EPA-R 3-73-032
FEBRUARY 1973
Ecological Research Series
Fish and Food Organisms
in Acid Mine
Waters of Pennsylvania
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, 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:
1. 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.
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EPA-R3-73-032
February 19T3
FISH AND FOOD ORGANISMS IN ACID MINE
WATERS OF PENNSYLVANIA
By
Robert L. Butler
Edwin L. Cooper
J. Kent Crawford
Donald C. Hales
William G. Kimmel
Charles C. Wagn«r
Pennsylvania State University
208 Life Sciences I
University Park, Pennsylvania 16802
Grant # WP-01539-Ol(N)l
Project 18050 DOG
Project Officer
Quentin H. Pickering
Newtown Fish. Toxicology Laboratory
Environmental Protection Ageney
3^11 Church Street
Cincinnati, Ohio
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20^60
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 30403
Price $2.10 domestic postpaid or $1.75 QPO Bookstore
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EPA Review Notice
This report has been reviewed by the EPA and approved for
publication. Approval does not signify that the contents
necessarily reflect the views and policies of the EPA, nor fdoes
mention of trade names or commercial products constitute endorse-
ment or recommendation for use.
ii
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ABSTRACT
The three parts of this project relate respectively to the three
objectives: 1) develop a rapid and non-lethal bioassay for acid
water using changes in utilization of cover and activity of fish,
2) determine the effect of different levels of acid mine drainage
on the presence or absence of fish populations in the watersheds
of Pennsylvania, 3) determine the median tolerance limits to low
levels of pH of five aquatic insects chosen on the basis of their
wide occurrence and common association in soft-water streams.
Analysis of variance revealed there was no relationship between
cover utilization and pH levels or between activity and pH levels
for four species of fish (smallmouth bass, longnose dace, rock
bass and brook trout). The failure of cover utilization and
activity to reflect changes in water quality conditions makes
this bioassay technique as tested unsuitable for the establishment
of water quality criteria.
In part II of the project it was found that common fish species
normally distributed over several watersheds were absent where
there was severe acid mine drainage. Of the 116 species of fishes
found 10 species exhibited some tolerance to acid mine drainage
(values of pH 5.5 or less). An additional 38 species were found
at pH values between 5.6 and 6.4 with the remaining 68 species at
pH levels above 6.4. Severe degradation occurred at pH levels
between 4.5 and 5.6.
In part III all five aquatic species survived exposure for four
days to pH levels from 6.5 to 4.0. The 96-hour TLm values ranged
from 3.31 for the most sensitive animal, Stenonema sp., to 1.72
for the most tolerant insect, Nigronia fasciata.
iii
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CONTENTS
Section
PART I: COVER RESPONSE OF FISH IN BIOASSAY OF ACID
WATER
Abstract
Contents
Figures
Tables
I Conclusions
II Recommendations
III Introduction
IV Materials and Methods
Apparatus
Bioassays
V Results
VI Discussion
VII Acknowledgments
VIII References
IX Appendices
PART II: THE EFFECTS OF ACID MINE DRAINAGE ON FISH
POPULATIONS
Abstract
Contents
Figures
Tables
I Conclusions
Page
1
3
5
6
8
11
13
15
17
29
55
59
61
65
75
77
79
81
83
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CONTENTS
Section Page
II Recommendations 85
III Introduction 87
IV Methods and Materials 89
Sources of Information
Fish Collections
Water Analyses
Computer Analyses
V Results and Discussion 95
Distribution of Fishes in Pennsylvania
Distribution of Acid Mine Drainage
Effect of pH and Acidity on Fish Populations
Tolerance of Individual Fish Species to pH
Levels
General Discussion of Acid Tolerance by
Fishes
VI Acknowledgments 121
VII References 123
PART III: ACUTE TOXICITY OF LOW pH TO AQUATIC INSECTS
Abstract 127
Contents 129
Figures 131
Tables 133
I Conclusions 135
II Recommendations 137
III Introduction 139
IV Methods and Materials 141
Apparatus
Bioassays
Test Organisms
vi
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CONTENTS
Section Page
V Results 147
VI Acknowledgments 155
VII References 157
vii
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PART I: COVERT RESPONSE OF FISH IN
BIOASSAY OF ACID WATER
by
J. Kent Crawford and Robert L. Butler
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ABSTRACT
The objective of these studies was to develop a rapid bioassay for
acid water using changes in utilization of cover and activity of
fish. Four species of fish (smallmouth bass, longnose dace, rock
bass and brook trout) were chosen for testing that were known to
be cover seekers and which included representatives of warm and cold
water streams of Pennsylvania. Observations of behavior were made
in a stream aquarium after a 100-hour period of acclimation to the
test water (pH levels 7.0 through 4.0) and either 1/2 or 24-hour
adjustment to the test area of the stream aquarium. Temperature,
velocity of water, and light period were controlled.
Analysis of variance revealed there was no relationship between
cover utilization and pH levels or between activity and pH levels
for any of the four species tested. The failure of cover utilization
and activity to reflect changes in water quality conditions makes
this bioassay technique as tested unsuitable for the establishment
of water quality criteria.
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CONTENTS
Section
I Conclusions
II Recommendations
III Introduction
IV Materials and Methods
Apparatus
Bioassays
V Results
VI Discussion
VII Acknowledgments
VIII References
IX Appendices
Page
11
13
15
17
29
55
59
61
65
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FIGURES
PAGE
1 Diagram of the stream aquarium used for
observing fish behavior 18
2 Test chamber of the stream aquarium 19
3 Artificial cover used in the behavioral
study 20
4 Cover utilization of age 0 smallmouth bass
in water acidified in the laboratory 30
5 Cover utilization of age I smallmouth bass in
water acidified in the laboratory 30
6 Cover utilization of age I, II and III smallmouth
bass in water acidified in the laboratory 31
7 Cover utilization of longnose dace in water
acidified in the laboratory 31
8 Cover utilization of brook trout in water
acidified in the laboratory 32
9 Cover utilization of rock bass in water
acidified in the laboratory 32
10 Activity of age I, II and III smallmouth bass in
water acidified in the laboratory 33
11 Activity of longnose dace in water acidified in
the laboratory 33
12 Activity of brook trout in water acidified in
the laboratory 34
13 Activity of rock bass in water acidified in
the laboratory 34
14 Cover utilization of age 0 and age I smallmouth
bass in unpolluted water and in water polluted
with acid mine drainage 41
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FIGURES
PAGE
15 Cover utilization and activity of age I, II
and III smallmouth bass in unpolluted water
and in water polluted with acid mine drainage 42
16 Cover utilization and activity of longnose
dace in unpolluted water and in water polluted
with acid mine drainage 43
17 Cover utilization and activity of brook trout
in unpolluted water and in water polluted
with acid mine drainage 44
18 Cover utilization and activity of rock bass
in unpolluted water and in water polluted
with acid mine drainage 45
19 Cover utilization for age 0 smallmouth bass in
unpolluted water and in treated acid mine water 46
20 Cover utilization and activity for age I, II and
III smallmouth bass in unpolluted water and in
treated acid mine water 47
21 Cover utilization and activity for brook trout
in unpolluted water and in treated acid mine
water 48
22 Cover utilization and activity for rock bass in
unpolluted water and in treated acid mine water 49
!
23 Cover utilization for age 0 and age I smallmouth
bass held for less than 15 days and for more than
15 days 51
24 Cover utilization and activity for longnose dace
with either a 1/2-hour or a 24-hour period of
adjustment to the test chamber 52
25 Cover utilization for smallmouth bass of ages I
and older with either a 1/2-hour or a 24-hour
period of adjustment to the test chamber 53
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TABLES
No.
1 Representative water chemistry of source
water used in behavioral tests 22
2 Fish ages and sizes and other specifics of
test variations used in the behavioral studies 26
3 Correlation coefficients and coefficients of
determination between cover utilization and
pH and between activity and pH for fish
tested in water acidified in the laboratory 35
4 Variables measured for tests of cover
utilization and activity 36
5 Independent variables included in the regression
analysis for each species 38
6 Results of multiple regression analysis using
cover utilization as the dependent variable 39
7 Results of multiple regression analysis using
activity as the dependent variable 40
8 Average velocities in the test chamber for each
species tested 40
9 Average cover utilization in seconds ± one
standard error of the mean for fish tested at
different pH levels in water acidified in the
laboratory 66
10 Average activity in movements per hour ± one
standard error of the mean for fish tested at
different pH levels in water acidified in the
laboratory 67
11 Average cover utilization and activity ± one
standard error of the mean for fish tested in
unpolluted water and in water polluted with acid
mine drainage and diluted to the desired pH
with unpolluted water 68
8
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TABLES
No. Page
12 Average cover utilization and activity ± one
standard error of the mean for fish tested
in unpolluted water and in treated acid mine
water 69
13 Average cover utilization in seconds ± one
standard error of the mean for smallmouth
bass age 0 and age I held in captivity for
less than 15 days and for greater than 15
days 70
14 Average cover utilization and activity ± one
standard error of the mean for longnose dace
and smallmouth bass with either a 1/2-hour
or a 24-hour period of adjustment to the
test chamber. Longnose dace were tested in
unpolluted water at pH 7 and smallmouth
bass were tested in water acidified in the
laboratory to pH 7 through 4 71
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SECTION I
CONCLUSIONS
1. Utilization of cover and activity of fish under successive
levels of pH (7 through 4) are not changed under the experimental
conditions and methods of this study. In the tests conducted no more
than nine per cent of the variation in the above behavior could be
accounted for by changes in pH. Utilization of cover and activity
of fish as tested are unsuitable as bioassays for establishment of
pH water quality criteria.
2. Of the 16 variables measured for relationships with cover
utilization and activity none exerted a significant controlling
influence over utilization of cover and activity of all species.
The one variable having the greatest affect was the test chamber.
Differences as small as 15 to 17 per cent in velocity between the
two chambers were related to the difference in behavior. In light
of published material, this is a very small change in velocity.
3. There was no significant difference in the utilization of cover
and activity of fish in treated acid mine drainage water as compared
to that of fish tested in water from the home stream. In view of the
first conclusion nothing can be said for or against treated water.
4. Although there was no significant difference in utilization of
cover between smallmouth bass of age I held less than 15 days versus
those held greater than 15 days, those held longer than 15 days
had a lower mean time of cover utilization and a smaller variance.
Smallmouth bass of age 0 showed a significantly lower response in
utilization of cover when held longer than 15 days. Holding wild
fish for extended periods modifies the behavioral parameters of
these studies and may likewise affect other types of behavior.
5. Although there was no significant difference in cover utilization
and activity with two different periods of adjustment to the aquarium
(1/2 hour and 24 hour), there was an increase in the mean utilization
of cover and decrease in mean activity with smaller variance in
tests with the longnose dace. There was a significant increase in
utilization of cover for smallmouth bass age I and older that were
allowed an adjustment period of 24hhours rather than 1/2 hour.
Therefore, acclimation to a test apparatus such as used in these
studies should be longer than is commonly used in studies of
behavior.
11
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SECTION II
RECOMMENDATIONS
1. The techniques and methodology used in this study should not
be followed for establishment of water quality criteria on pH.
2. If further work on utilization of cover and activity of fish
under successive levels of pH are to be made, the following results
of this study should be kept in mind.
a. Young-of-the-year and older fish if tested should be tested
separately. Response to cover is related to age.
b. The period of adjustment to the aquarium appears to be
important. The longer period of adjustment (24 hours)
appears to have advantage over the 1/2-hour period of
adjustment.
c. Attempts should be made in studies using stream aquaria
to obtain velocities of less difference than 15 to 17
per cent for paired chambers.
3. In these studies escape routes along gradients of pH were not
provided. Furthermore, the area for movement of fish was restricted.
It is assumed that once fish learn of these restrictions and a lack
of pH gradient, there will be no change in behavior. An experimental
stream of larger size may have taken care of this objection. The
development of a stream with a built in system for gradients of
pH would be highly desirable. Perhaps an open stream system could
be developed with acid introduction at one or more points and
gradient monitoring of acid conditions coupled with position and
activity of fish.
i
4. The fact remains that response to cover is still highly
predictable in some fish, especially the rock bass. If this behavior
is truly responsive under exposure to higher acidities, this species
should be considered in future testing.
13
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SECTION III
INTRODUCTION
When natural water comes in contact with tailings from abandoned
or operative coal mines, chemical reactions occur that can render
the water unsuitable for aquatic life. This drainage water is
characterized by a low pH, high concentrations of certain heavy
metals, and often, a heavy silt load. These water conditions are
common where coal is mined, whether by underground or surface
methods, and therefore constitute a massive pollution problem in
coal mining areas.
An inventory assessing the extent of the effects of mining showed
that 10,500 miles of streams "are significantly affected by coal
mine drainage pollution, 6,700 of these on a continuous basis"
(U. S. Department of the Interior, 1969). In Pennsylvania alone,
2,300 miles of streams are polluted by coal mine drainage (Pennsylvania
State Planning Board, 1968). Included in this mileage are parts
of all the major river systems in the state. The recreation
potential that is lost to pollution from coal mine drainage on
streams such as the Susquehanna, the Monongahela, the Kiskiminetas,
the Clarion and the Casselman makes acid mine drainage one of the
most extensive water problems in Pennsylvania. Knowledge of the
toxicity of mine drainage water to aquatic organisms is necessary
to direct programs of abatement and treatment.
There is reason to believe that behavioral bioassays can provide
a method for measuring non-lethal toxicity of pollutants such as
acid mine drainage. Jones (1962) has shown that fish avoid a
toxic pollutant when given a choice between toxic and non-toxic
concentrations. In fact, he could detect an avoidance reaction by
the fish being tested at sublethal concentrations of the toxicant.
He has thus shown behavioral'tests to be more sensitive than the
traditional time-to-death tests used in lethal toxicity studies.
Sprague (1964, 1968) and Ishio (1965) have operationalized the
avoidance reaction in successful sublethal toxicity tests. Waller
and Cairns (1972) have used fish movement as an indicator of zinc
toxicity. Warner et al. (1966) have quantified behavioral responses
of fish at sublethal toxicant concentrations and have shown fish
to be sensitive to varying concentrations of pesticides. Fish in
their experiments were trained to respond to a conditioned stimulus
in order to avoid an electrical shock. After exposure to sublethal
doses of toxaphene, the fish become hypersensitive to external
stimuli. Warner (1967) concluded in a review of literature on
bioassays that "quantitative behavioral change is the most sensitive
indicator yet developed of toxicant induced change in living systems."
Warner et^ al. (1966) and Warren (1971) also contended that the
behavior of an organism is an integrated reflection of the several
15
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bodily systems and therefore is a comprehensive parameter.
This is not true of other sublethal responses such as histochemical,
biochemical, or physiological change.
There is an apparent need for the establishment of reliable water
quality criteria for waters receiving acid mine drainage. Further,
it seems that a behavioral bioassay may be a quick and sensitive
way of establishing some of these criteria. This study evaluates
the use of change in cover utilization and change in activity of
fish as a behavioral bioassay.
16
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SECTION IV
MATERIALS AND METHODS
Apparatus
Observations of fish behavior were made in a stream aquarium with
water quality, temperature, depth and velocity controlled. The
stream aquarium (Figure 1) was constructed of plywood coated with
epoxy. Outer dimensions were 420 cm by 240 cm. The aquarium was
60 cm wide. Water temperature was controlled at a level known to
be near the preferred temperature of the species being tested by
two 1/2 horsepower Min-0-Cool coolers (Frigid Units Incorporated,
Toledo, Ohio) and by a 120 volt Indeecc (Industrial Engineering
and Equipment Company, St. Louis, Missouri) coil heater with an
accompanying thermostat. The copper coil of the heater was coated
with epoxy. Tests were conducted within 3 C for all but 4.4 per
cent of the fish used in the study. Maximum variation of water
temperature for testing was ± 6 C of the desired temperature.
Water depth was maintained at 12 cm with some variation due to
evaporation. At a depth of 12 cm the stream aquarium was
calculated to contain approximately 777 liters (206 gallons) of
water.
Water was forced in a counter-clockwise direction around the stream
aquarium by the force of recirculating water coming through the
nozzles from the pump. Water velocity was controlled by adjusting
the valves of the nozzles. The pump was a Vanton Chem Guard
centrifugal model (Vanton Pump and Equipment Corporation, Hillside,
New Jersey) with those parts of the pump in contact with the water
made of plastic. Deterioration of an 0 ring during the study
caused some metal-water contact. This was not noted until the end
of the experiment. This part, a mechanical seal assembly, was
heavily corroded, but it'is not known to what extent that corrosion
may have contributed toxic ions to the test water. All pipes
leading to and from the stream aquarium were made of polyvinyl-
chloride. Coils of the heating and cooling units and screens
dividing sections of the stream aquarium were coated with epoxy
to prevent contamination of the water. The stream aquarium was
lighted by twelve 40-watt fluorescent lights automatically controlled
by a Tork time switch (Time Controls Incorporated, Mt. Vernon,
New York) preset to turn on at daylight and off at darkness.
Two identical test chambers were set up by placing divider screens
in the stream aquarium (Figure 1). Each chamber was divided into
eighteen 20 cm x 20 cm quadrats (Figure 2). An artificial
cover 20 cm x 20 cm x 12 cm (Figure 3) was placed in one of the
center sections of the test chamber. The cover offered darkness,
17
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to nozzles
f
PUMP
z=n from drain
HEATER
o
O
COOLERS
O
TEST CHAMBER
#2
._ .. 1 7O .. n i
OBSERVE
6
0
VTION
* from pump ,_
NOZZLES
AREA
TEST CHAMBER
v^y
DRAIN
c
240
420
Figure 1. Diagram of stream aquarium used for observing fish behavior.
Distances in cm.
18
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120
VO
Observation Window
Figure 2. Test chamber of the stream aquarium. Distances in cm. Quadrat where artificial
cover was placed is marked X.
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20 cm
12 cm
ARTIFICIAL COVER
Figure 3. Artificial cover used in behavioral study. Cover was made
of plexiglass painted flat black.
20
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quiet water, visual reference and tactual reference, four factors
identified by Raines and Butler (1969) as being important in
eliciting cover usage by fish. From the dark observation area of
the stream aquarium (Figure 1), an observer noted the position of
the fish being tested. Movements made by the observer could not be
detected by the fish.
Recordings of fish behavior were made during observation periods
using an Esterline Angus 20 pen event recorder (Esterline Angus,
Glenside, Pennsylvania). One pen was used for each of the 18
quadrats. When the test fish was present in a quadrat, the pen
corresponding to that quadrat was activated by depressing a switch.
The switch was held depressed, and thus the pen activated, until
the fish moved to a different quadrat. Then, the switch was
released and the pen deactivated and another pen corresponding to
the new quadrat position of. the fish was activated. This procedure
gave a continuous recording of the position of the fish and a
count of the number of times a fish moved from one quadrat to
another.
Water sources for the bioassays ranged from a soft water stream to
a stream polluted with mine acid drainage. Galbraith Run, a small
stream draining sparsely populated forest lands, was used as the
standard water (Table 1) for the first series of smallmouth bass
tests and all the tests with longnose dace. Galbraith Run water
was chosen because it is low in total acidity, iron, sulfates
and aluminum. For the second series of smallmouth bass tests
and for the brook trout and rock bass tests water was taken from
the home stream of the fish being tested. This means that water
from Standing Stone Creek was used for the smallmouth bass, water
from Big Fishing Creek was used for brook trout, and water
from the Juniata River was used for rock bass (Table 1). Water
from the home stream of the test fish was used to avoid any shock
or physiological imbalance in the fish that could result from
being placed in water to which the fish was not accustomed.
Acid mine drainage water for the bioassays was taken from Beech
Creek, a stream with heavy mining activity in its upper reaches.
Acid mine water that had been treated to remove toxic quantities
of acid, iron and sulfate was taken from the mine drainage treatment
plant at Hollywood, Pennsylvania (Lovell, MS). The data of
table 1 are not averages, but are considered to be representative
of normal to low water flow conditions.
Water for the bioassays was transported from the source to the
laboratory in a tank painted with epoxy resin to insure that no
foreign toxicants were introduced. A gasoline operated portable
water pump was used to transfer the water from the stream to the
tank and then from the tank to the stream aquarium.
21
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Table 1. Representative water chemistry of source water used in behavioral tests.
Nb
Galbraith Run
Standing Stone Creek
Big Fishing Creek
Juniata River
Spruce Creek
Beech Creek
Treated acid mine
Acidity
(mg/1
CaO>3)
3
7
0
19
5
10
10
Alkalinity
(mg/1
CaC03)
28
70
93
65
93
10
10
Specific
Conductance
vinoho/ .
cm)
30
390
455
470
221
XL500
>1500
Total Sulfate
Iron (mg/1
(mg/1 SOrS)
-Fe) 4
<1.0 9.2
<1.0 22.2
<1.0 18.9
<1.0 21.4
<1.0 31.3
1.0 ^150.0
<1.0 >150.0
Aluminum
(mg/1 Al)
0.0
12.2
4.1
6.0
3.6
XLO.O
10.0
water from Hollywood,
Pennsylvania
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Four species of fish were chosen for testing which were known to
be cover-seekers and which included representatives of warm and
cold water streams of Pennsylvania. These species were: smallmouth
bass (Micropterus dolomieui), rock bass (Ambloplites rupestris),
brook trout (Salvelinus fontinalis), and longnose dace (Rhinichthys
cataractae). Each of these species is important in the overall
balance of Pennsylvania stream environments. Brook trout, smallmouth
bass, and rock bass are also important as sport fish.
Experimental fish for the study were taken from streams that were
known to have good populations of the species desired and that
were close to The Pennsylvania State University. Wild fish were
chosen over hatchery reared fish to avoid any existing behavioral
differences between wild and hatchery fish. Electrofishing was
used to catch fish for the study since this technique minimizes
injury and provides an easy method of capture. Nobrush Georator
generators (Georator Corporation, Manassas, Virginia) used to
capture the fish were either AC or DC, 115 or 230 volts depending
on the conductivity of the water. Generally, less conductive water
requires AC and high voltage to be effective in capture of fish.
Fish with any injuries or abnormalities were discarded.
Bioassays
Methods were chosen that measured fish activity and response to
cover with changes in water quality. Changes in fish behavior could
then be associated with changes in water quality, quantitative values
required of a useful bioassay.
Water quality levels used for the test were natural unpolluted
water, natural water acidified in the laboratory, diluted acid mine
water, and treated acid mine water. Tests conducted in natural
unpolluted water were considered controls. Water for these controls
was taken from Galbraith Run for the first series of smallmouth
bass tests and for the longnose dace tests. For the second series
on smallmouth bass and for brook trout and rock bass, control tests
were conducted in water from the home stream of the fish being
tested. Water from the same source as the control tests was also
used in tests of stress from low pH. Reagent grade sulfuric acid
was metered into the water to establish the pH levels of 6, 5, 4
and 3. These pH levels were maintained throughout the test by
adding sulfuric acid to decrease the pH to the level desired or by
adding calcium hydroxide to increase the pH to the desired level.
Acid mine drainage water from Beech Creek was too toxic to be
used directly without dilution as water for testing. The pH of
Beech Creek water ranges from 3.5 to 4.5 with total iron concentration
around 1 mg/liter. The acid mine drainage water was therefore
diluted with water from the same stream as the control water to pH
23
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levels of 5 and 6. Water obtained in this manner was similar
to that under natural conditions when a polluted stream is diluted
by confluence with a clean stream. Acid mine water that had been
treated at the experimental mine drainage treatment plant at
Hollywood, Pennsylvania, was tested to determine if the quality of
treated effluent was acceptable as judged by the criteria of the
behavioral bioassay.
One-hour observations of cover utilization and activity were made
for each individual fish at each water quality level. During the
tests, an observer recorded the presence or absence of the fish
being tested in each of the 18 quadrats. After the observation
period, the total number of seconds the fish spent in the quadrat
containing the artificial cover could be counted. This parameter
was used as a dependent variable hereafter called cover utilization
and was measured in seconds. Also counted was the number of times
each fish moved from one quadrat to another. This served as a
second dependent variable, activity, measured in movements per hour.
The research design called for each species of fish to be tested
at the following water quality levels:
(1) natural unpolluted water, pH approximately 7 (control)
(2) unpolluted water acidified in the laboratory to pH 6
(3) unpolluted water acidified in the laboratory to pH 5
(4) unpolluted water acidified in the laboratory to pH 4
(5) unpolluted water acidified in the laboratory to pH 3
(6) acid mine water diluted with unpolluted water to pH 6
(7) acid mine water diluted with unpolluted water to pH 5
(8) treated acid mine water
This series of tests was completed for all species except longnose
dace and yearling smallmouth bass which were not tested in treated
acid mine water. Activity was not recorded for smallmouth bass in
the first series of tests. Eight fish of each species were tested
at each water quality level.
Prior to observation each fish was allowed a 100-hour period of
acclimation to test water. The purpose of this acclimation period
was two-fold: (1) To assure that any initial shock caused by
transfer of the fish into a water of different quality than normal
would have passed and would no longer affect the behavior of the
fish; (2) To begin the tests after an acute toxicity period of 100
hours (Warner, 1967). By holding the fish for 100 hours and then
conducting the tests, it was assumed that only sublethal concentrations
were being tested. All species exposed to unpolluted water acidified
to pH 3, and in acid mine water at pH 5 died before the end of the
100-hour period of acclimation.
Included in the 100-hour acclimation period was a 1/2-hour or a
24-hour period of adjustment to the test area of the stream aquarium.
24
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The purpose of this adjustment period was to give the fish time to
become familiar with the new surroundings of the test chamber before
behavioral observations were made. The 1/2-hour adjustment period
was used for the first series of smallmouth bass and for longnose
dace. The erratic behavioral responses exhibited in these tests
suggested that the 1/2-hour adjustment period was not long enough.
The 24-hour adjustment period was used in all subsequent tests.
All fish were placed in the test chamber near the rear of the chamber.
By placing the fish at the rear of the test chamber, each fish
faced upstream to hold its position in the current and was
confronted immediately with the artificial cover.
Before being placed in the test chamber for the adjustment period,
fish were held in separate sections of nearby holding tanks during
the 100-hour period of acclimation to the test water. The purpose
of holding the fish in individual sections was to reduce the effects
of dominant and submissive behavioral patterns that had been
established before capture in the native streams or after capture
in the laboratory holding tank. Fish for testing were captured
as close as practical to the time of the test in order to avoid
holding fish in captivity for extended periods of time. No fish was
used for more than one test. While held in captivity, fish were
supplied natural foods, but food to which the fish may not have
been accustomed. Food was seldom eaten.
Fish as nearly uniform as possible in age and size were selected
to reduce behavioral variability due to differences in age and size.
Age and size ranges for each species as well as the tests conducted,
identification of source water, and time of adjustment to the test
chamber, are listed in table 2.
Water quality conditions were monitored throughout the study for
identification of conditions associated with non-normal fish behavior.
Measurements of pH were made with a Beckman Zerometic pH meter
(Beckman Instruments Incorporated, Fullerton, California). Readings
of pH and temperature were made before and after each test and the
average of the two readings used as the pH or temperature for the
test. Specific conductance was measured with a Beckman conductivity
meter on a sample of water taken either before or after each test.
Acidity and alkalinity were also measured from water samples taken
either before or after each test. Sulfates were measured once on
each day of testing. Total iron, ferrous iron, and aluminum were
measured once for each different water quality level used.
Chemical analyses were carried out using widely accepted methods.
Acidity and alkalinity were determined by titration with a standard
base or acid to an end point denoted by the color change of an
25
-------
Table 2. Fish ages and sizes and other specifics of test variations used in the behavioral
studies.
to
Species Ages
Smallmouth 0
bass
Smallmouth I
bass
Smallmouth I, II,
bass III
Longnose 0, I,
dace II
Brook I, II
trout
Range
of
Lengths
(mm)
76-135
108-165
126-221
68-92
115-170
Range
of
Weights
(Km)
4-28
16-69
26-128
2-7
14-59
Tests
Conducted
Complete series
(activity not
recorded)
All except
treated acid
mine water
(activity not
recorded)
Complete series
All except
treated acid
mine water
Complete series
Water
Source
Galbraith Run
Galbraith Run
Standing Stone
Creek
Galbraith Run
Big Fishing
Creek
Period of
Adjustment
to Test
Chamber
(hrs)
1/2
1/2
24
1/2
24
Period of
Acclimation
to Test
Water
(hrs)
100
100
100
100
100
Rock bass I, II.... 82-133 11-41 Complete series Juniata River 24
100
-------
indicator. Sulfates were precipitated with barium chloride, filtered,
ignited, and the residue weighed. The colorimetric phenanthroline
method was used to determine total iron. The procedures followed
for these tests are described in Standard Methods for the Examination
of Water and Wastewater (A.P.H.A. et_ al^. , 1971). Ferric iron and
aluminum were determined as described by Kolthoff et al. (1969)
using ammonium hydroxide to precipitate the oxide of the ion.
27
-------
SECTION V
RESULTS
Analyses of variance revealed there was no relationship between
cover utilization and pH or between activity and pH for any species
tested (Figures 4 through 9 and Appendix Table 9). Both young-of-
the-year and yearling smallmouth bass appeared to decrease cover
utilization with lower pH (Figures 4 and 5). Conversely, age I,
II and III smallmouth bass appeared to increase their cover
utilization with lower pH (Figure 6). Cover utilization of long-
nose dace fluctuated widely at different pH levels (Figure 7).
Cover utilization of brook trout remained approximately constant
through pH 7, 6 and 5 to pH 4 (Figure 8) when it dropped markedly
(although non-significant). Cover utilization of rock bass
remained consistently high and with small variance at all pH
levels (Figure 9).
Lower pH values also failed to produce any change in the activity
patterns of the fish. Smallmouth bass, longnose dace and brook
trout showed widely varying activity levels (Figures 10 through 12
and Appendix Table 10). Rock bass had consistently low activities
and low variance at all pH values (Figure 13 and Appendix Table 10).
A correlation analysis of these data confirms the independence of
each of the two measured behavioral parameters and pH. Correlation
coefficients and coefficients of determination were calculated for
cover utilization and pH and for activity and pH for all species
tested. No significant linear correlations were found (Table 3).
This means that there is no significant linear relationship that
exists between the behavioral parameters measured and pH. The
coefficient of determination gives the fraction of variation in
the dependent variable that can be accounted for by variation
in the independent variable. In the tests conducted no more than
9 per cent of the variation i in the behavioral parameter could be
accounted for by changes in pH (Table 3).
These statistical tests have shown that the aspects of fish behavior
used in the experiments under the testing conditions are not controlled
by pH. In order to determine what variable is controlling fish
behavior, a regression analysis was conducted. Sixteen independent
variables (Table 4) and two dependent variables were measured for
each test as described in the methods section of this paper. Some
independent variables were significantly correlated with other
independent variables. Correlation between independent variables
makes separation of the effects of each of the correlated variables
impossible by multiple regression. Therefore, when independent
variables were significantly correlated, one was chosen for the
analysis and the others were omitted. For instance, length, weight,
29
-------
23
O X-N
M W
^ % "*
& < C
K N O
> M O
O h-) (1)
U M (0
H ^
Figure 4.
4000
3000 -
2000 -
1000 -
0
0.92
T^
5
pH
Cover utilization of age 0 smallmouth bass in water
acidified in the laboratory. F -5 = 2.95. L Ninety- five
per cent confidence limits are shown.
2
O <-N
l-l W
H T3
(X < G
UNO
> M O
O hJ 0)
O M (0
Figure 5.
4000 -\
3000 -
2000
1000 -
F » 2.00
i
6
pH
Cover utilization of age I smallmouth bass in water
acidified in the laboratory. F _5 » 2.95. Ninety-five
per cent confidence limits are shown.
30
-------
z.
o
CO
W N O
> IH O
O J 0)
O M 0)
4000 -:
1
3000 -j J-
2000 -
1000 -
t
1
o i ,
U r^
7
"
r
i
6
-f
___]._ -
F »
1
5
a."
1.04
i
4
pH
Figure 6. Cover utilization of age I, II and III smallmouth bass
.05
2.95.
in water acidified in the laboratory. F
Ninety-five per cent confidence limits are°shown.
4000 n
W
UNO
O i-J fl)
O M (0
1000
pH
Figure 7. Cover utilization of longnose dace in water acidified in
the laboratory. F
.05
2.95. Ninety-five per cent
confidence limits are shown.
31
-------
M W
H *t)
Pi M O
O hJ M O
O iJ 0)
O M (0
4000
3000
2000 -
1000 -
_L
o
1.16
pH
Figure 9. Cover utilization of rock bass in water acidified in the
laboratory. F fl_ - 2.95. Ninety-five per cent confidence
limits are shown.
32
-------
> 4)
M 6
H (1)
O >
< Q
400 -1
300 -
200
100
F - 1.53
Figure 10. Average activity of age I, II and III smallmouth bass in
water acidified in the laboratory.a F
five per cent confidence limits are snown.
05
2.95. Ninety-
g
400
300 -
200 -
100
F - 1.22
pH
Figure 11. Average activity of longnose dace in water acidified in
the laboratory. F « 2.95. Ninety-five per cent
confidence limits are shown.
33
-------
400 -i
F = 2.35
CO
4-1
C
§
0)
300
200 -
100 -
0
T
o
PH
Figure 12. Average activity of brook.trout in water acidified in the
laboratory. F «5 » 2.95. Ninety-five per cent confidence
limits are shown.
>-i CD
H 4J
M C
> CU
M
y >
^ i
400
300
200 -
100 -
F = 0.40
-o-
T-
PH
Figure 13. Average activity of rock bass in water acidified in the
laboratory. F Qg - 2.95.
limits are shown.
Ninety-five per cent confidence
34
-------
Table 3. Correlation coefficients (r) and coefficients of
determination (r ) between cover utilization and pH
and between activity and pH for fish tested in water
acidified in the laboratory. Significant r C = .349.
Cover utilization and pH Activity and pH
Species
r r2 r r2
Smallmouth bass .256 .066
Age 0
Smallmouth bass .279 .078
Age I
Smallmouth bass -.282 .080 .281 .079
Age I, II and
III
Longnose dace -.282 .054 .272 .074
Brook trout .307 .094 .092 .008
Rock bass .085 .007 -.222 .049
35
-------
Table 4. Variables measured for tests of cover utilization and
activity.
1. pH of the test water
2. temperature of the test water
3. specific conductance of the test water
4. acidity of the test water
5. alkalinity of the test water
6. total iron concentration in the test water
7. ferrous iron concentration in the test water
8. ferric iron concentration in the test water
9. sulfate concentration in the test water
10. aluminum concentration in the test water
11. time of the test
12. chamber of the stream aquarium where the test was conducted
13. age of the test fish
14. length of the test fish
15. weight of the test fish
16. time the fish was held in captivity
36
-------
and age were significantly correlated for each species of fish.
In the multiple regression analysis, length was chosen to represent
the effects of these three variables and the other two were
omitted from the analysis. Likewise, total iron was chosen to
represent the significantly correlated variables total iron,
ferrous iron, and ferric iron. Independent variables included
in the regression analysis for each species are listed in table 5.
With all the included variables remaining in the multiple regression
equation, no significant reduction in the total sum of squares
of the dependent variables could be attributed to the combined
effects of the independent variables (Table 6 and Table 7). This
indicates that variables measured in the tests are not exerting
a significant controlling influence on cover utilization or activity.
Although established as a control variable, the chamber in which a
fish was tested emerged as the most important variable affecting
cover utilization for three species (Table 6) and as the most
important variable affecting activity for one species (Table 7).
The test chamber significantly influenced the cover utilization of
brook trout. A paired T-test was used to determine if the velocities
in test chamber no. 1 differed from the velocities in test chamber
no. 2. Velocities were found to be significantly higher in chamber
no. 1 for tests conducted with smallmouth bass ages I, II and III,
brook trout, rock bass and longnose dace, but not for smallmouth
bass age 0 or smallmouth bass age I (Table 8). Higher velocities
were accompanied by higher cover utilization and lower activity.
The only other variable to emerge more than once as a most important
variable controlling fish behavior was pH. Cover utilization of
three species and activity of one species was most strongly though
not significantly influenced by pH (Table 6 and Table 7).
Cover utilization and activity of fish were measured in acid mine
water taken from Beech Creek and diluted to the desired pH levels
with water from the home stream of the test fish or with water from
Galbraith Run. With the exception of results obtained for brook
trout, cover utilization and activity did not differ significantly
from fish tested in the home water (control) alone (Figures 14
through 18 and Appendix Table 11). The figures include the F-
values obtained in the analysis of variance.
Cover utilization and activity of fish in treated acid mine water
did not vary from cover utilization and activity of fish tested
in water from the home stream of the test fish (Figures 19 through 22
and Appendix Table 12). The treated water was taken from the mine
drainage treatment plant at Hollywood, Pennsylvania. Treatment
methods used to purify the raw acid mine water varied for each
species tested, but each included a neutralization process, an
oxidization process and a settling period.
37
-------
Table 5. Independent variables included in the regression analysis
for each species. Inclusion of a variable is denoted by X.
Species
X
O.
(U
3
4J
CO
M
0)
a.
£
0)
O
O CO
H 4->
UH O
H 3
CJ tJ
0) C
P. O
CO U
^
CO
4J
fi
c
o
M
M
0)
4-1
CO
U-l
rH
3
CO
4J 0)
0) 0
Q) -H
H H
}^
0)
43
4J 0
CO CO
0) f
H U
4-1
00
a
a)
(U.
J3
(!)
ti
r(
H
>>
4-1
H
H
4->
tx
CO
O
c
H
Smallmouth bass XX X XXX
Age 0
Smallmouth bass XX X XXX
Age I
Smallmouth bass XX X X X X
Ages I, II and
III
Longnose dace XXX X X X X
Brook trout XX XX XXX
Rock bass XX X X X X
38
-------
Table 6. Results of multiple regression analysis using cover
utilization as the dependent variable.
Species
Number of Regression
independent analysis r
variables results
Most
important
independent
variable
Smallmouth bass
Age 0
Smallmouth bass
Age I
Smallmouth bass
Age I, II and
III
Longnose dace
Brook trout
Rock bass
7
7
6
n.s.
n.s.
n.s.
.409 .167
.439 .193
PH
pH
.480 .230 Test chamber
n.s. .431 .186 pH
n.s. .577 .333 Test chamber
n.s. .530 .281 Test chamber
39
-------
Table 7. Results of multiple regression analysis using activity
as the dependent variable.
Species
Number of Regression
independent analysis r
variables results
Most
important
independent
variable
StrialImouth bass
Ages I, II and
III
n.s.
.456 .208 Test chamber
Longnose dace
Brook trout
Rock bass
7
7
6
n.s.
n.s.
n.s.
.515
.546
.402
.265
.298
.161
PH
Length
Test time
Table 8. Average velocities (cm/sec.) in the test chamber for each
species tested.
Species
Chamber 1
Chamber 2
Faired T-test
results
Smallmouth bass 16.3
Age 0
Smallmouth bass 16.3
Age I
Smallmouth bass 13.6
Ages I, II and
III
Longnose dace 9.7
Brook trout 13.6
Rock bass 13.6
16.7
16.7
11.5
8.1
11.5
11.5
n.s.
n.s.
p < .01
p < .05
p < .01
p < .01
40
-------
4000 -i
F = 0.18
55 x-s
O M O
8n3 0)'
H (0
4000 -
3000 -I
2000 -
1000 -
0.28
pH 7
unpolluted
pH 6
polluted
WATER TYPE
Figujre 14. Average cover utilization of age 0 and age I smallmouth
bass in unpolluted water (control) and in water polluted
with acid mine drainage and diluted to pH 6 with unpolluted
water. F Q_ - 4.60. Ninety-five per cent confidence limits
are shown1
41
-------
6 /-s
M W
H T3
W N) O
> M O
O J Q)
O M W
4000 -
3000 -
2000 -
1000 -
0.12
pH 7
unpolluted
pH 6
polluted
WATER TYPE
M C
> 0)
M B
B 5!
< §
AOO -
300 -
200 -
100 -
1.99
pH 7
unpolluted
pH 6
polluted
WATER TYPE
Figure 15. Average cover utilization and activity of age I, II and
III smallmouth bass in unpolluted water (control) and in
water pplluted with acid mine drainage and diluted to
pH 6 with unpolluted water. F Q_ = 4.60. Ninety-five
per cent confidence limits are*shown.
42
-------
O *->
M 0)
t"* *XD
W NI O
> l-i O
O i-J (U
O M W
4000 n
3000 -
2000 -
1000 -
F = 0.58
pH 7
unpolluted
pH 6
polluted
WATER TYPE
>* V)
H 4J
g g
H 0)
O >
400
300 -
200 -
100 -
0
F - 0.00
pH 7
unpolluted
pH 6
polluted
WATER TYPE
Figure 16. Average cover utilization and activity of longnose dace in
unpolluted water (control) and in water polluted with acid
mine drainage and diluted to pH 6 with unpolluted water.
F __ » 4.60. Ninety-five per cent confidence limits are
05
shown.
43
-------
n uuu -
3000 -
COVER
UTILIZATION
(seconds)
M N3
O 0
o o
o o o
i i i
-L
X
i i
pH 7 pH 6
unpolluted polluted
6.36
WATER TYPE
>
400 -i
300 -
200 -
100-
0
F - 3.73
pH 7
unpolluted
pH 6
polluted
WATER TYPE
Figure 17. Average cover utilization and activity of brook trout
in unpolluted water (control) and in water polluted with
acid mine drainage and diluted to pH 6 with unpolluted
water. F fl_ =» 4.60. Ninety-five per cent confidence
limits are shown.
-------
M W
H *O
< C
N O
M O
nJ (U
M M
4000 -|
3000-
2000-
1000-
n
F
-3T
pH 7 pH 6
unpolluted . polluted
WATER TYPE
>
B §
o >
4001
300-
200-
100-
F - 1.87
pH 7
unpolluted
pH 6
polluted
WATER TYPE
Figure 18. Average cover utilization and activity of rock bass in
unpolluted water (control) and in water polluted with acid
mine drainage and diluted to pH 6 with unpolluted water.
F nc " 4.60* Ninety-five per cent confidence limits are
u j
shown.
45
-------
IS
O x-x
M W
H «
« M O
O J Q)
U M (0
40001
3000 -I
2000 -I
1000-1
F = 0.45
unpolluted treated acid
water mine water
WATER TYPE
Figure 19. Average cover utilization for age 0 smallmouth bass in
unpolluted water (control) and in treated acid mine
water. F 0_ « 4.60. Ninety-five per cent confidence
limits are shown.
46
-------
M CO
-> ^ *°
os <: c
W N O
> M O
O hJ 0)
U M (0
4000
3000 -
2000 -
1000
0
1.55
f
unpolluted
water
treated acid
mine water
WATER TYPE
> ID
M a
400-
300-
200-
100-
0-
F - 1,01
_[
1 rF,
' I
unpolluted treated ac]
water mine water
WATER TYPE
Figure 20. Average cover utilization and activity for age I, II and
III smallmouth bass in unpolluted water (control) and in
treated acid mine water. F ._ = 4.60. Ninety-five per
cent confidence limits are snown.
47
-------
4000 i
3000 H
53
O
W
« 3 § 2000
UNO
> M O
O n3 w
H *J
> Q)
400 =
300i
200 n
i 100-
F - 2.95
unpolluted treated acid
water mine water
WATER TYPE
Figure 21. Average cover utilization and activity for brook trout in
unpolluted water (control) and in treated acid mine water.
F _, - 4.60. Ninety-five per cent confidence limits are
\JJ
shown.
48
-------
O ^
JH W
H t3
os < e
W N O
> M O
O >-) 0)
O M 03
KS
&!g
M S
4000-
3000-
2000-
1000-
o-1
-£-
F =
= 1.93
unpolluted treated acid
water mine water
400
300-
200-
100-
WATER TYPE
F 1.80
unpolluted treated acid
water mine water
WATER TYPE
Figure 22. Average cover utilization and activity for rock bass in
unpolluted water (control) and in treated acid mine
water. F Q5 = 4.60. Ninety-five per cent confic'ence
limits are shown.
49
-------
It was necessary to hold some age 0 and age I smallmouth bass for
a period of one month or more prior to using them in a behavioral
test. This was necessary because in cold water smallmouth bass
go beneath the rock substrate in streams (Munther, 1970). During
this period of inactivity, the fish can be taken with an electro-
shocker only with great difficulty. Therefore, before the onset
of cold weather a supply of fish was taken to be tested during the
period when freshly caught fish were not available. These bass
held more than 15 days utilized the cover less than bass held less
than 15 days (Figure 23 and Appendix Table 13). The fish held more
than 15 days were tested in laboratory acidified water at pH 6.
When it was found that the behavior of fish held in captivity was
not comparable to those held a short time, the tests were deemed
unacceptable and new tests were conducted with fresh fish.
The behavioral tests described were originally set up to maximize
the number of fish tested with the equipment available. The
critical item determining the number of fish that could be tested
was the stream aquarium. Using a 1/2-hour period of adjustment to
the test chamber and a 1-hour period of observation, five fish per
day could be tested in one test chamber by one observer. However,
using only a 1/2-hour adjustment period, a fish tested under
identical conditions displayed widely varying behavior. In an
effort to reduce that variation, a series of eight longnose dace
were allowed a 24-hour period of adjustment within the test chamber
of the stream aquarium. Cover utilization of these fish increased
and activity decreased (Figure 24 and Appendix Table 14). Although
the change in behavior was not statistically significant, it was
felt that the 24-hour period of adjustment before making behavior
observations would allow a more characteristic measure of the
"normal" behavior of the fish. Therefore, the entire series of
smallmouth bass tests was repeated for bass ages I and older using a
24-hour period of adjustment rather than a 1/2-hour period of
adjustment. These tests confirmed that cover utilization is
higher and activity lower for fish allowed 24 hours to adjust to
the test chamber (Figure 25 and Appendix Table 14). The 24-hour
adjustment period was then used for all subsequent tests to gain
reliability at the expense of time.
50
-------
O X-x
M (/)
£-H *O
OS < C
W N O
> M O
O hJ 0)
O M (0
4000 1
3000
2000-
1000
F - 5.62
HOLDING TIME
(days)
8
O -^
M M
H -0
< d
NO
M O
hJ 0)
M CD
4000-
3000-
2000-
1000-
F = 3.56
HOLDING TIME
(days)
Figure 23. Average cover utilization for age 0 and age I smallmouth
bass held for less than 15 days and for more than 15 days.
Tests were conducted in unpolluted water acidified to pH 6
in the laboratory. F
.05
4.60. Ninety-five per cent
confidence limits are shown.
51
-------
O /-N
M CO
H "O
BJ << a
UNO
> M O
O hJ 41
O M 0)
> 0)
M 6
H 0)
O >
40001
3000-
2000-
1000-
3.39
1/2-hour 24-hour
i
ADJUSTMENT PERIOD
400-i
300 H
200 H
100 H
4.39
1/2-hour
24-hour
ADJUSTMENT PERIOD
Figure 24. Average cover utilization and activity for longnose dace
with either a 1/2-hour or a 24-hour period of adjustment
to the test .chamber. Fish were tested in unpolluted water
at pH 7. F 05 * 4<6°* Ninety-five per cent confidence
limits are shown.
52
-------
53
O x-v
M (0
H t3
tf < C
UNO
> M O
O >J Q>
O M CO
H v-'
4000 1
3000 -
2000 -
1000 -
15.33
1/2-hour 24-hour
ADJUSTMENT PERIOD
Figure 25. Average cover utilization for smallmouth bass of ages I
and older with either a 1/2-hour or a 24-hour period of
adjustment to the test chamber. Fish were tested in
unpolluted water acidified in the laboratory to pH 7
through 4. F ni- 2.08. Ninety-five per cent confidence
. uj.
limits are shown.
53
-------
SECTION VI
DISCUSSION
Prior to conducting the tests, three possible behavioral responses
to increasing acidity could have been hypothesized. The fish
may gradually alter its behavioral patterns under more acid conditions
or a sudden, large change in behavior could occur at some threshold
level. Gradual alteration in behavior under increasing acidity
could occur in two directions. The response to increased acidity
could be an increase in cover utilization and decreased activity.
Such a response could be expected to conserve the energy of the
fish for physiological adjustment to acidity. If the response to
increased acidity was one of trying to escape, cover utilization
would decrease and activity increase. The avoidance tests that
have been done by Jones (196A), Ishio (1965), Sprague (1964, 1968)
and others indicate this latter type of response.
A third possibility is that instead of a gradual change in behavior
fish would exhibit a sudden behavioral change at some threshold
concentration. If the fish were physiologically capable of
adjustment, slight change in acidity should cause no change in
behavior but instead would be compensated for by physiological
mechanisms. At the point where physiological adjustment could no
longer compensate, a breakdown of normal activity patterns would
occur. In this case, cover utilization and activity would remain
at normal levels until some threshold of toxicity was reached when
cover utilization would increase or decrease and be accompanied
by a decrease or increase respectively in activity.
Fish in these experiments were observed to follow the behavioral
patterns above when exposed to low pH. Age 0 and age I smallmouth
bass exhibited decreasing cover utilization with increase in
acidity. Age I, II and III smallmouth bass showed increasing
cover utilization. Brook trout maintained a consistent cover
utilization until pH 4, then, as hypothesized cover utilization
decreased. Unexpected behavioral patterns also were exhibited.
Longnose dace showed widely fluctuating cover utilization with
changing pH and rock bass maintained an unchanging and consistently
high cover utilization regardless of the pH.
Activity patterns of fish tested also varied. Age I, II and III
smallmouth bass and longnose dace displayed a decrease in activity
with increasing acidity. Brook trout activity was erratic with
increasing acidity. Rock bass activity did not change from its
very low initial level even when the pH was reduced to 4.0.
55
-------
Longer acclimation times (24 versus 1/2 hours) failed to reduce
dramatically the variation of behavior exhibited by fish tested
under identical circumstances. Coefficients of variability,
the sample standard deviation expressed as a per cent of the
sample mean, for fish tested in unpolluted water generally fell
within a range of 50 to 75 per cent. This is a much higher
value than Haines and Butler (1969) found with juvenile smallmouth
bass under similar conditions. Since all fish were treated equally,
it must be assumed the high variability shown in these tests is
a reflection of normal biological variability present in a wild
population. That variability could have been magnified to a higher
level than observed by Haines and Butler (1969) by the imposition
of a more artificail testing apparatus. Also, Haines and Butler
held fish longer than in these studies and used the same fish
repeatedly with a series of different cover types. Perhaps their
treatment made their fish somewhat removed from the wild state.
Normal variability may have been suppressed.
High variability within treatments is undesirable. Future experi-
ments along these lines may avoid high variability by using fish
taken from a hatchery rather than wild fish. Hatchery fish
would also be disturbed less by the artificial testing environment
and handling than would wild fish. However, the possibility
must be considered that some strains of hatchery trout would not
respond to cover.
Utilization of cover, except for brook trout, and activity for
all species were not significantly changed from normal when fish
were exposed to water polluted with acid mine drainage and diluted
to pH 6. Treated acid mine water also failed to cause a change in
the behavior of the fish. These results must be interpreted to
mean that the polluted and treated waters were not toxic enough to
cause a behavioral change.
Warner et al. (1966) built a strong case for the use of behavioral
criteria. Their argument was stated in the introduction of this
paper. Sprague (1971) has not agreed with Warner et_ aL^. (1966).
He questioned the ecological significance of behavioral changes.
His contention is that a behavioral change may not be harmful to
the population. Instead, it may be within the range of normal1
adjustments of the fish and not ecologically detrimental. A
counter argument can be made that cover utilization and activity
are behavioral responses of ecological significance. Any
reduction in cover utilization or increase in activity would be
detrimental to a fish for at least two reasons. First, reduced
use of cover means a stream fish must spend more time holding against
the current. This requires energy which must be compensated for
by increased food intake. In some cases food is the limiting
56
-------
factor to a fish population. Any increased need for food would,
therefore, be detrimental to the population. Increased activity
would also come at a greater cost in energy. Second, anything
which requires a fish to leave cover makes that fish more
susceptible to predators. Increased activity, therefore,
enhances the prey-predator encounters. Objections of some
authors to using change in a single index as a bioassay criterion
because it may or may not have ecological significance, is not
applicable in the case of cover utilization and activity. Future
behavioral tests should attempt to demonstrate the ecological
significance of the behavioral parameters being used.
Behavioral bioassay is not applicable for substances to which
fish do not respond. Certain fish poisons such as antimycin would
fall into this category (Lennon and Berger, 1970). The fish are
unable to detect the poison, do not try to escape from it, and
are killed by it. This may be the reason that behavior in tests
described here failed to change. Perhaps the fish failed to
reflect any behavioral stress even though physiological stress
was occurring. This must be considered unlikely in light of
Ishio's work (Ishio, 1965). He found that fish easily detected
and avoided hydrogen ions in both high and low concentrations.
57
-------
SECTION VII
ACKNOWLEDGMENTS
We wish to express appreciation to graduate students especially
Ron Klauda, and to George Kauffman for their help in this research.
Dr. H. L. Lovell, Director of Mine Drainage Research at The Pennsylvania
State University, cooperated fully in supplying treated acid mine water
needed for the study.
Appreciation is also extended to Elaine Heilman of the Mine Drainage
Research Laboratory who assisted in the water analysis.
59
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SECTION VIII
REFERENCES
1. American Public Health Association, American Water Works
Association, and Water Pollution Control Federation. Standard
methods for the examination of water and wastewater. 13th ed.,
Washington, D. C. 874 p. (1971).
2. Appalachian Regional Commission. Acid mine drainage in Appalachia,
Appendix C: The incidence and formation of mine drainage
pollution. 45 p.; Attachments A-E (1969).
3. Appalachian Regional Commission. Acid mine drainage in
Appalachia. U. S. Government Printing Office. 126 p. (1969).
4. Baker, Robert A. and Albert G. Wilshire. Microbiological factor
in acid mine drainage formation: a pilot plant study.
Environmental Science and Technology 4: 401-407 (1970).
5. Biesecker, J. E. and J. R. George. Stream quality in Appalachia
as related to coal mine drainage, 1965. U.S.G.S. Circular 526,
27 p. (1966).
6. Burdick, G. E. Use of bioassays in determining levels of toxic
wastes harmful to aquatic organisms. Trans. Amer. Fish. Soc.
96(Suppl.): 7-12 (1967).
7. Doudoroff, P. and Max Katz. Critical review of the literature
on the toxicity of industrial wastes to fish. I. Alkalies, acids,
and inorganic gases. Sewage and Industrial Wastes 22(11):
1432-1458 (1950).
8. Doudoroff, P. and Max Katz. Critical review of literature on the
toxicity of industrial wastes to fish. II. The metals as salts.
Sewage and Industrial Wastes 25(7): 802-839 (1953).
9. Doudoroff, P. et^ ail. Bioassay methods for the evaluation of acute
toxicity of industrial wastes to fish. Sewage and Industrial
Wastes 23: 1380-1397 (1951).
10. Ellis, M. M. Detection and measurement of stream pollution.
U. S. Department of Commerce, Bull. Bureau of Fisheries, No. 22,
28: 365-437 (1937).
11. European Inland Fisheries Advisory Commission Working Party on
Water Quality Criteria for European Freshwater Fish. Water
quality criteria for European freshwater fish. Report on extreme
pH values and inland fisheries. EIFAC Tech. Paper 4: 1-24 (1968).
61
-------
12. Raines, Terry A. and Robert L. Butler. Responses of yearling
smallmouth bass (Micropterus dolomieui) to artificial shelter in
a stream aquarium. J. Fish. Res. Bd. Can. 26: 21-31 (1969).
13. Hartman, G. F. Observations on behavior of juvenile brown trout in
a stream aquarium during winter and spring. J. Fish. Res. Bd.
Can. 20: 769-787 (1963).
14. Henderson, Croswell. Application factors to be applied to
bioassays for the safe disposal of toxic wastes, p. 31-37.
In U.S.D.H.E.W. Biological problems in water pollution. Public
Health Service (1957).
15. Henderson, Croswell and Clarence M. Tarzwell. Bio-assays for
control of industrial effluents. Proc. 12th Industrial Waste
Conference. Engineering Bulletin, Purdue University 42: 123-144
(1957).
16. Hill, Ronald D. Mine drainage treatment: state of the art and
research needs. U.S.D.I. Federal Water Pollution Control
Administration. 99 p. (1968)
17. Ishio, S. Behavior of fish exposed to toxic substances. Adv.
in Water Pol. Res. 1: 19-33 (1965).
18. Jones, J. R. E. Fish and river pollution, p. 254-310, In
Louis Klein, River Pollution. II. Causes and Effects, Butterworths,
London (1962).
19. Jones, J. R. E. Fish and river pollution. Butterworths and
Company, London 203 p. (1964).
20. Katz, Max. The biological and ecological effects of acid mine
drainage with particular emphasis to the waters of the Appalachian
Region. Acid mine drainage in Appalachia, Appendix F. 65 p.
(1969).
21. Kim, A. G. An experimental study of ferrous iron oxidation in
acid mine water, p. 40-46. In ORSANCO, Second Symposium on coal
mine drainage research. Carnegie Mellon Univ. (1968).
22. Kolthoff, I. M. _et _al. Quantitative chemical analysis. 4th ed.
The MacMillan Company, London 1199 p. (1969).
23. Lennon, Robert E. and Bernard L. Berger. A resume on field
applications of antimycin A to control fish. Investigations in
Fish Control, U.S.D.I. No. 40: 19 p. (1970).
62
-------
24. Lovell, Harold L. Experience with biochemical-iron-oxidation
limestone-neutralization process. In ORSANCO, Fourth Symposium
on coal mine drainage research. Carnegie Mellon Univ. (MS).
25. McKee, J. E. and H. W. Wolf. Water quality criteria. California
Water Quality Control Board, Sacramento. 548 p. (1963).
26. McKim, J. M. and D. A. Benoit. Effects of long-term exposures
to copper on survival, growth, and reproduction of brook trout
(Salvelinus fontinalis). J. Fish. Res. Bd. Can. 28: 655-662
(1971).
27. Mount, Donald I. and Charles E. Stephan. A method for establishing
acceptable toxicant limits for fish malathion and the butoxyethanol
ester of 24-D. Trans. Amer. Fish. Soc. 96: 185-193 (1967).
28. Mount, Donald I. and Charles E. Stephan. Chronic toxicity of copper
to the fathead minnow (Pimephales promelas) in soft water. J. Fish.
Res. Bd. Can. 26: 2449-2457 (1969).
29. Munther, G. L. Movement and distribution of smallmouth bass in
the middle Snake River. Trans. Amer. Fish. Soc. 99: 44-53 (1970).
30. National Technical Advisory Committee. Water quality criteria.
Federal Water Pollution Control Administration. Government
Printing Office, Washington, D. C. 243 p. (1968).
31. Pennsylvania State Planning Board. Pennsylvania water supplement.
In U.S. Army Corps of Engineers, Development of water resources
in Appalachia. Part V, Chapter 9: 98 p. (1968).
32. Shumate, K. S. et^ al. Development of a conceptual model for pyrite
oxidation systems, p. 7-22. In Ohio State University Research
Foundation, Acid mine drainage formation and abatement, Water
Pollution Control Research Series, E.P.A. (1971).
33. Singer, Philip C. and Werner Stumm. Kinetics of the oxidation of
ferrous iron, p. 12-34. In ORSANCO, Second Symposium on coal
mine drainage research. Carnegie Mellon Univ. (1968).
34. Singer, Philip C. and Werner Stumm. Oxygenation of ferrous iron:
the rate-determining step in the formation of acidic mine
drainage. Water Pollution Control Research Series, F.W.P.C.A.
(1969).
35. Singer, Philip C. and Werner Stumm. Acidic mine drainage: the
rate-determining step. Science 167: 1121-1123 (1970).
63
-------
36. Sprague, J. B. Avoidance of copper-zinc solutions by young salmon
in the laboratory. J. Water Pol. Cont. Fed. 36: 990-1004 (1964).
37. Sprague, J. B. Avoidance reactions of rainbow trout to zinc
sulphate solutions. Water Research 2: 367-372 (1968).
38. Sprague, J. B. Measurement of pollution toxicity to fish III
sublethal effects and "safe" concentrations. Water Research
5: 245-266 (1971).
39. Temple, Kenneth L. and W. A. Koehler. Drainage from bituminous
coal mines. West Virginia University Bull., Engineering Experiment
Station Research Bull. No. 25: 35 p. (1954).
40. U. S. Department of the Interior. Federal Water Pollution Control
Administration. Stream pollution by coal mine drainage in
Appalachia. 261 p. (1969).
41. Waller, William T. and John Cairns, Jr. The use of fish movement
patterns to monitor zinc in water. Water Research 6: 257-269 (1972)
42. Warner, Richard E. Bio-assays for microchemical environmental
contaminants with special reference to water supplies. Bull, of
the WHO 36: 181-207 (1967).
43. Warner, Richard E., Karen K. Peterson and Leon Borgman. Behavioral
pathology in fish: quantitative study of sublethal pesticide
toxication. J. Appl. Ecol. 3(Suppl.): 223-247 (1966).
44. Warren, Charles E. Biology and water pollution control. W. B.
Saunders Company, Philadelphia. 434 p. (1971).
64
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SECTION IX
APPENDICES
Page No.
Table 9 Average cover utilization in seconds ± one
standard error of the mean for fish tested
at different pH levels in water acidified
in the laboratory. 66
Table 10 Average activity in movements per hour ±
one standard error of the mean for fish
tested at different pH levels in water
acidified in the laboratory. 67
Table 11 Average cover utilization and activity ±
one standard error of the mean for fish
tested in unpolluted water and in water
polluted with acid tnine drainage and
diluted to the desired pH with unpolluted
water. 68
Table 12 Average cover utilization and activity ±
one standard error of the mean for fish
tested in unpolluted water and in treated
acid mine water. 69
Table 13 Average cover utilization in seconds ± one
standard error of the mean for smallmouth bass
age 0 and age I held in captivity for less
than 15 days and for greater than 15 days.
Tests were conducted in water acidified in
the laboratory to pH 6. 70
Table 14 Average cover utilization and activity ± one
standard error of the mean for longnose dace
and smallmouth bass with either a 1/2-hour
or a 24-hour period of adjustment to the test
chamber. Longnose dace were tested in
unpolluted water at pH 7 and smallmouth
bass were tested in water acidified in the
laboratory to pH 7 through 4. 71
65
-------
Table 9. Average cover utilization in seconds one standard error of the mean for fish
tested at different pH levels in water acidified in the laboratory.
pH
Species
Analysis
of variance
results
Smallmouth bass
Age 0
Smallmouth bass
Age I
Smallmouth bass
Age I, II
and III
Longnose dace
Brook trout
Rock bass'
1904 ± 480 1863 ± 500 1778 ± 455 1028 ± 233 a
2965 ± 257 1948 ± 446 1983 ± 387 1935 ± 311 a
2745 ± 366 3176 ± 403 3221 ± 316 3528 ±65 a
2052 ± 598 1329 ± 572 2782 ± 431 2625 ± 520 a
2040 ± 549 2178 ± 599 2010 ± 606 478 ± 447 a
3544 ± 40 3541 ± 56 3350 ± 163 3555 ±45 a
n.s.
n.s.
n.s.
n.s.
n.s.
a. Lethal to all fish within the 100-hour period of acclimation to the test watei.
-------
ON
-j
Table 10. Average activity in movements per hour ± one standard error of the mean for fish
tested at different pH levels in water acidified in the laboratory.
Species
Smallmouth bass
Ages I , II
and III
Longnose dace
Brook trout
Rock bass
7
71 ± 40
123 ± 46
178 ± 92
3 ± 2
6
14 ± 11
-"-
177 ± 84
14 ± 8
5 ± 4
PH
5
20 ± 15
79 ± 33
29 ± 13
8 ± 5
4 3
13 ± 10 a
43 ±24 a
141 ±51 a
13 ± 13 a
Analysis
of variance
results
n.s.
n.s.
n.s.
n.s.
a. Lethal to all fish within the 100-hour period of acclimation to the test water.
-------
Table 11. Average cover utilization and activity ± one standard error of the mean for fish
tested in unpolluted water and in water polluted with acid mine drainage and
diluted to the desired pH with unpolluted water.
Species
Cover Utilization
(seconds)
Water Type
pBTT pH~5
unpolluted polluted
(control)
Analysis
of variance
results
Activity
(movements per hour)
Water Type
pH 7 pH 6
unpolluted polluted
(control)
Analysis
of variance
results
cr>
oo
Smallmouth bass
Age 0
Smallmouth bass
Age I
Smallmouth bass
Ages I, 11 and
III
Longnose dace
Brook trout
Rock bass
1904 ± 480 1618 ±467 n.s.
2965 ± 257 2724 ± 372 n.s.
2745 ± 366 2938 ± 434 n.s.
2052 ± 598 1470 ± 465 n.s.
2040 ± 549 3449 ± 102
3544 ± 40 3600 ± 0 n.s.
71 ± 40
13 ± 7
n.s.
123 ± 46 125 ± 40 n.s.
p < .05 178 ± 92 1 ± 1 n.s.
3 ± 2
0 ± 0
n.s.
-------
ON
Table 12. Average cover utilization and activity ± one standard error of the mean for fish
tested in unpolluted water and in treated acid mine water.
Cover Utilization
(seconds)
Activity
(movements per hour)
Species
Water
pH 7
unpolluted
(control)
Type
Treated acid
mine water
Analysis
of variance
results
Water
pH 7
unpolluted
(control)
Type
Treated acid
mine water
Analysis
of variance
results
Smallmouth bass
Age 0
Smallmouth bass
Ages I, II
and III
Brook trout
Rock bass
1904 ± 480 1436 ± 1192 n.s.
2745 ± 366 3229 ± 308 n.s.
2040 ± 549 2811 ± 440 n.s.
3544 ± 40 3600 ± 0 n.s.
71 ± 40 28 ± 26
178 ±92 18 ± 17
3 ± 2 0 ± 0
n.s.
n.s.
n.s.
-------
Table 13. Average cover utilization in seconds ± one standard
error of the mean for smallmouth bass age 0 and age I
held in captivity for less than 15 days and for greater
than 15 days. Tests were conducted in water acidified
in the laboratory to pH 6.
Holding Time . . .
Species ! Analysis
v less than greater than of variance
15 days 15 -days results
Smallmouth bass 1863 ± 500 629 ± 1A5 p < .05
Age 0
Smallmouth bass 1948 ± 446 948 ±287 n.s.
Age I
70
-------
Table 14. Average cover utilization and activity ± one standard error of the mean for longnose
dace and smallmouth bass with either a 1/2-hour or a 24-hour period of adjustment
to the test chamber. Longnose dace were tested in unpolluted water at pH 7 and
smallmouth bass were tested in water acidified in the laboratory to pH 7 through 4.
Species
Cover Utilization
(seconds)
Adjustment Period
1/2-hour
24-hour
Analysis
of variance
results
Activity
(movements per hour)
Adjustment Period
1/2-hour
24-hour
Analysis
of variance
results
Longnose dace
Smallmouth bass
2052 ± 598
2207 ± 187
3264 ± 275
3167 ± 158
n.s.
p < .01
123 ±46 21 ± 15 n.s.
Ages I and
older
-------
PART II: THE EFFECTS OF ACID MINE
DRAINAGE ON FISH POPULATIONS
by
Edwin L. Cooper and Charles C. Wagner
73
-------
ABSTRACT
Fish collections and water quality analyses, taken at the same sites
which were distributed systematically throughout the watersheds in
Pennsylvania, were used to determine the effect of different levels
of acid mine drainage on the presence or absence of fish populations.
Common fish species, normally distributed over several watersheds,
were absent where there was severe acid mine drainage. Small clean-
water tributaries in these severely affected areas contained diverse
fish populations.
From the list of 116 species of fishes found in Pennsylvania, 10
species exhibited some tolerance to acid mine drainage (fish were
present at pH values of 5.5 or less). With the exception of chain
pickerel, brook trout and largemouth bass, these 10 species are
unimportant as game fishes. An additional 38 species have been
found at pH values between 5.6 and 6.4 and the remaining 68 species
were found only at pH levels above 6.4. Such a comparison indicates
a rather broad threshold level of tolerance to acid mine drainage,
with severe degradation occurring at pH levels between 4.5 and 5.6.
This report was submitted in partial fulfillment of project number
WP-01539-01(N)1 under the partial sponsorship of the Office of
Research and Monitoring, Environmental Protection Agency, Washington,
D. C.
75
-------
CONTENTS
Section
I Conclusions
II Recommendations 85
III Introduction 87
IV Methods and Materials 89
Sources of Information
Fish, Collections
Water Analyses
Computer Analyses
V Results and Discussion 95
Distribution of Fishes in Pennsylvania
Distribution of Acid Mine Drainage
Effect of pH and Acidity on Fish
Populations
Tolerance of Individual Fish Species
to pH Levels
General Discussion of Acid Tolerance
by Fishes
VI Acknowledgments 121
VII References 123
77
-------
FIGURES
PAGE
1 Watershed map of Pennsylvania showing stations
at which one or more fish species were collected
(1957 through 1970) 90
2 Watershed map of Pennsylvania showing stations
at which no fish were taken and in which the pH
was less than 5.5 (1957 through 1970) 91
3 Distribution map of the river chub (Nocomis
micropogon) in Pennsylvania by major watersheds 101
4 Distribution map of the satinfin shiner (Notropis
analostanus) in Pennsylvania by major watersheds 102
5 Distribution map of the common shiner (Notropis
cornutus) in Pennsylvania by major watersheds 103
6 Distribution map of the spotfin shiner (Notropis
spilopterus) in Pennsylvania by major watersheds 104
7 Distribution map of the blacknose dace (Rhinichthys
atratulus) in Pennsylvania by major watersheds 105
8 Distribution map of the creek chub (Semotilus
atromaculatus) in Pennsylvania by major
watersheds 106
9 Distribution map of the white sucker (Catostomus
commersoni) in Pennsylvania by major watersheds 107
10 Distribution map of the pumpkinseed (Lepomis
gibbosus) in Pennsylvania by major watersheds 108
11 Distribution map of the mottled sculpin (Cottus
bairdi) in Pennsylvania by major watersheds
12 Distribution map of the slimy sculpin (Cottus
cognatus) in Pennsylvania by major watersheds HO
13 The effect of pH on the presence or absence of
fishes in streams of Pennsylvania. Stations at
which fish were present are designated by
solid bars 112
79
-------
FIGURES
PAGE
14 The relationship between pH and acidity and
their combined effect on the presence or
absence of fishes in streams of
Pennsylvania 113
80
-------
TABLES
No. Page
1 List of fish species collected in Pennsylvania
waters during the period 1957 through 1970 96
2 Order of appearance of 48 fish species with
decreasing hydrogen-ion concentration. In
parentheses are the number of species present
up to and including the pH indicated. The 68
additional species listed in Table 1 were
never found at a pH below 6.5 114
3 Fishes occurring in waters at different pH
values, arranged in order of decreased
tolerance to hydrogen-ion concentration.
Based on 68 collection sites ranging from
pH 4.6 to 6.4; each species taken in at
least 9 sites 115
4 Fishes occurring in waters at different pH
values, arranged in order of decreased
tolerance to hydrogen-ion concentration.
Based on 68 collection sites ranging from
pH 4.6 to 6.4; each speciesstakes in 4 to
7 sites 116
5 Fishes occurring in waters at different pH
values, arranged in order of decreased
tolerance to hydrogen-ion concentration.
Based on 68 collection sites ranging from
pH 4.6 to 6.4; each species taken in 1 to
3 sites 117
81
-------
SECTION I
CONCLUSIONS
1. Mine acid drainage has severely affected fish populations in
several major watersheds in Pennsylvania, including the Monongahela,
Youghiogheny, Kiskiminitas, Clarion, West Branch of the Susquehanna,
Swatara, Mahanoy, Catawissa, Nescopeck and upper Schuylkill. However,
in all of these drainages there are small tributaries of clean water
harboring diverse fish populations.
2. Total acidity, pH, and probably heavy metals, are all involved in
the toxic action of mine acid drainage on fish populations. No
information was obtained on concentrations of metallic ions in these
discharges, but concurrent readings of pH as low as 4.5 and total
acidity as low as 15 ppm are sufficient to account for the complete
loss of fish populations at about 90 per cent of the stations
examined where no fishes were present.
3. Several species of native fishes exhibited some tolerance for
acid mine water. The ten most tolerant species found in water ranging
from pH 4.6 to 5.5, in order of highest tolerance, were white sucker,
brown bullhead, pumpkinseed, chain pickerel, golden shiner, creek
chubsucker, largemouth bass, brook trout, creek chub and yellow
perch.
83
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SECTION II
RECOMMENDATIONS
The reclamation of streams receiving acid mine drainage must be
sufficient to maintain concurrent readings of pH not less than 5.5
and a total mineral acidity not exceeding 5 ppm to assure a diverse
and productive fish population. The added toxicity of heavy metals will
be largely avoided if suitable pH and acidity levels are maintained.
Even though fish populations have been eliminated in large portions
of major watersheds, there are sufficient native populations present
in small isolated tributaries to restock most waters once they are
reclaimed to a suitable quality for fishes.
85
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SECTION III
INTRODUCTION
In Appalachia, 1.5 million acres of land had been affected by surface
mining for coal prior to 1968 (Boccardy and Spaulding, 1968). As
a direct consequence, more than 5,000 miles of stream (about 4,000
miles in Pennsylvania and West Virginia alone) were made uninhabitable
by fishes because of the acid mine drainage which resulted (Kinney,
1964). In addition to surface mining, deep mining in Appalachia has
contributed to this form of pollution.
However, coal is the fuel used to produce most of our electricity,
and is an important mineral used by the steel industry. The present
concern over saving the environment should emphasize the possibility
of exploiting one important natural resource (coal) without degrading
another resource (water). Both of these resources are necessary to
maintain our present high living standards.
There is an abundance of information on the mechanisms which result
in the formation of sulphuric acid during coal mining operations
(Parsons, 1957; Braley, 1951). There are also many studies reporting
on effects of the individual constituents of acid-mine drainage on
many different organisms (McKee and Wolf, 1963; Doudoroff and Katz,
1950 and 1953). Many of the common metallic ions associated with
acid mine drainage, such as iron, aluminum, zinc and copper are toxic
to fishes (Affleck, 1952) and their degree of toxicity is often related
to the level of acidity in the environment (Brown and Jewell, 1926).
In comparison to the amount of information available on acid formation
in coal mining operations, there have been very few attempts to
measure the effects of acid mine drainage on natural communities of
fishes, such as the report !by Parsons (1968). The present study serves
only to inventory the major effects of acid mine drainage on the
distribution of natural fish populations in Pennsylvania, and perhaps
suggests some variability in response of different fishes to selected
levels of acidity. No work was done on specific compounds of acid mine
water in relation to individual fish species, but this study does
make it possible to predict the amount of reclamation of acid water
that is necessary to permit different species of fishes or mixed
populations of fishes to flourish under natural conditions.
87
-------
SECTION IV
METHODS AND MATERIALS
Sources of Information
Several different sources of information have been added to the
field work sponsored by the present contract in order to present as
complete a picture as possible. Data on fish distribution and
some analyses of water quality in Pennsylvania were collected by
the writers as early as 1957. Fishes collected during the trout
surveys conducted cooperatively in 1961 through 1965 by the U. S.
Bureau of Sport Fisheries and Wildlife and the Pennsylvania Fish
Commission have all been identified by the writers, and added a
significant amount of information to this study. In 1966, contract
14-16-0005-2091 between BSF&W and Pennsylvania State University
provided for the collection and interpretation of additional
data. The present contract, agreed upon in December 1968, completes
the field work on which this report is based. In all, at 1,257
stations fish collections were attempted and a water quality analysis
made at most of these. Stations at which at least one fish was
collected are presented in Figure 1; acid stations at which no fish
were collected are presented in Figure 2. Stations were selected on
all major streams in Pennsylvania in which some drainage from coal
mining occurs. Also particular care was taken to include clean
stations in each watershed as a control for both normal water
quality and normal distribution of fishes.
Fish Collections
In many of the trout stream surveys done during 1961 through 1965,
fishes were collected from representative sections of streams with
rotenone. This method is an excellent one for estimating the numbers
of fishes present (Boccardy and Cooper, 1963), but the bad public
relations involved in killing even one game fish led us to adopt
electrofishing as our chief method of collecting subsequent to 1965.
In most instances, electrofishing was continued in a 200- to 500-foot
section of stream, sampling all available microhabitats until no new
species were found. Such sampling reveals only a list of species present,
and there were almost always a few rare species represented by a single
individual in any collection.
In a few cases, quantitative estimates of the species composition
were made by the removal method, employing at least three consecutive
runs with electrofishing gear (Zippin, 1958). However, only the
list of species found at these stations has been used in the present
study.
89
-------
. t
'".: ' !' -*'' : '' '
. * ...''.:'.::-:.y
">.!.:'i ":^f
v «'/' &'»fc?'>\&*'£:?'''* V'.'::
* * * * v *"* "* * *.I I* * * *. .
.* f * . \« ." *'*.! ..
\
r :
"/!.«
Figure 1. Watershed map of Pennsylvania showing stations at which one or more fish
species was collected (1957 through 1970).
-------
VO
I ! . ! * *
.'Il'«l '
Figure 2. Watershed map of Pennsylvania showing stations at which no fish were
taken and in which the pH was less than 5.5 (1957 through 1970).
-------
Most of the fishes collected, including all large game species, were
returned to the stream after noting their abundance in the field
records. A representative sample of fishes was then preserved in
10 per cent formalin and identified at a later date in the laboratory.
These samples have been added to the permanent fish collections at
Pennsylvania State University and are available to students and any
other investigators for future studies.
Water Analyses
One of the major objectives of this study was to determine the
species of fishes in waters exposed to different concentrations of
mine acids. Among the many chemical analyses that could be made of
this variable and complex source of water, we selected five routine
analyses for two principal reasons: 1) The results from these
analyses are relatively consistent when replicated, and 2) the
combination of these analyses yields a general picture of the
severity of pollution from acid mine drainage. The analysis of
these water samples follow standard methods except where noted below.
Toxicity from heavy metals is probably involved in acid mine drainage
pollution, but this initial inventory of effects did not attempt any
analysis of metallic ions or their complexes.
Alkalinity.The total alkalinity was titrated in the laboratory with
0.02 N sulfuric acid to a methyl-orange end point at approximately
4.3 pH.
Acidity.Acidity was titrated in the laboratory at room temperature
with 0.02 N sodium hydroxide to a phenophthalein end point at
approximately 8.3 pH.
Hydrogen-ion concentration.pH was measured in the laboratory with
a Corning Model 7 electric meter, or equivalent. Several different
pH meters were tried in an attempt to obtain stream readings in
situ, but the performance of these portable pH meters was so erratic
from one stream to another, and in a short time interval, that we
decided to bring all samples back to the laboratory for pH analysis.
A few replicate readings (field pH reading compared with sample returned
to laboratory) gave us confidence that such a procedure did not
result in erroneous readings of more than 0.2 of a pH unit in the
majority of cases.
Sulfate.Concentrations of this ion were determined by the turbidimetric
method with a Hach Chemical Company field kit, and are not considered
to be more accurate than plus or minus 20 per cent. Sulfates in
water do not become toxic to fishes until the concentrations approach
or even exceed the saturation level of several thousand ppm
(McKee and Wolf, 1963). This measurement, however, is a useful
92
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adjunct to the analyses of alkalinity, acidity, and pH in that
sulfate readings higher than about 50 ppm lead one to suspect
contamination of the stream by acid mine drainage that had
subsequently been neutralized by alkaline materials.
Specific conductivity.The electrical resistance of the water was
measured in the field or in the laboratory with a Wheatstone-type
Industrial Instruments or Beckman meter, and all readings converted
to conductivity in micromhos per c3 corrected to a temperature
of 25 C. This independent reading of the total ions in solution
serves as a general check on the accuracy of the other analyses,
and also has obvious value in selection of efficient electrofishing
gear for particular waters.
Computer Analyses
The large amount of data on fish species and water quality, and the
desirability of analysing individual streams or watersheds against
this array of fish species and water quality parameters led us to
develop a generalized computer program to accomplish any of these
possible comparisons. In addition, by assigning map coordinates to
each collection site, this computer program can sort the original
data cards for any desired combination of characteristics and print
any distribution map which is desired. For example, Figure 2 is a
machine plot of all locations where the pH of the water was below
5.5 at the time of sampling and no fishes were collected. A print
out of all tabular material associated with any group of characteristics
is also available from such a computer program. The deck of cards and
the computer program are available for further use.
93
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SECTION V
RESULTS AND DISCUSSION
Distribution of Fishes in Pennsylvania
During the period 1957 through 1970 we have confirmed the occurrence
of 116 species of fishes in Pennsylvania (Table 1). This list is
not complete since there are several species probably present which
we have not collected and identified. These probable additions to
the list include the northern brook lamprey, lake sturgeon, Atlantic
sturgeon, hickory shad, lake herring, blacknose shiner, blackchin
shiner, mountain madtom, brindled madtom, pirate perch, longear
sunfish, eastern sand darter, and the swamp darter. Throughout this
report, common and scientific names of fishes follow the list of the
American Fisheries Society, Special Publication No. 6, 3d ed. 1970,
prepared by the Committee on Names of Fishes, Reeve M. Bailey,
Chairman.
The latest published compilation of Pennsylvania fishes (Fowler,
1940) lists 175 species as having occurred at one time or another
in Pennsylvania waters. However, many fishes in Fowler's list which
we failed to collect are marine species (two sharks, a skate, men-
haden, two needlefishes, a sole, threespine stickleback, bluefish,
and the striped bass). Other species in Fowler's list include
rare forms reported many years ago and are not now likely to be
present (shovelnose sturgeon, paddlefish, goldeye, Atlantic salmon,
bullhead minnow, river shiner, smallmouth buffalo, blue sucker,
spotted sucker, and the blue catfish). We have added only one species
to Fowler's list and this is the parasitic Ohio lamprey which has
suddenly become rather abundant in northwestern Pennsylvania.
The major watersheds in Pennsylvania are boundary limits for a few
species of fishes. For example, several of the darters (rainbow,
Johnny, variegate, blackside) are restricted to the Allegheny-Ohio
drainage, while the shield darter and tessellated darter are found
only on the Atlantic slope. The tonguetied minnow is likewise
restricted to the Allegheny-Ohio drainage while its close relative,
the cutlips minnow is found abundantly in Atlantic slope streams
but not elsewhere in Pennsylvania.
Some species are rare or only locally abundant, such as the
Allegheny brook lamprey, bowfin, bridle shiner, longnose sucker,
fourspine stickleback, warmouth, channel darter and the Tippicanoe
darter. These species are not very useful, therefore, as biotic
indicators because of their very restricted occurrence. However,
a great many species are both abundant and widely distributed in
most watersheds. Examples are the river chub, satinfin shiner,
95
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Table 1. List of fish species collected in Pennsylvania waters
during the period 1957 through 1970.
Petromyzontidae
Ichthyomyzon bdellium, Ohio lamprey
Ich thyomy z on greeleyi, Allegheny brook lamprey
Ichthyomyzon unicuspis, Silver lamprey
Lampetra aepyptera, Least brook lamprey
Lampetra lamottei, American brook lamprey
Petromyzon marinus, Sea lamprey
Lepisosteidae
Lepisosteus oculatus, Spotted gar
Lepisosteus osseus, Longnose gar
Amiidae
Amia. calva, Bowf in
Anguillidae
Anguilla rostrata, American eel
Clupeidae
Alosa aestivalis, Blueback herring
Alosa pseudoharerigus, Alewife
Alosa sapidissima, American shad
Dorosoma cepedianum, Gizzard shad
Salmonidae
Salmo gairdneri, Rainbow trout
Salmo trutta, Brown trout
Salvelinus fontinalis, Brook trout
Osmeridae
Osmerus mordax, Rainbow smelt
Umbridae
Umbra limi, Central mudminnow
Umbra phygaea, Eastern mudminnow
96
-------
Table 1 continued.
Esocidae
Esox americanus, Redfin pickerel
Esox lucius, Northern pike
Esox masquinongy, Muskellunge
Esox niger, Chain pickerel
Cyprinidae
Campostoma anomalum, Stoneroller
Carassius auratus, Goldfish
Clinostomus elongatus, Redside dace
Clinostomus funduloides, Rosyside dace
Cyprinus carpio, Carp
Ericymba buccata, Silverjaw minnow
Exoglossum laurae, Tonguetied minnow
Exoglossum maxillingua, Cutlips minnow
Hydrognathus nuchalis, Silvery minnow
Hybopsis amblops, Bigeye chub
Hybopsis dissimilis, Streamline chub
Hybopsis storeriana, Silver chub
Nocomis biguttatus, Hornyhead chub
Nocomis micropogon, River chub
Notemigonus crysoleucas, Golden shiner
Notropis amoenus, Comely shiner
Notropis analostanus, Satinfin shiner
Notropis atherinoides, Emerald shiner
Notropis bifrenatus, Bridle shiner
Notropis cornutus, Common shiner
Notropis dorsalis, Bigmouth shiner
Notropis hudsonius, Spottail shiner
Notropis photogenis, Silver shiner
Notropis procne, Swallowtail shiner
Notropis rubellus, Rosyface shiner
Notropis spilopterus, Spotfin shiner
Notropis stramineus, Sand shiner
Notropis volucellus, Mimic shiner
Phoxinus erythro gas t er, Southern redbelly dace
Pimephales notatus, Bluntnose minnow
Pimephales promelas, lathead minnow
Rhinichthys atratulus, Blacknose dace
Rhinichthys cataractae, Longnose dace
Semotilus atromaculatus, Creek chub
Semotilus corporalis, Fallfish
Semotilus margarita, Pearl dace
Catostomidae
Carpiodes Cyprinus, Quillback
Catostomus catostomus, Longnose sucker
97
-------
Table 1 continued.
Catostomus commersoni, White sucker
Erimyzon oblongus, Creek chubsucker
Hypentelium nigricans, Northern hog sucker
Moxostoma anisurum, Silver redhorse
Moxostoma duquesnei, Black redhorse
Moxostoma erythrurum, Golden redhorse
Moxostoma macrolepidotum, Shorthead redhorse
Ictaluridae
Ictalurus catus, White catfish
Ictalurus melas, Black bullhead
Ictalurus natalis, Yellow bullhead
Ictalurus nebulosus, Brown bullhead
Ictalurus punctatus, Channel catfish
Noturus flavus, Stonecat
Noturus gyrinus, Tadpole madtom
Noturus insignis, Margined madtom
Pilodictis olivaris, Flathead catfish
Percopsidea
Percopsis omiscomaycus, Trout-perch
Gadidae
Lota lota, Burbot
Cyprinodontidae
Fundulus diaphanus, Banded killifish
Fundulus heteroclitus, Mummichog
Atherinidae
Labidesthes sicculus, Brook silverside
Gasterosteidae
Apeltes quadracus, Fourspine stickleback
Culaea inconstans, Brook stickleback
Centrarchidae
Ambloplites rupestris, Rock bass
Enneacanthus gloriosus, Bluespotted sunfish
Lepomis auritus, Redbreast sunfish
Lepomis cyanellus, Green sunfish
Lepomis gibbosus, Pumpkinseed
Lepomis gulosus, Warmouth
Lepomis macrochirus, Bluegill
Micropterus dolomieui, Smallmouth bass
98
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Table 1 continued.
Micropterus salmoides, Largemouth bass
Pomoxis annularis, White crappie
Pornoxis nigromaculatus, Black crappie
Percidae
Etheostoma blennioides, Greenside darter
Etheostoma caeruleum, Rainbow darter
Etheostoma camurum, Bluebrease darter
Etheostoma flabellare, Fantail darter
Etheostoma maculatum, Spotted darter
Etheostoma nigrum, Johnny darter
Etheostoma olmstedi, Tessellated darter
Etheostoma tippicanoe, Tippicanoe darter
Etheostoma variatum, Variegate darter
Etheostoma zonale, Banded darter
Ferca flavescens, Yellow perch
Percina caprodes, Logperch
Percina copelandi, Channel darter
Percina macrocephala, Longhead darter
Percina maculata, Blackside darter
Percina peltata, Shield darter
Stizostedion v_. vitreum, Walleye
Scianenidae
Aplodinotus grunniens, Freshwater drum
Cottidae
Cottus bairdi, Mottled sculpin
Cottus cognatus, Slimy sculpin
99
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common shiner, spotfin shiner, blacknose dace, creek chub, white
sucker, pumpkinseed, mottled sculpin and the slimy sculpin (Figures
3 through 12). Within this group of species which are abundant we
were hoping to find some evidence of difference in resistance to
acid-mine drainage. This would permit us to list species as acid-
tolerant or acid-resistant and use their absence in streams within
their normal range of distribution as an indication of stream
degradation. A few fish species exhibit differences in acid
tolerance, but in general there seems to be a threshold level of
acid pollution (narrow range of pH) above which all species of fishes
quickly disappear.
Distribution of Acid Mine Drainage
Acid mine drainage has affected some watersheds in Pennsylvania much
more than others. Our field collections and water quality analyses,
which identify areas of acid pollution (Figure 2) agree well with the
map of coal bearing strata published in Boccardy and Spaulding (1968).
This is related to the presence or absence of both pyritic materials
and alkaline materials in the soils of the watershed. It is
unfortunate that carbonates are seldom associated with coal-and-pyrite-
bearing rock layers in Pennsylvania; streams draining coal-mining
areas are thus highly vulnerable to acid pollution because of their
very low alkalinity.
Although acid pollution is extensive in some watersheds, it is
important to note that healthy and diverse fish populations occur
in isolated, clean-water tributaries of even the most severely affected
watersheds (Figure 1). One is tempted to consider entire stream
drainages in Clearfield, Cambria, Indiana, Westmoreland, and Clarion
Counties as completely lost to acid-mine pollution. Such is not the
case for we found diverse fish populations scattered throughout all
of these areas. If the major sources of acid pollution could be
identified and the problems corrected, these affected watersheds
would be rapidly invaded and repopulated with a diverse assemblage
of fishes coming from existing native stream populations.
Effect of pH and Acidity on Fish Populations
In the sampling procedure applied to this study, stations were
selected from a stream drainage map to assure equal distribution by
watersheds. Of the 1,257 stations selected, 187 harbored no fish
populations for a variety of reasons. At 10 stations, the stream
was dry at the time of inspection. At five stations, domestic
sewage was the apparent cause of no fish being present; one other
station, located immediately downstream from a sulfite paper mill,
contained no fish. But by far the greatest number of stations (171)
that were completely devoid of fishes were associated with acid
drainage from mining operations.
100
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Figure 3. Distribution map of the river chub (Nocomis micropogon) in Pennsylvania
by major watersheds.
-------
o
NJ
Figure 4 Distribution map of the satinfin shiner (Notropis analostanus) in
Pennsylvania by major watersheds.
-------
o
CO
Figure 5. Distribution map of the common shiner (Notropis cornutus) in Pennsylvania
by major watersheds.
-------
Figure 6. Distribution map of the spotfin shiner (Notropis spilopterus) in
Pennsylvania by major watersheds.
-------
H
O
Ui
Figure 7. Distribution map of the blacknose dace (Rhinichthys atratulus) in
Pennsylvania by major watersheds.
-------
7 '
» *
Figure 8. Distribution map of the creek chub (Semotilus atromaculatus) in
Pennsylvania by major watersheds.
-------
o
-J
. / I . , I
: x^. : ' . V ' '^^
C : «..!.:... Y
\*\ * . «. ». . * (*
\j .i: . . >... .
it my * u
' .... Ss . «" . *
i*' :"*. :-.: l.*r ' ' "' ' :
'./ .
.
. .
Figure 9. Distribution map of the white sucker (Catostomus coimnersoni) in
Pennsylvania by major watersheds.
-------
o
00
Figure 10. -Distribution map of the pumpkinseed (Lepomis gibbosus) in Pennsylvania
by major watersheds.
-------
m ^
» *
* .'....
Figure 11. Distribution map of the mottled sculpin (Cottus bairdi) in Pennsylvania
by major watersheds.
-------
Figure 12. Distribution map of the slimy sculpin (Cottus cognatus) in Pennsylvania
by major watersheds.
-------
The pH at the 155 stations for which we have pH data and in which no
fish were found ranged from 2.8 to 7.2. The majority of these
(107) were strongly acid at a pH of 2.8 to 4.5 inclusive, and no
stations in this pH range were found to contain any fish. Within
the pH range from 4.6 to 6.4, 48 stations contained no fish but there
were 54 stations with fish (Figure 13). At four stations where the
pH ranged from 6.6 to 7.2 the absence of fish appeared to be
associated with high acidity, or a poorly understood complex of
chemical compounds present following neutralization of acid drainage.
An example of this latter group of toxic, but high pH values is the
station on the Quittapahilla Creek in Lebanon County. At this station,
we could find no fish by electrofishing but the water chemistry
values were as follows: pH 7.2, alkalinity 196 ppm, acidity 27 ppm,
sulfate 50 ppm and specific conductance 636 jumhos at 25 C. It should
be remembered that our water quality samples were taken only once
at each station, and that toxic conditions for fishes could have
occurred sometime prior to our sampling.
Although pH and acidity are correlated to some degree, there is
evidence that a combination of both measurements may prove to be
a better predictor of toxic conditions than either measurement alone
(Figure 14). However, this survey was not designed to include other
toxic materials such as heavy metals which undoubtedly would increase
the predictability of water quality indices on absence of fish popu-
lations. There are examples in our data where neither acidity nor
pH, per se were in the range suspected to be toxic, but no fishes were
found (Figure 14). As a working rule, we have found that a pH
below 4.5 and/or an acidity level above 15 ppm would be sufficient
to account for the absence of fishes at 90 per cent or more of the
stations studied.
Other workers have reported that pH values should be maintained
between 5.6 and 8.5 to maintain diverse and productive fish populations
(Spaulding and Ogden, 1968), and that pH values below 6.0 are
considered unfavorable for sport fishes. This generalization would
appear to be consistent with the results we obtained in the
present study.
Tolerance £f_ Individual Fish Species to pH Levels
When fish taxa are arrayed in order of their occurrence in water of
decreasing hydrogen-ion concentration (increasing pH) it appears
that some species are more tolerant than others to the water quality
conditions that accompany this change in pH (Table 2). Only 10
species were found in waters at a pH of 5.5 or below, and none in
water where the pH was 4.5 or below.
Ill
-------
IS1
CO
z
o
o
IU
tO
111
oc.
15^
K
UJ
CO
5
la's'l "1 "I "l "U.ol '1 '1 "1 "U
HYDROOEN-ION CONCENTRATION (pH)
Figure 13. The effect of pH on the presence or absence of fishes in streams of
Pennsylvania. Stations at which fish were present are designated by
solid bats-
-------
E
a
a.
O
O
1,000
800 -
600-
400-
300-
200-
100
80 -
60-
40-
30-
20-
10
8-
6-
4-
3-
2-
".. ':
ft * |t *
ft r
M| I
loo
00 0
0000
000 000
§
FISH PRESENT
FISH ABSENT
0 0
00 0
0 0 0
I i | I I i i | I i I i | i
3.0 3.5 4.0
i I I I i I I i i i i I i i I I I I
4.5 5.0 5.5 6.0
1 I
6.5
HYDROGEN-ION CONCENTRATION (pH)
Figure 14. The relationship between pH and acidity and their
combined effect on the presence or absence of fishes
in streams of Pennsylvania.
113
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Table 2. Order of appearance of 48 fish species with decreasing
hydrogen-ion concentration. In parentheses are the
number of species present up to and including the pH
indicated. The 68 additional species listed on Table 1
were never found at a pH below 6.5.
4.5 (0)
4.6 (5)
Chain pickerel
Golden shiner
White sucker
Brown bullhead
Pumpkinseed
4.7 (7)
Creek chubsucker
Largemouth bass
5.0 (8)
Brook trout
5.2 (9)
Creek chub
5.5 (10)
Yellow perch
5.6 (12)
Bluntnose minnow
Blacknose dace
5.9 (18)
Brown trout
Eastern mudminnow
Longnose dace
Margined madtom
Tesselated darter
Slimy sculpin
6.0 (34)
Ohio lamprey
Stoneroller
Silverjaw minnow
River chub
Common shiner
Silver shiner
Rosyface shiner
Mimic shiner
Hog sucker
Rock bass
Smallmouth bass
Greenside darter
Fantail darter
Johnny darter
Banded darter
Blackside darter
6.1 (36)
Cutlips minnow
Fallfish
6.2 (41)
Redfin pickerel
Redbreast sunfish
Rainbow darter
Variegate darter
Mottled sculpin
6.4 (48)
American eel
Redside dace
Spotfin shiner
Spottail shiner
Pearl dace
Bluespot sunfish
Green sunfish
114
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Table 3. Fishes occurring in waters at different pH values, arranged
in order of decreased tolerance to hydrogen-ion concentration.
Based on 68 collection sites ranging from pH 4.6 to 6.4;
each species taken in at least 9 sites.
Frequency of occurrence
Species in water of pH:
4.6 to 5.9 6.0 to 6.4
Brook trout
Pumpkinseed
White sucker
Creek chub
Brown trout
Blacknose dace
Brown bullhead
Margined madtom
Slimy sculpin
Longnose dace
Tessellated
darter
Common shiner
Hog sucker
Cutlips minnow
Mottled sculpin
% occurrence
in water of
pH 4.6 to 5.9
Total frequency
in 68 collections
12
7
10
6
2
5
2
1
1
1
1
0
0
0
0
20
15
25
22
9
32
12
8
8
11
13
11
9
9
9
38
32
29
21
18
14
14
11
11
8
7
0
0
0
0
32
22
35
28
11
37
14
9
9
12
14
11
9
9
9
115
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Table 4. Fishes occurring in waters at different pH values,
arranged in order of decreased tolerance to hydrogen-ion
concentration. Based on 68 collection sites ranging
from pH 4.6 to 6.4; each species taken in 4 to 7 sites.
Frequency of occurrence % occurrence Total frequency
Species in water of pH: in water of in 68 collections
4.6 to 5.9 6.0 to 6.4 pH 4.6 to 5.9
Creek chubsucker
Chain pickerel
Yellow perch
Largemouth bass
Bluntnose
minnow
Golden shiner
Johnny darter
Stoneroller
Fallfish
River chub
Fantail darter
Blacks ide
darter
Redfin pickerel
American eel
Rosyface shiner
2
3
1
1
1
1
0
0
0
0
0
0
0
0
0
2
4
3
4
5
6
7
7
7
5
5
4
4
4
4
50
43
25
20
16
14
0
0
0
0
0
0
0
0
0
4
7
4
5
6
7
7
7
7
5
5
4
4
4
4
116
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Table 5. Fishes occurring in waters at different pH values, arranged
in order of decreased tolerance to hydrogen-ion concentration.
Based on 68 collection sites ranging from pH 4.6 to 6.4;
each species taken in 1 to 3 sites.
Species
Eastern mudminnow
Ohio lamprey
Redside dace
Spottail shiner
Spotfin shiner
Pearl dace
Bluespot sunfish
Green sunfish
Rainbow darter
Variegate darter
Silver shiner
Mimic shiner
Rock bass
Redbreast sunfish
Banded darter
Silverjaw minnow
Smallmouth bass
Greenside darter
Frequency of occurrence in
water of pH:
4.6 to 5.9 6.0 to 6.4
Total frequency
in 68 collections
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
3
3
2
1
1
1
1
1
1
1
1
1
2
2
2
2
2
3
3
3
117
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However, since some species are more common than others even under
good water quality conditions, we have divided this analysis of the
68 stations, where the pH ranged from 4.6 to 6.4, into three parts
as follows: '
Fishes captured in 9_ to 37 different collections.This group of 15
species is ranged in order of decreasing tolerance to low pH, with
the number of occurrences at stations of pH 4.6 to 5.9, and the number
of occurrences at stations of pH 6.0 to 6.4. The brook trout, pump-
kinseed, white sucker and creek chub are the most tolerant of this
group with the common shiner, hog sucker, cutlips minnow and
mottled sculpin never occurring at pH values below 6.0 (Table 3).
Fishes captured in tj_ to ]_ different collections. This group, also
of 15 species, shows differences in tolerance to low pH although
their relative rareness within this range of pH values leads to less
confidence in predicting their response to low pH. Among this group,
the creek chubsucker and the chain pickerel were quite tolerant to
low pH, while the Johnny darter, the stoneroller and the fallfish
never occurred at a pH below 6.0 (Table 4).
Fishes captured in ^ to _3_ different collections.Except for the
eastern mudminnow, none of these species was taken in water where the
pH was less than 6.0 (Table 5). However, this group was represented
so rarely in the 68 collections that we have decided not to draw
any inferences concerning their tolerance to acid conditions from
this analysis. There remains the possibility that several of these
species (as well as some in the other two groups) are normally
found at pH values higher than 6.4 since some of them (spottail
shiner, spotfin shiner, rock bass, smallmouth bass, greenside
darter) are commonly found in streams in Pennsylvania.
General Discussion of Acid Tolerance by Fishes
Of the species which appear to be most tolerant to low pH, more
information is available on the brook trout than other species.
Powers (1929) reported natural populations of brook trout living in
headwaters streams of the southern Appalachians at pH values ranging
from 4.1 to 5.9, with several populations below 4.5 pH. The lowest
pH at which we collected brook trout in Pennsylvania was 5.0, and at
12 stations where the pH ranged from 5.0 to 5.9. It should be pointed
out that in these situations the total acidity in the water was less
than 10 ppm and the fish were apparently able to adjust to this
continuing source of hydrogen ions.
Packer and Dunson (1970) reported an increasing loss of sodium
from the body of brook trout as pH of the water was lowered from 7.0
118
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to 3.0 with a very rapid change occurring at pH 5.0 to 4.0. They
explained the loss of sodium as having a secondary role in the
death of the fish. The primary cause of death was thought to be
due to lowered blood pH interfering with the ability of the blood
to pick up and transport oxygen.
Brown and Jewell (1926) reported on the ability of several fishes
to withstand sudden changes of pH. They captured yellow perch, brown
bullhead, northern pike, bluegill, brook stickleback, fathead minnow,
southern redbelly dace, mudminnow, Johnny darter and smallmouth
bass from a lake at pH 8.5 and transferred them to a lake at pH 4.4
where they survived up to 40 days. The test fish suffered no more
mortality than did control fish kept at the original pH. Such an
experiment indicates, according to Brown and Jewell, that it is not
necessary to assume that fishes found in these waters of extremely
low pH have gradually developed a resistance to low pH, or are
physiologically different from fishes living at higher pH values.
However, fishes differ in their selection of a preferred range of
pH and detect the difference between slightly alkaline, neutral,
and slightly acid water. The blood will maintain its normal chemical
reaction (slightly alkaline) in the face of relatively large changes
in the environment, yet we know that the physiological mechanism
breaks down when the change is too great or too long continued
(Wells, 1915).
It is impossible to separate the effects of toxic action of acidity
from that due to heavy metals, since the toxicity of copper and iron
is known to increase with increase in acidity (McKee and Wolf, 1963).
To complicate the fish toxicity picture still further, it is known
that the toxic effects of certain metals (copper and zinc) are
additive at low concentrations (the effect being described as similar
joint action, Herbert and Vandyke, 1964), but copper and zinc
mixtures are synergistic in response at higher concentrations
(Lloyd, 1961).
119
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SECTION VI
ACKNOWLEDGMENTS
Much of the data on which this report is based comes from cooperative
studies between personnel from The Pennsylvania State University,
the Pennsylvania Fish Commission and the U. S. Bureau of Sport
Fisheries and Wildlife. Special recognition is due to Joseph A. Boccardy
for his foresight in initiating a cooperative trout stream survey in
Pennsylvania, and the collection of fishes and water quality data
which add a great deal to our present analysis.
121
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SECTION VII
REFERENCES
1. Affleck, R. J. Zinc poisoning in a trout hatchery. Australian J.
Marine and Freshwater Research 3(2): 142-169 (1952).
2. Boccardy, Joseph A. and Edwin L. Cooper. The use of rotenone
and electrofishing in surveying small streams. Trans. Amer. Fish.
Soc. 92(3): 307-310 (1963).
3. Boccardy, Joseph A. and Willard M. Spaulding, Jr. Effects of
surface mining on fish and wildlife in Appalachia. U. S. Bureau
of Sport Fisheries and Wildlife. Resource Publ. 65, 20 p. (1968).
4. Braley, S. A. Acid drainage from coal mines. Trans. (AIME)
Amer. Inst. of Mining and Metallurgical Engineers (Mining Branch)
190: 703-707 (1951).
5. Brown, Harold W. and Minna E. Jewell. Further studies on the
fishes of an acid lake. Trans. Amer. Microscopical Soc. 45:
20-34 (1926).
6. Doudoroff, Peter and Max Katz. Critical review of literature on
the toxicity of industrial wastes and their components to fish.
I. Alkalies, acids, and inorganic gases. Sewage and Industrial
Wastes 22(11): 1432-1458 (1950).
7. Doudoroff, Peter and Max Katz. Critical review of literature on
the toxicity of industrial wastes and their components to fish.
II. The metals as salts. Sewage and Industrial Wastes 25(7) :
802-839 (1953).
8. Fowler, Henry W. A list of the fishes recorded from Pennsylvania.
Pa. Board of Fish Commissioners, Biennial Report for 1940,
p. 59-78 (1940).
9. Herbert, D. W. M. and Jennifer M. Vandyke. The toxicity to fish of
mixtures of poisons. II. Copper-ammonia and zinc-phenol mixtures.
Ann. Appl. Biol. 53: 415-421 (1964).
10. Kinney, Edward C. Extent of acid mine pollution in the United States
affecting fish and wildlife. U. S. Bureau of Sport Fisheries and
Wildlife, Circular 191, 27 p. (1964).
123
-------
11. Lloyd, R. The toxicity of mixtures of zinc and copper sulphates
to rainbow trout (Salmo gairdrieri Richardson). Ann. Appl. Biol.
49: 535-538 (1961).
12. McKee, J. E. and H. W. Wolf. Water quality criteria. California
Water Quality Control Board, 2d ed., Publ. 3A, 548 p. (1963).
13. Packer, Randall K. and William A. Dunson. Effects of low
environmental pH on blood pH and sodium balance of brook trout.
J. Exp. Zool. 174(1): 65-72 (1970).
14. Parsons, John D. Literature pertaining to formation of acid-mine
wastes and their effects on the chemistry and fauna of streams.
Trans. Illinois Acad. Sci. 50: 49-59 (1957).
15. Parsons, John D. The effects of acid strip-mine effluents on the
ecology of a stream. Archives Hydrobiology 65(1): 25-50 (1968).
16. Powers, Edwin B. Fresh water studies. I. The relative temperature,
oxygen content, alkali reserve, the carbon dioxide tension and
pH of the waters of certain mountain streams at different altitudes
in the Smoky Mountain National Park. Ecology 10(1): 97-111 (1929).
17. Spaulding, Willard M. Jr., and Ronald D. Ogden. Effects of surface
mining on the fish and wildlife resources of the United States.
U. S. Bureau of Sport Fisheries and Wildlife, Resource Publ.
68, 51 p. (1968).
18. Wells, Morris M. Reactions and resistance of fishes in their
natural environment to acidity, alkalinity, and neutrality.
Biological Bulletin 29: 221-257 (1915).
19. Zippin, C. The removal method of population estimation. J. Wildlife
Management 22: 82-90 (1958).
124
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PART III: ACUTE TOXICITY OF LOW pH
TO AQUATIC INSECTS
by
William G. Kimmel and Donald C. Hales
125
-------
ABSTRACT
The object of this study was to determine the median tolerance limits
of five aquatic insect species to low levels of pH. Test species
were chosen on the basis of their wide occurrence and common association
in soft-water streams. A continuous-flow bioassay system was designed
to overcome the objectionable features of static bioassays. All species
survived exposure for 4 days to pH levels from 6.5 to 4.0. The 96-hour
TLm values ranged from 3.31 for the most sensitive animal, Stenonema
sp., to 1.72 for the most tolerant animal, Nigronia fasciata.
Sensitivity appeared to increase during ecdysis.
127
-------
CONTENTS
Section Page
I Conclusions 135
II Recommendations 137
III Introduction 139
IV Methods and Materials 141
Apparatus
Bioassays
Test Organisms
V Results 147
VI Acknowledgments 155
VII References 157
129
-------
FIGURES
PAGE
1 CONTINUOUS-FLOW BIOASSAY SYSTEM 142
2 BIOASSAY CHAMBERS 144
131
-------
TABLES
No. page
1 Chemical features of Galbraith Run 141
2 Acidity, alkalinity, and sulfate concentrations
of test water at various pH levels employed in
bioassays 145
3 Insects used for acute toxicity bioassays 146
4 Number of survivors of Stenonema sp.
following 96-hour exposures to levels of
pH from 6.5 to 1.0 at 11 to 14 C. 148
5 Number of survivors of Acroneuria lycorias
following 96-hour exposures to levels of
pH from 6.5 to 1.0 at 11 to 14 C 149
6 Number of survivors of Pteronarcys proteus
following 96-hour exposures to levels of
pH from 6.5 to 1.0 at 11 to 14 C 150
7 Number of survivors of Boyeria vinosa
following 96-hour exposures to levels of
pH from 6.5 to 1.0 at 11 to 14 C 151
8 Number of survivors of Nigronia fasciata
following 96-hour exposures to levels of pH
from 6.5 to 1.0 at 11 to 14 C 152
9 Median tolerance limits of five aquatic insect
species to low pH 153
133
-------
SECTION I
CONCLUSIONS
The five species of insects studied were generally tolerant of
short-term exposures to the lowered pH levels induced by sulfuric
acid. A similar conclusion was reached by Bell and Nebeker (1969)
who subjected nymphs of 10 species of stream insects to hydrochloric
acid. Of these species, Acroneuria lycorias and Boyeria vinosa
were common to both studies. The 96-hour TLm values for these two
species were 3.32 and 3.25, respectively, in the Bell and Nebeker
(1969) study and 3.16 and 2.25 in this study.
Aquatic insects may be more sensitive to low pH during ecdysis.
Deaths of Acroneuria lycorias at time of molt suggest the possibility
that this may be a critical time for toxicant action. The waxy
epicuticle of terrestrial arthropods is believed to be responsible
for their relative impermeability to water. Studies on several of
these organisms have shown that following ecdysis there is a marked
increase in transpiration rate (Edney, 1957). In the crustacea,
ecdysis is followed by a rapid uptake of water either by an increased
permeability of the new integument or by actual drinking (Green,
1961). An increased uptake of water at time of molt could render
the aquatic insect more vulnerable to hydrogen ions. Some nymphs
of the genus Stenonema may pass through 30 nymphal instars before
emergence (Burks, 1953). Nymphal instars of some species of stone-
flies may number as many as 22, and some members of the genus
Acroneuria spend 2 or 3 years in the stream before emergence
(Claassen, 1931). An increased sensitivity to hydrogen ions during
edcysis could eliminate insects from streams at higher pH levels
than their acute toxicity values would indicate.
We doubt that low pH alone accounts for the sparse insect faunas
characteristic of streams polluted by mine effluent. Bick, Hornuff,
and Lambremont (1953) have collected Boyeria vinosa and the genus
Stenonema from naturally acid streams having pH values between
4.0 and 5.0. We have found each of the species tested in this study
in a naturally acid stream at pH 4.0. Parsons (1968) found that
mine acid pollution of a stream resulted in a marked decrease in
the diversity and abundance of the benthic fauna. We have observed
similar conditions in a soft-water stream which receives mine
effluent from a polluted tributary. A diverse insect fauna including
Acroneuria lycorias, Stenonema sp., Pteronarcys proteus, and
Nigronia fasciata was present above the point of entrance of the
tributary. Below the tributary, the pH had decreased from 6.8 to
5.0 and the bottom fauna was severely diminished. Of the insects
we studied, only Nigronia fasciata which proved to be extremely
135
-------
tolerant of low pH in the laboratory was present. The 96-hour TLm
values of all five species (Table 9) and their occurrence in
naturally acid streams at pH values between 4.0 and 5.0 indicate
that they could survive pH 5.0. Thus, it appears that other
substances acting alone or synergistically with hydrogen ions are
responsible for the severe depletion of benthos in streams which
receive mine drainage.
136
-------
SECTION II
RECOMMENDATIONS
Measurement of pH alone for chemical surveys of water quality is
of limited value. Chosen representatives of four orders of aquatic
insects (Ephemeroptera) Stenonema sp., (Plecoptera) Acroneuria
lycorias and Pteronarcys proteus, (Odonata) Boyeria vinosa and
(Megaloptera) Nigronia fasciata were able to exist for 96-hour TLm
values well below those found under natural conditions.
The greater sensitivity to pH during ecdysis adds a new consideration
for water quality criteria. Most studies have been made without
regard to this sensitive period.
137
-------
SECTION III
INTRODUCTION
One of the principal environmental changes resulting from pollution
by coal mine effluent is a decrease in the pH of a receiving stream.
The introduction of large quantities of sulfuric acid is particularly
damaging to streams which lack effective buffering capacities.
Other possible toxic components include metallic ions such as iron
and aluminum and the ferric hydroxide precipitate commonly seen in
those streams which receive mine acids. The problem of mine acid
drainage is widespread, and the State of Pennsylvania, alone, has
2,500 miles of streams seriously affected by this pollution (Barnes
and Romberger, 1968).
A reduction or absence of normal benthic macroinvertebrate populations
can be used as one indication of the degree of stream pollution
(Wilhm and Dorris, 1968). In those streams polluted by mine acids,
there is a notable lack of insect fauna. Although the toxicity of
low pH to fish has been extensively studied (Doudoroff and Katz,
1950), the sensitivity of aquatic insects to low pH has not been
developed so extensively. The object of this study was to investi-
gate one aspect of the mine acid problem: the acute tolerance
limits of selected aquatic insects to the lowered pH levels induced
by sulfuric acid. Aquatic insects represent a large segment of the
stream community and form a vital link in the food chain which
ultimately supports fish. Knowledge of the relative toxicities of
high acidity and the other components of mine effluent to aquatic
insects is necessary if the nature of the problem posed by this
pollution to the stream community is to be understood.
139
-------
SECTION IV
MATERIALS AND METHODS
The experimental system was designed to simulate the pollution of
a soft-water stream by the sulfuric acid component of mine
effluent.
Apparatus
A continuous-flow bioassay system (Figure 1) was developed to over-
come the objectionable feature of static bioassays summarized by
Burke and Ferguson (1968). Objectionable features of static bioassays
were a decline in toxicant concentration due to uptake by test animals
and adsorption onto the container surface, a reduction of dissolved
oxygen supply, and the accumulation of animal waste products.
The experimental apparatus was constructed of two subsystems, one
for delivery of water and the other for delivery of dilute sulfuric
acid. All components which came into contact with water or acid
were constructed of polyvinyl chloride, polypropylene, Tygon tubing,
Fiberglas, copper tubing coated internally with epoxy paint, or
Pyrex glass to eliminate contamination of the test medium due to
corrosion.
A stream having low alkalinity and low total hardness was selected
as the water source for the experiment (Table 1). Water from
Table 1. Chemical features of Galbraith Run. Alkalinity, acidity,
and hardness are expressed as parts per million of calcium
carbonate.
Chemistry ppm
Total alkalinity 2.2
Total acidity 2-0
Total hardness 4.5
Sulfate 3.0
Galbraith Run was transported by means of tank truck to two 500-gal
Fiberglas reservoir tanks. This water was pumped through a particle
filter and cooling unit into a 25-gal constant-head tank which was
141
-------
Figure 1. Continous-flow bioassay system,
142
-------
continuously aerated. From the constant-head tank, tubing lines
ran to 10 Gilmont flowmeters (Roger Gilmont Instruments, Inc.)
which were set by means of needle valves to deliver a constant
flow of 100 ml/min to each of 10 1,000-ml filtering flasks.
A similar system consisting of reservoir, pumps, and constant-head
tank was used to deliver dilute sulfuric acid through flowmeters
for mixing with test water. Stock solutions of dilute sulfuric acid
were prepared from concentrated sulfuric acid and distilled water
in proportions to make acid concentrations 1 pH unit lower than
the level to be tested. Needle valves on the acid flowmeters were
adjusted to give the proper flow of stock solution to maintain a
desired pH value.
Bioassay chambers (Figure 2) were used to test multiple experimental
and control groups simultaneously. Chambers were constructed of
polyvinyl chloride and measured 14.1 x 10.8 x 38.9 cm. Each chamber
received acid-water mixture from its corresponding filtering flask.
Flow through the chambers ranged from about 100 to 110 ml/min
depending on the quantity of acid injected and provided approxi-
mately two complete changes of test medium per hour. One Vibert
egg-hatching box screened with fine nylon mesh was placed in each
chamber so that the small nymphs of Sterionema sp. could be tested
concurrently with other species. The temperature in each chamber
ranged from 11 to 14 C. A Beckman Model 96A pH Meter (Beckman
Instruments, Inc.) standardized with known buffer solutions was
used to check the pH of each chamber every 24 hours during a test.
A Heath Recording Electrometer Model EU-301A (Heath Company) was
also used to check the accuracy of the bioassay system during the full
course of an experiment. Test pH values were normally maintained
within +0.15 unit of the desired value. Experiments were repeated
if the pH deviated by more than 0.25 unit. Total alkalinity, total
acidity, and sulfate concentrations were determined for the various
pH levels tested (Table 2). All chemical analyses except sulfates
were performed according to procedures outlined in the 12th edition
of Standard Methods for the Examination of_ Water and Wastewater
(American Public Health Association, American Water Works Association,
and Water Pollution Control Federation, 1965). Sulfate determinations
were conducted with a Hach Model DR-EL Portable Water Engineer's
Laboratory (Hach Chemical Company).
Bioassays
Acute toxicity bioassays are used to determine the concentrations of
a given substance which are lethal to test organisms during exposure
periods from 24 to 96 hours (Doudoroff e£ al., 1951). A 96-hour TLm,
that concentration of sulfuric acid at which 50 per cent of the test
animals are killed, was used to measure the acute toxicity for each
species. Groups of insects were exposed to controlled pH levels and
143
-------
Figure 2. Bioassay chambers,
144
-------
Table 2. Acidity, alkalinity, and sulfate concentrations of
test water at various pH levels employed in bioassays.
Acidity and alkalinity are expressed as parts per million
of calcium carbonate; sulfate is in parts per million.
pH
7.1 (control)
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Total acidity
2.0
2.5
3.5
4.5
5.0
7.5
15.0
46.0
133.0
159.0
1,253.0
5,350.0
10,200.0
Total alkalinity
2.2
1.7
1.6
1.0
0.5
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
Sulfate
0
4
8
12
11
12
17
38
90
120
425
3,000
5,500
145
-------
the number of survivors noted at the conclusion of an experiment.
Ten insects of the same species were placed in each of the
experimental and control groups. Separate experimental groups were
exposed for 96 hours to each one-half pH unit from 6.5 to 1.0.
One control group (pH 7.1) was tested concurrently with each of
three experimental groups at the same pH. It was planned to repeat
a test if any mortality occurred in the controls. An individual
96-hour TLm value was obtained from the percentage survival at each
of the 12 pH levels tested by a modification of the straight line
graphical interpolation method (American Public Health Association
et_ al^., 1965). Three individual 96-hour TLm values were obtained
for each species by replicating the experimental procedure three
times. The mean 96-hour TLm and standard error were determined
from the hydrogen ion concentrations of the individual 96-hour
TLm values and subsequently reconverted to pH units.
Test Organisms
Five species representing four insect orders were chosen as typical
representatives of the benthic community of a soft-water stream
(Table 3). All animals were collected by means of handscreening
Table 3. Insects used for acute toxicity bioassays.
Organism Total body length (mm)
Stenonema sp. 5-7
Acroneuria lycorias Newman 12-19
Pteronarcys proteus Newman 16-30
Boyeria vinosa (Say) 17-33
Nigronia fasciata (Walker) 20-32
from various soft-water streams in central Pennsylvania. Some
Nigronia fasciata were collected from a moderately hard-water stream
due to lack of sufficient numbers in the soft streams. All animals
were transported to the laboratory in large plastic pails and
acclimated to the test water at least 96 hours prior to the start of
an experiment. Hydropsyche sp. and Leuctra sp. were also collected
but could not be maintained under laboratory conditions.
146
-------
SECTION V
RESULTS
There was no mortality among all five species for pH values above
4.0 (Tables 4, 5, 6, 7, and 8). No mortality was observed in any
of the control groups at pH 7.1. The various species, however,
differed markedly in their relative abilities to tolerate exposure
to lower pH levels (Table 9). Stenonema sp. proved to be the most
sensitive to acid conditions (Tables 4 and 9). Acroneuria lycorias
was also relatively sensitive (Tables 5 and 9). Pteronarcys proteus
was a rather tolerant organism and declined from 100 to 0 per cent
survival over a span of one-half pH unit (Tables 6 and 9). Boyeria
vinosa was very tolerant of low pH, and all individuals of this
species survived pH values as low as 2.5 (Tables 7 and 9). Nigronia
fasciata was the most tolerant of the species studied (Tables 8 and
9). Several of these experimental animals which survived the test
at pH 1.5 remained alive in the laboratory for an additional week
at this pH level.
Acroneuria lycorias appeared to be more sensitive to low pH during
periods of molting. In two of the replicates, all animals survived
exposure to pH 3.5 (Table 5). In the other replicate, nine of the
animals survived exposure to pH 3.5 with the lone mortality being
an individual which had molted during the course of the experiment.
In the second replicate, three animals were alive after 72 hours
at pH 3.0. One of these animals died within several hours after
the exoskeleton had split in the initial phase of the molting
process.
147
-------
Table 4. Number of survivors of Sterioriema sp. following 96-hour
exposures to levels
of pH from 6.5~to 1,
.0 at 11 to 14 C.
Number of survivors
PH
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Test 1
10
10
10
10
10
8
6
2
0
0
0
0
Test 2
10
10
10
10
10
10
8
1
0
0
0
0
Test 3
10
10
10
10
10
10
9
0
0
0
0
0
148
-------
Table 5. Number of survivors of Acrorieuria lycorias following
pH
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
96-hour exposures
11 to 14 C.
Test 1
10
10
10
10
10
10
9
4
0
0
0
0
to levels of pH from 6.5
Number of survivors
Test 2
10
10
10
10
10
10
10
2
0
0
0
0
to 1.0 at
Test 3
10
10
10
10
10
10
10
2
0
0
0
0
149
-------
Table 6. Number of survivors of Pteronarcys proteus following
pH
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
96-hour exposures
11 to 14 C.
Test 1
10
10
10
10
10
10
10
10
0
0
0
0
to levels of pH from 6.5
Number of survivors
Test 2
10
10
10
10
10
10
10
10
0
0
0
0
to 1.0 at
Test 3
10
10
10
10
10
10
10
10
0
0
0
0
150
-------
Table 7. Number of survivors of Boyeria vinosa following 96-hour
exposures to levels
of pH from 6.5 to 1.
.0 at 11 to 14 C.
Number of survivors
PH
6.5
6.0
5.5
5.0
i
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Test 1
10
10
10
10
10
10
10
10
10
0
0
0
Test 2
10
10
10
10
10
10
10
10
10
0
0
0
Test 3
10
10
10
10
10
10
10
10
10
0
0
0
151
-------
Table 8. Number of survivors of Nigronia fasciata following 96-hour
exposures to levels of pH from 6.5 to 1.0 at 11 to 14 C.
PH
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
Test 1
10
10
10
10
10
10
10
10
10
9
2
0
Number of survivors
Test 2
10
10
10
10
10
10
10
10
10
10
2
0
Test 3
10
10
10
10
10
10
10
10
10
10
1
0
152
-------
Table 9. Median tolerance limits of five aquatic insect species
to low pH.
Species
Stenonema sp.
Acroneuria lycorias
Pteronarcys proteus
Boyeria vinosa
Nigronia fasciata
Individual Mean 96-hour TLm
96-hour TLm and standard error
3.35
3.30 3.31 ± .02
3.28
3.10
3.20 3.16 ± .03
3.20
2.75
2.75 2.75
2.75
2.25
2.25 2.25
2.25
1.75
1.70 1.72 ± .02
1.71
153
-------
SECTION VI
ACKNOWLEDGMENTS
Valuable criticisms of this manuscript were provided by
Drs. Edwin L. Cooper, H. Clark Dalton and W. C. Hymer.
155
-------
SECTION VII
REFERENCES
1. American Public Health Association, American Water Works Association,
and Water Pollution Control Federation. Standard Methods for
the Examination of Water and Wastewater. 12th ed. Amer. Pub.
Health Assoc. Inc., New York. 769 p. (1965).
2. Barnes, H. L. and S. B. Romberger. Chemical aspects of acid
mine drainage. J. Water Poll. Control Fed. 40: 371-384 (1968).
3. Bell, H. L. and A. V. Nebeker. Preliminary studies on the tolerance
of aquatic insects to low pH. J. Kans. Entomol. Soc. 42: 230-236
(1969).
4. Bick, G. H., L. E. Hornuff and E. N. Lambremont. An ecological
reconnaissance of a naturally acid stream in southern Louisiana.
J. Tenn. Acad. Sci. 28: 221-231 (1953).
5. Burke, W. D. and D. E. Ferguson. A simplified flow-through
apparatus for maintaining fixed concentrations of toxicants in
water. Trans. Amer. Fish. Soc. 97: 498-501 (1968).
6. Burks, B. D. The mayflies or Ephemeroptera of Illinois. Bull.
111. Nat. Hist. Surv. 26: 1-216 (1953).
7. Claassen, P. W. Plecoptera Nymphs of North America North of
Mexico. Charles C. Thomas Pub., Springfield, 111., and Baltimore
Md. 199 p. (1931).
8. Doudoroff, P. (Chairman), B. G. Anderson, G. E. Burdick,
P. S. Galtsoff, W. B. Hart, R. Patrick, E. R. Strong, E. W. Surber,
and W. M. Van Horn. Bio-assay methods for the evaluation of acute
toxicity of industrial wastes to fish. Sew. and Ind. Wastes
23: 1380-1397 (1951).
9. Doudoroff, P. and M. Katz. Critical review on the toxicity of
industrial wastes and their components to fish. I. Alkalies,
acids, and inorganic gases". Sew. and Ind. Wastes 22: 1432-1458
(1950).
10. Edney, E. B. The Water Relations of Terrestrial Arthropods.
Cambridge Univ. Press. 109 p. (1957).
11. Green, J. A Biology of Crustacea. Quadrangle Books, Inc.,
Chicago. 180 p. (1961).
157
-------
12. Parsons, J. D. The effects of acid strip-mine drainage on the
ecology of a stream. Arch. Hydrobiol. 65: 25-50 (1968).
13. Wilhm, J. L. and T. C. Dorris. Biological parameters for
water quality criteria. BioScience 18: 477-481 (1968).
158
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V
2
Subject Field & Group
05C
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Pennsylvania State University, Department of Biology and Cooperative Fishery
Unit, University Park, Pennsylvania 16802
Title
Fish and Food Organisms in Acid Mine Waters of Pennsylvania.
Butler, Robert L.
Cooper, Edwin L.
Hales, Donald C.
Wagner, Charles C.
Kimmel, William G.
Crawford, J. Kent
16 I Pt°ioot Dett&atlon
EPA Project #18050 DOG
Note
Citation
Environmental Protection Agency report
number, EPA-R3-T3-032j February 1973.
Descriptors (Starred First)
*Water Pollution Effects, *Acid Mine Drainage, *Bloassay, Water Quality
Identifiers (Starred First)
*Fish, Behavior, Distribution, *Invertebrates, Acidity, Bioassay
Abstract
Objectives were: 1) develop a rapid and non-lethal bioassay for acid water
using changes in utilization of cover and activity of fish, 2) determine the effect
of different levels of acid mine drainage on the presence or absence of fish
populations in the watersheds of Pennsylvania, 3) determine the median tolerance
limits to low levels of pH of five aquatic insects chosen on the basis of their wide
occurrence and common association in soft-water streams. Analysis of variance
revealed there was no relationship between cover utilization and pH levels or between
activity and pH levels for four species of fish (smallmouth bass, longnose dace, rock
bass and brook trout.
In part II of the project it was found that common fish species normally distri-
buted over several watersheds were absent where there was severe acid mine drainage.
Of the 116 species of fishes found 10 species exhibited some tolerance to acid mine
drainage (values of pH 5.5 or less).
In part III all aquatic species survived exposure for four days to pH levels
from 6.5 to 4.0. The 96-hour TLm values ranged from 3.31 for the most sensitive
animal, Stenonema sp., to 1.72 for the most tolerant insect, Nigronia fasciata.
actor
JO?
J:c
Robert
(REV. JULY
L.
Butler
Institution
1969) SEND.
WITH
The
COPY OF
Pennsvlvani
DOCUMENT. TO:
a State
TTn -i -neiyc-t 1-n _-.
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON. O. C. 2O240
GPOt t»7O - 407 -«91
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