,
A Modular Large-scale
LaboratorX System to Acclimate
and Test quatic Organisms
'
..
October 1977
Fisheries & Marine Service
Technical Report No. 728
Fisheries and Marine Service
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Fisheries and Marine Service
Technical Report 728
October 1977
TO ACCLIMATE AND TEST AQUATIC ORGANISMS
by
E. Scherer, K.R. Scott, and S.H. Nowak
Western Region
Fisheries and Marine Service
Department of Fisheries and the Environment
Winnipeg, Manitoba R3T 2N6
This is the one hundred and fourth
Technical Report from the Western Region, Winnipeg
ii
€)
Minister of Supply and Services Canada 1977
Cat. no. Fs 97-6/728
ISSN 0701-7626
iii
TABLE OF CONTENTS
List of Figures .
iv
Acknowledgements.
v
Abstract/Resume •
vi
Introduction.
1
The System and Its Elements •
1
The tank •
1
Lighting .
1
Water conditioning and circulation
1
Principles of operation
1
Measurement and test results
2
An Application:
Monitoring Locomotor Activity of Fish
2
List of Major Components.
3
References
4
iv
LIST OF FIGURES
figure
1
Tank with water supply and lighting. A:
B: side view •
top view,
Water conditioning and circulation unit.
See text
p. 1-2
5
.
6
Heating and cooling performance (tests 1 and 2);
maximum system capacity vs. make-up flow .
7
4
Temperature drop and control (test 3)
8
5
Temperature rise and control (test 4)
8
Sonar beam - interrupt technique to record locomotor
activity. See text p. 2-3
9
3
7
A record of locomotor activity, obtained by sonar beaminterrupt technique
10
v
ACKNOWLEDGEMENTS
We thank F.A.J. Armstrong and J.F. Klaverkamp for constructive
reviewing of the manuscript, l.C. Dugal for translating the abstr&ct,
and Carol Catt for typing.
vi
ABSTRACT
Scherer, E., K.R. Scott, and S.H. Nowak. 1977. A modular large-scale
laboratory system to acclimate and test aquatic organisms. Can.
Fish. Mar. Serv. Tech. Rep. 728: vii + 10 p.
The system consists of:
1) The water tank, a commercially available above-ground type
circular swimming pool (5 m diameter; capacity 18,000 L), equipped with
styrofoam insulation, exchangeable polyethylene liners and dimmable
fluorescent overhead lighting.
2) A specially adapted water conditioning assembly incorporating
filtering, make-up water and temperature control.
3) An apparatus to record the biological response, at present a
sonar-beam interrupt device to monitor continuously the swimming activity
of fish.
In cooling, with 7.5 L/min make-up water, the system required 50 h
to lower the temperature from 12° to 7°C and a further 30-40 h to stabilize
at 6.75±0~05°C.
In heating, it needed 30 h to raise the temperature from 7.3°C to
14.9°C and a further 15-20 h to stabilize at 15.15±0.05°C.
A two-beam sonar activity monitor was tested and found to function
reliably.
The design and construction of the system, with a list of
components, are given.
Key words:
water filtration, temperature control, light control, toxicants,
bioassays, fish, aquatic organisms, locomotor activity recording,
sonar.
Scherer, E. K.R. Scott, and S.H. Nowak. 1977. A modular large-scale
laboratory system to acclimate and test aquatic organisms. Can.
Fish. Mar. Serv. Tech. Rep. 728: vii + 10 p.
Le systeme comprend:
1) Un reservoir circulaire de 5 m de diqmetre et d'une capacite
de 18,000 L, avec eclairage a luminosite controlee de tubes fluorescents.
vii
Le reservoir est un piscine exterieure qu•on peut se procurer aisement
chez le marchand; elle est du genre sur~lev~e et le fond en polyethylene
peut se remplacer facilement.
2) Un assemblage d•appareils pour climatiser 1•eau, developpe
specialement pour le genre d op~ration; il inclut filtration addition
d•eau et controle de la temperature.
3) Un appareil pour enregistrer les reactions biologiques; pour
1 instant, c•est un appareil sonar qui decele 1•activite du poisson par
1 interruption du faisceau sonar.
1
1
1
Pour abaisser la temperature de 1•eau de l2°C a 7 C lorsqu•on
additionne 7.5 L par minute, le temps requis est de 50 heures; pour
atteindre et maintenir 6.75±0.05°C requiert 30 a 40 heures additionnelles.
et 15
Il faut 30 heures pour elever la temperature de 7.5°C a 14.9°C
heures additionnelles pour atteindre et maintenir 15.15±0.05°C.
a 20
Un moniteur sonar
satisfaisants.
a deux
rayons a ete essaye avec des resultats
L•article decrit les operations et enumere les principales parties
composantes du systeme. Un schema du systeme y est inclus.
Mot clefs:
filtration de 1•eau, controle de temperature, controle
d•eclairage, substances toxiques, essais biologiques, poisson,
organismes aquatiques, enregistrement des activites
locomotrices, sonar.
1
INTRODUCTION
!lATER COilDITIOtHNG ANO CIRCULATIO!I UtHT
Prbwiples Qf operation
The advantage of controlled laboratory
conditions in ecological research is often offset
by basic shortcomings; a common one is limited
test space. Experiments with fish are most frequently carried out in moderately sized aquaria;
the literature abounds with small species and
stages of less than 10 or 15 em length while
underrepresenting larger ones. Also, ecological
analysis should include experiments with a number
of species simultaneously (assemblages of food
chains. competitors for niches. etc.). This
requires. for instance, a sufficient tank area to
lay out a variety of substrates for bottomdwelling plants and animals.
In particular, the need to assess the effect
of pollutants in a realistic way calls for multispecies experiments to establish species-specific
differences in sensitivity, as well as intra- and
interspecific interactions, under truly identical
conditions as provided within one test set-up.
These considerations led to the development
of a large-scale laboratory system as described.
THE SYSTH1 AND ITS ELEMENTS
See Fi gure~l
THE
TAI~K
The tank is an above-ground type circular
swimming pool, 5 m diameter x 1.2 m high, filled with
18,000 L of water. The wall consists of a single
sheet of corrugated 12-gauge steel plate. Insulation is provided by 2.5 em (1") thick styrofoam
billets on the sides and bottom. A 10 ml thick
replaceable one-piece polyethylene liner (nontoxic, chemically inert) covers the styrofoam,
providing a watertight enclosure. A 12 em
diameter glass observation port installed at one
location half-way up the side wall allows direct
observation of test organisms.
LIGHTING
A white circular plywood ceiling is
installed above the entire pool. This ceiling is
divided into 8 equal (45•) sectors, each containing
9 radially aligned 4' fluorescent lamps. Lamps
with two different color temperatures, 55oo•K, and
?Ooo•K, are installed alternately to approximate
the spectral quality of natural daylight. The 8
sectors are individually light-controlled by
dimmable ballasts. To avoid light intensity
fluctuations and flicker, especially at low light
levels, a voltage regulator was incorporated.
The distance between ceiling and tank surface is
adjustable, allowing different light maxima. A
black side curtain suspended from the edge of the
ceiling all around the tank perimeter excludes
extraneous light.
The conditioning unit (Figs. 1&2) for the
tank water performs the three separate functions
of (1) biological and mechanical filtration, (2)
temperature control and (3) make-up water flow
rate control. All components are mounted in an
angle-iron frame in a separate small ante-room
adjacent to the main tank room. This arrangement
isolates pump noises and vibrations from the tank
and allows monitoring and maintenance work to be
performed without disturbing ongoing tests. For
better reliability the conditioning unit consists
of two identical modules. All pipihg used in the
system is non-toxic polyvinyl chloride (PVC).
In operation, water returning from the tank
flows through a 3.8 em {ll,") PVC pipe. The water
is conducted to the conditioning unit in the
ante-room where it is split into two parallel
streams, one for each of the two cond1tioning
modules, the pipe size for each circuit being
reduced to 2.5 em (1"). For each of these streams
the water flows through an isolating valve a.nd
then to a centrifugal nitrile rubber pump (A) which
maintains a circulation rate of 45 L/min. The
flow then passes through a biological and
mechanical filter (B) consisting of a 61 em
diameter x 30 em high bed of calcite. This
provides a substrate for bacteria to convert
ammonia nitrogen to nitrite and hence to nitrate
in a two-step sequence preventing a potentially
toxic rise in ammonia nitrogen level. The calcite
also provides pH buffering and mechanical filtration of faeces, food residues, etc. (Scott &
Gillespie 1972).
Periodical manual backwashing of the filters
is dor.e by turning off pump (A), setting the
filter selector valve to the backwash position to
reverse the flow direction in the filter and
opening manual backwash valve (N) to admit fresh
dechlorinated water from the building supply. The
waste water goes to drain via a 3.8 em {ll,")
overflow. Backwashing is assisted by the introduction of compressed air via a 1.3 em ('>") 1ine
teed into the water line at the inlet to the
filter.
After passing through the filter the recirculated water goes through a variable area rotameter
flow indicator (C) and thence to a custom
designed water cooler-heater (D) (Scott 1972)
which either raises or lowers the temperature
depending on the setting of an electronic temperatu~e controller (E) whose thermistor sensing bulb
(F) is inserted into the water stream just downstream from pump (A). Cooling is provided by 1 hp
belt-driven refrigeration condensing unit (G), and
heating by a 3 kW inconel (30% Ni, 20% Cr) calrod
heater (H).
After temperature conditioning, the water is
returned to the fish tank and distributed evenly
by means of a 3.8 em (llo") diameter feed tube
running completely around the top perimeter of the
tank (see Fig. 1) and equipped with eight ·equally
spaced 1.3 em (lo") diameter outlets discharging
via individual valves at a depth of 46 em below
the water surface, thus providing sufficient
mixing to virtually eliminate thermal gradients.
2
The make-up water flow rate is controlled by hand
valve (P) and indicated by flow meter (L). The
rate can be varied from 0 to 9.5 L/min in each of
the two sections giving a total range of 19 L/min.
The entire conditioning unit can be bypassed if
desired for maintenance purposes with make-up
reverting to a flow-through basis by opening the
normally closed (N.C.) make-up by pass valve (R)
and closing the normally open (N.O.) conditioning
unit bypass valve (S). Water will then flow
directly from the building supply through the
distribution header into the tank.
Four tests were performed on the conditioning
unit to evaluate its performance:
1. Maximum cooling capability (Fig. 3).
2. Maximum heating capability (Fig. 3).
3. Temperature lowering rate and control
stability (Fig. 4).
4. Temperature raising rate and control stability
(Fig. 5).
Test 1 measured the maximum cooling capacity
of the system over a range of make-up water flow
rates from 0-15 L/min. Test 2 measured the maximum heating capacity of the system under similar
conditions. Since the tank is insulated, the
major source or sink for heat being extracted
from or added to the conditioning unit is the
make-up water. Therefore it was decided to correlate the data by plotting the maximum steady
state temperature difference between the tank
temperature and the make-up water temperature as
a function of make-up water flow rate. This
technique allows prediction of system capability
for any given make-up temperature. For example,
at a make-up flow rate of 11.7 L/min (24-hr turnover rate) entering Fig. 3 at point A, the curve
is intersected at point B giving at point C 6T =
-3.5"C. Therefore for a make-up temperature of
ll.0°C, minimum tank temperature would be 11.03.5=7.50C. For the cooling test 1, both
refrigeration units were turned to maximum cooling and the heaters were turned off. Conversely,
for the heating test 2, both heater controllers
v1ere set to maximum and the refrigeration units
\•lf're switched off. Results of both tests 1 and
2 show (Fig. 3) that over the range of make-up
water flow rates from 0-15 L/min the data can be
correlated by straight lines.
Tests 3 (Fig. 4) and 4 (Fig. 5) measured the
capability of the unit to lower or raise the
tank temperature to a pre-determined set point
and then control at that point.
For both tests 3 and 4, 20-gauge copperconstantan thermocouples were placed in the makeJP line; at 3 locations in the tank 45 em below
the surface; and in the ambient air. Recordings
were printed on paper tape automatically by a
scanning digital data aquisition system.
For test 3 the make-up water flow rate was
set at 7.5 L/min total. The system was set for
maximum cooling and allowed to run until the set
point was approached. Then one heater controller
was set to begin applying electrical power to
the heater. The results (Fig. 4) show that for
typical ambient and make-up water temperatures
the time to lower the temperature from 12°C to
7.0"C was about 50 h, after which the temperature gradually reduced to 6.75°C and thereafter
remained within *0.05°C of this value. For test
4 the make-up water flow rate was set at 11.3 L/
min total. The system was set for maximum
heating with both refrigeration units off and
allowed to run until the set point was approached.
Then one heater controller was turned off and the
other set to begin controlling. Fig. 5 shows
that for typical ambient and make-up water
temperatures the time to raise the temperature
from 7.3°C to 14.9°C was about 30 h after which
temperature gradually rose to 15.15°C and held to
within ±0.05°C of this value.
AN APPLICATION: MONITORING
LOCOMOTOR ACTIVITY OF FISH
Species-specific patterns of locomotor
activity have attracted increased attention over
the past 10 to 20 years (cf. Schwassmann 1971).
It presently appears that continuous monitoring
of fish activity and its modification by environmental contaminants may develop into a sensitive
sublethal bioassay (cf. Waller &Cairns 1972;
Bengtsson 1974; Scherer 1977). Our tank, by
providing sufficient size (reducing the likelihood
of "wall effects"), and well-controllable conditions, appeared particularly suitable for experiments of this kind.
One traditional technique to register animal
activity is to record the frequency of light beam
interruptions within the experimental area. Preferably, this light should be invisible (UV or IR).
The poor penetration of long wavelength
light (IR) in water and UV absorption by organic
material render light unsuitable for large-scale
underwater application. Ultrasound appears very
favourable by comparison. We opted, therefore,
for a technique that employs the interruption of
sonar beams as the method to measure fish
activity.
The frequency selected was 200 kHz, transmitting at 63 dB (re 1 ~bar) on the beam axis. 200
kHz is considered to be 1 to 2 orders of magnitude
above the maximum frequency perceived by fish
(Popper & Fay 1973), and a 2 ml-1 drive level was
sufficient to detect fish passages without
producing excessive reverberation. The beam in
such a double-ended system (separate receiver
and transmitter) was found to be essentially
cylindrical with a target-sensitive diameter of
10 em, or approximately 3 degrees wide. The beam
was oriented along a radial line at the midpoint
of the water column. Beam length was 170 em.
Long-term stability of the acoustic field,
as measured by the voltage at the receiving
transducer, was ±10% over 2 weeks, with additional short-term (1-2 second) fluctuations of
±10%. About half of the short-term fluctuation
is attributable to air bubble noise from in-tank
aeration. The remaining ±5% short-term
fluctuation may be due to changes in the tank
standing wave pattern caused by fish movements
and building vibrations. The long term fluctuation is due to signal generator frequency shift,
3
causing tuning into and out of standing wave
modes. Different water temperatures will not
affect beam operation. Tests demonstrated that
rainbow trout from 20 to 60 em length caused
signal levels to drop by 90 to 99% when crossing
the beam. The electronic system was developed to
respond only to on-axis beam interruptions, while
rejecting noise effects and standing wave variations. Counts were recorded on a digital counter,
and were transferred in ASCII (American Standard
Code for Information Interchange) onto punched
paper type. Data were plotted on a Hewlett
Packard 91008 Calculator system which was interfaced with a paper tape reader (Scott 1976).
The timing diagram (Fig. 6) illustrates typical
wave forms for 2 significant beam interruptions.
At t1 we observe a clear pass through the beam.
At t2, a fish enters the beam, leaves partially
and returns, then swims completely out. The
programmable Schmitt trigger (comparator type)
has adjustable high/low thresholds to protect
against such events registering as 2 counts. With
our rainbows from 20-60 em length we used a 'low'
reference at 85% signal reduction, and a 'high'
reference at 65% reduction (e.g. 0.5 and 1.2 volts
respectively for a 3.5 volt steady state signal).
The Schmitt trigger is capacitively OR-coupled
with additional channels to a falling-edge-triggered monostable multivibrator, which drives the
counter. This allows interruptions of any beam
to register counts while another beam is still
blocked by a fish.
Electronic performance was checked
continually over several months by determining
the overall loop gain = D.C. output.
A.C. input
This varied only :!0.3%/24 hours in our case,
whereas T2% was considered to be acceptable.
Acoustic function was tested using standard
targets consisting of air-filled PVC pipe
sections, "'1 em (3/8") and 2 em (3/4") diameter,
sealed off at their ends with rubber stoppers.
When a pipe target entered the beam periphery, an
increase {"overshoot") in received energy was
observed. This was due to energy being reflected
by the pipe into the receiver. Changes in the
overall beam pattern will be roughly reflected in
the overshoot characteristics, while the state of
the transducer beam core may be tested by
observing the effects of targets on axis. The
PVC pipe targets of 1 and 2 em diameter placed
on the beam axis caused respectively 43% and 65%
reduction in received level, repeatable to ±7%.
Overshoot was +10% for 1 em pipe and + 20% for 2
em pipe repeatable to 1:5%.
Electrical noise immunity was tested by
injecting a standard 50 mV 200 kHz electrical
test signal. No counts were recorded over 48
hours of test duration.
Receiving transducers were parallel-tuned
using maximum sensitivity as the criterion. The
transmitters are untuned in this case.
The described system presently employs two
sound beams, but up to about four could be used.
A larger number of beams may result in beam
interactions caused by reverberation. To circumvent this limitation, pulsed techniques could be
applied allowing reverberations to die out before
the next search pulse is sent.
For pulsed applications transmitters should
be series-tuned to minimum phase for maximum
bandwidth and minimum rise and fall time.
Fig. 7 shows examples of activity recordings
achieved using the beam-interrupt technique with
9 rainbow trout, ranging in lengths from 20-45 em.
Although a priori we had considered 2 beams to be
rather marginal to cover the large tank volume
(i.e. we expected some count gaps to occur}, the
results provided uninterrupted recordings with
distinct circadian patterns.
In summary, we believe the system proved its
value and potential as a multi-purpose tool for
ecological analysis, the monitoring of locomotor
activity being but one of the possible applications, as outlined in the introduction of this
report. To conduct multi-species tests in wellcontrolled factorial experiments, something
comparable to phytotron techniques applied by
botanists and agriculturists is a long-term
goal for further research and development work to
be carried out with this system.
LIST OF MAJOR COMPONENTS
1. Paper Tape Punch, Digitec Model 671
$900.
2. Tape Punch Controller, Digitec
Model 623
825.
3. Digital Counter Digitec Model 8120-M
400.
4. Amplifiers &controlling circuitry
400.
5. Transducers, Linden Labs f1odel
60020 (4 required)
100.
6. Circulating Pump, Cole Parmer Model
7006-10 (2 required)
420.
7. Filter-Pacfab 61 em diam (2 required)
850.
8. Rotameter, Brooks t1odel 8-1305
(2 required)
200.
9. Rotameter, Brooks lbdel 10-1305
(2 required)
250.
10. Water cooler-heater, custom design
(2 required)
600.
11. Electric immersion heater, Red Devil
3 k\~ 200V (2 required)
100.
12. Water-cooled refrigeration unit
Copeland Model WSWH 0100 (2 required) 1,000.
13. Electronic temperature control
Multi State Devices STC-20 (2 required) 120.
150.
14. Pool, Doughboy Aqualine 5 m diam
15. Liner for above. Speers Petrochemicals,
Winnipeg
110.
16. Lamp Dimmers. Luxtrol LFD10 (8 required) 800.
17. Fluorescent Lamps, GE 40 watt F70/F55
(72 required)
170.
18. Ballasts for above (32 Bl023 + 8
81024 , Luxtrol)
1,215.
19. Voltage regulator. Sorensen f-1odel
ACR 5000
850.
Total=$9,460.
4
REFERENCES
Bengtsson, B.E. 1974. Effect of zinc on the
movement pattern of the minnow, Phoximus
phoximus. Water Res. 8: 825-833.
Popper, N.A., and R.R. Fay. 1973. Sound
detection and processing by teleost fishes:
a critical review. J. Acoust. Soc. Am.
53(6): 1515-1529.
Scherer, E. 1977. Behavioural assays principles, results, and problems. Proc.
3rd Aquatic Toxicity Workshop, Halifax,
N.S. Environ. Prot. Serv. Tech. Rep. EPS5AR-77-1: p. 33-40.
Schwassmann, H.O. 1971. Biological Rhythms,
p. 372-428. In Hoar &Randall, Fish
Physiology. Vol. 6. Academic Press, New
York &London.
Scott, K.R. 1972. Temperature control system
for recirculation fish holding facilities.
J. Fish. Res. Board Can. 29(7): 1082-1083.
Scott, K.R., and D.C. Gillespie. 1972. A
compact recirculation unit for the rearing
and maintenance of fish. J. Fish. Res.
Board Can. 29(7): 1071-1074.
Scott, K.R. 1976. Speeding up batch telemetry.
Can. Controls Instrum. 15(10): 36-37 and
15(11): 22-24.
Waller, W.T., and J. Cairns, Jr. 1972. The use
of fish movement patterns to monitor zinc
in water. Water Res. 6: 257-269.
WATER CONDITIONING
- TEMR CONTROL
-FILTERING
A
FEED TUBE WITH VALVES
OPENING INTO EACH
SECTOR
I
DRAIN
t
ARRANGEMENT OF
LAMPS IN EACH
SECTOR
B
t
~
---
~
r
t---
'.1M
~X
[1(
.xx
III.X.;
Fig. 1.
rY'
:'f'/
Tank With Water Supply and Lighting.
A:
Top View, B:
Side View.
BACKWASH
AIR LINE
L
A
F
·L I
-B
-------l~----P
L_~~~~~~----~--N
BACKWASH
WASTE TO
DRAIN
c
I
-------l)HI-t~•t----
0
N.O.
Nr:.
MAKE-UP &
BACKWASH
WATER LINE
I
I
-I-.
:;>
s
Fig. 2.
./"
...
0'\
G
TANK
R
Water Conditioning and Circulation Unit.
EH 0
See Text P. 1-2.
.
..
14
a..
10
w
8
<(
I
6
4
z
2
:::l
~
:E
~
<(
I-
u
•
12
HEATING -TEST 2
0
COOLING -TEST I
-2 c
I
-4 -----------------------------~
-6k ~ ::=::=
1<J -8
I
I
I
I
I
I
2 3 4 5 6 7 8 9 10 II
MAKE-UP WATER FLOW RATE
Fig. 3.
:
I
I
:;~
12 13 14 15 16
(L/min)
Heating and Cooling Performance (Tests 1 and 2); Maximum System Capacity Vs. Make-Up
Flow.
'-I
8
12
(..)
o
II
w
MAKE-UP WATER= 7.5 L/min- TEST 3
MAKE-UP TEMP= 10.5±0.1-c:
AMBIENT TEMP = 16.7±0.3-c
0:::
:::::>
1-10
<{
0:::
w
0...
~
9
w
1~
z
<{
8
17
10
20
30
40
50
60
70
80
90
ELAPSED Tl ME ( HRS)
Fig. 4.
Temperature Drop and Control (Test 3).
16
15
~ 14
w
0:::
:::::>
CONTROLLER
ON!.
MAKE-UP WATER= 11.3L/min- TEST 4
13
~ 12
MAKE- UP TEMP= 10.5±0.20C
~II
AMBIENT TEMP= 17.4 ±0.2-<:
0:::
~
~10
z~ 9
;:!
8
7~--~----~--~----~--~----~--~----~
I0
20
30
40
ELAPSED
Fig. 5.
50
TIME
60
70
80
(HRS)
Temperature Rise and Control (Test 4).
9
SIGNAL GENERATOR
TIMJNG
DIAGRAM
®
ENVELOPE
DETECTOR
D C AMPLIFIER
®
MONOSTABLE
=2
t,
MULTIVIBRATOR
COUNTS
t
COUNTER,
TAPE
Fig. 6.
PUNCH
Sonar Beam - Interrupt Technique to Record Locomotor Activity.
See Text P. 2-3.
Rainbow Trout
Locomotor activity recording
over 4 days
20
...
=>
0
J:
I
II
......
I I
\ 1\
0
j
-
';;; I 0
c
~
0
(.)
19=00
Fig. 7.
07=00
Dark
Light
A Record of Locomotor Activity, Obtained by Sonar Beam-Interrupt Technique.
'·
.,
l
...