Methods for Measuring the Acute Toxicity of Effluents and
Receiving Waters to Freshwater and Marine Organisms
Fifth Edition
October 2002
U.S. Environmental Protection AgencyOffice of Water (4303T)
1200 Pennsylvania Avenue, NW Washington, DC 20460
EPA-821-R-02-012
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.
INTRODUCTION
1.1
This manual describes acute toxicity tests for use in the
National Pollutant Discharge Elimination System (NPDES) Permits
Program to identify effluents and receiving waters containing toxic
materials in acutely toxic concentrations. With the exception of
the Holmesimysis costata Acute Test (Table 19), the methods
included in this manual are referenced in Table IA, 40 CFR Part 136
regulations and, therefore, constitute approved methods for acute
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. The Holmesimysis costata Acute Test (Table
19) is specific to Pacific Coast waters and is not listed at 40 CFR
Part 136 for nationwide use. This method has been proposed but not
yet approved at 40 CFR Part 136.
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 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 acute 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, 1988a; USEPA, 1988b; USEPA, 1989a; USEPA,
1989b; USEPA, 1991a).
1.4
This methods manual serves as a companion to the
short-term chronic toxicity test methods manuals for freshwater and
marine organisms (USEPA, 2002a; USEPA, 2002b), the NPDES compliance
inspection manual (USEPA, 1988c), and the manual for evaluation of
laboratories performing aquatic toxicity tests (USEPA, 1991b). 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, 1991c).
1.6
The use of any test species or test conditions other than
those described in Tables 12-18 in this manual and referenced in
Table 1A, 40 CFR 136.3, shall be considered a major modification to
the method and subject to application and approval of alternate
test procedures under 40 CFR 136.4 and 40 CFR 136.5.
1.7
These methods are restricted to use by, or under the
supervision of, analysts experience in the use or conduct of, and
interpretation of data from, aquatic toxicity tests. Each analyst
must demonstrate the ability to generate acceptable test results
with the methods using the procedures described in this methods
manual.
1.8
This manual was prepared in the established
EMSL-Cincinnati format (USEPA, 1983a).
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
Effluent acute 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 is lethal to 50% of the test
organisms (LC50) within the prescribed period of time (24-96 h), or
the highest effluent concentration in which survival is not
statistically significantly different from the control.
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%.
2.4
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.
2.5
A negative result from an acute toxicity test does not
preclude the presence of chronic 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
acute (or chronic) toxicity.
2.6
The frequency with which acute 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.
2.7
Tests may be static (static non-renewal or static
renewal), or flow-through.
2.7.1 STATIC TESTS
2.7.1.1
Static non-renewal tests - The test organisms are exposed
to the same test solution for the duration of the test.
2.7.1.2
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.
2.7.2
FLOW-THROUGH TESTS
2.7.2.1
Two types of flow-through tests are in common use: (1)
sample is pumped continuously from the sampling point directly to
the dilutor system; and (2) grab or composite samples are collected
periodically, placed in a tank adjacent to the test laboratory, and
pumped continuously from the tank to the dilutor system. The
flow-through method employing continuous sampling is the preferred
method for on-site tests. Because of the large volume (often 400
L/day) of effluent normally required for flow-through tests, it is
generally considered too costly and impractical to conduct these
tests off-site at a central laboratory.
2.8
Advantages and disadvantages of the types of tests are as
follows:
2.8.1 STATIC NON-RENEWAL TESTS
2.8.1.1 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.
2.8.1.2 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 or
flow-through tests, 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.
2.8.2 STATIC-RENEWAL, ACUTE TOXICITY TESTS
2.8.2.1 Advantages:
1.
Reduced possibility of dissolved oxygen (DO) depletion
from high chemical oxygen demand (COD) and/or biological oxygen
demand (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.
2.8.2.2 Disadvantages:
1.
Require greater volume of effluent that non-renewal
tests.
2.
Generally less sensitive than flow-through tests, 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.
2.8.3 FLOW-THROUGH TESTS
2.8.3.1 Advantages:
1.
Provide a more representative evaluation of the acute
toxicity of the source, especially if sample is pumped continuously
directly from the source and its toxicity varies with
time.
2.
DO concentrations are more easily maintained in the test
chambers.
3.
A higher loading factor (biomass) may be used.
4.
The possibility of loss of toxicant due to
volatilization, adsorption, degradation, and uptake is
reduced.
2.8.3.2 Disadvantages:
1.
Large volumes of sample and dilution water are
required.
2.
Test equipment is more complex and expensive, and
requires more maintenance and attention.
3.
More space is required to conduct tests.
4.
Because of the resources required, it would be very
difficult to perform multiple or overlapping sequential
tests.
HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1
Development and maintenance of an effective health and
safety program in the laboratory requires an ongoing commitment by
laboratory management, and includes (1) the appointment of a
laboratory health and 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 laboratory staff member, (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
must determine that all required 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 must use safety equipment, as required, such as
rubber aprons, laboratory coats, respirators, gloves, safety
glasses, hard hats, and safety shoes.
3.2.2
LABORATORY SAFETY EQUIPMENT
3.2.2.1
Each laboratory (including mobile laboratories) must be
provided with safety equipment such as first aid kits, fire
extinguishers, fire blankets, emergency showers, and eye
fountains.
3.2.2.2
Mobile laboratories should be equipped with a telephone
to enable personnel to summon help in case of emergency.
3.3
GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1
Guidance in Material Safety Data Sheets should be
followed for reagents and other chemicals purchased from supply
houses. Incompatible materials should not be stored
together.
3.3.2
Work with effluents must be performed in compliance with
accepted rules pertaining to the handling of hazardous materials
(see Safety Manuals, Subsection 3.5). Personnel collecting samples
and performing toxicity tests should not work alone.
3.3.3
Because the chemical composition of effluents is usually
only poorly known, they must be considered as potential health
hazards, and exposure to them should be minimized. Fume and canopy
hoods over the test areas must be used whenever
necessary.
3.3.4
It is advisable to cleanse exposed parts of the body
immediately after collecting effluent samples.
3.3.5
All containers must be adequately labeled to indicate
their contents.
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
Good housekeeping contributes to safety and reliable
results.
3.3.8
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.
3.3.9
Mobile laboratories must 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 hepatitis B,
tetanus, typhoid fever, and polio.
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 industrial safety manuals, including
USEPA (1986) 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 testing activities. Local fire officials
should be notified of any potentially hazardous conditions.
QUALITY ASSURANCE
4.1 INTRODUCTION
4.1.1 Development and maintenance of a toxicity test laboratory
quality assurance (QA) program requires an ongoing commitment by
laboratory management, and includes the following: (1) appointment
of a laboratory quality assurance officer with the responsibility
and authority to develop and maintain a QA program;
(2) preparation of a quality assurance plan with data quality
objectives; (3) preparation of written descriptions of laboratory
standard operating procedures (SOP's) for test organism culturing,
toxicity testing, instrument calibration, sample chain-of-custody,
laboratory sample tracking system, etc.; and (4) provision of
adequate, qualified technical staff and suitable space and
equipment to assure reliable data.
4.1.2 QA practices within an aquatic toxicology laboratory must
address all activities that affect the quality of the final
effluent toxicity data, such as: (1) effluent sampling and
handling; (2) the source and condition of the test organisms; (3)
condition and operation 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 related to toxicity testing, see: FDA, 1978; USEPA, 1975;
USEPA, 1979a; USEPA, 1980a; USEPA, 1980b; USEPA, 1991b; 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 1991b.
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 toxicity testing areas, and from toxicity
test laboratories 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 and
Equipment).
4.3
TEST ORGANISMS
4.3.1
The test organisms used in the procedures described in
this manual are listed in Section 6, Test Organisms. The organisms
should appear healthy, behave normally, feed well, and have low
mortality in cultures, during holding, and in test controls. Test
organisms should be positively identified to species.
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. 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 and Equipment. 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 µg/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 SAMPLING AND SAMPLE HANDLING
4.5.1
Sample holding times and temperatures must conform to
conditions described in Section 8, Effluent and Receiving Water
Sampling and Sample Handling.
4.6
TEST CONDITIONS
4.6.1
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 vessel during the duration of each
test. Test solution temperatures should be maintained within the
limits specified for each test. DO concentration and pH in test
chambers should be checked daily throughout the test period, as
prescribed in Section 9, Acute Toxicity Test Procedures.
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 4.14) and
to document ongoing laboratory performance (Subsection
4.15).
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 4.15). 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 preformed with a reference toxicant,
unless the test organism supplier provides control chart data from
at least the last five monthly acute toxicity tests using the same
reference toxicant and test conditions.
4.7.4
The supplier should also 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 a routine reference toxicant test fails 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.
Suitable trout chow, Artemia, and other foods must be obtained as
described in this manual.
4.8.2
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. 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 effect 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 acute toxicity tests can be
compared with a food of known quality in side-by-side,
multi-concentration acute 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 test acceptability criteria for
control survival and reproduction or growth. If the concentration
of total organochlorine pesticides exceeds 0.l5 µg/g wet weight, or
the concentration of the total organochlorine pesticides plus PCBs
exceeds 0.30 µg/g wet weight, or toxic metals (Al, As, Cr, Co, 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 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 ACUTE TOXICITY TEST RESULTS
4.9.1
For the test results to be acceptable, control survival
must equal or exceed 90%.
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 will depend on the experience and
professional judgment of the laboratory analyst and the reviewing
staff of the regulatory authority. Any deviation from test
specifications must be noted when reporting data from a
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 Agency methods manuals
(USEPA, 1979a; USEPA, 1993b).
4.10.2
Reagent containers should be dated 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, conductivity,
salinity, alkalinity, and hardness must be calibrated and
standardized prior to use each day according to the instrument
manufacturer's procedures as indicated in the general section on
quality assurance (see EPA Methods 150.1, 360.1, 170.1, and 120.1;
USEPA, 1979b). Calibration data are recorded in a permanent
log.
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 EPA
methods (see EPA Methods 130.2 and 310.1; USEPA 1979b).
4.12
REPLICATION AND TEST SENSITIVITY
4.12.1
The sensitivity of toxicity 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: the experience and skill of the laboratory analyst; test
organism age, condition, and sensitivity; dilution water quality;
temperature control; and 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 (numbers of fish)
and the standard error of the test, and considered 20 fish per
concentration as optimum for Probit Analysis.
4.13.2
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. The single-laboratory
(intra-laboratory) and multi-laboratory (inter-laboratory)
precision of acute toxicity tests with several common test species
and reference toxicants are listed in Tables 1-4. Intra- and
inter-laboratory precision are described by the mean, standard
deviation, and relative standard deviation (percent coefficient of
variation, or CV) of the calculated endpoints from the replicated
tests.
4.13.3
Intra-laboratory precision data from 268 acute toxicity
tests with four species and five reference toxicants are listed in
Tables 1 and 2. The precision, expressed as CV%, ranged from 3% to
86%. More recent CV values reported by Jop et al. (1986), Dorn and
Rogers (1989), Hall et al. (1989), and Cowgill et al. (1990), fell
in a somewhat lower range (8% to 41%).
4.13.4
Inter-laboratory precision of acute toxicity tests from
253 reference toxicant tests with seven species, listed in Tables
2, 3, 4, and 5 (expressed as CV% for LC50s), ranged from 11% to
167%. Table 6 shows interlaboratory precision data from a study of
acute toxicity test methods using reference toxicant, effluent, and
receiving water sample types (USEPA, 2001a; USEPA, 2001b). Averaged
across sample types, total interlaboratory precision (expressed as
CV% for LC50s) ranged from 13% to 38.5% for the acute
methods.
4.13.5
No clear pattern of differences were noted in the intra-
or inter-laboratory test precision with the species listed,
although the test results with some toxicants, such as cadmium,
appear to more variable than those with other reference
toxicants.
4.13.6
Additional information on toxicity test precision is
provided in the Technical Support Document for Water Quality-Based
Toxics Control (see pp. 2-4, and 11-15; USEPA, 1991c).
1
Precision expressed as percent coefficient of variation, where
CV% = (standard deviation X 100)/mean.
2
SDS = Sodium dodecyl (lauryl) sulfate; NAPCP = Sodium
pentachlorophenate; CD = Cadmium; N = Number of tests; toxicant
concentration in mg/L.
3
Pimephales promelas tests were performed in soft, synthetic
freshwater; total hardness, 40-48 mg/L as CaCO3, by J. Dryer,
Aquatic Biology Section, EMSL-Cincinnati.
4
Daphnia data from Lewis and Horning, 1991. Tests with D. magna
used hard reconstituted water (total
hardness, 180-200 mg/L as CaCO3); tests with D. pulex used
moderately-hard reconstituted water (total
hardness, 80-100 mg/L as CaCO3).
5
Mysid tests were performed in 25 ppt salinity, natural seawater.
Data were provided by Steve Ward,
Environmental Services Division, U.S. Environmental Protection
Agency, Edison, New Jersey. Personal
communication, November 14, 1990.
1
From Table 2, p. 191, Grothe and Kimerle, 1985. Tests performed
at 20EC ±2EC; dilution water hardness, 100mg/L as CaCO3; dilution
water alkalinity, 76 mg/L as CaCO3; effluent hardness, approx. 1000
mg/L as CaCO3; effluent alkalinity, 310 mg/L as CaCO3; effluent
dilutions - 56%, 32%, 18%, 10%, 5.6%, 3.1%, 1.7%.
2
LC50 expressed in percent effluent.
3
Intra-laboratory precision expressed as the weighted mean
CV(%).
REFERENCE TOXICANT
1
Data for Pimephales promelas (fathead minnow), Oncorhyncus
mykiss (rainbow trout), and Daphnia magna were taken from USEPA,
1983b.
Data for, Mysidopsis bahia, and Cyprinodon variegatus
(sheepshead minnow) were taken from USEPA, 1981. Six laboratories
participated in each study. Test salinity was 28‰.
LC50s expressed in µg/L.
In the studies with the freshwater organisms, the water hardness
for five of the six laboratories ranged between 36 and 75 mg/L.
However, the water hardness for the sixth laboratory was 255 mg/L,
resulting in LC50 values for silver more than an order of magnitude
larger than for the other five. These values were rejected in
calculating the CV%. The mean weights of test fish were from
0.05-0.26 g for fathead minnows, and 0.22-1.32 g for rainbow trout.
Daphnia were #24-h old.
In studies with the marine organisms, only one LC50 (presumably
the combined LC50 from duplicate tests) was reported for each
toxicity test. LC50s for flow-through tests with Mysidopsis bahia
and Cyprinodon variegatus were calculated two different ways -- (1)
on the basis of the nominal toxicant concentrations (Nom), and (2)
on the basis of measured (Meas) toxicant concentrations. Test
organism age was #2 days for Mysidopsis bahia, and 28 days for
Cyprinodon variegatus. The salinity of test solutions was 28‰.
N, the total number of LC50 values used in calculating the CV(%)
varied with organism and toxicant because some data were rejected
due to water hardness, lack of concentration measurements, and/or
because some of the LC50s were not calculable.
2
CV% = Percent coefficient of variation = (standard deviation x
100)/mean.
TEST PRECISION (CV%)2
GRAPH3STAT4NO. LABS METHOD METHOD TOTAL5
SUBMITTING TEST TYPE VALID DATA N LC50 CV% N LC50 CV% N LC50
CV%
Pimephales promelas (96 h, 22EC)6 17 Pimephales promelas (24 h,
25EC)7 6 Ceriodaphnia dubia (48 h, 25EC)7 11 Mysidopsis bahia (96
h, 22EC)8 14
6 944 28.8 13 832 27.8 17 864 29.6 6 83211.5 6 83211.5 - - - 11
256 53.111 264 48.5 - - - 7 292 32.9 11 250 36.0 14 268 37.3
1
Interlaboratory study of toxicity test precision conducted in
1990 by the Environmental Monitoring Systems Laboratory -
Cincinnati, U.S. Environmental Protection Agency, Cincinnati, Ohio
45268, in cooperation with the states of New Jersey and North
Carolina, and the Office of Water Enforcement and Permits, U.S.
Environmental Protection Agency, Washington, DC.
2
Percent coefficient of variation = (standard deviation X
100)/mean. Calculated for LC50 from acute tests. LC50s expressed as
mg/L KCl added to the dilution water.
3
LC50 estimated by the Graphical Method.
4
LC50 estimated by Probit, Litchfield-Wilcoxon, or Trimmed
Spearman-Karber method.
5
LC50 usually reported for only one method of analysis for each
test. Where more than one LC50 was reported for a test, the lowest
value was used to calculate the statistics for "Total."
6
Data from the New Jersey Department of Environmental Protection:
static daily-renewal tests, using moderately- hard synthetic
freshwater.
7
Data from North Carolina certified laboratories: static
non-renewal tests, using moderately-hard reconstituted
freshwater.
8
Data from the New Jersey Department of Environmental Protection:
static daily-renewal tests, using 25 ppt salinity, FORTY FATHOMS®
synthetic seawater.
No. Labs Test Type Submitting Data LC50 CV%2
Pimephales promelas (48 h, 25EC)3 Ceriodaphnia dubia (48 h,
25EC)3 Mysidopsis bahia (48 h, 25EC)5 Menidia beryllina (48 h,
25EC)5
203 8964 28.6 171 4324 39.8 61 5324 30.1 39 1646 42.2
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.
LC50s were estimated by the graphical or Spearman-Karber
method.
2
Percent coefficient of variation = (standard deviation X
100)/mean.
3
Static non-renewal tests, using moderately-hard synthetic
freshwater (total hardness = 80-100 mg/L as CaCO3).
4
Expressed as mg KCl added per liter of dilution water.
5
Static non-renewal tests, using 30 ppt modified GP2 artificial
seawater.
6
Expressed as Fg Cu++ added per liter of dilution water.
TABLE 6. NATIONAL INTERLABORATORY STUDY OF ACUTE TOXICITY TEST
PRECISION, 2000: PRECISION OF LC50 POINT ESTIMATES FOR REFERENCE
TOXICANT, EFFLUENT, AND RECEIVING WATER SAMPLE TYPES1.
CV (%)2
Method Sample Type
Within-lab3 Between-lab4 Total5
Pimephales promelas KCl Municipal effluent Receiving water
7.62 19.7 21.1 10.3 19.2 21.8 --17.2 Average 8.96 19.4 20.0
Ceriodaphnia dubia KCl Municipal effluent Receiving water
14.6 15.2 21.1 9.68 32.8 34.2 --31.8 Average 12.1 24.0 29.0
Cyprinodon variegatus KCl Municipal effluent Receiving water
--26.0 --19.4 --32.5 Average --26.0
6
Menidia beryllina CuSO4 Industrial effluent Receiving water
---9.91 49.7 50.7
--26.3 Average 9.91 49.7 38.5
Holmesimysis costata7 Zn (48 h test) Zn (96 h test) Zn
(interlaboratory trial 1) Zn (interlaboratory trial 2)
Average
19
23 ---24 --1
21 13
1
From EPA's WET Interlaboratory Variability Study (USEPA, 2001a;
USEPA, 2001b).
2
CVs were calculated based on the within-laboratory component of
variability, the between-laboratory component of variability, and
total interlaboratory variability (including both within-laboratory
and between-laboratory components). For the receiving water sample
type, within-laboratory and between-laboratory components of
variability could not be calculated since the study design did not
provide within-laboratory replication for this sample type. The
study design also did not provide within-laboratory replication for
the Cyprinodon variegatus Acute Method.
3
The within-laboratory (intralaboratory) component of variability
for duplicate samples tested at the same time in the same
laboratory.
4
The between-laboratory component of variability for duplicate
samples tested at different laboratories.
5
The total interlaboratory variability, including
within-laboratory and between-laboratory components of variability.
The total interlaboratory variability is synonymous with
interlaboratory variability reported from other studies where
individual variability components are not separated.
6
Precision estimates were not calculated for the reference
toxicant sample type since the majority of results for this sample
type were outside of the test concentration range (ie.,
>100).
7
Holmesimysis costata Acute Test data were from Martin et al.
(1989). Zn was tested in two intralaboratory trials and in two
interlaboratory trials. Data from this study was only reported to
two significant figures.
4.14 DEMONSTRATING ACCEPTABLE LABORATORY
PERFORMANCE
4.14.1
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
intra-laboratory precision, expressed as percent coefficient of
variation (CV%), of each type of test to be used in a 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 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.
4.15
DOCUMENTING ONGOING LABORATORY PERFORMANCE
4.15.1
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.
4.15.2
A control chart should be prepared for each combination
of reference toxicant, test species, test condition, and endpoint.
Toxicity endpoints from five or six tests are adequate for
establishing the control charts. In this technique, a running plot
is maintained for the toxicity values (Xi) from successive tests
with a given reference toxicant (Figure 1), and endpoints (LC50s)
are examined to determine if they are within prescribed limits. 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. The mean ( ¯ ) and upper and lower control limits
(±2S) are re-
X calculated with each successive test result. 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.
4.15.3
Laboratories should compare the calculated CV (i.e.,
standard deviation / mean) of the LC50 for the 20 most recent data
points to the distribution of laboratory CVs reported nationally
for reference toxicant testing (Table 3-3 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.
4.15.4
The outliers, which are values falling outside the upper
and lower control limits, and trends of increasing or decreasing
sensitivity, are readily identified. 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. 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.
4.15.5
If the toxicity value from a given test with the
reference toxicant falls well outside the expected range for the
test organisms when using the standard dilution water, 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 point estimates should gradually narrow. However,
control limits of ±2S, by definition, will be exceeded 5% of the
time, regardless of how well a laboratory performs. Highly
proficient laboratories which develop a very narrow control limit
may be unfairly penalized if a test 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 LC50 for the 20 most recent data points to the
distribution of laboratory CVs reported nationally for reference
toxicant testing (Table 3-3 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.
4.15.6
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.
4.16
REFERENCE TOXICANTS
4.16.1
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
and other Agency programs requiring aquatic toxicity tests.
EMSL-Cincinnati hopes to release EPAcertified solutions of cadmium
and copper, with accompanying toxicity data for the recommended
test species, 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.
4.17
RECORD KEEPING
4.17.1
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.
4.17.2
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)].
FACILITIES AND EQUIPMENT
5.1 GENERAL REQUIREMENTS
5.1.1
Effluent toxicity tests may be performed in a fixed or
mobile laboratory. Facilities should include equipment for rearing
and/or holding organisms.
5.1.2
The facilities must be well ventilated and free of toxic
fumes. Sample preparation, culturing, and toxicity testing areas
should be separated to avoid cross contamination of cultures or
toxicity test solutions with toxic fumes. Laboratory ventilation
systems should be checked to ensure that return air from chemistry
laboratories and/or sample handling 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. 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.
5.1.3
Control of test solution temperature can best be achieved
using circulating water baths, heat exchangers, or environmental
chambers. Photoperiod can be controlled using automatic timers in
the laboratory or environmental chambers.
5.1.4
Water used for rearing, holding, and testing organisms
may be reconstituted synthetic water, ground water, surface water,
or dechlorinated tap water. Dechlorination can be accomplished by
carbon filtration, laboratory water conditioning units, or the use
of sodium thiosulfate. After dechlorination, total residual
chlorine should be non-detectable. Sodium thiosulfate may be toxic
to the test organisms, and if used for dechlorination, paired
controls with and without sodium thiosulfate should be incorporated
in effluent toxicity tests. 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.
5.1.4.1
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 QPAKTM2 or equivalent system. If large quantities of high
quality deionized water are needed, it may be advisable to supply
the laboratory grade water deionizer with preconditioned water from
a CULLIGAN®, CONTINENTAL®, or equivalent, mixed-bed water treatment
system.
5.1.5
Air used for aeration must be free of oil and fumes.
Oil-free air pumps should be used where possible. Particulates can
be removed from the air using BALSTON® Grade BX or equivalent
filters (Balston, Inc., Lexington, MA), and oil and other organic
vapors can be removed using activated carbon filters (BALSTON®, C-1
filter, or equivalent).
5.1.6
During rearing, holding, and testing, test organisms
should be shielded from external disturbances such as rapidly
changing light conditions (especially salmonids) and pedestrian
traffic.
5.1.7
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, and may be reused after cleaning.
Containers made of plastics, such as polyethylene, polypropylene,
polyvinyl chloride, TYGON®, etc., may be used 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. 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 as test chambers. The use of large ($20 L) glass carboys is
discouraged for safety reasons.
5.1.8
New plastic products should be tested for toxicity before
general use by exposing organisms to them under ordinary test
conditions.
5.1.9
Equipment which cannot be discarded after each use
because of cost, must be decontaminated according to the cleaning
procedures listed below. Fiberglass, in addition to the previously
mentioned materials, can be used for holding and dilution water
storage tanks, and in the water delivery system. All material
should be flushed or rinsed thoroughly with dilution water before
using in the test.
5.1.10
Copper, galvanized material, rubber, brass, and lead must
not come in contact with holding or dilution water, or with
effluent samples and test solutions. Some materials, such as
neoprene rubber (commonly used for stoppers), may be toxic and
should be tested before use.
5.1.11
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
CLEANING TEST CHAMBERS AND LABORATORY
APPARATUS
5.2.1
New plasticware used for effluent or dilution water
collection or organism test chambers does not require thorough
cleaning before use. It is sufficient to rinse new sample
containers once with sample dilution water before use. New
glassware must be soaked overnight in 10% acid (see below) and
rinsed well in deionized water and dilution water.
5.2.2
All non-disposable sample containers, test vessels,
tanks, and other equipment that has come in contact with effluent
must be washed after use in the manner described below to remove
surface 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.
5.2.3
All test chambers and equipment should be thoroughly
rinsed with the dilution water immediately prior to use in each
test.
5.3
APPARATUS AND EQUIPMENT FOR CULTURING AND TOXICITY
TESTS
5.3.1
Culture units -- see Appendix. It is preferable to obtain
test organisms from in-house culture units. If it is not feasible
to maintain cultures in-house, test organisms can be obtained from
commercial sources, and should be shipped to the laboratory in well
oxygenated water in insulated containers to minimize excursions in
water temperature during shipment. The temperature of the water in
the shipping containers should be measured on arrival, to determine
if the organisms were subjected to obvious undue thermal
stress.
5.3.2
Samplers -- automatic samplers, preferably with sample
cooling capability, that can collect a 24-h composite sample of 2 L
or more.
5.3.3
Sample containers -- for sample shipment and storage (see
Section 8, Effluent and Receiving Water Sampling and Sample
Handling).
5.3.4
Environmental chamber or equivalent facility with
temperature control (20EC or 25EC)
5.3.5
Water purification system -- MILLIPORE® MILLI-Q®,
MILLIPORE® QPAK™2, or equivalent. Depending on the quantity of high
grade water needed, a first-stage pre-conditioner deionizer, such
as a Culligan® or Continental® System, or equivalent, may be needed
to provide feed water to the high-purity system.
5.3.6
Balance -- analytical, capable of accurately weighing to
0.0001 g.
5.3.7
Reference weights, Class S -- for documenting the
performance of the analytical balance(s). The balance(s) should be
checked with reference weights which are at the upper and lower
ends of the range of the weighings made when the balance is used. A
balance should be checked at the beginning of each series of
weighings, periodically (such as every tenth weight) during a long
series of weighings, and after the last weight of a series is
taken.
5.3.8
Test chambers -- borosilicate glass or non-toxic
disposable plastic test chambers are suitable. Test chamber volumes
are indicated in the method summaries. To avoid potential
contamination from the air and excessive evaporation of test
solutions during the test, the chambers should be covered with
safety glass plates or sheet plastic, 6 mm (¼ in) thick.
5.3.9
Volumetric flasks and graduated cylinders -- Class A,
borosilicate glass or non-toxic plastic labware, 10-1000 mL for
making test solutions.
5.3.10
Volumetric pipets -- Class A, 1-100 mL.
5.3.11
Serological pipets -- 1-10 mL, graduated.
5.3.12
Pipet bulbs and fillers -- PROPIPET®, or
equivalent.
5.3.13
Droppers, and glass tubing with fire polished edges, 4 mm
ID -- for transferring test organisms.
5.3.14
Wash bottles -- for rinsing small glassware and
instrument electrodes and probes.
5.3.15
Glass or electronic thermometers -- for measuring water
temperature.
5.3.16
Bulb-thermograph or electronic-chart type thermometers --
for continuously recording temperature.
5.3.17
National Bureau of Standards Certified thermometer (see
USEPA Method 170.1; USEPA 1979b).
5.3.18
pH, DO, and specific conductivity meters -- for routine
physical and chemical measurements. Unless the test is being
conducted to specifically measure the effect of one of the above
parameters, a portable, field-grade instrument is
acceptable.
5.3.19
Refractometer -- for measuring effluent, receiving, and
test solution salinity.
5.3.20
Amperometric titrator -- for measuring total residual
chlorine.
5.4
REAGENTS AND CONSUMABLE MATERIALS
5.4.1
Reagent water -- defined as MILLIPORE® MILLI-Q®,
MILLIPORE® QPAK™2 or equivalent water (see Subsection 5.3.5
above).
5.4.2
Effluent, dilution water, and receiving water -- see
Section 7, Dilution Water, and Section 8, Effluent and Receiving
Water Sampling and Sample Handling.
5.4.3
Reagents for hardness and alkalinity tests (see USEPA
Methods 130.2 and 310.1; USEPA l979b).
5.4.4
Standard pH buffers 4, 7, and 10 (or as per instructions
of instrument manufacturer) for instrument calibration (see USEPA
Method 150.1; USEPA 1979b).
5.4.5
Specific conductivity and salinity standards (see USEPA
Method 120.1; USEPA 1979b).
5.4.6
Laboratory quality control check samples and standards
for the above chemistry methods.
5.4.7
Reference toxicant solutions (see Section 4, Quality
Assurance).
5.4.8
Membranes and filling solutions for dissolved oxygen
probe (see USEPA Method 360.1; USEPA 1979b), or reagents for
modified Winkler analysis.
5.4.9
Sources of Food for Cultures and Toxicity
Tests.
5.4.9.1
All food should be tested for nutritional suitability,
and chemically analyzed for organic chlorine, PCBs, and toxic
metals (see Section 4, Quality Assurance).
5.4.9.2
Brine Shrimp (Artemia) -- see Appendix A.
1.
Brine Shrimp (Artemia) Cysts.
There are many commercial sources of brine shrimp cysts. The
quality of the cysts may vary from one batch to another, and the
cysts in each new batch (can or lot) should be evaluated for
nutritional suitability and chemical contamination. The nutritional
suitability (see Leger et al., 1985, 1986) of each new batch is
checked against known suitable reference cysts by performing a
side-by-side growth and/or reproduction tests using the "new" and
"reference" cysts. If the results of tests for nutritional
suitability or chemical contamination do not meet standards, the
Artemia should not be used.
2.
Frozen Adult Brine Shrimp Frozen adult brine shrimp are
available from pet stores and other commercial sources.
5.4.9.3 Trout Chow
Starter or No. 1 pellets, prepared according to current U.S.
Fish and Wildlife Service specifications, are available from
commercial sources. (The flake food, TETRAMIN® or BIORIL®, can be
used regularly as a substitute for trout chow in preparing food for
daphnids, and can be used as a short-term substitute for trout chow
in feeding fathead minnows.)
5.4.9.4 Dried, Powdered Leaves (CEROPHYLL®)
Dried, powdered, cereal leaves (e.g., CEROPHYLL® or equivalent)
are available from commercial suppliers. Dried, powdered, alfalfa
leaves obtained from health food stores have been found to be a
satisfactory substitute for cereal leaves.
5.4.9.5
Yeast Packaged dry yeast, such as Fleischmann's, or
equivalent, can be purchased at the local grocery store.
5.4.9.6
Flake Fish Food The flake foods, TETRAMIN® and BIORIL®,
are available at most pet supply shops.
5.5 TEST ORGANISMS
5.5.1 Test organisms are obtained from inhouse cultures or
commercial suppliers (see Section 6, Test Organisms).
TEST ORGANISMS
6.1 TEST SPECIES
6.1.1
The species used in characterizing the acute 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 toxicity tests
must be identified to species. If there is any doubt as to the
identity of the test organisms, representative specimens should be
sent to a taxonomic expert to confirm the
identification.
6.1.2
Toxicity test conditions and culture methods are provided
in this manual for the following principal test
organisms:
Freshwater Organisms:
1.
Ceriodaphnia dubia (daphnid) (Table 12).
2.
Daphnia pulex and D. magna (daphnids) (Table
13).
3.
Pimephales promelas (fathead minnow) (Table
14).
4.
Oncorhynchus mykiss (rainbow trout) and Salvelinus
fontinalis (brook trout) (Table 15).
Estuarine and Marine Organisms:
1.
Mysidopsis bahia (mysid) (Table 16).1
2.
Cyprinodon variegatus (sheepshead minnow) (Table
17).
3.
Menidia beryllina (inland silverside), M. menidia
(Atlantic silverside), and M. peninsulae (tidewater silverside)
(Table 18).
6.1.3
The test species (AFS, 1991) listed in Subsection 6.1.2
are the recommended acute toxicity test organisms. They are easily
cultured in the laboratory, are sensitive to a variety of
pollutants, and are generally available throughout the year from
commercial sources. Summaries of test conditions for these species
are provided in Tables 12-18. Guidelines for culturing and/or
holding the organisms are provided in Appendix A.
6.1.4
Additional species may be suitable for toxicity tests in
the NPDES Program. A list of alternative acute toxicity test
species and minimal testing requirements (i.e., temperature,
salinity, and life stage) for these species are provided in
Appendix B. Table 19 provides a summary of test conditions for
Holmesimysis costata, which should also be considered an
alternative acute toxicity test species. The Holmesimysis costata
Acute Test (Table 19) is specific to Pacific Coast waters and is
not listed at 40 CFR Part 136 for nationwide use. It is important
to note that these species may not be as easily cultured or tested
as the species on the list in 6.1.2, and may not be available from
commercial sources.
6.1.5
Some states have developed culturing and testing methods
for indigenous species that may be as sensitive or more sensitive
than the species recommended in 6.1.2. However, EPA allows the use
of indigenous species only where state regulations require their
use or prohibit importation of the species in 6.1.2. Where state
regulations prohibit importation or use of the recommended test
species, permission must be requested from the appropriate state
agency prior to their use.
1 The genus name of this organism was formally changed to
Americamysis (Price et al., 1994); however, the method manual will
continue to refer to Mysidopsis bahia to maintain consistency with
previous versions of the method.
6.1.6
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 one or
more of the 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. EPA 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.
6.1.7
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, 1991c).
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 INHOUSE CULTURES
6.2.1.1
Inhouse cultures should be established wherever it is
cost effective. If inhouse cultures cannot be maintained, test
organisms should be purchased from experienced commercial suppliers
(see Appendix for sources).
6.2.2
COMMERCIAL SUPPLIERS
6.2.2.1
All of the principal test organisms listed in Subsection
6.1.2 are available from commercial suppliers.
6.2.3
FERAL (NATURAL OCCURRING, WILD CAUGHT)
ORGANISMS
6.2.3.1 The use of test organisms taken from the receiving water
has strong appeal, and would seem to be the logical approach.
However, it is impractical 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 collection 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. Fish such as fathead minnows,
sheepshead minnows, and silversides, and invertebrates such as
daphnids and mysids, are easily reared in the laboratory or
purchased.
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 test organisms
is known to the species level, it would necessary to examine each
organism caught in the wild to confirm its identity, which 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.
6.2.3.2
Guidelines for collection of feral organisms are provided
in USEPA, 1973; USEPA 1990a.
6.2.4
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, such as trout, can be obtained from stocks 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 first instars of daphnids and juvenile mysids and 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 Appendix A.
6.5
HOLDING AND HANDLING TEST ORGANISMS
6.5.1
Test organisms should not be subjected to changes of more
than 3EC in water temperature or 3‰ in salinity in any 12 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 dry surfaces 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 bolting cloth, plankton netting, or similar
material. Wide-bore, smooth glass tubes (4 to 8 mm inside diameter)
with rubber bulbs or pipettors (such as a PROPIPETTE® or other
pipettor) should be used for transferring smaller organisms such as
daphnids, mysids, and larval fish.
6.5.3
Holding tanks for fish are supplied with a good quality
water (see Section 5, Facilities and Equipment) with a flow-through
rate of at least two tank-volumes per day. Otherwise, use a
recirculation system where the 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 should be avoided. The DO must be maintained at
a minimum of 4.0 mg/L for marine and warm water, freshwater
species, and 6.0 mg/L for cold-water, freshwater species. The
solubility of oxygen depends on temperature, salinity, and
altitude. Aerate if necessary.
6.5.5
Fish should be fed as much as they will eat at least once
a day with live or frozen brine shrimp or dry food (frozen food
should be completely thawed before use). 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.
6.5.6
Fish should be observed carefully each day for signs of
disease, stress, physical damage, and mortality. Dead and abnormal
specimens should be removed as soon as observed. It is not uncommon
to have some fish (5-10%) mortality during the first 48 h in a
holding tank because of individuals that refuse to feed on
artificial food and die of starvation.
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. Another method
commonly used to maintain sufficient DO during shipment is to
aerate with an airstone which is supplied from a portable pump. The
DO concentration must not fall below 4.0 mg/L for marine and
warm-water, freshwater species, and 6.0 mg/L for cold-water,
freshwater species.
6.6.2
Upon arrival at the test site, 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 min period with dilution water. If receiving water
is used as 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 are 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 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.
6.6.4
In static tests, marine organisms can be used at all
concentrations of effluent by adjusting the salinity of the
effluent to a standard salinity (such as 25‰) or to the salinity
approximating that of the receiving water, by adding sufficient dry
ocean salts, such as Forty Fathoms®, or equivalent, GP2 or
hypersaline brine.
6.6.5
Saline dilution water can be prepared with deionized
water or a freshwater such as well water or a suitable surface
water. If dry ocean salts are used, care must be taken to ensure
that the added salts are completely dissolved and the solution is
aerated 24 h before the test organisms are placed in the solutions.
The test organisms should be acclimated in synthetic saline water
prepared with the dry salts. Caution: addition of dry ocean salts
to dilution water may result in an increase in pH. (The pH of
estuarine and coastal saline waters is normally
7.5-8.3.)
6.6.6
All effluent concentrations and the control(s) used in a
test should have the same salinity. However, if this is impractical
because of the large volumes of water required, such as in
flow-through tests, the highest effluent concentration (lowest
salinity) that could be tested would depend upon the salinity of
the receiving water and the tolerance of the test organisms. The
required salinities for toxicity tests with estuarine and marine
species are listed in Tables 16-19. However, the tolerances of
other candidate test species would have to be determined by the
investigator in advance of the test.
6.6.7
Because of the circumstances described above, when
performing flow-through tests of effluents discharged to saline
waters, it is advisable to acclimate groups of test organisms to
each of three different salinities, such as 10, 20, and 30‰, prior
to transporting them to the test site. It may also be advisable to
maintain cultures of these test organisms at a series of salinity
levels, including at least 10, 20, and 30‰, so that the change in
salinity upon acclimation at the desired test dilutions does not
exceed 6‰.
6.7
TEST ORGANISM DISPOSAL
6.7.1 When the toxicity test is concluded, all test organisms
(including controls) should be humanely destroyed and disposed of
in an appropriate manner.
