SECTION 4 QUALITY ASSURANCE

4.1 INTRODUCTION


4.1.1
Development and maintenance of a toxicity test laboratory
quality assurance (QA) program (USEPA, 1991b) 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 written descriptions 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 toxicity 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 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); USEPA
(1991c); DeWoskin (1984); and Taylor (1987).


4.1.4
Guidelines for the evaluation of laboratory performing
toxicity tests and laboratory evaluation criteria are 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 sheepshead minnow, Cyprinodon variegatus; the
inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia;
the sea urchin, Arbacia punctulata; and the red macroalga, Champia
parvula. The organisms used should be disease-free and appear
healthy, behave normally, feed well, and have low mortality in
cultures, during holding, and in test control. 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 on the
objectives of the study and logistical constraints, as discussed in
Section 7, Dilution Water. The dilution water used in the toxicity
tests may be natural seawater, hypersaline brine (100‰) prepared
from natural seawater, or artificial seawater prepared from
commercial sea salts, such as FORTY FATHOMS® or HW MARINEMIX®, if
recommended in the method. GP2 synthetic seawater, made from
reagent grade chemical salts (30‰) in conjunction with natural
seawater, may also be used if recommended. Hypersaline brine and
artificial seawater can be used with Champia parvula only if they
are accompanied by at least 50% natural seawater. Types of water
are discussed in Section 5, Facilities, Equipment, and Supplies.
Water used for culturing and test dilution water 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, 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
National 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 and salinity 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 vessel during the duration of
each test. Test solution temperatures should be maintained within
the limits specified for each test. DO concentrations and pH should
be checked at the beginning of the 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 test 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 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.
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 µg/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).


4.84
For foods (e.g., 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 CHRONIC TOXICITY TESTS


4.9.1 The results of the sheepshead minnow, Cyprinodon
variegatus, inland silverside, Menidia beryllina, or mysid,
Mysidopsis bahia, tests are acceptable if survival in the controls
is 80% or greater. The sea urchin, Arbacia punctulata, test
requires control egg fertilization equal to or exceeding 70%.
However, greater than 90% fertilization may result in masking toxic
responses. The red macroalga, Champia parvula, test is acceptable
if survival is 100%, and the mean number of cystocarps per plant
should equal or exceed 10. If the sheepshead minnow, Cyprindon
variegatus, larval survival and growth test is begun with
less-than-24-h old larvae, the mean dry weight of the surviving
larvae in the control chambers at the end of the test must equal or
exceed 0.60 mg, if the weights are determined immediately, or


0.50
mg if the larvae are preserved in a 4% formalin or 70%
ethanol solution. If the inland silverside, Menidia beryllina,
larval survival and growth test is begun with larvae seven days
old, the mean dry weight of the surviving larvae in the control
chambers at the end of the test must equal or exceed 0.50 mg, if
the weights are determined immediately, or 0.43 mg if the larvae
are preserved in a 4% formalin or 70% ethanol solution. The mean
mysid dry weight of survivors must be at least 0.20 mg. Automatic
or hourly feeding will generally provide control mysids with a dry
weight of 0.30 mg. At least 50% of the females should bear eggs at
the end of the test, but mysid fecundity is not a factor in test
acceptability. However, fecundity must equal or exceed 50% to be
used as an endpoint in the test. 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 conditions and test acceptability
criteria summaries). The acceptability of the test will 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 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 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, conductivity, and
salinity, must be calibrated and standardized according to
instrument manufacturers 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; (5) 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
(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 or more
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 using two reference toxicants with the
mysid, Mysidopsis bahia, and the inland silverside, Menidia
beryllina, is listed 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). For the Mysidopsis bahia and
the Cyprinodon variegatus test methods, the effluent sample was a
municipal wastewater spiked with KCl, the receiving water sample
was a river water spiked with KCl, and the reference toxicant
sample was bioassay-grade FORTY FATHOMS®


synthetic seawater spiked with KCl. For the Menidia beryllina
test method, the effluent sample was an industrial wastewater
spiked with CuSO4, the receiving water sample was a natural
seawater spiked with CuSO4, and the reference toxicant sample was
bioassay-grade FORTY FATHOMS® synthetic seawater spiked with CuSO4.
Additional precision data for each of the tests described in this
manual are presented in the sections describing the individual test
methods.


4.14.4
Additional information on toxicity test precision is
provided in the Technical Support Document for Water Quality-based
Toxic 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 ± (LOEC minus NOEC).


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 uncertainty 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 a $ 0.5
dilution factor. Other factors which can affect test precision
include: test organism age, condition, and sensitivity; temperature
control; and feeding.


TABLE 1. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 1991: SUMMARY OF RESPONSES USING TWO REFERENCE
TOXICANTS1,2
4
Organism Endpoint No. Labs KCl(mg/L)SD CV(%)3
Mysidopsis Survival, NOEC
bahia Growth, IC25 Growth, IC50 Growth, NOEC Fecundity, NOEC 34
NA NANA 26 480 3.47 28.9 22 656 3.17 19.3 32 NA NANA 25 NA NANA
Organism Endpoint No. Labs Cu(mg/L)4 SD CV(%)3
Menidia Survival, NOEC
beryllina Growth, IC25 Growth, IC50 Growth, NOEC 19 NA NANA 13
0.144 1.56 43.5 12 0.180 1.87 41.6 17 NA NANA
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
Static renewal test, using 25 ‰ modified GP2 artificial
seawater.
3
Percent coefficient of variation = (standard deviation X
100)/mean.
4
Expressed as mean.
TABLE 2. NATIONAL INTERLABORATORY STUDY OF CHRONIC TOXICITY TEST
PRECISION, 2000: PRECISION OF RESPONSES USING EFFLUENT, RECEIVING
WATER, AND REFERENCE TOXICANT SAMPLE TYPES1

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.
4.15 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 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.


4.16
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 re-calculated 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 0.05 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 fall well outside the expected range for the 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
plans 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 must
be maintained for each individual toxicity test or group of tests
on closely related samples. This file must 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
natural seawater or water made up from hypersaline brine derived
from natural seawater, or water made up from reagent grade
chemicals (GP2) or commercial (FORTY FATHOMS® or HW MARINEMIX®)
artificial sea salts when specifically recommended in the method.
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 of 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. Sample preparation, culturing, and toxicity testing
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., which
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 might 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 as 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, 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 generally does not require thorough cleaning
before use. It is sufficient to rinse new sample containers once
with dilution water before use. New, disposable, plastic test
chambers may have to be rinsed with dilution water before use. New
glassware must be soaked overnight in 10% acid (see below) and also
should be rinsed well in deionized water and seawater.



5.3.2
All non-disposable sample containers, test vessels,
pumps, tanks, and other equipment that has come in contact with
effluent must be washed after use to remove surface contaminants,
as described below.



1.
Soak 15 minutes 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 acid 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
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
toxicity tests 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 water 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 Food -- TETRAMIN® and BIORIL® or equivalent are
available at most pet supply shops.


4.
Feeding requirements and other specific foods are
indicated in the specific toxicity test method.




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. Also, see Section 4, Quality
Assurance.


5.6
TEST ORGANISMS




5.6.1
Test organisms are obtained from inhouse cultures or
commercial suppliers (see specific toxicity test method; Sections
4, Quality Assurance and 6, Test Organisms).


5.7
SUPPLIES


5.7.1 See toxicity test methods (see Sections 11-16) 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 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 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 tests described in
this manual are the sheepshead minnow, Cyprinodon variegatus; the
inland silverside, Menidia beryllina; the mysid, Mysidopsis bahia;
the sea urchin, Arbacia punctulata; and the red macroalga, Champia
parvula.


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 species in
Subsection 6.1.3. Where state regulations prohibit importation of
non-native fishes or use of the 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 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. 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.
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 should be purchased from
experienced commercial suppliers (see USEPA, 1993b).


6.2.3
Sheepshead minnows, inland silversides, mysids, and sea
urchins may be purchased from commercial suppliers. However, some
of these organisms (e.g., adult sheepshead minnows or adult inland
silversides) may not always be available from commercial suppliers
and may have to be collected in the field and brought back to the
laboratory for spawning to obtain eggs and larvae.


6.2.4
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 toxicity test methods.


6.2.5
FERAL (NATURAL OCCURRING, WILD CAUGHT)
ORGANISMS


6.2.5.1 The use of test organisms taken from the receiving water
has strong appeal, and would seem to be the logical approach.
However, it is 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 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 sheepshead minnows
and silversides, and invertebrates such as 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 be 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 ensure
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.




6.2.5.2
Guidelines for collection of natural occurring organisms
are provided in USEPA (1973); USEPA (1990a); and USEPA
(1993b).


6.2.6
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 juvenile mysids and 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 specified 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 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 ID) with rubber
bulbs or pipettors (such as a PROPIPETTE® or other pipettor) should
be used for transferring smaller organisms such as mysids, and
larval fish.


6.5.3
Holding tanks for fish are supplied with a good quality
water (see Section 5, Facilities, Equipment, and Supplies) 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 photo-degrade
dissolved organics.


6.5.4
Crowding should be avoided because it will stress the
organisms and lower the DO concentrations to unacceptable levels.
The DO must be maintained at a minimum of 4.0 mg/L. The solubility
of oxygen depends on temperature, salinity, and altitude. 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 (510%) 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. 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.


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 minute 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
The marine organisms can be used at all concentrations of
effluent by adjusting the salinity of the effluent to salinities
specified for the appropriate species test condition 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. The change
in salinity upon acclimation at the desired test dilution should
not exceed 6‰. The required salinities for culturing and toxicity
tests with estuarine and marine species are listed in the test
method sections.


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.
SECTION 7
DILUTION WATER
7.1 TYPES OF DILUTION WATER
7.1.1 The type of dilution water used in effluent toxicity tests
will depend largely on the objectives of the study.


7.1.1.1
If the objective of the test is to estimate the absolute
chronic toxicity of the effluent, a synthetic (standard) dilution
water is used. If the test organisms have been cultured in water
which is different from the test dilution water, a second set of
controls, using culture water, should be included in the
test.


7.1.1.2
If the objective of the test is to estimate the chronic
toxicity of the effluent in uncontaminated receiving water, the
test may be conducted using dilution water consisting of a single
grab sample of receiving water (if non-toxic), collected outside
the influence of the outfall, or with other uncontaminated natural
water (surface water) or standard dilution water having
approximately the same salinity as the receiving water. Seasonal
variations in the quality of receiving waters may affect effluent
toxicity. Therefore, the salinity of saline receiving water samples
should be determined before each use. If the test organisms have
been cultured in water which is different from the test dilution
water, a second set of controls, using culture water, should be
included in the test.


7.1.1.3
If the objective of the test is to determine the additive
or mitigating effects of the discharge on already contaminated
receiving water, the test is performed using dilution water
consisting of receiving water collected outside the influence of
the outfall. A second set of controls, using culture water, should
be included in the test.


7.1.2
An acceptable dilution water is one which is appropriate
for the objectives of the test; supports adequate performance of
the test organisms with respect to survival, growth, reproduction,
or other responses that may be measured in the test (i.e.,
consistently meets test acceptability criteria for control
responses); is consistent in quality; and does not contain
contaminants that could produce toxicity. Receiving waters,
synthetic waters, or synthetic waters adjusted to approximate
receiving water characteristics may be used for dilution provided
that the water meets the above listed qualifications for an
acceptable dilution water. USEPA (2000a) provides additional
guidance on selecting appropriate dilution waters.


7.1.3
When dual controls (one control
using culture water and one control using dilution water) are used
(see Subsections 7.1.1.1 - 7.1.1.3 above), the dilution water
control should be used to determine test acceptability. It is also
the dilution water control that should be compared to effluent
treatments in the calculation and reporting of test results. The
culture water control should be used to evaluate the
appropriateness of the dilution water source. Significant
differences between organism responses in culture water and
dilution water controls could indicate toxicity in the dilution
water and may suggest an alternative dilution water source. USEPA
(2000a) provides additional guidance on dual controls.


7.2
STANDARD, SYNTHETIC DILUTION WATER




7.2.1
Standard, synthetic, dilution water is prepared with
deionized water and reagent grade chemicals (GP2) or commercial sea
salts (FORTY FATHOMS®, HW MARINEMIX®) (Table 3). The source water
for the deionizer can be ground water or tap water.


7.2.2
DEIONIZED WATER USED TO PREPARE STANDARD, SYNTHETIC,
DILUTION WATER




7.2.2.1
Deionized water is obtained from a MILLIPORE MILLI-Q®,
MILLIPORE® QPAK™2 or equivalent system. It is advisable to provide
a preconditioned (deionized) feed water by using a Culligan®,
Continental®, or equivalent system in front of the MILLI-Q® System
to extend the life of the MILLI-Q® cartridges (see Section 5,
Facilities, Equipment, and Supplies).


7.2.2.2
The recommended order of the cartridges in a
four-cartridge deionizer (i.e., MILLI-Q® System or equivalent) is:
(1) ion exchange, (2) ion exchange, (3) carbon, and (4) organic
cleanup (such as ORGANEX-Q®, or equivalent), followed by a final
bacteria filter. The QPAK™2 water system is a sealed system which
does not allow for the rearranging of the cartridges. However, the
final cartridge is an ORGANEX-Q® filter, followed by a final
bacteria filter. Commercial laboratories using this system have not
experienced any difficulty in using the water for culturing or
testing. Reference to the MILLI-Q® systems throughout the remainder
of the manual includes all MILLIPORE® or equivalent
systems.


7.2.3
STANDARD, SYNTHETIC SEAWATER


7.2.3.1 To prepare 20 L of a standard, synthetic, reconstituted
seawater (modified GP2), using reagent grade chemicals (Table 3),
with a salinity of 31‰, follow the instructions below. Other
salinities can be prepared by making the appropriate dilutions.
Larger or smaller volumes of modified GP2 can be prepared by using
proportionately larger or smaller amounts of salts and dilution
water.


1.
Place 20 L of MILLI-Q® or equivalent deionized water in a
properly cleaned plastic carboy.


2.
Weigh reagent grade salts listed in Table 3 and add, one
at a time, to the deionized water. Stir well after adding each
salt.


3.
Aerate the final solution at a rate of 1 L/h for 24
h.


4.
Check the pH and salinity.




7.2.3.2
Synthetic seawater can also be prepared by adding
commercial sea salts, such as FORTY FATHOMS®, HW MARINEMIX®, or
equivalent, to deionized water. For example, thirty-one parts per
thousand (31‰) FORTY FATHOMS® can be prepared by dissolving 31 g of
sea salts per liter of deionized water. The salinity of the
resulting solutions should be checked with a
refractometer.


7.2.4
Artificial seawater is to be used only if specified in
the method. EMSL-Cincinnati has found FORTY FATHOMS® artificial sea
salts suitable for maintaining and spawning the sheepshead minnow,
Cyprinodon variegatus, and for its use in the sheepshead minnow
larval survival and growth test, suitable for maintaining and
spawning the inland silverside, Menidia beryllina, and for its use
in the inland silverside larval survival and growth test, suitable
for culturing and maintaining mysid shrimp, Mysidopsis bahia, and
its use in the mysid shrimp survival, growth, and fecundity test,
and suitable for maintaining sea urchins, Arbacia punctulata, and
for its use in the sea urchin fertilization test. The USEPA Region
6 Houston Laboratory has successfully used HW MARINEMIX® sea salts
to maintain and spawn sheepshead minnows, and perform the larval
survival and growth test and the embryo-larval survival and
teratogenicity test. Also, HW MARINEMIX® sea salts has been used
successfully to culture and maintain the mysid brood stock and
perform the mysid survival, growth, fecundity test. An artificial
seawater formulation, GP2 (Spotte et al., 1984), Table 3, has been
used by the Environmental Research Laboratory-Narragansett, RI for
all but the embryolarval survival and teratogenicity test. The
suitability of GP2 as a medium for culturing organisms has not been
determined.


TABLE 3. PREPARATION OF GP2 ARTIFICIAL SEAWATER USING REAGENT
GRADE CHEMICALS1,2,3

1
Modified GP2 from Spotte et al. (1984).
2
The constituent salts and concentrations were taken from USEPA
(2002a). The salinity is 30.89 g/L.
3
GP2 can be diluted with deionized (DI) water to the desired test
salinity.
7.3 USE OF RECEIVING WATER AS DILUTION WATER


7.3.1
If the objectives of the test require the use of
uncontaminated receiving water as dilution water, and the receiving
water is uncontaminated, it may be possible to collect a sample of
the receiving water close to the outfall, but should be away from
or beyond the influence of the effluent. However, if the receiving
water is contaminated, it may be necessary to collect the sample in
an area "remote" from the discharge site, matching as closely as
possible the physical and chemical characteristics of the receiving
water near the outfall.


7.3.2
The sample should be collected immediately prior to the
test, but never more than 96 h before the test begins. Except where
it is used within 24 h, or in the case where large volumes are
required for flow through tests, the sample should be chilled to
0-6°C during or immediately following collection, and maintained at
that temperature prior to use in the test.


7.3.3
The investigator should collect uncontaminated water
having a salinity as near as possible to the salinity of the
receiving water at the discharge site. Water should be collected at
slack high tide, or within one hour after high tide. If there is
reason to suspect contamination of the water in the estuary, it is
advisable to collect uncontaminated water from an adjacent estuary.
At times it may be necessary to collect water at a location closer
to the open sea, where the salinity is relatively high. In such
cases, deionized water or uncontaminated freshwater is added to the
saline water to dilute it to the required test salinity. Where
necessary, the salinity of a surface water can be increased by the
addition of artificial sea salts, such as FORTY FATHOMS®, HW
MARINEMIX®, or equivalent, GP2, a


natural seawater of higher salinity, or hypersaline brine.
Instructions for the preparation of hypersaline brine by
concentrating natural seawater are provided below.


7.3.4
Receiving water containing debris or indigenous
organisms, that may be confused with or attack the test organisms,
should be filtered through a sieve having 60 µm mesh openings prior
to use.


7.3.5
HYPERSALINE BRINE




7.3.5.1
Hypersaline brine (HSB) has several advantages that make
it desirable for use in toxicity testing. It can be made from any
high quality, filtered seawater by evaporation, and can be added to
deionized water to prepare dilution water, or to effluents or
surface waters to increase their salinity.


7.3.5.2
The ideal container for making HSB from natural seawater
is one that (l) has a high surface to volume ratio,


(2) is made of a noncorrosive material, and (3) is easily
cleaned (fiberglass containers are ideal). Special care should be
used to prevent any toxic materials from coming in contact with the
seawater being used to generate the brine. If a heater is immersed
directly into the seawater, ensure that the heater materials do not
corrode or leach any substances that would contaminate the brine.
One successful method used is a thermostatically controlled heat
exchanger made from fiberglass. If aeration is used, use only
oil-free air compressors to prevent contamination.


7.3.5.3
Before adding seawater to the brine generator, thoroughly
clean the generator, aeration supply tube, heater, and any other
materials that will be in direct contact with the brine. A good
quality biodegradable detergent should be used, followed by several
thorough deionized water rinses. High quality (and preferably high
salinity) seawater should be filtered to at least 10 mm before
placing into the brine generator. Water should be collected on an
incoming tide to minimize the possibility of
contamination.


7.3.5.4
The temperature of the seawater is increased slowly to
40°C. The water should be aerated to prevent temperature
stratification and to increase water evaporation. The brine should
be checked daily (depending on the volume being generated) to
ensure that the salinity does not exceed 100‰ and that the
temperature does not exceed 40°C. Additional seawater may be added
to the brine to obtain the volume of brine required.


7.3.5.5
After the required salinity is attained, the HSB should
be filtered a second time through a l-µm filter and poured directly
into portable containers (20-L CUBITAINERS® or polycarbonate water
cooler jugs are suitable). The containers should be capped and
labelled with the date the brine was generated and its salinity.
Containers of HSB should be stored in the dark and maintained under
room temperature until used.


7.3.5.6
If a source of HSB is available, test solutions can be
made by following the directions below. Thoroughly mix together the
deionized water and brine before mixing in the effluent.


7.3.5.7
Divide the salinity of the HSB by the expected test
salinity to determine the proportion of deionized water to brine.
For example, if the salinity of the brine is 100‰ and the test is
to be conducted at 25‰, 100‰ divided by 25‰ = 4.0. The proportion
of brine is 1 part in 4 (one part brine to three parts deionized
water).


7.3.5.8
To make 1 L of seawater at 25‰ salinity from a
hypersaline brine of 100‰, 250 mL of brine and 750 mL of deionized
water are required.


7.4
USE OF TAP WATER AS DILUTION WATER


7.4.1 The use of tap water in the reconstituting of synthetic
(artificial) seawater as dilution water is discouraged unless it is
dechlorinated and fully treated. Tap water can be dechlorinated by
deionization, carbon filtration, or the use of sodium thiosulfate.
Use of 3.6 mg/L (anhydrous) sodium thiosulfate will reduce 1.0 mg
chlorine/L (APHA, 1992). Following dechlorination, total residual
chlorine should not exceed 0.01 mg/L. Because of the possible
toxicity of thiosulfate to test organisms, a control lacking
thiosulfate should be included in toxicity tests utilizing
thiosulfate-dechlorinated water.


7.4.2
To be adequate for general laboratory use following
dechlorination, the tap water is passed through a deionizer and
carbon filter to remove toxic metals and organics, and to control
hardness and alkalinity.


7.5
DILUTION WATER HOLDING


7.5.1 A given batch of dilution water should not be used for
more than 14 days following preparation because of the possible
build up of bacterial, fungal, or algal slime growth and the
problems associated with it. The container should be kept covered
and the contents should be protected from light.
SECTION 8
EFFLUENT AND RECEIVING WATER SAMPLING, SAMPLE HANDLING, AND
SAMPLE PREPARATION FOR TOXICITY TESTS
8.1 EFFLUENT SAMPLING


8.1.1
The effluent sampling point should be the same as that
specified in the NPDES discharge permit (USEPA, l988b). Conditions
for exception would be: (l) better access to a sampling point
between the final treatment and the discharge outfall; (2) if the
processed waste is chlorinated prior to discharge, it may also be
desirable to take samples prior to contact with the chlorine to
determine toxicity of the unchlorinated effluent; or (3) in the
event there is a desire to evaluate the toxicity of the influent to
municipal waste treatment plants or separate wastewater streams in
industrial facilities prior to their being combined with other
wastewater streams or non-contact cooling water, additional
sampling points may be chosen.


8.1.2
The decision on whether to collect grab or composite
samples is based on the objectives of the test and an understanding
of the short and long-term operations and schedules of the
discharger. If the effluent quality varies considerably with time,
which can occur where holding times are short, grab samples may
seem preferable because of the ease of collection and the potential
of observing peaks (spikes) in toxicity. However, the sampling
duration of a grab sample is so short that full characterization of
an effluent over a 24-h period would require a prohibitively large
number of separate samples and tests. Collection of a 24-h
composite sample, however, may dilute toxicity spikes, and average
the quality of the effluent over the sampling period. Sampling
recommendations are provided below (also see USEPA,
2002a).


8.1.3
Aeration during collection and transfer of effluents
should be minimized to reduce the loss of volatile
chemicals.


8.1.4
Details of date, time, location, duration, and procedures
used for effluent sample and dilution water collection should be
recorded.


8.2
EFFLUENT SAMPLE TYPES


8.2.1 The advantages and disadvantages of effluent grab and
composite samples are listed below:
8.2.1.1 GRAB SAMPLES Advantages:


1.
Easy to collect; require a minimum of equipment and
on-site time.


2.
Provide a measure of instantaneous toxicity. Toxicity
spikes are not masked by dilution. Disadvantages:


1. Samples are collected over a very short period of time and on
a relatively infrequent basis. The chances of detecting a spike in
toxicity would depend on the frequency of sampling, and the
probability of missing spikes is high.
8.2.1.2 COMPOSITE SAMPLES: Advantages:


1.
A single effluent sample is collected over a 24-h
period.


2.
The sample is collected over a much longer period of time
than grab samples and contains all toxicity spikes.


Disadvantages:


1.
Sampling equipment is more sophisticated and expensive,
and must be placed on-site for at least 24 h.


2.
Toxicity spikes may not be detected because they are
masked by dilution with less toxic wastes.


8.3 EFFLUENT SAMPLING RECOMMENDATIONS


8.3.1
When tests are conducted on-site, test solutions can be
renewed daily with freshly collected samples.


8.3.2
When tests are conducted off-site, a minimum of three
samples are collected. If these samples are collected on Test Days
1, 3, and 5, the first sample would be used for test initiation,
and for test solution renewal on Day 2. The second sample would be
used for test solution renewal on Days 3 and 4. The third sample
would be used for test solution renewal on Days 5, 6, and
7.


8.3.3
Sufficient sample must be collected to perform the
required toxicity and chemical tests. A 4-L (1-gal) CUBITAINER®
will provide sufficient sample volume for most tests.


8.3.4
THE FOLLOWING EFFLUENT SAMPLING METHODS ARE
RECOMMENDED:


8.3.4.1 Continuous Discharges


8.3.4.1.1
If the facility discharge is continuous, a single 24-h
composite sample is to be taken.


8.3.4.2
Intermittent Discharges




8.3.4.2.1
If the facility discharge is intermittent, a composite
sample is to be collected for the duration of the discharge but not
more than 24 hours.


8.4
RECEIVING WATER SAMPLING




8.4.1
Logistical problems and difficulty in securing sampling
equipment generally preclude the collection of composite receiving
water samples for toxicity tests. Therefore, based on the
requirements of the test, a single grab sample or daily grab
samples of receiving water is collected for use in the
test.


8.4.2
The sampling point is determined by the objectives of the
test. At estuarine and marine sites, samples should be collected at
mid-depth.


8.4.3
To determine the extent of the zone of toxicity in the
receiving water at estuarine and marine effluent sites, receiving
water samples are collected at several distances away from the
discharge. The time required for the effluent-receiving-water
mixture to travel to sampling points away from the effluent, and
the rate and degree of mixing, may be difficult to ascertain.
Therefore, it may not be possible to correlate receiving water
toxicity with effluent toxicity at the discharge point unless a dye
study is performed. The toxicity of receiving water samples from
five stations in the discharge plume can be evaluated using the
same number of test vessels and test organisms as used in one
effluent toxicity test with five effluent dilutions.


8.5
EFFLUENT AND RECEIVING WATER SAMPLE HANDLING,
PRESERVATION, AND SHIPPING




8.5.1
Unless the samples are used in an on-site toxicity test
the day of collection (or hand delivered to the testing laboratory
for use on the day of collection), it is recommended that they be
held at 0-6°C until used to inhibit microbial degradation, chemical
transformations, and loss of highly volatile toxic
substances.


8.5.2
Composite samples should be chilled as they are
collected. Grab samples should be chilled immediately following
collection.


8.5.3
If the effluent has been chlorinated, total residual
chlorine must be measured immediately following sample
collection.


8.5.4
Sample holding time begins when the last grab sample in a
series is taken (i.e., when a series of four grab samples are taken
over a 24-h period), or when a 24-h composite sampling period is
completed. If the data from the samples are to be acceptable for
use in the NPDES Program, the lapsed time (holding time) from
sample collection to first use of each grab or composite sample
must not exceed 36 h. EPA believes that 36 h is adequate time to
deliver the sample to the laboratories performing the test in most
cases. In the isolated cases, where the permittee can document that
this delivery time cannot be met, the permitting authority can
allow an option for on-site testing or a variance for an extension
of shipped sample holding time. The request for a variance in
sample holding time, directed to the USEPA Regional Administrator
under 40 CFR 136.3(e), should include supportive data which show
that the toxicity of the effluent sample is not reduced (e.g.,
because of volatilization and/or sorption of toxics on the sample
container surfaces) by extending the holding time beyond more than
36 h. However, in no case should more than 72 h elapse between
collection and first use of the sample. In static-renewal tests,
each grab or composite sample may also be used to prepare test
solutions for renewal at 24 h and/or 48 h after first use, if
stored at 0-6°C, with minimum head space, as described in
Subsection 8.5. If shipping problems (e.g., unsuccessful Saturday
delivery) are encountered with renewal samples after a test has
been initiated, the permitting authority may allow the continued
use of the most recently used sample for test renewal. Guidance for
determining the persistence of the sample is provided in Subsection
8.7.


8.5.5
To minimize the loss of toxicity due to volatilization of
toxic constituents, all sample containers should be "completely"
filled, leaving no air space between the contents and the
lid.


8.5.6
SAMPLES USED IN ON-SITE TESTS




8.5.6.1
Samples collected for on-site tests should be used within
24 h.


8.5.7
SAMPLES SHIPPED TO OFF SITE FACILITIES




8.5.7.1
Samples collected for off site toxicity testing are to be
chilled to 0-6°C during or immediately after collection, and
shipped iced to the performing laboratory. Sufficient ice should be
placed with the sample in the shipping container to ensure that ice
will still be present when the sample arrives at the laboratory and
is unpacked. Insulating material should not be placed between the
ice and the sample in the shipping container unless required to
prevent breakage of glass sample containers.


8.5.7.2
Samples may be shipped in one or more 4-L (l-gal)
CUBITAINERS® or new plastic "milk" jugs. All sample containers
should be rinsed with source water before being filled with sample.
After use with receiving water or effluents, CUBITAINERS® and
plastic jugs are punctured to prevent reuse.


8.5.7.3
Several sample shipping options are available, including
Express Mail, air express, bus, and courier service. Express Mail
is delivered seven days a week. Saturday and Sunday shipping and
receiving schedules of private carriers vary with the
carrier.


8.6
SAMPLE RECEIVING




8.6.1
Upon arrival at the laboratory, samples are logged in and
the temperature is measured and recorded. If the samples are not
immediately prepared for testing, they are stored at 0-6°C until
used.


8.6.2
Every effort must be made to initiate the test with an
effluent sample on the day of arrival in the laboratory, and the
sample holding time should not exceed 36 h unless a variance has
been granted by the NPDES permitting authority.


8.7
PERSISTENCE OF EFFLUENT TOXICITY DURING SAMPLE SHIPMENT
AND HOLDING




8.7.1
The persistence of the toxicity of
an effluent prior to its use in a toxicity test is of interest in
assessing the validity of toxicity test data, and in determining
the possible effects of allowing an extension of the holding time.
Where a variance in holding time (> 36 h, but # 72 h) is
requested by a permittee (See Subsection 8.5.4), information on the
effects of the extension in holding time on the toxicity of the
samples must be obtained by comparing the results of
multi-concentration chronic toxicity tests performed on effluent
samples held 36 h with toxicity test results using the same samples
after they were held for the requested, longer period. The portion
of the sample set aside for the second test must be held under the
same conditions as during shipment and holding.


8.8
PREPARATION OF EFFLUENT AND RECEIVING WATER SAMPLES FOR
TOXICITY TESTS




8.8.1
Adjust the sample salinity to the level appropriate for
objectives of the study using hypersaline brine or artificial sea
salts.


8.8.2
When aliquots are removed from the sample container, the
head space above the remaining sample should be held to a minimum.
Air which enters a container upon removal of sample should be
expelled by compressing the container before reclosing, if possible
(i.e., where a CUBITAINER® used), or by using an appropriate
discharge valve (spigot).


8.8.3
It may be necessary to first coarse-filter samples
through a NYLON® sieve having 2 to 4 mm mesh openings to remove
debris and/or break up large floating or suspended solids. If
samples contain indigenous organisms that may attack or be confused
with the test organisms, the samples should be filtered through a
sieve with 60-µm mesh openings. Since filtering may increase the
dissolved oxygen (DO) in an effluent, the DO should be checked both
before and after filtering. Low dissolved oxygen concentrations
will indicate a potential problem in performing the test. Caution:
filtration may remove some toxicity.


8.8.4
If the samples must be warmed to bring them to the
prescribed test temperature, supersaturation of the dissolved
oxygen and nitrogen may become a problem. To avoid this problem,
samples may be warmed slowly in open test containers. If DO is
still above 100% saturation, based on temperature and salinity
(Table 4), after warming to test temperature, samples should be
aerated moderately (approximately 500 mL/min) for a few minutes
using an airstone. If DO is below 4.0 mg/L, the solutions must be
aerated moderately (approximately 500 mL/min) for a few minutes,
using an airstone, until the DO is within the prescribed range ($
4.0 mg/L). Caution: avoid excessive aeration.




8.8.4.1
Aeration during the test may alter the results and should
be used only as a last resort to maintain the required DO. Aeration
can reduce the apparent toxicity of the test solutions by stripping
them of highly volatile toxic substances, or increase their
toxicity by altering the pH. However, the DO in the test solution
should not be permitted to fall below 4.0 mg/L.


8.8.4.2
In static tests (non-renewal or renewal) low DOs may
commonly occur in the higher concentrations of wastewater. Aeration
is accomplished by bubbling air through a pipet at the rate of 100
bubbles/min. If aeration is necessary, all test solutions must be
aerated. It is advisable to monitor the DO closely during the first
few hours of the test. Samples with a potential DO problem
generally show a downward trend in DO within 4 to 8 h after the
test


is started. Unless aeration is initiated during the first 8 h of
the test, the DO may be exhausted during an unattended period,
thereby invalidating the test.
8.8.5 At a minimum, pH, conductivity or salinity, and total
residual chlorine are measured in the undiluted effluent or
receiving water, and pH and conductivity are measured in the
dilution water.


8.8.5.1
It is recommended that total alkalinity and total
hardness also be measured in the undiluted effluent test water and
the dilution water.


8.8.6
Total ammonia is measured in effluent and receiving water
samples where toxicity may be contributed by unionized ammonia
(i.e., where total ammonia $ 5 mg/L). The concentration (mg/L) of
unionized (free) ammonia in a sample is a function of temperature
and pH, and is calculated using the percentage value obtained from
Table 5, under the appropriate pH and temperature, and multiplying
it by the concentration (mg/L) of total ammonia in the
sample.


8.8.7
Effluents and receiving waters can be dechlorinated using
6.7 mg/L anhydrous sodium thiosulfate to reduce 1 mg/L chlorine
(APHA, 1992). Note that the amount of thiosulfate required to
dechlorinate effluents is greater than the amount needed to
dechlorinate tap water, (see Section 7, Dilution Water). Since
thiosulfate may contribute to sample toxicity, a thiosulfate
control should be used in the test in addition to the normal
dilution water control.


8.8.8
The DO concentration in the samples should be near
saturation prior to use. Aeration will bring the DO and other gases
into equilibrium with air, minimize oxygen demand, and stabilize
the pH. However, aeration during collection, transfer, and
preparation of samples should be minimized to reduce the loss of
volatile chemicals.


8.8.9
Mortality or impairment of growth or reproduction due to
pH alone may occur if the pH of the sample falls outside the range
of 6.0 - 9.0. Thus, the presence of other forms of toxicity (metals
and organics) in the sample may be masked by the toxic effects of
low or high pH. The question about the presence of other toxicants
can be answered only by performing two parallel tests, one with an
adjusted pH, and one without an adjusted pH. Freshwater samples are
adjusted to pH 7.0, and marine samples are adjusted to pH 8.0, by
adding 1N NaOH or 1N HCl dropwise, as required, being careful to
avoid overadjustment.





Table provided by Teresa Norberg-King, Duluth, Minnesota. Also
see Emerson et al. (1975), Thurston et al. (1974), and USEPA
(1985a).
8.9 PRELIMINARY TOXICITY RANGE-FINDING
TESTS


8.9.1
USEPA Regional and State personnel generally have
observed that it is not necessary to conduct a toxicity
range-finding test prior to initiating a static, chronic,
definitive toxicity test. However, when preparing to perform a
static test with a sample of completely unknown quality, or before
initiating a flow-through test, it is advisable to conduct a
preliminary toxicity range-finding test.


8.9.2
A toxicity range-finding test ordinarily consists of a
down-scaled, abbreviated static acute test in which groups of five
organisms are exposed to several widely-spaced sample dilutions in
a logarithmic series, such as 100%, 10.0%, 1.00%, and 0.100%, and a
control, for 8-24 h. Caution: if the sample must also be used for
the fullscale definitive test, the 36-h limit on holding time (see
Subsection 8.5.4) must not be exceeded before the definitive test
is initiated.


8.9.3
It should be noted that the toxicity (LC50) of a sample
observed in a range-finding test may be significantly different
from the toxicity observed in the follow-up, chronic, definitive
test because: (1) the definitive test is longer; and (2) the test
may be performed with a sample collected at a different time, and
possibly differing significantly in the level of
toxicity.


8.10 MULTICONCENTRATION (DEFINITIVE)
EFFLUENT TOXICITY TESTS


8.10.1
The tests recommended for use in determining discharge
permit compliance in the NPDES program are multiconcentration, or
definitive, tests which provide (1) a point estimate of effluent
toxicity in terms of an IC25, IC50, or LC50, or (2) a
no-observed-effect-concentration (NOEC) defined in terms of
mortality, growth, reproduction, and/or teratogenicity and obtained
by hypothesis testing. The tests may be static renewal or static
non-renewal.


8.10.2
The tests consist of a control and a minimum of five
effluent concentrations. USEPA recommends the use of a $0.5
dilution factor for selecting effluent test concentrations.
Effluent test concentrations of 6.25%, 12.5%, 25%, 50%, and 100%
are commonly used, however, test concentrations should be selected
independently for each test based on the objective of the study,
the expected range of toxicity, the receiving water concentration,
and any available historical testing information on the effluent.
USEPA (2000a) provides additional guidance on choosing appropriate
test concentrations.


8.10.3
When these tests are used in
determining compliance with permit limits, effluent test
concentrations should be selected to bracket the receiving water
concentration. This may be achieved by selecting effluent test
concentrations in the following manner: (1) 100% effluent, (2) [RWC
+ 100]/2, (3) RWC, (4) RWC/2, and (5) RWC/4. For example, where the
RWC = 50%, appropriate effluent concentrations may be 100%, 75%,
50%, 25%, and 12.5%.


8.10.4
If acute/chronic ratios are to be determined by
simultaneous acute and short-term chronic tests with a single
species, using the same sample, both types of tests must use the
same test conditions, i.e., pH, temperature, water hardness,
salinity, etc.


8.11
RECEIVING WATER TESTS




8.11.1
Receiving water toxicity tests generally consist of 100%
receiving water and a control. The total salinity of the control
should be comparable to the receiving water.


8.11.2
The data from the two treatments are analyzed by
hypothesis testing to determine if test organism survival in the
receiving water differs significantly from the control. Four
replicates and 10 organisms per replicate are required for each
treatment (see Summary of Test Conditions and Test Acceptability
Criteria in the specific test method).


8.11.3
In cases where the objective of the test is to estimate
the degree of toxicity of the receiving water, a definitive,
multiconcentration test is performed by preparing dilutions of the
receiving water, using a $0.5 dilution series, with a suitable
control water.


SECTION 9
CHRONIC TOXICITY TEST ENDPOINTS AND DATA
ANALYSIS
9.1 ENDPOINTS
9.1.1 The objective of chronic aquatic toxicity tests with
effluents and pure compounds is to estimate the highest "safe" or
"no-effect concentration" of these substances. For practical
reasons, the responses observed in these tests are usually limited
to hatchability, gross morphological abnormalities, survival,
growth, and reproduction, and the results of the tests are usually
expressed in terms of the highest toxicant concentration that has
no statistically significant observed effect on these responses,
when compared to the controls. The terms currently used to define
the endpoints employed in the rapid, chronic and sub-chronic
toxicity tests have been derived from the terms previously used for
full life-cycle tests. As shorter chronic tests were developed, it
became common practice to apply the same terminology to the
endpoints. The terms used in this manual are as follows:


9.1.1.1
Safe Concentration - The highest concentration of
toxicant that will permit normal propagation of fish and other
aquatic life in receiving waters. The concept of a "safe
concentration" is a biological concept, whereas the
"no-observed-effect concentration" (below) is a statistically
defined concentration.


9.1.1.2
No-Observed-Effect-Concentration (NOEC) - The highest
concentration of toxicant to which organisms are exposed in a full
life-cycle or partial life-cycle (short-term) test, that causes no
observable adverse effects on the test organisms (i.e., the highest
concentration of toxicant in which the values for the observed
responses are not statistically significantly different from the
controls). This value is used, along with other factors, to
determine toxicity limits in permits.


9.1.1.3
Lowest-Observed-Effect-Concentration (LOEC) - The lowest
concentration of toxicant to which organisms are exposed in a
life-cycle or partial life-cycle (short-term) test, which causes
adverse effects on the test organisms (i.e., where the values for
the observed responses are statistically significantly different
from the controls).


9.1.1.4
Effective Concentration (EC) - A point estimate of the
toxicant concentration that would cause an observable adverse
affect on a quantal, "all or nothing," response (such as death,
immobilization, or serious incapacitation) in a given percent of
the test organisms, calculated by point estimation techniques. If
the observable effect is death or immobility, the term, Lethal
Concentration (LC), should be used (see Subsection 9.1.1.5). A
certain EC or LC value might be judged from a biological standpoint
to represent a threshold concentration, or lowest concentration
that would cause an adverse effect on the observed
response.


9.1.1.5
Lethal Concentration (LC) - The
toxicant concentration that would cause death in a given percent of
the test population. Identical to EC when the observable adverse
effect is death. For example, the LC50 is the concentration of
toxicant that would cause death in 50% of the test
population.


9.1.1.6
Inhibition Concentration (IC) - The toxicant
concentration that would cause a given percent reduction in a
nonquantal biological measurement for the test population. For
example, the IC25 is the concentration of toxicant that would cause
a 25% reduction in mean young per female or in growth for the test
population, and the IC50 is the concentration of toxicant that
would cause a 50% reduction in the mean population
responses.


9.2
RELATIONSHIP BETWEEN ENDPOINTS DETERMINED BY HYPOTHESIS
TESTING AND POINT ESTIMATION TECHNIQUES


9.2.1 If the objective of chronic aquatic toxicity tests with
effluents and pure compounds is to estimate the highest "safe or
no-effect concentration" of these substances, it is imperative to
understand how the statistical endpoints of these tests are related
to the "safe" or "no-effect" concentration. NOECs and LOECs are
determined by hypothesis testing (Dunnett's Test, a t test with the
Bonferroni adjustment, Steel's Many-One Rank Test, or the Wilcoxon
Rank Sum Test with Bonferroni adjustment), whereas LCs, ICs, and
ECs are determined by point estimation techniques (Probit Analysis,
the Spearman-Karber Method, the Trimmed Spearman-Karber Method, the
Graphical Method or Linear Interpolation Method). There are
inherent differences between the use of a NOEC or LOEC derived from
hypothesis testing to estimate a "safe" concentration, and the use
of a LC, IC, EC, or other point estimates derived from curve
fitting, interpolation, etc.


9.2.2
Most point estimates, such as the LC, IC, or EC are
derived from a mathematical model that assumes a continuous
dose-response relationship. By definition, any LC, IC, or EC value
is an estimate of some amount of adverse effect. Thus the
assessment of a "safe" concentration must be made from a biological
standpoint rather than with a statistical test. In this instance,
the biologist must determine some amount of adverse effect that is
deemed to be "safe," in the sense that from a practical biological
viewpoint it will not affect the normal propagation of fish and
other aquatic life in receiving waters.


9.2.3
The use of NOECs and LOECs, on the other hand, assumes
either (1) a continuous dose-response relationship, or (2) a
non-continuous (threshold) model of the dose-response
relationship.




9.2.3.1
In the case of a continuous dose-response relationship,
it is also assumed that adverse effects that are not "statistically
observable" are also not important from a biological standpoint,
since they are not pronounced enough to test as statistically
significant against some measure of the natural variability of the
responses.


9.2.3.2
In the case of non-continuous dose-response
relationships, it is assumed that there exists a true threshold, or
concentration below which there is no adverse effect on aquatic
life, and above which there is an adverse effect. The purpose of
the statistical analysis in this case is to estimate as closely as
possible where that threshold lies.


9.2.3.3
In either case, it is important to realize that the
amount of adverse effect that is statistically observable (LOEC) or
not observable (NOEC) is highly dependent on all aspects of the
experimental design, such as the number of concentrations of
toxicant, number of replicates per concentration, number of
organisms per replicate, and use of randomization. Other factors
that affect the sensitivity of the test include the choice of
statistical analysis, the choice of an alpha level, and the amount
of variability between responses at a given
concentration.


9.2.3.4
Where the assumption of a continuous dose-response
relationship is made, by definition some amount of adverse effect
might be present at the NOEC, but is not great enough to be
detected by hypothesis testing.


9.2.3.5
Where the assumption of a noncontinuous dose-response
relationship is made, the NOEC would indeed be an estimate of a
"safe" or "no-effect" concentration if the amount of adverse effect
that appears at the threshold is great enough to test as
statistically significantly different from the controls in the face
of all aspects of the experimental design mentioned above. If,
however, the amount of adverse effect at the threshold were not
great enough to test as statistically different, some amount of
adverse effect might be present at the NOEC. In any case, the
estimate of the NOEC with hypothesis testing is always dependent on
the aspects of the experimental design mentioned above. For this
reason, the reporting and examination of some measure of the
sensitivity of the test (either the minimum significant difference
or the percent change from the control that this minimum difference
represents) is extremely important.


9.2.4
In summary, the assessment of a "safe" or "no-effect"
concentration cannot be made from the results of statistical
analysis alone, unless (1) the assumptions of a strict threshold
model are accepted, and (2) it is assumed that the amount of
adverse effect present at the threshold is statistically detectable
by hypothesis testing. In this case, estimates obtained from a
statistical analysis are indeed estimates of a "no-effect"
concentration. If the assumptions are not deemed tenable, then
estimates from a statistical analysis can only be used in
conjunction with an assessment from a biological standpoint of what
magnitude of adverse effect constitutes a "safe" concentration. In
this instance, a "safe" concentration is not necessarily a truly
"no-effect" concentration, but rather a concentration at which the
effects are judged to be of no biological significance.


9.2.5
A better understanding of the relationship between
endpoints derived by hypothesis testing (NOECs) and point
estimation techniques (LCs, ICs, and ECs) would be very helpful in
choosing methods of data analysis. Norberg-King (1991) reported
that the IC25s were comparable to the NOECs for 23 effluent and
reference toxicant data sets analyzed. The data sets included
short-term chronic toxicity tests for the sea urchin, Arbacia
punctulata, the sheepshead minnow, Cyprinodon variegatus, and the
red macroalga, Champia parvula. Birge et al. (1985) reported that
LC1s derived from Probit Analyses of data from short-term
embryo-larval tests with reference toxicants were comparable to
NOECs for several organisms. Similarly, USEPA (1988d) reported that
the IC25s were comparable to the NOECs for a set of daphnia,
Ceriodaphnia dubia chronic tests with a single reference toxicant.
However, the scope of these comparisons was very limited, and
sufficient information is not yet available to establish an overall
relationship between these two types of endpoints, especially when
derived from effluent toxicity test data.


9.3
PRECISION


9.3.1 HYPOTHESIS TESTS
9.3.1.1 When hypothesis tests are used to analyze toxicity test
data, it is not possible to express precision in terms of a
commonly used statistic. The results of the test are given in terms
of two endpoints, the No-Observed-Effect Concentration (NOEC) and
the Lowest-Observed-Effect Concentration (LOEC). The NOEC and LOEC
are limited to the concentrations selected for the test. The width
of the NOEC-LOEC interval is a function of the dilution series, and
differs greatly depending on whether a dilution factor of 0.3 or
0.5 is used in the test design. Therefore, USEPA recommends the use
of the $ 0.5 dilution factor (see Section 4, Quality Assurance). It
is not possible to place confidence limits on the NOEC and LOEC
derived from a given test, and it is difficult to quantify the
precision of the NOEC-LOEC endpoints between tests. If the data
from a series of tests performed with the same toxicant, toxicant
concentrations, and test species, were analyzed with hypothesis
tests, precision could only be assessed by a qualitative comparison
of the NOEC-LOEC intervals, with the understanding that maximum
precision would be attained if all tests yielded the same NOEC-LOEC
interval. In practice, the precision of results of repetitive
chronic tests is considered acceptable if the NOECs vary by no more
than one concentration interval above or below a central tendency.
Using these guidelines, the "normal" range of NOECs from toxicity
tests using a
0.5 dilution factor (two-fold difference between adjacent
concentrations), would be four-fold.
9.3.2 POINT ESTIMATION TECHNIQUES


9.3.2.1
Point estimation techniques have the advantage of
providing a point estimate of the toxicant concentration causing a
given amount of adverse (inhibiting) effect, the precision of which
can be quantitatively assessed (1) within tests by calculation of
95% confidence limits, and (2) across tests by calculating a
standard deviation and coefficient of variation.


9.3.2.2
It should be noted that software
used to calculate point estimates occasionally may not provide
associated 95% confidence intervals. This situation may arise when
test data do not meet specific assumptions required by the
statistical methods, when point estimates are outside of the test
concentration range, and when specific limitations imposed by the
software are encountered. USEPA (2000a) provides guidance on
confidence intervals under these circumstances.


9.4
DATA ANALYSIS


9.4.1 ROLE OF THE STATISTICIAN
9.4.1.1 The use of the statistical methods described in this
manual for routine data analysis does not require the assistance of
a statistician. However, the interpretation of the results of the
analysis of the data from any of the toxicity tests described in
this manual can become problematic because of the inherent
variability and sometimes unavoidable anomalies in biological data.
If the data appear unusual in any way, or fail to meet the
necessary assumptions, a statistician should be consulted. Analysts
who are not proficient in statistics are strongly advised to seek
the assistance of a statistician before selecting the method of
analysis and using any of the results.


9.4.1.2
The statistical methods recommended in this manual are
not the only possible methods of statistical analysis. Many other
methods have been proposed and considered. Certainly there are
other reasonable and defensible methods of statistical analysis for
this kind of toxicity data. Among alternative hypothesis tests
some, like Williams' Test, require additional assumptions, while
others, like the bootstrap methods, require computerintensive
computations. Alternative point estimations approaches most
probably would require the services of a statistician to determine
the appropriateness of the model (goodness of fit), higher order
linear or nonlinear models, confidence intervals for estimates
generated by inverse regression, etc. In addition, point estimation
or regression approaches would require the specification by
biologists or toxicologists of some low level of adverse effect
that would be deemed acceptable or safe. The statistical methods
contained in this manual have been chosen because they are (1)
applicable to most of the different toxicity test data sets for
which they are recommended, (2) powerful statistical tests, (3)
hopefully "easily" understood by nonstatisticians, and (4) amenable
to use without a computer, if necessary.


9.4.2
PLOTTING THE DATA




9.4.2.1
The data should be plotted, both as a preliminary step to
help detect problems and unsuspected trends or patterns in the
responses, and as an aid in interpretation of the results. Further
discussion and plotted sets of data are included in the methods and
the Appendices.


9.4.3
DATA TRANSFORMATIONS




9.4.3.1
Transformations of the data, (e.g., arc sine square root
and logs), are used where necessary to meet assumptions of the
proposed analyses, such as the requirement for normally distributed
data.


9.4.4
INDEPENDENCE, RANDOMIZATION, AND OUTLIERS




9.4.4.1
Statistical independence among observations is a critical
assumption in all statistical analysis of toxicity data. One of the
best ways to ensure independence is to properly follow rigorous
randomization procedures. Randomization techniques should be
employed at the start of the test, including the randomization of
the placement of test organisms in the test chambers and
randomization of the test chamber location within the array of
chambers. Discussions of statistical independence, outliers and
randomization, and a sample randomization scheme, are included in
Appendix A.


9.4.5
REPLICATION AND SENSITIVITY




9.4.5.1
The number of replicates employed for each toxicant
concentration is an important factor in determining the sensitivity
of chronic toxicity tests. Test sensitivity generally increases as
the number of replicates is increased, but the point of diminishing
returns in sensitivity may be reached rather quickly. The level of
sensitivity required by a hypothesis test or the confidence
interval for a point estimate will determine the number of
replicates, and should be based on the objectives for obtaining the
toxicity data.


9.4.5.2
In a statistical analysis of toxicity data, the choice of
a particular analysis and the ability to detect departures from the
assumptions of the analysis, such as the normal distribution of the
data and homogeneity of variance, is also dependent on the number
of replicates. More than the minimum number of replicates may be
required in situations where it is imperative to obtain optimal
statistical results, such as with tests used in enforcement cases
or when it is not possible to repeat the tests. For example, when
the data are analyzed by hypothesis testing, the nonparametric
alternatives cannot be used unless there are at least four
replicates at each toxicant concentration.


9.4.6
RECOMMENDED ALPHA LEVELS




9.4.6.1
The data analysis examples included in the manual specify
an alpha level of 0.01 for testing the assumptions of hypothesis
tests and an alpha level of 0.05 for the hypothesis tests
themselves. These levels are common and well accepted levels for
this type of analysis and are presented as a recommended minimum
significance level for toxicity data analysis.


9.5
CHOICE OF ANALYSIS




9.5.1
The recommended statistical analysis of most data from
chronic toxicity tests with aquatic organisms follows a decision
process illustrated in the flowchart in Figure 2. An initial
decision is made to use point estimation techniques (the Probit
Analysis, the Spearman-Karber Method, the Trimmed Spearman-Karber
Method, the Graphical Method, or Linear Interpolation Method)
and/or to use hypothesis testing (Dunnett's Test, the t test with
the Bonferroni adjustment, Steel's Many-one Rank Test, or Wilcoxon
Rank Sum Test with the Bonferroni adjustment). NOTE: For the NPDES
Permit Program, the point estimation techniques are the preferred
statistical methods in calculating end points for effluent toxicity
tests. If hypothesis testing is chosen, subsequent decisions are
made on the appropriate procedure for a given set of data,
depending on the results of tests of assumptions, as illustrated in
the flowchart. A specific flow chart is included in the analysis
section for each test.


9.5.2
Since a single chronic toxicity test might yield
information on more than one parameter (such as survival, growth,
and reproduction), the lowest estimate of a "no-observed-effect
concentration" for any of the responses would be used as the "no
observed effect concentration" for each test. It follows logically
that in the statistical analysis of the data, concentrations that
had a significant toxic effect on one of the observed responses
would not be subsequently tested for an effect on some other
response. This is one reason for excluding concentrations that have
shown a statistically significant reduction in survival from a
subsequent hypothesis test for effects on another parameter such as
reproduction. A second reason is that the exclusion of such
concentrations usually results in a more powerful and appropriate
statistical analysis. In performing the point estimation techniques
recommended in this manual, an all-data approach is used. For
example, data from concentrations above the NOEC for survival are
included in determining ICp estimates using the Linear
Interpolation Method.


9.5.3
ANALYSIS OF GROWTH AND REPRODUCTION DATA




9.5.3.1
Growth data from the sheepshead minnow, Cyprinodon
variegatus, and inland silverside, Menidia beryllina, larval
survival and growth tests, and the mysid, Mysidopsis bahia,
survival, growth, and fecundity test, are analyzed using hypothesis
testing according to the flowchart in Figure 2. The above mentioned
growth data may also be analyzed by generating a point estimate
with the Linear Interpolation Method. Data from effluent
concentrations that have tested significantly different from the
control for survival are excluded from further hypothesis tests
concerning growth effects. Growth is defined as the change in dry
weight of the orginal number of test organisms when group weights
are obtained. When analyzing the data using point estimating
techniques, data from all concentrations are included in the
analysis.


9.5.3.2
Fecundity data from the mysid, Mysidopsis bahia, test may
be analyzed using hypothesis testing after an arc sine
transformation according to the flowchart in Figure 2. The
fecundity data from the mysid test may also be analyzed by
generating a point estimate with the Linear Interpolation
Method.


9.5.3.3
Reproduction data from the red macroalga, Champia
parvula, test are analyzed using hypothesis testing as illustrated
in Figure 2. The reproduction data from the red macroalga test may
also be analyzed by generating a point estimate with the Linear
Interpolation Method.


9.5.4
ANALYSIS OF THE SEA URCHIN, ARBACIA PUNCTULATA,
FERTILIZATION DATA





9.5.4.1
Data from the sea urchin, Arbacia punctulata,
fertilization test may be analyzed by hypothesis testing after an
arc sine transformation according to the flowchart in Figure 2. The
fertilization data from the sea urchin test may also be analyzed by
generating a point estimate with the Linear Interpolation
Method.


9.5.5
ANALYSIS OF MORTALITY DATA




9.5.5.1
Mortality data are analyzed by Probit Analysis, if
appropriate, or other point estimation techniques, (i.e., the
Spearman-Karber Method, the Trimmed Spearman-Karber Method, or the
Graphical Method) (see Appendices H-K) (see discussion below). The
mortality data can also be analyzed by hypothesis testing, after an
arc sine square root transformation (see Appendices B-F), according
to the flowchart in Figure 2.


9.6
HYPOTHESIS TESTS


9.6.1 DUNNETT'S PROCEDURE


9.6.1.1
Dunnett's Procedure is used to determine the NOEC. The
procedure consists of an analysis of variance (ANOVA) to determine
the error term, which is then used in a multiple comparison
procedure for comparing each of the treatment means with the
control mean, in a series of paired tests (see Appendix C). Use of
Dunnett's Procedure requires at least three replicates per
treatment to check the assumptions of the test. In cases where the
numbers of data points (replicates) for each concentration are not
equal, a t test may be performed with Bonferroni's adjustment for
multiple comparisons (see Appendix D), instead of using Dunnett's
Procedure.


9.6.1.2
The assumptions upon which the use of Dunnett's Procedure
is contingent are that the observations within treatments are
normally distributed, with homogeneity of variance. Before
analyzing the data, these assumptions must be tested using the
procedures provided in Appendix B.


9.6.1.3
If, after suitable transformations have been carried out,
the normality assumptions have not been met, Steel's Many-one Rank
Test should be used if there are four or more data points
(replicates) per toxicant concentration. If the numbers of data
points for each toxicant concentration are not equal, the Wilcoxon
Rank Sum Test with Bonferroni's adjustment should be used (see
Appendix F).


9.6.1.4
Some indication of the sensitivity of the analysis should
be provided by calculating (1) the minimum difference between means
that can be detected as statistically significant, and (2) the
percent change from the control mean that this minimum difference
represents for a given test.


9.6.1.5
A step-by-step example of the use of Dunnett's Procedure
is provided in Appendix C.


9.6.2
T TEST WITH THE BONFERRONI ADJUSTMENT




9.6.2.1
The t test with the Bonferroni adjustment is used as an
alternative to Dunnett's Procedure when the number of replicates is
not the same for all concentrations. This test sets an upper bound
of alpha on the overall error rate, in contrast to Dunnett's
Procedure, for which the overall error rate is fixed at alpha.
Thus, Dunnett's Procedure is a more powerful test.


9.6.2.2
The assumptions upon which the use of the t test with the
Bonferroni adjustment is contingent are that the observations
within treatments are normally distributed, with homogeneity of
variance. These assumptions must be tested using the procedures
provided in Appendix B.


9.6.2.3
The estimate of the safe concentration derived from this
test is reported in terms of the NOEC. A step-by-step example of
the use of a t-test with the Bonferroni adjustment is provided in
Appendix D.


9.6.3
STEEL'S MANY-ONE RANK TEST




9.6.3.1
Steel's Many-one Rank Test is a multiple comparison
procedure for comparing several treatments with a control. This
method is similar to Dunnett's procedure, except that it is not
necessary to meet the assumption of normality. The data are ranked,
and the analysis is performed on the ranks rather than on the data
themselves. If the data are normally or nearly normally
distributed, Dunnett's Procedure would be more sensitive (would
detect smaller differences between the treatments and control). For
data that are not normally distributed, Steel's Many-one Rank Test
can be much more efficient (Hodges and Lehmann, 1956).


9.6.3.2
It is necessary to have at least four replicates per
toxicant concentration to use Steel's test. Unlike Dunnett's
procedure, the sensitivity of this test cannot be stated in terms
of the minimum difference between treatment means and the control
mean that can be detected as statistically significant.


9.6.3.3
The estimate of the safe concentration is reported as the
NOEC. A step-by-step example of the use of Steel's Many-One Rank
Test is provided in Appendix E.


9.6.4
WILCOXON RANK SUM TEST WITH THE BONFERRONI
ADJUSTMENT




9.6.4.1
The Wilcoxon Rank Sum Test is a nonparametric test for
comparing a treatment with a control. The data are ranked and the
analysis proceeds exactly as in Steel's Test except that
Bonferroni's adjustment for multiple comparisons is used instead of
Steel's tables. When Steel's test can be used (i.e., when there are
equal numbers of data points per toxicant concentration), it will
be more powerful (able to detect smaller differences as
statistically significant) than the Wilcoxon Rank Sum Test with
Bonferroni's adjustment.


9.6.4.2
The estimate of the safe concentration is reported as the
NOEC. A step-by-step example of the use of the Wilcoxon Rank Sum
Test with Bonferroni adjustment is provided in Appendix
F.


9.6.5
A CAUTION IN THE USE OF HYPOTHESIS TESTING




9.6.5.1
If in the calculation of an NOEC by hypothesis testing,
two tested concentrations cause statistically significant adverse
effects, but an intermediate concentration did not cause
statistically significant effects, the results should be used with
extreme caution.


9.7
POINT ESTIMATION TECHNIQUES


9.7.1 PROBIT ANALYSIS


9.7.1.1
Probit Analysis is used to estimate an LC1, LC50, EC1, or
EC50 and the associated 95% confidence interval. The analysis
consists of adjusting the data for mortality in the control, and
then using a maximum likelihood technique to estimate the
parameters of the underlying log tolerance distribution, which is
assumed to have a particular shape.


9.7.1.2
The assumption upon which the use of Probit Analysis is
contingent is a normal distribution of log tolerances. If the
normality assumption is not met, and at least two partial
mortalities are not obtained, Probit Analysis should not be used.
It is important to check the results of Probit Analysis to
determine if use of the analysis is appropriate. The chi-square
test for heterogeneity provides a good test of appropriateness of
the analysis. The computer program (see discussion, Appendix H)
checks the chi-square statistic calculated for the data set against
the tabular value, and provides an error message if the calculated
value exceeds the tabular value.


9.7.1.3
A discussion of Probit Analysis, and examples of computer
program input and output, are found in Appendix H.


9.7.1.4
In cases where Probit Analysis is not appropriate, the
LC50 and confidence interval may be estimated by the
Spearman-Karber Method (Appendix I) or the Trimmed Spearman-Karber
Method (Appendix J). If a test results in 100% survival and 100%
mortality in adjacent treatments (all or nothing effect), the LC50
may be estimated using the Graphical Method (Appendix
K).


9.7.2
LINEAR INTERPOLATION METHOD




9.7.2.1
The Linear Interpolation Method (see Appendix L) is a
procedure to calculate a point estimate of the effluent or other
toxicant concentration [Inhibition Concentration, (IC)] that causes
a given percent reduction (e.g., 25%, 50%, etc.) in the
reproduction, growth, fertilization, or fecundity of the test
organisms. The procedure was designed for general applicability in
the analysis of data from short-term chronic toxicity
tests.


9.7.2.2
Use of the Linear Interpolation Method is based on the
assumptions that the responses (1) are monotonically non-increasing
(the mean response for each higher concentration is less than or
equal to the mean response for the previous concentration), (2)
follow a piece-wise linear response function, and (3) are from a
random, independent, and representative sample of test data. The
assumption for piece-wise linear response cannot be tested
statistically, and no defined statistical procedure is provided to
test the assumption for monotonicity. Where the observed means are
not strictly monotonic by examination, they are adjusted by
smoothing. In cases where the responses at the low toxicant
concentrations are much higher than in the controls, the smoothing
process may result in a large upward adjustment in the control
mean.


9.7.2.3
The inability to test the monotonicity and piece wise
linear response assumptions for this method makes it difficult to
assess when the method is, or is not, producing reliable results.
Therefore, the method should be used with caution when the results
of a toxicity test approach an "all or nothing" response from one
concentration to the next in the concentration series, and when it
appears that there is a large deviation from monotonicity. See
Appendix L for a more detailed discussion of the use of this method
and a computer program available for performing
calculations.


SECTION 10
REPORT PREPARATION AND TEST REVIEW
10.1 REPORT PREPARATION
The toxicity data are reported, together with other appropriate
data. The following general format and content are recommended for
the report:
10.1.1 INTRODUCTION


1.
Permit number


2.
Toxicity testing requirements of permit


3.
Plant location


4.
Name of receiving water body



5.
Contract Laboratory (if the test was performed under
contract)



a.
Name of firm


b.
Phone number


c.
Address




6. Objective of test
10.1.2 PLANT OPERATIONS


1.
Product(s)


2.
Raw materials


3.
Operating schedule


4.
Description of waste treatment


5.
Schematic of waste treatment


6.
Retention time (if applicable)


7.
Volume of waste flow (MGD, CFS, GPM)


8.
Design flow of treatment facility at time of
sampling


10.1.3 SOURCE OF EFFLUENT, RECEIVING WATER, AND DILUTION
WATER
1. Effluent Samples


a.
Sampling point (including latitude and
longitude)


b.
Collection dates and times


c.
Sample collection method


d.
Physical and chemical data


e.
Mean daily discharge on sample collection date


f.
Lapsed time from sample collection to delivery


g.
Sample temperature when received at the
laboratory


2. Receiving Water Samples


a.
Sampling point (including latitude and
longitude)


b.
Collection dates and times


c.
Sample collection method


d.
Physical and chemical data


e.
Tide stages


f.
Sample temperature when received at the
laboratory


g.
Lapsed time from sample collection to delivery


3. Dilution Water Samples


a.
Source


b.
Collection date and time


c.
Pretreatment


d.
Physical and chemical characteristics


10.1.4 TEST METHODS


1.
Toxicity test method used (title, number,
source)


2.
Endpoint(s) of test


3.
Deviation(s) from reference method, if any, and the
reason(s)


4.
Date and time test started


5.
Date and time test terminated


6.
Type of volume and test chambers


7.
Volume of solution used per chamber


8.
Number of organisms used per test chamber


9.
Number of replicate test chambers per
treatment


10.
Acclimation of test organisms (temperature and salinity
mean and range)


11.
Test temperature (mean and range)


12.
Specify if aeration was needed


13.
Feeding frequency, and amount and type of food


14.
Test salinity (mean and range)


15.
Specify if (and how) pH control measures were
implemented


10.1.5 TEST ORGANISMS


1.
Scientific name and how determined


2.
Age


3.
Life stage


4.
Mean length and weight (where applicable)


5.
Source


6.
Diseases and treatment (where applicable)


7.
Taxonomic key used for species identification


10.1.6 QUALITY ASSURANCE


1.
Reference toxicant used routinely; source


2.
Date and time of most recent reference toxicant test;
test results and current control (cusum) chart


3.
Dilution water used in reference toxicant test


4.
Results (NOEC or, where applicable, LOEC, LC50, EC50,
IC25 and/or IC50); report percent minimum significant difference
(PMSD) calculated for sublethal endpoints determined by hypothesis
testing in reference toxicant test


5.
Physical and chemical methods used


10.1.7 RESULTS


1.
Provide raw toxicity data in tabular form, including
daily records of affected organisms in each concentration
(including controls) and replicate, and in graphical form (plots of
toxicity data)


2.
Provide table of LC50s, NOECs, IC25, IC50, etc. (as
required in the applicable NPDES permit)


3.
Indicate statistical methods to calculate
endpoints


4.
Provide summary table of physical and chemical
data


5.
Tabulate QA data


6.
Provide percent minimum significant difference (PMSD)
calculated for sublethal endpoints


10.1.8 CONCLUSIONS AND RECOMMENDATIONS


1.
Relationship between test endpoints and permit
limits.


2.
Action to be taken.


10.2 TEST REVIEW


10.2.1
Test review is an important part of an overall quality
assurance program (Section 4) and is necessary for ensuring that
all test results are reported accurately. Test review should be
conducted on each test by both the testing laboratory and the
regulatory authority.


10.2.2
SAMPLING AND HANDLING




10.2.2.1
The collection and handling of samples are reviewed to
verify that the sampling and handling procedures given in Section 8
were followed. Chain-of-custody forms are reviewed to verify that
samples were tested within allowable sample holding times
(Subsection 8.5.4). Any deviations from the procedures given in
Section 8 should be documented and described in the data report
(Subsection 10.1).


10.2.3
TEST ACCEPTABILITY CRITERIA




10.2.3.1
Test data are reviewed to verify that test acceptability
criteria (TAC) requirements for a valid test have been met. Any
test not meeting the minimum test acceptability criteria is
considered invalid. All invalid tests must be repeated with a newly
collected sample.


10.2.4
TEST CONDITIONS




10.2.4.1
Test conditions are reviewed and compared to the
specifications listed in the summary of test condition tables
provided for each method. Physical and chemical measurements taken
during the test (e.g., temperature, pH, and DO) also are reviewed
and compared to specified ranges. Any deviations from
specifications should be documented and described in the data
report (Subsection 10.1).


10.2.4.2
The summary of test condition tables presented for each
method identify test conditions as required or recommended. For WET
test data submitted under NPDES permits, all required test
conditions must be met or the test is considered invalid and must
be repeated with a newly collected sample. Deviations from
recommended test conditions must be evaluated on a case-by-case
basis to determine the validity of test results. Deviations from
recommended test conditions may or may not invalidate a test result
depending on the degree of the departure and the objective of the
test. The reviewer should consider the degree of the deviation and
the potential or observed impact of the deviation on the test
result before rejecting or accepting a test result as valid. For
example, if dissolved oxygen is measured below 4.0 mg/L in one test
chamber, the reviewer should consider whether any observed
mortality in that test chamber corresponded with the drop in
dissolved oxygen.


10.2.4.3
Whereas slight deviations in test conditions may not
invalidate an individual test result, test condition deviations
that continue to occur frequently in a given laboratory may
indicate the need for improved quality control in that
laboratory.


10.2.5
STATISTICAL METHODS


10.2.5.1 The statistical methods used for analyzing test data
are reviewed to verify that the recommended flowcharts for
statistical analysis were followed. Any deviation from the
recommended flowcharts for selection of statistical methods should
be noted in the data report. Statistical methods other than those
recommended in the statistical flowcharts may be appropriate (see
Subsection 9.4.1.2), however, the laboratory must document the use
of and provide the rationale for the use of any alternate
statistical method. In all cases (flowchart recommended methods or
alternate methods), reviewers should verify that the necessary
assumptions are met for the statistical method used.
10.2.6 CONCENTRATION-RESPONSE RELATIONSHIPS


10.2.6.1
The concept of a concentration-response, or more
classically, a dose-response relationship is "the most fundamental
and pervasive one in toxicology" (Casarett and Doull, 1975). This
concept assumes that there is a causal relationship between the
dose of a toxicant (or concentration for toxicants in solution) and
a measured response. A response may be any measurable biochemical
or biological parameter that is correlated with exposure to the
toxicant. The classical concentration-response relationship is
depicted as a sigmoidal shaped curve, however, the particular shape
of the concentration-response curve may differ for each coupled
toxicant and response pair. In general, more severe responses (such
as acute effects) occur at higher concentrations of the toxicant,
and less severe responses (such as chronic effects) occur at lower
concentrations. A single toxicant also may produce multiple
responses, each characterized by a concentration-response
relationship. A corollary of the concentration-response concept is
that every toxicant should exhibit a concentration-response
relationship, given that the appropriate response is measured and
given that the concentration range evaluated is appropriate. Use of
this concept can be helpful in determining whether an effluent
possesses toxicity and in identifying anomalous test
results.


10.2.6.2
The concentration-response relationship generated for
each multi-concentration test must be reviewed to ensure that
calculated test results are interpreted appropriately. USEPA
(2000a) provides guidance on evaluating concentration-response
relationships to assist in determining the validity of WET test
results. All WET test results (from multi-concentration tests)
reported under the NPDES program should be reviewed and reported
according to USEPA guidance on the evaluation of
concentration-response relationships (USEPA, 2000a). This guidance
provides review steps for 10 different concentration-response
patterns that may be encountered in WET test data. Based on the
review, the guidance provides one of three determinations: that
calculated effect concentrations are reliable and should be
reported, that calculated effect concentrations are anomalous and
should be explained, or that the test was inconclusive and the test
should be repeated with a newly collected sample. It should be
noted that the determination of a valid concentration-response
relationship is not always clear cut. Data from some tests may
suggest consultation with professional toxicologists and/or
regulatory officials. Tests that exhibit unexpected
concentration-response relationships also may indicate a need for
further investigation and possible retesting.


10.2.7
REFERENCE TOXICANT TESTING




10.2.7.1
Test review of a given effluent or receiving water test
should include review of the associated reference toxicant test and
current control chart. Reference toxicant testing and control
charting is required for documenting the quality of test organisms
(Subsection 4.7) and ongoing laboratory performance (Subsection
4.16). The reviewer should verify that a quality control reference
toxicant test was conducted according to the specified frequency
required by the permitting authority or recommended by the method
(e.g., monthly). The test acceptability criteria, test conditions,
concentration-response relationship, and test sensitivity of the
reference toxicant test are reviewed to verify that the reference
toxicant test conducted was a valid test. The results of the
reference toxicant test are then plotted on a control chart (see
Subsection 4.16) and compared to the current control chart limits
(± 2 standard deviations).


10.2.7.2
Reference toxicant tests that fall outside of recommended
control chart limits are evaluated to determine the validity of
associated effluent and receiving water tests (see Subsection
4.16). An out of control reference toxicant test result does not
necessarily invalidate associated 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 increasing test organism
sensitivity or toward decreasing test organism sensitivity), the
test conditions of both the effluent test and the reference
toxicant test, and the objective of the test. More frequent and/or
concurrent reference toxicant testing may be advantageous if recent
problems (e.g., invalid tests, reference toxicant test results
outside of control chart limits, reduced health of organism
cultures, or increased within-test variability) have been
identified in testing.


10.2.8
TEST VARIABILITY




10.2.8.1
The within-test variability of individual tests should be
reviewed. Excessive within-test variability may invalidate a test
result and warrant retesting. For evaluating within-test
variability, reviewers should consult EPA guidance on upper and
lower percent minimum significant difference (PMSD) bounds (USEPA,
2000b).


10.2.8.2
When NPDES permits require sublethal hypothesis testing
endpoints from Methods 1006.0 or 1007.0 (e.g., growth NOECs and
LOECs), within-test variability must be reviewed and variability
criteria must be applied as described in this section (10.2.8.2).
When the methods are used for non-regulatory purposes, the
variability criteria herein are recommended but are not required,
and their use (or the use of alternative variability criteria) may
depend upon the intended uses of the test results and the
requirements of any applicable data quality objectives and quality
assurance plan.




10.2.8.2.1
To measure test variability, calculate the percent
minimum significant difference (PMSD) achieved in the test. The
PMSD is the smallest percentage decrease in growth or reproduction
from the control that could be determined as statistically
significant in the test. The PMSD is calculated as 100 times the
minimum significant difference (MSD) divided by the control mean.
The equation and examples of MSD calculations are shown in Appendix
C. PMSD may be calculated legitimately as a descriptive statistic
for within-test variability, even when the hypothesis test is
conducted using a non-parametric method. The PMSD bounds were based
on a representative set of tests, including tests for which a
non-parametric method was required for determining the NOEC or
LOEC. The conduct of hypothesis testing to determine test results
should follow the statistical flow charts provided for each method.
That is, when test data fail to meet assumptions of normality or
heterogeneity of variance, a non-parametric method (determined
following the statistical flowchart for the method) should be used
to calculate test results, but the PMSD may be calculated as
described above (using parametric methods) to provide a measure of
test variability.


10.2.8.2.2
Compare the PMSD measured in the test with the upper PMSD
bound variability criterion listed in Table 6. When the test PMSD
exceeds the upper bound, the variability among replicates is
unusually large for the test method. Such a test should be
considered insufficiently sensitive to detect toxic effects on
growth or reproduction of substantial magnitude. A finding of
toxicity at a particular concentration may be regarded as
trustworthy, but a finding of "no toxicity" or "no statistically
significant toxicity" at a particular concentration should not be
regarded as a reliable indication that there is no substantial
toxic effect on growth or reproduction at that
concentration.


10.2.8.2.3
If the PMSD measured for the test is less than or equal
to the upper PMSD bound variability criterion in Table 6, then the
test's variability measure lies within normal bounds and the effect
concentration estimate (e.g., NOEC or LOEC) would normally be
accepted unless other test review steps raise serious doubts about
its validity.


10.2.8.2.4
If the PMSD measured for the test exceeds the upper PMSD
bound variability criterion in Table 6, then one of the following
two cases applies (10.2.8.2.4.1, 10.2.8.2.4.2).




10.2.8.2.4.1
If toxicity is found at the permitted receiving water
concentration (RWC) based upon the value of the effect
concentration estimate (NOEC or LOEC), then the test shall be
accepted and the effect concentration estimate may be reported,
unless other test review steps raise serious doubts about its
validity.


10.2.8.2.4.2
If toxicity is not found at the permitted RWC based upon
the value of the effect concentration estimate (NOEC or LOEC) and
the PMSD measured for the test exceeds the upper PMSD bound, then
the test shall not be accepted, and a new test must be conducted
promptly on a newly collected sample.


10.2.8.2.5
To avoid penalizing laboratories that achieve unusually
high precision, lower PMSD bounds shall also be applied when a
hypothesis test result (e.g., NOEC or LOEC) is reported. Lower PMSD
bounds, which are based on the 10th percentiles of national PMSD
data, are presented in Table 6. The 10th percentile PMSD represents
a practical limit to the sensitivity of the test method because few
laboratories are able to achieve such precision on a


regular basis and most do not achieve it even occasionally. In
determining hypothesis test results (e.g., NOEC or LOEC), a test
concentration shall not be considered toxic (i.e., significantly
different from the control) if the relative difference from the
control is less than the lower PMSD bounds in Table 6. See USEPA,
2000b for specific examples of implementing lower PMSD bounds.
10.2.8.3 To assist in reviewing within-test variability, EPA
recommends maintaining control charts of PMSDs calculated for
successive effluent tests (USEPA, 2000b). A control chart of PMSD
values characterizes the range of variability observed within a
given laboratory, and allows comparison of individual test PMSDs
with the laboratory's typical range of variability. Control charts
of other variability and test performance measures, such as the
MSD, standard deviation or CV of control responses, or average
control response, also may be useful for reviewing tests and
minimizing variability. The log of PMSD will provide an
approximately normal variate useful for control charting.
TABLE 6. VARIABILITY CRITERIA (UPPER AND LOWER PMSD BOUNDS) FOR
SUBLETHAL HYPOTHESIS TESTING ENDPOINTS SUBMITTED UNDER NPDES
PERMITS.1

Lower and upper PMSD bounds were determined from the 10th and
90th percentile, respectively, of PMSD data from EPA's WET
Interlaboratory Variability Study (USEPA, 2001a; USEPA, 2000b).