2.1 INTRODUCTION
2.1.1
The objective of aquatic toxicity tests with effluents or
pure compounds is to estimate the "safe" or "no-effect"
concentration of these substances, which is defined as the
concentration which will permit normal propagation of fish and
other aquatic life in the receiving waters. The endpoints that have
been considered in tests to determine the adverse effects of
toxicants include death and survival, decreased reproduction and
growth, locomotor activity, gill ventilation rate, heart rate,
blood chemistry, histopathology, enzyme activity, olfactory
function, and terata. Since it is not feasible to detect and/or
measure all of these (and other possible) effects of toxic
substances on a routine basis, observations in toxicity tests
generally have been limited to only a few effects, such as
mortality, growth, and reproduction.
2.1.2
Acute lethality is an obvious and easily observed effect
which accounts for its wide use in the early period of evaluation
of the toxicity of pure compounds and complex effluents. The
results of these tests were usually expressed as the concentration
lethal to 50% of the test organisms (LC50) over relatively short
exposure periods (one-to-four days).
2.1.3
As exposure periods of acute tests were lengthened, the
LC50 and lethal threshold concentration were observed to decline
for many compounds. By lengthening the tests to include one or more
complete life cycles and observing the more subtle effects of the
toxicants, such as a reduction in growth and reproduction, more
accurate, direct, estimates of the threshold or safe concentration
of the toxicant could be obtained. However, laboratory life cycle
tests may not accurately estimate the "safe" concentration of
toxicants because they are conducted with a limited number of
species under highly controlled, steady state conditions, and the
results do not include the effects of the stresses to which the
organisms would ordinarily be exposed in the natural
environment.
2.1.4
An early published account of a full life cycle, fish
toxicity test was that of Mount and Stephan (1967). In this study,
fathead minnows, Pimephales promelas, were exposed to a graded
series of pesticide concentrations throughout their life cycle, and
the effects of the toxicant on survival, growth, and
reproduction were measured and evaluated. This work was soon
followed by full life cycle tests using other toxicants and fish
species.
2.1.5
McKim (1977) evaluated the data from 56 full life cycle
tests, 32 of which used the fathead minnow, Pimephales promelas,
and concluded that the embryo-larval and early juvenile life stages
were the most sensitive stages. He proposed the use of partial life
cycle toxicity tests with the early life stages (ELS) of fish to
establish water quality criteria.
2.1.6
Macek and Sleight (1977) found that exposure of critical
life stages of fish to toxicants provides estimates of chronically
safe concentrations remarkably similar to those derived from full
life cycle toxicity tests. They reported that "for a great majority
of toxicants, the concentration which will not be acutely toxic to
the most sensitive life stages is the chronically safe
concentration for fish, and that the most sensitive life stages are
the embryos and fry." Critical life stage exposure was considered
to be exposure of the embryos during most, preferably all, of the
embryogenic (incubation) period, and exposure of the fry for 30
days post-hatch for warm water fish with embryogenic periods
ranging from one-to-fourteen days, and for 60 days post-hatch for
fish with longer embryogenic periods. They concluded that in the
majority of cases, the maximum acceptable toxicant concentration
(MATC) could be estimated from the results of exposure of the
embryos during incubation, and the larvae for 30 days
post-hatch.
2.1.7
Because of the high cost of full life-cycle fish toxicity
tests and the emerging consensus that the ELS test data usually
would be adequate for estimating chronically safe concentrations,
there was a rapid shift by aquatic toxicologists to 30- to 90-day
ELS toxicity tests for estimating chronically safe concentrations
in the late 1970s. In 1980, USEPA adopted the policy that ELS test
data could be used in establishing water quality criteria if data
from full life-cycle tests were not available (USEPA,
1980a).
2.1.8
Published reports of the results of ELS tests indicate
that the relative sensitivity of growth and survival as endpoints
may be species dependent, toxicant dependent, or both. Ward and
Parrish (1980) examined the literature on ELS tests that used
embryos and juveniles of the sheepshead minnow, Cyprinodon
variegatus, and found that growth was not a statistically sensitive
indicator of toxicity in 16 of 18 tests. They suggested that the
ELS tests be shortened to 14 days posthatch and that growth be
eliminated as an indicator of toxic effects.
2.1.9
In a review of the literature on 173 fish full life-cycle
and ELS tests performed to determine the chronically safe
concentrations of a wide variety of toxicants, such as metals,
pesticides, organics, inorganics, detergents, and complex
effluents, Woltering (1984) found that at the lowest effect
concentration, significant reductions were observed in fry survival
in 57%, fry growth in 36%, and egg hatchability in 19% of the
tests. He also found that fry survival and growth were very often
equally sensitive, and concluded that the growth response could be
deleted from routine application of the ELS tests. The net result
would be a significant reduction in the duration and cost of
screening tests with no appreciable impact on estimating MATCs for
chemical hazard assessments. Benoit et al. (1982), however, found
larval growth to be the most significant measure of effect and
survival to be equally or less sensitive than growth in early
life-stage tests with four organic chemicals.
2.1.10
Efforts to further reduce the length of partial lifecycle
toxicity tests for fish without compromising their predictive value
have resulted in the development of an eight-day, embryo-larval
survival and teratogenicity test for fish and other aquatic
vertebrates (USEPA, 1981; Birge et al., 1985), and a seven-day
larval survival and growth test (Norberg and Mount,
1985).
2.1.11
The similarity of estimates of chronically safe
concentrations of toxicants derived from short-term, embryo-larval
survival and teratogenicity tests to those derived from full
life-cycle tests has been demonstrated by Birge et al. (1981),
Birge and Cassidy (1983), and Birge et al. (1985).
2.1.12
Use of a seven-day, fathead minnow, Pimephales promelas,
larval survival and growth test was first proposed by Norberg and
Mount at the 1983 annual meeting of the Society for Environmental
Toxicology and Chemistry (Norberg and Mount, 1983). This test was
subsequently used by Mount and associates in field demonstrations
at Lima, Ohio (USEPA, 1984), and at many other locations (USEPA,
1985c, USEPA, 1985d; USEPA, 1985e; USEPA, 1986a; USEPA, 1986b;
USEPA, 1986c; USEPA, 1986d). Growth was frequently found to be more
sensitive than survival in determining the effects of complex
effluents.
2.1.13
Norberg and Mount (1985) performed three single toxicant
fathead minnow larval growth tests with zinc, copper, and DURSBAN®,
using dilution water from Lake Superior. The results
were comparable to, and had confidence intervals that overlapped
with, chronic values reported in the literature for both ELS and
full life-cycle tests.
2.1.14
USEPA (1987b) and USEPA (1987c) adapted the fathead
minnow larval growth and survival test for use with the sheepshead
minnow and the inland silverside, respectively. When daily renewal
7-day sheepshead minnow larval growth and survival tests and 28-day
ELS tests were performed with industrial and municipal effluents,
growth was more sensitive than survival in seven out of 12 larval
growth and survival tests, equally sensitive in four tests, and
less sensitive in only one test. In four cases, the ELS test may
have been three to 10 times more sensitive to effluents than the
larval growth and survival test. In tests using copper, the No
Observable Effect Concentrations (NOECs) were the same for both
types of test, and growth was the most sensitive endpoint for both.
In a four laboratory comparison, six of seven tests produced
identical NOECs for survival and growth (USEPA, 1987a). Data
indicate that the inland silverside is at least equally sensitive
or more sensitive to effluents and single compounds than the
sheepshead minnow, and can be tested over a wider salinity range,
5-30‰ (USEPA, 1987a).
2.1.15
Lussier et al. (1985) and USEPA (1987e) determined that
survival and growth are often as sensitive as reproduction in
28-day life-cycle tests with the mysid, Mysidopsis
bahia.
2.1.16
Nacci and Jackim (1985) and USEPA (1987g) compared the
results from the sea urchin fertilization test, using organic
compounds, with results from acute toxicity tests using the
freshwater organisms, fathead minnows, Pimphales promelas, and
Daphnia magna. The test was also compared to acute toxicity tests
using Atlantic silverside, Menidia menidia, and the mysid,
Mysidopsis bahia, and five metals. For six of the eight organic
compounds, the results of the fertilization test and the acute
toxicity test correlated well (r2 = 0.85). However, the results of
the fertilization test with the five metals did not correlate well
with the results from the acute tests.
2.1.17
USEPA (1987f) evaluated two industrial effluents
containing heavy metals, five industrial effluents containing
organic chemicals (including dyes and pesticides), and 15 domestic
wastewaters using the two-day red macroalga, Champia parvula,
sexual reproduction test. Nine single compounds were used to
compare the effects on sexual reproduction using a
two-week exposure and a two-day exposure. For six of the nine
compounds tested, the chronic values were the same for both
tests.
2.1.18
The use of short-term toxicity tests in the NPDES Program
is especially attractive because they provide a more direct
estimate of the safe concentrations of effluents in receiving
waters than was provided by acute toxicity tests, at an only
slightly increased level of effort, compared to the fish full
life-cycle chronic and 28-day ELS tests and the 28-day mysid
life-cycle test.
2.2
TYPES OF TESTS
2.2.1
The selection of the test type will depend on the NPDES
permit requirements, the objectives of the test, the available
resources, the requirements of the test organisms, and effluent
characteristics such as fluctuations in effluent
toxicity.
2.2.2
Effluent chronic toxicity is generally measured using a
multi-concentration, or definitive test, consisting of a control
and a minimum of five effluent concentrations. The tests are
designed to provide dose-response information, expressed as the
percent effluent concentration that affects the survival,
fertilization, growth, and/or development within the prescribed
period of time (40 minutes to seven days). The results of the tests
are expressed in terms of either the highest concentration that has
no statistically significant observed effect on those responses
when compared to the controls or the estimated concentration that
causes a specified percent reduction in responses versus the
controls.
2.2.3
Use of pass/fail tests consisting of a single effluent
concentration (e.g., the receiving water concentration or RWC) and
a control is not recommended. If the NPDES permit has a whole
effluent toxicity limit for acute toxicity at the RWC, it is
prudent to use that permit limit as the midpoint of a series of
five effluent concentrations. This will ensure that there is
sufficient information on the dose-response relationship. For
example, if the RWC is >25% then, the effluent concentrations
utilized in a test may be: (1) 100% effluent, (2) (RWC +
100)/2,
(3) RWC, (4) RWC/2, and (5) RWC/4. More specifically, if the RWC
= 50%, the effluent concentrations used in the toxicity test would
be 100%, 75%, 50%, 25%, and 12.5%. If the RWC is <25% effluent
the concentrations may be: (1) 4 times the RWC, (2) 2 times the
RWC, (3) RWC, (4) RWC/2, and (5) RWC/4.
2.2.4
Receiving (ambient) water toxicity tests commonly employ
two treatments, a control and the undiluted receiving water, but
may also consist of a series of receiving water
dilutions.
2.2.5
A negative result from a chronic toxicity test does not
preclude the presence of toxicity. Also, because of the potential
temporal variability in the toxicity of effluents, a negative test
result with a particular sample does not preclude the possibility
that samples collected at some other time might exhibit chronic
toxicity.
2.2.6
The frequency with which chronic toxicity tests are
conducted under a given NPDES permit is determined by the
regulatory agency on the basis of factors such as the variability
and degree of toxicity of the waste, production schedules, and
process changes.
2.2.7
Tests recommended for use in this methods manual may be
static non-renewal or static renewal. Individual methods specify
which type of test is to be conducted.
2.3
STATIC TESTS
2.3.1
Static non-renewal tests - The test organisms are exposed
to the same test solution for the duration of the test.
2.3.2
Static-renewal tests - The test organisms are exposed to
a fresh solution of the same concentration of sample every 24 h or
other prescribed interval, either by transferring the test
organisms from one test chamber to another, or by replacing all or
a portion of solution in the test chambers.
2.4
ADVANTAGES AND DISADVANTAGES OF TOXICITY TEST
TYPES
2.4.1 STATIC NON-RENEWAL, SHORT-TERM TOXICITY TESTS:
Advantages:
1.
Simple and inexpensive.
2.
More cost effective in determining compliance with permit
conditions.
3.
Limited resources (space, manpower, equipment) required;
would permit staff to perform more tests in the same amount of
time.
4.
Smaller volume of effluent required than for static
renewal or flow-through tests.
Disadvantages:
1.
Dissolved oxygen (DO) depletion may result from high
chemical oxygen demand (COD), biological oxygen demand (BOD), or
metabolic wastes.
2.
Possible loss of toxicants through volatilization and/or
adsorption to the exposure vessels.
3.
Generally less sensitive than renewal because the toxic
substances may degrade or be adsorbed, thereby reducing the
apparent toxicity. Also, there is less chance of detecting slugs of
toxic wastes, or other temporal variations in waste
properties.
2.4.2 STATIC RENEWAL, SHORT-TERM TOXICITY TESTS:
Advantages:
1.
Reduced possibility of DO depletion from high COD and/or
BOD, or ill effects from metabolic wastes from organisms in the
test solutions.
2.
Reduced possibility of loss of toxicants through
volatilization and/or adsorption to the exposure
vessels.
3.
Test organisms that rapidly deplete energy reserves are
fed when the test solutions are renewed, and are maintained in a
healthier state.
Disadvantages:
1.
Require greater volume of effluent than non-renewal
tests.
2.
Generally less chance of temporal variations in waste
properties.
SECTION 3
HEALTH AND SAFETY
3.1 GENERAL PRECAUTIONS
3.1.1
Each laboratory should develop and maintain an effective
health and safety program, requiring an ongoing commitment by the
laboratory management and includes: (1) a safety officer with the
responsibility and authority to develop and maintain a safety
program; (2) the preparation of a formal, written, health and
safety plan, which is provided to the laboratory staff; (3) an
ongoing training program on laboratory safety; and (4) regularly
scheduled, documented, safety inspections.
3.1.2
Collection and use of effluents in toxicity tests may
involve significant risks to personal safety and health. Personnel
collecting effluent samples and conducting toxicity tests should
take all safety precautions necessary for the prevention of bodily
injury and illness which might result from ingestion or invasion of
infectious agents, inhalation or absorption of corrosive or toxic
substances through skin contact, and asphyxiation due to a lack of
oxygen or the presence of noxious gases.
3.1.3
Prior to sample collection and laboratory work, personnel
should determine that all necessary safety equipment and materials
have been obtained and are in good condition.
3.1.4
Guidelines for the handling and disposal of hazardous
materials must be strictly followed.
3.2
SAFETY EQUIPMENT
3.2.1 PERSONAL SAFETY GEAR
3.2.1.1
Personnel must use safety equipment, as required, such as
rubber aprons, laboratory coats, respirators, gloves, safety
glasses, hard hats, and safety shoes. Plastic netting on glass
beakers, flasks and other glassware minimizes breakage and
subsequent shattering of the glass.
3.2.2
LABORATORY SAFETY EQUIPMENT
3.2.2.1
Each laboratory (including mobile laboratories) should be
provided with safety equipment such as first aid kits, fire
extinguishers, fire blankets, emergency showers, chemical spill
clean-up kits, and eye fountains.
3.2.2.2
Mobile laboratories should be equipped with a telephone
to enable personnel to summon help in case of emergency.
3.3
GENERAL LABORATORY AND FIELD OPERATIONS
3.3.1
Work with effluents should be performed in compliance
with accepted rules pertaining to the handling of hazardous
materials (see safety manuals listed in Section 3, Health and
Safety, Subsection 3.5). It is recommended that personnel
collecting samples and performing toxicity tests should not work
alone.
3.3.2
Because the chemical composition of effluents is usually
only poorly known, they should be considered as potential health
hazards, and exposure to them should be minimized. Fume and canopy
hoods over the toxicity test areas must be used whenever
possible.
3.3.3
It is advisable to cleanse exposed parts of the body
immediately after collecting effluent samples.
3.3.4
All containers should be adequately labeled to indicate
their contents.
3.3.5
Staff should be familiar with safety guidelines on
Material Safety Data Sheets for reagents and other chemicals
purchased from suppliers. Incompatible materials should not be
stored together. Good housekeeping contributes to safety and
reliable results.
3.3.6
Strong acids and volatile organic solvents employed in
glassware cleaning must be used in a fume hood or under an exhaust
canopy over the work area.
3.3.7
Electrical equipment or extension cords not bearing the
approval of Underwriter Laboratories must not be used. Ground-fault
interrupters must be installed in all "wet" laboratories where
electrical equipment is used.
3.3.8
Mobile laboratories should be properly grounded to
protect against electrical shock.
3.4
DISEASE PREVENTION
3.4.1
Personnel handling samples which are known or suspected
to contain human wastes should be immunized against tetanus,
typhoid fever, polio, and hepatitis B.
3.5
SAFETY MANUALS
3.5.1
For further guidance on safe practices when collecting
effluent samples and conducting toxicity tests, check with the
permittee and consult general safety manuals, including USEPA
(1986e), and Walters and Jameson (1984).
3.6
WASTE DISPOSAL
3.6.1 Wastes generated during toxicity testing must be properly
handled and disposed of in an appropriate manner. Each testing
facility will have its own waste disposal requirements based on
local, state and Federal rules and regulations. It is extremely
important that these rules and regulations be known, understood,
and complied with by all persons responsible for, or otherwise
involved in, performing toxicity testing activities. Local fire
officials should be notified of any potentially hazardous
conditions.
