United States
Environmental Protection
Agency
Environmental Research
Laboratory
Duluth MN 55804
Research and Development
EPA-600/S3-81-003 Apr. 1981
Project Summary
Effects of Fluctuating,
Sublethal Applications of
Heavy Metal Solutions
Upon the Gill Ventilatory
Response of Bluegills
(Lepomis macrochirus)
John Cairns, Jr., Kenneth W. Thompson, and Albert C. Hendricks
This study demonstrates the use of
minicomputers for continuously
observing the ventilatory behavior of
fish in order to monitor the quality of
the water passing through fish sensing
chambers. The water source could be
an industrial effluent, agricultural
runoff, or a carefully controlled
laboratory source as in this study.
Situations of complex, fluctuating
toxic effluents were simulated in order
to describe responses that could be
expected. These results were then
compared with those of control fish
which were not exposed to toxic
effluents.
Not only did ventilatory rates
significantly increase in response to
laboratory effluents (sublethal con-
centrations of heavy metal solutions),
but also the amplitude of the ventila-
tory signal was reduced considerably.
This is a promising system that could
be developed into a useful, on-line
environmental monitor for industrial,
agricultural, or other purposes.
This Project Summary was devel-
oped by EPA's Environmental Re-
search Laboratory, Duluth, MN and
it's Newtown Fish Toxicology Station,
Cincinnati, OH, to announce key
findings of the research project that is
fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
This project was undertaken to
investigate the feasibility of using
ventilatory behavior as a monitor of
environmental quality. The weak electrical
signals produced by the movement of
the muscles in the branchial region as a
fish ventilates its gills can be detected
by submerged non-contact electrodes
placed at either end of a holding tank
(Figures 1 and 2). The primary objectives
were to describe the average ventilatory
behavior as well as the response to
sublethal amounts of various heavy
metal solutions.
Most previous studies of the effects of
environmental changes on the ventila-
tory behavior were carried out by visual
observation of short, intermittent strip
chart recordings. Interfacing with a
minicomputer allows the continuous
accumulation of data and, thus, a more
complete picture of natural variability as
well as of the response to toxicants.
In this study, the ventilatory signals
from 16 fish at a time were amplified
-------
AM
Figure 1.
B
A bluegill in a ventilatory sensing chamber. Water flows into the
receiving chamber (A), through the sensing chamber (Bl. and out the
drain (C). The elctrodes are the S-shaped wires at either end of the
sensing chamber. The signal is carried to an amplifier (AM).
Lepomis macrochirus
(Bluegill Sunfish)
+3 -i
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•2
Q)
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10
20
30
Seconds
Figure 2. An example of the amplified ventilatory signal of a bluegill ventilating at
approximately 20 times per minute.
using high-grain amplifiers which were
interfaced to a PDP8/E minicomputer.
These data were accumulated contin-
uously, and both the ventilatory signal
and the average amplitude of the
ventilatory signal were recorded on
magnetic tape for later analysis.
Most fish toxicity data are based on
96-hour, constant concentration
laboratory tests, usually for a single
toxicant at a time. Actual industrial or
agricultural effluents are usually
complex mixtures that vary in composi-
tion and concentration, and the present
study investigated the effects of
fluctuating as well as constant toxicant
applications.
The fish were exposed to sublethal
concentrations of divalent zinc, divalent
copper, to mixtures of zinc and copper,
or to mixtures of zinc, copper, divalent
nickel, and hexavalent chromium.
Median lethal concentrations (LC50)
were determined with 96-hour continu-
ous flow toxicity tests (Table 1). The
toxicity of the two mixtures was found to
be additive.
Table 1. Ninety-six Hour Median
Lethal Concentrations
(LC50) of the Various
Toxicants Used in This
Study
95%
LC50 Confidence
Toxicant* (mg/l) Limits
MixA
Zn*2
Cu*
/V/+2
Cr+6
Zn+2
Ci/2
MixB
Zn+*
C
-------
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Fish 9 Control
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Fish 13 Treated
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Figure 3. Examples of the variability seen in the ventilatory rate response of the bluegill to solutions of Zn++ and Cu++. The first
fish in all six cases are untreated controls while the remaining two were chosen to give examples of the range of the
variability of response to the toxicant solutions. The solid line on the fourth vertical plane in each case indicates the
shape, duration, and concentration of the toxicant application.
The addition of dissolved salts to the
water in the holding chamber caused a
reduction in the electrical resistance
and this, when carried to extremes (i.e.,
sea water), can cause a large reduction
in the signal amplitude. However, none
of the toxicant concentrations used in
this study could be shown to cause more
than a 10% reduction in amplitude, and
most would cause less than 2%
reduction. Thus, the observed amplitude
reduction indicates a significant
response to the toxicant solutions—a
response that can be used to monitor
water quality, or more significantly, to
enhance the use of ventilatory rate for
that purpose.
Chi squared goodness of fit tests and
Cochran's C statistic indicated that
within each interval (pre-exposure,
exposure, post-exposure) the average
rates and amplitudes were normally
distributed and the variances were
homogeneous. Thus, it was appropriate
to compare the results between fish
using an analysis of variance (ANOVA).
The indication from these tests was that
there was little or no statistical
difference between the fish before or
after exposure, but, during the exposure
period, a considerable difference
existed between the exposed and
control fish. This was true for both rate
and amplitude studies. Statistically
differentiating between the responses
to the various patterns of application
was difficult due to the extreme
smoothing of data. However, it is
significant to note that the fish are
capable of responding in similar manner
to repeated exposures to toxic effluents
(Figure 3).
Conclusions
1. The ventilatory response of the blue
gill was shown to be measurably
affected by sublethal concentrations
of zinc, copper, and complex mixtures
of heavy metals.
4 US GOVERNMENT POINTING OFFICE H61-757-OU/7055
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2. The typical response of this species
was an elevated ventilatory rate and
a reduced ventilatory amplitude in
the presence of sublethal concentra-
tions of the various toxicant solutions.
3. Fish are capable of responding
similarly to repeated exposures of
toxicant solutions, indicating the
feasibility of an environmental
monitoring system based on ventila-
tory behavior.
4. The amplitude response was primar-
ily due to a real change in ventilatory
behavior of the fish, not to a change
in electrical properties of the water
due to the addition of the toxicant
solutions.
5. The ventilatory data met the require-
ments for parametric statistical
analyses when comparing individ-
uals. Thus, when sample size is large
enough, conventional methods such
as the analysis of variance can be
employed.
John Cairns, Jr.. Kenneth W. Thompson, and Albert C. Hendricks are with the
University Center for Environmental Studies, Virginia Polytechnic Institute
and State University, Blacksburg, VA.
William B. Horning, II, is the EPA Project Officer (see below).
The complete report, entitled "Effects of Fluctuating, Sublethal Applications of
Heavy Metal Solutions Upon the Gill Ventilatory Response of Bluegills
(Lepomis macrochirus),"fO''Qte''/Vo. PB 81-150 997; Cost: $11.00, subjectto
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Newtown Fish Toxicology Station
Environmental Research Laboratory—Duluth
U.S. Environmental Protection Agency
Cincinnati, OH 45244
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
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Agency
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