United States
                                  Environmental Protection
                                  Agency
      Office of
      Solid Waste and
      Emergency Response
Publication 9345.0-051
EPA540-F-94-012
PB94-963303
September 1994
  vvEPA          ECO   Update
  Office of Emergency and Remedial Response
  Hazardous Site Evaluation Division (5204G)
                            Intermittent Bulletin
                            Volume 2, Number 1
Using  Toxicity Tests  in   Ecological
            Assessment
      Toxicity tests are used to expose test organisms to a
  medium—water, sediment, or soil—and evaluate the effects of
  contamination on the survival, growth, reproduction, behavior
  and/or other attributes of these organisms. These tests may help
  to determine whether the contaminant concentrations in a site's
  media are high enough to cause adverse effects in organisms.
  Generally, toxicity tests involve collecting samples of media
  from a site and sending them to a toxicity laboratory, where the
  tests are performed. On occasion, investigators' measure toxic-
  ity by exposing test organisms to soil or water on site—these are
  known as in situ tests.
      As the general  guidelines at the end of this  Bulletin
  indicate, not all sites require toxicity tests. But where they are
  used, toxicity tests can contribute to ecological risk assessments
  in specific ways and at different stages in the assessment.
      1. Toxicity tests can demonstrate whether contaminants
  are bioavailable.2 The presence of a contaminant does not of
  itself indicate a potential for adverse effects. A contaminant can
  have toxic effects only if it occurs in a bioavailable form.
  Sometimes the presence of abrasives, such as the talc in pesti-
  cides, can damage an  organism's body covering, thereby in-
  creasing the bioavailability of certain contaminants for that
  organism.
       2. Toxicity tests can evaluate the aggregate toxic effects
  of all contaminants in a medium. Many Superfund sites present
  a complex array of contaminants, with a mixture of potentially
  harmful substances present in the media. At such sites, chemi-
  cal data alone cannot accurately  predict the toxicity  of the
  contaminants. Rather, toxicity  tests measure  the aggregate
  effects of contaminated media on organisms. These effects
  result from characteristics of the medium itself (such as hard-
ness and pH, in the case of water), interactions among contami-
nants, and interactions between contaminants and media. Con-
sequently, observed toxicity test results may often vary from
those predicted by chemical data alone.
    3. Toxicity tests can evaluate the toxicity of substances
whose biological effects may not have been well characterized.
The contaminants at a Superfund site might include substances
that have not been previously investigated regarding their
toxicity to wildlife or other organisms. Consequently, the
scientific literature contains no relevant data concerning these
substances. At such sites, toxicity tests of media samples
     1 The term "investigator" refers to the individual charged with
 responsibility for designing and/or carry ing out any part of an ecologi-
 cal risk assessment. Investigators can include government scientists,
 contractors, or university scientists. However the site manager (reme-
 dial project manager or on-scene coordinator) retains ultimate respon-
 sibility for the quality of the ecological risk assessment.
     2 Bioavailability is the presence of a substance in a form that
 organisms can take up. (Note that specialized terms appear in boldface
 and are defined either in the text or in accompanying footnotes.)
            IN  THIS  BULLETIN

Measurement Endpoints in Toxicity Testing	  2
Elements in a Toxicity Assessment 	  3
General Guidelines for Choosing Toxicity Tests	  9
  ECO Update is a Bulletin series on ecological risk assessment of Superfund sites. These Bulletins serve as supplements to Risk Assessment Guidance
  for Superfund, Volume II: Environmental Evaluation Manual (EPA/540-1-89/001). The information presented is intended to provide technical
  information to EPA and other government employees. It does not constitute rulemaking by the Agency, and may not be relied on to create a
  substantive or procedural right enforceable by any other person. The Government may take action that is at variance with these Bulletins.

-------
 indicate the combined toxicity of all contaminants, including those
 that have not been previously tested.
     4. Toxicity tests can characterize the nature of a toxic effect.
 Investigators can use toxicity tests to learn whether  contaminant
 concentrations have lethal or sublethal effects. Some examples of
 sublethal effects include reduced growth, impaired reproduction,
 and behavioral changes.
     5. Toxicity tests can characterize the distribution of toxicity at
 a  site. An investigator can have  toxicity tests  performed  on
 samples from a variety of locations at the site. In some instances
 toxicity tests may be a cost-effective way to determine the spatial
 extent of toxicity and identify areas with high levels of toxicity.
     6. Toxicity tests  can be used  to develop remedial goals.
 Acceptable levels of toxicity, as measured by toxicity tests, can
 form a criterion for remedial goals. For example, a goal might  be
 to reduce the toxicity of pond water over a stated time period. The
 remedial goal would specify the level to which toxicity should  be
 reduced and the species in which toxicity should be measured. The
 species should be representative  of the site and sensitive to  its
 contaminants. The species also should relate to the overall assess-
 ment endpoints.3
     7. Toxicity tests have a role in monitoring. Toxicity tests can
 be used to monitor the remediation of a Superfund site. Specifi-
 cally, toxicity testing can indicate whether sources of contamina-
 tion have been contained and whether remedial measures are
 reducing toxicity.
     8. Toxicity tests have  a role in determining a site's post-
 remediation potential to support a viable ecological community.
 For example, if a stream or waterbody receives contaminants from
 numerous sources, including a Superfund site, upstream toxicity
 testing may help  to determine what the water's potential for
 supporting a viable ecological community might be if the Superfund
 loadings are removed and the other sources remain unchanged.
     Toxicity tests include a broad spectrum of tests, differing in
 the species and exposure media  they  use and  the  effects  they
 measure. In making decisions about  whether to  conduct toxicity
 tests, which tests to choose, and how many to perform, investiga-
 tors are well advised to seek advice from qualified experts, such  as
 those serving on  a Regional Biological  Technical Assistance
 Group (BTAG).4
    This Bulletin first describes two major classes of toxicity
 tests—acute and chronic—and then explores the elements that  an
 investigator needs to consider in planning toxicity tests. Finally,
 the Bulletin offers general guidance on when to use toxicity tests
and how to select those appropriate to different sites. The compan-
 ion document, "Catalogue of Standard Toxicity Tests for Ecologi-
cal Risk Assessment"  (ECO Update Vol. 2, No. 2),  provides  an
annotated list of standardized tests appropriate for use with differ-
 ent media.
    3 An assessment endpoint is an ecological characteristic that may be
adversely affected by site contamination and that, at a Superfund site, can
help to drive remedial decision making (U.S. EPA, 1992).
Measurement  Endpoints  In
Toxicity Testing: Acute Vs.
Chronic  Tests

     Toxicity tests can measure lethal  and/or sublethal effects.
These effects are known as measurement endpoints: that is, they
are ecological attributes that may be adversely affected by expo-
sure  to  site contaminants and  that are readily measurable. In
addition, each  measurement  endpoint  is closely  related to an
assessment endpoint. Because of this close relationship, a mea-
surement endpoint can approximate or  represent the assessment
endpoint if the assessment endpoint is not amenable to  direct
measurement (U.S. EPA, 1992).
     Acute toxicity tests are  short-term tests that measure the
effects of exposure to relatively high concentrations of chemi-
cals. The measurement endpoint generally reflects the extent of
lethality.
     Chronic toxicity tests, on the other hand, generally are longer-
term tests that measure the effects of exposure to relatively lower,
less toxic concentrations. For a chronic toxicity test, the measure-
ment endpoint  concerns a sublethal effect (e.g.,  reproduction,
growth) or both lethality and a sub-lethal effect.

Acute Toxicity Tests
     A typical acute toxicity test exposes test organisms to a series
of dilutions of a site's medium and records deaths occurring over
a specified period of time,  usually 24 to 96 hours. Results can be
analyzed by comparing percent mortality of organisms exposed to
site media to percent mortality for organisms exposed to uncon-
taminated  media. (See section below  entitled "The Reference
Site.")  Alternatively,  results  of an acute toxicity test can be
analyzed to estimate the dilution of the medium at which 50
percent of the organisms died. This dilution (also referred to as a
concentration), called the LC50, is the median lethal concentration.
When an acute toxicity test reports an LCJ(|, the test results usually
will specify the test duration, the test species, and the life cycle
stage of the test species (e.g., the fathead minnow 96 hour LCJO).
Since LC5(|s are point estimates, which are estimates of the effects
from specific concentrations of contaminants, coefficients of varia-
tion  can be calculated for them.  (See section below entitled
"Statistical Analysis.")
    4 These groups are sometimes known by different names, depending
on the Region. Readers  should check with the appropriate Superfunc*
manager for the name of the BTAG coordinator or other sources of
technical assistance in their Region.  A more complete description of
BTAG structure and function is available in "The Role of BTAGs in
Ecological Assessment" (ECO Update Vol. 1, No. 1).

-------
     With some test organisms, toxicologists find death difficult to
 letermine unequivocally. In tests using such organisms, toxicolo-
gists evaluate another effect, such as immobility, that correlates
closely with death. As with death for a measurement endpoint,
results can be analyzed by comparing percent effect for organisms
exposed to site media and those exposed to uncontami nated media.
Alternatively, data can  be analyzed  to estimate the dilution at
which 50 percent of the  organisms  displayed the effect. This
dilution (also referred to as a concentration), called the EC50, is the
median effective concentration. When an acute toxicity test reports
an ECM, the test results will specify the effect, the test duration, the
test species, and the  life cycle stage of the test species. Like the
         Acute toxicity tests are short-term
     tests that measure the effects of expo-
     sure to relatively high concentrations
     of chemicals.
          Chronic toxicity tests generally are
     longer-term  tests that  measure  the
     effects of exposure to relatively lower,
     less toxic concentrations.
LCjjj, the ECJO is a point estimate and a coefficient of variation can
be calculated for it.
    In still other approaches to evaluating results, the laboratory
analyzes the data for the Lowest Observed Effect Concentration
(LOEC), which is the highest dilution causing statistically signifi-
cant toxic effects, or the  No Observed Effect Concentration
(NOEC), which is the lowest dilution at which no statistically
significant toxic effects occurred.5 Statistically determined using
hypothesis testing, LOECs and NOECs are not point estimates and
consequently coefficients of variation cannot be calculated for them.

Chronic Toxicity Tests
    A chronic toxicity test exposes test organisms to a series of
dilutions of a site's medium and measures sub-lethal effects, and
in some cases lethal effects as well. Sublethal effects may  include
growth reduction, reproductive impairment, nerve function im-
pairment, lack of motility, behavioral changes, and the develop-
ment of terata, which are structural abnormalities.  Results can be
analyzed in several ways. One is simply by a direct  comparison
    5 As  used in  this Bulletin, LOEC  is synonymous with Lowest
Observed Adverse Effect Concentration (LOAEC) and Lowest Observed
Adverse Effect Level (LOAEL), and NOEC with No Observed Adverse
Effect Concentration (NOAEC) and No Observed Adverse Effect Level
(NOAEL).
between percent  effect occurring in organisms exposed to site
media and those exposed to uncontaminated media. Other ap-
proaches to analysis determine the ECJO, the LOEC, or the NOEC.

Ecological Significance of Sublethal Effects
    Although it  would be an  oversimplification to extrapolate
from the outcome of chronic toxicity tests to ecological conditions
at  a Superfund site, site managers  need to be aware that the
sublethal effects that chronic toxicity tests measure in laboratories
are ecologically significant effects when they occur in the environ-
ment. For example, reduced growth can lead to decreased produc-
tion, smaller size, lower fecundity (eggs or  young per female),
increased susceptibility to predation, and other effects. Reproduc-
tive impairment can reduce the  population size and also bring
about  changes in a population's age structure. Production  of
individuals with terata can adversely affect a population because
these individuals have a lower growth rate, are generally unable to
reproduce, and have an increased susceptibility to predation.

A  Comparison of Acute and Chronic Toxicity
Tests  with Respect to  Time, Cost, and
Resolution
    In general,  acute  and chronic toxicity  tests  differ in the
amount  of time  required to perform them, their cost,  and their
resolution.
  • Because chronic tests extend through either a life cycle or a
    critical  developmental phase, they generally require more
    time to  perform than acute tests with the same type of test
    organisms.

  • Requiring more time to complete than  acute tests, chronic
    tests also can require more  funds. A chronic test also may
    require  more resources and increased numbers of laboratory
    analyses, further increasing  the cost of the test.

  • Chronic tests have greater resolution than acute  tests. For
    example, consider a chronic test that exposes invertebrates to
    site surface  water and records the number of young they
    produce. In a highly toxic medium, the organisms will die. In
    a less toxic medium, they may survive, but their reproductive
    capacity may be impaired  when compared  with controls
    maintained in an uncontaminated medium.
Elements in a Toxicity
Assessment

    The investigator needs to consider many elements when
planning a toxicity assessment: the objective, the reference site, the
medium analyzed, the test organisms, the test methodology, the
level of effort, the test site, and quality assurance/quality control
(QA/QC) standards. By  the choices that he or she makes, the
investigator can tailor the toxicity assessment to meet the needs of
the site and its stage in the Superfund process.

-------
The Objective
     As with any study, before planning a toxicity assessment the
investigator needs  to set  clear objectives.  In  particular,  the
assessment's objectives need to include some that address  the
medium  of concern, the characteristics of the contaminants of
concern, and the potential ecological components.6 For example,
if the study asks whether  soil  on the  site  is  toxic to
macroinvertebrates, then the study will need to analyze bulk soil
rather than an elutriate7 and will need to use an appropriate test
organism.
     The objectives of a toxicity assessment should indicate  the
level of effort appropriate to the assessment. For example, deter-
mining whether a particular medium is toxic would generally
require a low level of effort. Such a study might specify only two
species of test organisms and undiluted medium collected from a
limited number of sampling locations. If the objective of a toxicity
assessment is to determine the appropriate range of dilutions for
conducting further  tests (if these prove necessary) at a highly
contaminated site, a higher level of effort would be necessary.
Such  a study might specify using a series of tenfold dilutions of
media collected from locations known to have high contaminant
concentrations. A reasonably detailed characterization of a site's
toxicity would imply a high level of effort. This type of study might
include test organisms at different trophic levels* (such as an alga,
a macroinvertebrate, and a fish), several sampling locations (pos-
sibly based on a grid or selected from upstream and downstream
areas), and several dilutions of medium.

The Reference Site
     When planning a toxicity assessment, an investigator selects
a reference site that as closely as possible mirrors the characteris-
tics of the site medium being analyzed but is  unaffected by site
contamination. Analyzing a sample from the reference site allows
the investigator to measure background conditions. The investiga-
tor should try to locate the reference site as close as possible to  the
Superfund site so that the reference site will accurately reflect  the
site's conditions. Yet the reference site should lie at a great enough
distance from the Superfund site to be unaffected by site contami-
nation. Provided that pollutant loading from other sources does  not
occur upstream, an upstream location may provide an appropriate
reference site for a Superfund  site with contaminated  surface
water. Soil type and texture, vegetation,  and slope are important
considerations in selecting  a reference site with the appropriate
terrestrial characteristics.
    6 An ecological component is an individual organism, a population,
a community, a habitat, or an ecosystem that may suffer adverse effects as
a result of site contamination.
    7 An elutriate (or eluate) is the solution obtained when water re-
moves substances adsorbed to sediment particles.
    8 A trophic level is a stage in the  flow of food from one population
to another. For example, as primary producers (organisms that convert the
energy from sunlight to chemical energy) plants occupy the  first trophic
level, and grazing organisms occupy the second trophic level.
The Medium
     Toxicity tests vary as to the media they analyze. Aquatic test?
evaluate freshwater, marine, or estuarine samples. A few tests are
designed specifically to analyze bulk sediment samples, and a few
are specific for bulk soil  samples.  Bulk sediment or soil tests
specifically address toxicity in the  test medium. Alternatively,
laboratory technicians can prepare elutriates of sediment or soil
samples and analyze the  elutriates  by means of aquatic  tests.
Toxicity tests using elutriates give information about the transfer
of contaminants from sediment or soil to water. Such information
is most valuable when predicting effects of runoff or leaching from
soil  or determining the advisability of remediating  a site by
dredging contaminated sediments.
     A toxicity test also  should  include  measurements of the
appropriate physical and chemical parameters of the sample me-
dium. For water, these parameters might include alkalinity, hard-
ness, pH, temperature,  dissolved oxygen, total dissolved solids,
and total organic carbon. For a sediment sample, grain size, percent
          A toxicity test should include mea-
      surements of the appropriate physical
      and chemical parameters of the sample
      medium.
water, pH, total organic carbon,  and/or other parameters may
prove important to know.
     In some cases the physical or chemical parameters of the test
medium require adjustment in order to meet the conditions of a test
protocol. Sediment or soil may requiredewatering. Water samples
may need to have their pH, hardness, or dissolved oxygen content
adjusted.  Such  adjustments can change  the  solubility,
bioavailability,  or toxic properties  of sample  constituents  and
therefore should be avoided or minimized wherever possible. If the
test medium requires adjustment, the investigator should allow a
portion of it to remain unadjusted. This unadjusted portion is used
in a parallel control that will indicate whether the adjustment
contributes to, masks, or has no effect on toxicity. In cases where
the test medium  requires adjustment,  the investigator should
evaluate the data quality objectives (DQOs)g to determine whether
the adjustments would interfere with the study's objectives.
     For many  toxicity tests investigators must dilute  sample
media to determine LC5()s, EC5()s, LOECs, or NOECs. Protocols for
aquatic tests generally specify using specially treated laboratory
water as a diluent, but natural water can be used as well. Diluting
material for soils or sediments can consist of artificial soil prepared
in the laboratory.
     * Data quality objectives (DQOs) are statements that define the leve.
of uncertainty that investigator is willing to accept in environmental data
used to support a remedial decision. DQOs address the purpose and use of
data, the resource constraints on data collection, and any calculations
based on the data.

-------
Test Organisms
     Toxicologists have based their selection of test organisms on
several factors: sensitivity to a variety of substances, availability,
representativeness of a variety of ecosystems, and case of mainte-
nance and culture under laboratory conditions. For aquatic tests,
the most frequently  used test organisms are those employed for
toxicity testing for National Pollutant Discharge Elimination Sys-
tem (NPDES) permits. Table 1 summarizes information about the
organisms used in the standardized tests, while Figure 1 illustrates
a few of these organisms.
     When choosing from among the available standard test or-
ganisms, the investigator should select a species that is represen-
tative of resident  organisms, sensitive to site contaminants, rel-
evant to the overall assessment endpoints, and consistent with
DQOs. In a toxicity test, the test organisms serve as surrogates for
organisms  present on the  site.  For  instance, although fathead
minnows (Pimephalespromelas), a common test organism, may
not occur on the site, they can serve  as surrogates for other fish.
Consequently an LC5() for fathead minnows can serve as a measure-
ment endpoint for the assessment endpoint "survival of the min-
now populations in a specific stream  that flows through the site."
In a broader context, fathead minnows might represent all warm-
water fish on a site, since research has shown that organisms at the
same taxonomic level (level of classification, such as genus or
family)  often respond similarly to a  contaminant (Baker, 1989).
When selecting test  organisms, the investigator should keep the
study's  DQOs in mind. If the investigator's selection  is not
consistent with the DQOs, the applicability of the test data to the
site is questionable.
     Although the existence of well-established protocols and
considerable historical data makes the standard test organisms
useful, in some cases investigators find that none of the standard
organisms is representative of a site's ecosystem. If this situation
          When  choosing from  among  the
      available standard test organisms, the
      investigator should select  a  species
      that  is  representative  of resident
      organisms,  sensitive  to site contami-
      nants, relevant to the overall assess-
      ment endpoints,  and  consistent with
      DQOs.
occurs, the investigator must account for this lack of representa-
tiveness when interpreting test results. Alternatively, the investi-
gator may decide to use a "non-standard" or alternative species
instead of the one specified in the test protocol. The alternative
species might better represent resident organisms, show greater
sensitivity to the site's contaminants, or be more consistent with
the study's DQOs. State resource agencies can readily provide
information  on resident species. Using resident species as  an
alternative species has the potential of providing direct informa-
tion about the toxic effects to site species. Several criteria must be
specified for the use of alternative test species, including the source
for the test organism, the age range suitable for the test, a means for
eliminating variability in the organism's condition, and conditions
suitable for the test. In addition, if the organism must be collected
rather than purchased, the investigator will have to establish
standards for ensuring accurate identification and also should meet
all local, state, and federal requirements concerning the collection
of organisms.
     Generally, using  alternative species  increases the  cost  of
conducting toxicity tests, especially when the investigator needs to
determine optimal conditions for conducting the test. However,
the investigator, in consultation with  the BTAG, may decide that
the added usefulness of the results justifies the extra expense. In
such a case, the investigator  may be able to reduce the added
expense by employing a laboratory experienced in the use of the
species selected for the study.

Test Method
     Standard toxicity tests can employ a variety of methods for
collecting samples and for exposing test organisms to media.
Designing a toxicity assessment for a site requires the investigator
to select the most appropriate methods for studying the issues for
that site.
     Field biologists can collect media samples  for testing either
by the grab or the composite method. As the name implies, a grab
sample is a single sample, usually entailing little time and minimal
equipment to collect.  When the investigator expects the site's
contaminant picture to change little over time, a single grab sample
per location may adequately represent contamination. A compos-
ite sample, on  the other hand, is a mixed sample, which may be
collected at a single  location over a specified period of time or at
multiple locations at one time. When sampling a stream with a
highly variable flow rate, the investigator can specify the collec-
tion of a flow weighted composite sample. The BTAG can advise
the investigator as to the preferred collection method for a particu-
lar site.
     Toxicity tests analyzing water or elutriates of soil or sediment
can expose test organisms using the same sample medium through-
out the test or arranging for limited replacement of medium. Those
using the same sample medium throughout are called static tests,
while static-renewal tests  are those that replace all or part of the
sample  medium at  specified times  during the test. Since this
approach requires little space, manpower, and equipment, static
tests are comparatively simple  and  inexpensive to perform. In
addition, static tests require only small  sample volumes of one to
20 liters.
     On the other hand, static tests, particularly those  without
renewal of media, do have certain limitations. Over the course of
a  non-renewal  test, test  organisms can deplete the dissolved
oxygen  in the sample and suffer adverse effects unrelated to
toxicity. Alternatively, contaminants can break  down, volatilize,
or adhere to the walls of the container. As a result, the test might
not accurately reflect the medium's toxicity. Finally, as organisms
metabolize they release substances,  such as carbon dioxide and

-------
                         Table 1. Plant and Animal Species Used in Standard Toxicity Tests"
Medium Test Organism
F
R
E
S
H
W
A
T
E
R
MARINE
and
ESTUA-
RINE
WATERS
FRESH-
WATER
SEDIMENT
MARINE
SEDIMENT
SOIL
Test Temp (°C)
Life Stage
VERTEBRATES
Brook trout (Salvelinus fontinalis)
Rainbow trout (Oncorhynchus mykiss)
Fathead minnow (Pimephales promelas)
12
12
20-25
30-60 days
15-30 days
1-14 days
INVERTEBRATES
Amphipod (Hyalella)
Waterflea (Daphnia magna, Daphnia pulex, Ceriodaphnia)
Mayfly (Hexagenia limbata, Hexagenia bilineata)
Midge (Chironomus)
20 or 25
20 or 25
17,20-22
20 or 25
7-14 days
1-24 hours
Young nymph
First to second instar
ALGA
Selenastrum capricornutum
25
4-7 day stock culture
VERTEBRATES
Sheepshead minnow (Cyprinodon variegatus)
Silverside (Menidia species)
20 or 25
20 or 25
1-14 days
9-14 days
INVERTEBRATES
Sea urchin (Arbacia punctulata)
Mysid shrimp (Mysidopsis)
20
20
< 1 hour old
1-5 days
ALGA
Champia parvula
Amphipod (Hyalella azteca)
Midge (Chironomus tentans and Chironomus riparius)
Amphipod (Rhepoxynius abronius)
Amphipod (Eohaustorius estuarius)
Amphipod (Ampelisca abdita)
Amphipod (Grandidierella japonica)
Earthworm (Eisenia foetida)
Lettuce (Latuca sativa)
23
20-25
20 or 25
15
15
20
15-19
22
24
Sexually mature
7-14 days
First to second instar
Mature 3-5 mm, mixed sex
Mature 3-5 mm, mixed sex
Immature, or mature females only
Immature 3-6 mm, no females with embryos
300-600 mg adult
Seed
'Compiled from ASTM, 1992c; Greene et al, 1989; US Army Corps of Engineers, 1993; Weber et al, 1988; Weber et al, 1989; Weber et al, 1991.

-------
           Figure 1.  Test Organisms Commonly Used in Toxicity Tests
                     The organisms shown in these figures are the adult life
                     stages of the test organisms, not necessarily those life
                     stages used in toxicity tests.
 Fathead minnow (Pimephales promelas)
 50 mm.
Waterflea (Daphnia)
up to 3.5 mm for Daphnia pulex
Purple sea urchin
(Stronglyocentrotus purpuratus)
6 to 12 cm
Macroalga (Champia)
Actual size of branch tip 25 mm.

Mysid shrimp (Mysidopsis)
4.4 to 9.4 mm for Mysidopsis bahia
Marine amphipods
Species used in toxicity tests usually
3-6 mm.
           Reproduced by courtesy of Aquatic Research Organisms.
           P.O.Box 1271, Hampton, NH 03842.

-------
wastes, called metabolites. As these metabolites accumulate, they
can prove toxic to the test organisms. Alternatively, they may
interact with contaminants and alter the medium's apparent toxic-
ity. The static-renewal design overcomes to a certain extent the
disadvantages of the nonrenewal design.
     In place of the static design, aquatic toxicity tests may use a
flow-through method, continuously pumping fresh sample me-
dium through test chambers. With this method, dissolved oxygen
remains relatively high and metabolites  are flushed away. The
flow-through approach also has the advantage of minimizing loss
of toxics to  degradation,  adsorption, and  volatilization.  When
conducted on-site,  flow-through  tests can  provide information
about fluctuations in toxicity.
     Disadvantages of the flow-through  method include its ex-
pense and inconvenience. This approach uses complex equipment
that requires more maintenance than the equipment used for static
tests. Large volumes of sample and diluent are needed for flow-
through tests. As a result, these tests are more  expensive than
equivalent static tests.

Level of Effort
     The quantity and nature of information provided by toxicity
tests varies considerably with the level of effort mandated by the
study's objective. One factor determining the level of effort is the
number of species used as test organisms. When  the investigator
selects a test  species that is quite sensitive to the site's contami-
nants relative to other types of organisms in the community, then
a single-species test can  suggest the community's  maximum
susceptibility to the site's contaminants. At a higher level of effort,
employing more than one test species may indicate whether single-
species tests  have  underestimated or overestimated the  site's
toxicity. However, when evaluating water, toxicity tests should
always include  at least two  species, a fish and an invertebrate,
unless the site has only one contaminant and either fish or inverte-
brates are known to be insensitive to that contaminant.
    The level  of effort also varies  with  the range of media
concentrations analyzed and the duration  of the tests.
  •  Screening tests, which may be either acute or chronic tests,
    evaluate only the undiluted sample. Positive values on screen-
    ing tests may indicate the need to proceed to definitive tests.

  •  Range finding tests are abbreviated static  acute tests that
    expose test organisms to a broad range of media dilutions for
    8  to 20  hours. Such tests identify  the dilutions to use for
    definitive tests.

  •  Definitive tests provide a dose-response curve and reduced
    variability. Both acute and chronic tests can be conducted as
    definitive tests.Investigators can use these three types of tests
    to translate "level of effort" objectives into appropriate tox-
     icity assessments.

Test Site: Laboratory or in situ
     In general, toxicity tests are conducted by collecting samples
from a site and sending them to a laboratory for testing. Laboratory
tests offer the advantage of standardized protocols. In addition,
laboratories experienced in performing these tests are generally
available.
     Alternatively, toxicity tests can occur in situ, which means "in
place." That is, the investigator exposes test organisms to soil or
water on the site. In situ tests give organisms continuous exposure
to the site media under actual environmental conditions such as
temperature, stream flow, and light.10 Consequently, data from in
situ tests provide a more realistic assessment of toxicity than do
data from laboratory tests. In situ tests also provide a more direct
means of  comparing  toxicity  data  with  estimates of exposure
derived from field data. Ultimately, such comparisons help to
characterize the site's  ecological risk.
     On the other hand, in situ tests have certain disadvantages. In
particular, the investigator lacks control over the conditions under
which an in situ test occurs. For example, temperature may vary
considerably over  the course of an in situ test, whereas in  the
laboratory temperature would be carefully regulated. When inter-
preting data from in situ tests, the investigator should consider how
the varying conditions of the test could affect results. Another issue
associated with in situ tests concerns logistics, which can prove
difficult in  adverse weather conditions.
     At the present time, in situ tests generally consist of standard
laboratory tests adapted for on-site use. Some commonly used tests
employ earthworms or lettuce seeds as standard test organisms for
soil toxicity tests. For in situ aquatic toxicity tests, fish, clams, and
oysters are often used. Tests may instead use alternative or resident
species. In practical terms, to conduct in situ tests, test vegetation
is maintained in plots and test animals are held in containers on site.
The  earthworm test, in particular, has been successfully adapted
for insitu use. In fact, some field biologists routinely perform this
test in situ rather than having personnel conduct it as a laboratory
test.

Statistical  Analysis
     The statistical analysis of toxicity test results depends upon
the measurement endpoint. As mentioned earlier, LCJ(Jsand ECJ(|s
are point estimates; that is, they are estimates of effects from
specific dilutions of contaminants.  To calculate point estimates,
test data are analyzed using regression models that assume the less
dilute the sample, the  greater will be the effects. Coefficients of
variation can be calculated for point estimates.
     LOECs and NOECs compare results at test dilutions with
controls to  determine whether the results are significantly differ-
ent. LOECs and NOECs are calculated by means of a statistical
method called hypothesis testing. Coefficients of variation cannot
be calculated for values determined using hypothesis testing. Note
too that the values obtained for LOECs and NOECs can vary
considerably depending on the specific series of dilutions used in
the test.
     For detailed information  concerning statistical  analysis of
toxicity test data, investigators can consult Weber et al. (1988,
1989, 1991).
     10 In situ tests should not be confused with tests performed in a
mobile laboratory brought to the site. A mobile laboratory performs
toxicity tests under standard laboratory conditions, not site conditions.

-------
Quality Assurance/Quality Control (QA/QC)
Standards
     The investigator must specify standards for quality assurance
and quality control (QA/QC) to ensure that toxicity tests produce
reliable results. QA/QC standards  describe appropriate sample
handling and collection. Such  matters  as  the container type,
storage temperature, and maximum permissible storage time af-
fect the reliability of data. For example, elapsed time and exposure
to air can alter sample properties or result in the loss of volatile
chemicals. Generally, aquatic samples should be stored at 4°C until
used. Although holding time limitations for sediments vary with
individual sediment properties and contaminant characteristics,
the American Society for Testing and Materials (ASTM) recom-
mends storing sediments at 4°C and using them within two weeks
of collection.
     Test parameters should specify which controls to perform and
what values to accept for controls. For example, in an acute toxicity
test with Daphnia pulex,  a  control consists of test organisms
exposed to dilution water only.  For test results to be considered
valid, at least 90% of the animals in the control must survive the
test.
     Finally, QA/QC measures  need to ensure that the data col-
lected can support the appropriate statistical analysis.
     The BTAG can advise the investigator as to whether  the
proposed QA/QC standards are adequate.
General  Guidelines for Choosing
Toxicity Tests

     In an  ecological risk assessment of a Superfund site, the
investigator must decide whether toxicity testing will contribute to
the assessment and, if so, which and how many tests to perform.
The great differences among Superfund sites—differences in size,
terrain, and contaminant profile, to name a few—make a rigidly
standardized approach to toxicity studies unworkable . However,
a few widely accepted general guidelines do exist:
  •  Do not perform toxicity studies at a site where the contami-
     nants  of concern do not cause effects measured by toxicity
     tests. For example, polychlorinated biphenyls (PCBs) bring
     about  reproductive effects that many toxicity tests do not
     detect. The investigator should consult the BTAG to find out
     whether toxicity tests can detect the type of effects caused by
     a site's contaminants.

  •  At a site where several substances have contaminated surface
     water, aquatic toxicity testing generally should include both
     a fish andan invertebrate species. At some sites, it may prove
     advisable to include additional test organisms, as well.

  •  Select test organisms that are sensitive to the site contami-
     nants. For example, among standard freshwater test organ-
     isms, water fleas of the genus Ceriodaphnia and the embryos
     and larvae of fathead  minnows are  sensitive  to  a  broad
     spectrum of contaminants. Of the two species of midges used
     to conduct standard tests of freshwater sediments, Chironomus
     riparius is the choice when metals are the contaminants of
    concern. When conducting initial tests of saltwater species,
    it is usually appropriate to use a grass shrimp, penaeid shrimp,
    or mysid, because these invertebrates often are more sensitive
    than fish. As a final example, algae should be used for initial
    tests when herbicides and materials with suspected phytotox-
    icity are detected in fresh or salt water.

    These are only a few examples of how test organisms differ
    in their sensitivity to contaminants. Again, investigators will
    want to consult the BTAG for assistance in selecting appro-
    priate test organisms.

  •  When testing water, select a test organism that can tolerate the
    water's condition. For example, some organisms, such as the
    waterflea Ceriodaphnia, are extremely sensitive to water hard-
    ness. Also, certain inland waters have  high enough salinity to
    make the use of freshwater test organisms inadvisable.

    To extend the limited guidelines offered above, several EPA
scientists were interviewed  to learn  how they design toxicity
assessments. Each scientist offered a somewhat different outline
and set of priorities. These differences reflect differences in site
characteristics and geography, such as degree of urbanization and
amount of rainfall. In spite of the great variety in hazardous waste
sites, three general designs for toxicity assessments emerged from
these  interviews. The following section  presents the designs.
Where a design refers to specific toxicity tests, these are described
in the companion Bulletin, "Catalogue of Standard Toxicity Tests
for Ecological Risk Assessment" (ECO Update Vol. 2, No. 2).

Design  1
    Design  1 is based on the premise that designing a meaningful
toxicity assessment requires considering a contaminant's mode of
action, the level  of site contamination, the sensitivity of the test
type (acute or chronic), and the sensitivity  of the test organism. In
particular, sites are first reviewed to determine whether contami-
nants are expected to cause effects detectable by toxicity tests that
meet the stated DQOs for that site. The investigator then matches
contaminant  levels  with test type. For example, in  a  heavily
industrialized site with high levels of contaminants, many of the
samples in a chronic test may give highly positive results. Conse-
quently, the  study would provide little information about differ-
ences in toxicity at various locations on the site. At such a site,
acute tests might distinguish the varying toxicity  of different
locations, thereby identifying the more highly impacted areas and
helping to establish priorities for remedial decisions.
     Similar concerns are addressed when choosing test organ-
isms: too sensitive a test organism results in a lack of discrimina-
tion and too insensitive a test organism can give negative results
that are misleading. Also, Design 1 uses screening tests sparingly,
because these tests may suffer from over-interpretation of results.

Design  2
     Design 2 focuses on planning toxicity assessments for sites
where chemical data do not clearly  indicate  whether  the site
contaminants represent an ecological  problem requiring further
action. The premise is that sites with  intermediate levels of con-
tamination will most likely require toxicity testing. Where little

-------
contamination has occurred, the  chemical data will generally
indicate that the site is not an ecological problem and requires no
further ecological risk assessment. A heavily contaminated site
will almost certainly require further action.
     With this design, the investigator decides on ecological
components and endpoints as early as possible, making use of all
the information available about the site and its contaminant his-
tory. The ecological components and endpoints then serve to guide
choices of both toxicity tests and field studies.
     If the chemical data indicate the need for toxicity tests, as
reflected by the literature, the investigator next  decides which
media to test. Contamination may  have migrated from the initial
contaminated medium into other site-associated media. Toxicity
test selection occurs next, with the choice between acute and
chronic tests depending on the objectives. In the long run, gauging
toxicity based on the sub-lethal effects measured by chronic tests
proves more protective. In addition, chronic tests are considered
more appropriate if an organism spends most of its time on-site.
Conversely, acute tests better mimic conditions for organisms that
spend a limited amount of time on-site, such as migratory animals
or those with a large home range. Acute tests also prove useful
when the investigator has concerns about significant differences in
toxicity at different  locations on the site. If initial tests show no
toxic "hot spots," then further testing may not be necessary.
     Design 2 puts toxicity testing in perspective by viewing the
ecological risk assessment of a Superfund site as a triad consisting
of chemical testing, toxicity testing, and field study:
  •  Chemical testing indicates the presence of contamination.

  • Toxicity tests then  explore whether biological  effects are
     possible.

  •  Field studies investigate whether actual harm has occurred at
    the site.

    At a highly contaminated site, each leg of the triad will most
likely give evidence of impact. At many sites, however, results are
not clear-cut. For example, toxicity results might not correlate wel 1
with chemical data. Alternatively, field studies might not demon-
strate adverse ecological effects. Such sites require careful profes-
sional judgment to make a decision regarding ecological  effects
and the need for remediation.

Design 3
    Design 3 begins with a site reconnaissance visit, followed by
a "desktop assessment" to determine whether a site requires an
ecological risk assessment. The desktop assessment considers the
site's background  from scoping and also its contaminants, their
environmental concentrations, theirphysical and chemical proper-
ties, and the nature of the surrounding area. A site located in an
urban industrial area, for example, may not require an ecological
risk assessment: the site itself may have no ecological components
of concern,  and  the contaminants may not have a means of
migrating to areas having potential ecological components. On the
other hand, the existence of a conduit—a stream, a drainage ditch,
ground-water gradient, or land grade—that could carry contami-
nants from the site to surface water, wetlands, or terrestrial habitats
would indicate the need for an ecological risk assessment. In this
design, toxicity studies are viewed as useful tools and an effective
use of Superfund  resources at sites requiring ecological risk
assessments.
     Selection of sampling locations is one of the first tasks
undertaken when designing a toxicity study using this design.
Especially at large sites, the investigator avoids random sampling,
which would generate an unmanageably large number of samples
to analyze. Instead, site terrain and contaminant history are care-
fully studied in order to place sampling points in areas that will
extend the knowledge of the site. As a general rule, at a site with
contaminated surface water, the sampling plan should specify
sufficient samples to characterize fully the potential ecological
effects.
     When  testing samples of  surface water,  the  investigator
selects an initial battery of screening tests from among the stan-
dardized acute tests used in the NPDES permitting program. These
initial tests usually include organisms at three trophic levels (e.g.,
an alga, an invertebrate, and a fish). If screening level tests show
that surface  water is toxic, further tests include surface water
dilutions  and additional test species to characterize the site's
toxicity more fully.
     This design favors performing initial soil or sediment evalu-
ations at the screening level using either bulk samples, laboratory-
prepared  elutriates, or  pore water, the water  located between
particles and obtained by either  centrifugation or filtration. Like
surface water samples, elutriates and pore water samples  are
analyzed using screening level NPDES aquatic tests. If these tests
give positive  results, the investigator reviews the chemical data
and contaminant history for the site before making the serious
commitment of resources that  testing of bulk  soil or sediment
entails.
Conclusion

    The differences in these three designs largely reflect the fact
that an ecological risk assessment of a Superfund site needs to
address the site's characteristics and contaminant history.  As
scientists gain more  experience in conducting toxicity assess-
ments, the several designs existing today may evolve toward a
common blueprint that will readily accommodate site differences.
In addition to reflecting site differences, the designs also reflect
strategic differences in the deployment of resources, time, and
          As scientists gain more experience
     in  conducting toxicity  assessments,
     the several designs existing today may
     evolve toward a  common  blueprint
     that  will readily  accommodate  site
     differences.
                                                            10

-------
personnel. Despite these differences, however, the designs have
the same end: to assess the toxicity of contaminated media from
Superfund sites.
    To learn about other designs for toxicity assessments, inves-
tigators may wish to consult the scientists in the EPA Environ-
mental Services Division (ESD) in  their Region.  In  making
decisions about toxicity testing at a specific site, an investigator
should consult with the Regional BTAG. The BTAG will be able
to tell the investigator whether the Region has a standard design for
toxicity assessments. If the Region does not, the BTAG can advise
the investigator whether one of the above designs, one offered by
the ESD,  or  a  modification of any  of these will  further the
ecological risk assessment of a particular site.  Alternatively, the
BTAG may suggest  another  design  that  is better  suited to a
particular site.
References

  American Society for Testing and Materials (ASTM). 1992a.
    Annual Book of ASTM Standards: Water and Environmental
    Technology, Vol. 11.04. American Society for Testing and
    Materials, Philadelphia, PA.

  American Society for Testing and Materials (ASTM). 1992b.
    Standard Guide for Conducting Sediment Toxicity Tests with
    Freshwater Invertebrates. American Society for Testing and
    Materials, Philadelphia, PA.

  American Society for Testing and Materials (ASTM). 1992c.
    Standard Guide for Conducting 10-day Static Sediment Tox-
    icity Tests with Marine and EstuarineAmphipods. American
    Society for Testing and Materials, Philadelphia, PA.

  Baker, J.P.  1989. "Assessment Strategies and Approaches" in
    Warren-Hicks, W., B.R. Parkhurst, and S.S. Baker Jr., cds.
    Ecological Assessment of Hazardous Waste Sites: A  Field
    and Laboratory Reference. EPA/600/3-89/013.  Environ-
    mental Research Laboratory, Office of Research and Develop-
    ment, U.S. Environmental Protection Agency, Corvallis, OR.

  Bitton, G., B.J. Dutka, and C.W. Hendricks.  1989. "Microbial
    Toxicity Tests" in Warren-Hicks, W., B.R. Parkhurst, and
    S.S. Baker Jr., eds. Ecological Assessment of Hazardous
    Waste Sites: A Field and Laboratory Reference. EPA/600/3-
    89/013. Environmental Research Laboratory, Office of Re-
    search and  Development,  U.S. Environmental Protection
    Agency, Corvallis, OR.
Greene, J.C., C.L. Bartels, W.J. Warren-Hicks, B.R. Parkhurst,
   G.L. Linder, S.A. Peterson, and W.E. Miller. 1989.Protocols
   forShort-Term Toxicity Screening of Hazardous WasteSites.
   EPA/600/3-88/029. Environmental Research Laboratory,
   Office of Research and Development, U.S. Environmental
   Protection Agency, Corvallis, OR.

Linder, G. et al. 1992. Evaluation of Terrestrial Indicators for
   Use in Ecological Assessments at Hazardous Waste Sites.
   EPA/600/R-92/183. Environmental Research Laboratory,
   Office of Research and Development, U.S. Environmental
   Protection Agency, Corvallis, OR.

U.S. Army Corps of Engineers.  1993. Evaluation of Dredged
   Material Proposed for Discharge in Inland and Near Coastal
   Waters — Testing Manual (Draft). Office of Water, Wash-
   ington, DC.

U.S. Environmental Protection Agency. 1992. Framework for
   Ecological Risk Assessment.  EPA/630/R-92/001. Risk As-
   sessment Forum, Washington, DC.

Weber, C.I. 1991. Methods for Measuring the Acute Toxicity of
   Effluents and Receiving Waters to Freshwater and Marine
   Organisms. 4th edition. EPA/600/4-90/027. Environmental
   Monitoring Systems Laboratory, Office  of  Research and
   Development, U.S. Environmental Protection Agency, Cincin-
   nati, OH.

Weber, C.I., W.I. Horning,  D.J. Claim, T.W. Neiheisel, P.A.
   Lewis, E.L Robinson, J.  Menkedick, and  F. Kessler. 1988.
   Short-term Methods for Estimating the Chronic Toxicity of
   Effluents and Receiving  Waters to Marine and Estuarine
   Organisms. EPA/600/4-87/028. Environmental Monitoring
   and Support Laboratory, Office of Research  and Develop-
   ment,  U.S. Environmental Protection Agency, Cincinnati,
   OH.

Weber, C.I., W.H. Peltier, T.J. Norberg-King, W.B. Horning II,
   F. Kessler, and J. Menkedick. 1989. Short-term Methods for
   Estimating the Chronic Toxicity of Effluents and Receiving
   Waters to Freshwater Organisms. 2nd edition. EPA/600/4-
   89/001. Environmental  Monitoring Systems  Laboratory,
   Office of Research and Development, U.S. Environmental
   Protection Agency, Cincinnati, OH.
                                                           77

-------