tXEAI
       WATER POLLUTION CONTROL RESEARCH SERIES
18050 EDii U/71
     The  Use  of Bluegill  Breathing
              to  Detect  Zinc
          U.S. ENVIRONMENTAL PROTECTION AGENCY

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          WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes the
results and progress in the control and abatement of pollution
in our Ration's waters.  They provide a central source of
information on the research, development and demonstration
activities in the Environmental Protection Agency, through
inhouse research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.

Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications Branch
(Water), Research Information Division, R&M, Environmental
Protection Agency, Washington,  B.C. 20k60.

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             THE USE OF BLUE6ILLS TO DETECT ZINC
                        John  Cairns, Jr.
                                and
                        Richard E. Sparks
      Virginia  Polytechnic Institute and  State University
    Biology Department and Center for Environmental Studies
                   Blacksburg,  Virginia 24061
                              for the

                 ENVIRONMENTAL  PROTECTION  AGENCY
                       Project  #18050 EDQ
                          December 1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402 - Price 55 cents

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               EPA Review Notice
This report has been reviewed by the Environ-
mental Protection Agency, and approved for
publication.  Approval does not signify that
the contents necessarily reflect the view and
policies of the Environmental Protection
Agency, nor does mention of trade names or
commercial products constitute endorsement or
recommendation for use.
                      ii

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                           ABSTRACT
The presence of zinc at concentrations of 8.7, 5.22, 4.16 and 2.55 mg/1
in dechlorinated municipal tapwater was detected by an increase in
breathing rate or a change in breathing rate variance of bluegills.
None of the fish exposed to the three lower concentrations died during
the experiments.  The criterion for detection was an arbitrary number
of responses occurring at the same time.  When the criterion was
changed from a single response to three responses occurring at the same
time, the number of false detections ("detections" occurring before zinc
addition) decreased, but the laq between zinc addition and detection
increased.

Zinc concentrations of .025 and .075 mg/1 (approximately 1/100 and 1/34
of 2.55 mg/1, respectively) did not appear to affect the reproduction
and growth of bluegills in the laboratory, but .250 mg/1 zinc (approx-
imately 1/10 of 2.55 mg/1) inhibited spawning in ripe bluegills and
killed newly-hatched fry:

An in-plant system for the prevention of fish kills caused by spills
could be developed by monitoring several biological functions of fish
simultaneously to obtain informational redundancy and reduce error;
by exposing test fish to hiqher waste concentrations than occur in
the receiving stream as a safety factor; automating the collection and
analysis of data to reduce laq time; and by choosing appropriate
criteria for detection.

This report was submitted in fulfillment of Project 18050 EDQ under the
sponsorship of the Water Quality Office, Environmental Protection
Agency.
                             m

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                            CONTENTS
Section                                                        Page
  I        Conclusions                                           1
  II       Recommendations                                       3
  III      Introduction                                          5
  IV       Effects of zinc on fish breathing                     7
  V        Fish reproduction and growth                         29
  VI       Acknowledgments                                      41
  VII      Literature cited                                     43
  VIII     Publications                                         45

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                              FIGURE
                                                                 Page
1     A monitoring unit for industrial use                        26
                                vi

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                              TABLES
 No.

 1     Experimental  conditions                                    9

 2     Method for analyzing the breathing  rates  of  bluegillSj
         experiment 8                                         10-11

 3     Routinely determined characteristics  of dilution water     14

 4     Flow rates and zinc concentrations  during experiments      15

 5     Number of fish showing responses, before and  after
         exposure to 5.22 mg/1  zinc                              17

 6     Number of fish showing responses, before  and after
         exposure to 4.16 mg/1  zinc                           18-19

 7     Number of fish showing hetergeneous variances,
         before and after exposure to  4.16 mg/1  zinc             20

 8     Number of fish showing responses, before  and after
         exposure to 2.55 mg/1  zinc                              22

 9     Effectiveness of zinc detection using increases in
         fish breathing rates                                    23

10     Effectiveness of zinc detection using successive
         comparisons of breathing rate variances                 24

11     Zinc and dissolved oxygen concentrations, and
         temperatures in breeding tanks                          33

12     Survival  of adult bluegills, and weights, lengths
         and conditions of gonads of adults  at end  of
         breeding experiment                                  34-35

13     Spawning of adult bluegills and percentage hatch
         of eggs at four zinc concentrations                     37

14     Survival  and growth of bluegills in four  zinc
         concentrations                                          38

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                          SECTION I

                         CONCLUSIONS

1.  Breathing signals of bluegills show diurnal changes in rate and
amp!i tude.

2.  The presence of zinc at concentrations of 8.7, 5.22, 4.16 and
2.55 mg/1 in dechlorinated municipal tapwater was detected by in-
creases in breathing rate.  The presence  of zinc at a concentration
of 4.16 mg/1 was also detected by changes in breathing rate variance.
Increasing the criterion for detection from a response by one fish
to simultaneous responses by three fish generally reduced the number
of false detections ("detections" occurring before zinc was added to
the water) and increased the lag between introduction of zinc and
detection.

3.  Zinc concentrations of .075 and .025 mq/1 (approximately 1/34
and 1/100 of 2.55 mg/1) in dechlorinated municipal tapwater did not
appear to affect the reproduction and growth of bluegills in the
laboratory, but spawning was markedly reduced and there was complete
mortality of fry in .250 mg/1 zinc (approximately 1/10 of 2.55 mg/1).
A zinc concentration of .250 mg/1 is not safe for chronic exposure
of bluegills under conditions used in these experiments.

4.  A workable biological monitoring system probably could be developed
for the prevention of fish kills caused by industrial spills by monitor-
ing two or more biological functions to increase informational redun-
dancy and reduce error, automating data collection and analysis, exposing
tesffish to higher waste concentrations than occur in the receiving
stream as a safety factor, and choosing appropriate criteria for detec-
tion.

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                           SECTION II

                         RECOMMENDATIONS

The technique for monitoring fish breathing could be useful in the pre-
vention of fish kills caused by industrial spills and as a source of
biological information on which decisions in a river management system
could be based.

V!e therefore recommend that a system for monitorina the resnonse of fish
to water quality be develooed as follows:  (1) by combining the technique
for monitoring fish breathing with a technique for monitoring fish activity,
already developed under Project 18050 EDP Water Quality Office, Environ-
mental Protection Agency, thereby increasing the reliability of the monitor-
ing system (2) reducing the time required for data analysis by converting
analog breathing signals to numbers and then processing by comouter (3)
by laboratory testing of the system under simulated industrial conditions
to determine whether water potentially toxic to fish can be detected fast
enough to permit survival and recovery of the fish in the monitorino
unit and fish maintained under simulated "downstream" conditions (4) sub-
sequent testing of the monitoring unit at an actual industrial site.

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                           SECTION III

                          INTRODUCTION

The purpose of this research was to develop a method for ram'dly detect-
inq nonlethal effects of toxicants on the breathing rate of fish.  In
conjunction with already existing methods for measuring the chemical
and physical characteristics of v/ater, methods for rapidly measuring
biological effects of water guality would be useful in industrial plants
to prevent fish kills caused by soil Is, in river basins to provide infor-
mation on which quality control measures could be based (Cairns, et al.,1970)
and in water quality laboratories to provide the same information on  a
toxicant as a chronic bioassay, but in less time, as Drummond and Spoor
(1971) have demonstrated.

With the above potential applications in mind, the research was directed
to the goal of simplifying data collection and speeding analysis as much
as possible.

This research was an extension of preliminary work on the effects of
lethal concentrations of zinc sulfate on the breathing rate and heart
rate of bluegills, Lepomis macrochirus Rafinesque  (Cairns, et al., 1970).
The electrocardiogram (ECG) was obtained by inserting a needle electrode
in the breast of the fish.  This technique had to be abandoned during
the course of the present experiments because the tissue around the site
of electrode insertion softened within two days, the electrode nulled
out, and ECG's could not be obtained for the four to eleven day du-
ration of the experiments.

For the purpose of developing a new technique, it seemed best to use a
toxicant that was easily dosrd and measured, and whose toxicity to fish
was well documented: zinc sulfate.  The test species was the bluegill.

The effects of zinc sulfate on the growth and reproduction of bluegills
was also determined in an attempt to relate the effects of various zinc
concentrations on the breathing rate to safe concentrations for chronic
exposure.

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                           SECTION IV

                EFFECTS OF ZINC ON FISH BREATHING

Materials and methods:  Blueqills were obtained from the McKinney Lake
National Fish Hatchery, Hoffman, North Carolina, and were held for at
least two months in 50-gallon aquaria in a stock room prior to being
used in an exoeriment. 'The mean weight of the 52 fish used in the
experiments was 86.4 gms (S.D. 21.4) and the mean standard length (the
distance from the tip of the snout to the origin of the caudal fin
rays) was 13.4 cm (S.D. 1.1).  Fish were fed frozen Gordon Formula
daily (Axelrod, 1952), while the room lights were on, at a time chosen
from a random number table, to keep from training the fish to feed at
a particular time.

The stock room and the experimental room received water from a 500-
gallon tank where the temperature was adjusted and the chlorine re-
moved from municipal tan water by dripping in sodium thiosulfate.  At
least once a day, the following characteristics of the water were
determined: phenolphthalein alkalinity, total alkalinity by the brom-
cresol green-methyl red indicator method (Standard Methods, 1965),
nH with a Fisher Accumet pH meter, total hardness with a Hach test kit,
temnerature with a mercury thermometer, and chlorine with a Hach model
CN-46 chlorine tester.  Dissolved oxygen concentrations were measured
at random intervals by the azide modification of the iodometric method
(Standard Methods, 1965), and Conner and zinc concentrations were
measured at random intervals by atomic absorption spectrophotometry.

Seven fish that were to be used in an experiment were netted from the
stock tanks and placed in separate 5-gallon tanks in the experimental
room for one week.  Each fish was then moved into another 5-gallon
tank in the same room for another week, before being used in an ex-
periment.  All the 5-gallon tanks were painted black on the outside
so the fish could not see each other.  By following this routine,
experiments using six fish (one extra fish was kept as a spare in
case of disease or injury) could be run once a week, while additional
grouns of seven fish were acclimating.  Fish were fed approximately
1/4 gram of frozen Gordon Formula at a time chosen from a random number
table once each day during the two-week additional acclimation period,
and were not fed during experiments, to keep feces from accumulating
in the test chambers.

The 8x8x8 foot experimental room was constructed to minimize
disturbances to the fish, because preliminary experiments had shown
that floor vibrations or neoole talking could cause the heart rate
of the fish to slow and breathing to cease for several seconds.  The
sides and ceiling of each room v/ere made of an outer wall of 1/2-inch
plywood, a 3-inch inner layer of fiberglass insulation and an inner
wall of 1/2-inch Celotex.  There was a door in one wall.  The table
which held the test chambers rested on foam rubber pads and all
electrical and water connections were flexible to minimize the trans-
mission of vibrations.

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 Durotest Optima fluorescent bulbs were used  to approximate  the spectrum
 of natural light.  A dimming unit simulated  dawn  and  dusk by turning on
 the lights and gradually increasing the intensity over  a half-hour
 period starting at 6:30 a.m., and gradually  decreasing  the  intensity
 to 0 over a half-hour period starting at 6:30  p.m.  The maximum light
 intensity was approximately 100 foot~candles,  and the minimum intensity
 before the lights went out was approximately 1/2  foot candle, measured
 at the surface of the aquaria.

 A Grass model 5-D polygraph was modified so  that  it would automatically
 make 15-minute recordings  of breathing sionals at half-hour
 intervals from 6 to 8 in the morning and evening  and  at one-hour in-
 tervals the rest of the day.  Shielded cables  running through the walls
 of the isolation room connected each test chamber to  a  oolygraph channel.

 The test chambers and the  methods of preparing the fish were the same
 as described by Cairns, et al_.  (1970)  for experiments with  lethal zinc
 concentrations.   The fish  were placed  in test  chambers  by 6 p.m. one
 day and the recording began at 6 a.m.  the next day.   The only changes
 were that six plastic test chambers  were used  at  a time, and each chamber
 was placed in a  compartment, open only at the  top.  Therefore, adjacent
 fish could not see each other,  and if  one became  disturbed  enough to
 swim,  the response would not pass from one fish to another.  Water or
 zinc solutions were pumped into the  test chambers by  Harvard variable
 speed  peristaltic pumps (model  number  1210).  Water and  test solutions
 flowed by gravity out of the test chambers and were not reused.  It
 was necessary to insert a  stopcock  and  a flow meter  in each line to
 control  the flow rates.

 Zinc solutions were  prepared by thoroughly dissolving a weiohed amount
 of reagent grade ZnSO^H^O (Fisher  catalog No. Z-68) in 95 liters of
 dechlorinated tap water drawn from the  500-qallon reservoir and stored
 in a plastic  garbage can.   The  amount  of ZnSO^'/^O was calculated to
yield  a  certain  nominal concentration  of Zn++.   Up to six garbage cans
were connected by siphons.   Fresh  zinc  solutions were prepared twice a
day.   The  peristaltic  pumps  withdrew the  solution from  the last garbage
can which  contained  a  temperature  controller.  Actual zinc concentrations
in  the garbage cans  and the  outflow from  each  test chamber were deter-
mined  daily by atomic  absorption  snectrophotometry, starting with experi-
ment 6.

Table  1  shows the  conditions  for  each of  nine  experiments.   An electrode
was inserted  in  each fish  for some of the experiments.  Breathing sig-
nals were  obtained even when  the  electrode pulled out of the fish, so
four experiments were  carried out with the electrode taped to the bottom
of the test chamber.  Tricaine methanesulfonate (MS-222) at a concentra-
tion of  100 mg/1 was used  to  anesthetize  fish when the electrode was
inserted,  and no  anesthetic was used in  the other experiments.

No zinc was added  to the water  during the first four experiments, to
determine whether  there were diurnal patterns  and day-to-day changes


                                8

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Table 1 .  Experimental conditions
No. of control Duration
fish exposed of control
to dilution No. of fish period

Experiment
1
2
^ 3
4
5
6
7
8
9
water with no
zinc added
6
6
6
6
1
1
1
2
1
exposed
to zinc
0
0
0
0
5
5
5
4
3
(prior to
zinc addition)
96 hours
146 hours
98 hours
120 hours
28 hours
28 hours
28 hours
148 hours
127 hours
Duration

of exposure Total
to zinc
0 hours
0 hours
0 hours
0 hours
96 hours
92 hours
92 hours
96 hours
48 hours
duration
96 hours
146 hours
98 hours
120 hours
124 hours
120 hours
120 hours
244 hours
175 hours
Active
electrode
in fish
yes
yes
yes
no
no
yes
yes
no
no

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            Table 2.   Method for  analyzing  the breathing
                      rates  of bluegills, experiment 8
                              JDay_I
Period
Hour
Fish 1
Fish 2
Fish 3
Fish 4
Fish 5C

Fish 1
Fish 2
Fish 3
Fish 4
Fish 5C
Total

Fish 1
Fish 2
Fish 3
Fish 4
Fish 5C
Total
6
27
29
11
11
21

20
12
9
17
0

19
14
11
16
0
6:30
30
20
12
11
21

21
9
8
18
0

20
16
16
9
16
0
Dawn
7
39
29
15
16
23

29
18
10
14
19
0

12
28
17
8
18
0
7:30
42
Day
27
24
12
10
26
0
Day
18
31
16
10
23
0
8
34
19
16
2
32
29
14
9
33
0
7
16
32
14
11
24
0
9 1
Light
0 11
42 40 42
§32 28
18 15
13 16
36 36


35 34 30
28 40 42
14 15 12
11 10 11
30 37 33
0
^
0 0
r
22 24 26
34 34 36
16 2
2 20
10 11 12
28 28 28
0
1 0
12
39
28
18
14
35

26
34
18
10
33
0

15
24
16
16
24
0
1
41
28
18
14
32

30
26
12
10
31
0

28
27
15
16
25
0
Note:   Blanks indicate that the amplitude of the breathing signal  was
        so low that the rate could not be determined.
      T Measured zinc concentration of 4.16 mg/1  introduced, except for
        Fish 5, which was not exposed to zinc.
     QMaximum breathing rate for each fish during each period of  the
        first day.
        Breathing rates on second and seventh days which exceeded first
        day maxima.  The total number of fish showing increased breath-
        ing rates is shown at the bottom of each  column.
                                10

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Table 2 (continued)
        Day 1
2
37
27
16
12
32
3
38
46
15
11
35
4
40
16
12
32
5
35
14
12
33
6
38
13
6:30
45
40
13
13
42
Dusk
7
27
16
13
37
7:30 8
25 23
24 16
© 10
12 9
27 21
9
20
12
9
9
19
10
22
11
8
20
11
20
10
10
18
Dark
12 1
21
10
10
19
25
9
7
18
2
24
12
8
16
3
24
8
8
4
26
12
10
19
5
14
16
Day 2
27
30
12
10
30
0
|62|
28
12
16
30
1
22
24
13
10
32
0
22
28
13
11
27
0
33
45
13
10
27
0
26
20
15
11
26
0
22
42
15
13
31
0
34
46
14
10
26
0
24
46
16
12
36
0
30
40
15
11
29
0
31
22
16
13
28
0
38
40
16
10
28
0
22
19
13
10
29
0
30
42
15
12
26
0
15 15
28 24
18 11
12 8
23 22
0 0
Day 7
57 52
18 10
11 10
11 15
1 0
16
15
10
7
21
0
50
20
16|
17
3
17
10
7
17
0
48
22
12
|62
16
3
14
10
8
16
0
48
28
|14|
61
14
4
15
11
8
16
1
46
23 1
9
59
14
3
13
12
9
20
0
47

|14
55
15
3
Ib
13
8
16
0
43

16
49
18
3
16
10
8
17
1
48

14|
59
15
3
13
15
10
10
18
0
49
9
54
15
2
16
15
10
10
21
0
46|
12
55|
15
2
          11

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 in the breathing rate.  The results showed that analysis  of variance
 techniques could not be used for statistical  comparisons  between  ex-
 periments with no added zinc and later experiments  with zinc because
 the breathing rate variances at different times of  day for the  same
 fish, and at the same time of day for different fish,  were heterogeneous.

 An alternative method of analyzing the data was based  on  the observed
 diurnal change in breathing rate (Sparks, et  a!., 1970),  and on the
 observation that the breathing rate at each hour during the first day
 of recording was generally greater than the breathing  rate at the
 same hour on subsequent days, perhaps due to  incomplete recovery
 from the stress of handling.  The experimental  day  was divided  into
 four periods:  (1) a period from 6 to 8 a.m.  (dawn), when the breathing
 rate changed markedly (2)  a period from 9 a.m.  to 5 p.m.  (light)  when
 the rate was comparatively high (3) another period  of  rapid change from
 6  to 8 p.m.  (dusk) and (4) a period from 9 p.m.  to  5 a.m.  (dark)  when
 the rate was comparatively low.   The maximum  breathing rate of  each fish
 during each  period of the  first day was circled, as in Table 2.   Any in-
 crease in a  fish's breathing rate during a subsequent  period that exceed-
 ed the maximum rate observed during the corresponding  period of the first
 day was considered to be a response to a stimulus greater than  the stim-
 ulus  of being netted and placed  in the test chamber.   A box is  drawn
 around the responses in Table 2  that occurred  on day  2,  before any zinc
 was  added to the water, and on day 7,  when zinc was added  at 10 a.m.
 The  total  number of experimental  fish  (fish exposed to zinc on  day 7)
 showing responses  at each  hour of day 2 and day 7 is shown.  The  control
 fish,  5C,  was not  exposed  to zinc and  showed  no responses.   There are
 missing observations in Table 2  because of a  diurnal breathing  ampli-
 tude  change.   The  amplitude of the breathing  signals of some fish was
 so low that  rates  during the dark and  dawn periods  could  not be de-
 termined every hour.  Maximum breathing  rates during each  period  of
 the first  day were  determined from whatever values  were available.  If
 no breathing  rates  could be determined for a  fish during  a whole  period
 of the  first  day,  no further analyses  were made using  that  fish.

The results also suggested  another method  of  analyzing the  data.  During
 the four periods of the day described  above,  the breathing  rate of a fish
exposed for several  hours to a sublethal concentration of  zinc  generally
would fluctuate between high  values and  the normal  values  for that fish.
The fluctuation in  breathing  rate  could  be  expressed quantitatively as
a variance, and a two-sample  test  for  heterogeneity of variances was used
to determine whether  a significant  difference existed  between breath-
ing rate variances  (Sokal and Rohlf, 1969).  The breathing rate variance
for each period of  the first  day was computed for each fish, and compared
to the  variance for  the corresponding  period of the second day  for the
same fish.  If the  variances were  not  significantly different,  then the
fish was considered  to be exhibiting normal variation  in breathing rate
on day 2, and the variance  for each period of day 2 then was compared to
the variance for the corresponding  period  of day 3.   If the  variances
for corresponding periods of  days  1 and  2 were significantly different,
                               12

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the fish was considered to be exhibiting a response on day 2, the variance
for that period of day 2 was dropped, and the next comparison was made
between corresponding periods of day 1 and day 3.  For examole, if the
breathing rate variance for fish 1 during the dawn period of day 1 was
significantly different from the variance during the dawn period of day
2, the fish was considered to have shown a response during the dawn
period of day 2.  The variance for the dawn period, day 2, was dropped,
and the breathing rate variance for the dawn period of day 3 was compared
to the breathing rate variance for the dawn period of day 1.  Experiment
8 was analyzed both by comparing breathing rates on each day to the
maximal rates on day 1 and by variance comoarisons.  The effect of using
two significance levels (a = .05, a = .01) on the number of responses
obtained by variance comparisons was determined.

As stated above, the response of an individual fish was defined as a
change in breathing rate variance or an increase in breathing rate.  The
number of fish showing responses at one time was used as the criterion
for detection of zinc, and the effect of requiring different numbers of
simultaneous responses was examined.  For example, experiment 8 was
analyzed using the rule that a response by a single fish would count as
a zinc detection.  The number of false detections ("detections" occurring
before any zinc was added to the water) and the lag time (the time be-
tween zinc addition and detection) were determined.  Experiment 8 was
analyzed again using the rule that responses by two fish at the same
time would count as a detection.  The number of simultaneous responses
required for a detection was increased up to the total number of fish
exposed to zinc (three or four), and the number of false detections
and the lag time determined in each case.

Unavoidably, various extraneous disturbances to the fish were included
in the experiments.  The door of the experimental room was opened for
three minutes once a day during each experiment in order to feed the
fish that were acclimating.  The only response by the fish was a pause
in breathing lasting up to half a minute, followed by rapid breathing
for a few more minutes.  Breathing rates were never taken from the dis-
turbed portion of the record.  An earthquake of sufficient magnitude to
rattle bookcases occurred during experiment!. Workmen spent two hours
breaking open a cement wall in an adjoining laboratory during exper-
iment 9.  The effects of the last two disburbances are discussed in the
next section.

Results:  The characteristics of the dilution water are shown in Table 3
and the flow rates and zinc concentrations for each experiment are shown
in Table 4.

One of five fish exposed to a zinc concentration of 8.7 mg/1 died after
77 hours of exposure and another died after 87 hours.  None of the fish
died during any of the other experiments described below.  Each of the
fish exposed to 8.7 mg/1 zinc showed increases in breathing rate after
5-16 hours of exposure.  Maximum breathing rate increases ranged from
                               13

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Table 3 .   Routinely determined characteristics
           of dilution water.
Water Characteristic
Total hardness (mg/1 as CaC03
Phenol phthalein alkalinity
(mg/1 as CaC03)
Total alkalinity (mg/1 as CaC
DH
Temperature (°C)
Dissolved oxygen
Chlorine
Zinc, copper
Number
of
Analyses
) 394
393

03) 393
397
396
Maintained
None
Mean
51
0.0

41.3
7.8
19.7
at air

Standard
Deviation
10
0.0

8.8
0.3
1.8
saturation

Zinc and copper concentrations were
                        less than  .03 mg/1 in random analyses

                        of the dilution water by atomic

                        absorption photospectrometry.
                     14

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Table  4 •   Flow rates and zinc concentrations during experiments.

              NominalMeasured
               zinc      concentration          Flow rates
          concentration     (mg/1)     No.  of    (ml/min)    No.  of
Experiment   (mg/1)	mean S.  D.    samples  mean S.  D.   samples
i n 	 	

2 n 	 	
•3 n 	 	

4 n 	 	
c 07 	 	

6 4.9 ;4.28 0.25 20
7 2.8 2.55 0.42 15
8 4.9 4.16 0.27 24
9 6.0 5.22 0.20 9


inn
1 UU
QP.
JO
Q7
y /

94
84
98
99
7
/

c
•j
1 0
7
/
7
12
5
2
49
tt.
._
1ft
10
•ag
ou
7?
/ &
42
36
54
40
Note:  Dashes indicate that no analyses were made.
                              15

-------
 51-73 breaths/min. and occurred after exposure times ranging from 5  to
 79 hours.  Further analysis was impossible because of inadequate  records.

 Table 5 shows the breathing rate response data for three fish exposed
 to a measured zinc concentration of 5.22 mg/1  and one control fish ex-
 posed at the same time to dilution water containing no added zinc.  The
 breathing rates of the control  fish exceeded the first-day maximal rates
 just once (12midnight,
-------
         Table   5.  Number  of  fish  showing  responses,  before  and  after  exposure  to  5.22 mg/1 zinc
Time
Day
2

3

4

"5

6

7

8


Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Gam
0
0
0
0
0
0
0
0
0
0
1
0
0
0
7
0
0
0
0
0
0
0
0
0
0
0
0
1
0
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
9
0
0
0
0
0
0
0
0
0
0
0
0
end

10 11
0 0
0 0
1* 1*
0 0
0 0
0 0
0 0
0 0
0 |l
0 0
1 2
0 0
12
0
0
0
0
0
0
0
0
0
0
1
0
1pm
1
0
0
0
0
0
0
0
0
0
1
0
2
0
0
0
0
0
0
0
0
0
0
1
0
3
0
0
0
0
0
0
0
0
2
0
1
0
4
1
0
0
0
0
0
0
0
2
0
1
0
5
0
0
0
0
0
0
0
0
0
0
2
0
6
0
0
0
0
0
0
1
0
1
0
1
0
7
1
0
0
0
0
0
0
0
0
0
2
0
8
1
0
0
0
0
0
0
0
1
0
0
0
9
0
0
0
0
0
0
0
0
0
0
1
0
10
0
0
0
0
0
0
0
0
1
0
1
u
11
0
0
0
0
0
0
1
0
1
0
2
u
12
0
0
1
0
1
0
0
0
1
1
1
0
lam
0
0
2
0
0
0
0
0
1
0
1
0
2
0
0
0
0
0
0
0
0
1
0
1
U
3
0
0
0
0
0
0
0
0
1
0
1
U
4 5
0 0
0 0
0 0
0 0
1 0
0 0
0 0
0 0
1 1
0 0
2 0
0 0
of experiment


















*Workmen  were  breaking  open  a  cemont wall
^Measured zinc concentration of 5.22  mg/1  introduced.  Responses obtained during zinc exposure are
  underlined.
  Note:  There  were  3  experimental  fish  (Ex)  from  Experiment  9  and  1  control fish  (Con) from Experiment 9.

-------
           Table  6  .  Number of fish showing resoonses, before
                     and after exposure to 4.16 mg/1  zinc.
                                Time

 Day 6   7  8   9  10 11 12 1  2  3  4  5  6  7  8  9  10 11  12 1   2345
     am	pm	am	

 2
 Ex   000000000000000000100100
 Con 000000000000000000000000

 3
 Exonooooooooooi  ooooooooi  oo
 Con 000000000000000000000000

 4
 Ex   0000000000010000  Recorder off-  -  -  -
 Con 0000000000000001---      ----

 5
Ex   ---000000100100110021110
 Con ---000000000000000000000

 6
Ex   Recorder 000000000000011012012
     Off-  -000000000000000000000
NOTE:  There were 4 experimental  fish (Ex)  from Experiment 8 and 1 control
       fish (Con) from Experiment 8.
                                18

-------
                      Table  6 .  (continued)
                              Time

Day 6  7  8  9  10 11  12 1   2  3  4  5  6  7  8  9  10 11  12 1   234
    am                   n                                  am
7             1
              T
Ex  0  0  0  OT1   0001   000000334333322
Con 000000000000000000000000

8
Ex  200102001112110443343431
Con 000000000000000000000000

9
Ex  001121311211122233333222
Con 000000000000000000000000

10
Ex  020000001   001001  2322331   22
Con 0  00000000000000000000000

n
Ex  00000  end of experiment
Con 0  0  0  0  0
   Measured zinc concentration of 4.16 mg/1  introduced.  Responses
   obtained during zinc exposure are  underlined.
                                19

-------
       Table 7.  Number of fish showing heterogeneous
                 variances, before and after exposure
                 to 4.16 mg/1  zinc.
               o = .05                   a =.01
Day      Dawn Light Dusk Dark      Dawn  Light Dusk Dark
2

3

4

5

6

7

8

9

10

11

Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
Ex
Con
0
0
0
0
0
0
_
-
0
0
0
0
2
0
0
0
2
0
0
o
0
0
0
0
1
0
0
0
1
0
•I*
0
1
0
3
0
3
0


0
0
1
0
2
0
0
0
0
0
1
0
2
0
1
0
1
0


0
0
2
0
1
0
1
0
1
0
2
0
3
0
2
0
2
0


0
0
0
0
0
0
—
-
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
1
0
0
0
1
0
[2
0
1
0
2
0
3
0


0
0
1
0
0
0
0
0
0
0
0
0
1
0
0
0
0
0


0
0
1
0
0
0
0
0
0
0
0
0
1
0
2
0
1
0


VMeasured zinc concentration of 4.16 introduced
  Responses obtained during zinc exposure are under-
  lined.

  Note:  There were 4 experimental fish (Ex) and 1
  control fish (Con).
                           20

-------
the zinc was added and on two more occasions before the experiment was
terminated 67 hours after zinc addition.

When an earthquake occurred at 8 p.m. during day 2 of experiment 1
(Table 8), the fish ceased breathing for 12 to 28 seconds.   After
approximately five minutes the breathing rates became normal  again,
and therefore no responses were recorded for experiment 1  at 8 p.m.
in Table 8.  Bluegills react similarly  when the room lights are
turned off abruptly or someone rapidly approaches the test chambers.

Tables 9 and 10 summarize information that indicates the effectiveness
of the two methods of analysis.  The tables show that changing the
criterion for detection from one to three responses per time period
generally increases the lag time and decreases the number of false
detections.  Table 10 also shows that there are fewer false detections
by the variance comparison method with a = .01 than with a = .05.
In addition, the lag time was no greater for a = .01 than for a = .05
when either one or two responses per time period counted as a detection.

If three simultaneous breathing rate increases counted as a detection,
Table 9 shows that no false detections would occur in the three ex-
periments reported, but that the highest zinc concentration (5.22 mg/1)
would not have been detected before experiment 9 ended.  The effect
of missing observations>on the results would be to underestimate the
number of responses occurring at a time, and although the diurnal de-
crease in breathing amplitude resulted in missing observations during
the dawn and dark periods for approximately one out of five fish, there
were no missing observations for the fish used in experiment 9.  As
only three fish were exposed to zinc in this experiment, it is possible
that one unusually zinc-tolerant individual determined the outcome of
the test for detection.

If the criterion for detection in experiment 8 were two simultaneous
responses, then no false detections would have been obtained by the
variance comparison method with a = .01 (Table 10), whereas three false
detections would have been obtained by measuring increases in breathing
rates (Table 9).  In addition, the lag time would be less using the
variance comparison method.

Discussion:  Of the two methods used to analyze fish breathing rates,
checking for increases in breathing rate is the most attractive be-
cause it permits a response test as each successive value is generated.
The first day, of course, must be used to obtain the maximal breathing
rates used in all subsequent comparisons.  The variance comparison
method at first appears limited because enough values must accumulate
to obtain estimates of variances.  For example, if zinc had been intro-
duced at the beginning of the light period of day 7, exoeriment 8 (Table
7), it would not have been possible in the present system to detect the
zinc until 5 p.m., when nine values had accumulated, and the variance
was computed and tested against the variance of the previous day.  However,
                               21

-------
         Table  8 .   Number of fish showing responses, before
                    and after exposure to 2.55 mg/1 zinc.
                               Time
 Day  6   7  8  9  10 11 12  I  2  3  4  5  b  7  8910 11  12 1 ^T3   4   5
     am                   pm                                  am
 2              I
 Ex   0   0  1  IT!  1  1   1  1  1  1  1  2  1  2  1  1   1   1   0   1   1   1   1
 Con  000000000000000* 01   0000000

 3
 Ex   12111111121111010001121-
 Con  000  T  1  T  1   000001  T  0000   T   000   1   0

 4
 Ex   recorder 110023321001121222223
       off
 Con  0000001   0000000001   0000000
  Measured zinc concentration of 2.55 mg/1  introduced.   Responses obtained
    during zinc exposure are underlined.

  Note:  There were 3 experimental  fish (Ex)  from Experiment  7  and 6
    control fish (Con) from Experiment 1.

* Earthquake occurred during the 8  D.m. recording, day  2, Experiment  1.


                                22

-------
Table 9 .   Effectiveness of zinc detection using
           increases in fish breathing rates
No. of Detection criterion:
Zinc fish minimum no. of
(mg/1) Exposed fish showing response
to zinc at one time
5.22 3 1
2
3
4.16 4 1
2
3
2.55 3 1
2
3
Lag time (Mrs
from addition
of zinc)
0
4
not detected
after 45 hrs.
0
11
11
0
8
52
Fal se
detections
12 in 100 hrs.
1 in 100 hrs.
0 in 100 hrs.
19 in 123 hrs.
3 in 123 hrs.
0 in 123 hrs.
2 in 4 hrs.
0 in 4 hrs.
0 in 4 hrs.
                    23

-------
            Table 10.  Effectiveness of zinc detection
                       using successive comparisons of
                       breathing rate variances.
                  Detection criterion:
                  minimum no. of fish
Zinc       No.    showing heterogeneous
(mg/1)    Fish    variances during one
	time period a = .05
                Lag time (Mrs.     False
                from addition   detections
                  of zinc)	
4.16
1

2
                  Detection criterion:
                  minimum no.  of fish
                  showing heterogeneous
                  variances during one
                  time period  a = .01
 7        8 in 122 hrs,

 7        2 in 122 hrs,

43        0 in 122 hrs,
1
2
3
7
7
79
4 in 122 hrs.
0 in 122 hrs.
0 in 122 hrs.
                                24

-------
breathing rates could be taken every minute and the variances estimated
and tested every 10 minutes by means of analog-to-digital converters,
mini-computers, and teleprinters.  As swimming masks the breathing signals,
the computer would have to be programmed to distinguish breathing from
swimming.  In addition, the amplification of the breathing signal might
be controlled to compensate for the diurnal fluctuation in signal ampli-
tude.  The system would be relatively immune to sudden extraneous dis-
turbances (such as floor vibrations or a loud noise) if the computer were
programmed to skip the transitory pauses in the breathing signal that
such disturbances produce.

One advantage of the method of variance comparisons is that a decrease
in breathing rate variance, as well as an increase, counts as a response
because the statistical test detects differences in variance, not the
direction of difference.  It is possible that some toxicants would re-
duce breathing rate variance.  The disadvantage of counting breathing
rate increases as responses is that some toxicants might act as respir-
atory depressants.  This objection could be overcome by establishing
minimal, as well as maximal breathing rates for each fish before the
fish were exposed to toxicants, and then checking for values which
fall outside this range on subsequent days.  The method of choice could
be determined by further testing of the breathing monitoring system
with a variety of toxicants, singly and in combination.

The results show that the criterion for detection should be simultaneous
responses by some proportion of the exposed fish, not by all of them,
to reduce the probability of having the outcome determined by an exception-
ally unresponsive fish.

It would certainly be desirable to have a redundant detection system
in an industrial situation, where water and waste qualities are apt to
vary unpredictably.  It is conceivable that some harmful combination of
environmental conditions and waste quality would be detected by monitor-
ing one biological function but not by monitoring another.  The present
system for monitoring fish breathing could be combined with another system
for monitoring fish movement (already developed as project 18050 EDP,
Water Quality Office, Environmental Protection Agency) by using test
chambers that permit breathing signals to be obtained from free-swimming
fish (Spoor, ejt al_., 1971).  It should be possible to feed the test fish
and use them as long as they live and do not become incapable of response
after long exposure to toxicants, due to impairment of sensory mechanisms
or development of resistance.  It would be desirable to have some control
fish exposed to upstream water containing no waste from the plant, in
order to evaluate the effects of upstream conditions on the fish, and to
detect extraneous effects such as floor vibrations or noises that con-
tinue for several hours and disturb the fish  (Figure 1).  The lag time
inherent in the technological portion of the system could be reduced by
automating data collection and analysis as described above and the lag
time inherent in the biological portion of the system probably could be
reduced by metering proportionally more waste into the dilution water
delivered to the test fish than is delivered to the stream.  The latter
method would also serve to introduce a safety factor.

                               25

-------
                  IN - PLANT  MONITORING  UNIT
                                      UPSTREAM
                                       WATER
             MIXING
          MOVEMENT


           MONITOR
          BREATHING

          MONITOR
         I
         TO CENTRAL
         PROCESSOR
                                DRAIN
Figure 1.  A monitoring  unit for industrial use, showing how the test
fish are exposed to waste diluted with  uostream water and the control
fish are exposed to upstream water alone.
                             26

-------
If industrial users of a river complied with water quality standards  and
also were able to guard against accidental  spills or a detrimental  change
in environmental conditions by means of a biological monitoring system,
then full use could be made of a river without damaging the fish life.
                               27

-------
                            SECTION V

                  FISH REPRODUCTION AND GROWTH

Methods and materials:  Bluegill sunfish were seined from a local
pond and held for several months in the laboratory in the same
dilution water, with the same photoperiod and water temperatures,
as for the experiments described in Section IV.

Starting April 13, approximately 200 fish (approximate total  lengths:
8-15 cm; weights: 10-80 gms.) were brought into  breeding condition
by exposing them to a photoperiod of 16 1/2 hours of light, water
temperatures of 31-32°C, and by feeding them twice daily with frozen
Gordon Formula (Axelrod, 1952) and once daily with live mealworms.
The dimming system described earlier simulated a 1/2 hour dawn
starting at 6 a.m. and a 1/2 hour dusk starting  at 10 p.m.

On May 4 most of the fish could be sexed by gently squeezing the sides
and observing whether eggs or milt was extruded, and three females
and one male were placed in each of twenty 20-gallon tanks (standard
aquaria, long type, Ramfab Aquarium Products cat. no. RA-20L).

One standard clay flowerpot (upper rim-to-rim diameter = 6 inches)  was
placed on its side in each tank for the females  to hide from the aggressive
attacks of the males.  An artificial nest, described by Eaton (1970)
was also placed in each tank and five smooth pebbles, 2-3 cm in diameter,
were scattered on the bottom of each nest.

One toxicant delivery apparatus was used for each set of five tanks
receiving one concentration, and one water delivery apparatus was  used
for five control  tanks which received no added zinc.  The toxicant
delivery apparatus combined a toxicant dipper and needle valve described
by Mount and Brungs (1967) with a water delivery system described  by
Brungs and Mount (1970).  The dilution water was the same as that  used
in the experiments described in Section IV.  The zinc concentrations  for
the reproduction study were based on the lowest concentration used in
the fish breathing experiments; i.e. 2.5mg/l.  Tanks 6-10 received
.250 mg/1 zinc (1/10 of 2.5 mg/1), tanks 16-20 received .025 mg/1  zinc
(1/100 of 2.5), and tanks 11-15 received no added zinc and served  as
controls.  In addition, tanks 1-5 received 1/100 the 96-hour TL50
(median tolerance limit) for adult bluegill sunfish exposed to zinc in
municipal tapwater:  .075 mg/1.  The initial zinc concentration used  in
the stock jug of each toxicant delivery apparatus was calculated to
yield the desired concentration in the tanks.  The concentrations  in  the
tanks were measured by atomic absorption spectrophotometry and were
lower than desired, so the concentrations of the stock solutions were
adjusted until the correct concentrations were obtained in the tanks.

The flow rate to each tank was approximately 100 ml/min.  The water
entered at the top and front of the tank and was removed from the  bottom
                                29

-------
 carrying some detritus  with  it,  by means of a sheathed standpipe
 at the rear of the  tank.

 A plastic egg hatching  box  (20.5 cm long, 7.0 cm wide, and 15.5 cm
 deep)  hung on the front of each  aquarium and was large enough to accept
 three  egg cups.  The  egg  cups were made from Turtox plastic jars (5.5 cm
 o.d.,  6.8 cm tall), with  the bottoms removed.  In use, each end of the
 cup was covered with  a  piece of  ladies' woven nylon hose held in place
 with rubber banks.  Each  cup rested on an airstone cemented to the bottom
 of the hatching box.  A piece of plexiglas (20 cm long, 6.3 cm wide, and
 0.6 cm thick)  fit into  grooves in the hatching box and rested on top
 of the cups to keep them  from floating.

 Water  siphoned from each  tank into the hatching boxes and was returned
 to the tank by an air lift.  Another air lift delivered water from the
 hatching box to a plastic rearing chamber (38.0 cm long, 30.5 cm wide,
 and 17.5 cm deep) for rearing newly-hatched fry.  Thus the eggs were
 hatched and the fry reared in the same water as their parents.  Water
 drained from each rearing chamber through a rectangular opening (8.8 cm
 wide,  1.8 cm high) into a trough.  The bottom of the opening was 14.5
 cm from the bottom of the pan, and the opening was covered by .8 mm
 mesh nylon  netting.

 The fish in each tank were fed two grams of frozen Gordon Formula
 (Axelrod,  1952) twice a day  and  eight live mealworms once a day.  The
 tanks  were  cleaned once a week by siphoning detritus from the bottom.

 At 1 p.m.  every day,  the pebbles in each nest were removed and examined
 closely for eggs.  If eggs were  present, a plastic chamber (same dimen-
 sions  as  above) was filled with water from the tank and the nest was
 removed from the tank and placed upside down in the chamber over an
 airstone.   A new nest was substituted immediately for the old one.

 A  subsample of 200 eggs was  removed from the nest and placed in a hatching
 cup, which  in  turn was placed in the hatching box.  After 48 hours, the
 number of fry  in both the egg cup and the chamber were counted, by
 pipetting them into petri dishes and using a Dazor model M209 fluorescent
 magnifier and  a hand  tally counter.  The hatch in the subsample of eggs
 in  the cup  was assumed to be proportional  to the hatch in the nest, and
 the  numbers  of fry and eggs  in the cup were used to back-calculate the
 number of eggs spawned  in the nest:

Total  No. Eggs    _   No. Fry  X  (No. Eggs in Cup-rNo. Fry in Cup) + No. Eggs
     In  Nest      ~   In Nest                                         In Cup

When less than 200 eggs were spawned, the number of eggs in the nest and
 the  number of  fry in  the hatching chamber were counted directly, without
 removing a  subsample.

 Fish that were dead or that had lost their equilibrium were removed as
soon as they were noticed.  In addition, six fish had an eye disease that


                                 30

-------
started as a white spot and gradually consumed the entire eye, and
these fish were also removed.  Fish that were removed before the end
of the experiment were weighed, measured and sexed—unless they were
too decomposed.  Fish that were removed and could be sexed were re-
placed by a fish of the same sex from a stock of ripe fish kept in
dechlorinated tapwater containing no added zinc, until  July 20, when
no further replacements were made.  The breeding portion of the ex-
periment terminated August 19, when all the remaining adult fish were
killed, weighed, measured and sexed.  The condition and weight of the
gonads was also recorded.

Fifty fry from the first spawning in each tank were placed in the
rearing chamber for that tank.  Newly-hatched brine shrimp were rinsed
in dechlorinated tapwater and placed in each rearing chamber twice a
day for the fish to feed upon.

A census of the rearing chambers on July 8 revealed that very few fish
were surviving, so changes were made in the apparatus and methods.  Some
of the fry may have washed through the netting of the rearing chambers,
so the chambers were modified by drilling five 1.1 cm holes on centers
11.5 cm above the bottom of the chambers and covering them with a piece
of woven nylon.  All surviving bluegills were transferred to the modi-
fied chambers on July 8 and 9, and all fry hatched after July 9 were
placed in chambers of the new design.

In addition, we learned that brine shrimp were too large for bluegill
fry and that the National Water Quality Laboratory, Duluth, Minnesota,
was successfully feeding plankton to baby bluegills (James  M. McKim, III,
personal communication).  Consequently, starting July 23, plankton was
obtained regularly from nearby ponds, and fed, after straining through
a 0.8 mm mesh net, twice a day to the fish.  Samples of the plankton
were examined regularly under the microscope and never appeared to be
very rich, so the diet was supplemented with a pinch of TetraMin pow-
dered baby fish food twice a day.  Newly-hatched brine shrimp were
fed to the fish starting approximately the third week of growth.

Since no spawnings ever occurred in some tanks, the rearing chambers
for these tanks received fry from other tanks.  In addition to transfers
of fry made within chambers at the same zinc concentration, some fry
were taken from high zinc concentrations and put into chambers containing
low concentrations, and vice-versa.

The fry in each chamber were counted and total lengths determined 30,
60 and 90 days after introduction to the rearing chamber.  Total lengths
were determined by placing each fish in a glass petri dish over a metric
ruler.

Dissolved oxygen concentrations in the breeding tanks were determined
by a YSI oxygen meter, and temperatures by a mercury thermometer.  Since
water from the breeding tanks was delivered directly to the rearing
chambers by air lifts, we assumed that the water characteristics  in  the


                                31

-------
 tanks and chambers were the  same.   This assumption was confirmed for
 zinc by measuring zinc  concentrations  in the tanks and chambers at
 random intervals, but D.O. and  temperature were not measured in the
 rearing chambers.

 Results and discussion:   Zinc concentrations, dissolved oxygen concen-
 trations (D.O.),  and  temperatures  in the breeding tanks are shown in
 Table 11.   On  August  4,  1971, a new central air conditioning system
 began operating,  and  the room temperature was lowered 5.6°C in 12 hours.
 As  a result,  the  temperatures in the breeding tanks reached a new
 steady state,  approximately  4°C lower  than the mean temperatures re-
 corded earlier.   Since  a spawning  occurred for the first time in tank
 9 after the drop  in temperature, and six additional spawnings occurred
 in  other tanks, the temperature drop did not seem to affect spawning.
 However,  eggs  from a  spawning on August 4 that were exposed to a drop
 in  water temperature  of 12°C showed a  very low percentage hatch (2%)
 and were not  included in the results.

 The dechlorinated tapwater delivered to the control tanks (tanks 11-15)
 contained  a zinc  concentration ranging from .002 - .062 mg/1.  The
 mean zinc  concentrations and the standard deviations in all the tanks
 are shown  in Table 11  .   Nominal zinc concentrations will be used in the
 rest of the text.

 Data on  the adult bluegills  used as breeders are shown in Table 12.
 Although males and females were approximately the same size when they
 were introduced to the  breeding tanks on May 4, Table  12shows that
 the surviving males were generally heavier and longer than the sur-
 viving  females when the  breeding portion of the experiment terminated
 August  19.  Also,  mortality  among  females was proportionally greater
 than  it was among  males.  The disparity in growth and survival between
 males and  females  was probably due to biting and butting attacks by
 the males.  Most  of the  dead females had tattered fins and scales missing
 from their sides.  Females that were in tanks with very aggressive males
 would feed hesitantly,  even  when food was placed near their flowerpot
 shelters.   Most of the  fish  at all  zinc concentrations were still ripe
 on  August  19, and  there  were no trends in adult fish weights, lengths,
 survival,  or gonad weights that could be attributed to the effects of
 zinc.

 The  variation in  female  mortality  from tank to tank probably indicates
 some  variation in  aggressiveness from male to male, and variation in
 aggressiveness among males may explain why there were some tanks at all
 concentrations where no  spawning occurred (Table 13).  For example, the
male  in tank 17 (a tank  receiving  .025 mg/1 zinc) killed, or contributed
 to  the death of eight females in succession, and no spawning ever occurred
 in  this tank.  Once a spawning did occur in a tank, it was highly likely
 that  several more would  occur.  In  contrast to the multiple spawnings
obtained in the control   tanks and  tanks receiving zinc concentrations of
 .075  and .025 mg/1, however, only  a single spawning in one tank occurred
                                32

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 1
 2
 3
 4
 5

 6
 7
 8
 9
10

11
12
13
14
15

16
17
18
19
20
       Table 11. Zinc and dissolved oxygen concentrations.
                 and temperatures in breeding  tanks
Tank  Zn*
.075
.075
.075
.075
.075

.250
.250
.250
.250
.250

.000
.000
.000
.000
.000

.025
.025
.025
.025
.025
              Measured zinc con-
              centrations jmg/1)
                               P.O.  (mg/1)
N
14
14
14
14
14
15
15
15
15
15
14
14
14
14
14
14
14
14
14
14
Mean
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
S.D.
.043
.047
.039
.032
.028
.055
.048
.045
.043
.044
.023
.007
.010
.014
.011
.012
.019
.018
.017
.012
N
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
Mean
6.1
6.3
5.9
6.0
5.8
5.5
5.3
5.4
5.1
5.3
5.5
5.7
5.5
5.7
5.4
5.6
5.4
5.8
5.7
5.4
S.D.
0.5
0.2
0.7
0.7
0.5
0.6
0.7
0.7
0.9
0.8
0.6
0.5
0.6
0.5
0.8
0.7
0.7
0.5
0.5
0.7
Temperature ( C)
                                                N    Mean
4
4
4
4
4

4
4
4
4
4

4
4
4
4
4

4
4
4
4
4
30.8
31.2
31.6
31.6
31.6

31.0
30.
30.
30,
30,

30,
30,
30,
30
30.4

30.5
30.4
30.4
30.4
29.9
S.D.

1.0
0.5
0.5
0.5
0.5

0.4
0.8
0.8
0.9
0.8

0.9
0.6
0.7
0.9
0.9

.0.6
0.5
0.5
0.8
0.5
* Nominal zinc concentrations (mg/1)

N-number of readings
                                33

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Table 12.  Survival  of adult  bluegills and weights, lengths,
           and condition  of gonads of adults at end of
           breeding  experiment
Tank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Zn
(mg/1 )
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
°No. males
removed be-
fore end of
experiment
0
0
*1
0
0
0
0
0
0
0
0
1
0
0
0
0
0
0
0
0
No. females
removed be-
fore end of
experiment
*3
4
0
*1
1
5
0
2
3
0
4
1
3
*2
*2
0
*9
2
2
5


Wts.
(gms)
61.8
69.6
48.2
60.9
37.6
69.7
71.7
34.0
62.7
26.1
61.3

78.4
67.6
72.6
72.5
70.0
93.4
84.8
31.6


Standard
lengths (cm)
11.5
11.5
10.5
11.6
10.2
12.1
12.0
10.0
11.7
8.9
11.7

11.9
12.1
12.3
12.3
11.8
12.9
13.0
9.6

Males
No.
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
   One fish removed because eye was diseased.
   per tank contracted this disease.
No more than one fish
  aFish with eye disease, dead fish, and fish that had lost their
   equilibrium were removed immediately.  A ratio of three females
   and one male was maintained per tank by replacement of fish until
   July 20, 1971.

  bMean values (with ranges in parentheses) are shown for tanks
   containing more than one female.
                              34

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 Fish removed at end of experiment
                                         Females1
Gonads
Wt(gms)
1.2
1.5
0.9
1.7
< .1
1.0
1.2
0.2
0.9
0.5
0.8
Condition1
R



N


Wts.
' (gms)
13.1

24.2 16.6-33.6
31.2 23.0-39.4
29.4 23.7-35.0

29.1(22.0-35.4
Standard
lengths(cm) No.
7.2 1
0
9.1(8.5-9.8) 3
9.6(8.9-10.2) 2
9.6(9.4-9.7) 2
0
) 9.4(8.3-10.0) 3
44.6(38.3-51 .0)10.4(10.0-10.8)2
R

R
35.2(29.2-41.3
36.2(25.2-46.4

I 9.9(9.5-10.3) 2
1 9.7(9.0-10.3) 3
0
31.7(24.6-35.2) 9.5(9.0-9.8) 4
0.9
1.6
1.0
0.8
2.1
1.0
1.0
< .1
R

R
R
R
R

N
38.4(34.2-42.7)10.1(9.6-10.6) 2
39.1
21.6
10.3 1
8.7 1
28.6(28.1-29.0) 9.5(9.3-9.7) 2

13.4
26.8
20.6(7.8-32.1)
0
7.7 1
9.3 1
8.5(6.6-9.7) 4
Gonads
Wt(gms)
1.6

1.6(0.1-2.6
2.3(1.3-3.3
2.8(1.5-4.1

1.3(0.5-2.2
2.7(0.6-4.8
2.4(2.3-2.6
3.2(2.4-4.1

2.6(2.0-3.3
3.0(3.0-3.1
1.7
0.6
Condition*-
1R

2R, IN
2R
2R

3R
1R, IN
2R
3R
j
) 4Rd
) 2R
1R
1R
1.6(1.3-1.9) 2R

0.1
2.9

IN
1R.
1.6(0.4-3.5) 4Rd
CR = ripe, and indicates that milt was extruded when the sides  of the
 males were squeezed;  or in the case of females,  that the ovaries were
 swollen and filled with pinhead-size eggs.   N = not ripe, and  indicates
 that the testes weighed less than .1  gm and no milt was extruded from
 males;  in the case of females, no eggs were visible.  The number of
 ripe and unripe females in each tank is shown.

°An extra female was added to tanks 11 and 20.
                                35

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 at a concentration of .250 mg/1 zinc (Table 13).   Since eggs  or milt
 could be extruded from all the breeders at the beginning of the exper-
 iment, and since most of the fish had ripe gonads when the experiment
 terminated, the results in Table 13 indicate that a zinc concentration
 of .250 inhibits spawning in ripe fish.

 Table 13 also shows the percentage hatch in each  tank.   Where more  than
 one hatching was used, the mean percentage hatch  is shown, with the
 range in parentheses.  Hatching data from eight spawnings were  not  used
 because some of the eggs were hatching in the breeding tanks  in less
 than 24 hours at temperatures of 30-31°C.  Attempts to remove eggs  from
 these nests by pipetting generally caused the egg membranes to  rupture,
 releasing the fry.   In these cases, the percentage hatch in 48  hours
 of the eggs that were transferred without rupturing was confounded  with
 the 48-hour survival  of the fry that were also unavoidably transferred
 to the egg hatching cups.   A hatch of 21% from one spawning in  tank 16
 that was heavily fungused was also excluded.   The hatch obtained from
 the one spawning at the highest zinc concentration was low (43%),
 but within the range of values in the other zinc  concentrations.

 The number of fry introduced to the old rearing chambers with 0.8 mm
 mesh outlets and the number introduced  to the modified chambers with
 smaller mesh outlets  are shown in Table 14.   The  introductions  are
 shown in chronological  sequence from left to  right across the rows.
 For example, two introductions of fry were made to rearing chamber  4:
 one introduction of 50 fry from tank 4,  and a later introduction of
 51  fry from tank 1,  after  inspection revealed that there were no sur-
 vivors from the  first introduction.   Growth and survival  data in the
 right-hand portion  of the  table were always obtained on  fish  from the
 last  introduction reported in the left  side of the table.   Survival  of
young bluegills  at  all  zinc  concentrations  was poor.  Mortality was
 highest  during the  first weeks,  and can  probably  be attributed  to
 starvation.  The plankton  collections that  were fed to  the young blue-
 gills starting August 24 were never very rich,  and the  powdered baby
 fish  food may  not have been  utilized.   Once the young were large enough
 to  feed  on  brine shrimp, survival  improved  as shown  by  the reduction
 in mortality between  30, 60  and  90 days  as  compared to  the mortality
 between  day 0  and day 30.  At a  zinc concentration of  .250 mg/1, however,
no  bluegills survived longer than  30 days.  Fry obtained  from eggs
spawned  in  a zinc concentration  of .250  mg/1  and  fry obtained from  eggs
spawned  in  other zinc concentrations  were placed  in  .250 mg/1 zinc.
Most  of  the  fry  from  all these sources  died within  three  days in 250 mg/1
zinc  and were  visible on the bottom of  the  rearing chambers.  In addition,
fry taken from .250 mg/1 zinc and  placed in control  tank  11 showed  poor
survival:   only  four  fish  survived  for 30 days, one for  60 days, and
none  for 90  days.

An experiment  on  the  effects  on young bluegill  of momentary exposure
to a  high zinc concentration  was  inadvertently conducted when a hose
separated from a  connector and fell  into  rearing  chamber  5  while a
stock bottle was  being filled with concentrated zinc solution.  The
                                36

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   Table  13.   Spawning  of adult bluegills and percentage
               hatch  of  eggs at four zinc concentrations
Tank
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Mean
zinc
concen-
tration
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
Total
no. of
eggs
spawned3
8414
0
10647
4736
0
0
0
0
1009
0
7188
0
0
227
5849
4274
0
0
10202
0
Total
no. of
spawnings
3
0
8
5
0
0
0
0
1
0
4
0
0
2
7
3
0
0
5
0
Mean no.
of eggs
per
spawning0
2805
0
1331
1184
0
0
0
0
1009
0
1797
0
0
114
985
1425
0
0
2040
0
Percen-
tage a h
hatch3' b
72 (71-72)
0
66 (49-78)
57 (44-68)
0
0
0
0
43
0
62 (35-76)
0
0
33
73 (47-96)
86


78 (65-90)

aNumber of eggs and percentage hatch were not determined for all
 spawnings because of premature hatching, fungus infestation,
 etc. (see text).

bWhere more than one hatch was used, the mean percentage hatch
 is shown, with the range in parentheses.
                               37

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       Table  14.  Survival and growth of bluegills in four
                 zinc  concentrations
Rearing
chamber
1
2
3
4
5
6
7
8
9
10
n
12
13
14
15
16
17
18
19
20
Zn
mg/1
.071
.081
.079
.076
.074
.231
.232
.230
.234
.249
.028
.019
.019
.020
.017
.028
.040
.041
.035
.033
No. of fry
introduced
to old
rearing
chambers
50[l]a

50[3]
50[4]

50[15],51[15]

50[11]
50[11]
50[15]
50[11]



50[15]
50[16]

50[19]

No. of fry
introduced
to new
rearing
chambers
50H"
50f4"
50f3],45[19]
51 fl"
50[3]
53[9]
58[9]
50[1],58[19]
51 [9]

50[11],51[9]


50[15],40[14]

56[16]

51 [19]
50[19]

Mean total lengths (mm)
30
21.8(5)b
13.2(4)
(0)
21.0(4)
12.2(19)c
(0)
(0)
(0)
(0)
(0)
6.8(4)


9.5(2)
	 d
(0)

(0)
14.4(7)

Days
60
90
28.8(5) 38.8(5)
20.3(3
(0
24.5(4
34.3(3)
(0)
36.6(4)
19.2(18) 26.9(14)
(0) (0)
(0) (0)
(0) (0)
(0) (0)
(0) (0)
10.0(1) (0)




21.2(2) 27.8(2)
20.0(2) 33.0(1)
(0) (0)

(0)
19.3(6)


30.6(6)

a Numbers in brackets indicate the number of the tank  where  the  fry
    were obtained.
b Numbers in parentheses indicate the number of fish.
c At least 2 fish died as a result of a zinc spill  on  day  22 (see
    text).
d The fish in chamber 15 were not counted or weighed on  day  30.
                              38

-------
hose was removed from the tank in a fraction of a second and the young
bluegill were transferred to the proper zinc concentration  within  two
minutes, but two of the 21 fish died within 15 minutes.   During  the
two minute period, the 22-day-old bluegills had been exposed to  a  zinc
concentration of 9.18 mg/1, although the concentration initially may
have been higher in portions of the chamber.  There were 19 survivors
on day 30, 18 on day 60, and 14 on day 90.  After the initial  deaths,
survival in chamber 5 was comparable to survival in other tanks  at
the same concentrations.

In summary, it appears that a zinc concentration of .250 mg/1  inhibits
spawning in adult bluegills brought into breeding condition in dechlor-
inated municipal water containing no added zinc, and causes complete
mortality of bluegill fry.  Water containing no added zinc and zinc
concentrations of .075 and .025 mg/1 do not have these effects.

The lowest zinc concentration tested in the apparatus for monitoring
fish breathing was 2.55 mg/1.  This concentration was detected by the
monitoring method, and the reproduction and growth experiment shows
that 1/100 or 1/34 of this concentration might be safe for chronic
exposure of bluegills, but that 1/10 of this concentration certainly
is not.
                                39

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                          SECTION VI

                        ACKNOWLEDGMENTS

This research was carried out in the Aquatic Biology Laboratory of
the Biology Department and Center for Environmental  Studies,  Virginia
Polytechnic Institute and State University, Blacksburg,  Virginia,  24061.

The advice and equipment furnished by Dr. Alan G. Heath, Associate
Professor of Zoology, Biology Department, Virginia Polytechnic In-
stitute and State Unviersity, is gratefully acknowledged.  Dr. William
T. Waller, Enforcement Division, Environmental Protection Agency,  Kansas
City, Missouri, suggested that the breathing rate data be analyzed by
variance comparisons, and worked on the reproduction and growth studies
while he was a graduate student at Virginia Polytechnic Institute  and
State University.  Dr. James M. McKim, National Water Quality Laboratory,
Environmental Protection Agency, Duluth, Minnesota, and Project Officer
for this grant, furnished much helpful advice during the course of the
reproduction and growth experiments.
                               41

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                          SECTION VII

                        LITERATURE CITED

American Public Health Association.  1967.   Standard methods  for  the
     examination of water and wastewater.  12th ed.  New York.   769 p.

Axelrod, H. R.  1952.  Tropical fish as a hobby.  George Allen  and
     Unwin, Ltd. London.  264 p.

Brungs, W. A., and D. I. Mount.  1970.  A water delivery system for
     small fish-holding tanks.  Trans. Amer. Fish. Soc.  99(4): 799-802.

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.  Journal  Water Pollution Control
     Federation.  42(5): 685-703.

Drummond, R. A., and W. A. Spoor.  1971.  A method for recording  the
     responses of free-swimming animals to toxicants and deleterious
     environmental conditions.  American Chemical Society, Division
     of Water, Air and Waste Chemistry.  Preprints of Papers  Pre-
     sented at 162nd National Meeting.  11(2): 122-124.

Eaton, S. G.  1970.  Chronic malathion toxicity to the bluegill
     (Lepomis macrochirus Rafinesque).  Water Research.  4:673-684.

Mount, D. I., and W. A. Brungs.  1967.  A simplified dosing apparatus
     for fish toxicology studies.  Water Research.  1:  21-29.

Sokal, R. R., and F. J. Rohlf.  1969.  Biometry.  W. H. Freeman and  Co.
     San Francisco.  776 p.

Sparks, R. E., W. T. Waller, J. Cairns, Jr., and A. G.  Heath.  1970.
     Diurnal variation in the behavior and physiology of bluegills
     (Lepomis macrochirus Rafinesque).  The ASB Bulletin 17(3): 90
     (Abstract)^

Spoor, W. A., T. W. Neiheisel and R. A. Druimnond.  1971.  An electrode
     chamber for recording respiratory and other movements of free-
     swimming animals.  Trans. Amer. Fish. Soc. 100 (1): 22-28.
                               43

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                          SECTION VIII

                          PUBLICATIONS
Sparks, R. E., W. T. Waller, J.  Cairns,  Jr.  and A.  6. Heath. 1970.
     Diurnal variation in the behavior and physiology of  blueqills
     (Lepomis macrochirus Rafinesque).  The ASB Bulletin.   17(3):
     90 (Abstract).

Cairns, J., Jr., R. E. Sparks and W.  T.  Waller.  1971.  The relation-
     ship between continuous biological  monitoring  and water quality
     standards for chronic exposure.   American Chemical Society,
     Division of Water, Air and  Waste Chemistry. Preprints of
     Papers Presented at the 162nd National  Meeting.  11(2): 55-62.

Cairns, J., Jr., R. E. Sparks and W.  T.  Waller.  1971.  The relation-
     ship between continuous biological  monitoring  and water quality
     standards for chronic exoosure.   Abstracts of  Papers.  162nd
     National Meeting, September 12-17,  1971.   Washington,  D. C.
     WATR division, abstract no. 19.

Cairns, J., Jr., R. E. Sparks and W.  T.  Waller.  1971.   (manuscript
     in press)  The relationship between continuous biological
     monitoring and water quality standards for chronic exposure.

Cairns, J., Jr., R. E. Soarks and W.  T.  Waller.  1971.   (manuscript
     in press)  The use of fish  as sensors in  industrial  waste  lines
     to prevent fish kills.
                               45

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  SELECTED WATER
  RESOURCES ABSTRACTS
  INPUT TRANSACTION FORM
   J. Report No.
                                2.
                                         3. Accession f/o.
                       w
  4.  Title
    THE USE  OF  BLUEGILLS TO DETECT ZINC
  7.  Authors)  Cairns, John, Jr.
              Sparks, Richard E.
  9.  Organization
     Virginia Polytechnic Institute and State  University
     Biology Department and Center for Environmental  Studies
  12.  Sponsoring Organization

  IS.  Supplementary Notes
                       5.  Report Date

                       6.

                       8.  Performing Organization
                          Report No.

                       10.  Project No.
                       11. Contract/Grant No.
                          18050  EDQ

                       13. Type of Report and
                          Period Covered
  16.  Abstract
 The presence  of zinc at concentrations of  8.7,  5.22, 4.16 and 2.55 mg/1 in dechlorinated
 municipal  tapwater was detected by an increase  in breathing rate or a change  in  breath-
 ing rate  variance of bluegills.  None of the  fish exposed to the three  lower  concentra-
 tions died during the experiments.  The criterion for detection was an  arbitrary number
 of responses  occurring at the same time.   When  the criterion was changed from a  single
 response  to three responses occurring at the  same time, the number of false detections
 ("detections" occurring before zinc addition) decreased, but the lag between  zinc addi-
 tion and  detection increased.  Zinc concentrations of .025 and  .075 mg/1  (approximately
 1/100 and 1/34 of 2.55 mg/1, respectively) did  not appear to affect the reproduction
 and growth of bluegills in the laboratory, but  .250 mg/1 zinc (approximately  1/10 of
 2.55 mg/1) inhibited spawning in ripe bluegills and killed newly-hatched fry.  An in-
 plant system  for the prevention of fish kills caused by spills  could be developed
 by monitoring several biological functions of fish simultaneously to obtain  informationa
 redundancy and reduce error; by exposing test fish to higher waste concentrations than
 occur in  the  receiving stream as a safety  factor; automating the collection  and
 analysis  of data to reduce lag time; and by choosing appropriate criteria  for
 detection.
  17a. Descriptors
    *Water pollution control, Industrial  wastes, *Bioindicators,  Fishkill, Fish
      physiology
  17b. Identifiers
    *Biological monitoring, Bluegill,  Zinc, Leppmis macrochirus  Rafinesque
  Uc.COWRRFieJd& Group   Q5G, 05C
  18. Availability
19. Security Class.
   (Report)

20. Security Class.
  Abstractor Richard E. Sparks
21. No. of
   Pages

22. Price
                                                       Send To:
                                                       WATER RESOURCES SCIENTIFIC INFORM ATION CENTER
                                                       U-S. DEPARTMENTOFTHEINTER1OR
                                                       WASHINGTON. D. C. 2O24O
    Virginia Polytechnic Institute and State
WRSIC 102 (REV. JUNE 1971)
    University
                                                                                  GPO 913.261

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