9357

• UCREFRP Catalog Number: 9357
• Author: Beyers, D. W. and M. S. Farmer
• Title: Effects of Copper on olfaction of Colorado Pikeminnow
• USFW Year: 2001
• USFW - Doc Type: Environmental Toxicology and Chemistry
• Copyright Material: YES

908

Environ. Toxicol. Chern. 20,2001

were studied for the 96-h exposure (<10, 15.0,30.0,60.0, and
120 ILg/L copper). The 266-lLg/L concentration was not used
in the 96-h exposure because toxic effects were severe at 24
h, and it was anticipated that too few fish would survive the
longer exposure. Hamilton and Buhl [13] reported a 96-h LC50
of 269 ILg/L for Colorado pikeminnow exposed to copper un-
der similar water-quality conditions. Concentrations were as-
signed to replicate (n = 10) 1-L exposure beakers using a
balanced, randomized design. Beakers were filled with test
solutions to a depth of about 8.5 cm. Exposure solutions were
renewed every 24 h. Five fish were randomly assigned to each
exposure beaker. Fish were not fed during chemical exposure.
Cool-white fluorescent lamps were the only source of illu-
mination, and a 16:8-h light:dark photoperiod was maintained.

Physical and chemical conditions

Dilution water for copper exposures was a mixture of well
water and deionized water prepared to match the average hard-
ness in July of the Yampa River near Maybell, Colorado, USA.
Water quality for this time and location were selected because
of correspondence with spawning activity and presence of a
spawning site approximately 95 river-km downstream. Aver-
age water quality was estimated by calculating mean hardness
of two low (1981 and 1989), medium (1980 and 1982), and
high (1984 and 1985) water years (data from U.S. Geological
Survey gage 09251000; [14-19]). Target water quality char-
acteristics were hardness, 124 mg/L as CaC03; alkalinity, 100
mg/L as CaC03; and pH, 8.3. Dilution water was vigorously
aerated for approximately 48 h while being maintained at the
test temperature of 20 2: 20C. Dissolved oxygen, hardness, pH,
alkalinity, and specific conductance were measured daily dur-
ing toxicant exposures. Water temperature was measured con-
tinuously with a temperature recorder. Means and ranges for
the measured dilution water characteristics were dissolved ox-
ygen, 6.9 (6.5-7.5) mg/L; hardness, 117 (113-120) mg/L as
CaC03; pH, 8.3 (8.1-8.5); alkalinity, 80 (71-88) mg/L as
CaC03; specific conductance, 240 ILS/cm, and temperature,
18.5 to 21.0oC.

Copper solutions and analytical procedures

Stock solutions of analytical-grade copper sulfate (Mal-
linckrodt, Paris, KY, USA) were prepared in deionized water.
Exposure concentrations were prepared by pipetting the de-
sired amount of toxicant stock into 1-L glass beakers con-
taining 0.75 L dilution water. Exposure solutions were stirred
and transferred to exposure beakers within 30 min of prepa-
ration.
Toxicant concentrations were measured at the beginning of
exposure periods. On each occasion, two 50-ml samples were
collected from separate beakers for each exposure concentra-
tion. Unfiltered samples were placed in acid-washed polyeth-
ylene bottles, acidified to pH < 2 with analytical-grade nitric
acid, and held at 40C until analyzed by inductively coupled
plasma emission spectroscopy (Soil, Water, and Plant Testing
Laboratory, Colorado State University, Fort Collins, CO,
USA). Chemical analysis confirmed accuracy of exposure con-
centrations. Measured copper concentrations averaged 92%
(standard error = 4.8) of nominal. Measured and nominal con-
centrations were in close agreement; consequently, statistical
analyses were based on nominal concentrations.

Behavioral assay

Skin homogenate that was assumed to contain fright-pher-
omone was prepared before each assay. Donor Colorado

D.W. Beyers and M.S. Farmer

pikeminnow were juveniles obtained from the same mass cul-
ture used for study fish. Homogenate preparation involved
removing skin posterior to the head and anterior to insertion
of the caudal fin. Skin was cut into pieces and homogenized
(Omni-mixer, Sorvall, Norwalk, CT, USA) in dilution water
to a final concentration of 12.5 g/L.
The acclimation period for each behavioral assay was ini-
tiated immediately after conclusion of the 24- or 96-h expo-
sures. Fish and solutions from each replicate were transferred
to flow-through observation aquaria receiving dilution water
at a rate of 50 ml/min. Observation aquaria were lOX 20 X
15 cm high, and depth of water was 12 cm. Each aquarium
was enclosed within a blind to visually isolate fish. An ob-
servation port allowed access for a video camera lens. Fish
were allowed to acclimate to observation aquaria for approx-
imately 90 min before being tested for presence of a fright
reaction. After the acclimation period, movements of fish in
each aquarium were video recorded for 1 min, followed by
introduction of skin homogenate and an additional 1 min of
video recording. Aliquots (0.1 ml) of skin homogenate were
introduced into observation aquaria through injection ports in
the water-delivery system, and final concentration of the ho-
mogenate in aquaria was 0.625 mg/L. Preliminary studies in
which dilution water was injected into the delivery system
showed that the procedure did not disturb the fish or elicit a
fright reaction. Behavioral assays were conducted so that per-
sons responsible for video camera operation and fright-pher-
omone injections were unaware (blind) of the experimental
treatment assigned to fish within each aquarium. Upon con-
clusion of behavioral assays, three fish from each experimental
treatment were preserved for SEM observations and the re-
mainder were returned to culture facilities. Remaining fish
from the 96-h exposure were cultured for 14 d, then were
reassayed using the procedure described above to evaluate the
potential for recovery of olfactory ability.

.(

.J
I
II

Video interpretation

The behavioral reaction to fright pheromone is facilitated
by social interactions between fish. Consequently, the com-
bined response of all fish within an observation aquarium (ex-
perimental unit) was the unit of measurement. Generally, un-
disturbed fish were dispersed and oriented in different direc-
tions within an aquarium and only occasionally changed their
position during the 1-min preexposure observation period. Cri-
teria used to identify a positive fright reaction response were
demonstration of one or more of the following behaviors: (1)
cover seeking, characterized by fish assembling into a polar-
ized school, then moving toward the bottom of the aquarium
and reducing movement; (2) agitation, characterized by rapid
movement and frequent turning; and (3) dashing, characterized
by one or more fish displaying frenzied behavior, including
jumping out of the water, repeated and rapid changes in di-
rection, and swimming against aquarium walls. The criteria
were used only to qualify presence or absence of a fright
reaction and not to quantify intensity of the response.
Video interpretation was conducted by an observer who
was unaware (blind) of the experimental treatment assigned
to fish within each aquarium. The criteria were used to interpret
behavior during the first minute of each 2-min recording in-
terval in order to identify fish that were likely to give a fa1se-
positive response. Fish that demonstrated cover seeking, ag-
itation, or dashing behaviors before introduction of the skin
homogenate were excluded from subsequent analysis. Presence

,
..