After the holes are drilled in the cell bottoms, they can be used as templates
to mark the position of the holes to be drilled in the wooden shelves of the
diluter board. These latter holes should be larger than those in the glass.

The most critical cutting is for the W and C dividers since leakage will
occur between individual cells if the width of the dividers varies.

GLASS ASSEMBLY

The most important construction material is the silicone sealant or glass
glue. Dow Corning Glass and Ceramic Cement and General Electric Corporation
RTV are both satisfactory. Glues that are listed as dish-water safe are
preferable so that cleaning the assembled diluter with hot water will not
cause the joints to fail. Disposable l0~ or 15-ml plastic syringes with
enlarged bores in the tips for faster application are useful for applying a
thin bead of glue as needed and can be used with one hand (Figure 5).
Application with the original collapsible tube requires two hands to maintain
a Steady and constant flow of glue from the tube to the edges of the glass. If
the bead of glue is too thin, any irregularities in glass cutting will not be
filled by glue and will leak.

Assembly should begin with the M and FS cells, since they are the simplest
in terms of number of pieces. Waxed or other paper is placed on the table top
to catch any excess glue. The paper can be removed easily after the glue has
dried.

Before placing glue on the glass edges, follow the assembly procedures in
a dry run. For the M and FS cells, glue is placed on all four edges of the
bottom before it is placed on the waxed paper. Next, one of the two longer
pieces (the right and left side of the FS cell or the front and back of the M
cell) is held and glue placed on the two end edges. This piece of glass is
placed against the bottom and propped in a vertical position. The opposing
piece is glued and placed similarly. The remaining two pieces are placed
against the glued edges already in place. Slight pressure at all glued joints
distributes the glue, helps prevent leaks, and places the glass surfaces in
closer contact. To ensure against leaks, a pencil eraser or rounded wooden
dowel may be used to spread the freshly applied, excess glue along each seam.
Use care to avoid moving the glass.

The fronts, backs, and bottoms of the W and C cells should first be placed
as shown in Figure 6 and the lines drawn as indicated to show the location of
the cell dividers during assembly. A wax pencil or felt pen can be used.

It is important to remember during assembly, however, that these marked surfaces
should be on the outside of the cells so that glue adhesion is not affected by
these lines.

The W and C cell assembly procedures are identical. The initial
difference between this procedure and that used for the M and FS cells is
that the W and C cells are built beginning with the back piece placed on the
table top.

13


The third system is the multiple syringe injector as described by DeFoe
(1975) and included in the Appendix. This system completely eliminates
vaporization problems and is useful for highly toxic chemicals dissolved in
organic solvents. An additional very useful aspect of this system is the
ability to change the dilution factor without recalibration of the diluter.
Since each concentration has its own stock bottle, dilution factors can be
changed by changing stock-solution concentrations.

Since two of these three systems require a Mariotte bottle, some advice
as to their construction and use is necessary. The glass bottle itself should
be of 1- to 5~gal capacity and have a narrow mouth for filling. The mouth will
be sealed with a neoprene stopper while in use. A small hole should be drilled
in the shoulder of the bottle into which will be placed a neoprene stopper with
a glass tube (not capillary), the bottom of which will determine the liquid
level in the metering system. An additional small hole near the bottom is
used for connection to the metering system. As the stock solution initially
drains from the bottle, a partial vacuum is produced and air will enter the
bottle through the air tube until the air pressure stabilizes. This initial
flow may exceed the capacity of the metering system and should not enter the
Mcell. After stabilization, air will enter the bottle whenever any liquid
is removed by diluter operation. Wide variation in room temperature causes
variation in the pressure in the bottle and subsequent delivery of the toxic
solution. In this case the bottle should be insulated in such a fashion that
the calibrations can be read daily for monitoring-diluter operation.

34


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30


3) A new C-1 is constructed and placed on the M cell shelf, It will
have a standpipe siphon to deliver 2 liters. A water block is connected to
the vacuum line. The vacuum line that had gone to the W-1 cell can be extended
to go to the new C-1 cell. The old C-1 cell will empty to waste. The new C-1
cell is built to overflow into the old C-2 cell. The tube from the new cell
C-1 water block will go to the same FS cell as did the old cell C-l. The
siphon standpipe in the new cell C-1 must be slightly higher than the over-
flow tube to cell C-2 to prevent a premature siphon.

4) An additional water valve is used to discharge effluent into the new
C-1 cell and fill the remaining cells. This valve is also connected to the
microswitch under the valve bucket. Consequently, both valves open and close
together. The effluent flow rate is set so that cell C-5 overflows into the
old cell C-1 and then to waste before the valve is turned off. If the flow
rate is too slow, one or more of the last C cells will not be full when the
diluter cycles. The dilution-water flow rate may need adjustment since the
large W-1 volume is not needed in this mode.

5) All changes can be made in a temporary fashion for easy conversion
back to the basic diluter.

If there is a continuing need for an effluent-testing diluter, the above
modifications can be incorporated into the original design.

Additions of food, suspended solids, or other similar materials in equal
concentrations to all of the test tanks (Brungs and Bailey, 1967) as well as
variable toxicant concentrations can be accomplished as follows. A special
chamber much like that described by Brungs and Mount (1967) is used. Each
diluter receiving the material requires one cell (Figure 18). An extra cell
is used for timing, and the water coming from it is sent to waste. Water
flowing from those cells used to supply the diluter is used to activate a
metering device that meters the desired amount of material into the mixing
chamber, which in turn empties into the W-1 cells of the diluters of standard
design. Operation of this system is quite similar to regular operation. This
modified system differs in two important ways: (1) each of the initial
chambers must be calibrated to deliver exactly the required amount of water
to feed the diluter including the control (i.e., 12 liters for five concentra-
tions and a control on a 2-liter diluter); and (2) a microswitch or dilution-
water valve is not needed on the diluter since the diluter itself does not
control the flow rate. If suspended solids are used and settling of these
solids is undesirable, the siphon tubes can be set to nearly empty the cells
and thus reduce settling.

The injector system of DeFoe (1975) described in Section 6 is an
additional useful modification. In the standard diluter, when materials with
high toxicity and low solubility are tested, acetone, ethyl alcohol, or similar
low-toxicity water~miscible organic solvents are used to disperse the low-
solubility material in the water. This dispersant is then diluted in the
Same proportions as the toxicant. In certain cases it is necessary to
maintain a constant dispersant concentration and vary only the toxicant
concentration, This system is a series of equal~sized chambers. As these
empty, a ratchet-driven syringe is activated for each concentration. The

39


TABLE 1. METRIC CONVERSION TABLE

Inches Centimeters
48 122
24 61
16 40.6
14 35.5
12 30.5
1l : 28.0
10 25.4
8 20.3
6 15.2
5 12.7
4 10.2
3 7.6
2 5.1
1 2.54
0.75 1.91
0.5 1.27
0.25 0.64



546 J, FISH. RES. BOARD CAN.. VOL. 324), 1975

foe srt tus.

Fic. 1. Front (A) and lateral (B) views of injector system.

was also used to transport the injected material. The The cost of this system contasming five units is
injection mixing cells are Erlenmeyer flasks; one approximately $600~700.

flask 15 mounted on each injector operating lever. A

spring 1s needed to return each musing cell and

operating lever to original position. Counterbalances Benoit. D A.. AND F. A PLoList 1973 A simphfied

can be used instead of springs, if smaller mixing ceils flow-sphitting chamber and siphon for proportional di-
are necessary. luters. Water Res. 7: 1915-1916.

The approximate cost for one complete injection Mount. D. L.. No W. A BRUNGS 1967 A simplified
unit 1 from $100 to $130, this includes the injector dosing apparatus for fish toxicology studies. Water
cost of $85 and syringe cost which varies with size. Res. 1: 21-29

67


BIBLIOGRAPHY

Abram, F. S. H. 1970. Automatic Water Mixing Device. Lab. Pract. 19:915-916.

Abram, F. S. H. 1973. Apparatus for Control of Poison Concentration in
Toxicity Studies with Fish. Water Res. 7:1875-1879.

Bahner, L. H., and D. R. Nimmo. 1975. A Salinity Controller for Flow-Through
Bioassays. Trans. Am. Fish. Soc. 104: 388-389.

Bahner, L. H., C. D. Craft, and D. R. Nimmo. 1975. A Saltwater Flow-Through
Bioassay Method with Controlled Temperature and Salinity. Prog. Fish-Cult.
37:126-129.

Ballard, J. A., and W. D. OLiff. 1969. A Rapid Method for Measuring the
Acute Toxicity of Dissolved Materials to Marine Fishes. Water Res.
3:313-333.

Bengtsson, B. E. 1972. A Simple Principle for Dosing Apparatus in Aquatic
Systems. Arch. Hydrobiol. 70:43-45.

Benville, P. E., Jr., and S. Korn. 1973. A Simple Apparatus for Metering
Volatile Liquids into Water. J. Fish. Res. Board Can. 31:367-368.

Betts, J. L., T. W. Beak, and G. G. Wilson. 1967. A Procedure for Small-
Scale Laboratory Bioassays. J. Water Poll. Cont. Fed. 39:89-96.

Borthwick, P. W., M. E. Tagatz, and J. Forester. 1975. A Gravity-Flow
Column to Provide Pesticide-Laden Water for Aquatic Bioassays. Bull.
Environ. Contam. Toxicol. 13:183-187.

Brenniman, G., R. Hartung, and W. J. Weber, Jr. 1976. A Continuous Flow
Bioassay Method to Evaluate the Effects of Outboard Motor Exhausts and
Selected Aromatic Toxicants on Fish. Water Res. 10:165-169.

Brown, V. M. 1973. Concepts and Outlook in Testing and Toxicity of
Substances to Fish, p. 73-95. In Bioassay Techniques and Environmental
Chemistry. Ann Arbor Science Publishers, Inc., Ann Arbor, Mich.

Brown, V. M. 1976. Advances in Testing the Toxicity of Substances to Fish.
Chem. Ind. 2143-149,

Brungs, W. A. 1973. Continuous-Flow Bioassays with Aquatic Organisms:
Procedures and Applications. Biological Methods for the Assessment of
Water Quality. American Society for Testing and Materials, STP 528.
p. 117-126.

49


REFERENCES

American Public Health Association, American Water Works Association, and Water
Pollution Control Federation. 1971. Standard Methods for the Examination
of Water and Wastewater. 13th ed. Washington, D.C. 874 p.

American Public Health Association, American Water Works Association, and Water
Pollution Control Federation. 1976. Standard Methods for the Examination
of Water and Wastewater. 14th ed. Washington, D.C. 1193 p.

American Society for Testing and Materials. 1976. Proposed Standard Practice
for Conducting Acute Toxicity Tests with Fish, Macroinvertebrates, and
Amphibians. Mimeo. 51 p.

Arthur, J. W., R. W. Andrew, V. R. Mattson, D. T. Olson, G. E. Glass, B. J.
Halligan, and C. T. Walbridge. 1975. Comparative Toxicity of Sewage-
Effluent Disinfection to Freshwater Aquatic Life. U.S. Environmental
Protection Agency, Ecol. Res. Ser. EPA-600/3-75-012. 71 p.

Benoit, D. A., and F. A. Puglisi. 1973. A Simplified Flow-Splitting Chamber
and Siphon for Proportional Diluters. Water Res. 7:1915-1916.

Brungs, W. A., and G. W. Bailey. 1967. Influence of Suspended Solids on the
Acute Toxicity of Endrin to Fathead Minnows. Proc. Purdue Industrial
Waste Conf. 21:4-12.

Brungs, W. A., and D. I. Mount. 1967. A Device for Continuous Treatment of
Fish Holding Chambers. Trans. Am. Fish. Soc. 96:55-57.

DeFoe, D. L. 1975. Multichannel Toxicant Injection System for Flow-Through
Bioassays. J. Fish. Res. Board Can. 32:544-546.

Doudoroff, P., G. B. Anderson, G. E. Burdick, P. S. Galtsoff, W. B. Hart, R.
Patrick, E. R. Strong, E. W. Surber, and W. M. Van Horn. 1951. Bioassay
Methods for the Evaluation of Acute Toxicity of Industrial Wastes to Fish.
Sew. Ind. Wastes 23:1380-1397.

Halligan, B. J., and J. G. Eaton. 1978. Survival and Reproduction of Gammarus
lacustris and G. pseudolimnaeus Under Two Experimental Conditions. Prog.
Fish-Cult. 40(2) :59-62.

Hart, W. B., P. Doudoroff, and J. Greenbank. 1945. The Evaluation of the
Toxicity of Industrial Wastes, Chemicals and Other Substances to Fresh-
Water Fishes. Waste Control Laboratory, The Atlantic Refining Co.,
Philadelphia, Pa. 347 p.

47


Roberts, R. F., and H. E. Allen. 1972. The Control of pH and Total
Alkalinity or Total Carbonate in Aquatic Bioassays. Trans. Am. Fish.
Soc. 101:752-756.

Scheier, A., and D. T. Burton. 1973. A Description of Bioassay Flow-
Through Techniques, and the Use of Bioassay to Measure the Effects
of Low Oxygen at the Whole~Animal and the Molecular Level, p. 335-344.
In Bioassay Techniques and Environmental Chemistry. Ann Arbor Science
Publishers, Inc., Ann Arbor, Mich.

Schimmel, S. C., D. J. Hansen, and J. Forester. 1974. Effects of Aroclor®
1254 on Laboratory-Reared Embryos and Fry of Sheepshead Minnows
(Cyprinodon variegatus). Trans. Am. Fish. Soc. 103:582-586.

Shumway, D. L., and J. R. Palensky. 1973. Impairment of the Flavor of
Fish by Water Pollutants. U.S. Environmental Protection Agency, Ecol.
Res. Ser. EPA-R3-73-010. 80 p.

Smith, A. D., J. R. Butter, and G. W. Ozburn. 1977. A Pneumatic Dosing
Apparatus for Flow-Through Bioassays. Water Res. 11:347-349.

Solon, J. M., J. L. Lincer, and J. H. Nair, III. 1968. A Continuous Flow,
Automatic Device for Short-Term Toxicity Experiments. Trans. Am. Fish.
Soc. 97:501-502.

Sprague, J. B. 1969. Measurement of Pollutant Toxicity to Fish. I. Bioassay
Methods for Acute Toxicity. Water Res. 3:793-821.

Sprague, J. B. 1970. Measurement of Pollutant Toxicity to Fish. II.
Utilizing and Applying Bioassay Results. Water Res. 4:3-32.

Sprague, J. B. 1971. Measurement of Pollutant Toxicity to Fish. III.
Sublethal Effects and "Safe" Concentrations. Water Res. 5:245-266.

Sprague, J. B. 1973. The ABC's of Pollutant Bioassay Using Fish. Biological
Methods for the Assessment of Water Quality, American Society for
Testing and Materials, STP 528. p. 6-30.

Stark, G. T. C. 1967. An Automatic Dosing Apparatus Made with Standard
Laboratory Ware. Lab. Pract. 16:594-595.

Stephan, C. E. 1973. Chemistry and Fish Toxicology, p. 97-105. In Bioassay
Techniques and Environmental Chemistry. Ann Arbor Science Publishers,
Inc., Ann Arbor, Mich.

Stephan, C. E., and D. I. Mount. 1973. Use of Toxicity Tests with Fish
in Water Pollution Control, p. 164-177. In Biological Methods for the
Assessment of Water Quality. American Society for Testing and Materials,
STP 528.

Syrett, R. F., and W. F. Dawson. 1972. An Inexpensive Electronic Relay
for Precise Water-Temperature Control. Prog. Fish-Cult. 34:241-242.

53


Fic. 1. Photograph of a proportional diluter built as suggested in this paper.

57


Figure 19, “Dipping~bird" safety device.

45


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é

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AN

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17


SECTION 7

DILUTER MODIFICATIONS

The proportional diluter lends itself to numerous modifications. An
experienced person who has built several diluters can usually modify them to
adapt to special cases. It is recommended that a regular diluter be made and
used first, before modifications are tried. ‘he following directions assume
familiarity with the basic diluter. Some of the modifications that we have
used are multiple synchronous cycle systems; low toxicity effluent, i.e., 100%
effluent; superimposed additions, i.e., turbidity or food added in equal amounts
to all concentrations and then a toxicant added and diluted; and equal solvent
concentrations with dilution of only the toxicant and including a solvent
control,

The use of multiple diluters in synchronous set (Figure 15) is accomplished
as follows (Arthur et al., 1975). An extra W cell is added and fitted with an
overflow to waste, and the regular sixth or control cell is fitted with a
water block and starting siphon. The other cells are the same as in an
unmodified diluter. The flow~adjusting valves on the separate diluters are
adjusted to fill the W cells just a little faster than the required cycle time.
All of the vacuum lines are connected together and are started by a side-arm
vacuum aspirator from a water line by having the valve~bucket switch turn on
the water as needed. Timing is accomplished by building a separate timing
chamber such as shown in Figure 16 (Halligan and Eaton, 1978). The fill
time for this upper bucket is slightly longer than that of the W cells. When
this chamber cycles into the valve bucket, the supply solenoids are shut off
and the vacuum aspirator is activated. The emptying time must be sufficient
to allow all diluters to cycle, and when the valve bucket finally empties the
cycle is repeated. Equal flows are not necessary once all diluters are started
as long as the valve bucket stays down until the last diluter completes its
cycle. We have successfully operated three 2-liter and three 1-liter diluters
synchronously for nearly a year (Arthur et al., 1975).

In many instances it will be necessary to determine the toxicity of
industrial and municipal wastes or other materials that may not be extremely
toxic. Mariotte bottles or similar stock solution supplies are inadequate
when toxicity is measured in percentage instead of milligrams per liter. The
details for a modified diluter whose highest test concentration is 100% effluen
are shown in Figure 17. Only a few changes need to be made on a standard
diluter to convert it to an effluent-testing diluter.

1) The vacuum tube to cell W-1 is clamped so that the cell cannot empty
2) The M cell and chemical-metering device may be removed.

35


NOTES

tions can be changed independently of each other
and to any proportion with only minor adjust-
ments.

2) The device can be used in bioassays where
measurement of toxicant in solution, such as a
polyelectrolyte, is impractical or difficult. The
toxicant concentrations of the test water can be
precisely calculated from the amount of toxicant
injected and the volume of water used.

3) The system operates mechanically by using
the force of falling water; the operation of each
injector unit ceases to operate if the water flow
stops. This reduces the possibility of toxicant over-
dosing or flushing of individual concentrations in
the event of a support-system failure.

4) The commercial availability of the injection
units minimizes the amount of construction done
by the researcher, and the durability and simplicity
reduce the maintenance demand.

During the 30-mo evaluation of the injector
system, the coefficient of variation (C.V.) of dis-
pensing water soluable toxicants into bioassay
systems ranged from 8 to 12%. The injected
volume of polyelectrolytes measured over a month
period showed a C.V. of 8%. The C.V. for a
4-mo cadmium zinc mixture study ranged from
10 to 12%. Concentrations ranged from 4.8 to 8.0
and 25-140 yg/liter, respectively, for the two
metals. These C.V.s represent injector error and
analytical error.

For insoluble compounds, such as PCBs and
methoxychlor, the C.V. increased. A 14-mo PCB
exposure with concentrations ranging from 0.1
to 10 yg/liter, resulted in C.V. of 24-42%.
Methoxychior measurements after a month period
showed a C.V of 20%. These C.V.s include
dissolution, sorption, and analytical error in addi-
tion to injector error.

Operation — The delivery system is diagrammed
in Fig. 1A. Incoming water controlled by a
solenoid enters water-metering cell 0. Consecu-
tively, each cell fills and overflows, and fills the
subsequent cell. The last cell, 7, has a self-starting
standpipe siphon described by Benoit and Puglisi
(1973). This siphon delivers water from cell 7
to a bucket suspended over a microswitch which
shuts off the incoming water. From this bucket
the water siphons off and flows through a U-tube
creating the venturi vacuum, which starts the
flow through the remaining metering cell siphons
(1-6). Equal volumes of water are discharged to
each of the mixing cells below.

Each mixing cell is mounted on the operating
lever of the injector (Fig. 1B) and triggers an
injection each time water enters it. The operating
lever turns a cogged wheel that drives a threaded

66

545

rod against the syringe plunger. The injected
liquid is transported by a teffon tube from the
syringe to the mixing cell, where the turbulence
of the incoming water ensures adequate mixing.
Proper calibration of the standpipe siphon in the
mixing cell ensures that nearly all of the toxicant
and water is present and mixed before being
discharged into the flow-splitting cell. This cell
is also equipped with a standpipe siphon for more
accurate flow splitting and easier cleaning.

Control water can come from any of the water-
metering cells: in this system ceil 2 was used
(Fig. 1A). An individual metering cell ensures
that the control will always receive an amount
of water (1 liter) equal to that of other test tanks.
The proportional diluter uses the water remaining
after all metering cells are filled to supply control
water. Therefore, if flow rates change for any
reason, the control water volume could vary as
much as 50% until adjustments are made.

The dispersant control has one injection unit
to dispense the dispersant only, while the control
water empties directly into the flow-splitting cell.

Total cycling time for this system depends on
the flow rate of incoming water: in practice the
fastest was approximately one cycle per minute

The toxicant concentrations can be varied by
one or all of the following adjustments:

1) The toxicant stock solution for the syringes
can be changed. This adjustment will vary each
concentration independently of the others.

2) The quantity of toxicant solution injected
can be controlled by syringe size (10-50 ml) and
by adjusting the set screws on the operating
lever (Fig. 1B). These screws control the amount
of pivot of the operating lever, which can turn
from [ to 10 cogs of the wheel per cycle. These
adjustments allow an injection volume range of
2.3-85 yl.

3) The amount of water receiving the injection
can be altered by recalibrating the siphon in the
water-metering cell.

Materials — Most of the materials needed are
similar to those described by Mount and Brungs
(1967) for the proportional diluter. The following
materials are needed for the injection units — one
mechanical injector for each toxicant concentration
and for the dispersant control. The injectors are
available commercially; however. there 1s no patent
on the injector so it can be copied or constructed by
any individual. For each injector one gas-tight gas-
chromatograph syringe is required. Sizes 10-0 ml
have been used successfully This type syringe was
used because of the resistance of the teflon plunger
tip to toxicants and their dispersants. Teflon tubing


Number
1

2

12

13
14
15
16
17

18

19

20

FIGURES

Completed mounted diluter . . 6 6 ee ee et ee ee ee ee
Diluter board with shelves mounted . . 1... 1 ee ee eens
Glass-cutting layout «2. 6 1 ee ee ee we we ee ee ww we
Glass-cutting layout «6 6 1 ee ee ew wee ee we we wt
Glue-application system

Gluing layout for W and C cells... 1 ee ee ee ee eee
Gluing procedure for Wcells 2... 2s. eee eee ee ene
Final assembly plan for all cells... . 2.2... peewee
Tubing and accessory plan for assembled diluter.......-.
Valve bucket assembly... 1... eee we eee ee te ee
Toxicant metering assembly plan for the "dipping bird" system .

Construction plan for a "dipping bird" toxicant metering system
using volumetric pipette . 2... ee eee ee ee eee ee

Toxicant-metering assembly using "McAllister system" .....
Safety system for the "McAllister toxicant-metering system" . .
General plan for synchronous multiple diluter system ....

Timing system for multiple diluter system... . 1... ewe
Diluter modifications for effluent dilutions .........

Diluter system modified to include the addition of a food supply
(microorganisms) . 6 6 ee ee ee te ee ee ee te

"Dipping bird" safety device . 2.1... 1 2 eee eee eee
Diluter malfunction safety system... 2. +e eee eevee

vi

1

12

14

15

v7

18

20

30

31
32
33
36
37

38

40

45

46


1916 Dvane A. Benoit and Frank A. PuGList

The apparatus consists of six identical chambers with four flow-splitting siphons per
chamber (Fic. 1). Glass flow-splitting chambers measured 10 x 15 » 18cm high.
Four 2:5 cm holes were drilled in the bottom of each chamber. Glass-tube standpipes,
with two notches cut in one end (0-6 cm wide by 1-6 cm deep) and a neoprene stopper
around the other end, were inserted in each of the four holes so the bottom of the
notches measured 15 cm above the bottom of the chamber. Glass-tube sleeves, with a
neoprene stopper around the outside of one end and the core from the hole bored in
the stopper inserted in the other end, were placed over each standpipe. The fiow-
splitting chambers with siphons were then positioned beneath the diluter so the
toxicant-bearing water and dilutant water fell directly into each chamber. This
arrangement allows additional mixing before delivery to each exposure tank. As the
test water rises slightly above the top of the sleeves in each chamber, water is forced
through the notches and down the standpipes. This action creates a siphon which
empties the chamber and delivers test water to each exposure tank.

The diameter of each standpipe and sleeve was determined by the flow rate of each
concentration delivered from the 2-1. diluter into each chamber. If the diameters of the
standpipes are too large, some siphons will not start: and if they are too small, some
siphons will start ahead of the others. Siphon tubes for each duplicate fry and adult
exposure tank delivered 150 and 850 ml per cycle, respectively, and were calibrated by
moving the stoppers, in or out, on either end of the siphon sleeve. Siphon sleeves for
the adult tanks must be kept as long as possible so each mixing chamber empties after
every cycle, Test water delivered to each exposure tank varied by only 5-10 per cent
of the calculated volumes.

Delivery tubes from the flow-splitting chamber to each exposure tank should be
large enough to fit loosely over the lower end of each standpipe. The air break between
standpipe and delivery tube eliminates back pressure which can cause the siphon to
maifunction. If delivery tubes must be attached directly to the standpipes, they must
slope downward toward the exposure tanks so each delivery tube empties after every
cycle.

REFERENCE

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

69


APPENDIX C

Water Research Pergamon Press 1973. Vol. 7, pp. 1915-1916. Printed in Great Britain

NOTE

A SIMPLIFIED FLOW-SPLITTING CHAMBER AND
SIPHON FOR PROPORTIONAL DILUTERS

Duane A. BeNoiT and FRANK A. PUGLISI

U.S. Environmental Protection Agency, National Water Quality Laboratory, 6201 Congdon
Boulevard, Duluth, Minnesota 55804, U.S.A.

(Received 9 May 1973)

Abstract—Simplified flow-splitting chambers and siphons were designed and tested for use
with proportional diluters in bioassay systems. The apparatus allows each concentration from
the diluter to be thoroughly mixed and divided four ways for delivery to duplicate fry and
adult exposure tanks. Test water delivered to each exposure tank varied by only 5-10 per cent
of the calculated volumes.

THE PROPORTIONAL diluter developed by Mount and BRuNGs (1967) provided a
dosing apparatus which can maintain a series of constant concentrations of toxicant in
flowing water for bioassay systems. A typical bioassay system usually requires that
each concentration be divided four ways for delivery to duplicate fry and adult
exposure tanks. Flow-splitting chambers and siphons described in this report were
designed to be used with a proportional diluter which delivers 2 1. per concentration,
but the system can be modified to fit a diluter of any size. The simplified siphon has
several advantages over the conventional U-tube siphon: difficult tube bending is
eliminated, water volumes siphoned through each flow-splitting tube are easily
adjusted, and tubes can be disassembled for cleaning simply by removing each sleeve
from the standpipe.

Fic. 1. Flow-splitting chamber and siphons for proportional diluters: 4, siphon sleeve; B,
siphon standpipe; C, flow-splitting chamber; D, delivery tube to exposure tank.

1915

68


should take place while the diluter is functioning but with the delivery tubes
to the test chambers themselves diverted so that any accidents during calibra-
tion while tests are ongoing will not adversely affect the test. The delivery
volume from each cell should be checked at least twice for proper accuracy.

The W-1 volume is collected by an appropriate graduate cylinder or other
container as it enters or leaves the M-l cell. Necessary adjustments are made
based on common sense by moving the neoprene stopper core up or down in the
siphon sleeve to increase or decrease, respectively, the volume transferred.

By common sense we mean that only the necessary level of accuracy (i to 5
percent usually) is desirable. Finer adjustment is only time consuming. Cells
W-2 through W-5 are calibrated one at a time by disconnecting at the C cell
venturi the tube that connects the water block to the C cell venturi. The
water from the M cells is then collected when the diluter functions. The
corresponding C cell will not empty during this calibration step. After some
experience, two W cells can be checked at once. The W-6 volume, as discussed
earlier, is flow dependent and should only be calibrated at the intended flow
tate. Its delivery volume can be taken at the discharge from the valve bucket
or where it enters the control flow-splitting cell. Do not take it before it
enters the valve bucket since the valve will not function. Adjustments in the
delivery volume are made by the neoprene stopper core in the siphon sleeve of
W-6. Once the transfer volumes of cells W-2 through W-5 are fixed, cells C-2
through C-5 are calibrated by collecting the total flow from the paired W and

C cells before it enters the flow-splitting cells. The C cell volumes are
calculated and adjusted by difference between the total volume of the two cells
and that corresponding W cell volume previously determined. The C-1 cell
volume is measured as a double check on the calibrations of cells W-l and C-2
through C-5. Finally, the flow-splitting cells are calibrated after the
delivery tubes are replaced to simulate normal operation. The transfer volumes
are collected at the test chambers, again to simulate normal operation. These
flow-splitting cell volumes should be checked weekly and after any cleaning or
other manipulation.

28


(SIDE VIEW)

|
| \\_raste TOP

(TOP VIEW)

GLASS ADHESIVE

N\N

TABLE TOP

Figure 5. Glue~application system. Syringe tip to be bored out to
approximately 4 mm to give sufficient bead size.

14


SWITCH

VACUUM
[LINE

TRIGGER
LINE

SPRING

je T VENTURI

—]

FS rs{]| [[ rs

Reames TEST Vue! } TY

Figure 9, Tubing and accessory plan for assembled diluter.

FS FS FS

20


A bead of glue is placed on the bottom edge and ends of the back piece of
the W or C cell, and it is placed on the paper covering the table top. Glue is
placed on each end of the cell bottom, which is then placed against the large
edge and supported as shown in Figure 7. If the angle between the back side
and bottom is kept at slightly more than 90°, the first divider can be placed
more easily. Beads of glue are placed on three edges of the first divider;
none is added to the top edge. This divider is placed on edge on one of the
lines on the back side of the cell so that the two bottom edges are in line
(Figure 7). The bottom of the cell is rotated up to a 90° angle with the back
so that the bottom edge of the divider matches the line drawn on the cell
bottom. Continue to support the bottom with a block. Glue is added to three
edges of each of the remaining dividers and each is carefully placed in
position. The next step is to place a bead of glue on the bottom edge and
ends of the front side. This edge is placed in the corner formed by the
bottom of the dividers and the bottom of the cell. The front side is then
lowered to rest on the dividers so that those edges match the lines drawn on
the front side. If difficulty is encountered with multiple gluing of the W
and C cell dividers, each may be glued on one short to long side only and set
individually, leaving a 2-hr setting period between each. After these are
dry, glue may be applied to the long exposed edge of each divider. The long
edge of the eventual front is also glued, and the front is placed on the glued
edges as above. The ends can now be put in place without glue since they will
contact previously glued edges. As with the M and FS cells, a pencil eraser
or wooden dowel can be used to smooth the glue before drying to help prevent
leaks if this is necessary. With practice, this procedure may not be necessary
as the glue beads should then be placed evenly enough and thick enough to
avoid leaks.

After all cells have dried overnight, they should be tested for leaks after
plugging the drilled holes. The exposed edges should be smoothed with a file
or sandpaper to eliminate cuts. All cells can now be placed on the diluter
board in the approximate positions shown in Figure 8.

Other assembly procedures may become more desirable after experience has
been gained. The above details should be acceptable for beginners.

16


SECTION 6

TOXICANT METERING

The bibliography to this report contains numerous citations for toxicant-
metering systems. Any system should be activated only by diluter operation to
avoid overuse of the toxicant. We have had significant experimental experience,
with three systems, and we will discuss them in the order of their development.

The "dipping-bird" system (Figure 11) was described by Mount and Brungs
(1967). Details can be found in the Appendix. This metering device is useful
for water-soluble, non-volatile toxicants and some suspended solids. It can
be made most easily from a volumetric pipette (Figure 12) of an appropriate
size, usually between 2 and 50 ml. The dipping bird is located between the
W-1 cell and the Mcell. The cup is filled when W~l empties and the weight
and force of water rotates the arm about the pivot to introduce a known and
constant volume of stock solution. A small-diameter tube from the side of the
W-l water block above its normal water level should be used to put water in
the cup. The force of all the water from cell W-1 could damage the dipping-bird
assembly. After cell W-l is empty, the cup contents empty through the drain
hole allowing the arm to return and refill. The adjustable weight is necessary
to ensure proper rotation. By adding successive known quantities of water
(100 ml) to the Mariotte bottle (2- to 5-gal) it can be calibrated by marking
with a diamond point pencil so that the amount of toxicant used each day can
be determined. When the amount of toxicant is divided by the number of
diluter cycles over a known time period, the mean toxicant-delivery volume per
cycle can be determined by calculation. An electric counter wired to the
microswitch will provide the number of cycles. Also, the mean cycle time can
be determined. For example, over a 24-hr period (1,440 min) there were 576
cycles and approximately 1,210 ml of stock solution were used. The mean cycle
time was 2.5 min, and the mean dipping-bird volume was 2.1 ml. This procedure
is used for calibration of the dipping bird, and it is required daily to
monitor dilution-water flow rate and nominal toxicant concentration.

The McAllister system (McAllister et al., 1972) is described in the
original paper. It also requires a Mariotte bottle system. The detail of its
relationship to a proportional diluter is shown in Figure 13. This device is
more useful for volatile toxicants or toxicants dissolved in a solvent as there
is reduced contact with air. Figure 14 shows a modification of the McAllister
system by Puglisi (unpublished data) to protect against a malfunction of the
Mariotte bottle that would permit the introduction of excessive amounts of the
stock solution. The McAllister system can be calibrated and monitored in the
same manner as the dipping bird. When constructing this device, the capillary
tube should be as small as possible to prevent variations in the addition of
the toxic solution if variations in siphoning by cell W-1 occur.

29


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1L


TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing}

T REPORT NO.
EPA-600/3-78-072

2.

3 RECIPIENT'S ACCESSION NO

) TITLE AND SUBTITLE

rs

Manual for Construction and Operation of Toxicity-
Testing Proportional Diluters

iS REPORT DATE
July 1978 issuing date

Js PERFORMING ORGANIZATION CODE

AUTHOR(S)

A. E. Lemke, W. A. Brungs, and B. J. Halligan

8. PERFORMING ORGANIZATION REPORT NO.

rr)

PERFORMING ORGANIZATION NAME ANO ADDRESS
U.S. Environmental Protection Agency

10. PROGRAM ELEMENT NO
1BA608

Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804

TT. CONTRACT/GRANT NO

12. SPONSORING AGENCY NAME ANDO ADDRESS
Environmental Research Laboratory - Duluth

13 TYPE OF REPORT AND PERIOD COVERED

Office of Research and Developrent
U.S. Environmental Protecrion Agency
Duluth, Minnesota 55804

14 SPONSORING AGENCY CODE

EPA /600/03

18 SUPPLEMENTARY NOTES

76 AesTaacT

This paper presents a discussion of the testing pro
diluters. The construction, calibration, and operation
and trouble shooting techniques necessary
given.

A bibliography includes many related
in the text but which should be useful to
on physical toxicity testing methods, but
procedures are not included.

the reader. I
papers on stat

for successful use of such equipment are

published materials that are not discussed

cedures using proportional
of the equipment is explained,

necluded are numerous citations
istics or biological test

KEY WORDS AND DOCUMENT ANALYSIS

DESCRIPTORS lb IOENTIFIERS/OPE

NENOEO TERMS |.. COSATI I icld Group

Dilution Testing procedures
Construction Dilution equipment
Toxicity

Diluters propurtional
Operation calibration

14-8

78. DISTRIBUTION STATEMENT 1S SECURITY CLASS This Report | 21 NO OF PAGES
UNCLASSIFIED 78
RELEASE TO PUBLIC 20 SECURITY CLASS (This page; 22 PRICE
UNCLASSIFIED

EPA Form 2220-1 (Rev. 4-77) PREVIOUS ETITION ‘s OBSOLE

70

1 US GOVERNMENT PRINTING OFFICE 1978— 787.140/1442


APPENDIX A

Stater Research, Pergamon Press 1967. Vol. 1, pp. 21-29. Printed in Great Britain.

A SIMPLIFIED DOSING APPARATUS FOR FISH
TOXICOLOGY STUDIES

Donato I. Mount* and WILLIAM A. BRUNGS*

‘Aquatic Biology Activities, Basic and Applied Sciences Program, Cincinnat: Water
Research Laboratory, Federal Water Pollutton Control Administration,
U.S. Department of the Interior

(Received 12 September 1966)

Abstract—A simplified diluter for maintammng a scenes of constant concentrations of a material
in flowing water is described. It depends on water flows, metering cells, and venturi tubes to.
proportion volumes of water and toxicant to give desired concentrations, Construction
requires less than 2 days, and only readily available materials are nccded. An injector for mixing
pesticides in water is also described.

INTRODUCTION

Mount and Warner (1965) have described a serial dilution apparatus suitable for
maintenance of constant concentrations of materials in flowing water. They have
discussed the need for reliable systems that cannot deliver an excessively high con-
centration of toxicant in long-term fish toxicity studies. At the Newtown Laboratory
of the Cincinnati Water Research Laboratory, Federal Water Pollution Control
Administration, Cincinnati, Ohio, we have used this system for several years in fish
toxicology studies and have been well satisfied with its performance. Because of a need
for more narrow concentration series, such as 1, 0.8. 0.64, 0.51, etc.. we have modified
the serial diluter in order to make it more suitable for such uses. We have also found
that some of those who have constructed serial diluters have had problems before they
were able to achieve satisfactory operation. Apparently selection of tubing sizes was
troublesome. Some of the components and principies herein described, particularly
the water delivery system, can be used advantageously on the serial diluter and are
discussed later.

The modified diluter, called a proportional diluter, is not based on serial dilution
but rather on simultaneous dilution of one concentration. It has these advantages
over the serial diluter : (1) water is delivered to each chamber each half cycle so that the
tlow rate can be twice as great ; (2) timing problems are minimal ; (3) operation is much
simpler and easier to understand ; (4) malfunctions are less frequent than in the serial
diluter system; (5) it can deliver a series of concentrations, each concentration as
much as 90 per cent of each preceding concentration ; and (6) much less vertical space
ty needed. The main disadvantage is that it is impractical to deliver a series of con-
ventrations with a dilution factor greater than 50 per cent between each concentration ;
© g. a concentration series such as 1, 0.1, 0.01, etc.

The proportional diluter shown in Fic. | and described in this paper is one that can
deliver 5 toxicant concentrations and a control at any desired flow rate per concen-
tration up to 400 ml/min, and with a dilution factor from SO to 25 per cent between

* Respectively Fisheries Research Biologist and Aquatic Biologist.

55


: SECTION 2

DILUTER OPERATION - GENERAL

The purpose of the proportional diluter (Mount and Brungs, 1967) is simply
to provide several different mixtures, or proportions, of a toxic solution
with dilution water. Such diluters typically provide five different toxicant
concentrations and a control, which is dilution water. The automatic and
consistent production of dilutions permits the conduct of continuous-flow
toxicity tests with aquatic organisms.

The following description and reference to Figure 1 will clarify the basic
components of an operational diluter and their function. Dilution water enters
the upper left of the top row of glass cells, called the W, or water, cells.
During operation these W cells will empty to mix with water from the lower
C, or chemical, cells, The left W cell will empty into the M, or mixing cell,
and cause the introduction of a small volume of the toxic solution. The two
volumes mix before overflowing into the third level of cells, the FS cells.
When the W cells empty, they cause the matching C cells to empty by venturi
action. Different proportions or volumes of dilution and toxic-solution waters
are mixed to provide the different toxicant concentrations to be tested.

These mixtures enter the fourth level of cells, the FS or flow-splitting cells,
that divide each concentration into equal volumes for distribution to the
replicate test chambers containing the organisms being exposed.

A more detailed discussion of the diluter operation will follow after the
basic components and construction details have been described.


SECTION 5

DILUTER CALIBRATION

PRECALIBRATION

Table 4 contains the necessary cell delivery volumes for 50% and 33%
dilution ratios for 2-liter diluters with five test concentrations and a
control. The easiest procedure for initial precalibration requires that the
W-1 through W-5 and C-2 through C-5 cells be filled with an excess that
overflows into the W-6 and C-l cells. A siphon emptying into a graduate
cylinder is used to remove the appropriate volume in a cell; the conterts of
the siphon itself are returned to the cell. A mark is then made at the
resultant water level. The siphon sleeve is cut to end at this mark. The
core from a neoprene stopper can be used in the top of the siphon sleeve (see
Benoit and Puglisi, 1973 in Appendix). Adjustments in the position of this
neoprene core in the siphon sleeve allow for final, more precise calibration
if necessary.

Calibration of the M cell and valve bucket is different since these
containers should be nearly empty after siphoning. The appropriate volume
(Table 4) is added, and a mark is made to represent the water level. The top
of the siphon tube is set at this level. The siphon sleeve is placed so that
it is near the bottom of the container, but not so near as to restrict the
water flow. Since the siphon tube is notched at the top, a siphon effect will
begin just before the total volume enters the container. The volume of control
water entering the valve bucket is dependent upon the flow rate, and any
variation in the latter will change this volume. If the valve bucket does not
empty completely, the spring may not be strong enough to reset the microswitch.

The top of the W-6 siphon tube is below the top of the cell divider. The
siphon sleeve should be set so that less than the required 2-liter volume is
needed to start the siphon to accommodate the time lapse between the beginning
of the siphon and the turning off of the valve. Also, after dilution water
stops entering*the W-1 cell, some water will continue to enter W-6 as it
drains down from the other W cells. As a start, have the W-6 siphon begin
after about 1.5 liters have entered the W-6 cell and adjust if necessary. The
reader is referred to Benoit and Puglisi (1973) in the Appendix for details
on calibration of the flow-splitting cells.

FINAL CALIBRATION

After precalibration has been completed, the diluter should operate at
least overnight before making final adjustments. These final adjustments

26


A Simplified Dosing Apparatus for Fish Toxicology Studies 27

and kill the animals.) For longer tests, a safer device is needed. FIGURE 4 (taken from
Mount and WaRNkr, 1965) illustrates the method of choice for highly water-soluble
materials. By this method, the water solution is kept at a constant level in the funnel
by a Mariotte bottle. (The bottle must be insulated against rapid air temperature
fluctuations or the funnel may overflow.)

amore
sorree

ww

Fic. 4. Detatl of chemical-metering apparatus.

When water enters the plastic bucket of the chemical-metering apparatus from cell
W-1 (labelled funnel No. | on drawing), the tube rotates and the toxicant solution runs
through the tube and into the mixing chamber (M-1) beneath. The tube is made by
heating and drawing an appropriate sized piece of glass tubing and then bending it to
the necessary angle. By experience, we have found that partially closing the funnel
end of the tube (by firepolishing) and cutting a hole in the top for filling and releasing
air gives slightly better accuracy. (Note: The Mariotte bottle is not drawn to scale.)

Fon organics that are slightly soluble in water, we have used an injector as sketched
in Fic. 5. It is simply a lever arm actuated by the water filling the plastic bucket and
causing the arm to rotate. On the end opposite the bucket, a small pawl advances a
gear, 1, 2, or 3 teeth; the gear wheel turns the nut a few degrees, advancing the bolt
and piston a very short distance and displacing a few ul of solution through the needle
into the water from cell W-1. We have used a gear with 42 teeth and a bolt with 40
threads/in. so that by advancing the gear, one tooth at a time, there are 1680 injections/
in. of piston travel. With a 1-ml syringe, this gives approximately 0.25 yl/injection;
this can be increased up to 30 ul if a 50-mi syringe is used and the gear is advanced
three teeth. Thus, one full syringe lasts for 3-10 days, depending on the cycle time of
the diluter. Although the injector may seem difficult to construct, if one has a suitable
gear and the bolt, the rest can be made from glass tubing, rubber stoppers and burette
clamps.

Acetone solutions of organics can be used in the syringe as a stock solution, or if
the toxicant is a liquid and is water miscible, it can be used without further dilution.

62


A Simplified Dosing Apparatus for Fish Toxicology Studies 2
(D) Modifications of the serial diluter

The type of water-metering cell described in this paper is superior to that described
by Mount and Warner (1965). The main advantage is that the problem of pushing
water over the water siphon tubes does not exist because the system is an open one
and no pressure can develop. In addition, volume adjustments can be made more
readily, either by moving the tubes or using volume displacers.

Only one minor change need be made to adapt the open cell system to the serial
diluter. The water siphon tubes WS-1 to 5 must be set so that the siphons start in
proper order. This is achieved by setting WS-1 and WS-5 as close to the cell edge as
possible (as shown in Fic. 2B) and then raising WS-4 approximately 5 mm, WS-3,
10 mm, and WS-2, 15 mm above the cell edge. Funnels may be used for water blocks
as previously described or plastic bottles may be used as shown in Fic. 1. It is neces-
sary, as for the proportional diluter, to slope the cell unit so that when the influent
water is shut off by the valve, the water will drain down quickly to the level of the
Fartition tops.

SUMMARY

The diluter herein described has been found by testing to be as dependable as or
more dependable than the serial diluter described by MouNT and Warner (1965).
It operates simply and is much easier to understand and construct. The diluter shown
in Fic. 1 was built in approximately 13 hr. For very wide concentration ranges with
very large dilution factors between each concentration, the serial diluter (MOUNT and
Warner, 1965) is best, but for dilution factors, 50 per cent and smaller, the one
described here is superior.

REFERENCE

Mount D. I. and Warner R. E. (1965) A Serial-dilution Apparatus for Continuous Delisery of Variow
Concentrations of Materials in Water, U.S. Public Health Service Publ. No. 999-WP-23, 16 p"

64


APPENDIX B

Multichannel Toxicant Injection System for Flow-Through Bioassays

Davin L. DeFor

U.S. Emironmental Protection Agency, National Water Quality Laboratory Duluth, Minn 55804, USA

DeFoe. D L. 1975. Multichannel toxicant injection system for flow-through bioassays. J Fish.
Res Board Can. 32 S44-S46,

A mechanical injection system needing little maintenance iy descnbed which offers a de-
pendable and flexible method for maintaining multuple toxicant concentrations in flow-through
bioassays. Falling water from a senes of water-metenng cells similar to those of a proportional
diluter, activates a senes of mechanical synnge injectors. The system dispenses equal amounts of
toxicant dispersants (solvents. wetting agents. or additives) to all toxicant concentrations and toa
dispersant control and allows changing unlimited toxicant concentrations independently of each
other

DeFor. D. L. 1975 Multichannel toxtcant mection system for flow-through bioassays J. Fish
Res. Board Can. 32. $44-546

L'auteur décnt un appareil d'imjection mécanique ne requierant que peu d'entretien et
fournissant une méthode fiable et flexible pour maintenir plusieurs concentrations de toxique lors
d’analyses biologiques dans un courant continu. L’eav qui tombe dune seme de cellules comp-
teuses d'eau semblables a celles d'un appareil a dilution proportionnelle active un groupe de
seringues d’inyection mécaniques Lappareil distribue d'égales quantités d'agents dispersifs du
toxique (solvants, agents mouillants ou additifs) a toutes concentrations de toxique. ainsi qu'a un.
agent dispersif temom. et permet de vaner a I'infini les concentrations de toxique indepen-
damment les unes des autres.

Recerved July 29, 1974 Regu le 29 juillet 1973
Accepted January 6, 1975 Accepte le 6 janvier 1975

THis report describes modifications made to the toxicant dispersant or additive to all toxicant con-
Proportional diluter and injector of Mount and centrations and to a dispersant control.

Brungs (1967) to dispense equal amounts of In addition to dispensing equal amounts of
toxicant dispersant, the system has the following

Printed in Canada (J3505) advantages and qualities:

Imprimé au Canada (53505) {) An unlimited number of toxicant concentra-

65


ACCESSORY PARTS

By now the reader should have an adequate general understanding of the
proportional dilurer and should be able to begin the connections between the
various W, M, C, and FS cells. The principal accessories are the valve
bucket, venturi T, microswitch, dilution-water valve, water blocks, siphon
tubes, neoprene stoppers, and miscellaneous glass and plastic fittings and
tubes (Table 3). The toxicant-metering devices will be discussed in a later
section.

DILUTER ASSEMBLY

First cut the required five tubes for cells W~2 through W-5 31.5 cm (1 ft)
long from 10-mm OD glass tubing. Notch one end of each tube about 12 mm deep
with the glass saw; remove about one-half of the wall. All tubes should be
notched approximately the same.

Bore stoppers to fit the tubes, and install into bored holes in W cells.
In chambers W-2 through W-5 the bottom of the tube notch must be above the
tops of the dividers to prevent premature siphoning. The siphon in cell W-6
is installed with all of the tube below the dividers so it is self initiating.

Cut the required five tubes 25.5 cm long from 10-mm OD tubing for the C
cells. Notch and install in cells C-1 through C-4 with notch bottoms above
the dividers and in cell C-5 with the complete tube below the dividers.

Cut the one required tube 25.0 cm long from 10-mm OD tubing. Notch and
install with all of the tube 25 mm below the top edge of the M cell.

Cut the required 12 tubes 23.0 cm long from 8-mm OD tubing. Notch and
install with all of the tubes 25 mm below the top edge of the FC cells. (Note:
If notches are not of identical depths, install with the bottom of all notches
35 mm below the top of FC cells.)

Cut the one required tube 34.0 cm long from 18-mm OD tubing; bore a 4-mm
hole in the side of the tube 48 mm from one end. Notch the opposite end to
install in cell W-l with the 4~mm side hole at a 45° angle to the divider
between cells W-1 and W-2 and just below the bottom of the chamber support.
Glue in place with silicone sealant.

Select and remove the tube restrictions from four 14-mm OD glass T's. Y's
may be used, -but one arm of the Y must be bent to clear the W cell supports.

Select four 10-mm OD glass T's, remove the tube restrictions from the
opposite ends, and fasten together with suitable Tygon pieces with all the
right-angle outlets in the same plane to make the vacuum manifold. Fasten to
the top of the W cells.

Cut 10-mm-diameter center holes in the bottom of four 125-ml plastic
bottles. Select and remove the tubing restrictions from four 14-mm OD T's
(1/2-inch). Drill a 14-mm hole in neoprene stoppers of suitable size to fit
the tops of the 125-ml bottles.

23


ACKNOWLEDGMENTS

We wish to thank those present and former associates who have, over the
years, provided suggestions for design changes, operation, and other features
of proportional diluters. Without such positive influence this report would
be no improvement on the original 1967 publication.

viii


Number

TABLES

Metric Conversion Table... 6 ee ee ee ee ee eee
Dimensions of Principal Glass Parts for the 2-Liter Diluter .
Accessory Parts Needed to Construct a Diluter......++.s-.

Cell Delivery Volumes for 2-Liter Diluters with Five Concentra-
tions and Control... 1 1 eee eee ee ee eh ee ee

vii

10

27


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12


and flow-through toxicity tests for chemicals and aqueous effluents. Methods

for both static and conti’.uous-flow toxicity tests are included in the 14th
edition of "Standard Methods for the Examination of Water and Wastewater
(American Public Health Association et al., 1976). This latest edition has
been expanded to contain procedures to test algal productivity, phytoplankton,
zooplankton, scleractinian coral, marine polychaete annelids, crustaceans,

aquatic insects, and molluscs.

As interest increases in the need for continuous-flow toxicity testing,
we believe that this manual will expedite the generation of data with which to

better protect the aquatic evosystem.


t2 tem x 254m,

DIVIDER (5)

3

a

76
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76. 7 7
76.44.7617 6.1.

L— 73

406em

|__ DIVIDER (4)

Tem x 20.3cm

76cm

=

oe

a

om

cm

em

cm

em

em

be 10210 2 10 210.2 of 10 2-10 2)

Final assembly plan for all cells.

Figure 8.

18


Environmental | Research EPA600/3-78-072
July 1978 -

Ouluth MN 55804.

SEPA Manual
for Construction
and Operation
of Toxicity-Testing
Proportional Diluters

>> 22>


syringes are set to deliver a constant amount of dispersant containing various
amounts of the toxicant. It is also possible with this system to test several
compounds simultaneously for screening purposes, if desired.

41


SECTION 3

DILUTER CONSTRUCTION

EQUIPMENT

Diluter construction is greatly simplified and much improved by having
the proper equipment, such as sharp glass cutters, a glass cutting table, a
glass saw, a set of glass drills, which are used on a standard heavy duty
drill press, and a power stopper borer.

Sharp glass cutters are needed to obtain straight, smooth cuts to prevent
leaks. An optional piece of equipment is the glass cutting board, similar to
those used by hardware stores to cut window panes. One style is available from
Fletcher Terry Co., Bristul, Conn. 06010. A large flat surface and a good
straight edge may be substituted. The glass saw is used for cutting glass
tubing and is generally useful for a variety of cutting purposes. It is used
to make cut ends on tubing, both square and angled, as required during diluter
construction. A rolling table model, such as the Model C manufactured by
Pistorius Machine Co., Hicksville, New York 11801, is desirable, but if only
diluter and other glass tubing is to be cut, their Model CC12, which has a
tilting table, is satisfactory. The glass drills are necessary to drill holes
in the various glass cells and are listed as diamond impregnated tube drills
in the catalog of Sommer and Maca, Glass Machinery Co., 5501 W. Ogden Ave.,
Chicago, Ill. 60650. These drills are relatively expensive, but enable the
diluter builder to also drill drain holes in aquaria and test chambers. The
drill press can be of any type, but should be sturdy and vibration free. Hand-
held drills may be used, but breakage is increased.

The boring of stoppers for various parts of a diluter is time consuming,
and a power stopper-borer, such as that manufactured by E. H. Sargent Co.
(Model No. S-232DT), is very useful. Some glass bending is necessary, and if
a glass shop is not available, an air-blast-type burner, such as that
manufactured by Fisher Scientific Co., is very convenient. This burner enables
the operator to apply sufficient heat to the tubing to allow uniform bending.
If much glass work is to be accomplished, a ribbon burner of at least 6 inches
flame length is also very useful.

Accurate rulers and steel tapes, a micrometer for inside and outside
measurement, felt marking pens, and a sufficiently large work area to prevent
moving of assembled parts during construction and assembly also save time and
increase efficiency.


Figure 1. Completed mounted diluter.


Syrett, R. F., and W. F. Dawson. 1975. An Inexpensive Solid-State Temperature
Controller. Prog. Fish-Cult. 37:171-172.

U.S. Environmental Protection Agency. 1976. Bioassay Procedures for the
Ocean Disposal Permit Program. Environmental Research Laboratory, Gulf
Breeze, Florida. EPA-600/9-76-010. 106 p.

Vanderhorst, J. R., C. I. Gibson, L. J. Moore, and P. Wilkinson. “1977.
Continuous-Flow Apparatus for Use in Petroleum Bioassay. Bull. Environ.
Contam. Toxicol. 17:577-584.

Veith, G. D., and V. M. Comstock. 1975. Apparatus for Continuously Saturating
Water with Hydrophobic Organic Chemicals. J. Fish. Res. Board Can. 32:
1849-1851,

Walden, C., D. McLeay, and D. Monteith. 1975. Comparing Bioassay
Procedures for Pulp and Paper Effluents. Pulp Pap. Mag. Can. 76:68-72.

Warner, R. E. 1967. Bio-assays for Microchemical Environmental Contaminants.
Bull. World Health Organ. 36:181-207.

Weiss, C. M. 1955. A Constant Temperature Tank for Fish Bioassay Aquaria.
Sew. Ind. Wastes 27:1399-1401.

Westlake, G. F., W. H. van der Schalie, J. Cairns, Jr., and K. L. Dickson.
1976. The Use of Fish to Continuously Monitor an Industrial Effluent.
The Institute of Electrical and Electronics Engineers, Inc. Annals
No. 75 CH 1004-1 18-4. 3p.

Wuerthele, M., J. Zillich, M. Newton, and C. Fetterolf. 1973. Descriptions
of a Continuous-Flow Bioassay Laboratory Trailer and the Michigan
Diluter, p. 345-354. In Bioassay Techniques and Environmental Chemistry.
Ann Arbor Science Publishers, Inc., Ann Arbor, Mich.

54


swaasks J9INT IP eTdTITNW 103 wWaIsks BuyuTy, gt aanBrg

m7 BULA

ae

fe

6ulids

/ 7 SdaINIIP 4

/ — auy winnon,

——yoyoudsyy

S

~~ plouajog ~

AIDA” i

~J8jDM ~~
uo!niG

37


inch) is positioned under the bucket (Figure 10). The venturi is prepared

by cutting off the tubing restrictions on two right-angle ends of the T and
mounting the T with the remaining restriction towards the bucket. The siphon
in the bucket is 10-mm tubing 15 cm long, and the outer tube is 15-mm tubing 10
cm long. (If very short or long cycle times are desired, these dimensions must
be modified to suit.) The bucket is hung from the upper bracket with a

spring and light stainless steel wire. The operating arm of the microswitch

is fastened to the bucket hanger by a piece of monofilament line set so that
the microswitch is on when the bucket is up and off when the bucket is resting
on the bucket stop. (See section on Mechanisms of Action for further
explanation.)

Care must be taken to leave enough slack in the exit tube of the valve
bucket where it is attached to the venturi T so that the tubing does not hinder
the action of the spring. If very cold water is run through the diluter this
becomes an important consideration as the tubing becomes very stiff and may
cause malfunction of the switch.

25


SECTION 8

TROUBLE-SHOOTING

Most diluter malfunctions occur during the first days of operation or
after modification or calibration. Listed below are some of the most common
problems (A-D) and their causes (1-4).

A)

B)

c)

Loss of water flow.

1)
2)
3)
4)

Electric power failure or accidental disconnection.
Defective microswitch or water valve.
Weak valve-bucket spring that will not lift valve bucket.

Too much water remaining in valve bucket after emptying. Adjust
siphon sleeve.

W cell siphons won't start.

1)
2)
3)

4)

No water in one or more of the water blocks.
Disconnected vacuum line.
Dirt or other debris in vacuum tube.

Venturi T not functioning properly.

Diluter-cell or test-chamber overflow.

1)

2)

W-1 cell siphon does not break before the dilution-water valve
opens, water will continuously siphon and the M cell will commonly
overflow (see Section 9 for safety devices). The timing needs
adjustment. The problem can usually be eliminated by slowing the
flow through the valve bucket.

C~2 or other C cell siphon not functioning properly. If the C-2
or other C cell siphon does not break before the M cell empties,
water will continuously siphon until the M cell is empty. The
timing needs adjustment. For example, the siphon tube from the
W-1 cell can be restricted to slow the filling of the M cell.

42


Insert one cut end of a T into each stopper, and add 40 mm of 14-mm (1/2-
inch) tubing to each to make a water block.

Cut four 46.0-cm lengths of 12-mm OD glass tubing. Heat the plastic
bottles slightly (hot water or careful use of burner or heat gun) and force
the tubing into the predrilled hole in each bottle to within 10 mm of the inside
top of the bottle.

Make four 90° smooth bends (90 mm around the curve) from 14-mm tubing.
Select and remove the tubing construction from two ends at the right angle of
14-mm T's. Connect the T to the C cell siphons with the right angles and
appropriate Tygon tubing. Attach the T water-block assembly with suitable
Tygon tubing to the standpipes of cells W-2 through W-5 and to the C cell T's.

Mount the valve-bucket stop 32 cm below and the spring support just below
the bottom of the W cells. Attach the microswitch to the spring support by
using a small block of wood. The spring should have just sufficient power to
lift the empty bucket.

Cut both ends from a 250-ml (6-ounce) polyethylene bottle leaving a
cylinder 70-75 mm long. Select two size 11 stoppers to fit the ends of the
cylinder and bore a 19-mm (3/4-inch) hole in each near one edge. Bore a
14~mm hole in a size 2 stopper, cut a piece’ of 14-mm glass tubing 90 mm long
and insert both into one large stopper. Mount the W-1 cell water block onto
the W-1 outlet to just below the 4-mm hole with the second large stopper.
Insert a short piece of 4-mm tubing into the hole and connect with suitable
tubing to the vacuum manifold.

The preceding assembly instructions result in a diluter system that will
function normally. The given dimensions, however, are not in most cases the
only ones that will work. The most usual and easiest changes are those required
by vertical space limitations found in many laboratories. All of the glass
chambers, W, M, C, and FC cells, can be made shorter and deeper from front
to rear if necessary. This, of course, makes the cutting of glass more
wasteful, but the diluter will function. The simplest procedure is to determine
the total height available. Distribute this between the four layers; then,
using that dimension vertically, use the given dimension horizontally to
determine the depth dimension by dividing these into the volume. All vertical
tube lengths must then be adjusted to fit depending upon the amount of change
decided upon. One precaution that mst be taken is to adjust the siphon
lengths carefully as a small vertical dimension change has a large effect
because the two horizontal dimensions contribute much more to the volume.

When the diluter has been assembled and all tubing is connected, the
equipment should be checked for leaks. When it is leak free, all of the
chambers should be fastened to the diluter board with small clips and screws
(mirror clips work well) to prevent breakage during cleaning or other manipulation.

The valve bucket (either a cut-off 2-liter bottle or a 2-quart plastic

freezer box is satisfactory) is mounted by putting a stainless steel light rod
or heavy wire across the bucket as a hanger. The siphon venturi of 10 m (3/8

24


26 Downatp I, Mount and Wittiam A. BRUNGS:
DETAIL FOR SPECIFIC COMPONENTS

(A) Needle valve

Ficure 3, from Mount and WarRNER (1965), is reproduced here for convenience in
constructing the needle valve. For the diluter described in this paper, an inlet and outlet
rubber tube of j-in. id. is suggested and a needle made of 13-mm glass tubing. The
glass rod should be approximately 5 mm in dia. The taper below the vacuum venturi
should be from 5-8 mm in a distance of 1.5-2.5 cm. A string, pulley, and bucket
filled with sand makes a fine counterbalance weight to replace the valve spring.

mer

storage

rea
‘Sean

‘SLICED STOPPER

tr
puastic To

BEE
ventan stoswes

2” comrnon war ro

Fic. 3. Needle valve and vacuum venturi detail.

(B) Vacuum connexion for WS-\ tube

Since “‘U” shaped connecting tubes are not easily obtainable in 15 mm o.d., the
vacuum line Va-! is best connected to the WS-1 tube by blowing or grinding a small
hole in the side of the tube and gluing over the hole a short piece of 3 mm o.d. glass
tubing. Care must be taken to keep the A and A‘ distances in the proper relationship
as discussed earlier.

(C) Chemical-metering apparatus

Many types of metering apparatus can be used to introduce the toxicant; the
specific choice depends on the chemical characteristics of the toxicant. Pumps can be
used satisfactorily for short-duration tests in which no great damage will occur if the
water flow fails or slows drastically. (The pump would continue to introduce to~*-

61


SECTION 1

INTRODUCTION

The physical aspects or characteristics of testing toxicity of chemicals
and aqueous effluents with aquatic organisms were minor considerations before
the initiation of continuous-flow procedures. Initial recommendations called
for the use of “round (cylindrical) glass jars 9 to 12 inches in diameter, 10
to 12 inches high, of good quality clear glass 1/8 to 1/4 inch thick and with
a capacity of 2 1/2 to 5 gallons" (Hart et al., 1945). Equipment for
temperature control and oxygenation completed the procedures. Similar
recommendations persisted (Doudoroff et al., 1951) until more details were
presented by Henderson and Tarzwell (1957) that included a floor plan of a
proposed toxicity-testing laboratory for static procedures with industrial
effluents.

A serial-dilution apparatus was described by Mount and Warner (1965) that
delivered continuously a series of different concentrations of a material
dissolved in water. The proportional diluter, to be discussed in detail
throughout this report, was developed shortly thereafter (Mount and Brungs,
1967). This system is simpler to build and operate than the serial diluter
and its use is more flexible. These systems and others (see Bibliography)
helped initiate the conversion to continuous-flow testing procedures to
alleviate some of the toxicity~testing problems inherent in the static
procedures.

The use of continuous-flow techniques received added impetus with the
addition of general procedures to the 13th edition of "Standard Methods for
the Examination of Water and Wastewater" (American Public Health Association
et al., 1971). Only static tests had been described in earlier editions.
After 1971, numerous detailed toxicity test procedures were developed that
used or required continuous-flow techniques. The Environmental Research
Laboratory-Duluth (1971, 1972a, 1972b) recommended chronic toxicity test
procedures for the fathead minnow (Pimephales promelas), the brook trout
(Salvelinus fontinalis), and the flagfish (Jordanella floridae). The U.S.
Environmental Protection Agency (1973) published methods that included, among
others, those just cited for the fathead minnow and the brook trout. The
European Inland Fisheries Advisory Commission (1975) in its discussion of fish
toxicity-testing procedures concluded that a continuous-flow procedure was
ideal for obtaining reproducible results for a wide variety of substances.

The Committee on Methods for Toxicity Tests with Aquatic Organisms of the U.S.
Environmental Protection Agency (1975) published both static and continuous~
flow methods for toxicity tests with aquatic organisms. The American Society
for Testing and Materials (1976) is drafting standard procedures for static


W CELLS

4

XN
a
3

~T

12 7em| 8OTTOM

FRONT

x
8

ee eee

|
0.3 76_j 76 176 | 761 102
an a a a a ka
6iem Af
¢ CELLS
|
22 9em{ BACK
|
76cm] BOTTOM
eo {| eo jfofol o
22.9em| FRONT
: a
H 77, so Leis 63]
ban eS tea cn
—
i 40.6em al

Figure 6. Gluing layout for W and C celis.

15


Jackson, H. W., and W. A. Brungs. 1967. Biomonitoring of Industrial
Effluents. Proc. Purdue Industrial Waste Conf, 21(1966):117-124.

Lake, W., and J. S. Loch. A Mobile Field Laboratory for Aquatic Toxicity
Studies. Environ. Canada Resour. Manage., Tech. Rep. Ser. CEN/T-73-13.
52 p.

LaRoche, G., R. Eisler, and C. M. Tarzwell. 1970. Bioassay Procedures for .
Oil and Oil Dispersant Toxicity Evaluation. J. Water Poll. Control
Fed. 42:1982-1989.

Lemke, A. E. 1964. A New Design for Constant-Flow Test Chambers. Prog.
Fish-Cult. 26:136-138.

Lemke, A. E. 1969. A Water Hardener for Experimental Use. J. Am. Water
Works Assoc. 61:415-416.

Lennon, R. E., and C. R. Walker. 1964. 1. Laboratories and Methods for
Screening Fish-Control Chemicals. U.S, Fish and Wildlife Service,
Investigation in Fish Control 1. p. 1-15.

Lichatowich, J. A., P. W. O'Keefe, J. A. Strand, and W. L. Templeton. 1973.
Development of Methodology and Apparatus for the Bioassay of Oil,
p. 659-666. In Proc. of Joint Conference on Prevention and Control of
Oil Spills. American Petroleum Institute, Environmental Protection
Agency, and U.S. Coast Guard, Washington, D.C.

Little, L. W. 1976. Bioassays-Procedures and Results. J. Water Poll.
Control Fed. 48:1356-1367.

Lowe, J. I. 1964. Chronic Exposure of Spot, Leiostomus xanthurus, to
Sublethal Concentrations of Toxaphene in Seawater. Trans. Am. Fish.
Soc. 93:396~399,

Maciorowski, A. F. 1975. An Inexpensive Macroinvertebrate Bioassay Table
for Use in Continuous~Flow Toxicity Tests. Bull. Environ. Contam.
Toxicol. 13:420-423.

Marking, L. L. 1969. Toxicological Assays with Fish. Bull. Wildlife
Disease Assoc. 5:291-294.

Myers, R. L. -1977. Modifications of the SERL Proporitional Diluter. J.
Water Pollut. Control Fed. 49:859-861.

Ohio River Valley Water Sanitation Commission (ORSANCO). 1974. ORSANCO
24~hr Bioassay. Cincinnati, Ohio. 21 p.

Poels, C. L. M. 1975. Continuous Automatic Monitoring of Surface Water
with Fish. Water Treat. Exam. 29:46-56.

Riley, C. W. 1975. Proportional Diluter for Effluent Bioassays. J.
Water Pollut. Control Fed. 47:2620-2626.

52 (


24 Donato I. Mount and Wittiam A. BRUNGS

water will enter the vacuum tubes Va-! to 5. As the water passes the chemical venturi
tubes, CV-2 to 5, the chemical siphons, CS-2 to 5, are started and chemical cells C-2
to 5 are emptied.

Only two timing adjustments must be checked: (1) the ‘vater flow through the
vacuum venturi (VaV) must be fast enough to produce sufficient vacuum to start the
water siphons but slow enough so that the water valve remains closed sufficiently
long to allow water siphon WS-1 to empty cell W-1 before the influent water again
enters W-1 from tube IT; and (2) cells W-2 and C-2 must be emptied and the siphon
in tube T-2 broken before water from mixing cell M-1 enters cell C-2 through tube
S-7. Obviously, the siphon in T-3, T-4, and T-5 must also be broken before their
respective ceils fill. This latter problem should not occur if the tube sizes suggested in

Taste 2. TUBE USED ON DILUTER

od,

Tube No. (mm) Material
WS-1 15 Glass
WS-2 to 5 8 Glass
WV-2t0 5 8 Glass
WS-2A to SA. 8 Glass
WS-1A 15 Glass
‘WS-6 8 Glass
S-7 10 Glass
CS-2 to 5 10 Giass
CV-2 to § 10 Glass
T-ltoS 10 Glass
VaV 5 Glass
T-6 8 Glass
Va-l to 5 5 Plastic
VaMa 5 Plastic
VIBuT 7 Rubber
IT 10 Glass
NVI 25 (L in.) Plastic hose “T”
VIBuS 6 Glass

TaBLe 2 are used. If water enters C-2 too soon, the flow rate through tube WS-IA
can be slowed by restricting the opening. The siphon WS-6 is adjusted in height so
that the total vol. delivered from cell W-6 is 500 ml. The valve bucket (VIBu) should
have a capacity of approximately 500 ml and is best made from a polyethylene bottle.
The tube WS-6 must fill the valve bucket at a rate so that the valve closes quickly,
giving ample time for the water level in the water cells to drain down to the top of the
cell partitions. This drainage must be completed before the water begins flowing
through the valve venturi. The time required for drainage is reduced by sloping the
cells approximately 1 cm in 10 cm. The chemical cells should be sloped as well. This
can be accomplished by sloping the back board of the diluter or by sloping the two
shelves on the board as shown in Fic. 1.

CALIBRATION

The vol. delivered from water cell W-1 can best be measured by catching the delivery
from tube WS-1A. The delivery vol. from cells W-2 to 5 can be measured by opening

59


+, Waysks JsASTTTVON,, ursn A[quesse Bupjazauw—JueITxXo],

i

"ET aandty

/ Jani AYVMMdVvd

SESS

7739 W
— ir
an
WNNDWA
OL 1739
IM

a1110a~"]

SLLOIMUVW

32


\ [-
\ LIQUID LEVEL
‘ : INVERTED BOTTLE
| a ot BEAKER
ones VENT
| (cur OFF STOPPER
al ase ee MARIOTTE
ee pe 1 1 BOTTLE
CAPILLARY TUBING_— \ \ \
\ To. |
\ ORAIN |
\ \
1
\
|
|

DELIVERS V2mi

Figure 14. Safety system for the "McAllister toxicant-metering system".

33


Henderson, C., and C. M. Tarzwell. 1957. Bioassays for Control of Industrial
Effluents. Sew. Ind. Wastes 29:1002~1017.

McAllister, W. A., Jr., W. L. Mauck, and F. L. Mayer, Jr. 1972. A Simplified
Device for Metering Chemicals in Intermittent-Flow Bioassays. Trans.
Am. Fish. Soc. 101:555-557.

Mount, D. I., and W. A. Brungs. 1967. A Simplified Dosing Apparatus for
Fish Toxicology Studies. Water Res. 1:21-29.

Mount, D. I., and R. E. Warner. 1965. A Serial-Dilution Apparatus for
Continuous Delivery of Various Concentrations of Materials in Water.
U.S. Public Health Service Publ. 999-WP-33. 16 p.

U.S. Environmental Protection Agency. 1973. Biological Field and Laboratory
Methods for Measuring the Quality of Surface Waters and Effluents. Enviror
Monitor, Ser. EPA-670/4-73-001.

U.S. Environmental Protection Agency. Committee on Methods for Toxicity Tests
with Aquatic Organisms. 1975. Methods for Acute Toxicity Tests with
Fish, Macroinvertebrates, and Amphibians. Ecol. Res. Ser. EPA-660/3-75-
009. 67 p.

U.S. Environmental Protection Agency. Environmental Research Laboratory-Duluth.
1971. Recommended Procedure for Brook Trout Salvelinus fontinalis
(Mitchill) Partial Chronic Tests. Mimeo. 12 p.

U.S. Environmental Protection Agency. Environmental Research Laboratory~Duluth.
1972a. Recommended Bioassay Procedure for Fathead Minnows Pimephales
promelas (Rafinesque) Chronic Tests. Mimeo. 13 p.

U.S. Environmental Protection Agency. Environmental Research Laboratory-Duluth
1972b. Recommended Bioassay Procedures for Jordanella floridae (Good of
Bean) Chronic Tests. Mimeo. 9 p.

48


TRIGGER LINE

FROM @
MICROSWITCH a SPRING
z
VALVE
BUCKET __,-]
| }L- FLow NoTcH
H
|) La |--SIPHON ASSEMBLY
VACUUM
\ LINE
BUCKET STOP___/
VENTURI T
wager

Figure 10. Valve bucket assembly.

21


Drummond, R. A., and W. F. Dawson. 1970. An Inexpensive Method for
Simulating Diel Patterns of Lighting in the Laboratory. Trans.
Am. Fish. Soc. 99:434-435.

Eaton, J. G. 1973. Recent Development in the Use of Laboratory Bioassays
to Determine "Safe" Levels of Toxicants for Fish, p. 107-115. In
Bioassay Techniques and Environmental Chemistry. Ann Arbor Science
Publishers, Inc., Ann Arbor, Mich.

Esvelt, L. A., and J. D. Conners. 1971. Continuous-Flow Fish Bioassay
Apparatus for Municipal and Industrial Effluents, p. 159-224. In
Toxicity Removal from Municipal Wastewaters. Sanitary Engineering
Research Laboratory, Univ. California. SERL Report No. 71-7, Vol. IV.

European Inland Fisheries Advisory Commission. 1975. Report on Fish Toxicity
Testing Procedures. Food and Agriculture Organization of the United
Nations, Rome. Tech. Pap. 24. 25 p.

Falk, M. R. 1973. Simple Apparatus for Conducting Acute Toxicity Bioassays
Under Field Conditions. Water Res. 7:821-822,

Freeman, R. A. 1971. A Constant Flow Delivery Device for Chronic Bioassay.
Trans. Am. Fish. Soc. 100:135-136

Granmo, A., and S. 0. Kollberg. 1972. A New Simple Water Flow System for
Accurate Continuous Flow Tests. Water Res. 6:1597-1599.

Gregg, B. C., and A. G. Heath. 1975. A Method for Intermittent Chlorine
Dosing in Continuous-Flow Toxicity Tests. Bull. Environ. Contam.
Toxicol. 13:588-592.

Grenier, F. 1960. A Constant Flow Apparatus for Toxicity Experiments on
Fish. J. Water Poll. Control Fed. 32:1117~1119.

Henderson, C. 1957. Application Factors to be Applied to Bioassays for the
Safe Disposal of Toxic Wastes. Biology of Water Pollution. Seminar
on Biological Programs in Water Pollution, Cincinnati, Ohio (1956).
p. 31-37.

Henderson, C., and Q. H. Pickering. 1963. Use of Fish in the Detection of
Contaminants in Water Supplies. J. Am. Water Works Assoc. 55:715-720.

Herbert, D. W. M. 1952. Measurement of the Toxicity of Substances to
Fish. Inst. Sewage Purification J. and Proc. Part I. p. 60-66.

Hublou, W. F. 1959. A Plexiglas Constant-Flow Siphon. Prog. Fish-Cult.
21:47-48.

Hughes, J. R., T. H. Blahm, and D. R. Craddock. 1976. A Mobile Laboratory

with Flow-Through Capability for Thermal Tolerance Studies of Aquatic
Organisms. Marine Fish. Rev. 38:24-27.

51


A Simplified Dosing Apparatus for Fish Toxicology Studies 25

the bypass (By) on the water blocks WB-2 to 5 (shown only on Fic. 1 and 2A) and
catching the flow. The bypass must be sufficiently large and positioned so that no
water goes down WS-2A to 5A. These vols. should be checked while the diluter is
cycling normally in the event that the drain-down is not entirely complete. The
delivery vol. of cells C-2 through C-5 are determined as follows. The influent water to
the diluter is stopped just as the water cells begin to empty. After the “C” cells have
been filled and the overfiow into cell C-1 has stopped, 5-10 ml of water should be
added to cells W-2 through W-5 to prevent air from entering WS-1 to 5. Suction should
then be applied to tube T-2 with a suction bulb and the water delivered caught in a
graduated cylinder. This procedure should be repeated for cells C-3 to 5 and then the
water flow restored.

The volumes in W-1 through W-5 and C-2 through C-5 cells are adjusted as needed
by increasing or decreasing the depth (o which the siphon tubes extend into the cell.
The WS-1 to 5 tubes and CS-2 to 5 tubes should be glued to the outside of the water
and chemical cells so they are rigid, but they should be cut off approximately at the top
level of the cell partition and then an adjustable extension added to furnish the desired
length. This arrangement allows for maximum adjustment of vol. The cell ends of the
WS-I to 5 and CS-2 to 5 tubes should be exactly parallel to the water surface in the
cell so that the siphon breaks abruptly. Placing a funnel-shaped flare on the end of
the tube enhances abrupt breaking.

Taste 3. REQUIRED WORKING VOLUMES OF EACH CELL FOR DILUTION
FACTORS OF 50 AND 25 PER CENT BETWEEN CONCENTRATIONS

Vol.
(ml)

Cell No. 50 % Factor 25% Factor
Wel 968 1525
Ww-2 250 125
we 37s 219
wa 438 289
W-5 469 342
ws 500 500
Mel 968 1525
C2 250 375
C3 12s 281
C4 62 2
CS 31 158
Cl 500 500

TABLE 3 lists the requisite working volumes for 50 per cent and 25 per cent dilution
factors between concentrations. These two series of concentration intervals represent
the recommended extremes for this particular diluter. One should construct water
and chemical cells of different dimensions for better accuracy for greater or smaller
dilution factors.

60


Foreword
Preface
Abstract
Figures
Tables .

CONTENTS

i >
ee ew ee eee ee ee iv
cS Vv
Se ee ee eee EE

ee ee ee ee ee ee ee ee ee ee WILT

AcknowledgmentS 2. 6 6 ee ee ee et ee ee ee ee we et et ix

1.
2.
3.

Introduction «6 6 ee ee ee ee ee ee ee ee ee ee 1
Diluter Operation - General. 2 2. wee ee ee ee te ee ee 3
Diluter Construction . 6 6 ee ee eee ee ee ee ee 5
Equipment 2.6 ee ee ee ee et ee ee ee 5
Diluter-board construction... 6. 6 1s ee ee ee eens 6
Glass cutting . 61. 6 ee we eee ww ee ee ew ee 6
Glass assembly . 2 we ee ee ee ee ee ee et ee 13
Diluter Operation... 1 eee we eee ee ee ee 19
Mechanisms of action». 1 se ee ee wee ee eee ene 19
Flow rate requirements . .. 1 6 6 te wee eee ee eee 22
Accessory parts 2.6 6 ee ee ee et 23
Diluter assembly . 1. 6 we ee et ee ee ee ee ee ee) 2B
Diluter Calibration . 6 6 6 ee ee we ee ee ee ee ee ee ew) 26
Precalibration . . 1 1 6 1 ee ee eee ee ee ee ee 26
Final calibration 2. 1. 6 eee ee eee ee ee ee ee ee) 26
Toxicant Metering . 2. ee ee ee et ee eee ee ee ee 29
Diluter Modifications . 6 6 6 6 ee ee ee ee eh ee ee ee 35
Trouble~Shooting . 2. 1 eee ee eee ee ee ee he ee 42
Diluter Safety Devices «6 1 ee ee eee ee ee ew ee ew 4a

References . 6 6 6 ee ee ee ee ee ee ee ee ee ee eee AD
Bibliography . 6 6 1 1 6 ee ew we ewe ee ee ee ee ee ee 49

Appendices

A.
B
Cc.


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40


|
DILUTION
WATER

CLAMP

EFFLWENT
WATER

Figure 17. Diluter modifications for effluent dilutions.

38


28 Donatp I. Mount and Wittiam A, BRUNGS:

c.e88 Tose ae

LE ar
F —
i /

4

tm

sesh
cHetaas
ean

Sanco ase PLATE

SIDE_vieW

Fic. 5. Injector system for adding yl quantities to the water.

For such toxicants as pesticides we have found the following procedure to be the
only way in which we can measure as much pesticide in the water as is introduced.

First the injector is used to inject air into a small closed vessel such as a 60 mi
stoppered bottle. The air then forces the slurry through a capillary tube from the
bottle into the water in chamber M-1. (The syringe and bottle must be insulated
against sudden temperature changes.) The bottle is placed on a magnetic stirrer
located slightly below the M-1 cell, and the slurry is stirred continuously.

The slurry is made as follows: (1) 25 mg of Triton X-100 is dissolved in 15-25 ml
of water; (2) 1-2 ml of acetone containing the requisite amount of pesticide is then
added, or if the pesticide is a liquid, it is added directly without being dissolved
first in acetone; (3) the mixture is shaken vigorously and then made up to 50 mi
for use. Depending on the amount of pesticide present, the slurry is usually
white.

We have successfully maintained as much as 10 g of parathion in suspension in 50
ml of slurry in this way without exceeding 10.0 ppb of Triton in the test water, and
no acetone was present. The decided advantage of this slurry is that it is a micro-
suspension that disperses readily in the water and then goes into true solution, whereas
when pesticides are dissolved in acetone and introduced directly into water, they
usually precipitate and only violent agitation will disperse them in the water. Ludzack
(personal communication) stated that in tests he performed there was a marked
tendency for aldrin and dieldrin to appear in the surface film or above the water surface
on the sides of the container, when they were dissolved in organic solvents before they
were introduced into water.

63


DILUTER BOARD
2em x 6lem x 122 cm
27cm
W CELL SHELF 1.3em
'2.7em x 6iem
oy wee 22227 ooo EOE
voVv UW
30.5cm
M CELL SHELF
\Sem x 20cm
27cm
C CELL SHELF :
Semx4icm : fem
yO Vv
30cm
FS CELL SHELF
\Sem x 6icm
TZE.
fv VY WV
¥

Figure 2.
8

Diluter board with shelves mounted.


Brungs, W. A., and D. I. Mount. 1970. A Water Delivery System for Small
Fish-Holding Tanks. Trans. Am. Fish. Soc. 99:799-802.

Buikema, A. L., Jr., D. R. Lee, and J. Cairns, Jr. 1976. A Screening
Bioassay Using Daphnia pulex for Refinery Wastes Discharged into
Freshwater. J. Test. Eval. 4:119-125.

Burke, W. D., and D. E. Ferguson.- 1968. A Simplified Flow-Through
Apparatus for Maintaining Fixed Concentrations of Toxicants in Water.
Trans. Am. Fish. Soc. 97:498-501.

Burress, R. M. 1975, Development and Evaluation of On-site Toxicity Test
Procedures for Fishery Investigations. U.S. Fish and Wildlife Service,
Investigations in Fish Control 638. 8 p.

Cairns, J., Jr. 1969. Fish Bioassays ~ Reproducability and Rating. Revist.
Biol. 7:7-12.

Cairns, J., Jr., K. L. Dickson, and G. Lanza. 1973. Rapid Biological
Monitoring System for Determining Aquatic Community Structure in
Receiving Systems. Biological Methods for the Assessment of Water
Quality. American Society for Testing and Materials, STP 528. p. 148-163.

Cairns, J., Jr., J. W. Hall, E. L. Morgan, R. E. Sparks, W. T. Waller, and
G. F. Westlake, 1973. The Development of an Automated Biological
Monitoring System for Water Quality. Virginia Water Resour. Res. Center
Bull. 59. 50 p.

Cairns, J., Jr., R. E. Sparks, and W. T. Waller. 1973. The Design of a
Continuous Flow Biological Early Warning System for Industrial Use. Proc.
Purdue Industrial Waste Conf. 27(1972): 34 p.

Chandler, J. H., Jr., and S. K. Partridge. 1975. A Solenoid-Actuated
Chemical-Metering Apparatus for Use in Flow-Through Toxicity Tests.
Prog. Fish-Cult. 37:93-95.

Chandler, J. H., Jr., H. O. Sanders, and D. F. Walsh. 1974. An Improved
Chemical Delivery Apparatus for Use in Intermittent-Flow Bioassays.
Bull. Environ. Contam. Toxicol. 12:123-128.

Cherry, D. S., K. L. Dickson, and J. Cairns, Jr. 1975. The Use of a Mobile
Laboratory to Study Temperature Response of Fish. Proc. Pusdue Industrial
Waste Conf. 29(1974): 28 p.

Cline, T. F., and G. Post. 1972. Therapy for Trout Eggs Infected with
Saprolegnia. Prog. Fish-Cult. 34:148-151.

Davis, J. C., and B. J. Mason. 1973. Bioassay Procedures to Evaluate Acute

Toxicity of Neutralized Bleached Kraft Pulp Mill Effluent to Pacific
Salmon. J. Fish. Res. Board Can. 30:1565-1573.

50


2 Donato I. Mount and Wituiam A. BruNos

successive concentrations, Metering and chemical cells can be exchanged so that the
dilution interval between successive concentrations can be decreased down to 10 per
cent, that is, a concentration sequence such as 1, 0.9, 0.81, 0.73, etc. Because persons
have requested additional details of the serial diluter, more specifications are given in
this paper. Throughout the following description, a delivery vol. of 500 ml per
concentration is assumed with a maximum flow of 400 ml/min per concentration.

MATERIALS

As before (Mount and Warner, 1965), every effort has been made to utilize
materials readily available. Four sheets of 12 x 24-in. single-strength window glass,
appropriate glass tubing, glass glue, a hand glass cutter, rubber stoppers, a 1-in.
plastic hose “T”, plastic bottles, and optionally a mechanical counter, constitute the
materials needed. If one wishes, local glass stores will cut the glass to desired sizes,
and for a very modest price they will cut the necessary three holes. The availability
of an excellent silicone rubber glass glue (Clear Seal produced ty General Electric
or Glass and Ceramic glue produced by Dow-Corning*) has made the construction
of the chambers extremely simple. Clean glass can be glued without etching or scratch-
ing, and the pieces can be assembled by simply pressing the pre-glued edges together.
Taste I lists the recommended cell sizes for the diluter described in this paper.

TABLE 1. DIMENSIONS AND CAPACITIES OF METERING CELLS

Size Maximum

(cm) capacity
Cell No. HWL (ml)
Wel 12x 6x 23 1656
Ww-2 12x6x4 288
w-3 12 6x6 432
wt 12x 6x7 54
W-5 12x67 204
w+ 7 12x 6x8 576
M-1 10x 11x 16 1760
C2 12x3xt 396
C3 12x3x9 324
C4 I2x3x7 252
C5 12x3x5 180
C1 12x 3x6 216

Height does not include 3 cm of freeboard for sides and ends.

PRINCIPLES OF OPERATION

A series of water-metering cells are filled, the water is turned off, the cells are
emptied, and the water flow is restored. (The reader is referred to Fic. 2 for a better
understanding of the following.) Cell W-1 fills first from IT then overflows into W-2,
etc. When cells W-2 to 5 are emptied, appropriate quantities of a higher concentration

* Mention of commercial products does not constitute endorsement by Federal Water Pollution
Control Administration.

56


TABLE 3. ACCESSORY PARTS NEEDED TO CONSTRUCT A DILUTER

Item Dimension Number
Glass tubing 8-mm 1 1-m length
10-tam 6 l-m lengths
12m 5 Ism lengths
Lé-um 2 1-m lengths
16-mm 1 1lsm length
18-mm 4 ism lengths
25-mm 1 lm length
(or pve pipe
of same size)
Glass T's 10-mm (3/8-inch) 6
Glass U's 10-mm (3/8~inch) 4
Tygon Y's or glass 12~um (1/2~inch) 5
T's as available 10~mm (3/8~inch) 4
Glass sheets 30.5~ X 61- X 0.3-cm il
Tygon tubing 8-mm (5/16-inch) 10 fe
10-mm (3/3-inch) 10 fr
Box of tissues 1
Neoprene stoppers For 19=mm (3/4-inch) holes 3 dozen
Size 1l 2
Size 8 1
Polyethylene plastic bottles 125-ml 5
150-11 L
Plastic container l-qt 1
Glass cutter 1
Glass glue 2 tubes
Plastic syringes 10-mL as needed
20-ral a
30-m1
Microswitch (Burgess CT2 KRA2 is
satisfactory) Ll
Solenoid valve, stainless steel
(Valcor 15P19C8-4 is satisfactory) L

10


ABSTRACT

This paper presents a discussion of the testing procedures using
proportional diluters. The construction, calibration, and operation of the
equipment is explained, and trouble shooting techniques necessary for success~
ful use of such equipment are given.

A bibliography includes many related published materials that are not
discussed in the text but which should be useful to the reader. Included are
numerous citations on physical toxicity testing methods, but papers on statistics
or biological test procedures are not included.

iv


NOTCH

v Sy Sa

NE
cur) ‘ ane seno_/

VOLUMETRIC PIPETTE

FIRE POLISH
TO FILLING NOTCH

ates

FINAL PRODUCT

Figure 12. Construction plan for a “dipping bird" toxicant metering
system using volumetric pipette.

31


SECTION 4

DILUTER OPERATION

MECHANISMS OF ACTION

The operation of the proportional diluter (Figures 1, 9, and 10) depends
on the venturi action of water flowing past a tube which is part of a closed
air system. The principal is the reverse of that of a carburetor in which
incoming air sucks gasoline from the carburetor jets. As water flows through
the W cells and overflows by siphon by siphon W-6 into the valve bucket, the
weight of the valve bucket increases and operates the microswitch, which causes
the valve bucket to come to rest on the bucket stop. This closes the diluent
water valve allowing the water levels in the W cells to drain down to the tops
of the dividers. As the valve bucket fills, its siphon begins to empty
through the venturi T (Figure.10). As water passes the opening of the vacuum
line, air is removed resulting in a reduction in air pressure in the vacuum
line. This water from W-6 passes through the venturi T and to FS~6 as the
control test water. The vacuum line connects to the siphon U's.in cells W-1
through W-5 (Figure 9). Each siphon U is covered on one end by water in its
corresponding W cell and on the other end by water in the water block.
Consequently, the air volume within the vacuum line is closed to the atmosphere
at the time the vacuum is applied by the venturi action of the venturi T. As
the air pressure decreases in the vacuum line, water is pushed into each end
of each siphon tube in cells W-1 through W-5 by the atmospheric pressure, which
exceeds the pressure in the vacuum line. As the water enters the top of the
siphon tube a siphon begins, which empties these five cells. The W-1 cell
empties into the M cell where the toxic solution is added, resulting in the
highest toxicant test concentration. Cells W-2 through W-5 empty through the
C cell venturis from cells C-2 through C-5. Again, the pressure inside the C
siphon tubes is reduced, and those cells containing the toxic solution are
emptied into the FS cells together with the dilution water from the corresponding
W cells. Since the W-l1 cell contains the greatest volume of water, the
remaining W and C cells should be empty before the M cell containing the water
from W-l1 and the added toxic solution overflows to refill the C cells. (If
the M cell begins to empty before the siphons are broken in W-2 and C-2, the
water from the M cell will be siphoned out through the C-2 siphon.) Cells
C-2 through C-5 are filled, and the remaining 2 liters of the highest toxicant
test concentration water enters C-1 to flow to FS~l1. All the tubes emptying
into and from the M cell and into the valve bucket and into the FS cells should
be cut at about a 45° angle so that the tube drains completely after siphoning.

The selection of the tubing sizes controls the flow rate of water from
W-6 through the venturi T to FS-6 so that the valve bucket contains water long
enough to keep the dilution-water flow from entering W-l before W-1 has

19


EPA-600/3~-78-072
July 1978

MANUAL FOR CONSTRUCTION AND OPERATION OF

TOXICITY-TESTING PROPORTIONAL DILUTERS

by
aA. E. Lemke, W. A. Brungs, and 3. J. Halligan

Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804

ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
OFFICE OF RES*ARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804


D) Premature siphon.

1)
2)

Top of W or C siphons too low as compared to the cell dividers.
Water tubes not totally emptied by previous siphon action. This

difficulty should be eliminated by the 45° angle cuts at the ends
of the water tubes. The FS siphons may also function improperly.

43


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36


completely emptied into the Mcell. If dilution-water flow begins before the
W-1 siphon is broken, water will continuously flow directly through W-1
preventing the filling of the remaining W cells and subsequent proper
functioning of the diluter,

When most of the control test water from W-6 has flowed out of the valve
bucket allowing it to rise from the bucket stop and causing the microswitch to
open the dilution-water valve, the diluter should be in the following mode:
(1) All W cells are empty and their siphons broken; and (2) the M cell has
emptied to fill cells C-2 through C-5. The cycle will begin again at this
time.

FLOW RATE REQUIREMENTS

The flow rate of the dilution water determines the length of each cycle
of operation, and this flow rate is adjusted t» obtain the desired flow to the
test chambers. If duplicate test chambers are used, as this design allows,

1 liter of test solution will enter each test chamber each cycle from the
2-liter diluter. The total test solution for the diluter is 12 liters per
cycle. If each test chamber contains 20 liters and the experimental design
requires 10 volume exchanges per day, a total of 200 liters is necessary for
each chamber each day. Consequently, 200 diluter cycles per day are required;
this is just over 7 min per cycle (1,440 min + 200). The dilution-water flow
rate is then set to provide 12 liters of water plus the rest of the cycle in
7 min.

Coordination between flow rate and volume is necessary for the diluter
to function. The flow rate of dilution water and the height and size of the
siphon cell W-6 must be such that the amount of water flowing between the
time the valve bucket turns off the dilution water and cell W-6 empties is
equal to the delivery volume (2 liters) of one cycle to the rest of the pairs
of cells. The size of the bucket and the height of the siphon must be such
that the water begins to flow out of the valve bucket just before the water
stops flowing from cell W-6. The line from the valve bucket must be just small
enough so that all of the other operations of the cycle will be completed
before it empties. A smaller line will work, but it will lengthen the cycle
time needlessly. The venturi T must be sized so that it will draw a stream of
bubbles from the vacuum line during the flow from the valve bucket. The
siphon in the M cell must be adjusted so that nearly all of the water from the
W-1 cell has entered before the siphon starts the flow into cell C-2. This
siphon should be:rather large so that after the flow starts it will empty fast
and not delay the cycle time. The last consideration for smooth operation is
at the venturi below the C cells. In this case, the size of the pipe from the
W cells must be such that a good vacuum is formed. The main consideration
here is that the tube from the W cells is not too large causing the water to
back up into the C cell outlet tubing rather than forming the necessary vacuum.

22


DILUTER-BOARD CONSTRUCTION

We'll begin our detailed procedures with the simplest part of the diluter,
the board and shelves on which the glass parts for the 2-liter diluter will be
placed.

The pieces are most precisely cut on a table saw, but hand-held saws may be
used as necessary. Although metric measurements are used in this report, some
of the materials used in the construction of a diluter are not yet available
commercially in metric dimensions (see Table 1 for conversions). All pieces
should be cut from 2-cm exterior plywood and assembled as shown in Figure 2.
The holes necessary to permit exit of the tubing from the bottom of the various
chambers should be marked accurately with the drilled glass bottom (to be
discussed later) as a pattern, They should be larger than the holes in the
glass to permit manipulation of the stoppers or plastic tubing. The bracing
also must be placed so as not to interfere with the glass tubing, but at the
same time to prevent sagging in the shelf.

The board size shown and the placement of the shelves do not need to be
exact, but the W and C cell shelves should slope down to the right about 1.3-1.5
cm.

We have modified these dimensions in various ways where space, especially
height, is a limiting factor. The dimensions shown have been found to be
useful with regard to cleaning, calibration, and construction ease.

GLASS CUTTING

The primary skill needed to be successful in building a diluter is the
ability to cut glass with straight edges and parallel sides. A commercial
glass cutting board, if well maintained, is particularly good for long cuts. A
second technique is to use a large flat sturdy table and a heavy ruler or other
straight edge to guide the cutter. This latter technique is faster and more
versatile once mastered. All pieces should be cut with minimum tolerance.
After cutting, all edges should be dulled with a stone or fine-grit sand paper
to prevent hand cuts. The pieces should be cleaned by washing in a detergent
solution and then rinsed thoroughly and dried. Removal of grime is necessary
to ensure good glue adhesion. Glass should be double strength, but the "B"
or second grade is satisfactory. Flint glass tubing is preferred to pyrex
because the lower melting point of the former makes bending and cutting the
glass easier.

The dimensions for all the flat glass and tubing used to construct a
diluter are listed in Table 2; a list of accessory parts is given in Table 3.
Figures 3 and 4 contain layouts to guide the builder toward an efficient use of
30.5- by 61-cm glass sheets, a readily available and handy size to use. These
layouts will ensure that the proper number of pieces are available before
assembly begins. The crosshatched areas are waste. The 2-cm drain holes
should be cut before assembly as shown in Figures 3 and 4. The exact location
of each hole in the W, C, and FS cells should be marked so that they are
centered between the cell dividers or cell ends (in the case of the FS cells).

6


A Simplified Dosing Apparatus for Fish Toxicology Studies B

(concentration 1) from cells C-2 to 5 are mixed with the diluent water to give the
desired lower concentrations. While the water from cells W-2 to 5 is being emptied
through tubes WS-2 to 5 and WS-2A to SA, the water from W-1 is emptied through
WS-I into mixing chamber M-1 where the toxicant is added, and then the chemical
cells C-2 to 5 are filled from cell M-1 through tube S-7. Cell C-2 fills first then over-
flows into cell C-3, etc. The vol. of W-1 is adjusted so that after cells C-2 through
C-5 are filled, 500 ml flows into C-1 and then to the test chamber to furnish test water
for the high concentration. Water for a control test chamber is emptied from W-6 and
operates the water valve (NVI) to turn off the influent water from tube IT while
cells W-1 to 5 empty. It also flows through the vacuum venturi, (VaV) to produce a
partial vacuum in the vacuum manifold (VaMa), which is connected to each water
venturi (WV-1 to 5) by the tubes Va-1 to 5. The partial vacuum applied by the water
venturi causes water from the water cells to rise through the water siphon tubes
(WS-1 to 5) and start the siphoning action to empty the water cells. The water blocks
(WBel to 5) serve to prevent air from entering the system through water siphon tubes
WS-IA to 5A. The distance from the water level of each filled water-metering cell to
the top of its water siphon tube, distance “A” (Fic. 2A), must be less than the distance.
from the water level in its respective water block to the bottom of the “U” in its
water venturi, distance ‘A’. Otherwise the water siphons will not start but rather

CROSS SECTION aT CELL wa 8.C4 FRONT view
Figuee 27a Fiquee 2-8

Fic. 2. Semi-schematic scale drawing of diluter.
Legend: B—block ; Bu—bucket ; By—by pass spout ; C—chemical ; I—influent; M—mixing;
Ma—manifold; N—needle; S—siphon; T—tube; V—venturi; Va—vacuum; Vl—valve;
W—water.

58



SECTION 9

DILUTER SAFETY DEVICES

Several procedures or devices will alleviate problems that occur during
diluter operation and increase the probability of conducting successful toxicity
tests. If no tests are expected to last more than a few days, then the
importance of these devices is reduced.

The use of a calibrated stock-solution container and an electric counter
connected to the microswitch (see Section 6) will monitor the chemical-metering
apparatus with regard to diluter cycle time and toxicant introduction.

The dipping-bird metering device can occassionally overflow into the M
cell if the air-pressure changes significantly in the Mariotte bottle or if
air leaks into this bottle. A two-chambered toxicant enclosure (Figure 19)
can be used with a central divider over which excess toxicant can flow to a
drain tube. This overflow commonly occurs when the Mariotte bottle is refilled
and the air pressure inside is stabilized.

A safety device to indicate the continued operation of a proportional
diluter is very important. This device can initiate an audible or visual
signal, turn on aeration in the test chambers to protect against lowering
dissolved oxygen concentrations, or perform any other important function. We
have used the W-6 cell most frequently with a small capillary drain at the
bottom of this cell (Figure 20). This permits the water level in W-6 to fall
below the bottom of the siphon sleeve. Since the flow is slow through the
capillary, several minutes or more are required for this level to drop very
far. A float level switch (FPC level switch, Fluid Products Co., Hopkins,
Minn. 55343) located in the W-6 cell is used to activate alarms and aeration
when the water level drops significantly as the result of an elimination of
dilution-water flow to the diluter. This capillary drains to waste. The
capillary tube should be checked daily and frequently cleaned as clogging can
occur. -

44


WIRES TO

/___—ALARM SYSTEM
L___I/2" PIPE
w U
CELLS
=

be COUPLING
FLOAT
SWITCH

=n wy,
CAPILLARY

oRIP TuBe_/

Figure 20. Diluter malfunction safety system.

46


TABLE 2. DIMENSIONS OF PRINCIPAL GLASS PARTS FOR THE 2-LITER DILUTER

W Cells 1 bottom 12.7 em X61 cm 3 in XK 24 in
2 sides 26.7 em X61 cm Ll in X 26 tn
2 ends 12.7 om X27 em 3 in X11 in
5 dividers 11.3 cm X 24.1. cm 4 3/4 in X 10 in
¢ Cells 1 bottom 7.0 cm X 40.6 cm Jin X16 in
2 sides 21.6 cm X 40.6 cm 9 in X16 in
2 ends 7.0 em X 21.6 cm Bin X 9in
4 dividers 6.0 cm X20 cm 21/2inX 810
M Cells 1 boctom 21.6 em x 18.7 cm 6inX 8.n
2 sides 21.6 cm X 20.3 cm 6inX 8 in
2 ends 17.8 om X 21.6 cm 6 in X 61n
FS Celis 6 bottoms 10.2 cm X 21.6 cm Sin X 6 in
12 sides 10.2 cm X 17.8 cm San X 7 in
12 ends 15.2 cm X 17.8 cm 6 in X 7 in
Siphon tube Length (Wel through W-6) - 30.5 cm
(C-2 through C-5) - 25.4 cm
(e3) - 20.3 cm
(valve bucket) - dependent upon
bucket
dimensions
(Fs) - 17.8 cm
Siphon tube diameter (WoL) - 16 mm OD
(We2 through W-6) - 12 mm 0p
(C-2 through C-5) - 14 mm 0D
c - 12 mm OD
(valve bucket) - 10 mm OD
(FS) - 10 mm oD
Siphon tube U's glass (We2 through W=5) - 10 mm 3/8 in OD
(C-2 through C-3) - 12 mm 1/2 in OD
Venturi and siphon cube T's
connecting tubes (flint
glass) (Water blocks to C siphon U's) 12 mm
(C siphon T's to FS cells) 13 om
Tygon tubing (miscellaneous) - as needed to
connect glass
tubing
Siphon sleeve diameter (all) ID area co be at lease double of OD area

of siphon tube as A = vr? = satisfactory
sizes below

Outer siphon tubes; size and Length

WL 40 mm 18.5 cm

wW2 18 om 11.8 cm

W3 18 om 19.8 cm

We 18 mm 21.2 cm

WS 18 om variable depending
on cycle time

M

cL 18 mm 19 cm

2 18 mm 11.4 cm

C3 18 mm 11.5 cm

ca 18 om 10.8 cm

cs 18 mm 7.7 cm

FS 12 each 146 om 13 cm