PB81-150997
Effects of Fluctuating, Sublethal
Applications of Heavy Metal Solutions
Upon the Gill Ventilation Response of
Bluegills (Leporais macrochirus)
Virginia Polytechnic Inst, and State Univ.
Blacksburg
Prepared for
Environmental Research Lab.-Duluth
Cincinnati, OH
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
NTIS
-------
EPA-600/3-81-003
January 1981
PP81-15CQP7
EFFECTS OF FLUCTUATING, SUBLETHAL APPLICATIONS
OF HEAVY METAL SOLUTIONS UPON THE GILL VENTILATION
RESPONSE OF BLUEGILLS (Lepomis macrochirus)
by
John Cairns, Jr.
Kenneth W. Thompson
Albert C. Hendricks
Biology Department and
University Center for Environmental Studies
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061
R 80 52740 10
Project Officer
William B. Horning, II
Newtown Fish Toxicology Station
Cincinnati, Ohio 45244
0V
NATIONAL TECHNICAL
INFORMATION SERVICE
OS. DEPARTED! OF COBSEBCE
itio, va 22161
-------
NOTICE Vr
THIS DO C UMi'NT HAS B E'EN RE PROD U C ED
F;R.OM THE BE;ST" COP? FURNISHED us BY
THE; sPON s;oRI NG A;G E-N-G^, "; A tTTOuGH'"IT•'_';
is RECOGNIZED 'THAT CERTAIN PORTIONS
ARE ILLEGIBLE, IT IS BEING RELEASED
IN THE INTEREST O-F/MAKING"AVAILABLE
AS MUCH INFORMATION AS. POSSIBLE.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-81-003
3. RECIPIENT'S ACCESSION NO.
L
4. TITLE AND SUBTITLE
Effects of Fluctuating, Sublethal Applications of Heavy
Metal Solutions upon the Gill Ventilation Response of
Bluegills (Lepomis macrochirus)
5. REPORT DATE
January 1981 Issuing Date.
8. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. Cairns, Jr., K. W. Thompson, and A. C. Hendricks
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Biology Department and University Center for
Environmental Studies
Virginia Polytechnica Ins. and State University
Blacksburg, Virginia 24061
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
R805274010
12. SPONSORING AGENCY NAME AND ADDRESS
U.So Environmental Protection Agency
Newtown Fish Toxicology Station
3411 Church Street
Cincinnati,, Ohio 45244
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
EPA-600/03 •'••
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The ventilatory response of the bluegill to fluctuating, sublethal amounts of heavy- «
metals was investigated. Non-contact submerged, stainless steel electrodes were
used to detect the weak electrical potentials that are produced when fish ventilate '•'
their gills. These signals were amplified using high-gain amplifiers which were
interfaced with a minicomputer. These ventilatory data were accumulated continuously,
and both the ventilatory rates and average signal amplitudes were recorded on
electronic tape for later analysis.
The response to these toxic solutions was an increase in rate as well as a decrease
in signal amplitude. The latter response was shown to be a real response of the fish
and not due to changes in the electrical properties of the water when toxicant was
added. Due to the extreme smoothing of the data, the analysis of variance was unable
to detect any differences between the toxicant application patterns. It was signi- -
ficant to note, however, that the fish were capable of reacting to each subsequent
pulse of toxicant in a similar manner.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF.PAGES
20. SECURITY CLASS (This page)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (Rov. 4-77)
PREVIOUS EDITION IS OBSOLETE
-------
DISCLAIMER
This report has been reviewed by the Environmental Research
Laboratory-Duluth, U. S. Environmental Protection Agency, and
approved for publication. Approval does not signify that the .•
contents necessarily reflect the views and policies of the U. S.
Environmental Protection ,Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation
for use. . ...
ii
-------
FOREWARD
The Federal Water Pollution Control Amendments of 1972, the
1977 Clean Water Act, and the Toxic Substances Act all require
control of the discharge of toxic substances. A reasonable data
base exists on toxicity of known chemical compounds to aquatic
organisms. However, information is limited concerning the toxicity
or potential hazard of complex wastes that contain numerous
chemicals in varying concentrations and combinations. Because
of the increasing number and complexity of chemical compounds
being manufactured and the fact that aquatic organisms are not .....
usually exposed to individual chemicals, but rather to mixtures
of several or more, it is obvious that we cannot control toxic
discharges on a chemical-by-chemical basis. Thus, a rapid "'•"••..
biological test method is needed by regulatory agencies and dis-
chargers to evaluate the potential hazards of complex and variable
wastes prior to discharge into receiving waters. The biological
system must integrate the effects of these complex mixtures over-
time.
This report provides information concerning the acute
toxicity of several known heavy metals as well as mixtures of these
toxicants. The report also provides information regarding the
use of fish respiratory impairment as a measure of toxicant stress
when subjected to sublethal fluctuating concentrations of indi-
vidual heavy metals and mixtures of these toxicants.
William B. Horning
Chief
Newtown Fish Toxicology Station
Cincinnati, Ohio
iii
-------
PREFACE
The original proposal, forwarded January 28, 1977, listed
John Cairns, Jr. as the principal investigator. Kenneth L.
Dickson and W. H. van der Schalie were to be- staff members on
the project. Van der Schalie left before the project was funded
and was replaced by Kenneth W. Thompson when the project was
funded. Dickson's responsibilities were taken over by Albert C.
Hendricks in June, 1978.
IV
-------
ABSTRACT
The ventilatory response of the bluegill to fluctuating,
sublethal amounts of heavy metals was investigated. Non-contact,
submerged, stainless steel electrodes were used to detect the
weak electrical potentials that are produced when fish ventilate
their gills. These signals were amplified using high-gain
amplifiers which were interfaced with a minicomputer. These
ventilatory data were accumulated continuously, and both the
ventilatory rates and average signal amplitudes were recorded on
electronic tape for later analysis.
Acute toxicity of each of the heavy metal solutions used
was determined as well as the various mixtures used. The com.......
pounds used and the LCSOs determined (in parentheses) were ZnS04
(3.2 mg/1), CuCl2 (1.0 mg/1), NiCl2 (21.2 mg/1), and K2Cr04 *
(132.9 mg/1). The toxicity of zinc-copper and zinc-copper-nickel-
chromium mixtures was found to be additive under the test condi-
tions.
Use of conventional parametric procedures is inappropriate
for analysis of ventilatory data from an individual fish as
successive observations are not independent. This report deter-
mined that such analyses are appropriate when comparing data
between individuals.
The ventilatory behavior of bluegills (Lepomis macrochirus)
receiving sublethal exposures of heavy metal solutions was com-
pared to unexposed controls. The toxicant solutions were
delivered in short, intermittent 5-hour pulses, longer 20-hour
pulses, and conventional 96-hour applications. .
The response to these toxic solutions was an increase in
rate as well as a decrease in signal amplitude. The latter
response was shown to be a real response of the fish and not due
to changes in the electrical properties of the water when toxicant
was added. Due to the extreme smoothing of the data, the analysis
of variance was unable to detect any differences between the
toxicant application patterns. It was significant to note,
however, that the fish were capable of reacting to each subse-
quent pulse of toxicant in a similar manner.
... 'Both ventilatory rate and ventilatory amplitude, especially
when used in conjunction, can be powerful tools for the continuous
monitoring of surface water quality.
-------
This report was submitted in fulfillment of Contract No.
R 80 52 740 10 by the University Center for Environmental Studies
under the sponsorship of the U. S. Environmental Protection
Agency. This report covers the period July 11, 1977 to April 11,
1980.
vi
-------
CONTENTS
Foreward iii
Abstract v
Figures viii
Tables xi
Acknowledgment xii
1. Introduction 1
2. Conclusions 5
3. Materials and Methods 6
Test specimens 6
Toxicity tests 6
Water characteristics 9
Ventilatory studies 12
4. Results and Discussion 32
Toxicity tests 32
Ventilatory studies 34
References 44
Appendices
A. Results of Water Analyses and Individual Toxicity
Tests 51
B. Graphic representation of the average Ventilatory
rates for all 12 Ventilatory tests 56
C. Graphic representation, of the average signal
amplitude for all 12 ventilatory tests ...... 69
D. Examples of stressed and unstressed inter- and
intra-individual variation as illustrated by strip
• chart recordings 82
vii
-------
FIGURES
Number
1 A bluegill in a ventilatory sensing chamber.
Water flows into the receiving chamber (A),
through the sensing chamber (B), and out the
drain (C). The electrodes are the S-shaped
wires at either end of the sensing chamber.
The signal is carried to an amplifier (AM) .
2 An example of the amplified ventilatory
signal of a bluegill ventilating at
approximatly 20 times per minute ......
The solenoid controlled, continuous flow,
proportional diluter. Th'e:' time delay! relay
simultaneously opens ,the.v:valves on the head
boxes and closes those on.-the proportioning
boxes allowing the proportioning boxes to
fill with diluent and toxicant solutions.
When the level of the liquid in the diluent
proportioning box reaches the top in
compartment 5, a float valve is tripped and
the solenoid valves are returned to their
normal state,, delivering'toxicant and
diluent first to the mixing chambers and
ultimately to the fish chambers. This also
resets the time delay relay, and the cycle.
is repeated . . . . . . . . . ...... ... .
An individual "ventilatory sensing chamber.
The dimensions of the fish holding chamber
are: length, 12 icm; width,-4 cm; height,
10 cm; and\water depth, 6 cm 13
Cutaway view of the plywood isolation
chamber to show the composition ventilatory
sensing chambers 13
The water delivery and data accumulation
system 15
The rate of increase of the concentration
in the ventilatory sensing chambers 16
viii
-------
Number
8 The rate of decrease of the concentration
in the ventilatory sensing chambers 16
9 Strip chart recording of the ventilatory
signal of a bluegill. A. The ventilatory
signal as taken directly from the amplifier.
B. The computer produced derivitive of
the signal in A. The very sharp negative
peaks indicate that a count has been
recorded 19
10 Strip chart recording of the venilatory
signal of a bluegill. A. The ventilatory
signal as taken directly from the amplifier.
Note the increased bimodality with respect
to Figure 9. B. The computer produced
derivitive of the signal in A 20
11 The average ventilatory rate of a bluegill.
The arrows indicate points at which the
DC voltage drift caused a loss of signal.
Replacing the electrodes and terminal
barrier strips eliminated this problem 22
12 Toxicant pattern 1. Toxicant was delivered
to fish 7 and fish 14 for 96 hr. The
shaded area on the fourth vertical plane
indicates the shape,, duration, and concentra-
tion of the application of the application
of the toxicant. Fish 9 was a control and
received no toxicant. Interval 2 = 0 - 96 hr
(X axis); Interval 3 = 96 - 192 hr; Interval
4 = 192 - 216 hr. Data were not recorded
during Interval 1 which was the 24 hr prior
to hour 0 (See text for explanation 25
13 Toxicant pattern 2. Toxicant was delivered
to fish 14 and fish 13. The shaded area on
the fourth vertical plane indicates the shape,
duration, and concentration of the toxicant
applications. Each application lasted for
20 hr; each increment was 4 hr. Interval 2 =
0 - 96 hr (X axis); Interval 3 = 96 - 156 hr;
Interval 4 = 156 - 216 hr. Data were not
recorded during Interval 1 which was the 24 hr
prior to hour 0 (see text for explanation) .... 26
IX
-------
Number Page
14 Toxicant pattern 3. Toxicant was delivered
to fish 16 and fish 8. The shaded area on
the fourth vertical plane indicates the
shape, duration, and concentration of the
toxicant applications. Each application
lasted for 5 hr, each increment was 1 hr.
Interval 2=0-4= 171 - 216 hr. Data
were not recorded during Interval 1 which
was the 24 hr prior to hour 0 (see text
for explanation) ..... 27
15 Average ventilatory rate for a bluegill
during Intervall 1 and the first three
days of Interval 2. The fish was placed
in the sensing chamber during the first
hour 27
: x
-------
TABLES
Number Page
1 Concentrations of heavy metals used in
ventilatory studies 24
2 Example of partitioning of ventilatory
rate data for analysis 31
3 Results of combined toxicity tests 33
4 Results of chi squared tests for goodness
of fit to the normal distribution 38
5 Cochran's C Statistic 39
6 Results of one-way ANOVA 40
7 Results of Duncan's Multiple Range test at
the 95 percent level of significance 41
APPENDIX A
A-l Results of chemical analyses given as the
average of the five analyses for each fish
bioassay chamber . 51
A-2 Toxicant concentrations and survival during
toxicity tests 54
XI
-------
ACKNOWLEDGMENTS
This study would not have been possible without the
cooperation and assistance of the following people: J. H. Alls,
D. D. Donald, B. F. Higginbotham, D. Hooley, J. D. Landers, Jr.,
G. L. Nunn, W. J. Showalter, and V. A. Wagner.
xii
-------
SECTION 1
INTRODUCTION
The increasing volume and complexity of industrial, agricul-
tural , and other wastewater effluents in the modern world make
imperative the development of rapid methods for early detection of
developing-toxic situations. Use of a living organism as a -:
generalized monitor capable of responding to the entire toxicant
load of its environment has been suggested by numerous authors
(see Cairns and van der Schalie, in press, for a review). A
primary requirement for such a living monitor would be a detectable
.response to sublethal conditions as a warning of the developing •.
lethal situation. Many physiological and behavioral parameters
might be used as indicators of stress situations. One that shows
considerable promise and has received attention by many workers
is the rate at which a fish passes water across its gills during
"breathing" activity (ventilation).
The ventilatory rates of fishes have been used as an indica-
tor of stress for many years, and numerous methods have been
employed for observing the ventilatory process. The oldest and
simplest method is visual counting of the opercular movements, a
method that has proven useful for short-term studies for many
years (Belding, 1929; Meuwis and Heuts, 1957; Chizar et al.,
1972; Gee et al., 1977; Sorensen and Burkett, 1978).
Many workers have measured ventilatory rates with surgically
attached or implanted sensors of various types (Saunders, 1961;
Pasztor and Kleerekoper, 1962; Schaumburg et al., 1967; Cairns
et al., 1970; Davis, 1972; Sparks et al., 1972; Young, 1971;
Hughs, 1975; Lunn et al., 1975; Sellers et al., 1975; Barnett
and Toews, 1978). These attachments may contribute to the stress
of the fish, and the necessity of making these attachments,
complicated or not, to each individual specimen precludes the use
of such methods for gathering large, long-term data sets from
large numbers of individuals and species. Such data,sets are an
absolute necessity if any validity is to be attached to pre-
dictions made with such information.
The most recently developed method for determining the
ventilatory rates of fishes is also the one that lends itself
best to the accumulation of a large data base from numerous
individuals and species. This method detects, by submerged,
noncontact electrodes in aquaria, the weak electrical signals
-------
produced by the movement of the muscles in the branchial region
as a fish ventilates the gills during "breathing" activity
(Figures 1 and 2).
Possibly the earliest workers to use such noncontact sensing
were Kleerekoper and Sibakin (1956) who investigated the weak
electrical signals produced by the lamprey; however, their subject
was physically restrained in a plastic tube. Camougis (I960)
used noncontact electrodes to sense the bioelectrical potentials
of fish and other aquatic animals that were unrestrained in an
aquarium, and thus presumably, were less stressed than when
restrained. Probably the earliest use of this type of apparatus
to describe the response of freshwater fish to environmental
variables was that of Roberts (1964) who studied the effects of
temperature and photoperiod on Lepomis gibbosus. This type of
apparatus has been used more recently to investigate ventilatory
response to various environmental contaminants (Spoor et al.,
1971; Drummond et al., 1973; Drummond et al., 1974; Thomas and
Rice, 1975; Drummond and Carlson, 1977; Carlson and Drummond,
1978; Maki, 1979; Thomas and Rice, 1979). While some of these
investigators reported the effects on ventilatory rates, the
majority emphasized the cough or "gill purge" frequencies with
little emphasis on actual ventilatory rates; or'their changes.
Most noncontact studies., .mentioned thus far'have ..relied -on the
analysis of short intermittent segments of strip chart recordings
which leave a large amount of the ventilatory behavior undescribed.
Recent technological advances in.the field of electronics
have made relatively low-cost, dependable minicpmputers, micro-
processors, or other electronic recording devices economically
feasible for laboratory use. These devices are easily adaptable
for the continuous accumulation of fish ventilatory data and make
possible the establishment of the*large'data bases necessary for
statistical predictions and valid long-term environmental moni-
toring. Results from such systems have, been reported by Morgan
and Kuhn (1974), Morgan (1977) y-Westlake and van der Schalie
C1977), Thompson et al. (1978), Morgan et al. (1979), van der
Schalie et al. (1979), and Cairns and Thompson (in press).
Most toxicity data are based on conventional tests in which
constant toxicant concentrations are maintained and in which the
time to death of the test organism^is; the measured parameter.
However, fluctuating conditions are the rule in the "real world"
and constancy the exception. In order to develop a living,
continually sensing, water quality monitor, controlled "real
world" conditions must be simulated in the laboratory for deter-
mining predictable stress responses. The non-stressed behavior
of the test organism must first be described for comparison.
The present study was instigated to investigate and describe
the ventilatory response of the bluegill (Lepomis macrochirus)
to simulated "real world" conditions (that is, to fluctuating,
-------
Figure 1. A bluegill in a ventilatory sensing chamber. Water
flows into the receiving chamber (A), through the sensing chamber
(B), and out the drain (C). The electrodes are the S-shaped
wires at either end of the sensing chamber. The signal is carried
to an amplifier (AM).
5
10
SECONDS
Figure 2. An example of the amplified ventilatory signal of a
bluegill ventilating at approximately 20 times per minute.
-------
complex, sublethal toxicant concentrations) and to accumulate a
large enough data base to allow valid statistical characteriza-
tion and analyses. The..toxicants used..are all common components
of industrial effluents. Standard" 96-h'r-median lethal concentra-
tions under conditions in this laboratory were established for
all the toxicants used in order to relate to conventional toxicity
data and determine the concentrations to be used in the ventila-
tory studies.
-------
SECTION 2
CONCLUSIONS
1. Ninety-six hour median lethal concentrations for divalent
zinc, copper, and nickel, and for hexavalent chromium were
determined to be 3.2, 1.0, 21.2, and 132.9 mg/1, respectively.
2. At the concentrations tested, the toxicities of mixtures of ........
zinc and copper and of zinc, copper, nickel, and chromium
. were determined to be additive.
3. The ventilatory response of the bluegill (Lepomis macrochirus)
was shown to be measurably affected by sublethal concentra-..
tions of zinc, copper, and the complex mixtures indicated
above. This effect can be used as an indication of stress
in an automated biological monitor.
4. Ventilatory data meet the requirements for parametric
statistical analyses when comparisons are between fish and,
thus, when sample size is large enough, can be analyzed by
. conventional methods.
5. The bluegill displays two sensitive ventilatory responses to
sublethal amounts of heavy metals: the ventilatory rate
increases and the amplitude of the ventilatory signal
decreases. Both of these changes are statistically detectable
and are reversible on removal of the toxicant within the
limits of this study.
6. Under the conditions of these tests, the addition of the
heavy metal compounds could account for only a small portion
of the reduction in signal amplitude indicating that the
observed reduction was primarily due to a real change in the
ventilatory behavior of the fish.
7. No statistical differences could be demonstrated in the
average ventilatory response with respect to the pattern in
which the toxicant was applied. However, graphic results
indicated that the fish tend to respond to each successive
toxicant application in a similar manner, even when only 5
hours recovery time was allowed.
5
-------
SECTION 3
MATERIALS AND METHODS
TEST SPECIMENS
The test organism was the bluegill, Lepomis macrpchirus,.
All specimens used during this study were obtained from Osage
Catfisheries, Inc. of Osage Beach, MO and were maintained in
450-& continuous flow aquaria. The light regimen was continuous
light, and the temperature was maintained at a constant 22.5+l°C.
These conditions were maintained for both acute toxicity.and
ventilatory studies. . . .
Acclimation and Health
The fish were acclimated to these holding conditions 'for at
least 2 weeks before being used for tbxi city..,test's and for 4 .to
6 weeks, and often as long as six months or more, before being
used for ventilatory studies. ,
All fish were free of any visible disease-(mortality
-------
Proportional Diluter
A solenoid valve controlled proportional diluter system
similar to that described by Hendricks et al. (1977) and Peltier
(1978) was used for the toxicity tests. In this system, six
fish holding chambers of 6.4 i capacity received 233 ml of solu-
tion every 3 min providing a flow rate of 4.66 I per hour
(Figure 3). This rate was equivalent to 17.5 chamber volumes
during each 24-hr period.
The head boxes, proportioning boxes, mixing chambers, and
fish holding chambers were constructed of 0.64 cm plexiglas with
methylene chloride fused joints. Delivery tubes and standpipes
were of teflon, and fittings were of glass. The toxicant
reservoir was polyethylene (Figure 3). The diluter was scrubbed...
out and then flushed with a solution of ethylenediamine tetraacetic
acid (EDTA) between toxicity tests.
The toxicant solutions were delivered to the mixing head box
by a variable speed peristaltic pump. The concentration of
toxicant in the fish chambers could be changed between tests by
one or a combination of the following four methods: the concen-
tration of the toxicant in the toxicant reservoir could be
changed, the flow rates of the diluent water or of the toxicant
solution could be changed, or the percentage of toxicant could be
adjusted. The latter could be accomplished by changing the
height of the adjustable standpipes in each compartment of the
proportioning boxes (Figure 3).
Protocol for Toxicity Tests
Ten fish were placed in each of the fish chambers at the
start of each toxicity test. The number of individuals surviving
in each chamber was recorded after 1, 2, 4, 8, 12, and 24 hr and
every 24 hr thereafter. All dead fish were removed at each
observation. Death was defined as the loss of equilibrium. Fish
that could not respond to prodding with a blunt rod by righting
themselves were considered dead.
Probit Analysis
Probit analysis as described by Finney (1971) was performed
to determine the 96-hr median lethal concentration (LC50) values
and their 95 percent confidence limits. A computer program
available on SAS79 was used to carry out this analysis (SAS
Institute, 1979).
Two toxicity tests were run with each toxicant solution.
Toxicant concentrations were adjusted slightly when necessary
for the second test to allow for better resolution at concentra-
tions near the LC50 since probit analyses are more meaningful
-------
OECHLORINATED
TAP WATER 22*C
HEAD BOXES
'SUSS
TOXICANT
OEUVERY
PUMP
MIXING CHAMBERS
FISH
CHAMBERS
i 22 3m 4m sn i
nOKOUENT WTOMCANT BKTOXCANT 3TBn»X»NT5BCTOqC4NT OOXTOOCM4T
TOXICANT RESEHVOR
Figure,3. The solenoid controlled, continuous flow, proportional
diluter. The time delay relay simultaneously opens the valves on
the head boxes and closes those on the proportioning boxes allowing
the proportioning boxes to fill with diluent and toxicant solutions.
When the level of .the liquid i-n the diluent proportioning box
reaches the'"tbp in compartment 5, a float. yaJ.ve is tripped and
the solenoid valves are returned to their normal state, delivering ..
toxicant and diluent first to the mixing chambers and ultimately
to the fish chambers. This also resets the time delay relay,
and the cycle is repeated.
-------
with mortalities other than 100 percent or 0 percent. The results
of successive tests with the same toxicants were in fairly close
agreement, and the results of tests using the same toxicant were
pooled to increase sample size. The probit analysis was run on
the pooled data.
Joint Toxicity
Since organisms may be more sensitive to some toxicants than
to others, a method of equating the toxicity of the various com-
ponents of a mixture was needed. Sprague (1973) recommended that
the concentration of a toxicant in a mixture be reported as a
proportion of the LC50 as determined singly. In this method, the
sum..of -the ratios for all components of a toxicant mixture is
determined. If this total is 1.0, the toxicity of the mixture is '
considered to be additive. A total of less than 1.0 is considered
to be synergistic (greater than additive), and a total greater
than 1.0 is considered to represent a mixture with antagonistic
(less than additive) toxicity. Marking (1977) used this concept
as a basis for the development of a linear additive index in which
values of zero indicate additive toxicity, positive values indicate
synergistic toxicity, and negative values indicate antagonistic
mixtures. Marking's additive index (MAI) has the advantage of
providing a method for establishing critical limits for values
that are near zero.
MAI can be calculated using the equation:
\ Ami/Ai - S .
where A ./A. = toxic unit, i = the total number of toxic components
in the mixture, A . = LC50 for a toxicant in the mixture, A. = LC50
for that toxicant as determined individually, and S = the sum of
the biological, effects or the sum of the toxic units. Then MAI =
(1/S) - 1.0 if S £ 1.0, and MAI = 1.0-SifS>^1.0.
The significance of the deviation from zero can be determined
by substituting values of the 95 percent confidence limits for
the LC50 values of both A. and A . into the formula for MAI to
establish a range for MAI. The values that yield the greatest
deviation from MAI are used to establish the range. If the range
includes zero, the deviation is not considered to be'significant.
WATER CHARACTERISTICS
Dechlorinated tap' water from a single source was used as
diluent water for all toxicity tests and ventilatory studies and
to supply the holding tanks.
-------
Temperature
Water.was maintained .at.a.constant, temperature with a Therma-
treck 34-E temperature controller. A malfunction of the tempera-
ture controller during the first toxicity test using copper
resulted in a temperature of 19 C +_ 2.5 C. In all other toxicity
tests and all ventilatory studies, the temperature was maintained
at 22.5°C +_ 1°C. A Tempscribe remote bulb temperature recorder
was used to monitor the temperature of the diluent water con-
tinuously. Additionally, daily temperature measurements were
taken from each fish chamber of the proportional diluter when
toxicity tests were being run.
Water. Chemistry
Dissolved oxygen, conductivity, pH, alkalinity, and total
hardness were measured in each of tjie six fish chambers at the
beginning of every toxicant test and once -during each subsequent
24-hr period for the duration of a test... Consequently, five
measurements were taken from each chamber during ;the tests. Since
the diluent water for both toxicity testing and ventilatory
studies 'was from the. same source, chemical data.::were not measured
for the ventilatory chambers; The chemical values of the diluent
water ic the ventilatory ^studies. Is assumed•.•• to be the same as
those measured from chamber 1 (control) of the toxicity tests.
Dissolved oxygen was measured with a YSI Model 54 A oxygen
meter. Conductivity in micromhos/em was. measured with .a Bepkman
Model SM-210 conductivity meter modified to read in the 0 - 500
micromho range. When concentrations of toxicant .were high enough
to cause values greater than 500, the samples were diluted with
distilled water (conductivity = 0 micromho/cm). A Corning Model
7 pH meter was used to determine the pH. Alkalinity and total
hardness were determined tltrimetrically according to methods
described by American Publi.e..J3ea.lth Ass.pciatlon (APHA) et al.
(1976). Total-'hardness could hot be detefml'he'd in thi;sv.manner
when nickel was present and was determined instead by measuring
the calcium.and magnesium/concentrations"by atomic absorption
spectrophotometry and calculating total hardness by;the methods
given by APHA et al.- (1976). - . ' "... "' r .
' • The dissolved oxygen concentration was.maintained kt'Tevels
above 6.6 mg/& in all fish chambers throughout the toxicity ' -.
testing. Dissolved oxygen during ventilatory studies was measured
only at the end of a test and averaged 7.7 mg/£.
Conductivity of the diluent water ranged from 100 to 145
micromhos/cm, pH from 6.7 to 7.6, alkalinity from 22 to 42 mg/Z,
and total hardness from 16 to 82 mg/Jl. High concentrations of
toxicants, especially of hexavalent chromium caused the con-
ductivity to go as high as 3352 micromhos/cm, the pH to reach 8.3,
and the alkalinity to reach 564 mg/fc. The average values for
10
-------
each fish chamber during each toxicity test are given in Appendix
A (Table A-l).
A Wallace and Tiernan Amperometric Titrator was used to
periodically monitor the total chlorine content of the diluent
water. The total chlorine level remained between 0.01 and 0.05
mg/1 throughout the course of these studies.
Toxicant Measurement
Toxicant concentrations, as mg/Jl of the metal ion, were
determined with a Perkin-Elmer model 460 atomic absorption
spectrophotometer. Analyses were done using an air-acetylene
flame except when both nickel and chromium were present. In ........
that case a nitrous oxide-acetylene flame was used for the
determination of the concentration of the hexavalent chromium :
since nickel blocks the detection of chromium. Water samples
for determination of the concentration of the toxicant were
acidified with concentrated nitric acid, sealed with parafilm, ..:,.-•
and held until the end of a test before being analyzed.
During toxicity testing, five water samples were taken as
indicated for water chemistry. During ventilatory studies, water
samples were collected below the "drain without disturbing the
fish. These samples were taken when appropriate depending on the
toxicant application pattern. This was arranged so at least four
samples were taken at each toxicant level, and at least one sample
before and one after the toxicant was applied. While each sample
was analyzed separately the average concentration for each fish
chamber or ventilatory sensing chamber was used for final analyses.
Toxicant Solutions
The toxicant sources were reagent grade zinc sulfate (ZnSCK-
7H20), cupric chloride (CuCl2-2H20), nickelous chloride (NiCl2-
6H20), and potassium chromate (K2Cr04). To provide divalent zinc,
copper, nickel, and hexavalent chromium these compounds were
dissolved in distilled water. When chromium was one of the metals
in a toxicity test, an airstone was placed in the mixing head box
of the proportional diluter to facilitate mixing. The chromium
solution was so concentrated that a density gradient between the
diluent water and the chromium solution formed and resulted in a
very concentrated layer of toxicant solution in the bottom of the
mixing box. However, no precipitate was observed in'the mixing
head box.
When determining the toxicity of complex solutions, it is
desirable to control the ratios of the toxic strength of the
various toxicants so that one component is not the major contri-
butor of toxicity. Tsai and McKee (1980) defined the toxicity
ratio in the following manner:
11
-------
Toxicity ratio = *mI/\ : &m2/A2 "" Ami/A2 (2)
where the definitions of the terms are the same as for equation (1)
Due to interactions between the dissociation products of the
various chemical compounds and between these products and the
diluent water which resulted in various precipitates, separate
solutions of each compounds for use in joint toxicity tests were
prepared. Each was then pumped separately into the toxicant head
box where mixing occurred (Figure 3). Because of these complica-
tions, the desired toxicity ratio of 1:1 was achieved only for
MixA (Zn++, Cu++). The toxicity ratio ultimately achieved for
MixB (Zn++, Cu++, Ni++, Cr+6) was approximately 1:1.8:1.7:1.6.
However, since the primary purpose of these toxicity tests was
to indicate concentrations in the sublethal range for use in
ventilatory studies, these results were accepted and no further
toxicity tests were carried out.
VENTILATORY STUDIES .
The electrical action potential of muscle movement in the
branchial region of a fish during ventilatory or: "breathing11
activity produces an electronic signal. -'A system for recording
this signal continuously and-for applying toxicants to the fish
in varying, pre-selected patterns was constructed. • ., ;
Sensing Chambers •'•"'.
A fish ventilatory sensing chamber with a total of 16 :
individual 700 ml capacity monitoring compartments was constructed
of 6.35 mm transparent plexiglas. The ends and dividing baffles
were of 3.18 mm transparent plexiglas, and the sides of each
individual compartment were of 3.18 mm opaque white plexiglas
(Figures 4 and 5). All joints .w.ere fused.wi.th methylene chloride.
Electrodes :.. '' . •-,..,.
The submerged electrodes were of ...type 304 stainless steel
wire and were attached with silicone aquarium cement to t.he.-baffles
at either end of the monitoring compartment. The electrodes were
on the side .of the dividing baffles opposite the fish:to'prevent
contact between fish and electrode; 'Each eleetrode,,was 'formed :.V
in such a way that the wire never passed through the flow of
water from any of the perforations in the dividing baffles
(Figure 4). The electrode wire was insulated from just below the
water line, where the insulation was sealed to the wire with
silicone cement to prevent water intrusion, to the barrier strip
on the outside of the tank. The only exposed wire was below the
water surface and at the barrier strip connections.
Isolation Chamber
Since fish respond to visual stimuli and to vibrations in
12
-------
FISH HOLDING
^ELECTRODES
DRAIN
TO AMPLIFIER
Figure. 4.. An individual ventilatory sensing chamber. The
dimensions of the fish holding chamber are: length, 12 cm; width',
4 cm; height, 10 cm; and water depth, 6 cm.
Figure 5. Cutaway view of the plywood isolation chamber to show
the composite ventilatory sensing chambers.
13
-------
a manner similar to their response to chemical stimuli, the
entire fish holding chamber was enclosed in a plywood isolation
chamber. The supports of this enclosure were anchored in sand
filled containers to protect against vibration (Figures 5 and 6).
Water Delivery
Diluent water was delivered by gravity ,flow to three
plexiglas mixing chambers where the diluent could be mixed with
various toxicant solutions as desired (Figure 6). The water then
was passed into delivery chambers which were also of plexiglas
and equipped with 1 mm diameter standpipes. A constant head
pressure was maintained over these standpipes'by delivery in
excess with the excess drained through an adjustable standpipe.
This allowed delivery of water or water and toxicant at a constant
rate and concentration to each sensing chamber (Figure 6).
Toxicant Delivery
An Intergral Masterflex, variable speed, peristaltic pump
drive with remote control capability-.was used to introduce the
toxicant into the mixing chambers (Figure 6).- -As many as nine
pump heads could be used simultaneously for delivery of different
toxicant solutions tq.: each mixing chamber or for producing
various combinations in the mixing chambers. The toxicant solu-
tions were pumped through 1166 mm inside -diameter silicone tubing.
One of the three mixing chambers was used-'for control and," thus,
never received toxicant solution. Duplicate toxicant reservoirs,
pump heads, mixing chamberst and water delivery chambers (Figure
6) allowed for replicates of the treatments during each ventila-
tory test. Two different treatments during a single test could
have been carried out using this apparatus. Between ventilatory
runs, the system was rinsed with a solution of EDTA. The mini-
computer used a digital to analog converter to deliver a pre-
selected voltage to the pump drive. A computer-pump interface
was used to convert the output voltage from, the computer to the
optimum range for use by the pump. The.computer output voltage
could be changed by the computer at pre-selected 1-hr -intervals
to simulate fluctuating "spills" or releases. The concentration '
of toxicant in the individual sensing-compartments reached
maximum concentration in 15 - 30 minutes and dropped -to backgroud
level, at approximately the same" rate,'depending'on"the .flow, rate
through the compartment (Figures 7 and 8>. ,.,.,.,, :
Amplifiers
An amplifier designed for use with fish ventilatory signals
has been described in detail elsewhere (Gruber et al., 1977).
Sixteen of these high-gain noise immune amplifiers, one for each
of the sensing compartments, were constructed for this project.
The total gain of this amplifier is variable from 29,200 to
321,000 times. Due to individual variability among test subjects,
14
-------
Figure 6. The water delivery and data accumulation system.
15
-------
3.0
~2.5'
o>
J
Z
22.0
5
a:
1.3
8
1.0
0.0
137'ml/miii
108 trt/min.
0 IS 30 45 . ..6O
MINUTES AFTER TOXICANT FLOW.STARTS
Figure 7. The rate of increa.se of the concentration in the
ventilatory sensing chambers. •' ••'
3.On
z
S2.OH
<
i.s-
o
o
-f 1.0-
2o.5-
0.0
•;. .I37ml/min.
O8ml/mia :
0 15 30 45 60
MINUTES AFTER TOXICANT FLOW STOPS
Figure 8. The rate of decrease of the concentration in the
ventilatory sensing chambers.
16
-------
each amplifier was adjusted during the acclimation period to pro-
duce a ventilatory signal in the 4 to 8 volt range; consequently,
comparisons of absolute signal amplitude between fish are rela-
tive.
Design modifications that increase the maximum range of this
unit for use in marine situations have been described recently
by Cairns et al. (1980).
Data Accumulation
Each of the 16 ventilatory monitoring compartments was
connected to an amplifier. These in turn were interfaced to a
PDP-8E minicomputer through a 16-channel multiplexed analog to ...,
digital converter (Figure 6).
Ventilatory counts and an average scaling factor for each •
fish were accumulated for 15-min periods. The scaling factor is
a value from which an approximation of the average signal ampli-
tude can be recovered. - At the end of each of the 15-min periods,
the accumulated data were printed on a Decwriter II computer
terminal. At 30-min intervals, the two preceeding 15-min data
sets were stored on magnetic tape cassettes by a Decassette TU60
tape drive.
At the end of each ventilatory run, the minicomputer was
connected by telephone line to an IBM/370-1580, and data stored
on the cassettes were transcribed onto a Conversational Monitoring
System (CMS; an interactive programming system for the IBM
System/370). Transcription took approximately 1 hr. The mini-
computer could then be reset and another ventilatory study started
as desired.
Computer Program
The ventilatory signal is often bimodal (Thompson, et al.,
1978; Gruber et al., 1979). This bimodality is an artifact of
the amplifier design and is more pronounced at slower rates
(Thompson et al., 1978). The signal has been visually correlated
with the ventilatory activity of L. macrochirus, and each waveform,
bimodal or not, represents one complete ventilatory cycle (Gruber
et al., 1979).
The computer program developed for this project'is capable
of rejecting this artifact when it is present and of accumulating
the total ventilatory counts as well as an approximation of the
average amplitude of the signal. This program also uses a soft-
ware controlled variable threshold to reject low amplitude noise.
Signals with less than 1 volt peak-to-peak amplitude were con-
sidered to be noise.
17
-------
This program used the first derivative of the ventilatory
signal for determining the number of cycles or waveforms in a
reporting period. In each cycle there:.was at least one region
in which a relatively large change in the signal voltage resulted
in a large peak in the derivative signal at the point of maximum
change. A software Schmidt trigger was used to reduce the effect
of noise on the signal. This was done by establishing symmetrical
positive and negative thresholds for the derivative signal. When
the derivative signal exceeded the negative threshold, the count
logic was cleared. When a following positive signal exceeded the
positive threshold, the logic was clocked and a count registered.
Consecutive pulses of the same polarity did not produce counts
unless there was a pulse of opposite polarity occurring between
them. Examples of the computer produced derivative signal are
shown in Figures 9 and 10. The sharp negative pulse indicates
that a count has been registered and further counts will be
inhibited until the logic is reset by a negative pulse.
The similarity in shape of the electronic artifact to some
types of gill purge signals (van der Schalie.,.,:1980) precludes the
use of this program for detecting that response. Other types of
gill purges appear a,s: .sigrialis,'^pf.. .higher amplitude than the
associated ventilatory signals. These, as well as any body move-
ments that produce signals^ meeting the above criteria, would be
included as ventilatory. signals-. .
The amplitude of the input signal was variable over time and
between individual fish which limited the usefulness of the fixed
thresholds. To alleviate this problem, an automatic gain control
was used that kept track of the amplitude of the incoming signal
for 2 sec and adjusted the scaling factor for each succeeding
signal either up or down to result in a predetermined peak signal
level. A maximum scaling factor'prevented signals with amplitudes
below a specified level (1 volt peak-to-peak) from being amplified
beyond the threshold and,.thus,..producing false counts. This was,
in effect, a variable threshold which allowed a more accurate
interpretation, of the ventilatory events than was possible with
earlier biomonitoring programs that used'fixed thresholds with no
automatic gain control (Cairns et al. ,' 1977; van der Schalie,
1977). With those programs, large reductions in amplitude would
have registered as a reduction.In rate. With the present program,
real ventilatory--signaTs with amplitudes of less than 1 volt peak-
,to-peak would also be ignored, and a possible false redaction in '
rate would ;be;indicated.
The length of the time period during which the amplitude of
the signal was retained was variable and was determined by the
operator at the start of an experiment. This time period should
be similar to the period of the wave form being observed. If it
is too short, the smaller peaks may be amplified to the maximum
and result in false counts. If too long, a real cycle of low
amplitude would be lost if a very large peak immediately preceeded
18
-------
-i—r
—r-
is
Seconds
Figure 9. Strip chart recording of the ventilatory signal of a
bluegill. A. The ventilatory signal as taken directly from the
amplifier. B. The computer produced derivitive of 'the signal
in A. The very sharp negative peaks indicate that a count has
been recorded.
19
-------
10
Seconds
Figure 10. Strip chart recording of the ventilatory signal of a
bluegill. A. The ventilatory signal as taken directly from
amplifier. Note the increased bimodality with respect to Figure
9. B. The computer produced derivitive of the signal in A.
20
-------
it. Two seconds worked well for bluegills and was the value used
for all of the ventilatory studies reported here.
This program accumulates data with low error rates when in
use with input signals in the optimum range of 4 to 8 volts.
Normal cycles were missed less than 2 percent of the time, and
extra counts were added less than 1 percent of the time. These
levels remained below 10 percent even when the signals were
irregular and of low amplitude.
Major Equipment Problems
DC Voltage Drift—
The fish ventilatory signal is detected as a change from.,... .,.,„.,
zero voltage. From time to time, the background voltage drifted.,;
from zero to a positive or negative DC voltage. Detection of the
signal by the computer program is dependent on the signal breaking
both a positive and a negative threshold. These thresholds are..
set at absolute values around zero, and, if the background voltage"
departs greatly from zero, the signal is lost. When a sensing
chamber was empty or when a fish was between breaths, this
voltage should be zero in a properly adjusted system (Figure 2).
A 10,000 n loading resistor placed across the electrodes elimi- '
nated most of the voltage drift. However, as the equipment wore
out over time, the signal tended to drift from zero and data were
lost (Figure 11). The results from 10 fish during the first four
ventilatory studies were lost in this manner.
This problem was caused by corrosion of the terminal barrier
strips which were the connectors between the electrodes and the
amplifiers. These were inside the isolation chamber where the
atmosphere was very humid. The problem was solved by replacing
all electrodes and barrier strips. In practice, this is necessary
about every 18 months under conditions of continuous use.
Electronic Noise—
While shielded cable was used for all wiring and proper
grounding techniques were used, a considerable problem with
electrical interference or "noise" still existed that could mask
the ventilatory signals. The source of this noise appeared to
be ground currents flowing from the water supply to the drain.
Water dripped into the sensing chambers from a height of 10
cm above the water surface provided a broken stream of water and
eliminated this noise (Figure 6). The noise occurred only when
a continuous stream of water was present to carry ground currents.
Water Splash—
The solution to the noise problem caused the lesser problem
21
-------
24 48 72 96 120 144
TIME(HOURS)
168
192 216
Figure 11. The average ventilatory rate-of a-'bluegill. The
arrows indicate points at which.the DC voltage drift caused a loss
: of-signal. ' Replacing the' electrodes.' and -terminal barrier strips
eliminated this problem. .
22
-------
of splash from the dripping water sources. Since controls and
treated fish occupied alternate compartments, sometimes toxicant
splashed into control compartments. This was a minor problem,
and only one control fish was influenced by this; the data for
that fish was invalidated (#6, run 6).
Ventilatory Tests
Twelve, ten-day ventilatory studies were carried out, three
with each of the four toxicants. Each of the three toxicant
application patterns was included among these three tests, thus
all patterns were run with each toxicant (Table 1).
Toxicants-r
The toxicants.used for the ventilatory studies were divalent;
zinc, divalent copper, MixA and MixB. Neither divalent nickel
nor hexayalent chromium was tested separately. All toxicants were
used at concentrations that were indicated as being sublethal
by the toxicity tests. The amount of toxicant solution delivered
was approximately equivalent with respect to toxicity for each
test with a single toxicant; however, this was not true between
toxicants, and, consequently, the most valid comparisons are
between patterns for each toxicant (Table 1).
Application Patterns—
In the first application pattern, toxicant was delivered
to the sensing chambers at a constant level for 96 hr (Figure
12). In the second pattern, toxicant was delivered in two
variable 20-hr applications separated by a 20-hr interval. The
third pattern consisted of 8 variable 5-hr applications, each
separated by a 5-hr interval. The 20-hour variable applications
were delivered in five 4-hr increments: the first at the same
concentrations as the constant 96-hr application, the second
at approximately twice.that concentration, and the third at three
times that concentration. The concentrations then were reduced
in reverse order for the fourth and fifth increments, respec-
tively (Figure 13). The 5-hr variable applications were incre-
mented in the same manner, but the duration of each increment
was only 1 hr (.Figure 14.). A fourth pattern, zero, was designated
for statistical analyses. Pattern zero was that of the control
fish which received no toxicant.
Test Protocol—
Five of the 16 fish in each ventilatory test (3, 6, 9, 12,
15) were not exposed to toxicants and were used as controls.
The remaining 11 fish were exposed to various toxicants.
Each ventilatory test was run for 102 days and consisted of
4 time intervals. Ventilatory counts and average amplitudes were
23
-------
to
TABLE 1. CONCENTRATIONS OF HEAVY METALS USED IN VENTILATORY STUDIES
Toxicant
applications
One, 96 hr,
constant
Two, 20 hr,
variable
Toxicant
Zn++ Cu++ MixA
Run 1 Run 4 Run 7
Zn 2.5* Cu 0.5 Zn 0.6
Cu 0.2
Run 2 Run 5 Run 8
Zn 2.5, 5.7, 8.1 Cu 0.5, 1.0, 1.5 Zn 0.2, 0.4, 0.6
•
MixB
Run 10
Zn 0.3
Cu 0.1
Ni 2.5
Cr 25.5
Run 11
Zn 0.4, 0.
Cu 0.1, 0.
Ni 3.3, 5.
Cr 22.5,44.
8, 1.1
2, 0.2+
1, 8.0
9,58.7
Eight, 5 hr,
variable
Run 3
Zn 2.9, 5.7, 7.7
Run 6
Cu 0.5, 1.1, 1.3
Run 9
Zn 0.6, 1.4, 1.8
CuO.2, 0.5, 0.6
Run 12
Zn 0.4, 0.8, 1.1
Cu 0.1, 0.2, 6.2+
Ni 2.3, 5.3, 6.9
Cr 22.3,60.6,77.0
*A11 concentrations are given as the average mg/1 of all fish treated during that run.
-------
FISH 7 TREATED
26
FISH 14 TREATED
FISH 9 CONTROL
72 36 120 141 168 192 216
TIMEtHOURS)
Figure 12, Toxicant pattern 1. Toxicant was delivered to fish
7 and fish 14 for 96 hr. The shaded area on the fourth vertical ,:
plane indicates the shape, duration, and concentration of the
application of the toxicant. Fish 9 was a control and received.no
toxicant. Interval 2 = 0 - 96 hr (X axis); Interval 3 = 96 - 192
hr; Interval 4 = 192 - 216 hr. Data were not recorded during
Interval 1 which was the 24 hr prior to hour 0 (see text for
explanation).
25
-------
a jo
i6o;
120-
eo
80
40
\ y j •.•'••;' . . j
Y7__ y^
w« —. —^-^^—^ .x .
'VU^ju^/^^
S3
FISH 14 TPIEATED
'as ,.,..
F\SH 13. TREATED
jus- '•
OSH 9 CONTROL .
24 48 72 96 120 144 168 IB3 216
TIME (HOURS)
Figure 13. Toxicant pattern 2. Toxicant was delivered to fish ,
14 and fish 13. The shaded area on the.-fourth vertical plane
indicates the shape, duration, and concentration of the toxicant
applications. Each application lasted for 20 hr; each increment
was 4 hr. Interval 2 = 0- 96 hr. .(X axis); Interval -3 = 96 - 156-
hr; Interval 4 = 156-216 hr. Data were not recorded during
Interval i.which was the 24 hr prior to hour 0 (see text, for
explantion)
26
-------
FISH 16 TREATED
FISH 8 TREATED
216
FISH 12 CONTROL
24
48
72 36 120 144 168
TIME (HOURS)
216
Figure 14. Toxicant pattern 3. Toxicant was delivered to fish
16 and fish 8. The shaded area on the fourth vertical plane
indicates the shape, duration, and concentration of the toxicant
applications. Each application lasted for 5 hr, each increment
was 1 hr. Interval 2 = 0 - 96 hr (X axis); Interval 3 = 96 - 171
hr; Interval 4 = 171 - 216 hr. Data were not recorded during
Interval 1 which was the 24 hr prior to hour 0 (see text for
explanation).
27
-------
recorded only during the last 3 intervals, a total of 216 hr
(Figures 12, 13, and 14).
Interval 1. acclimation—The fish were placed in the sensing
chambers and allowed to acclimate for 24 hr. This 24-hr period
was usually sufficient time to allow stabilization of ventilatory
rates (Figure 15). Amplifiers could be adjusted during this
interval to produce signals in the optimum range (4-8 volts
peak-to-peak) and, if necessary, the rate of water flow to the
chambers could be reduced to eliminate electronic noise. No
adjustments were permitted during the remaining three intervals.
Interval 2. pretreatment—The ventilatory rate and average
amplitude were accumulated for 15-min intervals for 96 hr. No
toxicant was delivered during this interval which was to provide
control data for each individual fish.
Interval 3. treatment—Toxicant was delivered. This
interval varied in duration depending on the pattern of applica-
tion and continued only until the toxicant delivery ceased for
the final time. For pattern 1, this was 96 hr; for pattern 2,
60 hr; and for pattern 3, 75 hr.
Interval 4. post-treatment—The recovery period was monitored
from the end of interval 3 to the end of the ventilatory study.
This was 24 hr for pattern 1; 60 hr for pattern 2; and 45 hr for
pattern 3.
The duration of all intervals for pattern zero (controls)
was the same as for the treated fish in each ventilatory test,
respectively.
Data Treatment and Analysis
Data from four consecutive, 15-min reporting periods were
summed. The ventilatory counts were then divided by 60 to yield
the average counts per minute for each 1-hr period. Since the
signal amplitude was reported as an average, the hourly sums
were divided by 4 to produce an average voltage for each 1-hr
period. Nunn et al. (in press) have shown that such ventilatory
rate data are approximately normally distributed, and, thus,
standard parameteric analyses performed on them are statistically
valid.
These averages then were used to calculate an interval
mean for each fish during each of intervals 2, 3, and 4. Thus,
an average rate and amplitude were produced for each fish during
the pretreatment, treatment, and post-treatment intervals. For
example, the internal average rate for fish 12 (Figure 14) during
the pretreatment interval was 31.0 cycles per minute; during the
treatment interval, the average was 31.2 cycles per minute; and
28
-------
n 1 1 1 1 1 1 1
12 24 36 48 60 72 84 96
TIME (HOURS)
Figure 15. Average ventilatory rate for a bluegill during
Interval 1 and the first three days of Interval 2. The fish was
placed in the sensing chamber during the first hour.
29
-------
during the post-treatment interval 32.5 cycles per minute. These
interval means were then used for statistical analyses.
Since the amounts of toxicant delivered were equivalent for
each test with a single toxicant but were not equivalent between
toxicants, it was decided to analyze for differences between
patterns for each toxicant separately. The interval means from
all ventilatory tests with a single toxicant were assembled into
data sets. Each data set consisted of all the averages (interval
means) from one of the intervals (2, 3, or 4) for all three tests
with that toxicant. For example, the first data set was assembled
from the pretreatment interval for all three runs that used Zn"1"1"
as the toxicant. This was then partitioned into four cells
corresponding to the four patterns in which the toxicant was
applied (Table 2). This was done for the ventilatory rates for
each time interval for each of the 4 toxicants resulting in 12
partitioned data sets. In similar fashion, 12 data sets were
assembled for the average amplitude data. Thus, each of the
various analyses were performed on a total of 24 data sets. Even
though replicate treatment data were available, Treatments A and
B (Appendices B and C) were combined for analysis.
A chi squared test for goodness of fit was performed to com-
pare the distribution of the interval means in each data set to
the normal distribution (Siegel, 1956). Cochran's C statistic
was used to test for homogeneity of variances (Winer, 1971).
One-way analyses of variances for unequal sample sizes were then
used to test for differences due to the pattern in which toxicant
was applied, A post-mortem Duncan's Multiple Range test was then
used to locate the major sources of difference (Winer, 1971).
Computer programs available commercially were used to carry out
the latter two analyses (SAS Institute, 1979).
In addition to the application pattern and the toxicant, four
other independent variables that may have affected the ventila-
tory rate were measured for each individual fish. The standard
length of each fish was recorded in millimeters. The average
toxicity ratio was calculated by dividing the average concentra-
tion of toxicant by the LC50 of that toxicant and multiplying by
the total time that the toxicant was delivered. When fluctuating
applications were used, this was done for each concentration level,
and the three values were summed. For MixA and MixB, the values
for each individual component of the toxicant mixture were calcu-
lated and these values were summed. To determine the total dose
delivered, the average toxicity ratio was multiplied by the aver-
age rate of flow through the chamber in liters per minute. The
fourth variable was the maximum concentration reached (expressed
in terms of toxicity ratios) during the treatment interval. For
pattern 1, this was the average concentration; for patterns 2 and
3, it was the average concentration reached at the peaks of the
variable applications.
30
-------
TABLE 2. EXAMPLE OF PARTITIONING OF VENTILATORY RATE DATA FOR
, ANALYSIS
"Pattern 0Pattern 1Pattern 2Pattern 3
Toxicant Interval (control). (1 x 96 hr.). (2 x 20 hr) (8 x 5 hr)
Zinc
run 1
Zinc
run 2
Zinc
run 3
4 means* 9 means
3 means
5 means
10 means
10 means
Totals for 12 means 9 means 10 means 10 means
analysis
DC voltage drift resulted in loss of 3 fish from run 1; 3 from
run 2; and 1 from run 3.
A forward stepwise, multiple regression was then used to
indicate which, if any, of the independent variables, pattern,
standard length, average toxicity ratio, total dose, or maximum
concentration might have a significant effect on the ventilatory
rate during the treatment interval. This analysis produces an
F ratio to indicate the significance of each variable. An R-
Square value indicates the amount of the total variability
accounted for by the independent variables.
Only the data for the treated individuals from the treatment
intervals were used for this analysis. Separate analyses were
carried out for each of the four toxicants.
31
-------
SECTION 4
RESULTS AND DISCUSSION
TOXICITY TESTS
The results of the 96-hr continuous flow toxicity tests are
presented in Table 3.
Zinc
The acute toxicity of zinc to bluegills under conditions
similar to those in the present study (temperature 19 - 22.5°C in
soft water) has been reported by others. All the studies used
either zinc sulfate or zinc chloride as the source of zinc.
Pickering and Henderson (1966) reported 96-hr LC50 values of 4.85
(C.L. 3.55 - 6.32), 5.37 (C.L. = 3.84 - 7.00), 5.46 (C.L. = 4.29 -
6.94), and 5.82 rng/A (C.L. = 4.61 - 7.48) in soft water at 25°C.
Cairns and Scheier (1957a) reported values between 2.86 and 3.78
tag/H in soft water at 18°C and 1.93 - 3.63 mg/X, at 30°C. The work
of Cairns and Scheier (1957b) resulted in the highest reported
96-hr median lethal concentration (8.02 mg/2.) for these conditions.
The 96-hr LC50 of 3.21 mg/l (C.L. = 2.10 - 4.61) determined during
this study is comparable to the values reported in the literature.
Copper
The acute toxicity of copper to bluegills under similar
conditions has also been reported using copper sulfate or cupric
chloride as a source of copper. Pickering and Henderson (1966)
reported a 96-hr LC50 of 0.66 rag/I (C. L. = 0.52 - 0.85) at 25°C.
Trama (1954) reported a value of 0.74 mg/fc at 20°C; Patrick et
al. (1968) reported a value of 1.25 mg/Z at 18°C; and Benoit
(1975) reported a value of 1.1 mg/X, at 20°C. The value of 1.0
mg/fc (C.L. = 0.9 - 1.2) determined here compares favorably with
those values.
Nickel .
Few reports of the acute toxicity of nickel to bluegills
under similar conditions are to be found in the literature.
Pickering and Henderson (1966) used nickelous chloride and reported
96-hr LCSOs of 5.18 mg/£ (C.L. = 2.02 - 6.88) and 5.36 mg/SL (C.L. =
4.4 - 6.74) at 25°C in water of 20 mg/X, total hardness. In water
of 360 mg/X., the same authors reported an LC50 of 39.6 mg/X, (C.L. =
33.7 - 45.3) for nickel.
32
-------
Toxicant
Zn++
Cu++
Cr+6
K Zn++
a Cu++
Zn++
O3 ++
S Cr+6
TABLE 3. RESULTS
LC50
3.2
1.0
21.2
132.9
1.4
0.4
0.9
0.5
10.0
61.6
OF COMBINED
TOXICITY TESTS
95 percent C. L.
2.1
0.9
18.1
113.6
1.0
0.3
0.7
0.2
4.6
47.3
4.6
1.2
- 25.5
- 155.7
2.0
0.6
1.3
1.2
- 23.1
- 82.4
Total
fish
120
120
120
117*
119t
118* '
Three and two fish, respectively, died of behavioral causes and
were disregarded.
Only 119 fish at start due to counting error.
The LC50 of 21.2 mg/X, reported here falls between those ....
two values. Our tests were carried out in water with a total
hardness of 32 - 71 mg/i, values that are closer to the soft
water of Pickering and Henderson than to their hard water.
Pickering and Henderson used static tests and reported only the
calculated concentrations of Ni++ while the present tests were
continuous flow and the Ni++ concentrations were actually measured
five times during the course of the tests. It is also of interest
that the measurements are for total nickel in acidified samples.
Chromium
The acute toxicity of chromium (potassium chromate) has also
been reported in the literature. Trama and Benoit (1960) reported
an LC50 of 170 mg/S, for hexavalent chromium in water with a total
hardness of 45 mg/fc. The LC50 of 132.9 mg/5, (C.L. = 113.6 -
155.7) determined for hexavalent chromium during this study agrees
fairly well with those results. Trama and Benoit used static
toxicity tests and apparently reported calculated concentrations
rather than measured concentrations for chromium. Again, it is
well to remember that the concentrations given in the present
report are measures of the total chromium present in the acidified
sample.
33
-------
MixA
The acute toxicity of zinc-copper., mixtures to bluegills has
apparently not been reported in the literature.
An MAI of +0.218 was calculated with the data in Table 3.
The calculated index range (-0.644 to- +1.304) includes 0, and,
thus, we conclude that the toxicity of zinc-copper mixtures to
bluegills is additive under the test conditions.
MixB
The acute toxicity of zinc-copper-nickel-chromium mixtures
to bluegills has not been reported in.the literature.
MAI for this mixture is -0.738, which would seem to indicate
a tendency toward antagonistic toxicity. However, the calculated
index range of -2.982 to +0.193 indicates that this is not a
significant departure from additive toxicity. It should be kept
in mind that the actual toxicity ratio during these tests was
1:1.8:1.7:1.6, not the desired 1:1:1:1. Before discounting the
possibility that these metals display reduced-:;toxicity in mixture,
additional toxicity tests should be carried out.
VENTILATORY STUDIES
All living organisms display considerable natural variability
in morphological, physiological, and behavioral parameters. Vari-1-
ability in the ventilatory behavior of fish was both expected and
found; this quality makes change and adaptability possible—and
makes analysis of biological data difficult.
Strip chart figures illustrating variability in ventilatory
rates as well as in the shape and amplitude of the signal"are
included in Appendix D. These figures, .especially those repre-
senting interval 2 (the pretreatment interval), show that the
analysis of short intermittent segments of such recordings make
characterizing ventilatory behavior difficult. .The figures in
Appendix D can be placed in perspective by comparison with
Figures B-5 and G.-5 which indicate the average rates and. amplitudes,
respectively, for. these fish« The strip chart recordings from
all 4 fish were taken at the same points during run 5 (copper,
pattern 2).
Diurnal Variability
It has been shown that natural diurnal light cycles have a
definite effect on the ventilatory behavior of fishes (Morgan et
al., 1979; van der Schalie, 1977). By exposing the fish to
continuous 24-hr light, it was hoped that these effects would be
removed to simplify analysis of data. The graphic data for the
34
-------
pretreatment intervals in Appendices B and C show no obviously
identifiable cycles.
Other Variability
It can also be seen from Appendices B, C, and D that a great
deal of unexplained individual variability as well as variability
between individuals was apparent. One example of variation that
can be explained can be noted in run 1 (Figs. B-l, C-l). At
about hour 48, a malfunction of the temperature controller caused
the temperature to drop from 22°C to 16°C in less than 15 min and
remain there for approximately 6 hr at which point the tempera-
ture was returned to 22°C at the same rate. A rather high peak
for 4 or 5 hr is apparent for many of the fish. This corresponds
to the return to 22°C and is the result of both increased rates
and amplitudes. The rather quick return to prestress levels and...
the maintenance of those levels among the controls indicate, that
there was no deleterious effect on the fish, at least for the .......
duration of the test.
General Trends
A total of 55 control fish were monitored during this study.
The.average ventilatory rate for these fish during the pretreatment
interval was 31.7 counts per minute (range, 15.9 - 51.0). During
the treatment interval, the average rate was 31.0 counts per
minute (15.9 - 45.8), and during the post-treatment interval, it
was 28.7 counts per minute (14.2 - 43.8). The average rate for
the other 125 fish during the pretreatment period was 28.5 counts
per minute (.14.3 - 55.4). It is possible that the small downward
trend in rate is related to physical condition over time since
the fish were not fed during the tests. Van der Schalie (1977)
visually observed rates of 34.8 counts per minute (28.0 - 48.0)
during daytime observation.
The average amplitude during pretreatment for the controls
was 5.5 volts (2.5 - 9.9); during the treatment interval, it was
6.0 volts (1.8 - 12.3); and during post-treatment, 5.4 volts
(1.8. - 14.2). The average pretreatment value for the treated
fish was 5.8 volts (2.5 - 14.2). No obvious trends are apparent.
Changes in Rate
During the treatment interval, 26 control fish increased
their average rate with respect to the pretreatment interval
while 29 decreased. The average change was a decrease of only
0.7 counts per minute (2.2 percent). Average ventilatory rate
for 115 fish that received a toxicant treatment increased during
this interval while only 10 decreased. The average change here
was an increase of 15.5 counts per minute (54.4 percent).
35
-------
During the post-treatment interval, 39 of the control fish
decreased in average rate with respect to the treatment interval
while 16 showed an increase. The average change was a decrease
of 2.3 counts per minute (7.4 percent). Of the 125 treated fish,
123 survived the treatment interval, and 111 of these decreased
in average rate during the post-treatment interval while 12 showed
an increase. The average change for these fish was a decrease of
13.1 counts per minute. This was a 29.8 percent decrease with
respect to the treatment interval but 46.0 percent with respect
to the pretreatment interval. This can be compared with the 54.4
percent increase during the treatment interval.
The obvious conclusion is that sublethal, heavy metal stress
causes increased ventilation rates in bluegills while the removal
of that stress results in a return to rates near the pretreatment
levels.
Changes in Amplitude
"During the treatment interval, 27 of the control fish
decreased in average amplitude with respect to the pretreatment
interval and 28 increased. The average change was an increase
of 0.4 volts (7.3 percent). Average amplitude decreased in 107
treated fish while 18 responded with an increase. The average
change for these fish was a decrease of 2.1 volts (36.2 percent).
During the post-treatment interval, 21 of the control fish
increased in average amplitude and 34.decreased. The average
change was a decrease of 0.6 volts (10.0 percent) with respect
to the treatment interval. Of the 123 surviving toxicant exposed
fish, 112 increased their average amplitude during this interval
while only 11 decreased. The average change for these fish was
an increase of'2.4 volts. This was a 64.9 percent increase with
respect to the treatment interval and a 41.4 percent increase with
respect to the pretreatment interval. This value can be com-
pared with the 36.2 percent decrease during the treatment interval.
It is apparent from this that the application of these
toxicants caused a marked decrease in the average amplitude of
the ventilatory signals. It is further apparent that recovery
to near pretreatment levels is indicated with the cessation of
toxicant application.
Toxicant Effects on the System
It is known that large increases in conductivity or, corre-
spondingly, reductions in resistance can greatly reduce the
amplitude of the ventilatory signal. Cairns et al. (1980) reported
that 29 /oo sea water reduced the signal amplitude so much that
a considerable increase in amplification was necessary in order
to adapt this system to marine conditions. This raises a question
with regard to the source of the reduction in signal amplitude
36
-------
observed when toxicant was applied. Since any addition of dis-
solved salts can reduce the electrical resistance of the water
between the electrodes (see conductivity values, Table A-l), it
is possible that the observed change in amplitude is due to the
effect of the toxicant on the electrical resistance of the system
and not to an effect on the fish.
It is possible to measure the total resistance of a ventila-
tory sensing chamber by measuring the resistance when the loading
resistor is removed and a volt-ohm meter is attached in its
place. The total resistance for a normally operating ventilatory
sensing chamber is then the inverse of the inverse of the sum of
the measured resistance and the 10,0000 loading resistor. The
total resistance was measured for a chamber filled with diluent
water, various concentrations of the individual toxicants up to
120 mg/Jl (dissolved in diluent), and diluent water with a fish
in place. The fish itself had no measurable effect on the total
resistance, but the dissolved salt content did. The values ranged
from a high of 8389fi with diluent or diluent and fish to a low
of 714312 with a solution of 120 mg/fc Cu++.
It was then possible to simulate the change in total system
resistance that would occur with the addition of various concen-
trations of toxicant by changing the values of the loading resistor
while only diluent was passed through the chamber. Preliminary
data from an acclimated fish indicated that in order to reduce
the signal amplitude by 24.8 percent it is necessary to reduce
the total system resistance to approximately 6000J2. The amplitude
reduction indicated for the measured solutions (100 - 200 mg/fc)
was 12-13 percent. The maximum concentration delivered during
this study, test 12, would cause an amplitude reduction of less
than 10 percent. Thus, the average amplitude reduction of 36.2
percent would be due primarily to the effects of the toxicant on
the fish, not on the electronic system. It is also significant
to note that only in tests 11 and 12 did the toxicant concentration
reach levels that would depress the amplitude by more than 2
percent.
Distribution of Interval Means
The distribution of each of the 12 sets of interval means
for the ventilatory rates was compared with the normal distribu-
tion using a chi squared goodness of fit test. The results of
these analyses are presented in Table 4. The null hypothesis
that no difference exists could be accepted at a traditional
alpha level of 0.05 for nine of these distributions. Two of the
remaining three had probabilities for acceptance of null greater
than 0.01 while only one had a probability for acceptance of less
than 0.01.
The distribution of each of the 12 sets of interval means
for average amplitude was also compared with the normal distribu-
37
-------
TABLE 4. RESULTS OF CHI SQUARED TESTS FOR GOODNESS OF FIT TO
THE NORMAL DISTRIBUTION ...
Interval
Toxicant
Zinc
Zinc
Copper
Copper
MixA
MixA
MixB
MixB
Data set
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
Pretreatment. .
0.80 -
0.20 -
0.10 -
0.05 -
0.01 -
0.05 -
0.10 -
0.50 .-.
0.90*
0.30
0.30
0.10
0.02
0.10
0.20
0.70
. Treatment. .
.0.20 -
0.20 -
0.05 -
0.02 -
0.001-
0.001>
0.70 -
0.02 -
0.30
0.30
0.10
0.05
0.01
0.80
0.05
Post.-.treatment
0.70
0.30
0.20
0.70
0.10
0.02
0.01
0.30
-0.80
-0.50
-0.30
-0.80
-0.20
-0.05
-0.02
-0.50
Probability for obtaining a value this large or larger due to
chance.
tion using the chi squared goodness of fit test (Table 4). The
null hypothesis of no difference could be accepted at the 0.05
alpha level for 8 of these 12 data sets. Three of the remaining
four sets had probabilities for acceptance of null that were
greater than 0.02, and only one was less than 0.01 (P<0.001).
Homogeneity of Variances
Cochran's C statistic was calculated for the same data sets
indicated above (Table 5). Only 1 of the 12 data sets for
ventilatory rate exceeded the tabulated values at the 0.05 alpha
level. This indicated that, at that level, the null hypothesis
(that the cells have homogeneous variances) was acceptable for 11
of the 12 intervals tested.
The same calculations were carried out for the corresponding
amplitude data sets. It was possible to accept the null hypothesis
at the 0.05 alpha level for 8 of the 12.
Acceptability of ANOVA
Two-thirds (16) of the 24 data sets tested met both criteria
(normal distribution and homogeneity of variances) for the
acceptability of parametric analyses, and only 3 failed both. As
this indicates only minor or random departure from normality, the
one-way ANOVA with its relatively robust F test appears to be an
appropriate test for the analysis of these data.
38
-------
TABLE. 5... . CQCHRAN'S. C STATISTIC.
Interval
Pre-
Post-
Table
Toxicant. Data Set. . treatment. Treatment, .treatment Value* df
Zinc
Zinc
Copper
Copper
MixA
MixA
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
0.457
0.694
0.429
0.356
0.259
0.514
0.492
0.627
0.468
0.511
0.335
0.588
0.298
0.286
0.565
0.497
0.340
0.506
0.5967 11/3
0.5867 12/3
0.5667 13/3
MixB
MixB
Rate
Amplitude.
0.
0.
477
503. .
0
... o
.389
.429
0
0
.717
.648
0.
5667
13/3
Values that do not exceed the table value indicate homogeneous
varianceso
Largest N-I/Number of variances - 1.
Results of ANOVA
The results of the one-way analysis of variance tests are
presented in Tables 6 and 7. In Table 6, the smaller the
probability (P) values are the greater the chance that there is
a detected significant difference among the ventilatory rates or
the average amplitudes with respect to the patterns of applica-
tion of toxicant.
The Duncan's Multiple Range test was used in an attempt to
better define the indicated differences. In Table 7, the detected
differences are set off by commas. However, if one pattern appears
in two or more different groups within a single cell in this
table, these differences are blurred. The most significant
differences here are for patterns that are set apart from all
other patterns. For example, during the treatment interval for
the average ventilatory rates for MixB (Table 7), Duncan's
Multiple Range test indicates that the controls are significantly
different from all other patterns and that no difference can be
detected between patterns 1, 2, and 3, a conclusion that can be
confirmed from Figures B-10, B-ll, and B-12.
It can be seen from the F values in Table 6 that, with one
exception, there is no significant difference indicated between
ventilatory rates for any of the four patterns at the traditonal
alpha level of 0.05 during either the pretreatment or the post-
treatment time intervals. With only two exceptions, this holds
39
-------
TABLE 6. RESULTS OF ONE-WAY ANOVA.
Interval
Pretreatment
Toxicant
Zinc
Zinc
Copper
Copper
MixA
MixA
MixB
MixB
Data Set
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
F
2.77
7.57
1.26
0.50
1.54
1.53
3.58
3.07
P
-------
TABLE 7. RESULTS OF DUNCAN'S MULTIPLE RANGE TEST AT THE 95 PERCENT LEVEL
OF SIGNIFICANCE.
Interval
Toxicant
Zinc
Zinc
Copper
Copper
MixA
MixA
MixB
MixB
Data Set
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
Rate
Amplitude
Pretreatment
0 =
0 =
0 =
0 =
0 =
0 =
0 =
0 =
1
1
1
1
1
1
3
1
=3, 0=2*
= 3, 3 = 1, 2
= 2 = 3t
= 2 = 3
= 2 = 3
= 2 = 3
, 3 = 1 = 2
= 2, 3
Treatment
0, 1 =
0 = 3,
0 = 3,
0, 1 =
0 = 3,
0 = 3,
0, 1 =
0, 1 =
2
3
1
2
3
2
2
2
= 3t
= 2, 1
= 2 = 3
= 3
= 2 = 1
= 1
= 3
= 3
Post-treatment
0 =
0 =
0 =
0 =
0 =
0 =
0 =
0 =
3
1
1
2
1
1
1
1
,1 = 2 =
= 2 = 3
,0 = 3 =
- 3, 0 =
= 2 = 3
= 2 = 3
= 2 = 3
= 2v 1 =
3
2
1 = 2
2 = 3
0 = pattern 0 (control); 1 = pattern 1; 2 = pattern 2; 3 = pattern 3.
Significantly different patterns are set off by commas; however, if one pattern
appears in two or more different groups within a single cell, the differences are
less sharp. For example 0, 1=2=3 indicates that pattern 0 is significantly different
from all other patterns while patterns 1, 2, and 3 cannot be statistically
differentiated. 0=1=2=3 indicates no differences are detectable.
-------
true for the average amplitudes as well.
It is significant to note that in all instances the pro-
abilities that no differences exist are much smaller (in most
cases an order of magnitude) during the treatment intervals than
during the corresponding pre- and post-treatment intervals. The
indication being that the significant differences are much
greater during the treatment period, again a conclusion that can
be verified from the graphed data Appendices B and C.
The results of the Duncan's Multiple Range test (Table 7)
reveal no clear cut pattern during pretreatment and post-treatment
periods. While differences are indicated for 8 of the 16 data
sets, only 2 of these set a single pattern off from all others,
and these are different patterns in each case.
During the treatment interval, however, differences are
indicated for all eight data sets with pattern 0 (controls being
differentiated frora: all others most often (four times). When 0
was not differentiated by itself, it was always (four times)
included in a group with pattern 3,. and. in three of these cases
pattern 3 was also included in other sets.
It can be concluded that while these results supply no
absolutes, there is a definite trend that indicates statistically
that the effects of sublethal doses of these toxicants are
definitely detectable using, either average ventilatory rates or
average ventilatory amplitudes or preferably both. -
The various application patterns chosen for this study cannot
be differentiated as easily. When toxicant was applied in pattern
1, apparent acclimation occurred, especially during run 4 (Fig.
B-4, C-4). However, when the toxicant was applied in intermittent
bursts, the reaction to subsequent exposures was often as strong
as it was to the first exposure. These differences have been
statistically obscured by the extreme smoothing of data' (calcula-
tion of interval means) necessary for these analyses. Appropriate
methods do exist for the analysis of the ventilatory data for an
individual fish (Box and Tiao, 1975) but are extremely complicated
and time consuming.and are, at present, not practical for quick
analysis of large data sets such as these.
Mu1tip1e Re gr e s s i6n
The forward stepwise, multiple regression indicates little
or no predictability for any of the five variables used in the
analysis: pattern, standard length, average toxicity ratio, total
dose, or maximum concentration. In all cases', less than 25 percent
of the variability in ventilatory rate was accounted for by these
independent variables. None of the one variable models for all
toxicants yielded a significant F ratio. Furthermore, only
42
-------
application pattern in the copper analysis had a significant F
ratio in any of the multiple regression models (two). However,
this was of no real importance as the overall F ratios for these
two models were not significant.
It appears, from examination of the figures in Appendices
B and C, that the fish responded more sharply to the higher con-
concentrations of toxicant encountered in patterns 2 and 3 than
to the lower concentrations encountered during pattern 1. It is
possible that the failure of the statistical analyses to detect
differences due to either application pattern or the maximum
toxicant concentration was due to the extreme smoothing of the
data before it was analyzed.
Possible Causes
Some workers have indicated that the mechanism by which
heavy metals cause death is related to reduced diffusion capacity
of the gills with resulting lower oxygen levels in the blood
(Skidmore, 1970; Burton et al., 1972). Jt would seem reasonable
to hypothesize that such hypoxia might be the cause of the
increased ventilatory rates seen during this study. Others have
reported increased ventilatory rates with hypoxia (Skidmore,
1964; Gee et al., 1977). When the water supply was halted to one
of the sensing chambers, the dissolved oxygen level quickly fell
(3 hours) to less than 3.5 mg/fc. The ventilatory rate increased
from 45 counts per minute to 130-140 counts per minute; however,
the amplitude also increased nearly 300 percent, the opposite of -
the demonstrated amplitude reaction to sublethal levels of heavy
metals. While this is only preliminary data, it seems to indicate
some other physiological reason for the increased ventilatory
rates.
The studies indicated above were all carried out with very
concentrated solutions, concentrated enough to cause rapid death,
and emphasized gill damage and hypoxia (Skidmore, 1970; Burton
et al., 1972). Most chronic studies have emphasized very low
concentrations and have measured parameters such as growth,
reproductive success, and various physiological responses
(Christensen et al., 1972; Brungs et al., 1973; Benoit, 1975).
None of these can presently be correlated with the short term,
sublethal ventilatory effects described here. Perhaps an investi-
gation of the osmoregulatory changes indicated by Lewis and Lewis
(1971) and Katz (1979) might give a better insight into the
physiological mechanisms involved.
43
-------
REFERENCES
American Public Health Association (;APHA), American Water Works
Association, and Water Pollution Control Federation. 1976.
Standard Methods for the Examination of Water and Wastewater,
14th Ed. American Public Health Association, New York.
Barnett, J., and D. Toews. 1978. The Effects of Crude Oil and
the Dispersant, Oilsperse 43, on respiration and coughing
rates in Atlantic salmon (Salmo salar) Can. J. Zool. 56:
307-310.
fielding, D. L. 1929. The Respiratory Movements of Fish as an
Indicator of a Toxic Environment. Trans. Am. Fish. Soc.
59:238-245.
Benoit, D. A. 1975. Chronic Effects of Copper on Survival,
Growth, and Reproduction of the Bluegill (Lepomis macrochirus)
Trans. Am. Fish. Soc. 104:353-358.
Box, G. E. P., and G. C. Tiao. 1975. intervention Analysis with
Applications to Economic and- Envirpnniental- Problems. J. Am.
Stat. Assoc. 70:70-79".
Brungs, W. A., E. N. Leonard, and J. M. McKim. 1973. Acute and
Long-Term Accumulation .of Copper by the Brown Bullhead,
Ictalurus riebulosus. J. Fish. Res. Board Can. 30:583-586.
Burton, D. T., A. HI Jones)'and'J. Cairns, Jrv 1972. ..Acute Zinc
Toxicity to Rainbow Trout (Salmo gairdneri): Confirmation
of the Hypothesis that Death.is Related to Tissue Hypoxia.
J. Fish. Res. Board Can. 29': 1463-1466.
Cairns, J., Jr., K. L. Dickson, R. E. Sparks, and W."T. Waller.
1970. A Preliminary Report on Rapid Biological Information
Systems for Water Pollution Control. J. Water Pollut.
Control Fed. 42:685-703.
Cairns, J., Jr., D. Gruber, K. L. Dickson, A. C. Hendricks
and W. H. van der Schalie. 1977. Developing an On-Site
Continuous Biological Monitoring System for the Chemical
Industry. Pages 285-294 in L. Delpino and A. Krigman,
Eds. ,. Proceedings of Fifth Annual Industrial Pollution
Conference, Water and Wastewater Equipment Manufacturers
Assoc., Inc., McLean, Virginia.
Cairns, J., Jr., and A. Scheier. 1957a. The Effects of Tempera-
ture and Hardness of Water upon the Toxicity of Zinc to the
Common Bluegill (Lepomis macrochirus Raf.). Notulae
Naturae 299.
44
-------
Cairns, J., Jr., and A. Scheier. 1957b. The Effects of Periodic
Low Oxygen upon the Toxicity of Various Chemicals to Aquatic
Organisms. Proceedings of the 12th Industrial Waste Confer-
ence, Purdue Univ. Eng. Bull. 94:165-176.
Cairns, J., Jr., and K. W. Thompson. In press. A Computer Inter-
faced Toxicity Testing System for Simulating Variable
Effluent Loading. Proceedings of the Second Symposium on
Process Measurements for Environmental Assessment.
Cairns, J., Jr., K. W. Thompson, J. D. Landers, Jr., M. T. McKee,
and A. C. Hendricks. 1980. Suitability of Some Freshwater
and Marine Fishes for Use with a Minicomputer Interfaced
Biological Monitoring System. Water Resour. Bull. 16:
421-427.
Cairns, J., Jr., and W. H. van der Schalie. In press. Biological
Monitoring, Part I: Early Warning Systems. Water Research.
Camougis, G. 1960. Recording Bioelectrical Potential from
Aquatic Animals. Turtox News 38:156-157.
Carlson, R. W., and R. A. Drummond. 1978. Fish Cough Response -
a Method for Evaluating Quality of Treated Complex Effluents.
Water Res. 12:1-6.
Chizar, D., M. Moody, and J. T. Windell. 1972. Failure of
Bluegill Sunfish, Lepomis macrochirus. to Habituate to
Handling. J. Fish. Res. Board Can. 29:576-578.
Christensen, G. M., J. M. McKee, W. A. Brungs, and E. P. Hunt.
1972. Changes in the Blood of the Brown Bullhead (Ictalurus
nebulosus (Lesueur)) Following Short and Long Term Exposure
to Copper (II). Toxicol. Appl. Pharmacol. 23:417-427.
Davis, J. C. 1972. Sublethal Effects of Bleached Zraft Pulp
Mill Effluents on Respiration and Circulation in Sockeye
Salmon. J. Fish. Res. Board Can. 30:369-377.
Drummond, R. A., and R. W. Carlson. 1977. Procedures for
Measuring Cough (gill purge) Rates of Fish. Ecol. Res.
Ser. EPA-600/3-77-133.
Drummond, R. A., G. F. Olson, and A. R. Batterman. 1974. Cough
Response and Uptake of Mercury by Brook Trout, Salvelinus
fontinalis, Exposed to Mercuric Compounds at Different
Hydrogen-Ion Concentrations. Trans. Am. Fish. Soc. 103:
244-249. .
Drummond, R. A., W. A. Spoor, and G. F. Olson. 1973. Some
Short-Term Indicators of Sublethal Effects of Copper on
45
-------
Brook Trout, Salvelinus fontinalis. J. Fish. Res. Board
Can. 30:698-701.
Finney, D. J. 1971. Statistical Methods in Biological Assay,
2nd Ed. Griffin Press, London, England.
Fee, J. H., R. F. Tillman, and H. J. Smart. 1977. Reactions
of Some Great Plains Fishes to Progressive Hypoxia. Can.
J. Zool. 56:1962-1966.'
Gruber, D., J. Cairns, J; , K. L. Dickson, and A. C. Hendricks.
1979. A Cinematographic Investigation into the Fish's
Bioelectric Breathing Signal. J. Fish. Biol. 14:429-436.
Gruber, D., J. Cairns, Jr., K. L. Dickson, R. Hummel, III,
A. Maciorowski, and W. H. van. der. Schalie. 1977. An
Inexpensive, Noise-Immune Amplifier Designed for Computer
Monitoring of Ventilatory Movements of Fish arid Other
Biological Events. Trans. Am. Fish. Soc. 106:497-499.
Hendricks, A-.--C. ,.X. L. Di-cksony and J. Cairns, Jr. 1:977. A
Mobile Fish Bioassay 'Unit for Effluent Testing. Report
to Manufacturing Chemists Association.
Hughes, G. M. 1975. Coughing in the Rainbow Trout (Salmo
gairdneri) and the Influence of Pollutants. Revue Suisse
Zool. 82:47-64.
Katz, B. 1979. Relationship of the Physiology of Aquatic
Organisms to the Lethality of Toxicants: A Broad Overview
with Emphasis on Membrane Permeability. Pages 62-76 in
L. L. Marking and R. A. Kimerle, Eds. Aquatic Toxicology,
STP 607, American Society fpr testing and Materials,
Philadelphia, Pa.
Kleerekoper, H., and K. Sibakin. 1956. An Investigation of
the Electrical "Spike" Potentials Produced by the Sea
Lampregy (Petromyzon marinus) in the Water Surrounding
••• the Head Region. J.''Fish. Res'. Board Can. 13:375-383.
Lewis,; S. D., and W. M. Lewis. 1971. The Effect of Zinc and
Copper on the Osmolality of Blood Serum of the Channel
Catfish, Totalurns' puhctatus Rafinesque, and Golden Shiner,
Notemigonus crysbleucas Mitchill. Trans. Am. Fish. Soc.
100:639-643.
Lunn, C. R., D. P. Toews, and D. J. Pree. 1975; Effects of
Three Pesticides on Respiration, Coughing, and Heart
Rates of Rainbow Trout (Salmo gairdneri Richardson).
Can. J. Zool. 54:214-219.
46
-------
Maki, A. W. 1979. Respiratory Activity of Fish as a Predictor
of Chronic Fish Toxicity Values for Surfactants. Pages
77-95 in L. L. Marking and R. A. Kimerle, Eds., Aquatic
Toxicology, STP 667, American Society for Testing and
Materials, Philadelphia, Pa.
Marking, L. L. 1977. Method for Assessing Additive Toxicity
of Chemical Mixtures. Pages 99-108 iji F. L. Mayer and
J. L. Hamelink, Eds., Aquatic Toxicology and Hazard
Evaluation, STP 634, American Society for Testing and
Materials, Philadelphia, Pa. 307 pp.
Meuwis, A. L., and M. J. Heuts. 1957. Temperature Dependence
of Breathing Rate in Carp. Biol. Bull., Woods Hole
112:97-107.
Morgan, E. L., K. W. Eagleson, R. Herrmann, and N. D. McCollough,
Jr., 1979. Automated Biomonitoring Applications to Remote
Water Quality Stations and Satellite Data Retrieval: New
Developments in Acheiving Real-Time Biosensing for Watershed
Management. Ninth Intersociety Conference in Environmental
Systems.
Morgan, W. S. G. 1977. An Electronic System to Monitor the
Effects of Changes in Water Quality on Fish Opercular Rhythms.
Pages 38-55 in J. Cairns, Jr., K. L. Dickson and G. F.
Westlake, Eds., Biological Monitoring of Water and Effluent
Quality, STP 607, American Society for Testing and Materials,
Philadelphia, Pa.
Morgan, W. S. G., and P. C. Kuhn. 1974. A Method to Monitor
the Effects of Toxicants upon Breathing Rate of Largemouth
Bass (Micropterus salmoides Lacepede). Water Res. 8:67-77.
Nunn, G. L., J. Cairns, Jr., and D. Gruber. In press. The
Distribution of Ventilatory Frequencies Over Time of the
Bluegill, Lepomis macrochirus. J. Interdiscipl. Cycle Res.
Pasztor, V. M., and H. Kleerekoper. 1962. The Role of the
Gill Filament Musculature in Teleosts. Can. J. Zool.
40:785-806.
Patrick, R., J. Cairns, Jr., and A. Scheier. 1968. The
Relative Sensitivity of Diatoms, Snails and Fish to
Twenty Common Constituents of Industrial Wastes. Prog.
Fish-Cult. 31:137-140.
Peltier, W. 1978. Methods for Measuring the Acute Toxicity
of Effluents to Aquatic Organisms. EPA-600/4-78-012,
U. S. Environmental Protection Agency.
47
-------
Pickering, Q. H., and C. Henderson. 1966. The Acute Toxicity
of Some Heavy Metals to Different Species of Warmwater
Fishes. Air Water Pollut. Int. J. 10:435-463.
Roberts, J. L. 1964. Metabolic Responses of Freshwater Sunfish
to Seasonal Photoperiods and Temperatures. Helgoloender
Wissenschafteiche Meeresuntersuchunger 9:459-473.
SAS Institute, Inc. 1979. SAS User's Guide. SAS Institute,
Raleigh, N.C.
Saunders, R. L. 1961. The Irrigation of the Gills in Fishes,
I. Studies of the Mechanism of Bronchial Irrigation.
Can. J. Zool. 39:637-653.
Schaumburg, F. D., T. E. Howard, and C. C. Walden. 1967. A
Method to Evaluate the Effects of Water Pollutants on
Fish Respiration. Water Res. 1:731-737.
Sellers, C. M., A. G. Heath, and M. L. Bass. 1975. The Effect
of Sublethal Concentrations of Copper and Zinc on
Ventilatory Activity, Blood Oxygen and pH in Rainbow Trout
(Salmo gairdneri). Water Res. 9:401-408.
Siegle, S. 1956. Nonparametric Statistics for the Behavioral
Sciences. McGraw-Hill, New York. 312 pp.
Skidmore, J. F. 1964. Toxicity of Zinc Compounds to Aquatic..
Animals, with Special Reference to Fish. Quart. Rev. Biol.
39:227-248.
Skidmore, J. F. 1972. Respiration and Osmoregulation in Rainbow
Trout with gills damaged by Zinc Sulphate. J. Exp. Biol.
52:484-494.
Sorensen, E. M. B., and R. D. Burkett. 1978. Respiratory
Responses to Instant Thermal Stress Under Oxygen- Enriched
Conditions in Centrarchids. Texas J. Sci. 30:337-345.
Sparks, R. E., J. Cairns, Jr., R. A. McNabb, and G. Suter, II.
1972. Monitoring Zinc Concentrations in Water Using the
Respiratory Response of Bluegills (Lepomis macrochirus
Rafinesque). Hydrobiologia. 40:361-369.
Spoor, W. A., T. W. Neiheisel, and R. A. Drummond. 1971. An
Electrode Chamber for Recording Respiratory and Other
Movements of Free-Swimming Animals. Trans. Am. Fish. Soc.
100:22-28.
48
-------
Sprague, J. B. 1973. The ABC's of Pollutant Bioassay Using Fish.
Pages 6-30 in J. Cairns, Jr., and K. L. Dickson, Eds.,
Biological Methods for the Assessment of Water Quality, STP
528, American Society for Testing and Materials, Philadelphia,
Pa. 256 pp.
Thomas, R. E., and S. D. Rice. 1975. Increased Opercular Rates
of Pink Salmon (Oncorhynchus gorluscha) Fry after Exposure
to the Water Soluble Fraction of Prudhoe Bay Crude Oil. J.
Fish. Res. Board Can. 32:2221-2224.
Thomas, R. E., and S. D. Rice. 1979. The Effect of Exposure
Temperature on Oxygen Consumption and Opercular Breathing
Rates of Pink Salmon Fry Exposed to Toluene, Naphthalene, and
Water-Soluble Fractions of Cook Inlet Crude Oil and No. 2 Fuel
Oil. Pages 39-52 in W. B. Vernberg, F. P. Thurberg, A.
Calabrese, and F. J. Vernberg, Eds., Functional Responses.
Academic Press.
Thompson, K. W., K. L. Dickson, D. Hooley, J. Cairns, Jr., and
W. H. van der Schalie. 1978. A Computer Monitored Aquatic
Bioassay System. Pages 61-66 iji J. B. Carberry, Ed.,
Proceedings of Tenth Mid-Atlantic Industrial Waste Conference,
University of Delaware.
Trama, F. B. 1957. The Acute Toxicity of Copper to the Common
Bluegill (Lepomis macrochirus Raginesque). Notulae Naturae 257.
Trama, F. B., and R. J. Benoit. 1960. Toxicity of Hexavalent
Chromium to Bluegills. J. Water Pollut. Control Fed.
32(8):868-877.
Tsai, C. and J. A. McKee. 1980. Acute Toxicity to Goldfish of
Mixtures of Chloramines, Copper, and Linear Alkylate
Sulfonate. Trans. Am. Fish. Soc. 109:132-141.
van der Schalie, W. H. 1977. The Utilization of Aquatic
Organisms for Continuous and Automatic Monitoring of the
Toxicity of Industrial Waste Effluents. Ph.D. Dissertation.
Virginia Polytechnic Institute and State University,
Blacksburg, Va.
van der Schalie, W. H., 1980. A New Technique for Automatic
Monitoring of Fish Ventilatory Patterns and Its' Possible
Use in Screening Tests for Chlorine Toxicity. Pages 233-242
in J. G. Eaton, P. R. Parrish, and A. C. Hendricks, Eds.,
Aquatic Toxicology, STP 707, American Society for Testing
and Materials, Philadelphia, Pa.
van der Schalie, W. H., K. L. Dickson, G. F. Westlake, and
J. Cairns, Jr. 1979. Fish Bioassay Monitoring of Waste
Effluents. Environ. Manage. 3:217-235.
49
-------
Westlake, G. F., and W. H. van der Schalie. 1977. Evaluation
of an Automatic Biological Monitoring System at an
Industrial Site. Pages 30-37 in J. Cairns, Jr., K. L.
Dickson, and G. F. Westlake, Eds., Biological Monitoring
of Water and Effluent Quality, STP 607, American Society
for Testing and Materials, Philadelphia, Pa.
Winer, B. J.
Design.
1971. Statistical Principles in Experimental
McGraw Hill, New York.
Young, Y. 1971. EMG activity in Tench (Tinea tinea, L.)
Gill Lamellae and its Association with Coughing. J.
Phys. 215:37P-38P.
50
-------
APPENDIX A
RESULTS OF WATER ANALYSES AND INDIVIDUAL TOXICITY TESTS
TABLE A-l. RESULTS OF CHEMICAL ANALYSES GIVEN AS THE AVERAGE
OF THE FIVE ANALYSES FOR EACH FISH BIOASSAY CHAMBER
Test
number
1
(Zinc) '
2
(Zinc)
3
(Copper)
4
(Copper)
5
(Nickel)
Tank Conductivity Alkalinity
number micromhos/cm pH (mg/1)
1
2V
3IV
4III
511
I
1
2V
3IV
4III
511
I
1
2V
3IV
4III
511
I
1
2V
3IV
4III
511
I
1
2V
3IV
4III
511
I
110
123
131
150
179
204
109
115
118
121
129
139
106
106
106
106
106
106
110
111
112
113
115
117
118
128
134
147
162
194
7.5
7.4
7.4
7.3
7.3
7.2
7.2
7.2
7.1
7.0
7.0
6.9
7.1
7.1
7.1
7.0
6.9
6.9
7.1
7.1
7.1
7.1
6.9
6.9
7.1
7.1
7.1
7.1
7.1
7.1
26.8
28.0
28.4
29.2
29.2
29.2
31.6
32.0
31.2
30.0'
30.4
30.4
23.2
24.0
24.0
24.0
24.0
24.0
28.0
31.2
30.4
30.4
30.8
30.8
30.0
32.8
33.2
33.2
32.8
32.8
Total
hardness
(mg/1 CaC03)
22.4
28.8
32.8
39.6
48.0
59.2
23.6
27.2
28.4
31.2
35.6
45.6
28.8
. 28.8
28.8
28.4
28.0
28.8
22.0
21.2
23.3
23.6
24.4
24.8
36.6
34.6
39.0
32.2
31.8
33.2
51
-------
TABLE A-l (cont.)
Test
number
6
(Nickel)
7
(Chromium)
8
(Chromium)
9
(MixA)
10
(MixA)
11
(MixB)
Tank Conductivity
number micromhos/cm pH
1 .
2V
3IV
4III
511
I
1
2V
31 V
4III
511
I
1
2V
3IV
41 II
511
I
1
2V
3IV
: 41 IT "
511
I
1
" 2V
' 3IV
4III .
511- :
I
1
2V
3IV
4III
511
I
125
149
163
189
225
320
117
565
876
1496
2052
.3352
111
256
385
628
946
1198
111
114
114
117 .
120
126
118
123
125
.... "127
130
136
131
263
332
506
732
1106
7,1.
7.1
7.1
7.1
7.1
7.0
7.1
7.6
7.8
7.9
8.2.
..8.3.
..7.1
7.2
7.4
7.5
7.6
7.8
7.0
7.0
7.0
7.0
6.9
'•*' 6.8
••" 7.1
7.1
7.1
'-....• ••-•7.1"
7.0
6.9
6.8
7.1
7.2
7.4
7.5
7.7
Alkalinity
(mg/1)
36.0
35.2
35.6
36.0
35.8
35. 8
30.4
97.2
139.6
221.2
361.6
551.0
40.8
63.6
83.8
110.8
180.4
.268.0
30.4
30.0
30.0
30.0
30.0
" 30.0
•--•- 30.4
30.8
31.6
32.8
32.4 -.
32.8
31.6
53.8
65.0
94.8
131.6
218.0
Total
hardness
(mg/1 CaC03)
71.4
76.0
65.2
67.6
68.4
70.8
22.0
19.6
22.4
20.8
20.8
20.8
44 . 4
43.6
42.4
42.8
42.4
42.0
21.6
21.2
21.4
22.2
24.6
.. 26-6
29.2
32.4
32.8
32.8
34 . 8
34.8
44.8
40.4
38.8
41.6
43.6
44.6
52
-------
TABLE A-l (cont.)
Total
Test Tank Conductivity Alkalinity hardness
number number micromhos/cm pH (mg/1) (mg/1 CaCO.,)
«J
12 1 119 7.1 33.0 35.0
(MixB) 2V 310 7.3 57.2 43.0
3IV 421 7.4 75.2 41.0
4III 668 7.5 114.0 37.6
511 1106 7.6 171.6 38.6
I 1719 7.6 269.2 35.6
53
-------
TABLE A-2. TOXICANT CONCENTRATIONS AND SURVIVAL DURING TOXICITY
TESTS ....•• . " .
Test
number
1
(Zinc)
2
(Zinc)
3
(Copper)
4
(Copper)
5
(Nickel)
6
(Nickel)
Tank
number
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
•-, I . .
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
41 1 1
5LI- -- -
" I •
1 (Control)
2V
3IV
4III
511
I
Average
Zn++
0.0
2.6
3.6
6.2
10.9
23.2
0.1
1.2
2.1
3.5
5.7
9v9 ••
_ ..•_..
••'• -..,. _ ..
—
—
. -
-• *-.-->•".'"'.• .
_
- '
-
-
-
—
_
. - •
-
-
—
-
_
-
—
_
—
-
concentration (mg/1)
++ ++ +6
Cu Ni Cr
_ — • «
_ . _ _
_ _ _
_ _ _
_ _. _
_ _ _
_ ' _ _
.•*•'.— -
_ _ _
_ _ _
_ • _ _
".. - • _ : _
• o.o : -
0.3 , - -
0.5
•0.8..-
1.4
V-2.4,..,,., ,.,.,, ........ .f-,,.,.
o.o ' "•••'•--. _ • ': :
0.5 - -
0.7 - -
1.3
2.3 -
4.0 •;..' - ....'•"' \ -
0.0
2.3
3 . 3
7.7
-12.0
20.9
0.1
7.1
10.0
20 . 3
28.4
50.0 -
# Surviving
at 96 hr
(of 10)
10 '
9
5
0
0
0
10
7
7
8
1
0
10
10
10
10
0
• - . ,0,..
10
9
7
3
0
o
10
10
10
10
9
6
10
10
10
4
3
0
54
-------
TABLE A-2 (cont.)
Average concentration
Test
number
7
(Chromium)
8
(Chromium)
9
(MixA)
•? '.-lo'1:.
(MixA)
11 •",.
(MixB) '
12
(MixB)
Tank
number
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
I
1 (Control)
2V
,3IV
4III
511
I
1 (Control)
2V
3IV
4III
511
I
Zn++
_
-
-
-
—
-
_
-
-
-
-
-
0.0
0.4
0.6
1.2
1.8
3.1
0.0
0.4
0.6
1.2
2.0
3.7
0.0
0.4
0.6
1.0
1.9
3.0
0.0
0.4
0.7
1.3
2.0
3.1
Cu++
_
-
-
-
-
-
_
-
-
-
-
-
0.0
0.1
0.2
0.3
0.5
0.9
0.0
0.1
0.2
0.4
0.5
1.0
0.0
0.1
0.2
0.5
0.8
1.4
0.0
0.3
0.5
0.8
1.6
2.6
Ni++
_
-
-
-
—
-
_
-
-
-
-
-
_
-
-
_
-
- •-
; '• _
-
' - •
-
-
-
0.0
3.0
4.8
8.9
14.6
24.3
0.0
5.9
10.3
16.9
31.6
46.7
(mg/1)
Cr+6
0.1
37.6
62.8
119.8
181.0
289.0
0.1
66.8
109.6
199.8
331.2
525.2 ...
_
-
-
—
-
- •
• '_ / ;"'
. :_
-
- '
-
- ' '•
0.0
29.3
36.6
72.4
99.6
181.8
0.0
30.2 ,
49.9
78.1
135.1
226.0
# Surviving
at 96 hr
(of 10)
8*
9*
10
8
3
0
10
.. -.8
8
.0 . . ...
0
• .,..- .
10
10
10
5
0
0
v . VLO •••
': -lo - '
'9
-6t
8
0
10
8
10
7
0
0
8*
9
9
1
0
0
*Two, one, and two disregarded due to behavioral death
tOnly nine fish at the start, counting error
55
-------
APPENDIX B
Graphic representation of the average ventilatory ra-tes
for all 12 ventilatory tests. See text Figures 12, 13, and 14
and text table 1 for further details.
56
-------
01,
0 24 48 72
86 120 144 168 192 216
TIME (HOURS)
Figure B-l Average ventilatory rates for ventilatory test 1. Toxicant = Zn
pattern = 1, concentration = 2.5 mg/1. .'..;.
++
-------
Oi !
ooi
k*K^CJC?y*Fi
Tl. M 120 .' 144
TIME (HOURS)
Figure B-2. Average ventilatory rates for ventilatory test 2. Toxicant = Zn ,
pattern = 2, concentrations = 2.5, 5.7, 8.1 mg/1.
-------
01 I
CD j
, . I
72 M
TIME (HOURS)
Figure B-3, Average ventilatory rates for ventilatory test. 3. Toxicant = Zn
pattern = 3, concentrations = 2.9, 5.7, 7.7 mg/1.
-------
46 72 96 120 144 166 192 216
TIME (HOURS)
Figure B-4. Average ventilatory rates for ventilatory test 4. toxicant = Cu++,
pattern = 1, concentration = 0.5 mg/l.
-------
Ci;
M,
^(*>^i^j<^^
>O£ZL' ' '^6
^?"~^~—7~/ • HSH.i.
72 ' 96 120 M«
TIME (HOURS)
Figure B-5. Average ventilatory rates for ventilatory test 5. Toxicant = Cu++>
pattern = 2, concentrations = 0.5, 1.0, 1.5 rng/1. Representative strip chart records
for number 5, 6, 7, and 8 can be found in Appendix D. .-: '
-------
CO
itellL^Xl
72 96 120 . 144 ISO , 192
TIME (HOURS)
Figure B-6. Average ventilatory rates for ventilatory test 6. Toxicant = Cu++,
pattern = 3, concentrations = 0.5, 1.1, 1.3 tng/1. *Fish 6, a control, received a
partial dose of toxicant due to splash and was not included in the statistical analysis,
-------
GO I
2A 46 72
96 l» 144
TIME (HOURS)
KB 192 36
Figure B-7. Average ventilatory rates for.ventilatory test 7
pattern = 1, concentrations =0.6 mg/1 Zh ; 0.2 mg/1 Cu .
Toxicant = MixA,
-------
cn
-^-V^^^
M 43 72
W 120 144 oa 192 2K
TIME(HOURS)
Figure B-8. Average ventilatory rates for ventilatory test 8 Toxicant - MixA,
pattern = 2, concentrations = 0.6, 1.3, 1.8 mg/1 Zn++; 0.2, 0.4, 0.6 mg/1 Cu .
-------
O) '
01,
•%W'Wty&7 ' A«
X&^^ E
O SO 40 72 96 120 144 168 192
TIME (HOURS)
Figure B-9. Average ventilatory rates for ventilatory test 9. Toxicant = MixA,
pattern = 3, concentrations = 0.6, 1.4, 1.8 mg/1 Zn++; 0.2, 0.5, 0.6 mg/1 Cu++.
-------
05]
01'
ISO .,192 218
TIME (HOURS)
Figure B-10. Average ventilatory rates for ventilatory test 10. Toxicant
pattern = 1, concentrations = 0.3 mg/1 Zn++; 0.1 mg/1 Cu++; 2.5 mg/1 Ni**;
25.5 mg/1 Cr+6.
= MixB,
-------
7^r^>
72 W I20 144 08 192
TIME (HOURS)
Figure B-ll. Average ventilatory rates for ventilatory test 11. Toxicant - MixB,
pattern = 2, concentrations = 0.4, 0.8, 1.1 mg/1 Zn++; 0.1, 0.2, 0.2+ mg/1 Cu++;
3.3, 5.1, 8.0 mg/1 Ni++; 22.5, 44.9, 58.7 mg/1 Cr+6.
-------
72 96 120 144 ISO 192
TIME(HOURS)
Figure B-12. Average ventilatory rates for ventilatory test 12. Toxicant = MixB,
pattern = 3, concentrations = 0.4, 0.8, 1.1 mg/1 Zn++; 0.1, 0.2, 0.2+ mg/1 Cu++;
2.3, 5.3, 6.9 mg/1 Ni++; 22.3, 60.6, 77.0 mg/1 CrT6.
-------
APPENDIX C
Graphic representation of the average signal amplitude
for all 12 ventilatory tests. See text Figures 12, 13, and
14 and text table 1 for further details.
69
-------
•v)
o;
46 72 96 120 144
TIME (HOURS)
Figure C-l. Average ventilatory signal amplitudes for ventilatory test 1,
Toxicant = Zn++, pattern =1.
-------
TIME (HOURS)
Figure C-2. Average ventilatory signal amplitudes for ventilatpry test 2.
Toxicant = Zn++, pattern = 2. . : .''.'••
-------
-J
to
'/^mzw A*, =./
i^^^xov^^x / v.
86 120 144
TIME (HOURS)
Figure C-3. Average ventilatory signal amplitudes for ventilatory test 3.
Toxicant = Zn++, pattern =3.
-------
CO!
120 144
TIME (HOURS)
Figure C-4 Average ventilatory signal amplitudes for ventilatory test 4
Toxicant = Cu++, pattern =1. •
-------
S& . 120 144
; TIME (HOURS)
Figure C-5. Average ventilatory signal amplitudes for ventilatory test 5,
Toxicant = Cu , pattern = 2.
-------
en!
2K>
£00
ISO
^^^_^^^
S^&J^J^^^
2 86 120 144 168
TIME (HOURS)
Figure C-6. Average ventilatory signal amplitudes for ventilatory test 6.
Toxicant = Cu++, pattern = 3. *Fish 6, a control, received.a partial dose of
toxicant due to water splash and was not included in the' statistical analysis,
-------
.46 .-72 9S IEO 144 KS . 192 2t6
I' : TIME (HOURS)
Figure C-7. Average ventilatory signal amplitudes for ventilatory test 7.
Toxicant = MixA, pattern = 1.
-------
TIME (HOURS)
Figure C-8. Average ventilatory signal amplitudes for v>entilatory test 8.
Toxicant = MixA, pattern = 2. ..'; / | '-. .:; .
-------
2OO
ISO
^^
00 ,
72 . 86
TIME (HOURS)
Figure C-9. Average ventilatory signal amplitudes for ventilatory test 9.
Toxicant = MixA, pattern = 3.
-------
96 120 144
TIME (HOURS)
Figure C-10. Average ventilatory signal amplitudes for ventilatory test 10,
Toxicant = MixB, pattern =1.
-------
CO,
O!
TIME (HOURS)
Figure C-ll. Average ventilatory signal amplitudes for ventilatory test 11.
Toxicant = MixB, pattern = 2.
-------
Hi
TIME (HOURS)
Figure C-12. Average veiitilatory signal amplitudes for ventilatory test 12.
Toxicant = MixB, pattern =? 3.
-------
APPENDIX D
Examples of stressed and unstressed inter- and intra-
individual variation as illustrated by strip chart recordings,
These figures can be coordinated with the graphs of average
ventilatory rate in Figure B-5 and the average signal ampli-
tude in Figure C-5.
82
-------
0-
-3
o-
O
>
I- '
<
o-
-3J
0 -
-3J
10 20
SECONDS
30
Figure D-l. Examples of the appearance of the ventilatory signals
as they appear on a strip chart recorder. These recordings are
for fish 6 (control) from run 5. The voltages indicated are
arbitrary since the strip chart recorder further amplifies the
signal that is being sent to the computer. The average rates and
amplitudes recorded by the computer during the 15-min period from
which these recordings were made and the total hours elapsed since
the beginning of interval 2 are: A. 14 cts/min, 3.6 volts, 22.5
hr; B. 27, 2.8, 44.5; C. 49, 3.1, 74.5; D. 25, 5.3, 94.5.
83
-------
0-
-3J
0-
UJ
IT
0-
-3J
»3
0-
-3J
10 20
SECONDS
30
Figure D-l (cont.). Ventilatory signals for fish 6 (control) from
run 5. The computer values for the average rates and amplitudes
and the elapsed time are: E. 35 cts/min, 6.7 volts, 94.5 hr;
F. 25, 5.9, 97; G. 21, 5.8, 115.5; H. 31, 4.7, 123.
84
-------
*5 n
0-
-5
«5
O
-------
-3
•2 -i
0 •
en -2
o
J«ll^^
0-
-2-
*3 -
0 -
10 20
SECONDS
30
Figure D-2 (cont.). Ventilatory signals for fish 5 (treated) from
run 5. The computer values for the average rates and amplitudes
and the elapsed time are: D. 25 cts/min, 4.4 volts, 94.5 hr;
E. 56, 1.8, 97 (after 1 hr of toxicant exposure, concentration =
0.6 mg/1 Cu++); F. 127, 3.8, 107.5 (after 11.5 hr of exposure,
1.9 mg/1); G. 32, 3.4, 115.5 (after 19.5 hr exposure, 0.6 mg/1);
H. 42, 6.1, 123 (between toxicant applications after 8 hr of
recovery time).
86
-------
*3 1
0-
-3
»3 1
0 -
UJ
en
-3
o-
10 20
SECONDS
A
30
Figure D-3. Ventilatory signals for fish 7 (treated) from run 5.
The computer values for the average rates and amplitudes and the
elapsed time are: A. 18 cts/min, 6.1 volts, 22.5 hr; B. 18,
6.0, 44.5; C. 21, 6.3, 74.5; D. 20, 5.3, 94.5.
87
-------
+ 3 n
0-
'-3J
+ 3 -i
|UU \J\tAs-Wfs**^fff^ k/v ryy* ^
A>^W\fV^Nv-^^
UJ
cc
0-
-3
+ 3 i
0 •
-3 J
10
. 2 Q
30
SECONDS
Figure D-3 (cont.). Ventilatory signals for fish 7 (treated) from
run 5. The computer values for the average rates and amplitudes
and the elapsed time are: E. 46 cts/min, 2.1 volts, 97 hr (after
1 hr of toxicant exposure, concnetration = 0.5 mg/1 Cu"1"1"); F. 75,
2.2, 107.5 (after 11.5 hr of exposure, 1.4 mg/1); G. 13, 1.7,
115.5 (after 19.5 hr exposure, 0.5 mg/1); H. 36, 6.7, 123 (between
applications after 8 hr of recovery time).
88
-------
+5 n
-5
OT + 3
O
>
-5
10
20
30
SECONDS
Figure D-4. Ventilatory signals for fish 8 (treated) from run 5,
The computer values for the average rates and amplitudes and the
elapsed time are: A, 37 cts/min, 7,7 volts, 22.5 hr; B. 40,
5.8, 44.5; C. 68, 13.9, 74.5.
89
-------
10
SECONDS
30
Figure D-r4 (cont.). Ventilatory signals for fish 8 (treated) from
run 5. The computer values for the average rates and amplitudes
and the elapsed time are: D. 38 cts/min, 7.7 volts, 94«5 hr;
E. 57, 3,6, 97 (after 1 hr of toxicant exposure, concentration =
0.5 mg/1 Cu++); F. 20, 1.8, 107.5 (after 11.5 hr of exposure,
1.3 mg/1); G. 26, 1.8, 115.5 (after 19.5 hr exposure, 0.5 mg/1);
H. 44, 6.9, 123 (between toxicant applications after 8 hr of
recovery time).
90
-------
------- |