Short-term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Freshwater Organisms
Fourth Edition
October 2002
U.S. Environmental Protection AgencyOffice of Water (4303T)
1200 Pennsylvania Avenue, NW Washington, DC 20460
EPA-821-R-02-013
DISCLAIMER
The Engineering and Analysis Division, of the Office of Science
and Technology, has reviewed and approved this report for
publication. Neither the United States Government nor any of its
employees, contractors, or their employees make any warranty,
expressed or implied, or assumes any legal liability or
responsibility for any third party's use of or the results of such
use of any information, apparatus, product, or process discussed in
this report, or represents that its use by such party would not
infringe on privately owned rights.
CONTENTS
Page
FIGURES
SECTION 11 (Continued) Number Page
SECTION 13 (Continued) Number Page
TABLES
SECTION 11 (Continued) Number Page
SECTION 12 (Continued) Number Page
SECTION 13 (Continued) Number Page
SECTION 1
INTRODUCTION
1.1
This manual describes chronic toxicity tests for use in
the National Pollutant Discharge Elimination System (NPDES) Permits
Program to identify effluents and receiving waters containing toxic
materials in chronically toxic concentrations. The methods included
in this manual are referenced in Table IA, 40 CFR Part 136
regulations and, therefore, constitute approved methods for chronic
toxicity tests. They are also suitable for determining the toxicity
of specific compounds contained in discharges. The tests may be
conducted in a central laboratory or on-site, by the regulatory
agency or the permittee.
1.2
The data are used for NPDES permits development and to
determine compliance with permit toxicity limits. Data can also be
used to predict potential acute and chronic toxicity in the
receiving water, based on the LC50, NOEC, IC50 or IC25 (see Section
9, Chronic Toxicity Endpoints and Data Analysis) and appropriate
dilution, application, and persistence factors. The tests are
performed as a part of self-monitoring permit requirements,
compliance biomonitoring inspections, toxics sampling inspections,
and special investigations. Data from chronic toxicity tests
performed as part of permit requirements are evaluated during
compliance evaluation inspections and performance audit
inspections.
1.3
Modifications of these tests are also used in toxicity
reduction evaluations and toxicity identification evaluations to
identify the toxic components of an effluent, to aid in the
development and implementation of toxicity reduction plans, and to
compare and control the effectiveness of various treatment
technologies for a given type of industry, irrespective of the
receiving water (USEPA, 1988c; USEPA, 1989b; USEPA 1989c; USEPA,
1989d; USEPA, 1989e; USEPA, 1991a; USEPA, 1991b; and USEPA,
1992).
1.4
This methods manual serves as a companion to the acute
toxicity test methods for freshwater and marine organisms (USEPA,
2002a), the short-term chronic toxicity test methods for marine and
estuarine organisms (USEPA, 2002b), and the manual for evaluation
of laboratories performing aquatic toxicity tests (USEPA, 1991c).
In 2002, EPA revised previous editions of each of the three methods
manuals (USEPA, 1993a; USEPA, 1994a; USEPA, 1994b).
1.5
Guidance for the implementation of toxicity tests in the
NPDES program is provided in the Technical Support Document for
Water Quality-based Toxics Control (USEPA, 1991a).
1.6
These freshwater short-term toxicity tests are similar to
those developed for marine and estuarine organisms to evaluate the
toxicity of effluents discharged to marine and estuarine waters
under the NPDES permit program. Methods are presented in this
manual for three species of freshwater organisms from three
phylogenetic groups. The methods are all static renewal type
seven-day tests except the green alga, Selenastrum capricornutum,
test which lasts four days.
1.7
The three species for which test methods are provided are
the fathead minnow, Pimephales promelas; the daphnid, Ceriodaphnia
dubia; and the green alga, Selenastrum capricornutum.
1.7.1
Two of the methods incorporate the chronic endpoint of
growth in addition to lethality and one incorporates reproduction.
The fathead minnow, Pimephales promelas, embryo-larval survival and
teratogenicity test incorporates teratogenic effects in addition to
lethality. The green alga, Selenastrum capricornutum, growth test
has the advantage of a relatively short exposure period (96
h).
1.8
The validity of the freshwater chronic methods in
predicting adverse ecological impacts of toxic discharges was
demonstrated in field studies (USEPA, 1984; USEPA, 1985b; USEPA,
1985c; USEPA, 1985d; USEPA, 1986a; USEPA, 1986b; USEPA, 1986c;
USEPA, 1986d; Birge et al., 1989; and Eagleson et al.,
1990).
1.9
The use of any test species or test conditions other than
those described in the methods summary tables in this manual shall
be subject to application and approval of alternate test procedures
under 40 CFR 136.4 and 40 CFR 136.5.
1.10
These methods are restricted to use by, or under the
supervision of, analysts experienced in the use or conduct of
aquatic toxicity tests and the interpretation of data from aquatic
toxicity testing. Each analyst must demonstrate the ability to
generate acceptable test results with these methods using the
procedures described in this methods manual.
1.11
This manual was prepared in the established
EMSL-Cincinnati format (USEPA, 1983).
SECTION 2
SHORT-TERM METHODS FOR ESTIMATING CHRONIC TOXICITY
2.1 INTRODUCTION
2.1.1
The objective of aquatic toxicity tests with effluents or
pure compounds is to estimate the "safe" or "no effect"
concentration of these substances, which is defined as the
concentration which will permit normal propagation of fish and
other aquatic life in the receiving waters. The endpoints that have
been considered in tests to determine the adverse effects of
toxicants include death and survival, decreased reproduction and
growth, locomotor activity, gill ventilation rate, heart rate,
blood chemistry, histopathology, enzyme activity, olfactory
function, and terata. Since it is not feasible to detect and/or
measure all of these (and other possible) effects of toxic
substances on a routine basis, observations in toxicity tests
generally have been limited to only a few effects, such as
mortality, growth, and reproduction.
2.1.2
Acute lethality is an obvious and easily observed effect
which accounts for its wide use in the early period of evaluation
of the toxicity of pure compounds and complex effluents. The
results of these tests were usually expressed as the concentration
lethal to 50% of the test organisms (LC50) over relatively short
exposure periods (one-to-four days).
2.1.3
As exposure periods of acute tests were lengthened, the
LC50 and lethal threshold concentration were observed to decline
for many compounds. By lengthening the tests to include one or more
complete life cycles and observing the more subtle effects of the
toxicants, such as a reduction in growth and reproduction, more
accurate, direct, estimates of the threshold or safe concentration
of the toxicant could be obtained. However, laboratory life-cycle
tests may not accurately estimate the "safe" concentration of
toxicants because they are conducted with a limited number of
species under highly controlled, steady-state conditions, and the
results do not include the effects of the stresses to which the
organisms would ordinarily be exposed in the natural
environment.
2.1.4
An early published account of a full life-cycle, fish
toxicity test was that of Mount and Stephan (1967). In this study,
fathead minnows, Pimephales promelas, were exposed to a graded
series of pesticide concentrations throughout their life cycle, and
the effects of the toxicant on survival, growth, and reproduction
were measured and evaluated. This work was soon followed by full
life-cycle tests using other toxicants and fish species.
2.1.5
McKim (1977) evaluated the data from 56 full life-cycle
tests, 32 of which used the fathead minnow, Pimephales promelas,
and concluded that the embryo-larval and early juvenile life-stages
were the most sensitive stages. He proposed the use of partial
life-cycle toxicity tests with the early life-stages (ELS) of fish
to establish water quality criteria.
2.1.6
Macek and Sleight (1977) found that exposure of critical
life-stages of fish to toxicants provides estimates of chronically
safe concentrations remarkably similar to those derived from full
life-cycle toxicity tests. They reported that "for a great majority
of toxicants, the concentration which will not be acutely toxic to
the most sensitive life stages is the chronically safe
concentration for fish, and that the most sensitive life stages are
the embryos and fry". Critical life-stage exposure was considered
to be exposure of the embryos during most, preferably all, of the
embryogenic (incubation) period, and exposure of the fry for 30
days post-hatch for warm water fish with embryogenic periods
ranging from one-to-fourteen days, and for 60 days post-hatch for
fish with longer embryogenic periods. They concluded that in the
majority of cases, the maximum acceptable toxicant concentration
(MATC) could be estimated from the results of exposure of the
embryos during incubation, and the larvae for 30 days
post-hatch.
2.1.7
Because of the high cost of full life-cycle fish toxicity
tests and the emerging consensus that the ELS test data usually
would be adequate for estimating chronically safe concentrations,
there was a rapid shift by aquatic toxicologists to 30 - 90-day ELS
toxicity tests for estimating chronically safe concentrations in
the late 1970s. In
1980, USEPA adopted the policy that ELS test data could be used
in establishing water quality criteria if data from full life-cycle
tests were not available (USEPA, 1980a).
2.1.8
Published reports of the results of ELS tests indicate
that the relative sensitivity of growth and survival as endpoints
may be species dependent, toxicant dependent, or both. Ward and
Parrish (1980) examined the literature on ELS tests that used
embryos and juveniles of the sheepshead minnow, Cyprinodon
variegatus, and found that growth was not a statistically sensitive
indicator of toxicity in 16 of 18 tests. They suggested that the
ELS tests be shortened to 14 days posthatch and that growth be
eliminated as an indicator of toxic effects.
2.1.9
In a review of the literature on 173 fish full life-cycle
and ELS tests performed to determine the chronically safe
concentrations of a wide variety of toxicants, such as metals,
pesticides, organics, inorganics, detergents, and complex
effluents, Woltering (1984) found that at the lowest effect
concentration, significant reductions were observed in fry survival
in 57%, fry growth in 36%, and egg hatchability in 19% of the
tests. He also found that fry survival and growth were very often
equally sensitive, and concluded that the growth response could be
deleted from routine application of the ELS tests. The net result
would be a significant reduction in the duration and cost of
screening tests with no appreciable impact on estimating MATCs for
chemical hazard assessments. Benoit et al. (1982), however, found
larval growth to be the most significant measure of effect, and
survival to be equally or less sensitive than growth in early
life-stage tests with four organic chemicals.
2.1.10
Efforts to further reduce the length of partial
life-cycle toxicity tests for fish without compromising their
predictive value have resulted in the development of an eight-day,
embryo-larval survival and teratogenicity test for fish and other
aquatic vertebrates (USEPA, 1981; Birge et al., 1985), and a
seven-day larval survival and growth test (Norberg and Mount,
1985).
2.1.11
The similarity of estimates of chronically safe
concentrations of toxicants derived from short-term, embryo-larval
survival and teratogenicity tests to those derived from full
life-cycle tests has been demonstrated by Birge et al. (1981),
Birge and Cassidy (1983), and Birge et al. (1985).
2.1.12
Use of a seven-day, fathead minnow, Pimephales promelas,
larval survival and growth test was first proposed by Norberg and
Mount at the 1983 annual meeting of the Society for Environmental
Toxicology and Chemistry (Norberg and Mount, 1983). This test was
subsequently used by Mount and associates in field demonstrations
at Lima, OH (USEPA, 1984), and at many other locations. Growth was
frequently found to be more sensitive than survival in determining
the effects of complex effluents.
2.1.13
Norberg and Mount (1985) performed three single toxicant
fathead minnow larval growth tests with zinc, copper, and DURSBAN®,
using dilution water from Lake Superior. The results were
comparable to, and had confidence intervals that overlapped with,
chronic values reported in the literature for both ELS and full
life-cycle tests.
2.1.14
Mount and Norberg (1984) developed a seven-day cladoceran
partial life-cycle test and experimented with a number of diets for
use in culturing and testing the daphnid, Ceriodaphnia reticulata
(Norberg and Mount, 1985). As different laboratories began to use
this cladoceran test, it was discovered that apparently more than
one species was involved in the tests conducted by the same
laboratory. Berner (1986) studied the problem and determined that
perhaps as many as three variant forms were involved and it was
decided to recommend the use of the more common Ceriodaphnia dubia
rather than the originally reported Ceriodaphnia reticulata. The
method was adopted for use in the first edition of the freshwater
short-term chronic methods (USEPA, 1985e).
2.1.15
The green alga, Selenastrum capricornutum, bottle test
was developed, after extensive design, evaluation, and application,
for the National Eutrophication Research Program (USEPA, 1971). The
test was later modified for use in the assessment of receiving
waters and the effects of wastes originating from industrial,
municipal, and agricultural point and non-point sources (USEPA,
1978a).
2.1.16
The use of short-term toxicity tests including subchronic
and chronic tests in the NPDES Program is especially attractive
because they provide a more direct estimate of the safe
concentrations of effluents in receiving waters than was provided
by acute toxicity tests, at an only slightly increased level of
effort, compared to the fish full life-cycle chronic and 28-day ELS
tests and the 21-day daphnid, Daphnia magna, life-cycle
test.
2.2
TYPES OF TESTS
2.2.1
The selection of the test type will depend on the NPDES
permit requirements, the objectives of the test, the available
resources, the requirements of the test organisms, and effluent
characteristics such as fluctuations in effluent
toxicity.
2.2.2
Effluent chronic toxicity is generally measured using a
multi-concentration, or definitive test, consisting of a control
and a minimum of five effluent concentrations. The tests are
designed to provide dose-response information, expressed as the
percent effluent concentration that affects the hatchability, gross
morphological abnormalities, survival, growth, and/or reproduction
within the prescribed period of time (four to seven days). The
results of the tests are expressed in terms of the highest
concentration that has no statistically significant observed effect
on those responses when compared to the controls or the estimated
concentration that causes a specified percent reduction in
responses versus the controls.
2.2.3
Use of pass/fail tests consisting of a single effluent
concentration (e.g., the receiving water concentration or RWC) and
a control is not recommended. If the NPDES permit has a whole
effluent toxicity limit for acute toxicity at the RWC, it is
prudent to use that permit limit as the midpoint of a series of
five effluent concentrations. This will ensure that there is
sufficient information on the dose-response relationship. For
example, the effluent concentrations utilized in a test may be: (1)
100% effluent, (2) (RWC + 100)/2, (3) RWC, (4) RWC/2, and (5)
RWC/4. More specifically, if the RWC = 50%, appropriate effluent
concentrations may be 100%, 75%, 50%, 25%, and 12.5%.
2.2.4
Receiving (ambient) water toxicity tests commonly employ
two treatments, a control and the undiluted receiving water, but
may also consist of a series of receiving water
dilutions.
2.2.5
A negative result from a chronic toxicity test does not
preclude the presence of toxicity. Also, because of the potential
temporal variability in the toxicity of effluents, a negative test
result with a particular sample does not preclude the possibility
that samples collected at some other time might exhibit chronic
toxicity.
2.2.6
The frequency with which chronic toxicity tests are
conducted under a given NPDES permit is determined by the
regulatory agency on the basis of factors such as the variability
and degree of toxicity of the waste, production schedules, and
process changes.
2.2.7
Tests recommended for use in this methods manual may be
static non-renewal or static renewal. Individual methods specify
which static type of test is to be conducted.
2.3
STATIC TESTS
2.3.1
Static non-renewal tests - The test organisms are exposed
to the same test solution for the duration of the test.
2.3.2
Static-renewal tests - The test organisms are exposed to
a fresh solution of the same concentration of sample every 24 h or
other prescribed interval, either by transferring the test
organisms from one test chamber to another, or by replacing all or
a portion of solution in the test chambers.
2.4
ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST
TYPES
2.4.1 STATIC NON-RENEWAL, SHORT-TERM TOXICITY TESTS:
Advantages:
1.
Simple and inexpensive.
2.
Very cost effective in determining compliance with permit
conditions.
3.
Limited resources (space, manpower, equipment) required;
would permit staff to perform many more tests in the same amount of
time.
4.
Smaller volume of effluent required than for static
renewal or flow-through tests. Disadvantages:
1.
Dissolved oxygen (DO) depletion may result from high
chemical oxygen demand (COD), biological oxygen demand (BOD), or
metabolic wastes.
2.
Possible loss of toxicants through volatilization and/or
adsorption to the exposure vessels.
3.
Generally less sensitive than static renewal, because the
toxic substances may degrade or be adsorbed, thereby reducing the
apparent toxicity. Also, there is less chance of detecting slugs of
toxic wastes, or other temporal variations in waste
properties.
2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS: Advantages:
1.
Reduced possibility of DO depletion from high COD and/or
BOD, or ill effects from metabolic wastes from organisms in the
test solutions.
2.
Reduced possibility of loss of toxicants through
volatilization and/or adsorption to the exposure
vessels.
3.
Test organisms that rapidly deplete energy reserves are
fed when the test solutions are renewed, and are maintained in a
healthier state.
Disadvantages:
1.
Require greater volume of effluent than non-renewal
tests.
2.
Generally less chance of temporal variations in waste
properties.
SECTION 3 HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1
Each laboratory should develop and maintain an effective
health and safety program, requiring an ongoing commitment by the
laboratory management. This program should include (1) a safety
officer with the responsibility and authority to develop and
maintain a safety program, (2) the preparation of a formal,
written, health and safety plan, which is provided to each of the
laboratory staff, (3) an ongoing training program on laboratory
safety, and (4) regularly scheduled, documented, safety
inspections.
3.1.2
Collection and use of effluents in toxicity tests may
involve significant risks to personal safety and health. Personnel
collecting effluent samples and conducting toxicity tests should
take all safety precautions necessary for the prevention of bodily
injury and illness which might result from ingestion or invasion of
infectious agents, inhalation or absorption of corrosive or toxic
substances through skin contact, and asphyxiation due to lack of
oxygen or presence of noxious gases.
3.1.3
Prior to sample collection and laboratory work, personnel
will determine that all necessary safety equipment and materials
have been obtained and are in good condition.
3.1.4
Guidelines for the handling and disposal of hazardous
materials must be strictly followed.
3.2
SAFETY EQUIPMENT
3.2.1 PERSONAL SAFETY GEAR
3.2.1.1
Personnel should use safety equipment, as required, such
as rubber aprons, laboratory coats, respirators, gloves, safety
glasses, hard hats, and safety shoes. Plastic netting on glass
beakers, flasks, and other glassware minimizes breakage and
subsequent shattering of the glass.
3.2.2
LABORATORY SAFETY EQUIPMENT
3.2.2.1
Each laboratory (including mobile
laboratories) should be provided with safety equipment such as
first aid kits, fire extinguishers, fire blankets, emergency
showers, chemical spill clean up kits, and eye
fountains.
3.2.2.2
Mobile laboratories should be equipped with a telephone
or other means to enable personnel to summon help in case of
emergency.
3.3
GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1
Work with effluents should be performed in compliance
with accepted rules pertaining to the handling of hazardous
materials (see safety manuals listed in Section 3, Health and
Safety, Subsection 3.5). It is recommended that personnel
collecting samples and performing toxicity tests not work
alone.
3.3.2
Because the chemical composition of effluents is usually
only poorly known, they should be considered as potential health
hazards, and exposure to them should be minimized. Fume and canopy
hoods over the toxicity test areas must be used whenever
possible.
3.3.3
It is advisable to cleanse exposed parts of the body
immediately after collecting effluent samples.
3.3.4
All containers are to be adequately labeled to indicate
their contents.
3.3.5
Staff should be familiar with safety guidelines on
Material Safety Data Sheets for reagents and other chemicals
purchased from suppliers. Incompatible materials should not be
stored together. Good housekeeping contributes to safety and
reliable results.
3.3.6
Strong acids and volatile organic solvents employed in
glassware cleaning must be used in a fume hood or under an exhaust
canopy over the work area.
3.3.7
Electrical equipment or extension cords not bearing the
approval of Underwriter Laboratories must not be used. Ground-fault
interrupters must be installed in all "wet" laboratories where
electrical equipment is used.
3.3.8
Mobile laboratories should be properly grounded to
protect against electrical shock.
3.4
DISEASE PREVENTION
3.4.1
Personnel handling samples which are known or suspected
to contain human wastes should be immunized against tetanus,
typhoid fever, polio, and hepatitis B.
3.5
SAFETY MANUALS
3.5.1
For further guidance on safe practices when collecting
effluent samples and conducting toxicity tests, check with the
permittee and consult general safety manuals, including USEPA
(1986e) and Walters and Jameson (1984).
3.6
WASTE DISPOSAL
3.6.1 Wastes generated during toxicity testing must be properly
handled and disposed of in an appropriate manner. Each testing
facility will have its own waste disposal requirements based on
local, state, and Federal rules and regulations. It is extremely
important that these rules and regulations be known, understood,
and complied with by all persons responsible for, or otherwise
involved in performing the toxicity testing activities. Local fire
officials should be notified of any potentially hazardous
conditions.
SECTION 4
QUALITY ASSURANCE
4.1 INTRODUCTION
4.1.1
Development and maintenance of a toxicity test laboratory
quality assurance (QA) program (USEPA, 1991a) requires an ongoing
commitment by laboratory management. Each toxicity test laboratory
should (1) appoint a quality assurance officer with the
responsibility and authority to develop and maintain a QA program;
(2) prepare a quality assurance plan with stated data quality
objectives (DQOs); (3) prepare a written description of laboratory
standard operating procedures (SOPs) for culturing, toxicity
testing, instrument calibration, sample chain-of-custody
procedures, laboratory sample tracking system, glassware cleaning,
etc.; and (4) provide an adequate, qualified technical staff for
culturing and testing the organisms, and suitable space and
equipment to assure reliable data.
4.1.2
QA practices for toxicity testing laboratories must
address all activities that affect the quality of the final
effluent toxicity test data, such as: (1) effluent sampling and
handling; (2) the source and condition of the test organisms; (3)
condition of equipment; (4) test conditions; (5) instrument
calibration; (6) replication; (7) use of reference toxicants; (8)
record keeping; and (9) data evaluation.
4.1.3
Quality control practices, on the other hand, consist of
the more focused, routine, day-to-day activities carried out within
the scope of the overall QA program. For more detailed discussion
of quality assurance and general guidance on good laboratory
practices and laboratory evaluation related to toxicity testing,
see FDA, (1978); USEPA, (1979d), USEPA (1980b), USEPA (1980c), and
USEPA (1991c); DeWoskin (1984); and Taylor (1987).
4.1.4
Guidance for the evaluation of laboratories performing
toxicity tests and laboratory evaluation criteria may be found in
USEPA (1991c).
4.2
FACILITIES, EQUIPMENT, AND TEST CHAMBERS
4.2.1
Separate test organism culturing and toxicity testing
areas should be provided to avoid possible loss of cultures due to
cross-contamination. Ventilation systems should be designed and
operated to prevent recirculation or leakage of air from chemical
analysis laboratories or sample storage and preparation areas into
organism culturing or testing areas, and from testing and sample
preparation areas into culture rooms.
4.2.2
Laboratory and toxicity test
temperature control equipment must be adequate to maintain
recommended test water temperatures. Recommended materials must be
used in the fabrication of the test equipment which comes in
contact with the effluent (see Section 5, Facilities, Equipment and
Supplies; and specific toxicity test method).
4.3
TEST ORGANISMS
4.3.1
The test organisms used in the procedures described in
this manual are the fathead minnow, Pimephales promelas, the
daphnid, Ceriodaphnia dubia, and the green alga, Selenastrum
capricornutum. The fish and invertebrates should appear healthy,
behave normally, feed well, and have low mortality in the cultures,
during holding, and in test controls. Test organisms should be
positively identified to species (see Section 6, Test
Organisms).
4.4
LABORATORY WATER USED FOR CULTURING AND TEST DILUTION
WATER
4.4.1 The quality of water used for test organism culturing and
for dilution water used in toxicity tests is extremely important.
Water for these two uses should come from the same source. The
dilution water used in effluent toxicity tests will depend in part
on the objectives of the study and logistical constraints, as
discussed in detail in Section 7, Dilution Water. For tests
performed to meet NPDES objectives, synthetic, moderately hard
water should be used.
The dilution water used for internal quality assurance tests
with organisms, food, and reference toxicants should be the water
routinely used with success in the laboratory. Types of water are
discussed in Section 5, Facilities, Equipment and Supplies. Water
used for culturing and test dilution should be analyzed for toxic
metals and organics at least annually or whenever difficulty is
encountered in meeting minimum acceptability criteria for control
survival and reproduction or growth. The concentration of the
metals Al, As, Cr, Co, Cu, Fe, Pb, Ni, and Zn, expressed as total
metal, should not exceed 1 mg/L each, and Cd, Hg, and Ag, expressed
as total metal, should not exceed 100 ng/L each. Total
organochlorine pesticides plus PCBs should be less than 50 ng/L
(APHA, 1992). Pesticide concentrations should not exceed USEPA's
Ambient Water Quality chronic criteria values where available.
4.5 EFFLUENT AND RECEIVING WATER SAMPLING
AND HANDLING
4.5.1
Sample holding times and temperatures of effluent samples
collected for on-site and off-site testing must conform to
conditions described in Section 8, Effluent and Receiving Water
Sampling, Sample Handling, and Sample Preparation for Toxicity
Tests.
4.6
TEST CONDITIONS
4.6.1
Water temperature should be maintained within the limits
specified for each test. The temperature of test solutions must be
measured by placing the thermometer or probe directly into the test
solutions, or by placing the thermometer in equivalent volumes of
water in surrogate vessels positioned at appropriate locations
among the test vessels. Temperature should be recorded continuously
in at least one test vessel for the duration of each test. Test
solution temperatures should be maintained within the limits
specified for each test. DO concentration and pH should be checked
at the beginning of each test and daily throughout the test
period.
4.7
QUALITY OF TEST ORGANISMS
4.7.1
The health of test organisms is primarily assessed by the
performance (survival, growth, and/or reproduction) of organisms in
control treatments of individual tests. The health and sensitivity
of test organisms is also assessed by reference toxicant testing.
In addition to documenting the sensitivity and health of test
organisms, reference toxicant testing is used to initially
demonstrate acceptable laboratory performance (Subsection 4.15) and
to document ongoing laboratory performance (Subsection
4.16).
4.7.2
Regardless of the source of test organisms (in-house
cultures or purchased from external suppliers), the testing
laboratory must perform at least one acceptable reference toxicant
test per month for each toxicity test method conducted in that
month (Subsection 4.16). If a test method is conducted only
monthly, or less frequently, a reference toxicant test must be
performed concurrently with each effluent toxicity test.
4.7.3
When acute or short-term chronic toxicity tests are
performed with effluents or receiving waters using test organisms
obtained from outside the test laboratory, concurrent toxicity
tests of the same type must be performed with a reference toxicant,
unless the test organism supplier provides control chart data from
at least the last five monthly short-term chronic toxicity tests
using the same reference toxicant and control conditions (see
Section 6, Test Organisms).
4.7.4
The supplier should certify the species identification of
the test organisms, and provide the taxonomic reference (citation
and page) or name(s) of the taxonomic expert(s)
consulted.
4.7.5
If routine reference toxicant tests fail to meet test
acceptability criteria, then the reference toxicant test must be
immediately repeated.
4.8
FOOD QUALITY
4.8.1
The nutritional quality of the food used in culturing and
testing fish and invertebrates is an important factor in the
quality of the toxicity test data. This is especially true for the
unsaturated fatty acid content of brine shrimp nauplii, Artemia.
Problems with the nutritional suitability of the food will be
reflected in the survival, growth, and reproduction of the test
organisms in cultures and toxicity tests. Artemia cysts, and other
foods must be obtained as described in Section 5, Facilities,
Equipment, and Supplies.
4.8.2
Problems with the nutritional suitability of food will be
reflected in the survival, growth, and reproduction of the test
organisms in cultures and toxicity tests. If a batch of food is
suspected to be defective, the performance of organisms fed with
the new food can be compared with the performance of organisms fed
with a food of known quality in side-by-side tests. If the food is
used for culturing, its suitability should be determined using a
short-term chronic test which will determine the affect of food
quality on growth or reproduction of each of the relevant test
species in culture, using four replicates with each food source.
Where applicable, foods used only in chronic toxicity tests can be
compared with a food of known quality in side-by-side,
multi-concentration chronic tests, using the reference toxicant
regularly employed in the laboratory QA program.
4.8.3
New batches of food used in
culturing and testing should be analyzed for toxic organics and
metals or whenever difficulty is encountered in meeting minimum
acceptability criteria for control survival and reproduction or
growth. If the concentration of total organochlorine pesticides
exceeds 0.15 mg/g wet weight, or the concentration of total
organochlorine pesticides plus PCBs exceeds 0.30 µg/g wet weight,
or toxic metals (Al, As, Cr, Cd, Cu, Pb, Ni, Zn, expressed as total
metal) exceed 20 µg/g wet weight, the food should not be used (for
analytical methods see AOAC, 1990 and USDA, 1989). For foods (e.g.,
such as YCT) which are used to culture and test organisms, the
quality of the food should meet the requirements for the laboratory
water used for culturing and test dilution water as described in
Section 4.4 above.
4.9
ACCEPTABILITY OF SHORT-TERM CHRONIC TOXICITY
TESTS
4.9.1
For the tests to be acceptable, control survival in
fathead minnow, Pimephales promelas, and the daphnid, Ceriodaphnia
dubia, tests must be 80% or greater. At the end of the test, the
average dry weight of surviving seven-day-old fathead minnows in
control chambers must equal or exceed 0.25 mg. In Ceriodaphnia
dubia controls, 60% or more of the surviving females must have
produced their third brood in 7 ± 1 days, and the number of young
per surviving female must be 15 or greater. In algal toxicity
tests, the mean cell density in the controls after 96 h must equal
or exceed 1 x 106 cells/mL and not vary more than 20% among
replicates. If these criteria are not met, the test must be
repeated.
4.9.2
An individual test may be conditionally acceptable if
temperature, DO, and other specified conditions fall outside
specifications, depending on the degree of the departure and the
objectives of the tests (see test condition summaries). The
acceptability of the test would depend on the experience and
professional judgment of the laboratory investigator and the
reviewing staff of the regulatory authority. Any deviation from
test specifications must be noted when reporting data from the
test.
4.10 ANALYTICAL METHODS
4.10.1
Routine chemical and physical analyses for culture and
dilution water, food, and test solutions must include established
quality assurance practices outlined in USEPA methods manuals
(USEPA, 1979a and USEPA, 1979b).
4.10.2
Reagent containers should be dated and catalogued when
received from the supplier, and the shelf life should not be
exceeded. Also, working solutions should be dated when prepared,
and the recommended shelf life should be observed.
4.11
CALIBRATION AND STANDARDIZATION
4.11.1
Instruments used for routine measurements of chemical and
physical parameters such as pH, DO, temperature, and conductivity,
must be calibrated and standardized according to instrument
manufacturer's procedures as indicated in the general section on
quality assurance (see USEPA Methods 150.1, 360.1, 170.1, and 120.1
in USEPA, 1979b). Calibration data are recorded in a permanent log
book.
4.11.2
Wet chemical methods used to measure hardness, alkalinity
and total residual chlorine must be standardized prior to use each
day according to the procedures for those specific USEPA methods
(see USEPA Methods 130.2 and 310.1 in USEPA, 1979b).
4.12
REPLICATION AND TEST SENSITIVITY
4.12.1
The sensitivity of the tests will depend in part on the
number of replicates per concentration, the significance level
selected, and the type of statistical analysis. If the variability
remains constant, the sensitivity of the test will increase as the
number of replicates is increased. The minimum recommended number
of replicates varies with the objectives of the test and the
statistical method used for analysis of the data.
4.13
VARIABILITY IN TOXICITY TEST RESULTS
4.13.1
Factors which can affect test success and precision
include (1) the experience and skill of the laboratory analyst; (2)
test organism age, condition, and sensitivity; (3) dilution water
quality; (4) temperature control; and (5) the quality and quantity
of food provided. The results will depend upon the species used and
the strain or source of the test organisms, and test conditions,
such as temperature, DO, food, and water quality. The repeatability
or precision of toxicity tests is also a function of the number of
test organisms used at each toxicant concentration. Jensen (1972)
discussed the relationship between sample size (number of fish) and
the standard error of the test, and considered 20 fish per
concentration as optimum for Probit Analysis.
4.14
TEST PRECISION
4.14.1
The ability of the laboratory personnel to obtain
consistent, precise results must be demonstrated with reference
toxicants before they attempt to measure effluent toxicity. The
single-laboratory precision of each type of test to be used in a
laboratory should be determined by performing at least five tests
with a reference toxicant.
4.14.2
Test precision can be estimated by using the same strain
of organisms under the same test conditions and employing a known
toxicant, such as a reference toxicant.
4.14.3
Interlaboratory precision data from a 1991 study of
chronic toxicity tests with two species using the reference
toxicants potassium chloride and copper sulfate are shown in Table
1. Table 2 shows interlaboratory precision data from a study of
three chronic toxicity test methods using effluent, receiving
water, and reference toxicant sample types (USEPA, 2001a; USEPA,
2001b). The effluent sample was a municipal wastewater spiked with
KCl, the receiving waster sample was a river water spiked with KCl,
and the reference toxicant sample consisted of moderately-hard
synthetic freshwater spiked with KCl. Additional precision data for
each of the tests described in this manual are presented in the
sections describing the individual test methods.
TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 1991: SUMMARY OF RESPONSES USING A REFERENCE
TOXICANT1
Organism Endpoint No. Labs % Effluent2 SD CV(%)
Pimephales Survival, NOEC 146
promelas Growth, IC25 124 Growth, IC50 117 Growth, NOEC 142
NA NA NA
4.67 1.87 40.0
6.36 2.04 32.1 NA NA NA
Ceriodaphnia Survival, NOEC
dubia Reproduction, IC25 Reproduction, IC50 Reproduction, NOEC
162NA NA NA 155 2.69 1.96 72.9 150 3.99 2.35 58.9156NA NA NA
1
From a national study of interlaboratory precision of toxicity
test data performed in 1991 by the Environmental Monitoring Systems
Laboratory-Cincinnati, U.S. Environmental Protection Agency,
Cincinnati, OH 45268. Participants included Federal, state, and
private laboratories engaged in NPDES permit compliance
monitoring.
2
Expressed as % effluent; in reality it was a reference toxicant
(KCl) but was not known by the persons conducting the tests.
TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING
WATER, AND REFERENCE TOXICANT SAMPLE TYPES1.
Organism Endpoint Number of Tests2 CV (%)3
Pimephales promelas Growth, IC25 73 20.9
Ceriodaphnia dubia Reproduction, IC25 34 35.0
Selenastrum capricornutum
(with EDTA) Growth, IC25 21 34.3 Growth, IC50 22 32.2
Selenastrum capricornutum (without EDTA) Growth, IC25 21
58.5
Growth, IC50 22 58.5
1
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a;
USEPA, 2001b).
2
Represents the number of valid tests (i.e., those that met test
acceptability criteria) that were used in the analysis of
precision. Invalid tests were not used.
3
CVs based on total interlaboratory variability (including both
within-laboratory and between-laboratory components of variability)
and averaged across sample types. IC25s or IC50s were pooled for
all laboratories to calculate the CV for each sample type. The
resulting CVs were then averaged across sample types.
4.14.4
Additional information on toxicity test precision is
provided in the Technical Support Document for Water Quality-based
Control (see pp. 2-4, and 11-15 in USEPA, 1991a).
4.14.5
In cases where the test data are used in Probit Analysis
or other point estimation techniques (see Section 9, Chronic
Toxicity Test Endpoints and Data Analysis), precision can be
described by the mean, standard deviation, and relative standard
deviation (percent coefficient of variation, or CV) of the
calculated endpoints from the replicated tests. In cases where the
test data are used in the Linear Interpolation Method, precision
can be estimated by empirical confidence intervals derived by using
the ICPIN Method (see Section 9, Chronic Toxicity Test Endpoints
and Data Analysis). However, in cases where the results are
reported in terms of the No-Observed-Effect Concentration (NOEC)
and Lowest-Observed-Effect Concentration (LOEC) (see Section 9,
Chronic Toxicity Test Endpoints and Data Analysis) precision can
only be described by listing the NOEC-LOEC interval for each test.
It is not possible to express precision in terms of a commonly used
statistic. However, when all tests of the same toxicant yield the
same NOEC-LOEC interval, maximum precision has been attained. The
"true" no effect concentration could fall anywhere within the
interval, NOEC ± (NOEC minus LOEC).
4.14.6
It should be noted here that the dilution factor selected
for a test determines the width of the NOEC-LOEC interval and the
inherent maximum precision of the test. As the absolute value of
the dilution factor decreases, the width of the NOEC-LOEC interval
increases, and the inherent maximum precision of the test
decreases. When a dilution factor of 0.3 is used, the NOEC could be
considered to have a relative variability as high as ± 300%. With a
dilution factor of 0.5, the NOEC could be considered to have a
relative variability of ± 100%. As a result of the variability of
different dilution factors, USEPA recommends the use of the
dilution factor of 0.5 or greater. Other factors which can affect
test precision include: test organism age, condition, and
sensitivity; temperature
control; and feeding.
4.15 DEMONSTRATING ACCEPTABLE LABORATORY
PERFORMANCE
4.15.1
It is a laboratory's responsibility to demonstrate its
ability to obtain consistent, precise results with reference
toxicants before it performs toxicity tests with effluents for
permit compliance purposes. To meet this requirement, the
intralaboratory precision, expressed as percent coefficient of
variation (CV%), of each type of test to be used in the laboratory
should be determined by performing five or more tests with
different batches of test organisms, using the same reference
toxicant, at the same concentrations, with the same test conditions
(i.e., the same test duration, type of dilution water, age of test
organisms, feeding, etc.), and the same data analysis methods. A
reference toxicant concentration series (0.5 or higher) should be
selected that will consistently provide partial mortalities at two
or more concentrations.
4.16
DOCUMENTING ONGOING LABORATORY PERFORMANCE
4.16.1
Satisfactory laboratory performance is demonstrated by
performing at least one acceptable test per month with a reference
toxicant for each toxicity test method conducted in the laboratory
during that month. For a given test method, successive tests must
be performed with the same reference toxicant, at the same
concentrations, in the same dilution water, using the same data
analysis methods. Precision may vary with the test species,
reference toxicant, and type of test. Each laboratory's reference
toxicity data will reflect conditions unique to that facility,
including dilution water, culturing, and other variables; however,
each laboratory's reference toxicity results should reflect good
repeatability.
4.16.2
A control chart should be prepared for each combination
of reference toxicant, test species, test conditions, and
endpoints. Toxicity endpoints from five or six tests are adequate
for establishing the control charts. Successive toxicity endpoints
(NOECs, IC25s, LC50s, etc.) should be plotted and examined to
determine if the results (X1) are within prescribed limits (Figure
1). The chart should plot logarithm of concentration on the
vertical axis against the date of the test or test number on the
horizontal axis. The types of control charts illustrated (see
USEPA, 1979a) are used to evaluate the cumulative trend of results
from a series of samples, thus reference toxicant test results
should not be used as a de facto criterion for rejection of
individual effluent or receiving water tests. For endpoints that
are
¯
point estimates (LC50s and IC25s), the cumulative mean (X) and
upper and lower control limits (± 2S) are recalculated with each
successive test result. Endpoints from hypothesis tests (NOEC,
NOAEC) from each test are plotted directly on the control chart.
The control limits would consist of one concentration interval
above and below the concentration representing the central
tendency. After two years of data collection, or a minimum of 20
data points, the control chart should be maintained using only the
20 most recent data points.
4.16.3
Laboratories should compare the calculated CV (i.e.,
standard deviation / mean) of the IC25 for the 20 most recent data
points to the distribution of laboratory CVs reported nationally
for reference toxicant testing (Table 3-2 in USEPA, 2000b). If the
calculated CV exceeds the 75th percentile of CVs reported
nationally, the laboratory should use the 75th and 90th percentiles
to calculate warning and control limits, respectively, and the
laboratory should investigate options for reducing variability.
Note: Because NOECs can only be a fixed number of discrete values,
the mean, standard deviation, and CV cannot be interpreted and
applied in the same way that these descriptive statistics are
interpreted and applied for continuous variables such as the IC25
or LC50.
4.16.4
The outliers, which are values falling outside the upper
and lower control limits, and trends of increasing or decreasing
sensitivity, are readily identified. In the case of endpoints that
are point estimates (LC50s and IC25s), at the P0.05 probability
level, one in 20 tests would be expected to fall outside of the
control limits by chance alone. If more than one out of 20
reference toxicant tests fall outside the control limits, the
laboratory should investigate sources of variability, take
corrective actions to reduce identified sources of variability, and
perform an additional reference toxicant test during the same
month. Control limits for the NOECs will also be exceeded
occasionally, regardless of how well a laboratory performs. In
those instances when the laboratory can document the cause for the
outlier (e.g., operator error, culture health or test system
failure), the outlier should be excluded from the future
calculations of the control limits. If two or more consecutive
tests do not fall within the control limits, the results
must be explained and the reference toxicant test must be
immediately repeated. Actions taken to correct the problem must be
reported.
4.16.5
If the toxicity value from a given test with a reference
toxicant falls well outside the expected range for the other test
organisms when using the standard dilution water and other test
conditions, the laboratory should investigate sources of
variability, take corrective actions to reduce identified sources
of variability, and perform an additional reference toxicant test
during the same month. Performance should improve with experience,
and the control limits for endpoints that are point estimates
should gradually narrow. However, control limits of ± 2S will be
exceeded 5% of the time by chance alone, regardless of how well a
laboratory performs. Highly proficient laboratories which develop
very narrow control limits may be unfairly penalized if a test
result which falls just outside the control limits is rejected de
facto. For this reason, the width of the control limits should be
considered in determining whether or not a reference toxicant test
result falls "well" outside the expected range. The width of the
control limits may be evaluated by comparing the calculated CV
(i.e., standard deviation / mean) of the IC25 for the 20 most
recent data points to the distribution of laboratory CVs reported
nationally for reference toxicant testing (Table 3-2 in USEPA,
2000b). In determining whether or not a reference toxicant test
result falls "well" outside the expected range, the result also may
be compared with upper and lower bounds for ±3S, as any result
outside these control limits would be expected to occur by chance
only 1 out of 100 tests (Environment Canada, 1990). When a result
from a reference toxicant test is outside the 99% confidence
intervals, the laboratory must conduct an immediate investigation
to assess the possible causes for the outlier.
4.16.6
Reference toxicant test results should not be used as a
de facto criterion for rejection of individual effluent or
receiving water tests. Reference toxicant testing is used for
evaluating the health and sensitivity of organisms over time and
for documenting initial and ongoing laboratory performance. While
reference toxicant test results should not be used as a de facto
criterion for test rejection, effluent and receiving water test
results should be reviewed and interpreted in the light of
reference toxicant test results. The reviewer should consider the
degree to which the reference toxicant test result fell outside of
control chart limits, the width of the limits, the direction of the
deviation (toward increased test organism sensitivity or toward
decreased test organism sensitivity), the test conditions of both
the effluent test and the reference toxicant test, and the
objective of the test.
4.17
REFERENCE TOXICANTS
4.17.1
Reference toxicants such as sodium chloride (NaCl),
potassium chloride (KCl), cadmium chloride (CdCl2), copper sulfate
(CuSO4), sodium dodecyl sulfate (SDS), and potassium dichromate
(K2Cr2O7), are suitable for use in the NPDES Program and other
Agency programs requiring aquatic toxicity tests. EMSL-Cincinnati
hopes to release USEPA-certified solutions of cadmium and copper
for use as reference toxicants through cooperative research and
development agreements with commercial suppliers, and will continue
to develop additional reference toxicants for future release.
Standard reference materials can be obtained from commercial supply
houses, or can be prepared inhouse using reagent grade chemicals.
The regulatory agency should be consulted before reference
toxicant(s) are selected and used.
4.18
RECORD KEEPING
4.18.1
Proper record keeping is important. A complete file
should be maintained for each individual toxicity test or group of
tests on closely related samples. This file should contain a record
of the sample chain-of-custody; a copy of the sample log sheet; the
original bench sheets for the test organism responses during the
toxicity test(s); chemical analysis data on the sample(s); detailed
records of the test organisms used in the test(s), such as species,
source, age, date of receipt, and other pertinent information
relating to their history and health; information on the
calibration of equipment and instruments; test conditions employed;
and results of reference toxicant tests. Laboratory data should be
recorded on a real-time basis to prevent the loss of information or
inadvertent introduction of errors into the record. Original data
sheets should be signed and dated by the laboratory personnel
performing the tests.
4.18.2
The regulatory authority should retain records pertaining
to discharge permits. Permittees are required to retain records
pertaining to permit applications and compliance for a minimum of 3
years [40 CFR 122.41(j)(2)].
SECTION 5
FACILITIES, EQUIPMENT, AND SUPPLIES
5.1 GENERAL REQUIREMENTS
5.1.1
Effluent toxicity tests may be performed in a fixed or
mobile laboratory. Facilities must include equipment for rearing
and/or holding organisms. Culturing facilities for test organisms
may be desirable in fixed laboratories which perform large numbers
of tests. Temperature control can be achieved using circulating
water baths, heat exchangers, or environmental chambers. Water used
for rearing, holding, acclimating, and testing organisms may be
ground water, receiving water, dechlorinated tap water, or
reconstituted synthetic water. Dechlorination can be accomplished
by carbon filtration, or the use of sodium thiosulfate. Use of 3.6
mg (anhydrous) sodium thiosulfate/L will reduce l.0 mg chlorine/L.
After dechlorination, total residual chlorine should be
non-detectable. Air used for aeration must be free of oil and toxic
vapors. Oil-free air pumps should be used where possible.
Particulates can be removed from the air using BALSTON® Grade BX or
equivalent filters, and oil and other organic vapors can be removed
using activated carbon filters (BALSTON®, C-1 filter, or
equivalent).
5.1.2
The facilities must be well ventilated and free from
fumes. Laboratory ventilation systems should be checked to ensure
that return air from chemistry laboratories and/or sample holding
areas is not circulated to test organism culture rooms or toxicity
test rooms, or that air from toxicity test rooms does not
contaminate culture areas. Sample preparation, culturing, and
toxicity test areas should be separated to avoid cross
contamination of cultures or toxicity test solutions with toxic
fumes. Air pressure differentials between such rooms should not
result in a net flow of potentially contaminated air to sensitive
areas through open or loosely- fitting doors. Organisms should be
shielded from external disturbances.
5.1.3
Materials used for exposure chambers, tubing, etc., that
come in contact with the effluent and dilution water should be
carefully chosen. Tempered glass and perfluorocarbon plastics
(TEFLON®) should be used whenever possible to minimize sorption and
leaching of toxic substances. These materials may be reused
following decontamination. Containers made of plastics, such as
polyethylene, polypropylene, polyvinyl chloride, TYGON®, etc., may
be used as test chambers or to ship, store and transfer effluents
and receiving waters, but they should not be reused unless
absolutely necessary, because they could carry over adsorbed
toxicants from one test to another, if reused. However, these
containers may be repeatedly reused for storing uncontaminated
waters, such as deionized or laboratory-prepared dilution waters
and receiving waters. Glass or disposable polystyrene containers
can be used for test chambers. The use of large ($ 20 L) glass
carboys is discouraged for safety reasons.
5.1.4
New plastic products of a type not previously used should
be tested for toxicity before initial use by exposing the test
organisms in the test system where the material is used. Equipment
(pumps, valves, etc.) which cannot be discarded after each use
because of cost, must be decontaminated according to the cleaning
procedures listed below (see Section 5, Facilities, Equipment and
Supplies, Subsection 5.3.2). Fiberglass and stainless steel, in
addition to the previously mentioned materials, can be used for
holding, acclimating, and dilution water storage tanks, and in the
water delivery system, but once contaminated with pollutants the
fiberglass should not be reused. All material should be flushed or
rinsed thoroughly with the test media before using in the
test.
5.1.5
Copper, galvanized material, rubber, brass, and lead must
not come in contact with culturing, holding, acclimation, or
dilution water, or with effluent samples and test solutions. Some
materials, such as several types of neoprene rubber (commonly used
for stoppers), may be toxic and should be tested before
use.
5.1.6
Silicone adhesive used to construct glass test chambers
absorbs some organochlorine and organophosphorus pesticides, which
are 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.
5.2
TEST CHAMBERS
5.2.1
Test chamber size and shape are
varied according to size of the test organism. Requirements are
specified in each toxicity test method.
5.3
CLEANING TEST CHAMBERS AND LABORATORY
APPARATUS
5.3.1
New plasticware used for sample collection or organism
exposure vessels does not require thorough cleaning before use. It
is sufficient to rinse new sample containers once with dilution
water before use. New glassware must be soaked overnight in 10%
acid (see below) and rinsed well in deionized water and dilution
water.
5.3.2
All non-disposable sample containers, test vessels,
tanks, and other equipment that have come in contact with effluent
must be washed after use to remove contaminants as described
below.
1.
Soak 15 min in tap water and scrub with detergent, or
clean in an automatic dishwasher.
2.
Rinse twice with tap water.
3.
Carefully rinse once with fresh, dilute (10%, V:V)
hydrochloric or nitric acid to remove scale, metals and bases. To
prepare a 10% solution of acid, add 10 mL of concentrated acid to
90 mL of deionized water.
4.
Rinse twice with deionized water.
5.
Rinse once with full-strength, pesticide-grade acetone to
remove organic compounds (use a fume hood or canopy).
6.
Rinse three times with deionized water.
5.3.3 Special requirements for cleaning glassware used in the
green alga, Selenastrum capricornutum, toxicity tests (Method
1003.0, Section 14). Prepare all graduated cylinders, test flasks,
bottles, volumetric flasks, centrifuge tubes and vials used in
algal assays as follows:
1.
Wash with non-phosphate detergent solution, preferably
heated to $ 50°C. Brush the inside of flasks with a stiff-bristle
brush to loosen any attached material. The use of a commercial
laboratory glassware washer or heavy-duty kitchen dishwasher
(under-counter type) is highly recommended.
2.
Rinse with tap water.
3.
Test flasks should be thoroughly rinsed with acetone and
a 10% solution (by volume) of reagent grade hydrochloric acid
(HCl). It may be advantageous to soak the flasks in 10% HCl for
several days. Fill vials and centrifuge tubes with the 10% HCl
solution and allow to stand a few minutes; fill all larger
containers to about one-tenth capacity with HCl solution and swirl
so that the entire surface is bathed.
4.
Rinse twice with MILLIPORE® MILLI-Q® OR QPAK™2, or
equivalent, water.
5.
New test flasks, and all flasks which through use may
become contaminated with toxic organic substances, must be rinsed
with pesticide-grade acetone or heat-treated before use. To
thermally degrade organics, place glassware in a high temperature
oven at 400°C for 30 min. After cooling, go to
7. If acetone is used, go to 6.
6.
Rinse thoroughly with MILLIPORE® MILLI-Q® or QPAK™2, or
equivalent water, and dry in an 105°C oven. All glassware should be
autoclaved before use and between uses.
7.
Cover the mouth of each chamber with aluminum foil or
other closure, as appropriate, before storing.
5.3.4
The use of sterile, disposable pipets will eliminate the
need for pipet washing and minimize the possibility of
contaminating the cultures with toxic substances.
5.3.5
All test chambers and equipment must be thoroughly rinsed
with the dilution water immediately prior to use in each
test.
5.4
APPARATUS AND EQUIPMENT FOR CULTURING AND TOXICITY
TESTS
5.4.1
Apparatus and equipment requirements for culturing and
testing are specified in each toxicity test method. Also, see
USEPA, 2002a.
5.4.2
WATER PURIFICATION SYSTEM
5.4.2.1
A good quality, laboratory grade deionized water,
providing a resistance of 18 megaohm-cm, must be available in the
laboratory and in sufficient quantity for laboratory needs.
Deionized water may be obtained from MILLIPORE® Milli-Q®,
MILLIPORE® QPAK™2 or equivalent system. If large quantities of high
quality deionized water are needed, it may be advisable to supply
the laboratory grade deionizer with preconditioned water from a
Culligan®, Continental®, or equivalent mixed-bed water treatment
system.
5.5
REAGENTS AND CONSUMABLE MATERIALS
5.5.1 SOURCES OF FOOD FOR CULTURE AND TOXICITY TESTS
1.
Brine shrimp, Artemia sp., cysts -- Many commercial
sources of brine shrimp cysts are available.
2.
Frozen adult brine shrimp, Artemia -- Available from most
pet supply shops or other commercial sources.
3.
Flake fish food -- TETRAMIN® and BIORIL® are available
from most pet shops.
4.
Trout chow -- Available from commercial
sources.
5.
Cereal leaves, CEROPHYLL® or equivalent -- Available from
commercial sources.
6.
Yeast -- Packaged dry yeast, such as Fleischmann's, or
equivalent, can be purchased at the local grocery store or
commercial sources.
7.
Alfalfa Rabbit Pellets -- Available from feed stores as
Purina rabbit chow.
8.
Algae - Available from commercial sources.
5.5.1.1
All food should be tested for nutritional suitability and
chemically analyzed for organochlorine pesticides, PCBs, and toxic
metals (see Section 4, Quality Assurance).
5.5.2
Reagents and consumable materials are specified in each
toxicity test method section. Also, see Section 4, Quality
Assurance.
5.6
TEST ORGANISMS
5.6.1
Test organisms should be obtained from inhouse cultures
or from commercial suppliers (see specific test method; Section 4,
Quality Assurance; and Section 6, Test Organisms).
5.7
SUPPLIES
5.7.1 See test methods (see Sections 11-14) for specific
supplies.
SECTION 6
TEST ORGANISMS
6.1 TEST SPECIES
6.1.1
The species used in characterizing the chronic toxicity
of effluents and/or receiving waters will depend on the
requirements of the regulatory authority and the objectives of the
test. It is essential that good quality test organisms be readily
available throughout the year from inhouse or commercial sources to
meet NPDES monitoring requirements. The organisms used in the
toxicity tests must be identified to species. If there is any doubt
as to the identity of the test organism, representative specimens
should be sent to a taxonomic expert to confirm the
identification.
6.1.2
Toxicity test conditions and culture methods for the
species listed in Subsection 6.1.3 are provided in this manual
also, see USEPA, 2002a.
6.1.3
The organisms used in the short-term chronic toxicity
tests described in this manual are the fathead minnow, Pimephales
promelas, the daphnid, Ceriodaphnia dubia (Berner, 1986), and the
green alga, Selenastrum capricornutum.
6.1.4
Some states have developed culturing and testing methods
for indigenous species that may be as sensitive, or more sensitive,
than the species recommended in Subsection 6.1.3. However, USEPA
allows the use of indigenous species only where state regulations
require their use or prohibit importation of the recommended
species in Subsection 6.1.3. Where state regulations prohibit
importation of non-native fishes or the use of recommended test
species, permission must be requested from the appropriate state
agency prior to their use.
6.1.5
Where states have developed culturing and testing methods
for indigenous species other than those recommended in this manual,
data comparing the sensitivity of the substitute species and the
one or more recommended species must be obtained in side-by-side
toxicity tests with reference toxicants and/or effluents, to ensure
that the species selected are at least as sensitive as the
recommended species. These data must be submitted to the permitting
authority (State or Region) if required. USEPA acknowledges that
reference toxicants prepared from pure chemicals may not always be
representative of effluents. However, because of the observed
and/or potential variability in the quality and toxicity of
effluents, it is not possible to specify a representative
effluent.
6.1.6
Guidance for the selection of test
organisms where the salinity of the effluent and/or receiving water
requires special consideration is provided in the Technical Support
Document for Water Quality-based Toxics Control (USEPA,
1991a).
1.
Where the salinity of the receiving water is < 1‰,
freshwater organisms are used regardless of the salinity of the
effluent.
2.
Where the salinity of the receiving water is $ 1‰, the
choice of organisms depends on state water quality standards and/or
permit requirements.
6.2
SOURCES OF TEST ORGANISMS
6.2.1
The test organisms recommended in this manual can be
cultured in the laboratory using culturing and handling methods for
each organism described in the respective test method sections. The
fathead minnow, Pimephales promelas, culture method is given in
Section 11 and not repeated in Section 12. Also, see USEPA
(2002a).
6.2.2
Inhouse cultures should be established wherever it is
cost effective. If inhouse cultures cannot be maintained or it is
not cost effective, test organisms or starter cultures should be
purchased from experienced commercial suppliers (see USEPA,
2002a).
6.2.3
Starter cultures of the green algae, Selenastrum
capricornutum, S. minutum, and Chlamydomonas reinhardti are
available from commercial suppliers.
6.2.4
Because the daphnid, Ceriodaphnia dubia, must be cultured
individually in the laboratory for at least seven days before the
test begins, it will be necessary to obtain a starter culture from
a commercial source at least three weeks before the test is to
begin if they are not being cultured inhouse.
6.2.5
If, because of their source, there is any uncertainty
concerning the identity of the organisms, it is advisable to have
them examined by a taxonomic specialist to confirm their
identification. For detailed guidance on identification, see the
individual test methods.
6.2.6
FERAL (NATURAL OCCURRING, WILD CAUGHT)
ORGANISMS
6.2.6.1 The use of test organisms taken from the receiving water
has strong appeal, and would seem to be a logical approach.
However, it is generally impractical and not recommended for the
following reasons:
1.
Sensitive organisms may not be present in the receiving
water because of previous exposure to the effluent or other
pollutants.
2.
It is often difficult to collect organisms of the
required age and quality from the receiving water.
3.
Most states require collecting permits, which may be
difficult to obtain. Therefore, it is usually more cost effective
to culture the organisms in the laboratory or obtain them from
private, state, or Federal sources. The fathead minnow, Pimephales
promelas, the daphnid, Ceriodaphnia dubia, and the green alga,
Selenastrum capricornutum, are easily cultured in the laboratory or
readily available commercially.
4.
The required QA/QC records, such as the single laboratory
precision data, would not be available.
5.
Since it is mandatory that the identity of the test
organism be known to species level, it would be necessary to
examine each organism caught in the wild to confirm its identity.
This would usually be impractical or, at the least, very stressful
to the organisms.
6.
Test organisms obtained from the wild must be observed in
the laboratory for a minimum of one week prior to use, to assure
that they are free of signs of parasitic or bacterial infections
and other adverse effects. Fish captured by electroshocking must
not be used in toxicity testing.
6.2.6.2
Guidelines for collecting natural occurring organisms are
provided in USEPA (1973), USEPA (1990) and USEPA
(1993b).
6.2.7
Regardless of their source, test organisms should be
carefully observed to ensure that they are free of signs of stress
and disease, and in good physical condition. Some species of test
organisms can be obtained from commercial stock certified as
"disease-free".
6.3
LIFE STAGE
6.3.1 Young organisms are often more sensitive to toxicants than
are adults. For this reason, the use of early life stages, such as
larval fish, is required for all tests. In a given test, all
organisms should be approximately the same age and should be taken
from the same source. Since age may affect the results of the
tests, it would enhance the value and comparability of the data if
the same species in the same life stages were used throughout a
monitoring program at a given facility.
6.4 LABORATORY CULTURING
6.4.1
Instructions for culturing and/or
holding the recommended test organisms are included in the
respective test methods (also, see USEPA, 2002a).
6.5
HOLDING AND HANDLING TEST ORGANISMS
6.5.1
Test organisms should not be subjected to changes of more
than 3°C in water temperature in any 12 h period or 2 units of pH
in any 24-h period.
6.5.2
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 are dropped
or touch a dry surface or are injured during handling must be
discarded. Dipnets are best for handling larger organisms. These
nets are commercially available or can be made from small-mesh
nylon netting, silk batting cloth, plankton netting, or similar
material. Wide-bore, smooth glass tubes (4 to 8 mm ID) with rubber
bulbs or pipettors (such as PROPIPETTE®) should be used for
transferring smaller organisms such as larval fish.
6.5.3
Holding tanks for fish are supplied with good quality
water (see Section 5, Facilities, Equipment, and Supplies) with
flow-through rate of at least two tank volumes per day. Otherwise
use a recirculation system where water flows through an activated
carbon or undergravel filter to remove dissolved metabolites.
Culture water can also be piped through high intensity ultraviolet
light sources for disinfection, and to photodegrade dissolved
organics.
6.5.4
Crowding must be avoided because it will stress the
organisms and lower the DO concentrations to unacceptable levels.
The solution of oxygen depends on temperature and altitude. The DO
must be maintained at a minimum of 4.0 mg/L. Aerate gently if
necessary.
6.5.5
The organisms should be observed carefully each day for
signs of disease, stress, physical damage, or mortality. Dead and
abnormal organisms should be removed as soon as observed. It is not
uncommon for some fish mortality (5-10%) to occur during the first
48 h in a holding tank because of individuals that refuse to feed
on artificial food and die of starvation. Organisms in the holding
tanks should generally be fed as in the cultures (see culturing
methods in the respective methods).
6.5.6
Fish should be fed as much as they will eat at least once
a day with live brine shrimp nauplii, Artemia, or frozen adult
brine shrimp, or dry food (frozen food should be completely thawed
before use). Adult brine shrimp can be supplemented with
commercially prepared food such as TETRAMIN® or BIORIL® flake food,
or equivalent. Excess food and fecal material should be removed
from the bottom of the tanks at least twice a week by
siphoning.
6.5.7
A daily record of feeding, behavioral observations, and
mortality should be maintained.
6.6
TRANSPORTATION TO THE TEST SITE
6.6.1 Organisms are transported from the base or supply
laboratory to a remote test site in culture water or standard
dilution water in plastic bags or large-mouth screw-cap (500 mL)
plastic bottles in styrofoam coolers. Adequate DO is maintained by
replacing the air above the water in the bags with oxygen from a
compressed gas cylinder, and sealing the bags or by use of an
airstone supplied by a portable pump. The DO concentration must not
fall below
4.0 mg/L.
6.6.2 Upon arrival at the test site, the organisms are
transferred to receiving water if receiving water is to be used as
the test dilution water. All but a small volume of the holding
water (approximately 5%) is removed by siphoning and replaced
slowly over a 10 to 15 minute period with dilution water. If
receiving water is to be used as the dilution water, caution must
be exercised in exposing the test organisms to it, because of the
possibility that it might be toxic. For this reason, it is
recommended that only approximately 10% of the test organisms be
exposed initially to the dilution water. If this group does not
show excessive mortality or obvious signs of stress in a few hours,
the remainder of the test organisms may be transferred to the
dilution water.
6.6.3
A group of organisms must not be
used for a test if they appear to be unhealthy, discolored, or
otherwise stressed, or if mortality appears to exceed 10% preceding
the test. If the organisms fail to meet these criteria, the entire
group must be discarded and a new group obtained. The mortality may
be due to the presence of toxicity, if the receiving water is used
as dilution water, rather than a diseased condition of the test
organisms. If the acclimation process is repeated with a new group
of test organisms and excessive mortality occurs, it is recommended
that an alternative source of dilution water be used.
6.7
TEST ORGANISM DISPOSAL
6.7.1 When the toxicity test(s) is concluded, all test organisms
(including controls) should be humanely destroyed and disposed of
in an appropriate manner.
