EPA-600/4-78-012
January 1978
METHODS FOR MEASURING THE ACUTE TOXICITY OF EFFLUENTS
TO AQUATIC ORGANISMS
by
William Peltier
Bioassay Subcommittee
EPA Biological Advisory Committee
Environmental Research Laboratory
Athens, Georgia 30601
U.S. Environmental Protection Agency
Region 5, Library (PL-12J)
77 West Jackson Boulevard. 12th Floor
Chicago, IL 60604-3590
Project Officer
Cornelius I. Weber
Aquatic Biology Section
Environmental Monitoring & Support Laboratory
Cincinnati, Ohio 45268
ENVIRONMENTAL MONITORING & SUPPORT LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
V
This report has been reviewed by the Environmental Monitoring and
Support Laboratory, U.S. Environmental Protection Agency, and approved
for publication. The mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
11
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FOREWORD
Environmental measurements are required to determine the quality of
ambient water, the character of effluents, and the effects of pollutants
on aquatic life. The Environmental Monitoring and Support Laboratory-
Cincinnati conducts research to develop, evaluate, and promulgate methods
to:
Measure the presence and concentration of physical, chemical and
radiological pollutants in water, wastewater, bottom sediments,
and solid waste.
Concentrate, recover, and identify enteric viruses, bacteria, and
other microorganisms in water.
Measure the effects of pollution on freshwater, estuarine, and
marine organisms, including the phytoplankton, zooplankton, peri-
phyton, macrophyton, macroinvertebrates, and fish.
Automate the measurement of the physical, chemical, and biological
quality of water.
Conduct an Agency-wide quality assurance program to assure stand-
ardization and quality control of systems for monitoring water and
wastewater.
The Federal Water Pollution Control Act Amendments of 1972 (PL 92-500)
explicitly state that it is the national policy that the discharge of
toxic substances in toxic amounts be prohibited. Determination of the
toxicity of effluents, therefore, has high priority in the EPA water pol-
lution control program. However, suitable, standardized methodology
for effluent bioassays has not been available to EPA regional and state
programs. This report fills an urgent current need for standardized
methods to measure the toxicity of effluents to aquatic life.
Dwight G. Ballinger
Director
Environmental Monitoring and
Support Laboratory - Cincinnati
iii
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PREFACE
The effluent toxicity tests described in this document were prepared
by the Bioassay Subcommittee of the EPA Biological Advisory Committee
to provide methods needed for effluent monitoring by EPA Regional and
State NPDES programs and for self-monitoring by NPDES permit holders.
Two types of toxicity tests are described:
1. A preliminary, short-term, static, range-finding test for use in
determining the concentrations of effluent to be used in a "defini-
tive" test.
2. A long-term (generally 96-hr), flow-through, definitive test for
use in determining the acute toxicity of the effluent, expressed
as a LC50 or EC50.
These methods will be included in the second edition of the EPA manual,
"Biological Field and Laboratory Methods for Measuring the Quality of
Surface Waters and Effluents," but were printed separately in limited
quantity to make them available prior to the publication of the manual.
Bioassay Subcommittee Members
William Peltier, Chairman, S&A Division, Region IV
John Arthur, Environmental Research Laboratory, Duluth
Bruce Binkley, National Enforcement Investigation Center, Denver
Stanley Hegre, Environmental Research Laboratory, Narragansett
William Horning, Newtown Fish Toxicology Laboratory
Philip Lewis, Environmental Monitoring & Support Lab, Cincinnati
Royal Nadeau, S&A Division, Region II
Ronald Preston, S&A Division, Region III
Steven Schimmel, Environmental Research Laboratory, Gulf Breeze
Milton Tunzi, S&A Division, Region IX
Contributors
Ronald Eisler, Environmental Research Lab, Narragansett
Charles Stephan, " " " , Duluth
Lee Tebo, S&A Division, Region IV
Cornelius I. Weber, Environmental Monitoring & Support Lab., Cincinnati
Cornelius I. Weber, Ph.D.
Chairman, Biological Advisory Committee
Chief, Aquatic Biology Section
Biological Methods Branch
Environmental Monitoring & Support Laboratory
IV
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ABSTRACT
This report describes methods for the measurement of the acute
toxicity of effluents to macroinvertebrates and fish. The methods include
a preliminary short-term (8-24 hr.)» range-finding test and a
long term (96 hr.), flow-through (or alternate static) definitive test for
use in determining the LC50 or EC50 of the effluent. The report includes
guidelines on effluent sampling and holding, facilities and equipment,
dilution water, test species selection and handling, and data interpretation.
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CONTENTS
Foreword
Preface iv
Abstract v
Figures viii
Tables ix
1. Introduction 1
2. Facilities and Equipment 4
General Requirements 4
Construction Materials 4
Effluent (Toxicant) Delivery System 5
Test Chambers 6
Type 6
Cleaning 6
3. Test Organisms 7
Species 7
Source 7
Size 7
Holding and Handling Care 10
Disease Treatment 10
Transportation and Acclimation 13
4. Dilution Water 14
5. Effluent Sampling and Holding 17
Sampling 17
Holding 18
6. Test Procedure 19
Range-Finding Test 19
Definitive Test 19
Test Conditions 19
Number of Test Organisms 20
Loading of Test Organisms 20
Test temperature 20
Oxygen requirements and aeration 21
Beginning the test 21
Feeding 21
Duration 22
7. Test Results 22
Biological data 22
Chemical and physical data 22
Calculation of LC50 and EC50 24
Reports 24
References 26
Appendices
A. Litchfield and Wilcoxon abbreviated method 29
B. Log-concentration versus percent survival method .... 37
C. Dilutor systems, control panel and equipment lists ... 39
vii
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FIGURES
Number page
1 Data Sheet for Effluent Toxicity Test 33
2 Line fitted to data, and LC16, LC50 and LC84 as read
from the line 34
2
3 Nomograph for obtaining Chi from expected-percent-
affected and observed-percent-affected minus the
expected-percent-affected .... 35
4 Nomograph for raising base S to a fractional exponent . 36
5 Plotted data and fitted line for log-concentration
versus percent survival method 38
6 Photographs of dilutor systems. A. Solenoid valve
dilutor system. B. Vacuum siphon dilutor system .... 40
7 Solenoid valve dilutor system; general diagram .... 41
8 Solenoid valve dilutor system; detailed diagram .... 42
9 Vacuum siphon dilutor system; general diagram 44
10 Vacuum siphon dilutor system; detailed diagram .... 45
11 Dilution water chambers 47
12 Effluent chambers 48
13 Mixing chamber 49
14 Dilutor control panel 50
viii
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TABLES
Number Page
1 Recommended species and test temperatures 8
2 Recommended prophylatic and therapeutic treatments
for freshwater fish 11
3 Preparation of reconstituted fresh waters 15
4 Preparation of reconstituted sea water 16
5 Percentage of un-ionized ammonia in distilled water
at various temperatures and pH's 23
6 Salinity correction factor 23
7 Corrected values for 0% or 100% effect 32
8 Values of Chi 32
IX
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SECTION 1
INTRODUCTION
The Declaration of Goals and Policy of the Federal Water Pollution
Control Act Amendments of 1972, Section 101(a)(3), states that "it is
the national goal that the discharge of toxic pollutants in toxic amounts
be prohibited". Current Agency programs for the protection of aquatic
life in receiving waters are based in part on effluent limitations for
individual chemicals. However, toxicity data are available for only a
limited number of compounds. The effluent limitations, therefore, may not
provide adequate protection where the toxicity of the components in the
effluent is not known, where there are synergistic effects between toxic
substances in complex effluents, and/or where a complete chemical charac-
terization of the effluent has not been carried out. Since it is not
economically feasible to determine the toxicity of each of the thousands
of potentially toxic substances in complex effluents or to conduct
an exhaustive chemical analysis of the effluent, the most direct and cost-
effective approach to the measurement of the toxicity of effluents is to
conduct a bioassay with aquatic organisms representative of indigeneous
populations. For this reason, the use of effluent bioassays to identify
and control toxic discharges is rapidly increasing within the Agency and
state NPDES programs.
The lack of standardized bioassay methodology developed explicitly
for effluents has delayed the implementation of EPA regional and state
effluent toxicity testing programs and has resulted in a lack of uniformity
in test procedures. In response to this problem, a subcommittee was
organized within the EPA Aquatic Biology Methods Advisory Committee,
sponsored by the Environmental Monitoring and Support Laboratory, Cincinnati,
to prepare effluent bioassay methods for the NPDES program. To provide
valid methods, it was essential to include EPA Regional and Enforcement
programs personnel with extensive experience in conducting effluent toxi-
city tests. In completing their task, the subcommittee members drew from
their own experience and borrowed heavily from the report, "Methods for
Acute Toxicity Tests with Fish, Macroinvertebrates and Amphibians,"
(EPA 660/3-75-009), previously prepared primarily to standardize basic
laboratory methods for determining the toxicity of pure compounds.
The acute toxicity tests for effluents described in this report are
used to determine the effluent concentration, expressed as a percent volume,
that is lethal to 50 percent of the organisms within 96 hrs or some other
prescribed period of time. This value is termed the median lethal concen-
tration or LC50. Where death is not easily detected, for example, with
some invertebrates, other indicators such as immobilization must be used
as the "adverse effect." Blood chemistry, biochemical measurements or
histological examinations can also be employed to measure the toxicity of
an effluent. The concentration of effluent, expressed as a percent
volume, that causes such a defined adverse effect (other than death) in
50 percent of the test organisms within the prescribed exposure period is
termed the median effective concentration or EC50.
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A two-test sequence is generally required to estimate the acute
toxicity of an effluent: (1) a preliminary, short-term (8-24 hr),
range-finding test is conducted to define the range of effluent dilutions
to be used in the definitive test, and (2) a more rigorous long-term
definitive test is conducted (using the range of effluent dilutions
determined by the range-finding test) over a 96-hour time period to arrive
at the acute toxicity of the effluent which is expressed as a LC50 or EC50.
One of the following procedures shall be used for the range-finding
test:
A. Test organisms are placed in suitable containers and exposed
under static conditions to 3-5 widely-spaced dilutions of the
effluent for a period of 8-24 hours.
B. If the effluent has a high dissolved oxygen demand, or the
organisms require flowing water, a flow-through test is used
to define the range of toxicity of the effluent.
One of the following procedures shall be used for the definitive test:
A. Preferred procedures (flow-through tests)
1. Test organisms are exposed to effluent solutions flowing
into and out of test chambers on a once-through basis for
the duration of the test. The effluent is conveyed directly
and continuously from the source to the dilutor system.
2. Test organisms are exposed to effluent solutions flowing into
and out of test chambers on a once-through basis for the
duration of the test as in A.I above. However, the effluent
is supplied to the dilutor system from discrete effluent samples
collected periodically. The interval at which samples are
collected is based on the variability of the effluent charac-
teristics, production schedule, batch processes, retention
time, etc. (see p. 17 - Effluent Sampling and Holding).
The continuous effluent sampling technique (A.I) is the best of two
methods described.
B. Alternative procedures (static tests)
1. Test organisms are exposed to a fresh solution of the same
concentration of effluent every 24 hours, either by trans-
ferring the test organisms from one test chamber to another,
or by replacing the effluent solution in the test chambers.
2. Test organisms are exposed to the same effluent solution for
the duration of the test. However, the effluent solution in
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each test chamber may be filtered, aerated, or sterlized by
continuous circulation through an appropriate apparatus.
3. Test organisms are exposed to the original effluent solution
for the duration of the test without being continuously
circulated through an apparatus as in B.2.
The alternative procedures may be used in emergency situations or
where adequate facilities are not available to the investigator. However,
it must be understood that it is not a valid test unless it can be con-
clusively demonstrated that the chemical characteristics and toxicity of
the effluent do not change over time. Because of toxicant adsorption on
the test chambers, uptake by test organisms and the effect of metabolites
on toxicity, it is preferred that the effluent solution be renewed at
least once every 24 hours as described in B.I above.
The special environmental requirements of some organisms, such as
flowing water, fluctuating water levels, or substrate may preclude the use
of static testing.
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SECTION 2
FACILITIES AND EQUIPMENT
GENERAL REQUIREMENTS
Effluent toxicity tests may be carried out in a fixed or mobile lab.
Depending upon the scope of the bioassay program, facilities may include
equipment for rearing, holding and acclimating organisms. Temperature
control is achieved using circulating water baths, or environmental
chambers. Dilution water may be ground water, surface water, reconstituted
water, or dechlorinated tap water. Holding, acclimation, and dilution
water should be temperature controlled and aerated whenever possible.
Air used for aeration must be free of oil and fumes; filters to remove
oil in water are desirable. Test facilities must be well ventilated and
free of fumes. During holding, acclimating, and testing, test: organisms
should be shielded from disturbances.
Some organisms may have special environmental requirements such as
f lowing-^water, fluctuating water levels, or substrate which must be
provided. During holding, acclimating, and testing, immature stream
insects should always be in flowing water as described by Nebeker and
Lemke (1968); penaeid shrimp and bottom-dwelling fish should be provided
a silica sand substrate. Since cannibalism can occur among many species
of arthropods, they should be isolated by some means (e.g., with screened
compartments), or the claws of crabs and crayfish should be bound.
CONSTRUCTION MATERIALS
Materials used in the construction of the test equipment which
come in contact with the effluent should be carefully chosen. Glass,
No. 316 stainless steel, and perfluorocarbon plastics (TEFLONR) should
be used whenever possible to minimize leaching, dissolution, and sorption.
Linear polyethylene may also be used with some types of industrial
and municipal effluents, but should be avoided with those containing
synthetic organic compounds or pesticides. Unplasticized plastics such as
polyethylene, polypropylene, TYGONR and fiberglass can be used for holding,
acclimating, and dilution-water storage tanks, and in the water delivery
system. Copper, galvanized material, rubber, brass, and lead must not come
in contact with holding, acclimation, or dilution water, or with effluent
samples and test solutions.
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EFFLUENT (TOXICANT) DELIVERY SYSTEM (Flow-through test only)
Although many types of toxicant delivery systems have been designed*,
the flow-through, proportional-dilutor delivery system has proven to be
the best and the preferred system for routine effluent toxicity tests
conducted in, both fixed and mobile laboratories.
The following factors should be considered in designing the system:
(1) whether the apparatus will be installed and used in a fixed or mobile
laboratory; (2) the existence of adequate space and/or structural require-
ments for the delivery system, test chambers, effluent and dilution-water
storage; (3) the applicability of the delivery system to specific effluent
characteristics (high suspended solids, volatiles, etc.); (4) the system's
dependability, durability, flexibility, and ease of maintenance and replace-
ment; (5) the ability to perform within the flow rate and concentration
accuracy limitations; and (6) the cost of the system.
Two types of proportional dilutors are described in the appendix. The
solenoid value system is preferred, but the vacuum siphon system is
acceptable, if funds are limited.
The flow rate through the proportional dilutor must provide for
at least five complete water volume changes in 24 hours in each test
chamber, plus sufficient flow to maintain an adequate concentration of
dissolved oxygen. It is often desirable to construct the dilutor with
an additional reserve flow capacity, depending on its application and/or
special effluent characteristics. The flow rates through the test
chamber should not vary by more than 10 percent among test chambers at
any time during any test. The dilutor should also be capable of maintain-
ing the test concentration in each test chamber within 5 percent of the
starting concentration for the duration of the test.
The calibration of the dilutor should be checked carefully before
and after each test. This check should determine the volume of effluent
and dilution water used in each portion of the effluent delivery system
and the flow rate through each test chamber. The general operation of
the dilutor should be checked at least at the beginning and end of each day
during the test.
*(Lowe, 1964; Mount and Brungs, 1967; Sprague, 1969; Freeman, 1971; Cline
and Post, 1972; Granmo and Kollberg, 1972; Bengtsson, 1972; Lichatowich,
ej^ al^. , 1973; Schumway and Palensky, 1973; Abram, 1973; Schimmel, Hansen,
and Forester, 1974; DeFoe, 1975; Riley, 1975).
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TEST CHAMBERS
Type
Test chambers used in flow-through tests are constructed of 1/4 inch
plate glass held together with (GE) clear silicone adhesive. Silicone
adhesive absorbs some organochlorine and organophosphorus pesticides which
are then difficult to remove. Therefore, as little of the adhesive as
possible should be in contact with water; extra beads of adhesive inside
the containers should be removed. Stainless steel (#316) can be used
in the construction of test chambers, but must be of welded, not soldered,
construction.
The size of the chambers may vary according to the size of the test
organism and/or the facilities, but the test solution should have a mini-
mum depth of 5 cm. All chambers should have either a glass or screen
cover to prevent organisms from jumping out.
The test chambers most commonly used in static tests are wide-mouth,
3.9 liter (1-gallon) or 19.6 liter (5-gallon) soft-glass bottles. Con-
tainers such as 1-liter battery jars or 250-ml beakers are often more
suitable as test chambers for fish eggs and/or larvae and small Crustacea.
Special glass or stainless steel test chambers can be constructed to
accommodate a test organism which requires special physical conditions.
Cleaning
All test chambers, whether new or used, must be washed in the follow-
ing manner to remove surface contaminants:
A. Soak and wash in suitable detergent in water preferably heated to
a temperature of 50°C or higher. The detergent (powder or liquid)
should not be a fatty-acid base but entirely synthetic (SPARKLEENR
or ALCONOXR).
B. Rinse with water (preferably heated to 50°C or higher).
C. Rinse with a fresh, dilute (5 percent) hydrochloric acid for
removing metals and bases.
D. Rinse with water (preferably heated to 50°C or higher).
E. Rinse with acetone to remove organic compounds. When contaminated
with a pesticide, test chambers must be rinsed with acetone
before they are placed in the hot detergent soak (Item A. above).
F. Rinse twice with water.
When feasible, the outlined cleaning procedure must be used for other
equipment that comes in contact with the dilutor system, pumps, tanks, etc.
All test chambers and equipment must be thoroughly rinsed with the dilution
water prior to each test.
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SECTION 3
TEST ORGANISMS
SPECIES
Whenever possible, effluent toxicity tests should be conducted with a
sensitive species that is indigenous to the receiving water, readily
available, and either commercially or recreationally important. Acceptable
species include those listed in Table 1, which are sensitive to most
toxicants. The test organisms must be identified to species.
SOURCE
Although effluent tests should be conducted with species that are
indigenous to, or stocked into, the receiving water, the test organisms
do not have to be taken from the receiving water. It is often difficult
to obtain fish of the desired size and condition from the receiving
water. Collection permits are often difficult to obtain, and the
organisms in the receiving water may have been previously exposed to the
effluent. Fish captured by electroshocking must not be used in testing.
The usual sources of freshwater fish used for toxicity tests are private,
state, and Federal hatcheries. If trout are used as test organisms,
they should be obtained from stock that has been certified as disease-
free.
Some fish, such as Fathead and Sheepshead minnows, are easily reared
under laboratory conditions (USEPA, 1972; Schimmel and Hansen, 1974).
However, it is more practical to collect most marine species from the
indigenous population.
Some invertebrates such as daphnids, midges, and shrimp may be
reared in laboratory cultures. Care must be taken to insure that only
young age groups and early instars are used in testing. Daphnids from
cultures in which ephippia are being produced should not be used in the
tests. Invertebrates not amenable to laboratory rearing are usually
obtained directly from wild populations.
SIZE
Very immature fish (not actively feeding on exogenous food), spawning
fish, or recently spent fish should not be used. Fish weighing between
0.5 and 5.0 grams each are preferred. In any single test, all fish
should be taken from the same year class, and the total length (tip of
snout to end of tail) of the longest fish should be no more than 1-1/2
times that of the shortest one. Immature invertebrates should be used
whenever possible.
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TABLE 1. RECOMMENDED SPECIES AND TEST TEMPERATURES
o
Species Test temperature (°C)
Freshwater
Vertebrates
Coho salmon, Oncorhynchus kisutch 12
Rainbow trout, Salmo gairdneri 12
Brook trout, Salvelinus fontinalis 12
Goldfish, Carassius auratus 22
Fathead minnow, Pimephales promelas 22
Channel catfish, Ictalurus punctatus 22
Bluegill, Lepomis macrochirus 22
o
Invertebrates
Daphnids, Daphnia magna or I), pulex 17
Amphipods, Gammarus lacustris, (2. fasciatus, or 17
G_. pseudolimnaeus 17
Crayfish, Orconectes sp., Cambarus sp., Procambarus 22
sp., or Pacifastacus leniusculus 22
Stoneflies, Pteronarcys sp. 12
Mayflies, Baetis sp. or Ephemerella sp. 17
Hexagenia limbata or tl. bilineata 22
Midges, Chironomus sp. 22
Marine and estuarine
Vertebrates
Sheepshead minnow, Cyprinodon variegatus 22
Mummichog, Fundulus heteroclitus 22
Longnose killifish, Fundulus similis 22
Silverside, Menidia sp. 22
Threespine stickleback, Casterosteus aculeatus 22
Pinfish, Lagodon rhomboides 22
Spot, Leiostomus xanthurus 22
Shiner perch, Cymatogaster aggregata 12
Pacific staghorn sculpin, Leptocottus armatus 12
Sanddab, Citharichthys stigmaeus 12
Flounder, Paralichthys dentatus, P_. lethostigma 22
English sole, Parophrys vetulus 12
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TABLE 1. (Cont'd)
o
Species Test Temperature (°C)
Marine and estuarine
3.
Invertebrates
Shrimp, Penaeus setiferus, P_. duorarum, or 22
P_. aztecus
Grass shrimp, Palaemonetes sp. 22
Shrimp, Crangon sp. 22
Oceanic shrimp, Pandalus jordani 12
Blue crab, Callinectes sapidus 22
Dungeness crab, Cancer magister 12
Mysid shrimp, Mysidopsis sp., Neomysis sp. 22
Q
Freshwater amphipods, daphnids, and midge larvae and shrimp should be
cultured and tested at the recommended test temperature. Other in-
vertebrates should be held and tested within 5°C of the temperature of
the water from which they were obtained. They should be tested at the
recommended test temperature if it is within this range; otherwise, they
should be tested at the temperature from the series 7, 12, 17, 22, and
27°C that is closest to the recommended test temperature and is within
the allowed range.
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HOLDING AND HANDLING
Disinfect holding tanks or chambers with 0.5 percent commercial
bleach for one hour. Brush thoroughly with the disinfectant. Rinsed well
between groups of organisms. Other equipment used to handle organisms
must be disinfected with 0.5 percent commercial bleach, or 30-percent
formalin.
When feasible, tanks must receive an uncontaminated, constant-quality
water in a flow-through system with a flow rate of at least 2 tank-volumes
per day. Otherwise, a recirculation system where the water flows through
a charcoal filter to remove dissolved metabolites or passes by an ultra-
violet light for disinfection is necessary. Only as a last resort should
a dechlorinated tap water be used for freshwater organisms or a synthetic
salt water for marine organisms.
When organisms are first brought into the facility, they must be
quarantined for a minimum of 10 days. To avoid unnecessary stress after
collection and transportation, organisms should not be subjected to more
than a 3°C change in water temperature or 3-ppt change in salinity in any
12-hour period. Invertebrates should be held within 5°C of the temperature
of the water from which they were obtained. To maintain organisms in good
condition during holding, crowding should be avoided. Dissolved oxygen
must be greater than 40 percent saturation for warm water species and
greater than 60 percent saturation for cold water species. Aeration may
be used if necessary. Organisms should be fed at least once a day, and
excess food and fecal material should be removed from the bottom of the
tanks at least twice a week by siphoning. Organisms should be observed care-
fully for signs of disease, stress, physical damage, and mortality. Dead and
abnormal specimens should be removed as soon as observed. A daily log of
feeding, behavioral observations, and mortality must be maintained.
Organisms should be handled as little as possible. When handling is
necessary, it should be done as gently, carefully, and quickly as possible
to minimize stress. Organisms that touch dry surfaces or are dropped or
injured during handling must be discarded. Dipnets are best for handling
larger organisms. Such nets are commercially available or can be
made from small-mesh nylon netting, nylon or silk bolting cloth, plankton
netting, or similar material. Nets coated with urethane resin are best
for handling catfish. Smooth glass tubes with rubber bulbs should be used
for transferring smaller organisms such as daphnids and midge larvae.
DISEASE TREATMENT
During holding, fresh and salt water fish should be chemically treated
to cure or prevent disease as recommended in Table 2. However, if the fish
are severely diseased, it is advisable to discard the entire lot. When
invertebrates become diseased, they should be discarded. Tanks which are
contaminated with disease-causing microorganisms must be disinfected with
0.5 percent commercial bleach.
10
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TABLE 2. RECOMMENDED PROPHYLACTIC AND THERAPEUTIC TREATMENTS FOR
FRESHWATER FISH3
Disease
Chemical
Concentration Duration of
(mg/1) Treatment
External Oxytetracycline hydrochloride
Bacteria (water soluble)
Procaine Penicillin G in
Dihydrostreptomycin sulfate
solution (Franklin Lab)
Benzalkonium chloride
(HYAMLNE 1622 )
Nitrofurazone (water mix)
Neomycin sulfate
25
30-60 min
Monogenetic
trematodes
fungi, and
external,
d
protozoa
Formalin plus zinc-free
malachite green oxalate
Formalin
Potassium permanganate
Sodium chloride
DEXONR (35% Active Ingredient)
(3ml/100 gal) 48-72 hrs
1-2
3-5b
25
25
0.1
150-250
2-6
15000-30000
2000-4000
20
30-60 min
30-60 min
30-60 min
1-2 hrs
30-60 min
30-60 min
5-10 min dip
(e)
30-60 min
Parasitic
copepods
Trichlorf on
(MASOTEN )
0.25 Continuous
This table indicates the order of preference of treatments that have been
reported to be effective,but their efficacy against diseases and toxicity
to fish may be altered by temperature or water quality. Caution; test
treatments on small lots of fish before making large-scale applications.
Fish should not be treated the first day they are in the facility.
11
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TABLE 2 (continued).
Before using a treatment other than those listed in this table, addi-
tional information should be obtained from sources such as Davis (1953),
Hoffman and Meyer (1974), Reichenbach-Klinke and Elkan (1965), Snieszko
(1970), and van Duijn (1973).
b. Active ingredient.
c. Treatment may be accomplished by (1) transferring the fish to a static
treatment tank and back to a holding tank; (2) temporarily stopping
the flow in a flow-through system, treating the fish in a static
manner, and then resuming the flow to flush out the chemical; or (3)
continuously adding a stock solution of the chemical to a flow-through
system by means of a metered flow or the technique of Brungs and Mount
(1967).
d. One treatment is usually sufficient except for Ichthyophthirius ("Ich"),
which must be treated daily or every other day until no sign of the
protozoan remains. This may take 4-5 weeks at 5-10°C and 11-13 days
at 15-21°C. A temperature of 32°C is lethal to "Ich" in one week.
e. Minimum of 24 hours, but may be continued indefinitely.
f. Continuous treatment should be employed in static or flow-through
systems until no copepods remain, except that treatment should not be
continued for more than 4 weeks and should not be used above 27°C.
12
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TRANSPORTATION AND ACCLIMATION
Organisms reared and held at a fixed facility are transported to the
test site in the water in which they were reared and held. If the labora-
tory is mobile, the acclimation tank can be used in transporting organisms
from the rearing and holding facilities to the test site. At the test site,
dilution water (receiving water) is pumped to the laboratory for use in the
acclimation of the organisms. If dilution water is not readily accessible,
it can be transported to the laboratory and stored in a tank for use in
the acclimation procedure and toxicity test. During transport and acclimation
the change in water temperature must not exceed 3°C within 12 hours, and the
concentration of dissolved oxygen must not fall below 40 percent of saturation
for warm water species and 60 percent of saturation for cold water species.
Upon arriving at the test site, the organisms are acclimated to the test
dilution water and temperature by gradually changing from 100-percent holding
water to 100-percent dilution water over a period of 24 hours. All organisms
must be exposed to 100-percent dilution water for at least 24 hours before
they are used for the tests, and must be held at the test temperature (±2°C)
for at least 24 hours before tests are begun.
A group of organisms must not be used for a test if they appear to be
diseased or otherwise stressed. They should not be used if more than
5 percent die during the 48 hours immediately preceding the test. If a group
of organisms fail to meet these criteria, all organisms must be discarded
and a new group obtained. The same acclimation procedure must be followed
for the new group of organisms.
Marine organisms, at times, may require acclimation to a series of
salinities ranging from 5 to 35 ppt. The reason for such ranges is that
most effluents discharged into the marine environment consist of adulter-
ated freshwater. Therefore, when diluted with the salt water, the higher-
percent effluent volumes will usually have a low salinity. In these cases,
the salinity will be inversely proportional to the percent effluent
volume.
13
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SECTION 4
DILUTION WATER
A dilution water is acceptable if healthy test organisms survive
in it through the acclimation period and toxicity test without showing signs
of stress, such as discoloration or unusual behavior. For efEluent toxi-
city testing, the dilution water should be a representative sample of the
receiving water, and should be obtained from a point as close as possible
to, but upstream of or outside of, the zone influenced by the effluent.
It is preferable to pump the dilution water continuously to the acclima-
tion tank and dilutor. However, it may be more practical to transport
batches of water in tanks to the testing site as the need arises, and
then continuously pump water to these systems.
In an estuarine environment, the investigator should collect uncon-
taminated water having a salinity as near as possible to the salinity of
the water at the receiving site.
Pretreatment of the dilution water should be limited to filtration
through a nylon sieve having 2-mm or larger holes to remove debris
and/or break up large floating or suspended solids. The water should be
obtained from the receiving water as close as possible to the time the
test begins. It should not be obtained more than 96 hours prior to
testing. If acceptable dilution water cannot be obtained from the
receiving water, some other uncontaminated, well-aerated surface or
ground water, or commercially available media can be used. This water
must have a total hardness, total alkalinity, and specific conductance
within 25 percent, and pH within 0.2 units, of the receiving water at
the time of testing.
If the substitute dilution water must be modified, reconstituted
water must be prepared for use as the diluent. Recommended procedures
are given in Tables 3 and 4. There are also commercially available salt
water media such as INSTANT OCEANR and RILA SALTSR.
With highly toxic effluents requiring very large volumes of dilu-
tion water, it may be convenient to locate the testing facility near the
source of the dilution water, and transport the effluent.
14
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TABLE 3. PREPARATION OF RECONSTITUTED FRESH WATERS
3a. Quantities of (mg/1) reagent grade chemicals required to prepare
recommended reconstituted fresh waters and the resulting water
qualities.
Water
Type
Reagent Added
NaHCO. CaSO. .2H00 MgSO. KCL
3 42 4
Final Water Quality
, Alka-
b c c
pH Hardness linity
Very
Soft
Hard
Very
soft
hard
12
48
192
384
7.
30.
120.
240.
5
0
0
0
7.
30.
120.
240.
5
0
0
0
0.5
2.0
8.0
16.0
6.
7.
7.
8.
4-6.8
2-7.6
6-8.0
0-8.4
10-13
40-48
160-180
280-320
10-13
30-35
110-120
225-245
3b. Quantities of reagent-grade chemicals to be added to aerated, soft
reconstituted freshwater for buffering pH. The solutions should not
be aerated after addition of these chemicals.
Volume (ml) of solution added to 15 liters of water
l.ON NaOH
1.0 m KH0PO.
2 4
0.5 m
6.0
6.5
7.0
7 c
/ . J
8.0
8.5
9.0
9.5
10.0
1.3
5.0
19.0
19.0
6.5
8.8
11.0
16.0
80.0
30.0
30.0
20.0
____
40.0
30.0
20.0
18.0
a. From Marking and Dawson (1973).
b. Approximate equilibrium pH after aeration and with fish in water.
c. Expressed in mg/1 as CaCO .
d. Approximate equilibrium pH with fish in water.
15
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TABLE 4. PREPARATION OF RECONSTITUTED SEA WATERa'b
Add the following reagent-grade chemicals in the amounts and order listed
to 890 ml water. Each chemical must be dissolved before another is added.
Chemical Amount
3 mg
SrCl2'6H20 20 mg
H3B03 30 mg
KBr 100 mg
KC1 700 mg
CaCl2-2H20 1.47 g
Na2S04 4.00 g
MgCl2'6H20 10.78 g
NaCl 23.50 g
Na2Si03'9H20 20 mg
Na4EDTA(c) 1 mg
NaHCO 200 mg
a. If the resulting solution is diluted to 1 liter, the salinity should be
34±0.5 g/kg, and the pH 8.0±0.2. The desired salinity is attained by
dilution at time of use.
b. From Kester et^ a±. (1967), Zaroogian et^ al. (1969), and Zillioux
et al. (1973).
c. Tetrasodium ethylenediaminetetraacetate. This should be omitted when
toxicity tests are conducted with metals. When tests are conducted
with plankton or larvae, the EDTA should be omitted and the medium
should be stripped of trace metals (Davey et^ al_. , 1970).
16
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SECTION 5
EFFLUENT SAMPLING AND HOLDING
SAMPLING
The effluent sampling point must be the same as that specified in the
NPDES discharge permit. Conditions for exception would be: (1) better
access to a sampling point between the final treatment and the discharge
outfall, or (2) if the processed waste is chlorinated prior to discharge
to the receiving waters, the sampling point may be located prior to con-
tact with the chlorine if the purpose of the test is to determine toxicity
levels of the unchlorinated effluent. Sampling should be based on an under-
standing of the short and long-term operations and schedules of the dis-
charger. It is desirable to evaluate an effluent sample that most closely
represents the "normal" or "typical" discharge and operating conditions
of the plant in question. The retention time of the effluent in the waste
water treatment facility, as indicated in Par. A.l.a-c and B.l-3 below,
must be measured using dye studies. The only way in which the sample may
be altered prior to testing is by filtering through a TEFLON or stainless
steel screen with 2-mm or larger holes.
A. Flow-through test
1. If the industrial or municipal facility discharges continu-
ously, the effluent should be pumped directly and continuously
from the discharge line to the dilutor system for the
duration of the test. The use of the effluent grab samples
should be avoided. However, if the effluent cannot be pumped
directly and continuously to the dilutor system, the fol-
lowing alternative methods may be employed for collection
of the effluent:
a. When the measured minimum retention time of the effluent
is less than 96 hours, as determined from dye studies, a
6hour flowproportional composite sample must be col-
lected and transported to the dilutor every 6 hours for
the duration of the test.
b. When the measured minimum retention time of the effluent
is between 4 days (96 hours) and 14 days, as determined
from dye studies, then a 24-hour flow-proportional com-
posite sample may be collected daily for the duration
of the test.
17
-------
c. When the measured minimum retention time of the effluent
is greater than 14 days, as determined from dye studies,
a single grab sample may be collected daily for the
duration of the test.
2. If the industrial or municipal facility discharges inter-
mittently (i.e., less than 96 hours of continuous flow),
a flow-proportional composite sample may be taken for
an 8-hour operating period or for the duration of the plant
operating schedule.
B. Static test
If a flow-through test cannot be used, a static test may be
conducted with effluent collected by one of the following methods:
1. When the measured minimum retention time of the effluent is
less than 96 hours, as determined with dye studies, a flow
proportional composite sampler may be employed to collect the
samples. Four consecutive 6-hour flow-proportional composite
samples may be collected and used in setting up 4 separate
static tests.
2. When the measured minimum retention time of the effluent is
between 4 days (96 hours) and 14 days, as determined with dye
studies, a 24-hour flow-proportional composite sample may be
used in the test.
3. When the measured minimum retention time of the effluent is
greater than 14 days, as determined with dye studies, a
single grab sample may be collected and used in the test.
HOLDING
Effluent grab samples must be stored in covered, unsealed containers.
Violent agitation must be avoided. However, undissolved materials must be
uniformly dispersed by gentle agitation. This agitation must immediately
precede adjustment of any aliquot of the effluent to test temperatures
before adding it to the dilution water. Although it is desirable to
refrigerate samples prior to the test, it is often convenient to store
samples in a constant-temperature water bath or controlled-environment
room at the temperature at which the test is conducted. The test should
be initiated as soon as possible, but no later than 8 hours after collection
of the effluent.
The persistence of the toxicity of an effluent may be a factor in deter-
mining specific toxicity limits in an NPDES permit, and is determined by
measuring its toxicity upon collection and again after holding 96 hours.
If after holding the effluent 96 hours, its toxicity has not decreased 50%
or more, it is classified as persistent. (When special tests, such as
persistence are conducted, the exact methodology must be detailed in the
report.)
18
-------
SECTION 6
TEST PROCEDURE
RANGE-FINDING TEST
Unless the approximate toxicity of the effluent is already known, it
is necessary to conduct an abbreviated, preliminary, range-finding test to
determine the concentrations that should be used in the definitive tests.
This test can be either a static or flow-through test. However, the test
most often used is an abbreviated static test in which groups of 5
organisms are exposed to three to five widely-spaced effluent dilutions,
and a control, for 8 to 24 hours.
Because the characteristics of the effluent and the receiving water
may vary significantly within short periods of time, the toxicity observed
in a range-finding test may not be representative of the toxicity of the
effluent. If the range-finding test is to be conducted with the same
sample of the effluent with which the definitive test is to be conducted,
the duration of the range-finding test cannot exceed 8 hours (see limits
in holding time for effluents, p. 18).
DEFINITIVE TEST
Test Conditions-
The determination of a LC50 or EC50 must employ a control and at
least five concentrations of effluent in an exponential series. To
calculate the LC50 or EC50 with reasonable accuracy, a definitive test
must meet both of the following criteria:
A. Each concentration of the effluent must be at least 50
percent of the preceding concentration.
B. One concentration must have killed (or affected) more than 65
percent of the organisms exposed to it, and one concentration other
than the control must have killed (or affected) less than 35
percent of the organisms.
If 100-percent effluent does not kill (or affect) more than 65 percent
of the organisms exposed to it, the percentage of organisms killed (or
affected) by various levels of the effluent in the receiving water must be
reported.
The control shall consist of the same dilution water, conditions,
procedures, and organisms used in testing the effluent. A test is not
acceptable if more than ten percent of the organisms die in the control.
Number of Test Organisms
At least 20 organisms of a given species must be exposed to each treat-
ment. More than one species may be used in the same test chamber in a
19
-------
given test, if segregated. One-half of the organisms of each species
exposed to each treatment should be placed in separate test chambers to
serve as replicates. To qualify as true replicates, no water connections
can exist between replicate test chambers. Randomization of treatments is
desirable.
Test animals are normally captured for transfer from acclimation tanks
to test chambers by dip netting. No more than 20 percent of the total
number of organisms transferred to each chamber should be added from a
given net capture.
Loading of Test Organisms
For all tests, a limit must be placed on the weight of organisms
per liter of test solution. This will minimize the depletion of dissolved
oxygen, the metabolic conversion of effluent constituents, the accumu-
lation of metabolic waste products, and/or stress induced by crowding, any
of which could significantly affect the test results.
For flow-through tests, loading in the test chambers must not exceed
5 grams per liter at temperatures of 20°C or less, or 2.5 grams per liter
at temperatures above 20°C.
For static tests, loading in the test chambers must not exceed 0.8
grams per liter at temperatures of 20°C or less and 0.4 grams per liter at
temperatures above 20°C.
Test Temperature
For flow-through tests, it is desirable to hold the temperature
within ± 2.0°C of the acclimation temperature throughout the test. This
can be accomplished by passing the effluent and/or dilution water through
separate stainless steel coils immersed in a heating or cooling water bath
prior to entering the dilutor system.
For static tests, the temperature may be that at which the test
organisms were held prior to transportation or acclimation at the site.
The instantaneous ambient temperature should not vary more than ±2°C at any
time during the test.
Oxygen Requirements and Aeration
Aeration may alter the results of toxicity tests and, as a general
rule, should not be employed. It can reduce the apparent toxicity of an
effluent by stripping it of highly volatile toxic substances, or increase
its toxicity by altering the pH. However, the dissolved oxygen (DO) in the
test solution should not be permitted to fall below 40 percent saturation
for warm water species and 60 percent saturation for cold water species.
In most flow-through tests, DO depletion is not a problem in the test
chambers because aeration occurs as the liquids pass through the dilutor
system.
20
-------
If the DO concentration decreases to a level that would be a
source of additional stress, the turnover rate of the solutions in the
test chambers must be increased sufficiently to maintain acceptable DO
levels. If the increased turnover rate does not maintain adequate DO
levels, aerate the dilution water prior to the addition of the effluent,
and aerate all test solutions.
Caution must be exercised to avoid excessive aeration, and it
should be used only as a last resort in maintaining adequate DO levels.
When aeration is used, the exact methodology must be detailed in the report.
Beginning the Test
The test begins when the test organisms are first exposed to the
effluent.
A. Flow-through test
The dilutor system should be in operation 24 hours prior to
the addition of the test organisms and the beginning of the test.
During this period, necessary adjustments can be made in the
percent effluent volumes, temperature, and flow rate through
the test chambers.
B. Static test
The effluent is added to the dilution water and mixed well
by stirring with a glass rod. The test organisms are placed
in the chambers within 30 minutes. This procedure conserves DO
and is sufficient for the effluent to become evenly dispersed
in the dilution water.
Feeding
Organisms should not be fed during the test. However, feeding is per-
mitted when using newly hatched or very young organisms. Feeding in
a flow-through system minimizes problems encountered in a static system,
such as alteration of test dilution and build-up of food and metabolic
waste products, resulting in a greater oxygen demand.
Duration
The test duration may range from a minimum of 8 hours to 96 hours,
depending on whether it is a range-finding test or a definitive test and
the purpose of the test.
21
-------
SECTION 7
TEST RESULTS
BIOLOGICAL DATA
The lengths and weights of the test organisms should be determined by
sacrificing and measuring representative organisms before the test or
by obtaining the lengths and weights of all surviving organisms at the
end of the test. The number of dead (or affected) organisms in each
test container should be counted 24, 48, 72, and 96 hours after the
beginning of the test. (See data sheet in Appendix, Fig. 1, p. 33).
Dead organisms must be removed at least once every 24 hours.
Death is the indicator most frequently used for studying toxicity
to aquatic organisms. The criteria for death are usually no movement
(especially no gill movement in fish) and no reaction to gentle prod-
ding. Death is not easily determined for some invertebrates.
The effect generally used for determining toxicity to daphnids and midge
larvae is immobilization, which is defined as the inability to move
except for minor activity of appendages, and with crabs, crayfish, and
shrimp the effects used are immobilization and loss of equilibrium.
Other effects can be used for determining an EC50, but the effect and
its definition must always be reported. General observations on such
things as erratic swimming, loss of reflex, discoloration, changes in
behavior, excessive mucus production, hyper-ventilation, opaque eyes,
curved spine, hemorrhaging, molting, and cannibalism should be reported.
CHEMICAL AND PHYSICAL DATA
The dissolved oxygen concentration and pH must be measured at the
beginning of the test, and every 24 hours thereafter, in the control
and in the high, medium, and low effluent concentrations, for the
duration of the test. The specific conductance, total alkalinity,
total hardness, and salinity, where applicable, should be measured at
the beginning of the test in the control and the high, medium and low
effluent concentrations. There may be a build-up of ammonia in the static
toxicity tests. It is advisable, therefore, to measure the concentration
of total ammonia nitrogen in the control, high, medium, and low effluent
concentrations at the beginning and end of each static test. The percentage
of un-ionized ammonia in the test containers can be determined from
Tables 5 and 6. Temperature should be recorded at least hourly in at least
one container during the acclimation period and test.
22
-------
TABLE 5. PERCENTAGE OF AMMONIA THAT IS UN-IONIZED IN DISTILLED
WATER AT VARIOUS TEMPERATURES AND pH'Sa
PERCENT UN-IONIZED AMMONIA
Temperature
CO
pH
6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5
10.0
7
12
17
22
27
0.01 0.05 0.15 0.46 1.45 4.44 12.8 31.7 59.5
0.02 0.07 0.22 0.68 2.13 6.44 17.9 40.8 68.5
0.03 0.10 0.32 1.00 3.08 9.14 24.1 50.1 76.1
0.04 0.14 0.45 1.43 4.39 12.7 31.5 59.2 82.1
0.06 0.21 0.65 2.03 6.15 17.2 39.6 67.4 86.8
TABLE 6. SALINITY CORRECTION FACTOR
Salinity (g/kg)
Factor
5
10
15
20
25
30
35
0.82
0.80
0.77
0.75
0.73
0.72
0.72
a. Skarheim (1973), and Thurston et al.(1974).
b. These values can be corrected for salinity by multiplying by the approp-
riate factor from Table 6.
c. The resulting values should be within about ±5% over the range of pH
from 6 to 9, and temperature from 0° to 30°C.
23
-------
Water samples collected for chemical analysis should be taken
midway between the top, bottom, and sides of the test containers
and should not include any surface scum or material stirred up from
the bottom or sides.
Methods used for chemical analysis must be those specified in
Section 304(g) of the Federal Water Pollution Control Act
Amendments of 1972 (Chemical Methods Manual, USEPA, 1977).
CALCULATION OF LC50 AND EC50
For each set of data, the 96-hr LC50 or EC50 and its 95-percent
confidence limits must be calculated on the basis of the initial volume
percent of the effluent in the test solutions. The "volume percent"
equals "(100 x volume of effluent)/(volume of effluent + volume of
dilution water)." If other (24-,48-,72-hr) LC and EC values are
calculated, their 95 percent confidence limits must also be determined.
A variety of methods are available to calculate a LC and EC (Finney,
1964, 1971). The most widely used are the log-concentration versus-
percent-survival, probit, logit, moving-average, and Litchfield-Wilcoxon
(1949) methods.
Two examples of calculating a LC50 using a hypothetical set of
data have been provided in the Appendix. The two methods used in the
calculations are: (1) the Litchfield-Wilcoxon and, (2) the log-
concentration-versus-percent-survival. If more than 10 percent of the
control organisms die, none of the previously mentioned methods may
be used in calculating LC and EC values, and the remaining test
results must be used with caution in evaluating the toxic effect.
REPORTS
A report of the results of a test must include the following:
A. The name of the test method, investigator and laboratory, and
the date the test was conducted.
B. A detailed description of the effluent, including its source,
date and time of collection, composition, known physical and
chemical properties, and variability.
C. The source of the dilution water, the date and time of its
collection, its chemical characteristics, and a description of
any pretreatment.
D. Detailed information about the test organisms, including scientific
name, length and weight, age, life stage, source, history, observed
diseases, treatments, and acclimation procedure used.
24
-------
E. A description of the test procedure: the test chambers, including
the depth and volume of solution; the way the test was begun; the
number of organisms per treatment; and the loading. For the flow-
through system, the water volume changes per 24 hours in each test
chamber must be calculated and reported.
F. The definition of the adverse effect (death, immobility, etc.) used
in the test, and a summary of general observations on other effects or
symptoms.
G. The number and percentage of organisms in each test chamber (including
the control chambers) that died or showed the "effect" used to measure
the toxicity of the effluent.
H. A 24-, 48-, 72-, and 96-hr LC50 or EC50 value for the test
organisms, depending on the duration of exposure. If 100 percent
effluent did not kill or affect more than 65 percent of the test
organisms, report the percentage of the test organisms killed or
affected by various concentrations of the effluent.
I. The 95-percent confidence limits for the LC50 and EC50 values and the
method used to calculate them.
J. The methods used for and the results of all chemical analyses.
K. The average and range of the acclimation temperature and the test
temperature.
L. Any deviation from this method.
M. Any other relevant information.
25
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for fish toxicological studies. Water Res. 1:21-29.
19. Nebeker, A. V. and A. E. Lemke. 1968. Preliminary studies on the
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30. U.S. Environmental Protection Agency. 1974. Methods for chemical
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2396.
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APPENDIX
A. LITCHFIELD AND WILCOXON ABBREVIATED METHOD OF DETERMINING THE LC50
General Procedure
Step 1: Tabulate the data (see sample data sheet, Fig. 1, p. 33) showing
the percent-effluent volumes used, the total number of organisms exposed to
each percent-effluent volume, the number of affected organisms, and the
observed percent-affected organisms (see Example 1 below). Do not list
more than 2 consecutive 100 percent affects at the higher percent-effluent
volumes or more than two consecutive 0 percent affects at the lower percent-
effluent volumes.
Step 2: Plot the percent-affected organisms against the percent-effluent
volume on 2 cycle, logarithmic probability paper (Fig. 2), except for
0 percent or 100 percent affect values. With a straight edge, fit a
temporary line through the points, particularly those in the region of
40 percent to 60 percent affects.
Step 3: Using the line drawn through the points, read and list an
"expected" percent affect for each percent-effluent volume tested. Disre-
gard the "expected" percent value for any of the percent volumes less than
0.01 or greater than 99.99. Using the expected-percent-affect, calculate
from Table 7 a "corrected" value for each 0 percent or 100 percent affect
obtained in the test. (Since the expected values in the table are whole
numbers, it will be necessary to obtain intermediate values by interpolation.)
Plot these values on the logarithmic probability paper (Fig. 2) used in
Step 2 and inspect the fit of the line to the completely plotted data. If
after plotting the corrected expected values for 0 percent and 100 percent
affected, the fit is obviously unsatisfactory, redraw the line and obtain
a new set of expected values.
Step 4: List the difference between each observed (or corrected) value and
the corresponding expected value. Using each difference and the corres-
ponding expected value, read and list the contributions to Chi-square (Chi2)
from Fig. 3 ( a straight edge connecting a value on the Expected-Percent
Affected scale with a value on the Observed-Minus-Expected scale, will
indicate at the point of intersection of the Chi^ scale, the contribution to
Chi2. Sum the contributions to Chi2 and multiply the total by the average
number of organisms per effluent volume, i.e., the number of organisms used
in K concentrations divided by K, where K is the number of percent-affected
organism values plotted. The product is the "calculated" Chi2 of the line.
The degrees of freedom (N) are 2 less than the number of points plotted,
i.e., N =K-2. If the calculated Chi2 is less than the Chi2 given in Table 8
for N degrees of freedom, the data are non-heterogeneous and the line is
a good fit. However, if the calculated Chi2 is greater than the Chi2 given
in Table 8 for N degrees of freedom, the data are heterogeneous and the line
is not a good fit. In the event a line cannot be fitted (the calculated
Chi2 is greater than the tabular Chi2), the data can not be used to calcu-
late a LC50 or EC50. Litchfield and Wilcoxon provided an alternate
method for calculating the 95 percent confidence limits under these cir-
cumstances. However, the toxicity test should be repeated.
29
-------
Step 5: Determine the confidence limits of the LC50.
a. Read from the fitted line (Fig. 2), the percent effluent volumes for
the corresponding 16, 50, 84 percent affects (LC16, LC50 and LC84).
b. Calculate the slope function, S, as:
S = LC84/LC50 + LC50/LC16
2
c. From the tabulation of the data determine N', which is defined as the
total number of test organisms used within the percent-affected-
organism interval of 16 percent and 84 percent. Calculate the exponent
(2.77//N1) for the slope function and the factor, f;LC50» used to
establish the confidence limits for the LC50 (or EC50).
_ (2.77//N1)
£LC50 b
^ne ^LC50 can k£ obtained directly from the nomogram in Fig.4 by laying
a straight-edge across the appropriate base and exponent values and
reading the resultant "f" value.
e. Calculate the confidence limits of the LC50 as follows:
(1) Upper limit for 95% probability = LC50 X
(2) Lower limit for 95% probability = LC50/fLC5()
Example
Steps 1-4: The data were tabulated and plotted (Fig. 2) and the
expected values were read from the graph.
STEP ONESTEP THREESTEP FOUR
%
Effluent
Volume
3.2
5.6
10.0
18.0
32.0
56.0
100.0
Number of
Organisms
20
20
20
20
20
20
20
Number of
Affected
Organisms
0
1
11
7
12
18
20
Observed %
Affected
Organisms
0(.2)b
5
55
35
60
90
100 (99.0)
Expected %
(Fig. 2)
.6
3'5 a
(14.5)a
38.0
67.0
87.5
97.0
Observed
Minus
Expected
0.4
1.5
Aberrant
3.0
7.0.
2.5
2.0
Chi2
0.005
0.006
Value
0.004
0.024
0.006
0.014
Total 0.059
a. Percent-affected organisms at the 10 percent effluent volume is obviously
an aberrant value and should be omitted when fitting the line in Step 2.
b. Step 3 "Corrected" affected values from Table 7.
30
-------
Step 4 (Cont.);
2
Calculation of Chi
a. Total number of organisms used in 'K' concentrations = 120 = 20
6
b. Calculated Chi2 = 20 x 0.059 = 1.18
c. Degrees of Freedom (N)=K-2=6-2=4
2
d. From Table 8, the Chi for 4 degrees of freedom = 9.49
2 2
e. The calculated Chi is less than the tabular Chi . Therefore, it is
assumed the line is a good fit, and the data are non-heterogeneous.
Step 5:
a. From the fitted line in Fig. 2, determine the (percent) effluent
concentrations corresponding to the 16%, 50% and 84% affected
organism values:
b. LC84 effluent concentrations = 50.0%
LC50 " " = 23.0%
LC16 " " 10.0%
c. Calculate the slope function, 'S', as:
S = LC84/LC50 + LC50/LC16 = 50.0/23.0 + 23.0/10.5
2 2
= 2.17 + 2.19 = 4.36 = 2.18
2 2
d. N' = 40 (From Figure 2)
e. Calculate the exponent (N1) and factor, £,._,._
= S2'77//N'= 2.182'77/1/4° = 2.182'77/6'32 - 2.18°'438 - 1.41 -
f. Calculate the confidence limits of the LC50
(1) Upper limit for 95% probability = LC50 X fTr,_ = 23.0 X 1.4 = 32.2%
(2) Lower limit for 95% probability = LCSO/f.^n = 23.0/1.4 = 16.4%
.LLOU
31
-------
TABLE 7. CORRECTED VALUES OF 0% OR 100% EFFECT
Expected
Value
Corrected Value
0
0
10 3.2
20 6.0
30 8.3
40 9.9
50
60 90.1
70 91.7
80 94.0
90 96.8
1
0.3
3.5
6.2
8.4
10.0
89.5
90.2
91.9
94.3
97.1
2
0.7
3.8
6.5
8.6
10.1
89.6
90.4
92.2
94.5
97.4
3
1.0
4.1
6.7
8.8
10.2
89.6
90.5
92.4
94.8
97.7
4
1.3
4.4
7.0
9.0
10.3
89.6
90.7
92.6
95.1
98.0
5
1.6
4.7
7.2
9.2
10.4
89.7
90.8
92.8
95.3
98.4
2
4
7
9
10
89
91
93
95
98
6
.0
.9
.4
.3
.4
.7
.0
.0
.6
.7
7
2.3
5.2
7.0
9.4
10.4
89.8
91.2
93.3
95.9
99.0
8
2.6
5.5
7.8
9.6
10.4
89.9
91.4
93.5
96.2
99.3
9
2.9
5.7
8.1
9.8
10.5
90.0
91.6
93.8
96.5
99.7
TABLE 8. VALUES OF Chi (p = 0.05)
Degrees of Freedom (N)
1
2
3
4
5
6
7
8
9
10
Chi2
3.84
5.99
7.82
9.49
11.1
12.6
14.1
15.5
16.9
18.8
32
-------
FIG. I. DATA SHEET FOR EFFLUENT TOXICITY TEST.
Industry/Toxicant.
Address
Contact
Effluent Serial Number .
NPDES Permit Number
Beginning' Date.
Ending: Date.
Test Organism
.Time.
.Time.
Test Temperature Range.
Cone.
or
%
Test
Container
Number
Number of
Live Organisms
0
24
48
72
96
Dissolved Oxygen
(mg/l)
0
24
48
72
96
PH
0
24
48
72
96
Total Alkalinity
(mg/l as CaCOs)
0
24
48
72
96
Total Hardness
(mg/l as CaCO 3)
0
24
48
72
96
0
24
48
72
96
OJ
LO
-------
u>
-P-
p
ro
O
m z Zj
a m
ro >
- CO ^
y* 7J >
^ rn -
> > >
i°g
H!3n
O O
rn *-
So o »
% ORGANISMS AFFECTED
-------
EXPECTED
%
AFFECTED
50 =50
70 30
80 20
Chi
. 2
90
10
95
96
E
97 -3
98
-1.0
99.5
99.6-44
99.7
99.8- -.2
99.9-
99.95
99.96-
9 ft 97-
99.98 .02
-.3
05
.04
.03
OBSERVED MINUS
EXPECTED
- 50
40
30
20
-10
5
4
3
- 2
- -.3
.2
- -.OS
2.0
1.0
.50
.40
.30
.20
.10
.05
.04
.03
.02
.01
.005
.004
.003
.002
.001
FIG. 3. NOMOGRAPH FOR OBTAINING Chi2 FROM
EXPECTED % AFFECTED AND
OBSERVED-MINUS-EXPECTED (STEP 4).
35
-------
-p
'
EXPONENT
LC50
10
5.0
4X>
3.0
-
2.0
-
1.50
1.40
1.30
-
~
1.20
_
-
1.10
ta
m +*+* +*
- 100
- 50
10.0
5.0
4.0
3.0
-2.0
15
- 1.4
-1.3
1.2
1 10
- 1.05
1.04
- 1.03
- 1.02
4.0
-
3.0
-
2.0
1.5
-
1.0
.90
.80
.70
.60
.50
-
.40
-
.30
-
_.20
FIG. 4. NOMOGRAPH FOR RAISING BASE S TO
A FRACTIONAL EXPONENT
36
-------
B. LOG-CONCENTRATION VERSUS PERCENT-SURVIVAL METHOD OF DETERMINING
THE LC50
General Procedure
Step 1: Plot the percent effluent volumes and the corresponding
percent survival on semi-logarithmic paper (Fig. 5).
Step 2: Locate the 2 highest points on the graph which are separated
by the 50 percent survival line and connect them with a diagonal straight
line. However, if one of the points is an aberrant value, the next
lowest or highest percent-effluent volume is used.
Step 3: Read on the scale for percent-effluent volume, the value of
the point where the diagonal line and the 50 percent survival line
intersect. This value is the LC50 percent-effluent volume for the
test. If by chance one of the effluent concentrations happens to have
50 percent survival, no graphing is necessary.
Example
Step 1: The percent-effluent volumes and the corresponding percent
survival data from the Litchfield and Wilcoxon example are plotted in
Fig. 5.
Step 2: The two highest points which are separated by the 50 percent
survival line (65 percent and 40 percent) are located and connected with a diagonal
diagonal straight line. The percent survival in the 10 percent-effluent
volume was considered an aberrant value and, therefore, was omitted from the
evaluation.
Step 3: An LC50 of 25.4 percent-effluent volume for the test was
derived from the point where the diagonal line and the 50 percent
survival line intersected in Fig.5.
37
-------
0 10 20 30 40 50 60 70 80 90 100
% SURVIVAL
FIG. 5. PLOTTED DATA AND FITTED LINE FOR
LOG-CONCENTRATION VERSUS
% SURVIVAL METHOD
38
-------
C. DILUTOR SYSTEMS, CONTROL PANEL AND EQUIPMENT LISTS
Two proportional dilutor systems are illustrated: (1) The Solenoid
Valve System, and (2) the Vacuum Siphon System. The designs incorporate
features from devices developed by many other Federal and State pro-
grams, and have been shown to be very versatile for on-site bioassays
in mobile labs as well as in fixed (central) labs. The Solenoid Valve
System is fully controlled by solenoids (Figs. 6, 7, & 8), and is the
preferred system. The Vacuum Siphon System (Figs. 6, 9, & 10), how-
ever is acceptable. Both systems employ the same control panel
(Fig. 14).
If in the range-finding test, the LC50 of the effluent falls in the
concentration range, 5.6% to 100%, premixing is not required, and the
mixing chamber is by-passed by running a TEFLONR tube directly from
the effluent in-flow pipe to chamber E-l. Chambers D-l and D-2 and the
mixing chamber are deactivated.
To provide the capability of using the dilutor system to carry out
tests of the toxicity of pure compounds, the control panel is equipped
with an auxiliary power receptacle to operate a metering pump to deliver
an aliquot of the stock solution of the pure compound directly to the
mixing chamber during each cycle. In this case, chamber D-l is
deactivated and chamber D-2 is calibrated to deliver a volume of
2000 ml, which is used to dilute the aliquot to the highest concen-
tration used in the toxicity test.
39
-------
FIG. 6. PHOTOGRAPHS OF DILUTOR SYSTEMS:
SOLENOID VALVE SYSTEM (LEFT), AND
VACUUM SIPHON SYSTEM (RIGHT).
40
-------
FLOW CONTROL
VALVES-
NORMALLY OPEN
SOLENOID VALVES
DILUTION WATER
INFLOW
EFFLUENT
INFLOW
CYCLE
COUNTER
LAPSE
TIME
CLOCK
LIQUID LEVEL
SWITCH
DILUTION WATER CHAMBERS
MIXING CHAMBER
NORMALLY OPEN
SOLENOID VALVE
MAGNETIC
STIRRER
EFFLUENT
CHAMBERS
MIXING CHAMBERS
TEST CHAMBERS 420 LITERS CAPACITY
FIG. 7. SOLENOID VALVE DILUTOR SYSTEM,
GENERAL DIAGRAM.
41
-------
n
D-l
n
D-2
n
II
D-3
n
ADJUSTABLE
STANOPIPE
DRAIN
-DILUTION WATER CHAMBERS
NORMALLY CLOSED
SOLENOID VALVES
6 mm 0 D DELIVERY TUBE
NORMALLY OPEN SOLENOID VALVE
6 mm O.D. DELIVERY TUBE
EFFLUENT CHAMBERS
NORMALLY CLOSED
SOLENOID VALVE
« 6 mm OD DELIVERY TUBE
-MIXING CHAMBER
IEOO ml CAPACITY
10 mm OD DELIVERY TUBE
-TEST CHAMBER
4-20 LITER CAPACITY
FIG. 8. SOLENOID VALVE DILUTOR SYSTEM,
DETAILED DIAGRAM.
42
-------
for dilution water and
Solenoid System Equipment List
1. Diluter Glass.
2. Stainless Steel Solenoid Valves
a. 3 - normally open, two-way, 55 psi, water, 1/4" pipe size,
9/32" orifice size, ASCO 8262152, for incoming effluent and
dilution water pipes and mixing chamber pipe.
b. 1 - normally closed, two-way, 15 psi, water, 3/8" pipe size,
3/8" orifice size, ASCO 8030B65, for D-2 chamber evacuation
pipe.
c. 12 - normally closed, two-way, 36 psi, water, 1/4" pipe
size, 9/32" orifice size. ASCO 8262C38, for remaining dilution
chamber and effluent chamber evacuation pipes.
3. Stainless steel tubing, seamless, 316-grade, austenetic.
a. 10 ft - 3/8" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
b. 60 ft - 1/4" OD, 0.035" wall thickness,
effluent pipes.
c. 1 ft - 3/4" OD, 0.035"-wall thickness, for standpipe in D-l
chamber.
4. Swagelok tube connectors, stainless steel.
a. 4 - male tube connectors, male pipe size 1/4", tube OD 3/8".
b. 2 - male tube connectors, male pipe size 1/2", tube OD 3/8".
c. 26 - male tube connectors, male pipe size 1/4, tube OD 1/4"
d. 2 - male tube connectors, male pipe size 3/8", tube OD 3/8".
e. 2 - male adapter, tube to pipe, male size 1/2", tube OD 3/8".
5. 7 - 1200 ml stainless steel beakers.
6. Several Ibs each of Neoprene stoppers, size 00, 0, and 1, 1 Ib of siz 5.
7. ]4 - aquarium 2-5 gal.
8. Magnetic stirrer.
9. 2 - PVC ball valves, 1/2" pipe size.
10. Dilutor control panel - see Fig. 14 and equipment list.
11. Plywood sheeting, exterior grade: one - 4' x 8' x 3/4", one -
4' x 8' x 1/2".
12. Pine or redwood board, 1" x 8", 20 ft.
13. Epoxy paint, 1 gal.
14. Assorted wood screws, nails, etc.
15. 25 ft - 1/4" ID, TEFLONR tubing, to connect the mixing chambers
to the test chambers (see Fig. 10).
43
-------
NORMALLY OPEN
( SOLENOID VALVES
-FLOW CONTROL VALVES
^DILUTION INFLOW
DILUTION WATER CHAMBERS
-LIQUID LEVEL SWITCH
NORMALLY CLOSED
SOLENOID VALUE
TEST CHAMBERS 4-20 LITERS CAPACITY
FIG. 9. VACUUM SIPHON DILUTOR SYSTEM, GENERAL DIAGRAM.
44
-------
D-l
D-2
D-3
ADJUSTABLE
STANDPIPE
DRAIN
DILUTION WATER CHAMBERS
VACUUM LINE
6mm OD CONNECTING TUBE T FORM
10 mm OD U SHAPE SYPHON TUBE
6mm 0 D VACUUM LINE TUBE
STAINLESS STEEL HOSE CLAMP
10 mm ID CONNECTING TUBES Y FORM
10 mm 0 D DELIVERY TUBE
120 ml BOTTLE VACUUM BLOCK
10 mm 0 D. DELIVERY TUBE
10 mm O.D. DELIVERY TUBE
10 mm O.D. AUTOMATIC SYPHON TUBE
EFFLUENT CHAMBERS
10mm O.D. U SHAPE SYPHON TUBE
10 mm I.D CONNECTING TUBE Y FORM
10 mm 0 D. DELIVERY TUBE
MIXING CHAMBER I2OO ml CAPACITY
10 mm O.D. DELIVERY TUBE
TEST CHAMBERS CAPACITY
4- 20 LITERS
FIG. 10. VACUUM SIPHON DILUTOR SYSTEM, DETAILED DIAGRAM.
45
-------
Vacuum Siphon System Equipment List
1. Diluter Glass.
2. Stainless steel solenoid valves.
a. 2 - normally open, two-way, 55 psi, water, 1/4" pipe size,
9/32" orifice size, ASCO 8262152, for incoming effluent and
dilution water pipes.
b. 1 - normally closed, two-way, 15 psi, water, 3/8" pipe size,
3/8" orifice size, ASCO 8030B65, for dilution water evacuation
pipe.
3. Stainless steel tubing, seamless, 316-grade, austenetic.
a. 60 ft - 3/8" OD, 0.035" wall thickness, for dilution water and
effluent pipes.
b. 20 ft - 5/16" OD, 0.035" wall thickness, for standpipes in
mixing chambers.
c. 1 ft - 3/4" OD, 0.035" wall thickness, for standpipe in D-l
chamber.
4. Swagelok tube connectors, stainless steel
a. 4 - male tube connectors, male pipe size 1/4", tube OD 3/8".
b. 2 - male tube connectors, male pipe size 3/8", tube OD 3/8".
c. 2 - male adapter, tube to pipe, male pipe size 1/2", tube OD 3/8",
d. 2 - male tube connectors, male pipe size 1/2", tube OD 3/8".
5. 7 - 1,200 ml stainless steel beakers.
6. Several Ibs each of NEOPRENER stoppers, size 00, 0 and 1; 1 Ib of
size 5.
7. 14 - aquariums, 2-5 gal.
8. Magnetic stirrer.
9. 2 - PVC Ball valves, 1/2" pipe size.
10. Dilutor control panel equipment - see Fig. 14 and equipment list.
11. 7 - 120 ml NALGENER bottles.
12. 3 ft, l-in-2 aluminum bar, for siphon support brackets.
13. Stainless steel set screws, box of 50, for securing SS tubing in
siphon support brackets.
14. Stainless steel hose clamps, box of 10, size #4 or 5,(need 3 boxes).
15. 6 - NALGENER T's, 5/16" OD.
16. 12 - TYGONR Y connectors, 3/8" I.D.
17. TYGONR tubing, 3/8" OD, 10 ft.
18. Plywood sheeting, exterior grade: one - 4' x 8x x 3/4",
one - 4' x 8' x 1/2".
19. Pine or redwood board, 1" x 8", 20 ft.
20. Epoxy paint, 1 gal.
21. Assorted wood screws, nails, etc.
22. 25 ft - 5/16" ID, TEFLONR tubing, to connect the mixing chambers to
the test chambers.
46
-------
231 m m
A
iK N
D-l
D
B
(.
1
-2
D-3
B
D
4
D-5
B
D-6
D-7
1
L
D-8
609 m m
20mm HOLE CENTERED AT INDICATED DISTANCE
x 20 mm HOLE FOR VACUUM
.. Ll SIPHON SYSTEM ONLY.
- - (See Fig. 6)
oo ooooooo
33mm 70mm 165mm 248mm 297mm 350mm 411 mm 477mm 558mm
DRAIN HOLES IN BOTTOM PLATE 1C) SHOWN FOR SOLENOID VALVE DILUTOR SYSTEM. FOR VACUUM
SIPHON DILUTOR SYSTEM, SINGLE DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER D-1.
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6 mm (I/4")PLATE GLASS
A 225 mm X 95 mm - 3
B 200 mm X 95mm - 6
C 609 mm X 95mm - I (Bottom Plate)
D 609 mm X 23! mm - 2 (Side Panels)
INSIDE CELL MEASUREMENTS AND APPROXIMATE VOLUME
D-l 95 mm X 225 mm X 95 mm -2030ml
D - 2 IIS mm X 200 mm X 95 mm -2185ml
D-3 40 mm X 200mm X 95 mm - 760ml
D-4 45mm X 200 mm X 95mm- 855ml
D-5 50 mm X 200 mm X 95mm - 950 ml
D-6 60 mm X 200 mmX 95mm - 114 Oml
D-7 60mm X 200 mmX 95mm-ll40ml
D-8 90mm X 200mm X 95mm-l7IOml
NOTE: 1/8" - 316 GRADE AUSTENISTIC STAINLESS STEEL
MAY BE SUBSTITUTED FOR GLASS IN PART C
FIG. II. DILUTION WATER CHAMBERS
47
-------
180 mm
] E
E-l
. |_i
j E
E-2
U
3 E
E-3
M
i E
A
E-4
- M
i e
E-5
"
E-6
I I ...
t -y j-i H N h
20 mm HOLE
FOR VACUUM
SIPHON SYSTEM ONLY.
(See Fig. 6)
367 mm
20 mm HOLE CENTERED AT INDICATED DISTANCE
o
o o o o o
61mm
154 mm
213 mm 254mm 290mm 336mm
DRAIN HOLES IN BOTTOM PLATE (C) SHOWN FOR SOLENOID VALVE DILUTOR SYSTEM. FOR VACUUM
SIPHON DILUTOR SYSTEM, SINGLE DRAIN HOLE IS REQUIRED ONLY FOR CHAMBER D-1.
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6mm (l/4")PLATE GLASS
A I74 mm X 40 mm - Z
B I55 mm X 40 mm - 5
C 367mm X 40 mm - I (Bottom Plate)
0 367mm X 180mm- 2 (Side Panels)
INSIDE CELL MEASUREMENTS AND APPROXIMATE VOLUME
E-l NOmm X 155mm X40 mm - 682 ml
E- 2 65 mm X 155 mm X 40 mm - 403 ml
E-3 40 mm X l55mmX40mm-248ml
E;4 30mm X !55mmX 40mm- 186ml
E-5 30 mm X l55mmX40mm- 186ml
E-6 50mm XI55mmX40mm-3l8ml
NOTE: 1/8" - 316 GRADE AUSTENISTIC STAINLESS STEEL
MAY BE SUBSTITUTED FOR GLASS IN PART C.
FIG. 12. EFFLUENT CHAMBERS
48
-------
M 2
186mm
SIDE VIEW
M-l
I 90 mm
-M-3
180 m m
PART M-l
END VIEW
20 mm DIAMETER HOLE
105 mm
INDIVIDUAL PART SIZE AND NUMBER OF
PIECES USING 6mm (I/4")PLATE GLASS
M- I 180mm X 105 mm-l
M -Z 180mm X 105 mm-l
M-3 190mm X 105 mm-1 (BOTTOM PLATE)
M- 4 190mm X !86mm-2 (SIDE PANELS)
APPROXIMATE CAPACITY 3365 ml )
FIG. 13. MIXING CHAMBER
49
-------
TDR-I
ALL SOLENOIDS
FIG. 14. DILUTOR CONTROL PANEL.
-------
Dilutor Control Panel Equipment List*
Designation
Al
CTR-1
ET
F,
2
L.S.
SJ,
SJ,,
CRT Description
Encapsulated amplifier
Cycle counter
Elapsed time indicator
Input power fuse
Recepticle
Aux A.C. output jack
Main input power cord
Fill indicator light
Emptying indicator light
Level sensor (Dual Sensing Probe)
Plug
On-off main power switch (spst)
On-off aux power switch (spst)
Solenoid
Manufacturer
Cutler Hammer 1353H98C
Rodington #P2-1006
Courac #636W-AA H&T
Little fuse 342038
Amphenol 91PC4F
Stand. 3-prong AC Recpt.
Stand. 3-prong AC maleplug
Dialco 95-0408-09-241
Dialco 95-0408-09-241
Cutler Hammer 13653H2
Amphenol 91MC4M
Cutler Hammer 7580 K7
Cutler Hammer 7580 K7
(See Solenoid and Vacuum
System equipment lists)
SJ. - SJ,,
4 16
TDR-1
TDK- 2
Additional Solenoids for
Solenoid Valve System
Time delay relay
Aux time delay relay
ii it M
Dayton 5x829
Dayton 5x829
*Consult local electric supply house.
51
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/4-78-012
2.
3. RECIPIENT'S ACCESSIOI*NO.
4. TITLE AND SUBTITLE
METHODS FOR MEASURING THE ACUTE TOXICITY OF
EFFLUENTS TO AQUATIC ORGANISMS
5. REPORT DATE
January 1978 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William Peltier
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Bioassay Subcommittee
EPA Biological Advisory Committee
Environmental Research Laboratory
Athens, Georgia 30601
10. PROGRAM ELEMENT NO.
1BD612
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Monitoring & Support Lab.- Gin., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
In-House
14. SPONSORING AGENCY CODE
EPA/600/06
15. SUPPLEMENTARY NOTES
view i A*n T raw i co
Supplement to "Biological Field and Laboratory Methods for Measuring the
Quality of Surface Waters & Effluent"
16. ABSTRACT
This report describes methods for the measurement of the acute
toxicity of effluents to macroinvertebrates and fish. The methods include a
preliminary short-term (8-24 hr.)» range-finding test and a long-term (96 hr.),
or alternate static definitive test for use in determining the LC50 or EC50 of
the waste. The report includes guidelines on effluent sampling and holding,
facilties and equipment, dilution water, test species selection and handling,
and data interpretation.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Effluents
Bioassay
Toxicity
Industrial Wastes
Sewage
Water Pollution
Fishes
Invertebrates
Fresh Water Biology
Marine Biology
6C
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
62
2O. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
52
S. GOVERNMENT PRINTING OFFICE. 1378-757-1W6615 Region No. 5"
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