U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
PB-224 436
BIOASSAY DILUTER CONSTRUCTION, TRAINING MANUAL
HERBERT W, JACKSON
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO
June 1973
-------
PB 224 436
c. I
EPA-430/1-73-007
BIOASSAY DILUTER CONSTRUCTION
TRAINING MANUAL
U.S. ENVIRQNMENT^^ROTE
OFFICE OF
AGENCY
INFORMATION SERVICE
U S Department of Commerce
Springfield, VA. 22151
-------
NOTICE
THIS DOCUMENT HAS BEEN REPRODUCED FROM
THE BEST COPY FURNISHED US BY THE SPONSORING
AGENCY. ALTHOUGH IT IS RECOGNIZED THAT CER-
TAIN PORTIONS ARE ILLEGIBLE, IT IS BEING RE-
LEASED IN THE INTEREST OF MAKING AVAILABLE
AS MUCH INFORMATION AS POSSIBLE.
-------
BIBLIOGRAPHIC DATA
SHEET
1. Report No.
EPA-430/ 1-73-007
5. Report Date
June 1973
4. Title and Subtitle
Bioassay Diluter Construction (training course manual)
6.
7. Author(s)
Herbert W. Jackson, Ph. D. (manual production coordinator
8- Performing Organization Rept.
No.
9. Performing Organization Name and Address
U. S. Environmental Protection Agency, WPO
Manpower Development Staff, National Training Center
Cincinnati, OH 45268
10. Project/Task/Work Unit No.
11. Contract /Grant No.
12. Sponsoring Organization Name and Address
Same as #9
13. Type of Report & Period
Covered
14.
15. Supplementary Notes
16. Abstracts
This manual is designed to supplement tutorial instruction and published
literature describing the design and construction of various types of flow-through
bioassay and biomonitoring equipment. Illustrations, tables of design values,
list of materials needed, and reprints of significant publications from technical
journals are included. '
17. Key Words and Document Analysis. 17a. Descriptors
Bioassay, Diluters, Biomonitoring
17b. Identifiers/Open-Ended Terms
Diluter construction, Flow through, Bioassay
Details of illustrations in
this documant fnay be bettor
•fvdled on microfiche.
17c. COSATI Field/Group Q6F
18. Availability Statement
Release to public
19..Security Class (This
*"• Report)
" 'ASSIFII
of Pages
1EEL
(This
20. Security Class (This
Page
UNCLASSIFIED
NTIS-3S (REV. 3-72)
USCOMM-DC 14952-P72
-------
EPA-430/1-73-007
June 1973
BIOASSAY DILUTER CONSTRUCTION
This manual is designed to supplement tutorial instruction
and published literature describing the design and construction
of various types of flow-through bioassay and biomonitoring
equipment. Illustrations, tables of design values, and lists
of materials needed are included.
ENVIRONMENTAL PROTECTION AGENCY
Water Programs Operations
TRAINING PROGRAM
June, 1973
-------
AUTHOR'S PREFACE
This manual has been prepared to assist individuals in the construction of constant
flow bioassay and biomonitoring equipment. While it is written from the point of
view that such persons will come to Cincinnati in order to take advantage of the
construction tools available here, and also the personal counseling in regard to
needs and design; it can also be a useful supplement to the literature when used
alone in one's own home laboratory. Everi in Cincinnati, the individual is expected
to work largely by himself after the initial interviews and demonstrations are
completed, although an instructor is always available for questions and assistance.
It must be recognized that the design and details of equipment in this field are
still under development, and that what is "standard" today may readily be sup-
planted by a. new concept or device tomorrow. Consequently if use is made of
this manual more than a year or so after its date of issue, the worker should
either contact the author, or the National Water Quality Laboratories at
6201 Congdon Boulevard, Duluth, Minnesota 55804 to determine if significant
changes should be incorporated.
H. W. Jackson, Ph.D.
Cincinnati, Ohio
May 1973
a s
ill
-------
CONTENTS
The Construction of Proportional Dilutors for
Bioassay and Biomonitoring
Page No.
Introduction 1
Program in Cincinnati 1
Construction (procedures) 2
Toxicant Delivery Systems 5
Electrical Control Systems 5
Mixing and Flow- splitting Devices 5
Testing and Mounting 6
Biomonitoring ' 7
Special Design Problems 8
Experimental Tanks 8
Water Supply 8
Construction Hints (with limited equipment) 9
Boring Holes 9
Repairing Breaks 10
Acknowledgments 10
References 10
TABLES
1 Working Volumes for Standard 0. 5 Liter Diluter 11
2 Pieces Required for Diluter Tanks 12
3 Design Factors: Volumetric Equivalents and
Calculations 13
4 Pieces Required for Biomonitor Setup
(cf: Plates 14 and 15) 14
5 Materials for One Standard Diluter 15
6 A Short Table of Random Numbers 16
7 Potential Rate of Progress for a Skilled
Technician 17
-------
C o n t e n t s
PLATES
NOTE: A mixture of metric and English units is
employed due to the custom of employing
metric units for scientific volumetric
expressions, while the equipment is con-
structed of "double strength window
glass" which is 1/8 inch thick. See
Table 2.
1 Flow Plan for Standard Diluter
2 Stock Sheets
3 Cutting and Assembly: M-l
4 Cutting Plan for W Tank
5 Cutting Plan for C Tank
6 Cutting Plan for Flow-Splitter Tanks
7 Divider Spacing
8 Flow-Splitters and Siphon Breakers
9 Duluth Flow Distributors
10 Dipping Bird Chamber (optional)
11 Small Dipping Bird Chamber
12 Some Constant Level Devices
13 Suggested Layout for Proportional Diluter
14 A Flow Plan for Biomonitoring
15 Biomonitor Control Box
16 Test Tank
17 A Water Conditioning System
18 A Simple Toxicant Meter
APPENDICES
1 "A Simplified Dosing Apparatus for Fish
Toxicology Studies"
Mount, D. I., andW. M. Brungs. 1967
2 "A Water Delivery System for Small Fish-
Holding Tanks"
Brungs, W. A., and D. I. Mount. 1970
3 "Biomonitoring Industrial Effluents"
Jackson, H. W., and W. A. Brungs. 1966
4 "Continuous-Flow Fish Bioassay Apparatus
for Municipal and Industrial Effluents"
Esvelt, L. A., and J. D. Conners. 1971
I/
-------
THE CONSTRUCTION OF PROPORTIONAL DILUTEES
FOR BIOASSAY AND BIOMONITORING
I INTRODUCTION
A The following step-by-step instructions
are intended for use in the National
Training Center at Cincinnati, Ohio, .to
supplement .official publications. They
are designed to be carried out under the
general supervision of an instructor.
The trainee will be expected to work
largely by himself after an introductory
discussion with one or more consultants.
Occasional personal demonstrations are
given by the instructor of "tricks" and
procedures difficult to describe succinctly
in words.
B The equipment described is based on the
following publications which are attached
as Appendices 1 to 4.
1 Mount, D. I. and W. A. B'rungs.
A Simplified Dosing Apparatus for Fish
Toxicology Studies. Water Research,
1:21-29, 1967.
2 Brungs, W. A. and D. I. Mount.
A Water Delivery System for Small
Fish-holding Tanks. Trans. Am. 'Fish.
Soc., 99(4):799-802. October 1970.
3 Jackson, H. W. and W. A. Brungs.
Biomonitoring Industrial Effluents.
Industrial Water Engr., 14-18.
July 1966.
4 Esvelt, Jarry A. and Jerrold D.
Connors. Continuous-Flow Fish
Bioassay Apparatus for Municipal
and Industrial Effluents.
These papers are part of these instructions
and should be read before coming to
Cincinnati, although complete comprehension
of the operating mechanisms will probably
not be clear until working models are avail-
able for study.
Two additional references are cited.
-The reference to a paper by Mount and
Warner cited at the end of Mount and
Brungs '67 may be useful for "trouble
shooting. " It contains the original
description of this type of equipment.
For further information, current
refinements, and special methods,
contact the author or the Director,
National Water Quality Laboratory,
6201 Congdon Boulevard, Duluth,
Minnesota 55804.
H PROGRAM IN CINCINNATI
A Discuss needs and objectives with
instructor on arrival, including decision
as to whether or not to incorporate flow
splitter tanks.
B Observe flow-through equipment in
operation.
C Review the Mount and Brungs 1967 paper
again, hereafter referred to as "M&B '67."
D Study M&B '67, Figure 2 and the section
"Principles of Operation" beginning on
page 22. Also study Plate 1 of this
supplement until the operation of the
apparatus is understood.
E Remarks
1 For long term or larger scale operations,
it is desirable to scale up equipment
to deliver larger working volumes.
For example, the working volume can
be readily increased from 0.5 to 1 liter
by doubling the thickness of the W and C
tanks and increasing the capacity of the
M-1 tank in proportion. This is the
procedure currently in use by the
National Water Quality Laboratories
in Newtown, Ohio, and Duluth, Minnesota.
BI.BIO.met.23b.4.73
-------
The Construction of Proportional Diluters
Plans and procedures which follow are
for the original 0. 5 liter delivery size
as described in M&B '67. Note that
total flow per 24 hours can be increased
or decreased by appropriate adjustments.
(Discuss adjustment of the diameter of
the WS-6 siphon in particular [M&B '67,
Fig. 2-B and Table 2] with your
instructor.)
Mixing and Flow-splitting Tanks.
a Experience has shown that an
additional set of tanks is advisable
between the C tanks and the experimental
or test aquaria. Their main function
is to completely mix the discharge of
each C chamber with that of the corre-
sponding W chamber before passing
the entire volume on to the test tanks.
They are Incorporated in the designs
used in Appendix 4. Details of
construction are described below.
b In addition to mixing, they can
also be used to distribute and
adjust the flow between two or more
replicate tanks, as shown in Plates
8 and 9.
Improvement in statistical validity
can be achieved not only by operating
two or more replicate sets of test tanks
as suggested above, but also by arranging
these tanks on the laboratory bench in a
random manner, instead of in sequence
of concentrations. A short table of
random numbers is included as Table 6
for your convenience in this regard.
Test tanks shown in Plate 1 are so
arranged.
Ill CONSTRUCTION
It is important to begin the construction of
equipment as soon as possible in order to
provide time for overnight drying of the
cement if testing and/or assembly before
departure is anticipated.
A Obtain instruction in the use of the sheet
glass cutter and the carborundum wheel
from the instructor. (This is important
as both equipment and pperator may be
seriously injured through improper use.)
This will immediately be followed by a
demonstration of techniques for the
. assembly of glass cells by edge cementing.
'Items not described below are.covered in
M&B '67. Note Section X below for
suggestions as to how to proceed when
equipment used in this course is not
available in the home laboratory.
B Lay out, cut and label major blocks of
glass for the W, M, C and flow splitter
taijks (see Plate 2).
C Assembling the M-1 Tank
1 Subdivide the M sheet as shown in
Plate 3, and bend the support tube for
the discharge siphon. Cut a notch in
one end plate to receive same.
(Sections of glass Xed out are spare,
and may be used to replace pieces
broken, rniscut, etc.)
• 2 Assemble the pieces on the bench top
and determine exactly how they will be
put together. Cf: Plate 3. Always
. check pieces cut for each tank assembly
against Table 2 (or 4) to assure a com-
plete set before beginning assembly.
a It may be assumed that edges,
which have just been cut, are chemi-
cally clean. The glass surfaces to
which the edges will be cemented,
however, are seldom clean enough
to permit perfect adhesion by the
cement, and may contaminate the
experiment. Clean all surfaces
that will be "in" thoroughly with a
good laboratory solvent such as
acetone.
b Spread a sheet of protective paper
(such as newspaper) over the bench
top to protect it from the cement.
3 Cement the M-1 Chamber together.
a Fill a disposable plastic syringe
with cement, if not already done.
Clear "silicdne rubber" cement,
available in tubes over the counter
-------
The Construction of Proportional Diluters
g
at hardware stores has been found
best for this work. Disposable
plastic syringes in 10 or 20 cc sizes
are available from scientific supply
houses.
Consult your plans and be sure that
the notch for the siphon tube will be
properly placed.
Apply cement to the bottom edge of
the "front" piece and lay it flat on the
paper, top edge toward you.
Application of cement to the edges
of the glass plates is one of the most
critical procedures in the entire
operation, and should be practiced
until it can be executed with speed,
precision, and thoroughness. Neither
too much nor too little, and no gaps!
Joints should be filled so that internal
corners can be completely cleaned (no
pockets), but exposure of cement
inside chambers should be kept to an
absolute minimum.
Set the bottom piece lightly against
the coated edge and prop it roughly
in position with some object.
If siphon support is to be placed
in corner as shown on Plate 3 and
time is critical, apply a bed of
cement and place it in position.
Prop upper end 1/4 inch or more
away from front piece, and proceed
as in f. If time is not critical, wait
until cement on rest of M-1 has
dried for 1/2 to 1 hour before
setting regardless of position.
Coat the appropriate three edges
of the notched end piece (including
the inside of the notch) and set it
in position on the end of the side
piece. Bring the bottom piece up
until enough contact is made to hold
it in position. Carefully smooth
cement around siphon base, using
minimum necessary for strength.
Now coat the edges of the other end
piece, and set it in position.
h Set the remaining side piece in
position after applying cement to
the bottom edge, and press the
entire assembly gently but firmly
together. Be careful not to slide
cemented surfaces. After 5 or 10
minutes, turn the cell upright to
rest on its bottom for final alignment.
Do not attempt hard pressure to force
contact where there is insufficient
cement, or an improperly cut piece
of glass. If'such a problem appears,
fill in after partial drying.
NOTE: In fabricating the deeper
tanks (see below) the siphon base »
(or straight drain piece) may be set
before or after assembly as seems
most expedient. If set before allow
20 to 30 minutes for cement to stiffen
before assembling remaining parts.
i After cement has set (20 to 30 minutes)
have the instructor check and comment.
He will show you how to detect probable
pin-hole leaks visually, and how to '
correct them.
D Slack time can be used to lay out and cut
flat pieces for remaining tanks and to
prepare siphon pieces, valve bucket,
water blocks, valve, etc. The instructor
will be available for assistance as needed.
E The Larger Tank Assemblies
1 The W and C chambers of the standard
diluter were designed by M&B '67
with sufficient capacity to deliver any '
of the dilution ratios cited in Table 1
(Cf: M&B '67 Table 3).
e
The concept of the flow-splitter tanks
has developed more recently, but it
will be assumed from hereon that they
will be incorporated.
• 2 Unless special needs were determined
in the initial conference, cut the pieces
of the W, C, and flow-splitter tanks "
as diagrammed in Plates 4, 5, and 6,
and check them against the list (Table 2).
-------
The Construction of Proportional Diluters
F Assembling the Larger Tanks
1 Assemble the various pieces and
determine exactly how they will fit
together. Make sure that the notch
for the W-6 siphon support or the
C-1 drain is properly cut and
positioned. '
2 Mark the positions of the various
divider pieces as shown in Plate 7
on the outside of both sides and the
bottom. Lay these pieces out on
the bench top so that the markings
show through, but will be on the
outside in the finished assembly.
Place the bottom edge of the front
side on the bench, away from you.
3 Apply cement to proper edges as
before (III-C-3-h above). Apply cement
to all appropriate edges of a given
piece at one time. Cement dries
relatively slowly, so that a few minutes
delay before laying on matching piece
can be tolerated. Extreme delay
(10-15 minutes) will, however, result
in poor adhesion.
a Install in the following sequence:
bottom against "front" piece.
dividers over marks, "back" side
"on top" and against bottom, ends
on. Note that cement must be
applied to the ends of front, back
and bottom pieces (in contrast to
sequence in M-1 cell) in order for
the end piece to be properly sealed.
b The W and C tanks should be per-
mitted to set up for at least an hour
if possible before cementing the
siphon tupes (WS-1 to 5 and CS-2
to 5, M&B '67) in place. Tops of
all bends should be equidistant above
edge of tank. The completed units
must be permitted to dry overnight
before further assembly or testing.
As noted in the M-1 section (in-C-3-e),
siphon bases or drain tubes in W-6
or C-1 may be installed in advance,
or later. Not at this time. You
might prefer to mount siphons as
described in Appendix 4.
After overnight curing smooth all
exposed sharp edges with a
carborundum stone or flat file.
Provisional internal extensions of
siphons, and also the W-6 overflow
siphons may now be installed, using
a thin film of stopcock grease to
prevent permanent "locking" of
plastic tubing sleeves to glass.
1) Preliminary 'determination of
the depth of internal siphon
extensions may be made at this
time (or if preferred before
siphon tubes are cemented in
place). Refer to Table 1 (or
your own special design figures)
and note the working volume to
be delivered by each cell. Prop
the end of each tank in turn up
approximately one inch on the lab
bench (W-l or C-2), so that the
tank slopes down to the discharge
end as in Plate 13. Seal the
discharge tubes with rubber
tubing and a pinch clamp.
2) Fill the tanks with a rubber tube
led into the first chamber, allow-
ing the water to overflow naturally
into succeeding chambers.
3) Siphon off the desired working
volume from each chamber into
a graduated cylinder.
4) Mark the water level remaining
in the chamber on the outside
of the glass.
5) Extend the internal ends of the
siphons down to this mark (or
to the surface of the water) as
noted above.
6) In W-6 or any terminal chamber
to be emptied by an overflow
siphon draw the water level
down 1/2 to 1 inch below the
lip of the last divider and mark
this level. Now draw off the
working volume, and again make
a mark. The top of the overflow
siphon should be placed at the
-------
The Construction of Proportional Diluters
upper mark, the intake at the
lower mark.
7) All siphon extensions will
empty more cleanly if flared
as described in Appendix 4.
Ask your instructor for a demonstra-
tion, or consult Reference 1.
d For final "fine tuning" calibration,
see M&B '67.
G Toxicant Delivery Systems
1 If a dipping bird mechanism is desired
for chemical metering, suggested plans
for a cup and associated devices are
presented in Plates 10, 11. and 12.
These differ slightly from those
illustrated in M&B '67, but serve the
same purpose. Note: a safety factor
is introduced if the glass (back)
mounting piece is cemented to a piece
of wood or metal, or even wire loops,
for attachment to the diluter panel
(Plate 13).
2 The dipper itself can easily be
fabricated from a discarded volumetric
pipette as will be demonstrated by the
instructor (see also Plate 12, A).
3 Injector mechanisms for micro
quantities of toxicants are introduced
at the end of M&B '67. Further
developments are noted in Esvelt et al,
1971 (Appendix 4).
4 A simple toxicant metering device
which has no moving parts has recently
been developed at the Fish-Pesticide
Laboratory at Columbia. Missouri
(see reference: McAllister et al, 1972).
Structural features are shown in
Plate 18. For further information
contact the authors at the above
address, or consult the publication
cited.
H Electrical Control Systems
1 Solenoid valves activated by micro
switches and floats are more flexible
and efficient for larger systems, and
. can be readily wired to ensure
"fail-safe" operation.
2 An excellent description of one such
plan is given following page 163 of
SERL Report No. 71-7. See
Appendix 4.
I Mixing and Flow-splitting Devices
1 The general principle of flow-
splitter tanks is shown in Plate 8A.
The plan view (center of plate)
illustrates various possible arrange-
ments of nozzles or siphons.
Since many holes are required
for this plan, and since the test
solutions only dwell in these tanks
for a relatively few seconds per
cycle, some laboratories resort to
plexiglass for their construction.
Boring holes in glass (especially
large ones) is not as difficult as it
might seem, however, (see below)
and glass is still the recommended
material. Use smallest possible holes.
2 If distribution only is desired (and
mixing is no prdblem), simple small
bore glass nozzles may be employed
as shown in Plate 8B. Care must be
exercised, however, that the supply
.tube from the C and W tanks does not
'direct more liquid into one tube than •
the other. This should be checked
-by catching and measuring the discharge
'per cycle from each nozzle separately.
o
3 If complete mixing prior to splitting
is important (as is usually the case),
•" some device such as the Duluth Flow
Distributor (see Plate 9) is recommended.
This retains the discharge from the
C and W tanks until they are thoroughly
mixed. As the cycle is nearly finished,
both (or all) distributor siphon tubes'
tip over simultaneously, and the
completely mixed' liquid is distributed
to the various tanks.
4 Since the lengths of the inner siphon
tubes are identical, the rates of
discharge will likewise (supposedly)1
-------
The Construction of Proportional Diluters
be identical. However, if, on catching
and measuring the discharge from each
siphon tube as described above (Paragraph
2), it is found that one tube is delivering
more than another, the offending collar
tube can be slipped upward a few milli-
meters on the stopper seal. This will
break the siphon sooner and thus the
flow to that particular tank, and leave
more for the other(s). The principle
is clearly illustrated in Plate 9.
If glass-blowing equipment is available,
a much less fragile construction for the
Duluth Flow Distributor is as follows:
a Close the top of the central siphon
tube, leaving the tip with a short
spike (approximately 1/8 to 1/4
inch long).
b Blow a hole (or two) in the side of
the siphon tube, just below the tip, the
total cross sectional area of which is
approximately equal to the cross
sectional are a of the tube itself.
(If two holes are blown, they should
be at exactly the same level.)
c Fashion a central cavity in the
underside of the sliding rubber seal
of th« outer collar tube to receive
and
siphon tube.
"center" the spike of the central
d Install and adjust the central siphon
tube to the desired height in the mixing
chamber.
e Adjuvt the position of the sliding
rubber s«al in the outer collar tube so
that the bottom of the tube is at the
desired height when the outer tube
is set over the inner one. It is not
necessary that a rigid connection be
made.
The above system greatly simplifies
the adjustment of relative volumes
dispensed to the various replicate tanks.
J Siphon Breakers
1 The distributor devices for the flow
splitter tanks above all depend in one
way or another on an equal (or pro-
portional) delivery rate from each.
In order to accomplish this, there
must be no additional suction from
the supply lines which catch the
various discharges and deliver them
to the proper test chambers. This
is easily prevented by the use of
suction or "siphon breakers) " which
are simply devices for freely admitting
air into the delivery lines immediately
below the flow splitter. Probably the
simplest system is that illustrated
in Plate 8, B2. The plan suggested in
Plate 8, Bl permits the use of a
smaller size of tubing for distributing
the discharge to the test tanks.
IV TESTING AND MOUNTING
A Before assembling equipment on the
2-1/2' x 4' panel, each chamber should
be tested for leaks.
1 Place the tank on sheets of brown
paper toweling on the laboratory
bench. Close the outlets of W-6 or
C-1 with short pieces of rubber tubing
and pinch clamps.
2 Lead water from tap into first chamber
and fill within half an inch of the
divider top. Stop and look for leaks.
If any are noted, mark the point of
their first appearance.
3 Now overflow the first compartment
and allow the next one to nearly fill.
Mark leaks as before. Continue until
all chambers are tested.
B Stopping Leaks, Big and Little
1 "Pinhole" leaks between chambers
inside may be ignored. The chambers
empty so quickly when the siphons
start that these tiny leaks will be
inconsequential and will eventually
seal themselves with detritus.
Large leaks should be stopped.
2 External leaks of any size must be
plugged.
-------
The Construction of Proportional Diluters
a. Dump water out and wipe exterior
dry.
b Insert a 6-inch piece of glass
tubing in the end of a long piece
of rubber tubing, and attach to
the compressed air cock. Turn
air on hard, and blow water out of
the chambers at the points of
serious leakage.
c Force cement into crack from
outside with finger or disposable
syringe. Avoid adding to area of
exposed cement on inside of tanks,
if possible.
C Glass tubing should be well covered with
stopcock grease before slipping on plastic
tubing. This will prevent it from "freezing"
and will greatly facilitate testing and
adjustment.
D Plate 13 suggests a basic layout for a
2-1/21 x 4' panel. One half inch exterior
(or marine) plywood, painted on both
sides, is good for a permanent assembly.
If the equipment is to be disassembled
for shipment, 2 x 4 ft. pegboard panels
are provided for test assemblies in the
laboratory.
V BIOMONITORING
A If the need developed in the preliminary
conference is simply for surveillance
or monitoring to detect change (usually
deleterious) of an effluent, rather than
to assay or measure an exacting parameter,
the physical equipment may be somewhat
simplified. This is particularly true
of the toxicant administration, as the
degree of toxicity involved is usually much,
much less. Note paper by Jackson and
Brungs '66 (Reference 3).
B Before constructing such a system,
however, consideration should be given
to using the M&B '67 plan appropriately
modified. For example, waste could
be delivered directly to the C tanks,
by-passing M-1 and eliminating the W-1
siphon (simply clamp off the vacuum line
to W-l). A separate (probably solenoid)
valve, would need to be installed to cut
off the waste flow during1 cycling.
Proportional dilutions could then be
maintained, including full strength waste
(from C-1 chamber) and pure dilution
water (stream or lake) from W-6, as
described in Jackson and Brungs '66.
A simplified version of this equipment
which could meet biomonitoring require-
ments is shown in Plates 14 and 15.
1 Although a full range of dilutions from
full strength waste to pure dilution
water is shown here, special circum-
stances might indicate that pure
dilution water, pure waste water, and
perhaps one dilution only would meet
the needs of the moment. In this case,
the remaining chambers could either
be deleted from the original structure,
or not used.
2 A suggestion is offered in Plate 15 for
a system to permit continuous flow of
both effluent and dilution water. This
can be controlled "hydraulically" as
shown, or electrically. Such a flow-
shifting device is particularly helpful
where suspended solids are involved.
3 Plate 14 shows an extra overflow or
"waste" chamber on.the C tank in order
to permit exact metering of the 100%
waste. If this is not important, the C
tank structure may be shortened to
nine inches or less (7 inches, for
example), and an ample supply of
pure waste run through the C-1 chambers
on a simple excess flow-through basis.
This eliminates the necessity of a
water block and siphon setup for the
C-l chamber. One should realize,
however, in exercising this option,
that should C-1 metering ever become
desirable, a new tank would be needed".
4 The water block for C-1 must, of
course, be below the level of the C
tank, as the others are below the
level of the W tankj As a matter of ;
fact, the system generally works
-------
The Construction of Proportional Diluters
best if the W-2 water block is also
placed low, just above the entrance
of the C-2 siphon. '
This system will work faster if 10 and
12 mm tubing is used for the W-3 and
W-4 siphons, and 14 mm tubing for
W-2 and C-2.
VI SPECIAL DESIGN PROBLEMS
A In case special needs arise which cannot
be achieved by the standard (0.5 liter)
diluter, note the design factors in
Table 3. Factors for 1 liter diluters
are also cited. Proportional diluters
up to four liters per concentration per
cycle are now in use in some laboratories.
B Each flow-splitter chamber must hold the
combined flow of a W cell and the
corresponding C cell, leaving one or
more inches of freeboard for safety.
C The discharge from the W-6 and C-l
cells may be used as "volumetric cushions, "
since solutions passing through them are
not necessarily proportioned (mixed with)
to any other flow. However, the intent
of the original M&B '67 design was that
the siphon depth in W-6 and the working
volume discharged from W-1 would be so
adjusted that the W-6 and C-l working
volumes (discharges) would each be 0.5
liters, and thus equal to the other flows.
VII EXPERIMENTAL TANKS
A Tanks or aquaria as such are not strictly
speaking apart of the "proportional
diluter." However, they are so fundamental
to its proper use, that a brief description
is offered here of a type of in-house con-
struction found to be cheap and effective.
Construction details are shown in Plate 16.
B Nearly any size may be employed, but the
"workhorse" of the National Water Quality
Laboratories for fish bioassays has been
the two cubic foot size illustrated.
C The exact locations of overflows, screens,
use or nonuse of dividers, etc. are all
optional. Glass is ordered in bulk in a
ratio of three pieces of 12" x, 24" to two of
12" x 12". Half of the 12" x 12" pieces
have holes drilled as shown. All water is
delivered via glass tubing over the top.
D If additional dilutions are desired or if
it is desired to meter pure control and
pure M-l water from special chambers,
it is a relatively simple matter to add on
one or more chambers to both tank series.
For example, W-6 and C-l could be fitted
with siphons and vacuum lines, and
calibrated for desired working discharge
volumes. Both then might discharge
directly to test tanks (via flow-splitters
if desired), without being mixed with
any other solution. Overflow from W-6
could operate valve bucket through a
"W-7" chamber, and overflow from C-l
could be wasted. Variations are infinite.
VIE WATER SUPPLY
A A simple "head box" is not hard to devise,
but if a float chamber and valve is involved,
examine the fittings to assure that no metal
other than some acceptable form of stain-
less steel comes in contact with the water.
A constant overflow system may be safer.
B This is fine if an acceptable water supply
is available which requires no treatment
before use. However, s6me individuals
are faced with the alternatives of either
treating tap water, or not running
experiments. There is also the situation
where 'the temperature of the water
supply must be controlled.
C In either case, it is desirable to maintain
a constant head without losing any of the
treated water (whether chemical or
thermal treatment). One.approach is to
use a realtively small "box" composed
of two chambers, connected by a small
but adequate aperture or tube. The raw
water supply (valve or overflow) operates
in and out of one chamber, while the
e xperimental water supply is taken from
the other. No treated water ia thus wasted.
-------
The Construction of Proportional Diluters
D Plate 17 illustrates an arrangement
whereby not only the above results are
achieved, but the water is also mixed,
aged, and aerated (this would probably
not be desirable for thermal control).
The chemical treatment indicated might
be, for example, thiosulfate, to counteract
chlorine or an activated charcoal filter
might be inserted. In any event, a chemist
should calculate the amount and type of
treatment required in order to obtain
the desired quality.
E For information on acceptable water
quality for experimental use as such,
consult "Standard Methods" or other
references.
K CONSTRUCTION HINTS
A If a sheet glass cutter is not available
in the laboratory, order pre-cut sizes
of glass sheets (from Table 2) from local
hardware store. Specify approximately
l/32nd inch tolerances.
B Sheet glass may be cut by hand (especially
smaller pieces) using a hand glass cutter
and a meter stick, and extra thick yard-
stick, or other straight-edge. Be sure
edge is truly straight. Clamp, or have
another person hold straight edge in
position. Practice first on scrap pieces.
C Wire hacksaw baldes with silicon- carbide
grit advertised in hobby shops for cutting
glass bottles etc. are excellent for cutting
notches if a carborundum cutting wheel
is not available. Use long, slow strokes.
WEAR FULLY ENCLOSED SAFETY
GLASSES.
D Butane gas torches with wide wing tips
are excellent for bending glass tubing.
E Boring Holes
1 Best: Order holes to be bored as
specified, by glass supplier.
If you wish to bore your own, procure
a six-inch length of brass tubing
slightly smaller in diameter than you
want the hole to be. Cut the end of the
tubing off square, and file several
notches around the cutting edge. Chuck
the tubing in a drill press. If drill
chuck will not accept large enough
tubing, fit a short squat section of the
tubing desired with a one-hole rubber
stopper. Cut the head from a 1/4"
or 5/16" bolt, run a nut up the thread
end not farther than the length of the
rubber stopper, and slip on a washer
as large or larger than the diameter
of the cutting tube to be used. Force
the thread end of this assembly into
the stopper hole, and this in turn into
the cutter tube. Now chuck the bolt
shaft in the drill press as above. This
will not be as easy to line up and get
started as a straight piece of tubing but
once seated, grinding can proceed as
described below.
In order to reduce chipping when the
drill breaks through, support the glass
to be cut on a flat board (a scrap of
"Formica" shelf topping is excellent).
Even better: put a layer of plaster of
paris between the glass and the board
and let it set, or apply a small patch
of masking tape to the underside of the
glass.
Build a little coffer-dam around the
area where the hole is to be cut, using
putty. Pour in about one fourth of a
teaspoonful of No. 220 silicon carbide
grit or equivalent, and add a few drops '
of water to make a thin slurry. If
cutting action slows, add more grit
and water. Never let center get stiff.
With the drill press running about
300 revolutions per minute (faster
for small holes, slower for large '
holes), lower the end of the tubing !
gently into contact with the glass.
You will hear a grinding sound as
the action starts. Raise and lower
the tubing about once every five
seconds. You should be through a
piece of 1/8-ince glass in a few minutes.
-------
The Construction of Proportional Diluters
Take it easy as the drill comes through
the glass--to avoid splintering chips
off the edges of the hole on the bottom
as noted above. Drilling through rounded
objects is more difficult, but can be
accomplished with proper care.
3 Special steel drill bits for boring holes
in glass may be obtained from profes-
sional glass working equipment supply
houses. (Reference 1, page 189)
F Repairing Breaks
1 Glass cracked previous to assembly
(such as a "bottom" in which several
holes have already been notched or
drilled) may be "assembled" as any
other two pieces: 'apply cement to
broken edges and press them into
position (do not attempt if fragmented
into several pieces).
2 Glass cracked after assembly may be
waterproofed by a narrow bead of
cement applied to both sides. Be sure
preliminary surface cleaning is
thorough.
REFERENCES
1 Hammesfahr. J. E., and C. L. Stong.
Creative Glass Blowing. W. H.
Freeman & Company, San Francisco.
1968. A helpful section is included
on "Scientific Glassware."
2 McAllister, Jr., W. A., W. L. Mauck,
and F. L. Mayer, Jr. A Simplified
Device for Metering Chemicals in
Intermittent-Flow Bioassays. Trans.
Am. Fish. Soc. 101 (4):555. October,
1972.
A CKNOWLEDGMENT
This outline has been reviewed by personnel
of the National Water Quality Laboratory.
Special thanks are due to Mr. Timothy
Neiheisel, and Dr. W.A. Brungs. Plate 9
is from a personal communication from
Dr. Brungs. The procedure for hole boring
is in part from a personal communication
from Mr. C. L. Stong, Amateur Scientist
Editor, Scientific American.
This outline was prepared by H. W. Jackson,
Chief Biologist, National Training Center,
Direct Technical Training Branch, Manpower
Development Staff, WPO, Environmental
Protection Agency, Cincinnati, OH 45268.
10
-------
Table 1
WORKING VOLUMES IN STANDARD 0. 5 LITER DILUTEE
Cell No. 0. 5 Factor (1) Log Series (2) . 25 Factor (3) 1:2:1 (4)
Vol. Std. (5)
Diluter Chambers
Wl
W2
W3
W4
W5
W6
Ml
C2
C3
C4
C5
Cl
968
250
375
438
469
500
968
250
125
62
31
500
1080
220
340
410
450
500
1080
280
160
90
50
500
1525
125
219
289
342
500
1525
375
250
211
158
500
1250
125
250
375
X
500
1250
375
160
125
X
500
1580
375
470
750
750
930
1650
460
350
280
200
230 (6)
(1) Each successive dilution is half as strong as the one before.
(2) The ratio of the strength of each successive dilution to the one before it is
approximately 1:1.8. This is the logarithmic series recommended in
"Standard Methods" for static bioassays. Starting with C-l as = 100%, the
strength of C-2 + W-2 would thus be 56%. C-3 + W-3 = 32%, C-4 + W-4 = 18%,
and C-5 + W-5 = 10%.
(3) Each successive dilution is 3/4 as strong as the preceding one.
(4) Starting with 100% pure test solution from C-l, C-2 + W-2 = 75%,
C-3 + W-3 = 50%, and C-4 + W-4 = 25%. C-5 + W-5 would not be used, and
W-6 would deliver pure dilution water.
(5) It is evident by inspection that the Standard Diluter is so designed that any of
the ratios cited may be obtained without structure change.
11
-------
Table 2
PIECES REQUIRED FOR IMLUfFER TANKS (*)
Standard 0. 5 Liter Diluter
No. of Part
Tank pcs. Name
M 2 sides
2 ends
1 . bottom
W 2 sides
2 ends
5 dividers
1 bottom
C 2 sides
2 ends
4 dividers
*Vacuum and siphon
described in Table 2
here.
Dimensions
in inches Notes
4X7
3-3/4 X 4 one with notch
4X7
6 X24
2-1/2X6-1/8 one with notch
2-1/4 X approx. 5
2-1/2 X 24
6 X15
1-1/2X6-1/8 one with notch
1-1/4X5
systems for 0. 5 liter diluter are well
, page 24, M&B '67 and are not included
One Liter Diluter (selected items)
No. of Part
Tank pcs. Name
M 2 sides
2 ends
1 bottom
W 2 sides
2 ends
5 dividers
1 bottom
C 2 sides
2 ends
4 dividers
1 bottom
Siphon Systems
W 4 WS-2 to 5
1 WS-1
C 4 CS-2 to 5
1 S-7
5 T-l to 5
All U's
Dimensions
in inches Notes
6X7
6 X 5-3/4 one with notch
6X7
6 X24
4-3/4 X 6-1/8 one with notch
4-1/2 X 5
4-3/4X24
6 X15
2-3/4X6-1/8 one with notch
2-1/2X5
2-3/4X15
Dimensions
o. d. in mm
10
16
12
12
12
12 (or 1/2")
12
-------
Table 3
DESIGN FACTORS: VOLUMETRIC EQUIVALENTS AND CALCULATIONS
1 inch = 25. 4 mm = 2. 5 cm
1/4 inch = 6. 35 mm '
2 2
1 square inch = 642 mm = 6. 5 cm
g
1 cubic inch = 16. 4 cm (ml)
o
1 liter contains 61 in.
Factors for 0. 5 Liter Diluter
o
For partitions 1-1/4 inches wide, count 8 cm per inch of height*
or 40. 3 cm2 for 5 inches.
cc per running inch of C tank: 102. 5
2
For partitions 2-1/4 inches wide, count 14. 5 cm per inch of height,
or 72. 6 cm2 for 5 inches.
cc per running inch of W tank: 186
2
Ends of M chamber (per Table 2) contain 94 cm
cc per running inch of M tank: 235
13
-------
Table 4
PIECES REQUIRED FOR BIOMONITOR SETUP (cf: Plates 14, 15)
w
c
Control
Box
No. PCS.
2
2
3
1
2
2
4
1
1
1
1
3
2
1
Part Name
sides
ends
dividers
bottom
sides
ends
dividers
bottom
mounting plate
(back)
front
bottom
ends and tall
partitions
short partitions
slide plate*
Dimensions (in. )
6X9
2-1/2X6-1/8
2-1/4X5
2-1/2 X 9
6 X 10-1/2
2-1/2 X 6-1/8
2-1/4 X 5
2-1/2 X 10-1/2
2-3/8 X 7
2-3/8 X 5-5/8
1-5/16 X 5-5/8
1-1/16 X2
1-1/16 Xl-1/2
1 X8
Notes
one with notch
one with notch
2 notches or hi
l(or 2)
Miscellaneous 1
1
2
slide caps
bottom
wire U (or 1/8"
hole)
float chamber and
float
quadrant wheel and
bearings
control rods
1/2 X 1-5/16
1-5/16 X 5-5/8
to receive tubes
To receive control rod(s)
See Plate 14
See Plate 14
See Plate 14
*Could be of stainless steel.
14
-------
Table 5
MATERIALS FOR ONE 0. 5 LITER STANDARD DILUTER
1 Sheet double strength window glass, 36 x 42
Glass Tubing (not less than:)
OP 4 ft. lengths
8 mm 3
10 mm 3
14 mm 1/2
Capillary tubing or solid rod 1/2
(approx. 8 mm OD)
"Us"
1/4" (8 mm) 4
3/8" (10 mm) 4
"Ts"
3/16" OD, glass 4
1/4" (8 mm) OD, glass 1
3/8" ID, PVC or glass* 1
3/4" (or l") ID. PVC 1
Plastic Tubing
6' -1/8" aquarium air tubing, thick walled
assorted short lengths of 1/4, 3/8 and 1/2 inch Tygon
tubing (or equivalent) for connections
5 30 ml polyethylene bottles, vials, or equivalent (for water
blocks)
3 assorted larger polyethylene bottles
1 or more volumetric pipettes of selected sizes e. g., 5 ml
(for dipping bird)
1 Outdoor, or marine, plywood pc. 1/2" X 30" X 48" (optional
at Cincinnati)
assorted rubber stoppers
* "1/2 inch" PVC "street L" and "T11 may be substituted as demonstrated.
15
-------
Table 6
SHORT TABLE OF RANDOM NUMBERS
46 00 85 77 27 92 8G 26 45 21 89 91 71 42 64 64 58 22 75 81 74 91 48 46 18
44 19 15 32 63 55 87 77 33 29 45 00 31 34 84 05 72 90 44 27 78 22 07 62 17
34 39 80 62 24 33 81 67 28 11 34 79 26 35 34 23 09 94 00 80 55 31 63 27 91
74 97 80 30 65 07 71 30 01 84 47 45 89 70 74 13 04 90 51 27 61 34 63 87 44
22 14 61 60 86 38 33 71 13 33 72 08 16 13 50 56 48 51 29 48 30 93 45 66 29
40 03 96 40 03 47 24 60 09 21 21 18 00 05 86 52 85 40 73 73 57 68 36 33 91
52 33 76 44 56 15 47 75 78 73 78 19 87 06 98 47 48 02 62 03 42 05 32 55 02
37 59 20 40 93 17 82 24 19 90 80 87 32 74 59 84 24 49 79 17 23 75 83 42 00
11 02 55 57 48 84 74 36 22 67 19 20 15 92 53 37 13 75 54 89 56 73 23 39 07
10 33 79 26 34 54 71 33 89 74 68 48 23 17 49 18 81 05 52 85 70 05 73 11 17
67 59 28 25 47 89 11 65 65 20 42 23 96 41 64 20 30 89 87 64 37 93 36 96 35
93 50 75 20 09 18 54 34 68 02 54 87 23 05 43 36 98 29 97 93 87 08 30 92 98
24 43 23 72 80 64 34 27 23 46 15 36 10 63 21 59 69 76 02 62 31 62 47 60 34
39 91 63 18 38 27 10 78 88 84 42 32 00 97 92 00 04 94 50 05 75 82 70 80 35
74 62 19 67 54 18 28 92 33 69 98 96 74 35 72 11 68 25 08 95 31 79 11 79 54
91 03 35 60 81 16 61 97 25 14 78 21 22 05 25 47 26 37 80 39 19 06 41 02 00
42 57 66 76 72 91 03 63 48 46 44 01 33 53 62 28 80 59 55 05 02 16 13 17 54
06 36 63 06 15 03 72 38 01 58 25 37 66 48 56 19 56 41 29 28 76 49 74 39 50
92 70 96 70 89 80 87 14 25 49 25 94 62 78 26 15 41 39 48 75 64 69 61 06 38
91 08 88 53 52 13 04 82 23 00 26 36 47 44 04 08 84 80 07 44 76 51 52 41 59
68 85 97 74 47 53 90 05 90 84 87 48 25 01 11 05 45 11 43 15 60 40 31 84 59
59 54 13 09 13 80 42 29 63 03 24 64 12 43 28 10 01 65 62 07 79 83 05 59 61
39 18 32 69 33 46 58 19 34 03 59 28 97 31 02 65 47 47 70 39 74 17 30 22 65
67 43 31 09 12 60 19 57 63 78 11 80 10 97 15 70 04 89 81 78 54 84 87 83 42
61 75 37 19 56 90 75 39 03 56 49 92 72 95 27 52 87 47 12 52 54 62 43 23 13
78 10 91 11 00 63 19 63 74 58 69 03 51 38 60 36 53 56 77 06 69 03 89 91 24
93 23 71 58 09 78 08 03 07 71 79 32 25 19 61 04 40 33 12 06 78 91 97 88 95
37 55 48 82 63 89 92 59 14 72 19 17 22 51 90 20 03 64 96 60 48 01 95 44 84
62 13 11 71 17 23 29 25 13 85 33 35 07 69 25 68 57 92 57 11 84 44 01 33 66
29 89 97 47 03 13 20 86 22 45 59 98 64 53 89 64 94 81 55 87 73 81 58 46 42
16 94 85 82 89 07 17 30 29 89 89 80 98 36 25 36 53 02 49 14 34 03 52 09 20
04 93 10 59 75 12 98 84 60 93 68 16 87 60 11 50 46 56 58 45 88 72 50 46 11
95 71 43 68 97 18 85 17 13 08 00 50 77 50 46 92 45 26 97 21 48 22 23 08 32
86 05 39 14 35 48 68 18 36 57 09 62 40 28 87 08 74 79 91 08 27 12 43 32 03
59 30 60 10 41 31 00 69 63 77 01 89 94 60 19 02 70 88 72 33 38 88 20 60 86
05 45 35 40 54 03 98 96 76 27 77 84 80 08 64 60 44 34 54 24 85 20 85 77 32
71 85 17 74 66 27 85 19 55 56 51 36 48 92 32 44 40 47 10 38 22 52 42 29 96
80 20 32 80 98 00 40 92 57 51 52 83 14 55 31 99 73 23 40 07 64 54 44 99 21
13 50 78 02 73 39 66 82 01 28 67 51 75 66 33 97 47 58 42 44 88 09 28 58 06
67 92 65 41 45 36 77 96 46 21 14 39 56 36 70 15 74 43 62 69 82 30 77 28 77
72 56 73 44 26 04 62 81 15 35 79 26 99 57 28 22 25 94 80 62 95 48 98 23 86
28 86 85 64 94 11 58 78 45 36 34 45 91 38 51 10 68 36 87 81 16 77 30 19 36
69 57 40 80 44 94 60 82 94 93 98 01 48 50 57 69 60 77 69 60 74 22 05 77 17
71 20 03 30 79 25 74 17 78 34 54 45 04 77 42 59 75 78 64 99 37 03 18 03 36
89 98 55 98 22 45 12 49 82 71 57 33 28 69 50 59 15 09 25 79 39 42 84 18 70
58 74 82 81 14 02 01 05 77 94 65 57 70 39 42 48 56 84 31 59 18 70 41 74 60
50 54 73 81 91 07 81 26 25 45 49 61 22 88 41 20 00 15 59 93 51 60 65 65 63
49 33 72 90 10 20 65 28 44 63 95 86 75 78 69 24 41 65 86 10 34 10 32 00 93
11 85 01 43 65 02 85 69 56 88 34 29 64 35 48 15 70 11 77 83 01 34 82 91 04
34 22 46 41 84 74 27 02 57 77 47 93 72 02 95 63 75 74 69 69 61 34 31 92 13
Adapted with permission from A Million Random Digits by The Rand Corporation,
Copyright, i955, The Free Press.
-------
Table 7
POTENTIAL RATE OF PROGRESS FOR A SKILLED LABORATORY TECHNICIAN
At end of: Could expect to have:
Day 1 Discussed objectives. Visited working bioassay laboratory
and observed flow through equipment in action.
Studied working model in laboratory and learned its
operation.
Received instruction in the various laboratory procedures.
Cut out and assembled an M-l tank.
Day 2 Cut out and assemble W and C tanks and installed siphon
bases.
Day 3 Test tanks for water tightness and repair flaws.
Cut and assemble dipping bird and tank.
Cut and assemble assorted tubing and small fittings.
Mount equipment on temporary pegboard (or pack for
shipment). Or:
Day 4 Test and calibrate setup in A. M.
Disassemble and pack for shipment in P. M.
NOTE: Individuals not routinely engaged in constructing and operating
laboratory equipment of this type should plan on five days to accomplish
the above objectives. Flow-splitter tanks might not be completed in the
above timetable. •
17
-------
PLATES
-------
PLATE 1. FLOW PLAN FOR STANDARD
DILUTION WATER
W-l
METERED TEST
SUBSTANCE
W-2
W-3
W-4
W-5
M-l
TEST MIX
C-2
C-3
MIXING AND FLOW - SPLITTING
CHAMBERS TO PERMIT
REPLICATION (OPTIONAL,
ADVISABLE. SEE BELOW)
1 I
C-4
C-5
VACUUM
VENTURI
C-l
W2
+
C2
(2)
W3
+
C3
, (3l
W4
+
C4
• (4\
W5
+
C5
• (5),
PURE
TEST
MIX
i (Di
1 1
CONTROL
)
o (0).
'ANDOMLYARRANGED
CST TANKS
(TWO SETS OF I
DILUTIONS)
SIPHON- I
BREAKERS J
T
1
T
4
T
5
5
T
0
¥
2
V
0
V
1
2
T
3
-------
PLATE 2. STOCK SHEETS
(BASED ON "DOUBLE STRENGTH " WINDOW GLASS, 1/8"x36"x42")
NOTE SEQUENCE OF CUTS. LABEL EACH PIECE WHEN CUT.
36" :
4"
M-l TANK (PL. 3)
CUT#1
CUT#2
C-TANK BOTTOM AND ENDS (PL.5)
FLOW-SPLITTER TANK DIVIDERS (PL. 6,7)
42"
19%"
W-TANK (PL.4)
3
U
CUT #4
12"-
C-TANK (PL.5)
15'
FLOW-SPLITTER TANK (PL.7,8,9)
SCALE: 3/16" = l
-------
PLATE 3. CUTTING AND ASSEMBLY: M-l
7" »
7"-
7"
V
4"
BOTTOM
FRONT
BACK
TOP EDGE
END
TOP EDGE
END
NOTCH FOR SIPHON BASE
SCALE: 3/16" = l"
4"
ASSEMBLY
BACK
PLAN
FRONT
7"
'EXACT LOCATION
OPTIONAL
33/4"-
END VIEW
. NOTCH IN END
PLATETO RECEIVE
SIPHON BASE
^(LOCATION OPTIONAL)
'' '' ^^
LA jf ^
SBDE VIEW
OF END
SIPHON BASE
CEMENTED TO BASE
AND END
_D
SCALE: 1/2" = 1"
-------
PLATE 4. GUTTING PLAN FOR
(Cf: TABLE 3)
•19V4"
24"
6"
u
<
CQ
6"
O
06
a
o
9 >
^
O <*.
9 *
5%
>co-
ui _
0 T
r \
— co
o «*•
• S DIVIDERS @ SLIGHTLY OVER 4 3/4 " (4.8") =24"
"BE SURE THIS PIECE IS FULL 2 '/2 " WIDE, OR MORE.
SCALE
-------
PLATE 5. CUTTING PLAN FOR C-
.12"
6"-
FRONT
BACK
SVV
NOTES: A. FROM 6.QNG1 a" WIDE STiQP DESIGNATED O^ PLATE 2,
CUT: 1 PIECE 15 !/4" LONG FOR BOTTOM
2 PIECES SVg" LONG FOR ENDS. ONE WITH MOTCH
FOR DRAIN (FOR C-l CHAMBER)
B. DIVIDERS MUST BE EXACTLY RIGHT IN ORDER FOR
TANK TO FIT AND SEAL FREE OF LEAKS.
SCALE:3"=1'
-------
PLATE 6. CUTTING PLAN FOR
FLOW SPLITTER TANKS
NOTE: FOR DIVIDERS, SEE PLATE 2.
24"
SCALE: 3/16=i
-------
PLATE 7. D8VSDER SPACING
W TANKS
i
6
i
i i
W 1
W 2
W 3
W 4
W 5
W 6
INCHES FROM LEFT END: 8V2 1Ql/2 13
16 19
24
C TANKS
J
6
i
i >
C 2
C 3
C 4
C5
C 1
INCHESFROMLEFTEND:
(INSIDE)
103/4 123/4 15
FLOW-SPLITTER TANKS
T
5"
INCHES: 4
8
12 16 20 24
-------
PLATE 8.. FLOW-SPUTTERS AND SIPHON BREAKERS
A. FLOW-SPLITTER TANKS
DISCHARGES
W-2 + C-2 W-3 + C-3 W-4 + C-4 W-5 + C-5 C-l
W-6
1
1
*
^4"—
•i a
I
1 1
1
i
i
it
1
i
J USIPHON' 'BREAKER. '.DEVICES' 1 ^
/ X / X X \ XV XX / \
REPLICATETEST TANKS
-* - 94" ..*.
PLAN
21/4"
(BNSDDE)
.— LDEN
O
t
O
4
O
4
®
0 0
©©
©©
II V W A
TICAL FLOW -SPLJTTING NOZZELS. SEf RELttW_S^.A_ _.. •/.
(IN PRACTICE ALL TANKS WOULD BE SIMILAR)
B. DETAILS
GLASS RING
CEMENTED / •
INTO NOTCH |-
PLASTIC VIALS-i
PAIRSOF
IDENTICAL
NOZZELS
AIR
POSSIBLE ALTERNATIVE
ARRANGEMENTS
(Cff:PL. 9)
3
I '
9 IKI
INLETS
T"
LARGER
TUBING
a. b
4 SIPHON BREAKERS
STOPPERS THRUS1
^ THROUGH^
BORED HOLES
DIFFERENT FLOWS TO
DIFFERENT TANKS
NO SCALE.
-------
PLATE 9. DULUTH FLOW DISTRIBUTORS
THESE DEVICES WHEN INSTALLED IN FLOW-SPLITTER TANKS
CAN BE ADJUSTED TO DISTRIBUTE THE FLOW EVENLY OR DIFFERENTIALLY
BETWEEN TWO OR MORE TEST TANKS WITH GREAT PRECISION.
LEVEL TO WHICH FLOW-
SPLITTER TANK WOULD
FILL AT WHICH ALL
SIPHONS START A
SIMULTANEOUSLY
NOTCH
LEVEL AT
WH9CH SIPHON A
BREAKS, DELIVERING
LARGER QUANTITY
OF WATER
INNER SIPHON
TUBES, NOTCHED
AT TOP TO ADMIT
WATER, THRUST
INTO SHALLOW
BORER CUT IN
STOPPER CORES
LOOSE BOTTOM
COLLAR SERVES
AS GUIDE BUT
ADMITS WATER
(OPTIONAL)
ADJUSTABLE COLLAR
TUBE TO CONTROL
•*"~ HEIGHT AT
WHICH S8PHON
BREAKS, AND THUS
VOLUME DELIVERED
STOPPER CORE TO
SEAL TOP OF
INNER S5PHON
TUBE, AND HOLD
COLLAR TUBE AT
PROPER HEIGHT
LEVEL AT.WHBCH
SIPHON 3 BREAKS,
DELIVERING SMALLER
QUANTITY OF WATER
SIPHON BREAKER
= •== (SEE PLATE 8) =^~
TO TANK A
TO TANK B
-------
PLATE 10. DIPP5NG BBRD CHAMBER (OPTIONAL)
NOTE: DIMENSIONS SHOWN WILL ACCOMODATE A 40 ml
SCOOP, WHICH IS PROBABLY MAXIMUM THAT
SHOULD BE USED WITH STANDARD DILUTER.
SMALLER CHAMBER FOR SMALLER SCOOP SHOWN
ON PLATE 11.
PLAN
7"
L
u
"
o —
D-* OVERFLOW
j^ SUPPLY
W Id U
X * ^
BACK PIVOTS FOR
DIP. BIRD
FRONT PIVOTS
X { X
1 ui — in n i
FRONT VIEW
[NOTE: MOUNTING PLATEMAY DEEP NOTCHJ ] PIVOTSJ
BE GLUED TO WOODOR METAL FOR TOP >\J ON APPROX.
[SUPPLEMENTARYPLATE FOR SUPPORT SCREW i/2'' CENTERS
ACTUAL ATTACHMENT TO DILUTER BOARD.
OVERFLOW
SUPPLY
FRONT GLASS CUT ON
BEVEL (OPTIONAL), j
SHALLOW NOTCH FOR1 3
BOTTOM SUPPORT SCREW.
I 5
T
V2"
i
^ END VBEW
— I3
i i
SUPPLY
1 PC 3x7
,3
MOUNTING
PLATE
2PCSlJ/4xl3/ ENDS, ONE
WITH CORNERS
1 PC 2xsV4
1 PC 13/4X7
CUT OUT
BOTTOM
FRONT
LOWER RT. CORNER BEVELED
SCALE: 3/4"=l"
-------
PLATE 11. SMALL DIPPING BIRD CHAMBER
(FOR MORE TOXIC MATERIALS)
PLAN
Va" • -..-^|
2"
3"
+*
- SPACER
l"xiV4"x4V4" DIPPING
BIRD CHAMBER
\ t /
PIVOTS ON APPROX.
1/2"CENTERS
/
•TfT
1 I I
FRONT VIEW
NOTCH FOR UPPER SUPPORT SCRE
STANDARDIZED MOUNTING PIECE
!> SPACER PIECE INIS|
Jnj-—OVERFLOW B ACK CH AMBERJ51
|i'! SUPPLY COMMON END PIECi[l
n|, / FOR BOTH CHAMBE "
^"J) ^
OPTIONAL BEVEL
O.ON FRONT PIECE
NOTCH FOR LOWER SUPPORT SCREW
LEFT END VIEW
BACK
CHAMBER
.«.-*OVERH
| i FLOW
SUPPLY
2"
T
1 "
T
PIECES REQUIRED
l/_ MOUNTING PIECE
1 PC 3X&2
2 PCS lVxl3
2PCSl1Ax5/
ENDS
4- /g SPACERS
IPC^^xA1^ CENTER WALL
1 PClV4x6T/2 FRONT
(BEVELED CORNER OPT8ONAL)
1 PC 2x4^/2 BOTTOM
SCALE: 3/>," = r'
-------
PLATE 12. SOME CONSTANT LEVEL DEVICES
EXPERIMENTAL
STOCK
SOLUTION
APPROXIMATELY 5mm
GLASS TUBING, OPEN
TO AIR
PARTIAL VACUUM
ATMOSPHERIC PRESSURE
CAUSES LIQUID TO SEEK
SAME LEVEL
ASPIRATOR (OR
MARIOTT) BOTTLE
DIPPING BIRD
B.
~~ STOCK~
SOLUTION
AIR FROM
CONSTANT LEVEL
CHAMBER BUBBLES
UP THROUGH STOCK
CONTAINER
12 OR MORE mm ID
CONSTANT WATER
LEVEL
TO DIPPING BIRD
OR OTHER "CONSTANT--
DEMAND \
AIR SUPPLY
RISER DRAWN DOWN
TO CAPILLARY TIPTO
RESTRICT RATE OF
REPLENISHMENT OF
AIR IN CONSTANT
LEVEL CHAMBER.
PREVENTS-CHUG-
GING"
ATMOSPHERIC
PRESSURE IN
CONSTANT LEVEL
CHAMBER
CONSTANT LEVEL
CHAMBER (1 IN.
OR LESS ID)
30
(NO SCALE)
REMARKS:
"A" IS THE SIMPLEST
KNOWN CONSTANT LEVEL
DEVICE FOR THE TYPE OF
USE ILLUSTRATED. THE
ACTUAL LEVEL IS
ADJUSTED BY SLIDING
THE AIR SUPPLY TUBE
UP OR DOWN.
"B" IS A DEVICE WHICH
MAY BE USED IF AN
ASPIRATOR BOTTLE IS
NOT AVAILABLE, IF IT
CANNOT BE MOUNTED AT
THE SAME LEVEL AS THE
DIPPING BIRD, OR FOR
SOME OTHER USES IF
SETTLABLE SOLIDSARE
PRESENT WHICH NEED
SLIGHTLY MORE VIGOROUS
AGITATION. IF FASTER
DELIVERY IS REQUIRED,
CUT CAPILLARY RISER TIP
OFF AT POINT OF SLIGHTLY
LARGER DIAMETER.
-------
PLATE 13. SUGGESTED LAYOUT FOR
APPROX. PROPORTIONAL DBLUTER
INCHES
FROM TOP
0
30"-
APPROX.
WCHES
FROM TOP
HI
.SUPPLY^
DIP.; BIRD! {
APP:3"x6"-7" Ji
17-
37-
48-
M-l
4"x7"
VALVE
IOD—
TO W-l
VALVE
BUCKET
TANK
1
B
1-
S
D V ff
1 !
FLOW SPLITTER TANKS
•j i s
, 8 9
i
1
1
S
8
SIPHON BREAKERS
1/1 I 1 I
DISTRIBUTOR TUBES
II II II
h 32
-37
.48
31
SCALE: 2"=r
-------
PLATE 14. A FLOW PLAN FOR BlOMONITORING
(SEE ALSO PLATE15, AND TEXT SECTION Y)
WASTE DIL. WATER
A A CONTROL ^_^
WORKING
VOLUMES:
CONTROL BOX
(FOR DETAIL, SEE
PLATE 15)
PUSH-ROD i 0
QUADRANT
WHEEL
(Cf: PLATE 15)
WASTE
PARTITION
SPACING (IN.):
(BASED ON 2V4"
THICK CELLS)
WORKING
VOLUMES:
W-2
375ml.
/
W-3
250ml.
/
W-4
125ml,
/
W-l
500 ml.
n
\
0
\-
9
C-2
125 m
PARTION
SPACING(IN.. _
(2V4" THICK CELLS) Q
PROPORTIONS OF
MONITORED
SOLUTION IN
TEST TANKS
(£FIOW = 500ml
PER TANK PER CYCLE)
C-3
250ml
C-4
375ml
C-l
500ml
LU
h-
FLOAT
CHAMBER
r "i
FLOAT
VACUUM-
VENTURI
(CONTROL)
)
-------
PLATE 15. BSOMONITOR CONTROL
FLEXIBLE TUBE FLEXIBLE TUBE
FOR WASTE WATER FOR DILUTION WATEK
SLIDE CAP
PLATE
1
l/
c
I 1
8
i— i
D
/'/ \ T-2—^ — =}
-,'f r -=\ v ----- ---T£r_-_ft±- - _-J
I W£S/E WAJjER n DILUTION .WATER f
1 CHAMBER" n CHAMBER i
i n ,, " a
M " ii *
i || 11 n 8
1 TO ii TO H TO It TO [
1 WASTE n DILUTER|| WASTE n DILUTER j
1 ^r 9 1 m \ i if ^ 8 *? fl
f^\ 1 tf~*\ B B^I 1 Ir^^ B
. 55/ .- r
- 5 /g
' '"'
G
CONTROL
ROD
NOTCH FOR
^SUPPORT
SCREW
MOUNTING
* PLATE
SLIDE PLATE
(COULD BE STAINLESS STEEL
WITH HOLES FOR TUBES)
NOTCHES TO HOLD FLEXIBLE
\ SUPPLY TUBES
t
i"
i
i
0
4 1} ' " tit n3/ "
1 /4 ' 2/4
-I'n
I / ' ' ,,,
fc
WIRE
^LOOP
CEMENTED
TO GLASS
c
END VIEW
WHEEL
f:.PLATE 14)
•COUNTING
PLATE -
DRAIN(S)
c
-5LIUC rUMI E
»
••§••••
—
,77;
£
T2
i1/"
\
j
» r
2
i
3.
/8
HOLE FOR
CONTROL ROD'
SCALE: 3A"=1"
SLIDE PLATE WORKS BEST IF SUSPENDED BY SUPPLY TUBES
-------
12"
n
II
II
II
II
II
II
II
PLATE 16. TEST TANK.
(3PCS:12"x24", 2 PCS 12"xl2")
SIDE VIEW
24"
• •
II"
UK
III!
Illl
Hit
B"
PARALLEL
GLASS TUBES TO
SUPPORT DIVIDING
PARTITION IF NEEDED
12"
PLAN OF DISCHARGE END
END VIEW
GLASS TUBES TO
/SUPPORT STAINLESS
/STEEL MESH TO
5^ PROTECT OVERFLOW
\ / AND RETAIN
\/TEST ANIMALS
1
i n
i ii
1 81
n /2h- ii
H
99
811
II
II
II
L _ _Ji_
K/*j
EMERGENCY 1 |
OVERFLOW^ i |
x-^--s
NORMAL ' j
OVERFLOW / ,
_^ I \
_ 7 •
^^ (V2" HOLES) 1
1
[_
J_
SCALE: V4"=l
34
-------
PLATE 17. A WATER CONDITIONING SYSTE,
\ TANK AND OVERFLOW
[eg: 50 GAL. POLYETHYLENE)
RAW WATER
CONSTANT LEVEL
CHEMICAL FEED (Cf: PLATE 12)
SUPPLY *
AIR SUPPLY— »T\
-~
ff^
'• ^ ^ & TIP
OF F
j ° BEL«
POLYETHYLENE REAGENT
A BOTTL=
/" i! 1
-\J t V ' 6
iy Y CONSTANT _%«.
^Tfcr^ II WATER LEVEL 1
1
X
U
*>
^
^
N
i
1
/
CONSTANT
OVERFLOW
T
O DRAIN
1 xH
\*°'
^,
°::>^ *«sj
'. ', i , MULTIPLE SMALL (Vs
" , < , HOLES DISPERSE
',' , ' "MAKE-UP" WATER ONLY
.' ( , , ALL EXCESS SUPPLY
•' , , OVERFLOWS TO DRAIN.
i •
i ' ' i
4/\ r^ - • •
ADJUSTING HEIGHT
OF CAPILLARY DRIP
TIP DETERMINES RATE
FEED. SEE"B"
BELOW FOR DETAIL.
STREAM OF AIR BUBBLES^
-f* AERATES, AND CAUSES
'CONSTANT CIRCULATION
'AND MIXING
AIR STONE
B. CAPILLARY GL ASS TU BIN G DR AWN TREATED
AND BENT AS SHOWN WILL OPERATE WATER
FOR MONTHS WITHOUT ATTENTION TO
AS LONG AS FEED CONTAINS NO DILUTERS
SUSPENDED SOLIDS ETC.
DROPS FROM HERE
35
-------
PLATE 18. A SIMPLE TOXICANT
MAIN
VACUUM
WS-1 SYSTEM
BREATHING TUBE ADJUSTMENT
UP OR DOWN (a) WILL
CHANGE VOLUME OF
CHEMICAL DISPENSED
\\
\\
MARIOTTE
BOTTLE
RESERVOIRE OF CHEMICAL
DISPENSED EACH CYCLE
ADJUSTMENT "a"
: PLATE 12)
VACUUM
TEFLON TUBING
CAPILLARY TUBE SECTION.
LENGTH OF SECTION AND
DIAMETER OF BORE DETERMINE
RATE OF REFILL OF DISPENSING
RESERVOIRE BETWEEN CYCLES
*
• THE SHORTER THE DISTANCE "b",
AND THE SMALLER THE DIAMETER
OF THE TUBING USED, THE SMALLER
WILL BE THE WORKING VOLUME
DELIVERED.
PRIMARY
VENTURI
SECONDARY
VENTURI
M-l
CHAMBER
NO SCALE
AFTER MCALLISTER et ai.'72
36
-------
APPENDICES
37
-------
APPENDIX 1
Water Research, Pergamon Press 1967. Vol. 1, pp. 21-29. Printed in Great Britain. **
A SIMPLIFIED DOSING APPARATUS FOR FISH
TOXICOLOGY STUDIES
DONALD I. MOUNT* and WILLIAM A. BRUNGS*
Aquatic Biology Activities, Basic and Applied Sciences Program, Cincinnati Water
Research Laboratory, Federal Water Pollution Control Administration,
U.S. Department of the Interior
(Received 12 September 1966)
Abstract—A simplified diluter for maintaining a series 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 needed. 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 principles 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
flow 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
is needed. The main disadvantage is that it is impractical to deliver a series of con-
centrations with a dilution factor greater than 50 per cent between each concentration;
e.g. a concentration series such as 1, 0.1, 0.01, etc.
The proportional diluter shown in FIG. 1 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 50 to 25 per cent between
* Respectively Fisheries Research Biologist and Aquatic Biologist.
21
** Permission granted by Pergamon Press for reprinting
of this article.
-------
22 DONALD T. MOUNT and WILLIAM A. BRUNGS
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 by 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.
TABLE 1 lists the recommended cell sizes for the diluter described in this paper.
TABLE 1. DIMENSIONS AND CAPACITIES OF METERING CELLS
Cell No.
W-l
W-2
W-3
W-4
W-5
W-6
M-l
C-2
C-3
C-4
C-5
C-l
Size
(cm)
H W L
12x6x23
12x6x4
12x6x6
12x6x7
12x6x7
12x6x8
10x11x16
12x3x11
12x3x9
12x3x7
12x3x5
12x3x6
Maximum
capacity
(ml)
1656
288
432
504
504
576
1760
396
324
252
180
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 FIG. 2 for a better
understanding of the following.) Cell W-l 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.
-------
FIG. I. Photograph of a proportional diluter built as suggested in this paper.
(FaiiiiK p. 22)
40
-------
A Simplified Dosing Apparatus for Fish Toxicology Studies
23
(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 5A, the water from W-l is emptied through
WS-1 into mixing chamber M-l where the toxicant is added, and then the chemical
cells C-2 to 5 are filled from cell M-l through tube S-7. Cell C-2 fills first then over-
flows into cell C-3, etc. The vol. of W-l is adjusted so that after cells C-2 through
C-5 are filled, 500 ml flows into C-l 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 (NV1) to turn off the influent water from tube IT while
cells W-l 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
(WB-1 to 5) serve to prevent air from entering the system through water siphon tubes
WS-1A to 5A. The distance from the water level of each filled water-metering cell to
the top of its water siphon tube, distance "A" (Fio. 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
s
S-«
-y
1"
WV-4
KB
CROSS SECTION AT CELL W4 a C4
Figure 2-A
T-Z T-5 T-4 Ti T-l
FRONT VIEW
Figure 2-8
Fio. 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; VI—valve;
W—water.
-------
24 DONALD I. MOUNT and WILLIAM A. BRUNGS
water will enter the vacuum tubes Va-1 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 water 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-l before the influent water again
enters W-l 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-l 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 cells fill. This latter problem should not occur if the tube sizes suggested in
TABLE 2. TUBE USED ON DILUTER
Tube No.
WS-1
WS-2 to 5
WV-2 to 5
WS-2A to 5A
WS-1 A
WS-6
S-7
CS-2 to 5
CV-2 to 5
T-l to 5
VaV
T-6
Va-1 to 5
VaMa
VIBuT
IT
NV1
VI BuS
o.d.
(mm)
15
8
8
8
15
8
10
10
10
10
5
8
5
5
7
10
25 (1 in.)
6
Material
Class
Glass
Glass
Glass
Glass
Glass
Glass
Glass
Glass
Glass
Glass
Glass
Plastic
Plastic
Rubber
Glass
Plastic hose "T"
Glass
TABLE 2 are used. If water enters C-2 too soon, the flow rate through tube WS-1 A
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 FIG. 1.
CALIBRATION
The vol. delivered from water cell W-l can best be measured by catching the delivery
from tube WS-1 A. The delivery vol. from cells W-2 to 5 can be measured by opening
-------
A Simplified Dosing Apparatus for Fish Toxicology Studies 25
the bypass (By) on the water blocks WB-2 to 5 (shown only on FIG. 1 and 2A) and
catching the flow. The bypass must be sufficiently large and positioned so th*t no
water goes down WS-2A to 5A. These vols. should be checked while the diluier 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 overflow into cell C-l 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-l through W-5 and C-2 through C-5 cells are adjusted as needed
by increasing or decreasing the depth to 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-1 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.
TABLE 3. REQUIRED WORKING VOLUMES OF FACH CELL FOR DILUTION
FACTORS OF 50 AND 25 PER CENT BETWEEN CONCENTRATIONS
Cell No.
W-l
W-2
W-3
W-4
W-5
W-6
M-l
C-2
C-3
C-4
C-5
C-l
Vol.
(ml)
50 % Factor
968
250
375
438
469
500
968
250
125
62
31
500
25% Factor
1525
125
219
289
342
500
1525
375
281
211
158
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.
-------
26
DONALD I. MOUNT and WILLIAM A. BRUNCS
DETAIL FOR SPECIFIC COMPONENTS
(A) Needle valve
FIGURE 3, from MOUNT and WARNER (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 |-in. i.d. 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.
STOPPER
OUTLCT TO
WATER MANIFOLD
FIG. 3. Needle valve and vacuum venturi detail.
(B) Vacuum connexion for WS-l tube
Since "U" shaped connecting tubes are not easily obtainable in 15 mm o.d., the
vacuum line Va-1 is best connected to the WS-l 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 toxicant
-------
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 WARNER, 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.)
PIAST1C BUCKET
Fio. 4. Detail of chemical-metering apparatus.
When water enters the plastic bucket of the chemical-metering apparatus from cell
W-l (labelled funnel No. 1 on drawing), the tube rotates and the toxicant solution runs
through the tube and into the mixing chamber (M-l) 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.)
For organics that are slightly soluble in water, we have used an injector as sketched
in FIG. 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 /d of solution through the needle
into the water from cell W-l. 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 0.2S //I/injection;
this can be increased up to 30 n\ if a 50-ml 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.
-------
28
DONALD I. MOUNT and WILLIAM A. BRUNCS
WASHtP _
; ,F:V-iS
Cl SSI TUS< AXL»' I j'j| I \
• y
^-t —^ „..
i , i~..,...i i,
•CONCENTRIC S
PLASTIC TUBING PSION"--/
COLLARS
GLASS SYRINGE
TOP VIEW
TROW CELL W-l
HtAVY SHftr
METAL A»U
SUPPORT - --
SIDE VIEW
WOOD BASE PLATE
ici. 5. Injector system for adding//I 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 ml
stoppered bottle. The air then forces the slurry through a capillary tube from the
bottle into the water in chamber M-l. (The syringe and bottle must be insulated
against sudden temperature changes.) The bottle is placed on a magnetic stirrer
located slightly below the M-l 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 ml
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.
46
-------
A Simplified Dosing Apparatus for Fish Toxicology Studies 29
(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 FIG. 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 FIG. 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
partition 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 FIG. 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 Delivery of Various
Concentrations of Materials in Water. U.S. Public Health Service Publ. No. 999-WP-23, 16 pp.
-------
APPENDIX _2_
Made in United States of America
Reprinted from TRANSACTIONS OF THE AMERICAN FISHERIES SOCIETY
Vol. 99, No. 4, October 1970
pp. 799-802 *
A Water Delivery System for
Small Fish-Holding Tanks
INTRODUCTION
The necessity for maintaining small popula-
tions of fish and other aquatic life in holding
tanks for observation, acclimatization, and BO
forth is steadily increasing as more investi-
gators become involved in physiological, lexi-
cological, and other investigations of aquatic
life. The principal initial problems in utilizing
aquatic life in laboratory investigations are of
a facilities nature; for example, the design of
holding facilities. A more specific problem is
that of water flow control. Many of us have
regulated water flow to holding tanks by an
assortment of techniques such as valves, screw-
clamps, and small-bore tubing. Anyone who
has worked with such techniques has probably
had difficulty in maintaining uniform flow
rates because of clogging of the water lines at
the point of restriction. This can be especially
troublesome if a "natural" water is used.
Valuable lots of aquatic organisms can be
lost if the water flow stops or becomes insuf-
ficient.
The water delivery system described herein
is a modification of the proportional diluter
described in detail by Mount and Brungs
(1967). This system is almost free from
clogging caused by suspended solids, cladc-
cerans, snails, and so forth, since it avoids the
problem of restricted openings as a means of
providing controlled water flows to each hold-
ing tank. The particular design discussed here
(Figure 1) can be used to deliver 500 milli-
liters (ml) to each of six holding tanks as
often as every two minutes. Comparable
systems have been used in the Newtown Fish
Toxicology Laboratory for longer than one
year with rare malfunctions and little main-
tenance other than occasional cleaning.
MATERIALS
All materials used for construction of the
water delivery system are readily available.
Single- or double-strength window glass, ap-
Ficuu 1.
system.
-Photograph of operational water delivery
propriate glass and vinyl tubing, glass glue,
a hand glass cutter, a 1-inch plastic hose "T",
plastic bottles, rubber stoppers and, optionally,
a mechanical counter, constitute the necessary
materials. If one wishes, local glass stores
will cut the glass to desired sizes and cut the
necessary hole. An excellent silicone rubber
glass glue (Silicone Seal produced by General
Electric or Glass and Ceramic glue produced
by Dow-Corning) * now on the market has
made construction of the water delivery system
extremely simple. Clean glass can be glued
without etching and the pieces can be assem-
bled by simply pressing the edges together
with glue. Disposable plastic syringes of 10-ml
capacity filled with the glue are ideal for
depositing a fine bead of glue on the edges
to be glued. The water delivery system for
approximately a 500-ml delivery from each
of six cells would measure 24" wide X 6" high
X 2" deep, each cell being 4" wide. Individual
cell dividers would be 5" high. Minor varia-
tions in dimensions are unimportant as long
as the pieces can be easily assembled with
appropriate overlap. The water delivery sys-
tem is usually placed on a 2-inch deep shelf
attached to a piece of %-inch plywood.
* Mention of product and company name does not
constitute endorsement by the Federal Water Quality
Administration or the U. S. Department of the Interior.
799
* Permission granted by American Fisheries Society for reprinting
of this article.
48
-------
800
TRANS. AMER. FISH. SOC, WO, NO. 4
\
"I.
CROSS SECIION *l Clll W4
noun 7-a
(OONI V1IH
flOUOl 2-0
FIGURE 2.—Semi-schematic scale drawing of water delivery system. (Legend explained in text;
PRINCIPLES OF OPERATION
The details of operation are . thoroughly
discussed for the proportional diluter (Mount
and Brungs, 1967) but will be included here
for convenience. The series of water-metering
cells is filled, the water is turned off by the
valve, the cells are emptied, and the water
flow restored. Cell W-l (Figure 2B) fills first
from IT, W-l then overflows into W-2, and
so forth. As cell W-6 is filled it overflows
through siphon WS-6 into the valve bucket
(VIBu), which should also have a capacity
of about 500 ml. A 1-pint plastic refrigerator
dish is quite satisfactory. As VIBu fills, the
weight of the water causes the water valve
(NV1) to turn off the influent water from
tube IT while the water metering cells empty.
The tube WS-6 must fill the valve bucket at
a rate such 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
dividers. This drainage, which is quickened
by having the lefthand end of the system about
one inch higher than the righthand end, should
be completed before the water begins flowing
through the valve bucket water line (VIBuT)
to the vacuum venturi (VaV). This insures
delivery of uniform volumes from each water
cell every time the system cycles. As the water
originating from W-6 passes through the
vacuum venturi, a partial vacuum is produced
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 vacuum venturi causes water
to be pushed up the water siphon tubes (WS-1
to 5) by the greater atmospheric pressure.
This results in siphons being started in the
water siphon tubes. The water blocks (WB-1
to 5) serve to prevent air from entering the
system through water siphon tubes WS-1A to
5A. These latter siphons are connected to
drain lines to each holding tank. The lines
leading to the holding tanks should slope so
that they are completely drained after each
cycle. For easiest operation it is advisable
to have the flow from WS-6A drop directly
into the nearest holding tank. This will avoid
possible complications in the operation of the
vacuum venturi (VaV). It is absolutely neces-
sary that the distance from the water level of
each filled water cell to the top of its water
siphon tube, distance "A" (Figure 2A), 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 siphons will not start but rather
water will enter the vacuum tubes Va-1 to 5.
There is only one timing adjustment to be
-------
SHORT PAPERS AND NOTES
801
TABLE 1.—Tube sizes for water delivery system
Outside diameter
Number ( mm ) Material
WS-1 to 5
WS-6
WV-1 to S
WS-1A to 5A
WS-6A
VIBuS
VaV
Va-1 to 5
NV1
VIBuT
IT
10 Glass
8 Glass
10 Gla^s "U" connector
11 Glass
8 Class
8 Glass
8 Glass
5 Plastic
25 (1-inch) Plastic hose
6 Rubber
12 Glass
"T"
checked: the water flow through the vacuum
venturi (VaV) must be fast enough to produce
sufficient vacuum to start the siphons but
slow enough to allow the valve to remain
closed long enough to allow siphon WS-1 to
empty cell W-l before the influent water again
enters through tube IT.
The only calibration necessary can be made
by collecting the water from each water siphon
tube (WS-1 A to 5A) during normal operation.
The volumes in W-l through W-5 are adjusted
as necessary by raising or lowering the depths
to which the siphon tubes extend into the cell.
The WS-1 to 5 tubes should be glued to the
outside of the water cells so that they are
rigid, but they should be cut off approximately
at the top level of the cell dividers and then
an adjustable extension added to furnish the
desired length. The cell ends of the WS-1 to 5
tubes should be exactly parallel to the water
surface in the cell so that the siphon breaks
abruptly. The delivery volume of the W-6 cell
will vary with the flow rate through tube IT.
Therefore, it can be calibrated only after the
desired flow rate has been set. The height
of the siphon tube WS-6 is then adjusted
accordingly.
Tube sizes and other specifications are
included in Table 1.
Figure 3, from Mount and Warner (1965),
is reproduced here for convenience in con-
structing the needle valve (NV1 in Figure
2A). For the water delivery system described
in this paper, inlet and outlet tubes of %-inch
I. D., and a needle made of 13-mm glass tubing
are suggested. The glass rod should be about
6 mm in diameter. A string, a pulley, and a
small plastic cup filled with sand make a
suitable counterbalance weight should it be
necessary to substitute for the valve spring.
The tension of the valve spring should be
OUUII IU WAtll MkNIKXB
FIGURE 3.—Detail of the main water valve and
vacuum venturi.
adjusted so that the valve closes quickly as
water enters the valve bucket (VaBu), but
yet does not reopen before WS-1A is empty.
It is advisable to install a device such as an
oblong wire loop around the glass rod near
the valve bucket. This wire is adjusted so
that the full weight of the water rests at the
bottom of the wire loop when the valve is
closed. This protects against breaking of the
glass rod at the point at which the valve spring
attaches. This loop must not inhibit full open-
ing of the valve. The U-shaped vacuum mani-
fold venturi, valve venturi (VaV) in Figure
2B, is now replaced by a glass "T" connector
which operates more efficiently. It is sug-
gested that a mechanical or electrical counter
be incorporated to the movement of the valve
arm. The counter records the number of cycles
and daily determinations of the flow rate can
be made for further confidence in the water
delivery system.
When the delivery system becomes dirty
due to algal or other growth or organic mate-
rial, it can be cleaned by dissolving granular
calcium hypochlorite (such as that used for
swimming pools) and adding it into the W-l
cell as water is entering the system. The water
from the system must be diverted from the
holding tanks to avoid killing the organisms.
After all water cells are filled it is best to
stop the water flow into the system for several
minutes for more complete cleaning. It is
suggested that the system be allowed to run
50
-------
1502 TKANS. AMER. FISH. SOC, 1970, NO. 4
for 1-2 hours (with the water diverted from
tin; holding tanks) and that sodium thiosulfate
be added sufficient to neutralize any remain-
ing chlorine. If the water is sufficiently hard,
a calcium precipitate may coat the glass in
(he delivery system, in which case the above
procedure with a 10% nitric acid solution is
recommended. The acid or chlorine appears •
to have no effect on the silicone rubber glue
used in assembly of the system.
ADAPTATIONS
The water delivery system is an extremely
versatile device. If more than six water cells
are desired the system could be constructed
with additional cells by an appropriate in-
crease in width. In addition, any number of
cells, other than the one operating the water
valve and valve venturi, can be removed from
operation by clamping the appropriate vacuum
line(s) (Va-1 to 5). More than one water
cell may also be used to deliver water to an
individual holding tank. If it is desired to
deliver different volumes of water at the same
time, the length of the extensions of the water
siphons (WS) into the individual water cells
can be adjusted to provide more or less than
500 ml.
For applications that would require greater
maximum flow rates than that of the system
described (approximately 250 ml/min), an
increase in the depth of the system from 2 to 4
inches would double the potential flow rate.
If this is done water delivery tubes of in-
creased size are recommended.
LITEIIATUHE CITED
MOUNT, I). I.. AND W. A. BRUNCS. 1967. A simpli-
fied dosing apparatus for fish toxicology studies.
Water Research, vol. 1, pp. 21-29.
-, AMD l{. E. WAHNEK. 1965. A serial-dilution
apparatus (or continuous delivery of various con-
centrations of materials in water. U.S. Dept. of
Health, Education, and Welfare, Public Health
Service Publication No. 999-WP-23, 16 pp.
WILLIAM A. BRUNCS
Ni-wtiiwn Fish Toxicology Laboratory
•'1411 Chnn-h Street
I'.iniinuali, Ohio 4:>24-t
and
DONALD I. MOUNT
National Water Quality Laboratory
620I Congdon Boulevard
Duluth, Minnesota 55804
-------
APPENDIX 3
BIOMONITORING INDUSTRIAL EFFLUENTS
by
H. W. Jackson, Ph.D.
Water Programs Operations
Manpower Development Staff
Direct Training Branch
National Training Center
Cincinnati, OH 45268
and
W. A. Brungs, Jr., Ph.D.
Assistant for Water Quality Criteria
Environmental Protection Agency
National Water Quality Laboratory
6201 Congdon Blvd.
Duluth, MN 55804
Reprinted from Industrial Water Engineering
14-18, 45, July 1966 ~~
* Permission granted by Publicom, Inc., publishers
of INDUSTRIAL WATER ENGINEERING, for reprinting
of this article.
-------
BIOMONITORING
INDUSTRIAL
EFFLUENTS
by Herbert W. Jackson and
William A. Brungs, Jr.
The Toxicity of Industrial Effluents Can Be
Evaluated By Continuously Flowing Samples
Through Test Aquariums.
Apparatus use* in studying ejjects of toxicants in water on aquatic ttje. in
the study shown, paddlewher's in the exposure chambers circulate water over
fish eggs. Toxicant is added Ly yteans of a serial dilution apparatus.
Plant operating personnel need to
know the general quality of an effluent
being discharged at a fairly constant
rate. They also must be warned if a
slug of toxic material is released to
the receiving water. For example,
many of the fish kills resulting from
the release of slugs of highly toxic
substances could have been prevented
had these slugs been detected before
the effluent left the plant.
Conventional bioassay procedures
can evaluate only single samples taken
at particular times. Continuous-flow
bioassays of single grab samples over
a long period of time can be very
useful, but do not solve the problem
of transient variations. A technique
that does permit exercising continuous
surveillance over the toxicity of an
effluent is biomonitoring, a concept
similar to the one advanced by Hend-
erson and Pickering1 for water sup-
plies.
The Concept
Some progressive plants have met
this need to determine effluent Quality
by installing aquariums in which fish
are exposed to the plant effluent on
a continuous flow-through basis. A
"satisfactory" effluent quality is dej
termined by the survival of the fish.
Any deleterious change or effect is
evidenced either by the death of the
fish or a change in their behavior.
This is biomonitoring.
Conventional bioassays*'3'4 can
provide important information about
the actual toxicity of batches of the
effluent in terms of TLm's (that con-
centration which will kill half of the
test animals in some stipulated period
of time) and, if sufficient samples are
tested, about the range of variation.
This is a relatively slow process and
would be prohibitive on an hourly or
even a shift basis. Bioassays should
be run from time to time to ascertain
the exact toxicity of a waste even
.though it is being monitored as out-
lined below. Such tests also provide
essential guidance in setting up ap-
propriate dilutions • for continuous
monitoring.
The following procedures refer only
to toxic wastes having a relatively.
rapid action. Wastes such as cadmium
which have long delayed cumulative
effects at low concentrations5, oxygen-
demanding wastes, radioactive wastes,1
and others would either be inappropri-
ate or would not elicit a recognizable
reaction soon enough to be of use
in the following context.
While the procedures described here
14
INDUSTRIAL WATER ENGINEERING
53
-------
are for use with a final effluent, they
might a'so be applied to process wastes
within the plant.
Objectives of Biomonitoring
Three basic objectives of biomon-
itoring are:
(A) To demonstrate the continuous
suitability of an effluent for aquatic
life provided slow-acting or cumula-
tive toxins are not involved;
(B) To detect change (usually del-
eterious) in the biological accept-
ability of the effluent itself:
(C) To detect change in the effect
of the effluent on the biota of the re-
ceiving water.
The continuous testing of an undi-
luted effluent (Objective A) is usually
accomplished by leading a small
stream of the effluent through an
aquarium. This aquarium may be lo-
cated in a public lobby to enhance
public relations, or it may be in the
plant for operational use only. This
is a relatively simple and direct ap-
proach and needs no elaboration.
Objectives B and C are intrinsically
more difficult to accomplish. By def-
inition it is assumed that wastes re-
quiring biomonitoring may exhibit
acute toxicity; hence to achieve Ob-
jectives B and C, the effluent will prob-
ably require some dilution to support
aquatic life.
Equipment and Flow Plans
A single basic design of exposure
tanks and flow plan can be used to ac-
complish Objective B or C. With the
exception of a simple suggestion for
proportioning flow of effluent to dilu-
tion water, engineering devices for ac-
complishing the various needs out-
lined are not discussed. (See references
6, 7, and 8.)
Dimensions and arrangement should
be adapted to local circumstances.
Special care should be used to ensure
that all surfaces that come in contact
with the waste or the dilution water
are constructed of nontoxic and non-
corrosive materials. This precaution is
particularly necessary for marine wa-
ters, where bimetalic contacts are very
dangerous. An experienced aquatic
biologist should be consulted in the
preparation of plans. Settling in the
tanks will be minimized if fish are used
as the test organisms since their move-
ments will keep tank contents well
mixed except for heavy solids.
Exposure tanks (Figure 1) should
be large enoueh (10 to 20 gallons)
that the test organisms can live normal.
ly under plant conditions*. The larger
sizes are more stable, but also require
Figure 1 — Schematic flow plan for objectives B or C.
larger supplies of effluent and dilu-
tion water. Simple construction will
facilitate feeding, cleaning, and dis-
ease control. "Eye-appeal" is not nec-
essary unless public relations are in-
volved, but scrupulous sanitation is
essential as in all long-term animal
culture'-10. Tanks should be situated
in a lighted and well-ventilated room.
but not exposed to direct sunlight. Am-
bient room temperatures are generally
satisfactory, but should not be per-
mitted to go above or below limits
based on local biological experience.
Inlet and overflow should be similar
in all tanks so that conditions will be
the same except for the quality of the
water. The total flow of water through
the tank should be adjusted so that
the hydraulic retention times are equal,
whether the total flow is coming from
one source or two. There are no
standards for ideal hydraulic deten-
tion time, but generally it is advisable
to exchange the volume of each tank
at least once each shift, and preferably
more often.
Tank No. 1 (Figure 1) contains
only unadulterated dilution water to
establish that the test animals will live
in it. This tank is the control or "ref-
erence" to which the other tanks are
compared. Tank 2 and 3 (more may
be added at point 1) contain mixtures
of effluent and dilution water. If the
experimental animals die or show
distress in these tanks, a change for
the worse in the characteristics of the
effluent being monitored is indicated.
Note that Tanks 2 and 3 are con-
nected to both the effluent supply
line c and to d, the dilution water
supply line; i and j are mixing or
proportioning devices set to predeter-
mined amounts. In contrast, Tank 1 is
connected only to line d (the dilution
water); h is an adjustment valve or
device to regulate the flow. All tanks
overflow to the sewer through open-
ing k, which should be screened to
prevent escape of test animals and
clogging in outlet pipe.
Effluent Supply and Dilution
The supply of effluent should be
constant and controllable. The prime
requisite is that it be fresh so that
changes may be detected at the earli-
est possible moment. A constant-head
JULY, 7966 15
-------
Figure 2 — Suggested plan for proportioning flow between effluent and
dilution water. (See i and j Figure 1.)
reservoir is suggested as a relatively
simple device to achieve this, or a
simple tap on a waste line under pres-
sure might be adequate.
The source and quality of the di-
lution water is the most important
single factor in the system because
this is the scale or standard by which
the toxicity of the effluent is assayed.
Any available biologically accept-
able water may be used to detect sim-
ple change in the toxicity of the efflu-
ent itself (Objective B). Biological ac-
ceptability in this case is determined
simply by whether the test animals
will live in it under plant conditions.
Another consideration should be con-
stancy of chemical and physical char-
acteristics. Consideration might be'giv-
en, under certain circumstances, to a
standardized water prepared in
batches.
To test the effect of the effluent on
the receiving water (Objective C), the
dilution water obviously must be the
receiving water. In most cases the
water varies from time to time as run-
off water washes in different materials
from the surface of the land, or other
plants release various wastes. Plan C
(Objective C) automatically evaluates
the toxicity of the effluent when dis-
charged into this changing situation.
Thus it is important that Plan C dilu-
tion water should be obtained in such
a way that none of the waste to be
tested has been swirled to the point
of removal by an eddy or backwater.
This water should contain all of the
components present down to the out-
fall being monitored, but none of the
effluent itself.
In a stream situation, dilution water
can be taken well upstream from the
waste discharge. A pipeline with a con-
tinuous flow is ideal, since slugs of
material from upstream sources that
might modify the toxicity of the efflu-
ent being monitored would be taken
into the monitoring system and quick'y
distributed to the exposure tanks. Be-
cause such systems are notoriously sub-
ject to "problems," batch transporta-
tion of control water may have to be
employed. In this case, the interva's
between refills shou'd be as short as
possible to ensure that the water in
the monitoring system represents that
in the stream as closely as possible. In
lakes, estuarine, or coastal situations,
batch supply may be the only practical
solution.
If the receiving water is already
continuously toxic to aquatic life, it
is unsuitable for use in the system.
Under such circumstances Plan C
would be unworkable and the only
recourse wculd be Plan A or B. Treat-
ed water such as drinking water from
a city or industrial plant should never
be used for any of these plans, even
if dechlorinated, because the chemicals
used in treatment may react with the
waste being monitored. It is not un-
usual for even dechlorinated treated
water to be actually toxic to the ex-
perimental animals.
Proportioning Flow
The plan (Figure 2) by which eff'.u-
ent and dilution water are propor-
tioned to the test tanks (Tanks 2 and
3) determines what the system will ac-
complish. A suggested plan for propor-
tioning flow between effluent and di-
lution water (i and j) in Figure 1 is
given in Figure 2. The control valves
(m) are shown as if on a rigid pipe
to contain heavy pressure. If pressure
is low as from a nearby low-head res-
ervoir, laboratory-type pinch clamps
on rubber tubes (n) could serve the
same purpose. In case of clogging jet
nozzles may give less trouble than
valves. Sedimentation chambers ahead
of the valves (m) might also be useful.
Rubber tubes (n) can be momen-
tarily diverted to catch flow in an ap-
propriate sampling device such as a,
graduated cylinder. Based on the time
required to discharge some standard
volume, flow could be proportioned
to any desired ratio (for example,
one part effluent to two parts dilution
water).
After the effluent and dilution wa-
ter are mixed in and pass through the
funnel (o), the mixture enters an in-
verted polyethylene bottle (p) with
the bottom removed. This bottle is
equipped with an outlet (q) to dis-
charge excess water to waste. This de-
vice maintains a constant head dis-
charge to the tank through tube r, final
control is by a valve (s). Design of the
discharge mechanism should be identi-
cal for all tanks. If it is necessary to
lead the final discharge to tanks in
varying positions, a siphon-breaking
device should be included at the end of
16
INDUSTRIAL WATER ENGINEERING
55
-------
tube r to avoid modifying flows.
The still well (t) to collect precipi-
tates is optional. Material that would
settle out here would be kept from
settling out in the tanks. The sedi-
ment can be periodically removed
through drain u by releasing valve v.
The removal of such material, as well
as the use of a similar trap in the
effluent line ahead of valve m (where
indicated by suspended solids in the
effluent), is a policy matter that
should be resolved in the project-de-
sign statement.
Flow Plan for Objective B
If the system is to be operated to
detect only serious detrimental change
in the effluent itself, Tanks 2 and 3
should contain mixtures in such pro-
portions of effluent and dilution water
as to permit the test animals to live
as lung as the effluent is normal.
One possible combination would be
to adjust the mixing mechanism at i
(Figure 1) to admit such a proportion
of effluent that the test animals in
Tank No. 1 could barely survive. The
slightest increase in toxicity of the
effluent would then immediately be
made evident by the death of the test
animals and appropriate remedial ac-
tion could be taken. The mixing mech-
anism j in Tank No. 3 might be ad-
justed to provide a greater margin of
safety, for example 1/2 or I/10th
the toxicity of Tank 2. If Tank 2 ani-
mals then died, but Tank 3 animals
survived, it would presumably indi-
cate only a moderate increase in tox-
icity.
Flow Plan for Objective C
Monitoring the effect of the efflu-
ent on the receiving water, from the
point of view of protecting aquatic
life, is ideal, but may also be very
difficult. The reference tank (e, Figure
I) receives water fresh frcm the re-
ceiving body. This water is free of
any trace of the effluent being moni-
tored, but contains all substances, nat-
ural and artificial, presently in the
receiving water. These components
may change from time to time, and
one of these changes may increase the
toxic effect of the effluent (syn-
ergism).
Thus the death of animals in the
strongest test tank, but not in the
reference tank, may be the result of
an increase in the toxicity of the plant
waste or of a synergistic reaction of
the effluent with a material in the
receiving water. No matter what the
cause, the death itself serves as a warn-
ing and immediate action can be tak-
en on the discharge to protect aquatic
life.
On the other hand, if a slug of
strongly toxic matter enters the re-
ceiving water from some outside
source, the deaths of the animals in
the reference Tank 1 (and probably
also those in exposure Tanks 2 and 3)
would show that the "fish kill" pre-
sumably in progress in nature was not
the result of the effluent. A parallel
installation under Plan B might dem-
onstrate no change in the waste being
monitored.
Dilution Systems
Various dilution systems might be
employed under Plan C. One possible
system is to simply test two or more
constant proportions of effluent against
the (changeable) receiving water, and
then to observe the actual portions of
waste entering the receiving water. If
the highest known ratio of flow of ef-
fluent to stream is, for example, 1
to 10, and the strongest concentration
in the monitor system is 1 to 10,
then as long as the fish in the l-to-10
mixture live, no damage would be ex-
pected in the stream especially at high
flows.
If the stream flow should fall below
its base level, or the waste flow in-
crease, damage might be expected, and
preparations could be made for reme-
dial action. If the above conditions
were constant, and dead fish began to
appear in the tank with the highest
concentration of effluent (No. 2, for
example), this might indicate a rise in
the toxicity of the plant effluent it-
self.
A different approach is to propor-
tion the mixure to stimulate the actual
mixture taking place in the receiving
water. For this purpose if the location
were on a flowing Stream, the flow of
both the stream and final effluent
must be known. Periodic adjustments
might be made by. hand, or by auto-
matic equipment involving telemetry of
both effluent and stream flow.
Serial-dilution apparatus used for continuous delivery of -various concentra-
tions of materials in water.
Reproduced from
best available copy.
56
JOT.Y. 1966 17
-------
Test Animals
No universal recommendations can
be made about test animais to be used
although many suggestions are avail-
able1-". Irwin" investigated the suit-
ability of 57 species of freshwater
fishes for this purpose. Briefly, they
should be of local importance, they
must be a type that can be maintained
in good health in the laboratory in the
dilution water to be used, and enough
must be employed so that reasonable
statistical reliability is assured (for
example, 10 per tank).
Fish are usually employed as test
animals, although there is no reason
not to use any other organisms that
can be successfully kept alive in the
test tanks". A prime consideration
for public relations purposes might be
the local importance of the organisms
selected, for example, oysters, shrimp,
or mummichogs in coastal locations,
or young bluegills, trout, or minnows
in inland areas. The number and size
of test animals used may have to be
determined by experience. Such fac-
tors as temperature and oxygen con-
tent of the tanks will affect the num-
ber of experimental animals that can
be maintained.
Continuous availability is also im-
portant. It is always wise not to
change the test species after a pro-
gram has been established, since the
reactions of different species to the
waste being monitored may not be the
same". Whatever species is selected,
it should be one that is either avail-
able on a year-round basis, or one that
can be stockpiled at times of abund-
ance and successfully kept until
needed.
Animals in the exposure . tanks
should be fed the same as those in
the stock tanks. The same kind of
food in the same ratio of food to
weight of test fish should be added
to each tank at the same intervals. Un-
natural acceptance of food in ex-
posure tank may indicate a measure
of distress, even in the absence of
mortality.
Special Considerations
Oxygen determinations should be
run occasionally to ensure that any
deleterious symptoms are the result of
the effluent and not of oxygen defici-
ency. Total oxygen demand by fishes
is more nearly a function of the total
weight of fish than the number. Two
or three 6-inch fishes might demand
as much or more oxygen than a dozen
or more 2-inch fishes.
Actual minimum acceptable levels
will also depend on the temperature
and type of fish used: Carp or
Titapia might endure a minimum of
2 ppm of oxygen at 90° f, where
trout or salmon would require a;
least 5 ppm at 60° F.
Long-continued exposure to low
level concentrations in tanks- may
result in cumulative intoxication or
acclimatization. In most cases these
effects can probably be best counter-
acted by periodic renewal of the test
fishes, for example: At 60-day inter-
vals (as it is reported11 that at 60
days either acclimatization or in-
creased sensitivity may modify toxi-
city).
When obtaining stocks of test
animals from receiving waters, these
sa'me factors should be borne, in mind.
Fish or other organisms taken from
below the outfall might have acquired
some immunity or sensitivity to the
effluent being tested. Those taken
from well above the outfall probably
have not, unless they have. recently
migrated upstream.
Fish of a species normallv present
in the receiving stream, but imported
from some other (unpolluted) source.
will presumably exhibit a completely
unconditioned response. It is also
possible that the effluent being moni-
tored is one to which acclimatization
is so slow or slight as to be negligible
under the conditions of this test.
Generally, since t!ie aquatic life to
be protected is that already present in
the stream, the most logical source of
fish is the stream itself. Under oper-
ating conditions, however, it is not
always practicable to collect the ex-
perimental animals from this source
and imports from another area may
be necessary.
Selection of Dilutions
The "critical range" of toxicity
may be defined as the range between
the highest concentration that kills no
test animals and the lowest concen-
tration that kills all. The TLD. ot
the conventional bioassay3 is in the
middle portion of this range. In
general, as a test is prolonged, the
critical range is narrowed until a level
of relative stability is reached. The
slope and magnitude of the curve
thus represented are functions 01 "he
toxicant, species used, and environ-
mental conditions.
When an effluent is to be biomoni-
tored under Plan B, the selection ot
appropriate dilutions might be based
on the above concept of ''critical
range." If a "tight" control is desired.
the highest concentration might be
established near the TLm. When a
hatch of test animals is first placed in
such a dilution, approximately half
of them may (by design) be expected
to die. The survivors, however, would
constitute a rigorous control as any
increase in toxicity would he expected
to kill the remaining animals in order
of susceptibility until, is the top of the
critical range is reached, all would he
dead.
A somewhat less stringent control
would he effected with a dilution near
the lower end of the cri.ical range.
It is not generally practicable to
determine the lower end of the critical
. range precisely, but a reasonable esti-
mate can often be made. If the criti-
cal range in question happens to be
relatively wide, a moderate increase
in effluent toxicity would kill only
some of the test animals. If the criti-.
cal range is very narrow, there is little
choice between a dilu.ion set at the
lower end and one at the TLm.
' In any large population of test
animals kept in an exposure tank over
an extended period of time, an occa-
sional animal may be expected to die.
The mortality of significance then is
not the occasional individual death.
but' the sudden death of 25%, 50%,
or 100% of the test animals. When
this happens, biomonitoring has
sounded the alarm to take appropriate
action to detoxify the effluent or to
divert it from the receiving stream
until ii is again normal. H
ACKNO WLED CM EN 7 : This feature
is based on a paper presented by the
authors at Purdue Industrial Was'.e
Conference sponsored by Purdue Uni-
versity, May 3-5, 1966.
References
1. Henderson, Croswell and Picker-
ing. Quentin H.: "Use of Fish in the
Detection of Contaminants in Water
Supplies," Journ. AWWA 55(6):715-
720, 1963.
2. American Public Health Asso-
ciation, Inc.: Standard Methods for
the Examination of Water and Waste-
water. Part VI: "Bioassay Methods
for the Evaluation of Acute Toxicity
of Industrial Wastewaters and Other
Substances to Fish,". 12th Ed., New
York. i965.
3. Weiss, Charles M.: "Use of Fish
to Detect Organic Insecticides in
Water," Journ. WPCF 37(5) :647-
658, 1965.
4. Henderson, Croswell and Tarz-
well, Clarence M:. "Bio-Assays for
Continued on page 45
18
INDUSTRIAL WATER ENGINEERING
-------
BIOMONITORING Continued from pane IB
lilt- Control of IniluMrial Elilurnl-," Sewage anil Ind. Wastes
29(91:1002-1017. 1957.
5. Pirkrrinp. Qurnlin H.: Ki-Kurvh in l'roKrr»s I1**-
0. Mount, Donald I. and Warnvr. Richard £.: A Serial-Dilu-
tion Apparatus for ihr Cuntinuouf Delivery of I'ariout Conceit-
/rations of .Material in Vater. V. S. Dept. of HEW, PHS Puhli-
ration No. (\\): 1480-1485, 1963.
9. Emmens, C. W.: Keeping anil Breeding Aquarium Fishes.
Aradcmic Tress, Inc., New York, 1953.
10- Lewis, W. M.: Maintaining Fishes lor Experimental and
Instructional 1'itrposex. Southern Illinois I'niv. Frew, Carbondalr,
III.
11. Irwin, William H.-.Filty-sevi'n Species of Fish in Oil Re-
finery Waste Bioassay. Trans. 30th N. Am. Wild, and Nat. Ret.
Con I. March 8-10, 1965. Wild. Mgmt. Inst. Wire Bldg.. Wash-
ington, D. C.
12. Klork, John W. and Pcursoii, Erman A.: Engineering
Evaluation and Development of Bioassay Kinetics. Stale Water
I'ollu. Control lioard, Sacramento, Calif., September 1961.
13. Mount, Donald I.: Personal Communication, January, 1966.
58
JULY, /966 45
-------
APPENDIX
CONTINUOUS-FLOW FISH BIOASSAY APPARATUS
FOR MUNICIPAL AND INDUSTRIAL EFFLUENTS
Larry A. Esvelt
Jerrold D. Conners
February 1971
Sanitary Engineering Research Laboratory
College of Engineering
and
School of Public Health
University of California
Berkeley
SERL Report No. 71-3
59
-------
CONTINUOUS-FLOW FISH BIOASSAY APPARATUS FOR
MUNICIPAL AND INDUSTRIAL EFFLUENTS
SERL REPORT NO. 71-3
Appendix A consists of SERL Report No. 71-3 published in
February, 1971. This report describes the development, construction
and operation of the continuous-flow fish bioassay apparatus used
throughout this study.
155
-------
TABLE OF CONTENTS
Page
LIST OF TABLES 160
LIST OF FIGURES 160
Chapter
I. INTRODUCTION . . . l6l
II. THE DILUTING APPARATUS . . . 163
Operating Principle ......<, 163
Wastewater and Dilution Water Delivery 166
Electrical Control 167
Proportioning Boxes 169
Dilution Adjustment .„ 171
Fail-Safe Features ...... 171
Predilution for More Toxic Wastes . . 173
Other Diluter Designs „ 175
III. CONTINUOUS FLOW BIOASSAY APPARATUS .... 177
Operating Procedure 177
Operating Experience 178
REFERENCES 182
159
-------
LIST OF TABLES ,
Table Title . Page
A-l Diluter Adjustments For Three Logarithmic
Concentration Series , 172
A-2 Diluter Performance For Seven Weeks of
Operation Without Adjustment 181
LIST OF FIGURES
Figure Title Page
A-l Proportional Diluter 164
A-2 Continuous-Flow Fish Bioassay Apparatus
in Operation at SERL 165
A-3 Electrical Diagram 168
A-4 Diluter Proportioning Boxes 170
A-5 Predilution System 174
A-6 Thirty-Liter Assay Vessel . . . 179
... 62
160
-------
I. INTRODUCTION
There are several advantages to continuous-flow bioassay
techniques for evaluating toxicity of municipal and industrial effluents
over the traditional "batch" procedures as outlined in the Twelfth
Edition of Standard Methods [ ij. In 1965 Mount and Warner [2]
described a serial dilution apparatus for continuous-flow assays.
Subsequently, Mount and Brungs [3] described a proportional diluting
device providing a repeated dosing to assay vessels, which approxi-
mates continuous flow. Both devices were intended for "clean water"
systems dosed with small portions of highly toxic substances. They
were not intended to dilute partially-treated wastewaters of relatively
low toxicity. The design of a continuous-flow fish bioassay apparatus
applicable to treated or untreated domestic or industrial wastes was
the objective of the work described in this report,
Standard Agreement S-1956 between the California State
Department of Fish and Game and The Regents of the University of
California charged the University's Sanitary Engineering Research
Laboratory with cooperating with the State in the design of a continuous-
flow fish bioassay apparatus. In fulfillment of this agreement SERL
was to:
a. Prepare a list of design criteria which the bioassay apparatus .
must rneet to satisfy its needs in evaluating advanced waste
treatment.
b. Meet with the State Departments of Fish and Game (DFG)
and Water Resources (DWR) to select the criteria which
the apparatus must meet for the needs of the State.
c. Prepare designs of alternative assay systems which will
meet the selected criteria.
d. Meet with the Departments of Fish and Game and Water
Resources representatives to select the most suitable design.
e. Assist in the construction and evaluation of a prototype of
the selected design.
Items a, b, and c were covered at a meeting held at Nimbus,
California on April 16, 1970. It was decided at that meeting that
Item c could not be performed efficiently without first obtaining some
operating experience with preliminary designs. Thus, Items c and e
were treated as one task which had to be accomplished before proposing
the final design of the bioassay apparatus. This report is concerned :-'
primarily with the design selected and placed into use by SERL and J
DFG during subsequent studies of the San Francisco Bay-Delta system:
63
161
-------
; lf.2
Criteria for the design of the continuous or pulsating flow
hioassay apparatus included:
a. The apparatus must accurately and reliably deliver preset
dilutions of wastewater to assay vessels at a desired rate
of flow.
b. It should be sufficiently reliable to operate satisfactorily for
24 hours unattended and up to 120 hours without shutdown.
c. The apparatus should be of lightweight modular construction
for ease of portability.
d. Construction should be of nontoxic materials which are
resistant to corrosion and capable of being easily cleaned
mechanically or with chemical cleaning agents.
e. The apparatus should be capable of diluting primary domestic
wastewater effluents as well as wastewaters subjected to
more extensive treatment processes.
f. It should be flexible enough to provide a range of dilutions
from 100% wastewater to less than 10%.
g. It should be possible to control the assay vessel liquid
detention time at six hours or less and down to two hours if
desired.
64
-------
II. THE DILUTING APPARATUS
The diluting apparatus which was determined to give the best
performance according to the design criteria is shown in detail on
Figure A-l. The working principle of this device is similar to that
described by Mount and Brungs [3] in that a pulsating flow from
individual proportioning chambers is used to feed the bioassay vessels.
Figure A-2 is a photograph of the assembled bioassay apparatus in
operation.
The Mount and Brungs apparatus has only clean water intro-
duced which is hydraulically controlled and shut off when siphons are
flowing. A portion of the flow has toxicant added, then is rediluted
with the remaining water. The design described in this report accom-
modates separate wastewater and dilution water feed with electrical
control. Continuous flow of wastewater in this diluter prevents
occurrence of suspended solids problems in a shut-off valve. Fail-
safe features prevent operation in case of dilution or wastewater feed
failure. The electrical control system adds flexibility and reliability
of operation, especially in assay vessel detention time establishment.
OPERATING PRINCIPLE
The diluter (Figure A-l) consists of two proportioning boxes,
one for dilution water and one for wastewater. Five proportioning
chambers within each box operate on the fill-and-draw principle,
each discharging a preset volume once per diluter cycle. To achieve
a diluted wastewater flow to an assay vessel the discharge of two
proportioning chambers, one dilution water and one wastewater, is
combined. Four diluted wastewater streams flow from four pairs of
chambers. The single remaining chamber in each box discharges
directly to an assay vessel, resulting in one vessel receiving 100%
wastewater and one vessel receiving only dilution water which serves
as a control during the bioassay test. These are assay vessels one
and six, receiving discharges from Wl and D6 proportioning chambers,
respectively.
The wastewater concentration in the flow to vessels two,
three, four, and five is determined by the "working volumes" of the
pairs of dilution chambers discharging to each one. The working
volume is established by the chamber dimensions and its siphon depth
(see later section entitled PROPORTIONING BOXES). The flow rates
(and assay vessel detention times) are fixed by the dilution chamber
working volumes and diluter cycle frequency.
A dosing sequence is initiated when all proportioning chambers5
in both proportioning boxes are filled. They are filled sequentially by
overflow from the previous (uphill) chamber with the waste or dilution
water introduced to one end of the proportioning box. When all chambers
65
-------
TIME DELAY RELAY
DILUTION WATER
PROPORTIONING BOX
Sudlan monlfatd taMid backboard
(3/«'l 1/16" Tygon)
Mlcrocvftctt
ii ' ' M '
3 'p 04 i os lyo'* !i^ I
fe:^:^
WASTEWATER -
PROPORTIONING BOX
I All bottl*< art poly«thyl«ni with boltomi
cut out; fotlentd wilh pip* I trap*
Z Siphon apparaluf ihown for Dilutcr 3
typical for 2.4.5.
3. Apporatut mounted on plywood!,
exterior Qraoj* AC
I I 1 I I
TO FISH ASSAY VESSELS
© © (?) ® ®
12 mm 00 Gtan and
3/8* i VK" Tygan
SCALE
FIGURE A-l. PROPORTIONAL DILUTER
66
-------
Proportioning BOMS
FIGURE A-2. FOUR CONTINUOUS FLOW FISH BIOASSAY UNITS
IN OPERATION AT SERL
-------
166
of the waste water proportioning box are full, Wl overflows to waste.
When all chambers of the dilution water proportioning box have been
filled, the siphon tube in D6 begins to operate. The water flowing
from the D6 siphon fills the inverted 30-m£ plastic bottle, depressing
the micro switch lever which shuts off both wastewater and dilution
water flows (see section ELECTRICAL CONTROL). The overflow
and draining flow from the inverted 30-ml bottle (the bottom is cut
out and the neck plugged except for a small drain hole) is captured
in the 125-ml bottle and discharged through the aspirator and on to
fish assay vessel number six.
The flow through the aspirator creates a suction on the mani-
fold running above the dilution water proportioning box water level
(and behind the backboard). The manifold ia connected by Y's to the
siphons from Wl and D2 through D5 proportioning chambers. The
siphon for each of these chambers is necessarily submerged and the
discharge tube from each Y is led to a water seal such that a vacuum
can be drawn and thus initiate the attached siphons (D2 through D5 and
Wi). The water seals provided consist of inverted 125-m? polyethylene
bottles with the bottoms cut out. The outlet tubes (glass tubing) extend
up into the bottle through rubber stoppers thus providing a small
reservoir to act as a water seal (or trap) for the siphon discharge
tubes above.
Overflow from the water seals serving the D2 through D5
siphons passes through a second set of Y's which act as aspirators
to start th'e siphons from W2 through W5. Five hundred-mi poly-
ethylene bottles with bottoms removed act ae funnels and mixing
vessels (and a water seal for Wl) for the flow passing to the six assay
vessels.
All of the proportioning box sections are drained +o the depth
of the siphons during each cycle. Then each is refilled with waste-
water or water for the next cycle, this being initiated by a return of the
micro switch to the closed position.
WASTEWATER AND DILUTION
WATER DELIVERY
Wastewater is delivered continuously to the dilution apparatus
where it is used to fill the wastewater proportioning box chambers or
is bypassed to waste, according to the position of the solenoid. When
actuated, the solenoid pulls the waste flow switching lever to direct
the waste flow to the wastewater proportioning box (cf. Figure A-l).
When deenergized the solenoid drops the lever by gravity (or spring
return if gravity return is not sufficiently fast) to allow the waste-
water to bypass to the drain portion of the wastewater proportioning
box. The bypassed flow, as well as overflow from Wl, may pass to
waste or be recycled to a holding tank if the wastewater to be bio-
assayed is hauled in and in short supply.
68
-------
167
Dilution water may be obtained from various sources,
according to the objectives of the assay. When a noncorrosive,
suspended solids free water is used, a shut-off type of solenoid valve
of corrosion-resistant and nontoxic materials can be used to control
the flow, as is shown in Figure A-l. The flow should come from a
constant-head tank to minimize pressure fluctuation. If more
corrosive (e. g. , saline) water is to be used for the bioassay, a bypass
system for dilution water similar to that shown for wastewater in
Figure A-i can be used. A head tank would be less necessary for a
bypass system and water conservation could be practiced by returning
bypassed (and head tank overflow) water to a holding vessel. This
would be desirable in instances where dilution water is in short supply
or must be transported a considerable distance.
ELECTRICAL CONTROL
The control of wastewater and dilution water flows to the
respective proportioning boxes is accomplished by the two solenoids
shown in Figure A-l. Figure A-3 is a more complete schematic of
the electrical control system, showing the specific components used
in the prototype diluter constructed at SERL. Components include
two solenoids for wastewater and dilution water control, a microswitch,
and a time delay relay.
The electrical cycle is initiated when power is turned on or
the microswitch returns to its normally closed position at the end of
a dilution cycle. This provides power, through the normally closed
(NC) contacts, to the time delay relay. The microswitch achieves its
NC state when the 30-m£ bottle in the D6 discharge empties (i. e. , D6
siphon ceases to flow). A small spring is necessary for adjustment
of the critical balance of the microswitch so it moves to the depressed
condition when D6 siphon is flowing and returns to normal after flow
ceases and the bottle drains.
After the time delay relay (TDR) is energized, a time delay
period is initiated. After a preset time the normally open contact
is closed which actuates the solenoids, allowing liquid flow to the
proportioning boxes. The TDR used for the SERL prototypes is shown
in Figure A-3, along with two alternates. Solenoid and solenoid valve
models indicated are illustrations of workable alternates. Other
alternates are equally acceptable for these applications as long as the
time delay for closing the circuit to the solenoids is adjustable.
After the proportioning boxes are filled, the D6 siphon begins
to flow, filling the 30-m£ bottle which breaks the microswitch contact.
This breaks the circuit to the TDR and the solenoids, shutting off flow
to the proportioning boxes. The TDR automatically resets so that
after D6 empties the next cycle begins.
The setting on the TDR establishes the desired hydraulic
detention time in the assay vessels (time required to fill the empty
vessel to its overflow point). Knowing the vessel volume and volume
of discharge to each vessel per diluter cycle (500 ml for this system),
69
-------
Wastewater Solenoid
^-Continuous duty;
4x318 Dayton,
4x241 Dormeyer
Dilution Water Solenoid
Normally closed , st. stl. •,
Hoke S90A380C,
Asco 8262B38
Valve
Time Delay Relay - 5 min time range -
Cramer 471 or Agastat 2412 - Pull in
Delay or Eagle Cycleflex Hp 5
St. Stl. Screw,
Washer, and Nut
30ml Polyethylene Bottle
Connection from
Prediluter if used
(See Fig: 5)
Extension Spring
Nut brazed
to Switch Arm
Equipment shown
is for 110 vac.
Similar equipment
available for 12 vdc
Depress ~ to break contact
and shut off Solenoids
Release ~ to activate Time Delay Relay
MICROSWITCH
Connect normally closed
BZ2RW 80-A2
FIGURE A-3. ELECTRICAL DIAGRAM
lev
-------
169
the frequency of diluter cycling can be computed. Then the fill and
discharge time for the diluter proportioning hoxes (the time from
solenoid actuation to microswitch return to normal after the dilution
chambers empty) is determined. The TDR is set to delay the difference
between the desired cycle time and fill-empty time. As as example:
Fish assay vessel volume, 30 t.
Volume delivered per diluter cycle, 500 ml
Desired vessel detention time, 6 hr
Therefore:
= 60 cycles per detention time
6 hr x 60 min/hr
oO cycles/detention time
= 6 min/cycle
or 1 cycle of the diluter each 6 minutes. If the time (as measured
with all systems in operation) for fill and discharge of the diluter is
3-1/2 minutes, the TDR must be set for 2-1/2 minutes to achieve the
6-min cycle time.
PROPORTIONING BOXES
The proportioning boxes for the wastewater and dilution water.
were designed to deliver dilutions ranging from 100, 50, 25, 12, and
6% wastewater to 100, 80, 64, 51, and 41% wastewater. This range
of dilutions and other intermediate dilution ranges can be achieved by
adjusting the depth of the siphon in each proportioning chamber. Total
delivery (wastewater plus dilution water) to each assay vessel is
designed to be 500 m£ per cycle.
Details of construction of the plexiglass boxes are shown in
Figure A-4. The boxes constructed at SERL had 3/8-in. outside walls.
and bottom with 1/4-in. interior dividers. The plexiglass pieces can
be readily precut and assembled using acrylic solvent cement.
Plexiglass can be cut with a table saw using a sharp, fine tooth blade.
Solvent-cemented joints are most satisfactory when sawed edges are
sanded smooth and polished surfaces are sanded lightly to give them
texture prior to application of the solvent. The solvent application
should be continuous until the plexiglass surface becomes gelatinous,
then the pieces joined, aligned and held firmly in place for at least •
a one-hour curing time. Application of acrylic cement along the joint
during curing will help prevent bubble formation. Leaks may be
stopped using an acrylic cement which contains acrylic monomer.
A detail of the individual siphons is also shown in Figure A-4.
Wood strips across the front of each proportioning box (see Figure
A-l) serve the dual purpose of retaining the proportioning boxes on the
mounting board and providing mounting strips for the siphons, which
are held by short loops of Tygon tubing. The siphon depth into each
71
-------
3/8"
Typical Plexiglass Divider, 1/4" thick
/ Chamber
;; Width
/
/
j
/
\
4 1/2"
- 1 .!
see data oelow ^
-vl
o
DILUTION WATER
PROPORTIONING BOX
WASTEWATER
PROPORTIONING BOX
5 Chambers
02
D3
D4
D5
D6
Width, in.
1 3/4
2 3/8
2 5/8
2 7/8
31/8
6 Chambers
Drain
Wt
W2
W3
W4
W5
Width, in.
13/4
3
2V2
2
13/4
H/2
4 Dividers at 1/4" each, 5 Dividers at 1/4"each,
2 ends at 3/8*each.
Total length0141/2"
2 ends at 3/8 each.
Total length = 141/2"
Hole as reqd for drain
or D6 siphon
Plexiglass Box
Wood, Iax2'
Tygon loop attached
to wood on each
side of glass tube
8 or 10 mm OD
Gloss Tube
Note-
Lorger tubing shown
is for Wmtewater,
smaller size is • for
Dilution Water
5/16" x 1/16" or
3/8"xl/l6" Tygon
8 or 10 mm OD
Glass Tubs, flared
SIPHON DETAIL
FIGURE A-4. DILUTER PROPORTIONING BOXES AND SIPHON DETAIL
-------
171
chamber can be adjusted with different lengths of Tygon tubing between
the flared and bent glass tubes. Fine adjustment can be achieved by
changing the depth of glass tube insertion into the Tygon tubing. The
flared intake provides each siphon with a more reproducible end point
to the siphoning action and thus more consistent volumes.
DILUTION ADJUSTMENT
Selection of a particular dilution sequence must be made for
each waste bioassay. Municipal wastewaters following primary treat-
ment may have a toxicity such that the median lethal concentration
(LC-50, TL-50, or TLm) is in the range of 20 to 80%.* A dilution
range including the final LC-50 must be chosen. Industrial wastes are
much more variable in toxicity and a special knowledge of the applica-
tion or a trial and error selection must be made in these applications.
Treatment may of course alter the acute toxicity of wastewater
effluents.
The established practice is to select a. dilution series with c
logarithmically equivalent spacings between dilutions [1,4]. Having
selected the dilutions to be used, the volume required from each
proportioning chamber (working volume) can be computed. Subse-
quently, knowing the volume per unit depth for each chamber, siphon
depths are established. Dilution adjustment can be made with reason-
able accuracy by measuring the depth of the siphon in each proportioning
chamber. Table A-l contains chamber volumes per unit depth for
proportioning boxes constructed to the dimensions in Figure A-4.
Percentage of waste with working volumes and siphon depths is shown
for three logarithmic series of wastewater concentration. Actual
dilutions achieved are computed from measurement of dilution water
delivery and total delivery during operation.
FAIL-SAFE FEATURES
The diluter shown in Figure A-l contains fail-safe features. '
If the dilution water supply is interrupted, the dilution water siphons •
and the wastewater siphons will not function. As neither wastewater '
nor dilution water will be delivered to the assay vessels, the bioassays
will become "static" until the malfunction is detected and rectified.
A reduction in the rate of dilution water flow will result in
extended fill time for the dilution water proportioning box. This will
in turn result in a longer cycle time and increased detention periods
in the assay vessels. However, the dilutions will remain constant.
If electrical failure occurs, neither dilution nor wastewater
solenoids can be activated, therefore no water or wastewater is
*.
Values in this range have been experienced for continuous flow
96-hour bioassays of domestic wastewater utilizing the Golden Shiner.
Tap water was used as dilution water.
73
-------
TABLE A-l
DILUTEE ADJUSTMENTS FOR THREE LOGARITHMIC CONCENTRATION SERIES
Assay Vessel
Proportioning
Chamber
Chamber Volume,
ml/cm depth
Percent Waste
Working Volume, ml
Siphon Depth, cma
Percent Waste
Working Volume, ml
Siphon Depth, cm
Percent Waste
Working Volume, ml
Siphon Depth, cm
1
Wl
48.4
100
500
10. 3
100
500
10.3
100
500
10.3
2
DZ
28. 2
W2
40. 3
50
250
8.9
250
6.2
67
165 335
5. 9 8. 1
80
100
3.6
400
9.9
3
D3
38.3
W3
32. 2
25
375
9.8
125
3.9
45
275 225
7. 2 7. 0
64
180
4.7
320
10.0
4
D4
42.4
W4
28.2
12
438
10.3
62
2.2
30
350 150
8.3 5.3
5
244
5.8
1
256
9. 1
5
D5
46.3
W5
24. 2
6
469
10. 1
31
1.3
20
400 100
8. 6 4. 1
41
295
6.4
205
8. 5
6
control
D6
50.4
0
500
9.9
0
500
9.9
500
9.9
- -"
-------
173
delivered. Once again the bioassays become "static" tests until the
power is restored.
li the wastewatcr supply fails* the Wl proportioning chamber
will not receive wastewater. Each time a siphon empties a dilution
chamber, especially with the flared siphons as shown in Figure A-4,
it draws the water surface to slightly below the end of the siphon.
This creates an air break that stops the siphon from flowing. The
air break in chamber Wl, when the chamber is not refilled, prevents
a vacuum from being drawn in the manifold connecting siphons Wl,
D2, D3, D4, and D5 to the aspirator. Therefore, with a wastewater
supply failure, even though D6 fills and siphons, the aspirator fails
to start the other siphons and all assay vessels become "static" units
except vessel 6 (the control vessel) which has a slightly increased
flow and decreased detention time.
A reduction in the wastewater flow rate, such that the waste-
water proportioning chambers are not full when the dilution water
proportioning box is full and ready to discharge, results in a reduced :
flow to vessel 1 if Wl receives sufficient liquid to close the air break.
If the air break in Wl does not close, vessels 1 through 5 do not
receive flow during that cycle. This latter will occur when Wl
receives no waste by the time D6 fills and starts to siphon, shutting
off the waste flow to the wastewater proportioning box. None of the
siphons will start due to the air break in Wl. The wastewater pro-
portioning box will continue to fill during subsequent fill cycles of
D6 until Wl receives wastewater and then all siphons operate.
One deviation from fail-safe operation should be noted. If
the waste flow fails after only a small amount has entered the Wt
proportioning chamber such that the water surface is still well below
the top, the aspirator suction may not be sufficient to start the siphon
and empty Wl. This will allow all dilution water siphons to start on
subsequent cycles although no wastewater is flowing. A more efficient
aspirator may alleviate this problem.
PREDILTUIOH FOR MORE TOXIC WASTES
Wastewaters which exhibit greater toxicity than can be
accurately determined with the proportional diluter described herein
can be "prediluted" prior to testing. An apparatus for predilution on
a continuous basis for use with the proportional diluter system is
shown in Figure A-5. A pulsating flow principle with positive volumetric
dilution and wastewater control as used here is similar to that employed
for the individual dilutions in the proportional diluter. The predilution
electrical control system is tied into the one for diluter control only
for power. It is connected so the power is off and predilution flows
do not operate while the diluter siphons are working. It may be
connected to prevent flow to the prediluter while the diluter is filling
or siphoning.
75
-------
174
DILUTION WATER |
Pivot
Waste Flow
Switching Lever
Connect to
Suction Manifold
(See Fig. 1)
PREOILUTION
PROPORTIONING
BOX
Note • Drain Chamber Behind •
100% Waste Chamber
125 ml Bottle
(Water Seal)
10 mm 00 Gloss Tube
and 3/8" x 1/16" Tygon
Siphon
Microswitch
PREDILUTION
HOLDING BOX
Diluter Solenoid
Valve
^ Electrical
jf Connections
—--jr^
-------
175
In Figure 5 the two microswitches are wired in series using
the normally closed contacts. When power is applied with all pre-
diluter boxes empty (siphon microswitch up and float microswitch down
normally), the solenoid valve for dilution water and solenoid for waste
flow switching will be actuated. This will cause the predilution pro-
portioning boxes to fill with wastewater and dilution water. The
wastewater flow must be sufficient so its compartment fills first and
overflows.
When the dilution water box fills to the top of its siphon,
siphoning commences. The siphoning dilution water shuts off both
solenoids by depressing the siphon microswitch, and starts the siphon
from the wastewater box. When the predilution proportioning boxes
empty the siphon microswitch reengages, but the float attached to the
float microswitch is adjusted so it keeps the circuit broken until the
predilution holding box is emptied.
The solenoid valve for dilution water delivery to the dilution
water proportioning box (see Figure A-l) is connected to an aspirator
for use with the predilution apparatus. The aspirator starts the
siphon from the predilution holding box to the wastewater proportioning
box when the normal diluter cycle starts filling the dilution water
proportioning box (refer to section on WASTEWATER AND DILUTION
WATER DELIVERY). The line from the predilution box siphon to the
wastewater proportioning box must be extended down into chamber
W5 below the siphon level from that chamber to ensure that a vacuum
can be drawn by the aspirator to empty the predilution box.
The chamber dimensions and siphon depths in the predilution
proportioning box determine the predilution achieved from this
appurtenance to the basic diluter. About 2000 m? total delivery per
cycle is needed to assure adequate flow to the wastewater proportioning
box. A box 4 in. wide by 6 in. deep with 4-1/Z-in. high wastewater
overflow is satisfactory with 4-in. long wastewater chamber and 7-in.
long dilution water chamber. This provides a predilution range of
10% to 50% wastewater. The predilution holding box must have an
operating capacity equal to the total delivery per cycle. A box 4 in.
wide by 8 in. long by 6 in. deep provides a 2-liter working capacity.
The fail-safe features of the basic diluter can be retained by
providing a small chamber in the prediluter proportioning box between
the wastewater and overflow chambers. (On Figure A-5 they are shown
adjacent to each other perpendicular to the page. ) This additional
chamber contains a siphon to waste (through a water seal) and is started
by a main diluter manifold connection (refer to Figure A-l). If waste
flow should stop, the main diluter siphons would not start due to the
air break in this redundant siphon.
OTHER DILUTER DESIGNS
In arriving at the present design as the most appropriate,
several alternatives were considered. For example, proportioning
with a battery of small pumps was discussed and rejected because of
77
-------
176
the difficulty of incorporating fail-safe features, due to the problem
of achieving constant flows with variable discharge heads and the
relatively large initial expense which would be involved. There .are
undoubtedly applications where proportioning with timer-controlled
pumps would be acceptable or possibly even preferable. '
A design was constructed in which orifices discharged
vertically upward from tubes leading from constant head tanks for the
waste and dilution waters. The orifices could be adjusted to alter
the head and the flows from a wastewater and a dilution water orifice
combined for a particular dilution. Tests with the apparatus showed
that the low flows required were extremely difficult to obtain consist-
ently, that very minute head variations in the constant head tank
changed the flows significantly, and that suspended solids accumulation
in the aperture changed flows appreciably. In addition, the necessity
for a head tank is undesirable for wastewater because of solids
precipitation.
Another design of similar principle was also tested. This
design incorporated flow tubes leading directly from the sides of
constant head tanks for each waste and dilution water. The tubes
had lips over which the liquid flowed in proportion to its height
(adjustable) with respect to the tank liquid level. Once again solids
accumulation at the discharge point, where they dried and decreased
discharge with time, was the basis for rejection. Solids also accumu-
lated in the constant head vessel and fail-safe features could not be
incorporated. This design might be applicable for suspended solids
free wastes as it is easily adjustable. Two constant head tanks in
series would be desirable to control variations in discharge.
The design chosen was superior to all other designs considered
especially in the consistency of delivery of the preset dilution volumes
and the ease with which the delivery could be checked once it was
established. Periodic visual inspection of a single diluter cycle
ascertains whether it is (and has been) working correctly and if a full
working volume is delivered from each proportioning chamber each
cycle.
78
-------
III. CONTINUOUS FLOW BIOASSAY APPARATUS
The complete apparatus for bioassay testing includes the
diluter described above plus the assay vessels, a means for furnishing
waste and dilution water, and other appurtenances. Figure A-2 shows
4 bioassay setups operating on 4 separate waste streams. The diluters
are mounted on plywood (exterior grade, painted). A single diluter
can be mounted on a 30-in. wide by 4-ft high plywood piece. A
dilution apparatus including diluter and prediluter required a 3-ft wide
by 5-1/2-ft high piece. Two slotted angle frames are supporting
2 diluters each, back-to-back. The 6 fish assay vessels for each
diluter are located at ground level.
Cost of materials for the apparatus shown in Figure A-2 was
about $800. Three to four man weeks were necessary for assembling
the units. The assay vessels used by SERL are constructed of 1/4-in.
plexiglass with "welded" corners and have PVC pipe tees for overflows.
Figure A-6 shows the dimensions of these vessels. The lower end of
the tee is screened (nylon screen) to prevent fish escape.
Since domestic wastewater with varying degrees of treatment
may be assayed, supplemental oxygen is required to maintain adequate
dissolved oxygen in each assay vessel. Cylinder oxygen has been used
quite successfully and the apparatus for its administration can be
seen in Figure A-2. The oxygen from the cylinder is delivered through
a pressure regulating valve and a needle valve and metered through a
rotameter to a manifold (lower left corners of each diluter). From
the manifold, oxygen is regulated to each individual assay vessel
through aquarium air valves, and released at the bottom of the vessels
through air stones. Aeration •with pure oxygen reduces the gas flow
necessary to maintain a given DO and thereby reduces the likelihood
of stripping volatiles from solution in the assay vessel.
For the bioassay tests in progress and shown in Figure A-2,
tap water was used as dilution water. The tap water was passed
through a column of activated carbon to assure that no chlorine was
present. The columns containing the carbon can be seen fastened
vertically on the left side of the diluters.
OPERATING PROCEDURE
Bioassays run by SERL with the diluters, in connection with
the 1970 Bay-Delta project, used wastewater pumped directly from
the effluent stream of various treatment processes. The desired
dilution range was selected (no prediluters have been placed in operation
to date) and the siphon depths set to the nearest 0. 1 cm measured from
the top of the dilution box dividers. A total delivery per cycle of
500 m? was used and a total cycle time of 6 min accomplishes a 6-hr
detention time in the 30-£ assay vessels. This is the maximum detention
177
-------
17K
lime reported to be recommended in the forthcoming thirteenth edition
.•' Standard Methods [4].
The fish assay vessels were essentially "completely mixed"
due to the combined effects of the periodic discharges of wastewater/
dilution water and the oxygen bubbling through the tanks. This
provided a dampening effect on transients which may have occurred in
the waste stream. A shorter detention time would increase the effect
of concentration transients, although wastewater that has passed
through major treatment structures will have undergone some
dampening of peaking transients.
Normally 20 to 30 fish were used per assay vessel with
weights of 1 to 4 g per fish. The daily flow through each vessel
usually exceeded 3 liters per gram of fish, easily meeting or
exceeding recommended flows [1,4],
Administration of the bioassay tests generally followed
recommendations made in Standard Methods f 1], but with some
modifications based on the work of Sprague [4],
OPERATING EXPERIENCE
Operation of 4 continuous flow fish bioassay apparatus on a
nearly continuous basis has been very satisfactory. Wastes assayed
have included municipal primary effluent as well as secondary and
tertiary effluents. Periodic (each one or two weeks) flushing of
proportioning boxes with tap water under pressure has been sufficient
for operation of up to 8 consecutive assays each of 1-week duration.
Assay vessels have been drained and flushed at the same intervals.
No bioassay run has been terminated due to diluter failure.
Solids and grease accumulation in small amounts on the
interior of proportioning chambers has given the appearance of
fouling but closer inspection has revealed that the transparent con-
struction materials fostered an illusion.
The diluters have proven to be very consistent in proportioning
flows to the assay vessels. Small deviations may occur due to the
siphoning action and sequence of siphoning from the individual dilution
chambers. This is the result of a flow from one chamber to another
existing at the time chamber D6 begins to siphon. Even though the
solenoids shut off the feed, the water level in each chamber is slightly
above the dividers causing the first siphons which start operating to
pass slightly more liquid during that cycle due to overflow from
adjacent chambers. The rate of waste flow to the diluter will also
affect slightly the dilutions achieved as the flow rate between chambers
determines the depth of each dilution chamber above the dividers at
the start of siphoning.
Two diluters operated for 7 weeks at SERL without dilution
adjustment were checked weekly for performance. During these
.checks the total delivery per cycle and delivery per cycle from the
80
-------
IS
1
II
>
4 n" i.
/ / JT
'/ ^ '"
/ 7 s' \\
/' PVC Tee ' 1 ,
/ jWl '
Plastic Screen
// ^
12"
8"
',
FIGURE A-6. 30-LITER ASSAY VESSEL
-------
180
dilution water proportioning chamber were measured and the.waste
percentage computed. Table A-2 shows the waste percentages
measured each week arid the mean, standard deviation, and coefficient
of variation* for each dilution over the 7-week period. Diluter A
operated with, primary effluent while diluter B operated with treated
wastewater.
From Table A-2 no trend in standard deviation can be seen
although the coefficient of variation increased as the percentage
wastewater in the dilution decreased. This indicates the errors are
largely attributable to the siphoning deviations mentioned earlier
in this section. The average standard deviation of the 8 dilutions
was 0. 58%.
Standard deviation x 100, divided by the mean.
-------
TABLE A-2
DILUTER PERFORMANCE FOR SEVEN WEEKS
OF OPERATION WITHOUT ADJUSTMENT
GO
Week
1
2
3
4
5
6
7
Mean
Standard
Deviation
Coefficient
of
Variation,
Percent Waste Delivered to Assay Vessel
Dilute r A
A2
64.3
64. 1
64. 1
65.0
63.6
63. 1
63.0
63.9
0. 71
1. 1
A3
36.4
37. 3
37.7
37.0
36. 3
36.3
35.0
36.6
0.88
2.4
A4
19. 1
21.0
21.4
21.3
21. 5
20.8
20. 5
20. 8
0. 83
4.0
A5
14. 1
14.3
13. 7
14. 3
13.0
13.3
13.6
13.8
0. 50
3.7
Dilute r B
B2
81.3
82. 1
81.4
81.4
81.2
81. 1
81.4
81.4
0. 32
0.4
B3
68. 1
67.9
68.0
68. 1
67.3
67.4
67. 0
67. 7
0.45
0.7
B4
59.0
59.0
58.8
57.9
59.3
58.6
59.3
58.8
0.49
0.8
B5
46. 2
47.0
46.0
45.7
45.6
45.7
45. 6
46. 0
0. 51
1. 1
oo
-------
182
REFERENCES
3.
4.
American Public Health Association. Standard Methods of the
Examination of Water and Wastewater,12th Edition, 1965.
Mount, D. I. , and R. E. Warner. A Serial-Dilution Apparatus
for Continuous Delivery of Various Concentrations of
Materials in Water. U. S. Public Health Service Publ. No.
999-WP-23, 1965.
Mount, D. I. , and W. A. Brungs. "A Simplified Dosing Apparatus
For Fish Toxicology Studies, " in Water Research, New York:
Pergamon Press, 1967.
Sprague, J. B. "Review Paper, Measurement of Pollutant
Toxicity to Fish, I. Bioassay Methods for Acute Toxicity, "
in Water Research, New York: Pergamon Press, 1969.
84
-------
IlLILHIE!
FROM NATSONAL TECHNICAL JNFORMATJON SERV5CE
Government Reports Annual Index—11876
NTISANNI-7601 /PAT PCS345.00
Interagency Task Force on Product Liability. Insurance
Study
PB-263 600/PAT263p PC$9.00/MF$3.00
Engineering Design Handbook. Metric Conversion
Guide
ADA-029 902/PAT146p PC$9.00/MF$9.00
U.S. Technology Policy
PB-263 806/PAT182p PC$9.00/MF$3.00
Interagency Task Force on Product Liability
Briefing Report: Executive Summary
PB-262 515/PAT56p PC$4.50/MF$3.00
How to Obtain Information in Different Fields of
Science and Technology: A User's Guide
AD-780 061/PAT124p PC$5.50/MF$3.00
Buying Solar
PB-262 134/PAT81p PC$5.00/MF$3.00
Methanol: Its Synthesis, Use as a Fuel, Economics,
and Hazards
NP-21727/PAT186p PC$7.50/MF$3.00
Citizens Radio Service Transactions
PB-235 959/PATPC$100.00 (Mag. Tape )
Saudi Arabia Five Year Plan, 1075-1980
PB-246 572/PAT683p PC$16.25/MF$3.00
Engineering of Wind Energy SysSams
SAND-750 530/PAT24p PC$3.50/MF$3.00
Performance Measurement and She Criminal Justice
System: Four Conceptual Approaches
PB-262 196/PAT419p PC$11.00/MF$3.00
Outer Continental Shelf Oil and Gas Costs and
Production Volume: Their Impact on the Nation's
Energy Balance to 1990
PB-262 533/PAT241p PC$8.00/MF$3.00
Operational influences on Reliability
ADA-035 016/PAT210p PC$7.75/MF$3.00
Reducing Excess Hospital CajpaeJfly
HRP-0015 199/PAT236p PCS8.00/MFS3.00
Guide and Checklist for Development and Evaluation
of State and Local Government Radiological
Emergency Response Plans in Support of Fixed
Nuclear Facilities
PB-264 798/PAT82p PC$5.00/MF$3.00
HOW JO ORDER
When you indicate the method of pay-
ment, please note if a purchase order is not
accompanied by payment, you will be billed
an addition $5.00 ship and bill charge. And
please include the card expiration date when
using American Express.
Normal delivery time takes three to five
weeks. It is vital that you order by number
or your order will be manually filled, insur-
ing a delay. You can opt for airmail delivery
for $2.00 North American continent; $3.00
outside North American continent charge per
item. Just check the Airmail Service box. If
you're really pressed for time, call the NTIS
Rush Handling Service (703)557-4700. For a
$10.00 charge per item, your order will be
airmailed within 48 hours. Or, you can pick
up your order, in the Washington Informa-
tion Center & Bookstore or at our Springfield
Operations Center within 24 hours for a
$6.00 per item charge.
You may also place your order by tele-
phone or if you have an NTIS Deposit Ac-
count or an American Express card order
through TELEX. The order desk number is
(703) 557-4650 and the TELEX number is
89-9405.
Thank you for your interest in NTIS. We
appreciate your order.
METHOD OF PAYMENT
Q Charge my NTIS deposit account no.
Q Purchase order no. _
Check enclosed for $
NAME
Q Bill me. Add $5.00 per order and sign below. (Not avail-
able outside North American continent.) ADDRESS-
ED Charge to my American Express Card account number
Card expiration date-
Signature
Airmail Services requested
Clip and mail to:
National Technical Information Service
U.S. DEPARTMENT OF COMMERCE
Springfield. Va. 22161
(703) 357-4650 TELEX 89-9405
CITY. STATE. ZIP-
Item Number
Quantity
Paper Copy
(PC)
Microfiche
(MF)
Unit Price*
All prices subject to change. The prices
above are accurate as of 8/77
Foreign Prices on Request.
Sub Total
Additional Charge
Enter Grand Total
Total Price*
-------
EPA 430/1-73/007
June 1973 .
Bioassay diluter construction : train-
ing manual
US EPA
MID-CONTINENT
ECOLOGY DIVISION
LABORATORY LIBRARY
DULUTH, MN
------- |