead Minnow (Pimephales promelas). J. Fish. Res.
Bd. Canada. 28.: 1119-1123, August 1971.
9. Abbott, W. S. A Method of Computing the Effectiveness of an
Insecticide. J. Econ. Entomol. 1.8:265-267, April 1925.
10. Steel, R. G. D., and J. H. Torrie. Principles and Procedures of
Statistics. New York, McGraw-Hill, 1960. 481 p.
11. Sprague, J. B. Measurement of Pollutant Toxicity to Fish. I.
Bioassay Methods for Acute Toxicity. Water Res. 3^:793-821,
November 1969.
12. Shelford, V. E. An Experimental Study of the Effects of Gas Waste
upon Fishes, with Special Reference to Stream Pollution. Bull. 111.
State Lab. Nat. Hist. 11:380-412, March 1917.
13. Snedecor, G. W., and W. C. Cochran. Statistical Methods. Ames,
Iowa State Univ. Press, 1967. 593 p.
14. Brett, J. R. Rate of Gain of Heat Tolerance in Goldfish. Univ.
Toronto Stud. Biol. Ser. .53:5-28, 1946.
15. Scidmore, W. J. An Investigation of Carbon Dioxide, Ammonia, and
Hydrogen Sulfide as Factors Contributing to Fish Kills in Ice
Covered Lakes. Prog. Fish-Cult. 1SK3):124-127, July 1957.
16. Lemke, A. E., and D. I. Mount. Some Effects of Alkyl Benzene Sul-
fonate on the Bluegill Lepomis macrochirus. Trans. Amer. Fish.
Soc. 92_(4): 372-378, October 1963.
278
-------
17. Goodman, L., and A. Gillman. The Pharmacological Basis of Thera-
peutics. New York, MacMillan Co., 1955. 1831 p.
18. Smith, L. L., Jr., and D. M. Oseid. Effects of Hydrogen Sulfide
on Fish Eggs and Fry. Water Res. 6^:711-720, June 1972.
19. Olson, L. E., and L. L. Marking. Toxicity of TFM (lampricide) to
Six Early Life Stages of Rainbow Trout (Salmo gairdneri).
J. Fish. Res. Bd. Canada. .3(3:1047-1052, August 1973.
20. Smith, L. L., Jr., and D. M. Oseid. Toxic Effects of Hydrogen
Sulfide to Juvenile Fish and Fish Eggs. In: Proc. 25th Purdue
Ind. Waste Conf. Ext. Ser. No. 137, Purdue Univ., 1971. p. 739-744.
21. Eriksen, C. H. The Relation of Oxygen Consumption to Substrate
Particle Size in Two Burrowing Mayflies. J. Exp. Biol. 40:447-
453, September 1963.
22. Eriksen, C. H. Respiratory Regulation in Ephemera simulans
Walker and Hexagenia limbata (Serville) (Ephemeroptera). J.
Exp. Biol. 40^:455-467, September 1963.
23. Siesennop, G. M.S. Thesis. The Effect of Temperature on the
Toxicity of Copper, Phenol, and Hydrogen Sulfide to the Amphipod,
Gammarus £seudolimnaeus, Bousfield. St. Paul, University of
Minnesota, 1972. 145 p.
24. Bonn, E. W., and B. J. Follis. Effects of Hydrogen Sulfide on
Channel Catfish, Ictalurus punctatus. Trans. Amer. Fish. Soc.
96^:31-36, January 1967.
25. Eriksen, C. H. Ecological Significance of Respiration and Substrate
for Burrowing Ephemeroptera. Can. J. Zool. 4^:93-103, January 1968.
279
-------
26. Oseid, D. M., and L. L. Smith, Jr. Factors Influencing Acute
Toxicity Estimates of Hydrogen Sulfide to Freshwater Invertebrates.
Water Res. 8_: 739-746, August 1974.
27. Mount, D. I., and C. E. Stephan. A Method for Establishing Accep-
table Toxicant Limits for Fish - Malathion and the Butoxyethanol
Ester of 2,4-D. Trans. Amer. Fish. Soc. 96/2):185-193, April 1967.
28. National Academy of Science. Water Pollution Control in the United
States. A Panel Report. Arlington, Nat. Water Comm. MWC-EES-72-
059, 1972. 259 p.
280
-------
SECTION XVII
PUBLICATIONS
1. Smith, L. L., Jr., and D. M. Oseid. Toxic Effects of Hydrogen
Sulfide to Juvenile Fish and Fish Eggs. In: Proc. 25th Purdue
Ind. Waste Conf. Ext. Ser. No. 137, Purdue Univ., 1971. p. 739-744.
2. Smith, L. L., Jr., and D. M. Oseid. Effects of Hydrogen Sulfide on
Fish Eggs and Fry. Water Res. ^:711-720, June 1972.
3. Adelman, I. R., and L. L. Smith, Jr. Toxicity of Hydrogen Sulfide
to Goldfish (Carassius auratus) as Influenced by Temperature,
Oxygen, and Bioassay Techniques. J. Fish. Res. Board Can. 29:
1309-1317, September 1972.
4. Oseid, D. M., and L. L. Smith, Jr. Swimming Endurance and Resis-
tance to Copper and Malathion of Bluegills Treated by Long-term
Exposure to Sublethal Levels of Hydrogen Sulfide. Trans. Amer.
Fish. Soc. 3.01(4): 620-625, October 1972.
5. Smith, L. L., Jr., and D. M. Oseid. Effect of Hydrogen Sulfide on
Development and Survival of Eight Freshwater Fish Species. In: The
Early Life History of Fish, Blaxter, J. H. S. (ed.). New York,
Springer-Verlag, 1974. p. 415-430.
6. Oseid, D. M., and L. L. Smith, Jr. Chronic Toxicity of Hydrogen
Sulfide to Gammarus pseudolimnaeus. Trans. Amer. Fish. Soc.
103(4):819-822, October 1974.
281
-------
7. Smith, L. L., Jr., and D. M. Oseid. Chronic Effects of Low Levels
of Hydrogen Sulfide on Freshwater Fish. Presented at Conference of
International Assoc. Water Pollution Research, Paris, France,
September 1974.
8. Oseid, D. M., and L. L. Smith, Jr. Factors Influencing Acute
Toxicity Estimates of Hydrogen Sulfide to Freshwater Invertebrates.
Water Res. 8^:739-746, August 1974.
9. Oseid, D. M., and L. L. S ith, Jr. Long-term Effects of Hydrogen
Sulfide on Hexagenia limbata (Ephemeroptera). Environ. Entomol.
4.: 15-19, 1975.
10. Smith, L. L., Jr., D. M. Oseid, G. L. Kimball, and S. El-Kandelgy.
Toxicity of Hydrogen Sulfide to Various Life History Stages of
Bluegill (Lepomis macrochirus Rafinesque). Trans. Amer. Fish. Soc.
1976. In press.
11. Smith, L. L., Jr., D. M. Oseid, and L. E. Olson. Acute and Chronic
Toxicity of Hydrogen Sulfide to the Fathead Minnow (Pimephales
promelas). Environ. Sci. & Technol. 1976. In press.
282
-------
SECTION XVIII
GLOSSARY
Acute Test - Bioassay with duration less than 3 weeks with effect
measured by survival.
Acute Toxicity - Lethal toxicity occurring within 12 hr to 3 weeks.
Adults - Fish which have spawned or are capable of spawning during the
current reproductive season.
Application Factor - The quotient of the no-effect concentration of a
toxicant for a fish population divided by the LC50 concentration for
the same toxicant and population: no-effect concentration/ 96-hr LC50
concentration or LTC.
Chemical Metering Apparatus (CMA) - Sometimes referred to as "dipping
bird" apparatus - a dispensing system (Mount and Warner 1967) used to
inject toxicants to test chambers on a cyclic basis determined by
water discharge.
Chronic Test - A bioassay which exceeds 3 weeks with effects measured
by growth, fecundity, behavior, or survival.
Chronic Toxicity - Adverse effect on growth, fecundity, behavior, or
survival in tests exceeding 3 weeks in length.
283
-------
Crayfish, adult - Organisms which have spawned or exhibit reproductive
behavior during the normal spawning period.
Crayfish, berried female - Female with eggs attached to swimmerettes.
Crayfish, juvenile - Crayfish from 3rd instar to development of repro-
ductive capacity.
Crayfish, larvae - Newly hatched crayfish through the first two instars.
Crayfish, subadult - Organisms which will reproduce during the next
natural spawning season.
Eggs, eyed - Eggs in which embryos have eyes well pigmented and dis-
tinguishable without magnification and which will hatch in several
days under normal conditions.
Eggs, green - Eggs which have been fertilized but have not gone beyond
the primary cleavages.
Fingerlings - Fish after absorption of the yolk sac and assumption of
normal feeding patterns, usually reserved for fish through the first
season of growth.
Fry - Fish which have not yet reached fingerling stage and which may
have yolk sac still unabsorbed.
Juveniles - Usually synonymous with fingerlings but may be applied to
fish in the second season of growth.
LC50 - The concentration of a toxicant which will cause 50% of test
organisms to be dead at a specified time, commonly 24, 48, 96 hrs up
to 3 weeks.
284
-------
Lethal - Used with reference to toxicant concentrations which cause
death within short periods.
Lethal Threshold Concentration (LTC) - The calculated LC50 concentration
obtained in an acute test at a point in time when no change in LC50
has occurred for 48 hr (sometimes designated as asymptotic LC50).
Molecular H^S - That portion of dissolved sulfide (H2S + HS~ + S~) which
is unionized and does not include HS or S .
No-effect Concentration (Safe Level) - The concentration of a toxicant
which will permit organisms to complete various life history stages
without apparent adverse effects.
Sac Fry - Any fish still carrying unabsorbed yolk sac.
Safe Level - See "No-effect Concentration."
Subadults - Juvenile fish capable of spawning during their next repro-
ductive cycle but which have not yet shown reproductive behavior, colora-
tion, or secondary sex characteristics.
Sublethal - Used with reference to toxicant concentrations which permit
survival without reference to possible adverse effects on growth, repro-
duction, behavior, etc.
Swim-up Fry - Fry which have left the bottom and swim normally through
test chamber and accept food but still retain some of yolk sac.
Young-of-the-year (yy) - Fish during first season of growth.
285
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-062a
3. RECIPIENT'S ACCESSION* NO.
4. TITLE AND SUBTITLE
Effect of Hydrogen Sulfide on Fish and Invertebrates
Part I - Acute and Chronic Toxicity Studies
5. REPORT DATE
July 19T6 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Lloyd L. Smith, Jr., Donavon
and Steven J. Broderius
M. Oseid, Ira R. Adelman,
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Entomology, Fisheries, and Wildlife
University of Minnesota
St. Paul, Minnesota 55108
10. PROGRAM ELEMENT NO.
1BA608
11, CQWRWCT/GRANT NO.
R800992
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory
U.S. Environmental Protection Agency
Office of Research and Development
Duluth, Minnesota 558QH
13. TYPE OF REPORT AND PERIOD COVERED
'inal Report (Aug., 1969-May, 1
14. SPONSORING AGENCY CODE
EPA-ORD (OHEE)
15. SUPPLEMENTARY NOTES
See Part II, EPA-600/3-76-062b
16. ABSTRACT ~~~~~
Acute and chronic toxicity of hydrogen sulfide to seven fish species and eight
invertebrates were determined in continuous-flow bioassays. Fish species were
fathead minnows, goldfish, bluegill, walleye, white sucker, brook trout, and rain-
bow trout. Invertebrates were Asellus, Crangonyx, Gammarus, Baetis, Hexagenia,
Ephemera, Procambarus, and Cambarus. In 159 acute tests lethal threshold concen-
tration for juvenile fish varied from 0.0087 mg/liter in rainbow trout to O.OSHo
mg/liter in goldfish. Except in goldfish, fry stage was up to three times more
sensitive than the juvenile. In 96 tests on invertebrates the 96-hr LC50 ranged
from 0.020 mg/liter in Baetis to 1.070 mg/liter in Asellus. Acute toxicity of H2S
to fathead minnows varied 2^-fold between 6.5 and 2^.0 C. Temperature effects were
not as marked on invertebrates. In chronic exposure to HgS in 29 tests running up
to 825 days, maximum no-effect concentration to fish ranged from O.OOQil mg/liter in
bluegills to 0.0100 mg/liter in goldfish. No-effect level was determined from
growth, survival, reproduction, or swimming performance. In nine chronic tests
running up to 138 days, maximum safe levels ranged from 0.0012 mg/liter in Gammarus
to 0.0152 mg/liter in Hexagenia. Application factors relating acute toxic
(96-hr LC50 for juveniles) to no-effect levels varied from .231 in rainbow trout
to .013 in bluegills and from .091 in Gammarus to .OU8 in Procambarus.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. cos AT I Field/Group
*Toxicity
*Fresh water fishes
Temperature
*Invertebrates
Variability
Mortality
Dissolved gases
Fatigue (biology)
*Hydrogen Sulfide
*Life cycles
Reproduction (biology) Combined stress
Growth
Oxygen
Application factors
Acute toxicity
'Chronic toxicity
Seasonal effects
LC50
Sublethal
Bioassay methods
06/T
Release to Public
19. SECURITY CLASS (This Report)'
Unclassified
21. NO. OF PAGES
302
>0. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
286
*USGPO: 1976-657-695/5454 Region 5-11
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