United States
Environmental Protection
Agency
Environmental Monitoring and Support
Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S4-85/014 Aug. 1986
SERA Project Summary
RECEIVED
NOV211986
ENVIRONMENTAL PROTECTION AGENCY
LIBRARY, REGION V
Short-Term Methods for
Estimating the Chronic
Toxicity of Effluents and
Receiving Waters to Freshwater
Organisms
William B. Horning, II and Cornelius I. Weber
The first agency methods manual for
estimating the chronic toxicity of ef-
fluents and receiving waters describes
short-term (four- to eight-day) methods
for estimating the chronic toxicity of
effluents and receiving waters to a
freshwater fish, an invertebrate, and an
alga. Also included are guidelines on
laboratory safety, quality assurance,
facilities and equipment, dilution water,
effluent sampling and holding, data
analysis, report preparation, and organ-
ism culturing and handling. Listings of
computer programs for Dunnett's Pro-
cedure and Probit Analysis are provided
in an Appendix.
This Project Summary was developed
by EPA's Environmental Monitoring and
Support Laboratory, Cincinnati, OH, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
As a result of the increased awareness
of the value of effluent toxicity test data
for toxics control in the National Pollutant
Discharge Elimination System (NPDES)
'permit program, which emerged from the
extensive effluent toxicity monitoring
activities of the regions and states, and
the recent availability of short-term
chronic toxicity test methods, the U.S.
Environmental Protection Agency issued
a national policy statement entitled,
"Policy for the Development of Water
Quality-Based Permit Limitations for
Toxic Pollutants," in the Federal Register
Vol. 49, No. 48, Friday, March 9,1984. A
technical support document on the use of
effluent and receiving water toxicity data
also has been prepared by the Office of
Water Enforcement and Permits to pro-
vide additional guidance on the imple-
mentation of the biomonitoring policy.
This new agency policy proposes the
use of toxicity data to assess and control
the discharge of toxic substances to the
Nation's waters through the NPDES
permit program. The policy states that
"biological testing of effluents is an
important aspect of the water quality-
based approach for controlling toxic pol-
lutants. Effluent toxicity data, in conjunc-
tion with other data, can be used to
establish control priorities, assess com-
pliance with state water quality stand-
ards, and set permit limitations to achieve
those standards." All states have water
quality standards which include narrative
statements prohibiting the discharge of
toxic materials in toxic amounts.
Objective
The four short-term tests described in
the manual are for use in the NPDES
Program to estimate one or more of the
following: (1) the chronic toxicity of
effluents collected at the end of the
discharge pipe and tested with a standard
dil ution water (moderately hard synthetic
-------
freshwater); (2) the chronic toxicity of
effluents collected at the end of the
discharge pipe and tested with dilution
water consisting of non-toxic receiving
water collected upstream from the outfall,
or with other uncontaminated surface
water or standard dilution water having
approximately the same hardness as the
receiving water; (3) the toxicity of receiv-
ing water downstream ffom the outfall;
and (4) the effects of multiple discharges
on the quality of the receiving water. The
tests may also be useful in developing
site-specific water quality criteria.
These methods were developed to
provide the most favorable cost-benefit
relationship possible, and are intended
for use in on-site effluent toxicity tests,
and to determine the toxicity of effluent
samples shipped to central and distant
laboratories.
The tests include:
1. Seven-day, sub-chronic, fathead
minnow (Pimephales promelas),
static renewal, larval survival and
growth test.
2. Seven-day, (three-brood), chronic,
Ceriodaphnia dubia, static renewal,
survival and reproduction test.
3. Eight-day, sub-chronic, fathead
minnow (Pimephales promelas),
static renewal, embryo-larval sur-
vival and teratogenicity test.
4. Four-day, chronic, Selenastrum
capricornutum, static, growth test.
Short-Term Methods for
Estimating Chronic Toxicity
The purpose of aquatic toxicity tests
with effluents or pure compounds is to
estimate the "safe" or "no effect" con-
centration of these substances, which is
defined as the concentration which will
permit normal propagation of fish and
other aquatic life in the receiving waters.
The endpointsthat have been considered
in tests to determine the adverse effects
of toxicants generally have been limited
to only a few, such as mortality, growth,
and reproduction.
Acute mortality is an obvious and easily
observed effect, which accounts for its
wide use in the early period of evaluation
of the toxicity of pure compounds and
complex effluents. The results of these
tests were usually expressed as the
concentration lethal to 50% of the test
organisms (LC50) over relatively short
exposure periods (two to four days).
As exposure periods of acute tests
were lengthened, the LC50 and lethal
threshold concentration were observed
to decline for many compounds. By
lengthening the tests to include one or
more complete life cycles and observing
the more subtle effects of the toxicants,
such as a reduction in growth and repro-
duction, more accurate, direct estimates
of the threshold or safe concentration of
the toxicant could be obtained. However,
because of the high cost of full life cycle
toxicity tests and the discovery that early
life-stage test data are adequate to esti-
mate chemically safe concentrations,
there has been a shift to rapid, short-term
tests using growth and reproduction.
Health and Safety
Collection and use of effluents in
toxicity tests may involve significant risks
to personal safety and health. Personnel
collecting effluent samples and conduct-
ing toxicity tests should take all safety
precautions necessary for the prevention
of bodily injury and illness which might
result from ingestion or invasion of
infectious agents, inhalation or absorp-
tion of corrosive or toxic substances
through skin contact, and asphyxiation
due to lack of oxygen or presence of
noxious gases.
Prior to sample collection and labor-
atory work, personnel should determine
that all necessary safety equipment and
materials have been obtained and are in
good condition.
Quality Assurance
Quality Assurance practices for effluent
toxicity tests consist of all aspects of the
test that affect data quality, such as: (1)
effluent sampling and handling; (2) the
source and condition of the test organ-
isms; (3) condition of equipment; (4) test
conditions; (5) instrument calibration; (6)
replication; (7) use of reference toxicants;
(8) record keeping; and (9) data evaluation.
Effluent and Receiving Water
Sampling and Sample Handling
The effluent sampling point usually
should be the same as that specified in
the NPDES discharge permit, except
under the following conditions: (1) if there
is better access to a sampling point
between the final treatment and the
discharge outfall; (2) if the processed
waste is chlorinated'prior to discharge to
the receiving waters, 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 waste-
water streams or non-contact cooling
water, additional sampling points may be
chosen.
Whether to collect grab or composite
samples is decided upon based on the
objectives of the test and an understand-
ing 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 col-
lection 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 large number of separate samples and
tests. Collection of a 24-h composite
sample, however, may dilute toxicity
spikes, and averages the quality of the
effluent over the sampling period. Aera-
tion during collection and transfer of
effluents should be minimized to reduce
the loss of volatile chemicals. Definitive
tests performed for NPDES permit pur-
poses require daily effluent sample col-
lection and daily renewal of test solutions.
It is common practice to collect grab
samples for receiving water toxicity stud-
ies. When non-toxic receiving water is
required for a test, it may be collected
upstream from the outfall or from other
uncontaminated surface water having
approximately the same hardness (± 10%)
as the receiving water. If the objective of
the test is to determine the additive effects
of the discharge on receiving water which
may already be contaminated, the test is
performed using dilution water consisting
of receiving water collected daily up-
stream from the outfall.
To determine the extent of the zone of
toxicity in the receiving water down-
stream from the outfall, receiving water
samples are collected at several distances
downstream from the discharge. The time
required for the effluent-receiving-water
mixture to travel to sampling points
downstream from the outfall may be
difficult to ascertain, and it may not be
possible to correlate downstream toxicity
with effluent toxicity at the discharge
point unless a dye study is performed. The
toxicity of receiving water samples from
five stations downstream from the dis-
charge point can be evaluated using the
same number of test vessels and test
organisms as used in one effluent toxicity
test with five effluent dilutions.
-------
Sample Handling and
Preservation
If the data from the samples are to be
acceptable for use in the NPDES Program,
the lapsed time from collection of a grab
or composite sample and the initiation of
the test must not exceed 72 h. Composite
samples should be chilled during collec-
tion, where possible. Except when used
within 24 h of collection, samples must
be chilled after collection and maintained
at 4°C until used.
Samples collected for off-site toxicity
testing are to be chilled to 4°C when
collected, shipped iced to the central
laboratory, and there transferred to a
refrigerator (4°C) until used. Every effort
must be made to initiate the test with an
effluent sample on the day of arrival in the
laboratory.
Sample Preparation
With the Ceriodaphnia and fathead
minnow tests, effluents and surface
waters must be filtered through a (30 Aim)
plankton net to remove indigenous organ-
isms that may attack or be confused with
the test organisms. Surface waters used
in algal toxicity tests must be filtered
through a 0.45 ^m pore diameter before
use. It may be necessary to first coarse-
filter the dilution or waste water through
a nylon sieve having 2- to 4-mm holes to
remove debris or break up large floating
or suspended solids.
The dissolved oxygen (DO) concentra-
tion in the dilution water 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.
If the dilution water and effluent must
be warmed to bring them to the prescribed
test temperature, supersaturation of the
dissolved gases may become a problem.
To prevent this problem, the effluent and
dilution water are heated to 25°C and
checked for dissolved oxygen with a
probe. If the DO exceeds 8.5 mg/L (100%
saturation), the solutions are aerated
vigorously with an air stone (usually 1 -2
min) until the DO is lowered to 100%
saturation.
Chronic Toxicity Test End
Points and Data Analysis
Numerous terms are used to define the
end points employed in chronic toxicity
tests. The primary terms in current use
are listed below:
• Safe Concentration—The highest con-
centration which will permit normal
propagation of fish and other aquatic
life in receiving waters.
• No Observed Effect Concentration
(NOEQ—The highest concentration of
toxicant to which organisms are ex-
posed in a full life-cycle or partial life-
cycle test, which causes no statistically
significant adverse effect .on the ob-
served parameters (usually hatchabil-
ity, survival, growth, and reproduction).
• Lowest Observed Effect Concentration
(LOEC)—The lowest concentration of
toxicant to which organisms are ex-
posed in a life-cycle or partial life-cycle
test, which causes a statistically sig-
nificant adverse effect on the observed
parameters (usually hatchability, sur-
vival, growth, and reproduction).
• Maximum Acceptable Toxicant Con-
centration (MATC)—An undetermined
concentration within the interval
bounded by the NOEC and LOEC.
• Chronic Value (ChV)—A value lying
between the NOEC and LOEC, derived
by calculating the geometric mean of
the NOEC and LOEC. The term is
sometimes used interchangably with
MATC.
• Lethal Concentration (LC) or Effective
Concentration (EC)—A point estimate
of the toxicant concentration that
would adversely affect a given percent
of the test organisms, calculated by
regression (such as Probit Analysis).
The LCI (or ECI) is the estimated
concentration of toxicant that adverse-
ly affects 1% of the test population,
and is defined here as the threshold
concentration, or lowest concentration
that would cause an adverse effect on
the observed parameters. The LCI (ECI)
falls in the range of the NOEC and
LOEC.
It is recommended that the data always
be plotted as a preliminary step to help
spot problems and detect unsuspected
trends or patterns in the responses.
Transformations of the data, such as arc
sine and logs, can be used if they help the
data meet the assumptions of the pro-
posed analyses.
Growth data from the fathead minnow
larval survival and growth test, and
reproduction data from the Ceriodaphnia
survival and reproduction test, are ana-
lyzed using Dunnett's Procedure if the
assumptions of normality and homoge-
neity of variance are met. If the assump-
tions are not met, the data are analyzed
using Steel's Many-One Rank Test.
The growth response data from the
algal toxicity test may be (1) converted to a
proportion of the growth of the controls,
which may then be analyzed by Probit
Analysis or, (2) after an appropriate
transformation if necessary to meet the
assumptions of normality and homoge-
neity of variance, may be analyzed by
Dunnett's Procedure or Steel's Many-
One Rank Test.
Mortality data from the fathead minnow
larval survival and growth test and the
fathead minnow embryo-larval survival
and teratogenicity test are used in a
Probit Analysis to determine the LCI, if
Probit Analysis is appropriate.
Fisher's Exact Test is used to analyze
the mortality data from the Ceriodaphnia
survival and reproduction test prior to the
analysis of the reproduction data. Mor-
tality data from the fathead minnow larval
survival and growth test, and the fathead
minnow embryo-larval survival and tera-
togenicity test, can be analyzed by Dun-
nett's Procedure or Steel's Many-One
Rank Test after transforming the square
root of the proportion of dead organisms
to an arc sine value. This transformation
is performed by the computer program for
Dunnett's Procedure (see Appendix to full
report).
Dunnett's Procedure consists of an
analysis of variance (ANOVA) to deter-
mine the error term, which is then used in
a multiple method for comparing each of
the treatment means with the control
mean, in a series of paired tests. Use of
Dunnett's Procedure requires at least two
replicates per treatment. The use of
Dunnett's Procedure is contingent on the
assumption that the observations are
independent and normally distributed,
with homogeneity of variance. Before
analyzing the data, the assumptions are
checked using the procedures provided in
the Appendix to the full report.
Some indication of the sensitivity of the
analysis should be provided by calculat-
ing: (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.
Calculation of beta levels (Type II error,
which results when the null hypothesis is
not rejected when it should be) as an
indication of the power of the test would
be another alternative. The safe concen-
tration derived from this test is reported in
terms of the NOEC. If, after suitable
transformations have been carried out,
the normality assumptions have not been
-------
met, the Steel Many-One Rank Test
should be used.
Steel's Many-One Rank Test is a mul-
tiple method for comparing several treat-
ments with a control which is similar to
Dunnett's Procedure, except that the
assumption for normality need not be
met. The data are ranked, and the anal-
ysis 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 to detect smaller differences
between the treatments and control. For
data that are not normally distributed.
Steel's Many-One RankTest can be much
more efficient. It is necessary to have at
least four replicates to use Steel's test.
The sensitivity of this test cannot be
stated in terms of the minimum difference
between treatment means and the con-
trol mean. The safe concentration is
reported as the NOEC.
Probit Analysis is used to analyze
percentage data from concentration-re-
sponse tests. The analysis can provide an
estimate of the concentration of toxicant
lethal to a given percent of the test
organisms and provide a confidence
interval for the estimate. Probit Analysis
also assumes normal distribution of log
tolerances and independence of the indi-
vidual responses. To use Probit Analysis,
at least two partial mortalities must be
obtained.
Fisher's Exact Test is a statistical
method based on the hypergeometric
probability distribution that can be used
to test whether the probability of a
response is the same in two binomial
populations. When used with the Cerio-
daphnia data, it provides a conservative
test of the equality of any two survival
proportions assuming only the independ-
ence of responses from a binomial popu-
lation.
Summary of Test Methods
1. Fathead minnow (Pimephales pro-
melas) larvae (preferably less than
24-h old) are exposed in a static
renewal system for seven days to
different concentrations of effluent
or to receiving water. Test results
are based on the survival and
growth (increase in weight) of the
larvae.
2. Fathead minnow embryos and lar-
vae are exposed in a static renewal
system, from shortly after fertiliza-
tion of the eggs through four days
posthatching (total of eight days), to
different concentrations of effluent
or to receiving water. Test results
are based on the total frequency of
both mortality and gross morpho-
logical deformities (terata).
3. Cladocera (Ceriodaphnia dubia) ex-
posed in a static renewal system for
seven days to different concentra-
tions of effluent or to receiving
water. Test results are based on
survival and reproduction. If the
test is conducted as described, the
control organisms should produce
three broods of young during the
seven-day period.
4. Freshwater algae (Selenastrum
capricornutum) are exposed in a
static system to a series of concen-
trations of effluent or to receiving
water, for 96 h. The response of the
population is measured in terms of
changes in cell density (cell counts
per mL), biomass, chlorophyll con-
tent, or absorbance. If the test is
extended to 14 days, it may be used
to measure the algal growth poten-
tial of wastewaters and surface
waters.
The EPA authors, William B. Horning, II (also the EPA Project Officer, see below)
and Cornelius I. Weber, are with Environmental Monitoring and Support
Laboratory, Cincinnati, OH 45268.
The complete report, entitled "Short- Term Methods for Estimating the Chronic
Toxicity of Effluents and Receiving Waters to Freshwater Organisms," (Order
No. PB 86-158 474/AS; Cost: $16.95, subject to change) will be available only
from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring and Support Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S4-85/014
0000329 PS
U S ENVIR PROTECTION AGENCY
REGION 5 LIBRARY
230 S OEAR80RN STREET
CHICAGO IL 60604
------- |