United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-355
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1740
Whole Sediment Acute
Toxicity Invertebrates,
Marine
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Public Draft"

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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1740 Whole sediment acute toxicity invertebrates, ma-
rine.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.).
(2) [Reserved]
(b)	Objective. This guideline may be used to determine the toxicity
and bioaccumulation potential of chemicals in estuarine or marine sedi-
ments in marine invertebrates. Natural sediment is spiked with different
concentrations of pesticide or contaminant and the results from the sedi-
ment toxicity tests can be used to determine causal relationships between
the chemical and biological response. Reported endpoints from whole sedi-
ment toxicity tests include the LC50 (median lethal concentration), EC50
(median effective concentration), NOEC (no-observable-effect-concentra-
tion), or the LOEC (lowest-observable-effect-concentration).
(c)	Definitions.
Clean. Clean denotes a sediment or water that does not contain con-
centrations of test materials which cause apparent stress to the test orga-
nisms or reduce their survival.
Concentration. Concentration is the ratio of weight or volume of test
material(s) to the weight or volume of sediment.
Contaminated sediment. Contaminated sediment is sediment contain-
ing chemical substances at concentrations that pose a known or suspected
threat to environmental or human health.
Control sediment. Control sediment is sediment that is essentially free
of contaminants and is used routinely to assess the acceptability of a test.
Any contaminants in control sediment may originate from the global
spread of pollutants and does not reflect any substantial input from local
or non-point sources. Comparing test sediments to control sediments is
a measure of the toxicity of a test sediment beyond inevitable background
contamination.
Effect concentration (EC). Effect concentration is the toxicant con-
centration that would cause an effect in a given percent of the test popu-
lation. Identical to LC when the observable adverse effect is death. For
example, the EC50 is the concentration of toxicant that would cause death
in 50% of the test population.
Inhibition concentration (IC). Inhibition concentration is the toxicant
concentration that would cause a given percent reduction in a non-quantal
measurement for the test population. For example, the IC25 is the con-
centration of toxicant that would cause a 25% reduction in growth for
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the test population and the IC50 is the concentration of toxicant that would
cause a 50% reduction.
Interstitial water or pore water. Interstitial water or pore water is
water occupying space between sediment or soil particles.
Lethal concentration (LC). Lethal concentration is the toxicant con-
centration that would cause death in a given percent of the test population.
Identical to EC when the observable adverse effect is death. For example,
the LC50 is the concentration of toxicant that would cause death in 50%
of the test population.
Lowest observable effect concentration (LOEC). Lowest observable
effect concentration is the lowest concentration of a toxicant to which or-
ganisms are exposed in a test which causes an adverse effect on the test
organisms (i.e., where the value for the observed response is statistically
significant different from the controls).
No observable effect concentration (NOEC). No observable effect
concentration is the highest concentration of a toxicant to which organisms
are exposed in a test that causes no observable adverse effect on the test
organisms (i.e., the highest concentration of a toxicant in which the value
for the observed response is not statistically significant different from the
controls).
Overlying water. Overlying water is the water placed over sediment
in a test chamber during a test.
Reference sediment. Reference sediment is a whole sediment near an
area of concern used to assess sediment conditions exclusive of material(s)
of interest. The reference sediment may be used as an indicator of local-
ized sediment conditions exclusive of the specific pollutant input of con-
cern. Such sediment would be collected near the site of concern and would
represent the background conditions resulting from any localized pollutant
inputs as well as global pollutant input. This is the manner in which ref-
erence sediment is used in dredge material evaluations.
Reference-toxicity test. Reference-toxicity test is a test conducted in
conjunction with sediment tests to determine possible changes in condition
of the test organisms. Deviations outside an established normal range indi-
cate a change in the condition of the test organism population. Reference-
toxicity tests are most often performed in the absence of sediment.
Sediment. Sediment is particulate material that usually lies below
water. Formulated particulate material that is intended to lie below water
in a test.
Spiked sediment. Spiked sediment is a sediment to which a material
has been added for experimental purposes.
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Whole sediment. Whole sediment is sediment and associated pore
water which have had minimal manipulation. The term bulk sediment has
been used synonymously with whole sediment.
(d)	Test method. (1) Whole sediment toxicity tests are outlined for
four species of estuarine/marine amphipods, Ampelisca abdita,
Eohaustorius estuarius, Rhepoxynius abronius, and Leptocheirus
plumulosus. Whole sediment tests last 10 or more days, and are conducted
in in 1-L test chambers containing 175 mL (2 cm) of sediment and
800 mL of overlying water. The overlying water does not have to be re-
newed and test organisms do not have to be fed during the toxicity test.
The endpoint is survival, but reburial for E. estuarius, L. plumulosus, and
R. abronius is optional.
(2) A range-finding test to establish a suitable range of test concentra-
tions is recommended. If no toxicity is observed at concentrations of
100 mg/kg dry weight of sediment, a definitive test is not required.
(e)	Water, reagents, and standards—(1) Water, (i) Sea water used
in sediment toxicity test should be of uniform quality and allow satisfac-
tory survival, growth, or reproduction of the test organisms. Organisms
cultured and tested in the selected sea water should not show signs of
disease or stress.
(ii)	Natural sea water should be from uncontaminated surface-water
upstream of known discharges. Sea water should be collected at slack high
tide or within 1 h of high tide. Full strength sea water should be collected
from areas with salinities of 28 ppt. Sea water for estuarine test may be
collected from areas with salinities close to the test salinity or diluted with
freshwater. Water prepared from natural sea water should be covered,
maintained at 4 °C, and used with 2 days.
(iii)	Although natural sea water is preferable, reconstituted water is
acceptable. Reagent grade chemicals should be added to high-purity dis-
tilled or deionized water (1 MQ). Each batch of reconstituted water should
be measured for salinity, pH, and dissolved oxygen (DO). Suspended par-
ticles should be removed by filtration (<5 (im) from reconstituted water
at least 24 h before use.
(2)	Reagents. All reagents and chemicals purchased from supply
houses should be accompanied by appropriate data sheets. All test mate-
rials should be reagent grade. However, if specified as necessary, commer-
cial product, technical-grade, or use-grade materials may be used. Dates
for receipt, opening, and shelf-life should be logged and maintained for
all chemicals and reagents. Do not use reagents beyond shelf-life dates.
(3)	Standards. Acceptable standard methods for chemical and phys-
ical analyses should be used. When appropriate standard methods are not
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available or lack the required sensitivity, other sources should be consulted
for reliable methods.
(f) Sample collection, storage, manipulation, and characteriza-
tion—(1) Sample collection, (i) Procedures for handling natural sediments
should be established prior to collection. Pertinent data such as location,
time, core depth, water depth, and collection equipment should be re-
corded.
(ii) Replicate sampling should be used for the collection of natural
sediment to determine the variance in sediment characteristics. While some
disruption of the sediment is inevitable regardless of the sampling equip-
ment used, disruption of sediment should be kept to a minimum. Several
devices are available for collecting sediment, but benthic grab or core sam-
plers are recommended. The depth of sediment collected should reflect
the expected exposure. During sediment collection, exposure to direct sun-
light should be kept to a minimum. Cooling of sediment to 4 °C is rec-
ommended.
(2)	Storage. Storage of sediment may affect bioavailability and tox-
icity. Although nonionic and nonvolatile organic contaminates in sediment
may not result in substantive changes, metals and metalloids may affect
redox, oxidation, or microbial metabolism in sediment. It is best to hold
sediments at 4 °C in the dark and test within 2 to 8 weeks after collection.
Long storage may result in changes of sediment properties. Sediment tests,
and especially pore water tests, should be performed within 2 weeks of
collection to minimize property changes in the sediment.
(3)	Manipulation, (i) During homogenization, water above sediment
that may have settled during shipment should be mixed back into the sedi-
ment. Sieving should not be used to remove indigenous organisms, unless
an excessive number of oligochaetes are present. Because oligochaetes
may inhibit the growth of the test organisms, it may be advantageous to
remove them by sieving. If sieving is used, sediment samples should be
analyzed before and after sieving to document the influence of sieving
on sediment characteristics. Sediments collected from multiple locations
or sites may be pooled and mixed using suitable apparatus (e.g. stirring,
rolling mill, feed mixer, etc.).
(ii) The preparation of test sediment may be accomplished by the
spiking of natural or formulated sediments. Additional research is needed
before formulated sediments may be used routinely. The responses of
spiked sediment may be affected by mixing time and aging. Spiked sedi-
ment should be used immediately. Point estimates of toxicity or minimum
concentrations at which toxic effects are observed may be determined by
spiking natural sediments with a range of chemical concentrations. The
test material should be reagent grade unless there is a specific need to
use commercial product, technical-grade, or use-grade material. Specific
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information required for all test materials include but is not limited to
the following:
(A)	Identity and concentration of major ingredients and impurities.
(B)	Solubility in test water.
(C)	Estimated toxicity to the test organism and to humans.
(D)	When measured test concentrations are required, the precision
and bias of analytical method at the planned concentrations of test mate-
rial.
(E)	Recommended handling and disposal procedures.
(iii) Organic solvents should not be added to the sediment mixture
because they may affect the concentration of dissolved organic carbon in
pore water.
(4) Characterization, (i) The characteristics of all sediment should
be determined, and at a minimum the following factors should be meas-
ured: pH and ammonia concentration of pore water, organic carbon content
(total organic carbon, TOC), particle size distribution (percent sand, silt,
clay), and percent water content. Additional analyses are suggested and
include biological oxygen demand, chemical oxygen demand, cation ex-
change capacity, Eh, total inorganic carbon, total volatile solids, acid vola-
tile sulfides, metals, synthetic organic compounds, oil and grease, and pe-
troleum hydrocarbons. Various physicochemical parameters should also be
determined for interstitial water. Sediment characterization should also in-
clude qualitative parameters such as color, texture, and the presence of
macrophytes or animals.
(ii)	Standard analytical methods should be used to determine chemical
and physical data. For each analytical method, precision, accuracy, and
bias should be determined in sediment, water, and tissue. Analysis should
include analytical standards and reagent blanks as well as recovery calcula-
tions.
(iii)	Concentrations of spiked chemicals should be measured in sedi-
ment, interstitial water, and overlying water at the beginning and at the
end of the test. Degradation products should also be measured where ap-
propriate. Sediment chemistry should be monitored during and at the end
of a test. Separate replicates resembling the biological replicates and con-
taining organisms should be specified for chemical sampling. The con-
centration of test material in water is measured by pipetting water samples
from 1 to 2 cm above the sediment surface. Caution should be used to
eliminate the presence of any surface debris, material from the sides of
the chamber, or sediment in the overlying water sample. At the end of
the test, the test material may be removed for chemical analysis by siphon-
ing (without disturbing sediment) the overlying water. Appropriate samples
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of sediment can then be removed for chemical analysis. The suggested
method for isolation of interstitial water is by centrifugation without filtra-
tion.
(g)	Collection and maintainence of test organisms. (1) The methods
for collection of test organisms are species-specific. Subtidal species, such
as A. abdita and L. plumulosus, may be collected with a small dredge
or grab and may also be collected by skimming the sediment surface with
a long-handled, fine-mesh net. E. estuarius and R. abronius are found both
subtidally and intertidally. The aforementioned methods are suitable for
subtidal populations. Intertidal populations may be collected using a shov-
el. Approximately one-third more organisms should be collected than are
required for testing.
(2)	All collecting, sieving, and transporting equipment should be clean
and constructed of nontoxic material and clearly marked "live only". All
apparatus should be cleaned and rinsed with an appropriate water source
before use.
(3)	Collected organisms should be handled gently, carefully, quickly,
and no more than necessary. The sieving operation should be conducted
by slowly immersing the sieve into collection site water. Sieved test orga-
nisms should be kept submerged in ambient collection water at all times.
Direct exposure to sunlight of amphipods out of sediment must be avoided.
(4)	A. abdita and L. plumulosus should be isolated from collection
site sediment by using a 0.5 mm mesh sieve. A. abdita which remain in
tubes must be left undisturbed for 20 to 30 min to allow for natural exit
of the organisms. A 1.0 mm sieve should be used to isolate E. estuarius
andi?. abronius.
(5)	A. abdita and L. plumulosus may be collected with a small dredge
or grab apparatus (e.g. PONAR, Van Veen, etc.) and E. estuarius and
R. abronius may be collected with a shovel. Collected amphipods should
be sieved in the field by slow immersion in collection site water. Sieved
amphipods should be separated from detritus and predators and transferred
gently to transport containers containing 2 cm of collection site sediment.
Mesh sizes of 0.5 to 1.0 mm should be utilized. Salinity and temperature
of collection site sediment should be recorded surface and bottom loca-
tions. Amphipods should be transported in coolers with ice packs and held
in the collection-site sediment at or below the temperature at the collection
site. Aeration is recommended for transport times exceeding 1 h. Collec-
tion site sediment should be used as holding sediment in the laboratory
and as control test sediment.
(h)	Ampelisca abdita, Eohaustorius estuarius, Leptocheirus
plumulosus, or Rhepoxynius abronius 10- to 28-Day Survival Test For
Sediments—(1) Recommended test method. The recommended test tem-
peratures for conducting sediment toxicity tests with E. estuarius and R.
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abronius, A. abdita, and L. plumulosus are 15, 20, and 25 °C, respectively.
E. estuarius and L. plumulosus should be tested at a salinity of 20 ppt
and A. abdita and R. abronius at a salinity of 28 ppt. The recommended
photoperiod is 24 h of light with illumination of 500 to 1,000 lx. Sediment
(175 mL) and 800 mL of overlying seawater are placed in 1-L glass test
chambers. Twenty organisms are placed in each test chamber to begin the
test. Five replicates per treatment are recommended, however, this number
may vary depending upon need. The size and life stage of amphipod re-
quired for testing varies from 2-4 mm for L. plumulosus to 3-5 mm for
the three remaining species. Additionally, no mature male or female A.
abdita or L. plumulosus should be used for testing. Overlying water does
not have to be renewed and test organisms do not have to be fed during
the test.
(2)	General procedure—(i) Introduction of sediment. The test sedi-
ment should be homogenized one day before the test is to commence
(day--l) using a rolling mill, feed mixer, or other suitable apparatus. The
sediment should be observed for homogeneity visually and quantitatively
by measuring TOC, chemical concentrations, and particle size. A
175-mL aliquot of homogenized sediment should be added to each test
chamber, and settled with the use of a nylon, fluorocarbon, or polyethylene
spatula.
(ii)	Addition of overlying water. A turbulence reducer (a disk cut
from polyethylene, nylon, or Teflon, or a glass Petri dish attached to a
glass pipet) should be used when adding overlying water. Turbulence re-
ducers should be rinsed with seawater between replicates, and individual
turbulence reducers used between treatments. Test chambers should be
covered, immersed in a temperature bath, and gently aerated. The test com-
mences once the test organisms are added to the test chambers (day-0).
(iii)	Addition of amphipods. Twenty amphipods are randomly added
to each test chamber in batches of 5 to 10 on day--0 following the addi-
tion of sediment and overlying water. One-third more amphipods than nec-
essary are sieved from culture or control sediment and transferred to sort-
ing trays. Recommended sieve sizes are 0.5 mm for A. abdita and L.
plumulosus and 1.0 mm for E. estuarius and R. abronius. Isolated
amphipods are transferred from the sorting tray to 150 mL of test sea
water using pipets. The test organisms are observed for injury or stress
after addition. If any E. estuarius, E plumulosus, and R. abronius have
not burrowed within 5 to 10 min, they should be replaced. A. abdita that
have not burrowed within 1 h should also be replaced. Organisms express-
ing sediment avoidance, should be removed, recorded, but not replaced.
(3)	Test conditions—(i) Aeration. Overlying sea water should be
continuously aerated from day - 1 to day - 10 except when test organisms
are being added. DO should be maintained at approximately 90 percent
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saturation using gentle aeration without disturbing the sediment. Results
are unacceptable if DO falls to below 60 percent saturation.
(ii)	Lighting. Lights must be left on for the duration of the 10-day
testing period. The constant light assures that the test organisms to remain
burrowed during the test.
(iii)	Feeding. None of the four test species need to be fed during
the 10-day testing period.
(iv)	Water temperature. The respective selected test temperatures
are representative of the summertime thermal maximum for each species.
E. estuarius and R. abronius (Pacific Coast amphipods) must be tested
at 15 °C. A. abdita and L. plumulosus must be tested at 20 °C and
25 °C, respectively.
(v)	Salinity. Overlying water salinity should be 28 ppt for A. abdita
and R. abronius and 20 ppt for E. estuarius and L. plumulosus. Pore water
salinity must be measured prior to the start of the test.
(4)	Measurements and observations, (i) Temperature should be
measured daily from at least one replicate from each treatment. Tempera-
ture of the water bath or exposure chamber must be monitored continu-
ously.
(ii)	Salinity, DO, and pH should be measured in overlying water daily
in one test chamber in each treatment. These parameters should be meas-
ured in all test chambers at the beginning of the test and at termination.
(iii)	Ammonia concentration should be measured near day-2 and
day-8 during the 10-day test period. Ammonia concentration measure-
ments should be accompanied by pH and temperature measurements. pH,
temperature, and ammonia concentration should be measured in pore water
at the beginning of the test.
(iv)	Air-flow to overlying sea water must be monitored daily. Any
test organisms trapped in air-water interface must be gently pushed back
down using a glass rod or pipet.
(5)	Ending a test, (i) Recovery of organisms from control sediment
should equal or exceed 90 percent in a 10-day test or 80 percent in a
28-day test.
(ii) Test animals are isolated from the test chambers by sieving with
sea water. Sieves should not exceed 0.5 mm. Test organisms should be
washed into sorting trays containing sea water. Caution should be taken
that no tube-dwelling organisms remain trapped on the sieve. Slapping
the sieve forcefully against the surface of the water should successfully
dislodge all A. abdita. The remaining species should be easily separated
by the sieving process.
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(iii) Small portions of material should be washed into sorting trays
and should be examined carefully. The tubes of A. abdita should be teased
apart under a dissecting microscope to ensure that all organisms are ac-
counted for. The numbers of living, missing, or dead amphipods should
be observed and recorded for all test chambers. Missing animals and all
observed animals failing to respond to gentle prodding (i.e. neuromuscular
twitch of pleopods or antennae) are recorded as dead.
(6)	Test data. The primary endpoint for the 10-day sediment test
is survival. Effective mortality (the sum of dead animals plus survivors
that fail to rebury) may also be determined. To determine reburial, E.
estuarius, L. plumulosus, and R. abronius should be transferred to a
2-cm layer of 0.5 mm sieved control sediment and overlying test sea water
(2 cm).
(7)	Interpretation of results—(i) Influence of indigenous orga-
nisms. Because test sediments collected from the field may contain indige-
nous species, data interpretation may be complicated by the presence of
organisms similar to the test organism or predatory organisms.
(ii)	Effect of grain size. While the four estuarine/marine test species
are generally tolerable of a wide range of sediment types, grain size may
adversely affect some species of amphipod. When this possibility exists,
a clean control/reference sediment should be incorporated into the test de-
sign to facilitate distinction of contaminant effects versus particle size ef-
fects. Species-specific ranges of grain sizes are as follows.
(A)	A. abdita: Survival may be impacted in sediments containing 95
percent or more sand. Test sediment should contain less than 95 percent
sand.
(B)	L. plumulosus: Survival should not be impacted in clean sedi-
ments containing 100 percent sand to 100 percent sand + clay.
(C)	E. estuarius: Survival is unaffected by clean sediments containing
0.6 to 100 percent sand. However increased mortality may be associated
with increased proportions of fine-grained sediment. In these cases an ap-
propriate control/reference should be included.
(D)	R. abronius: Very fine grains, particularly silts and clays, may
reduce survival of this species. When test sediments contain silts and clays,
the use of control/reference groups with particle sizes characteristic of the
test sediment is recommended.
(iii)	Effects of pore water salinity. The range of salinity tolerance
is variable for the four amphipod species. For sediment testing, two sce-
narios for test salinity are acceptable given that appropriate conditions are
met:
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(A)	Salinity tolerance range is the range of salinity in which a given
species can survive for 10 days when the overlying water salinity is
matched to that of the pore water salinity. In laboratory sediment testing,
the overlying water salinity can be based on the standard salinity for each
test species, or adjusted to match the salinity of pore water. It is critical
that pore water salinity be measured prior to test initiation and that the
appropriate species be used. Salinity tolerance ranges for A. abdita, E.
estuarius, E plumulosus, and R. abronius are 20-32 ppt, 2-34 ppt, 1.5-
32 ppt and 25-32 ppt, respectively.
(B)	Salinity application range is the range of pore water salinities
in which a given species can survive for 10 days when using the species-
specific overlying water salinity. Salinity application ranges for A. abdita
with overlying water salinity of 28 to 32 ppt, E. estuarius with overlying
water salinity of 20 ppt, E plumulosus with overlying salinity of 20 ppt,
and R. abronius with overlying water salinity of 28 to 32 ppt are 0 to
34, <2 to 34, <1.5 to 32, and 25 to 34, respectively.
(iv) Effects of sediment-associated ammonia. Ammonia concentra-
tions in field-collected sediments may be toxic to amphipods. When am-
monia concentration exceeds the water column no-effect levels, any mor-
tality observed during the 10-day sediment test may be due to the ammo-
nia. Ammonia levels should be measured approximately 1 cm above the
sediment surface on day-0 and, if necessary, reduced prior to the addition
of test organisms by flushing the overlying water for up to two consecutive
24-h periods (six volume replacements per hour). Following flushing, the
overlaying water ammonia concentration should be remeasured. If ammo-
nia is at acceptable levels testing may be initiated but flushing at a rate
of sic volume changes per 24-h period must be maintained throughout
the test. Ammonia concentrations in overlaying water should be measured
again on day-10. If ammonia is not at acceptable levels, 24-h flushings
must continue at the six-volume change per 24 h rate and ammonia con-
centration measured every 24 h.
(i) Interferences. (1) Interferences are characteristics of sediment or
the sediment test system with potential to affect survival of test organisms
independent of sediment-associated contaminant affects. Interferences are
categorized into three categories: Noncontaminant factors causing reduced
survival, changes in bioavailability due to manipulation or storage, and
the presence of indigenous species. Noncontaminant factors can make ex-
trapolation of laboratory test results to the field difficult. Specifically, the
motility of organisms (i.e. escapism) and photoinduced toxicity due to UV
light in from the sun (e.g. UV light absent from fluorescent light) may
be markedly different between laboratory conditions and the natural envi-
ronment. Other noncontaminant factors include sediment particle size, pore
water salinity, and pore water ammonia concentration. The test conditions
must be matched appropriately with the tolerance limits of the four
amphipod test species (see paragraph (k)(l) of this guideline).
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(2)	Bioavailability of sediment-associated contaminants can be altered
by collection, handling, and storage. The handling, storage, and preparation
of test sediment should be as consistent as possible. Test sediments should
be presieved and rehomogenized prior to introduction into the test cham-
bers. Bioavailability may also be affected by temperature, salinity, the ratio
of sediment to overlying water, and the depletion of contaminant due to
organismal uptake. In some cases it is advantageous to normalize sediment
concentrations to dry weight, organic-carbon content, or acid volatile sul-
fides.
(3)	Test sediment collected from the field may contain indigenous
organisms, and can potentially make interpretation of treatment effects dif-
ficult. If the presence of indigenous or predatory organisms is suspected,
the test sediment should be press-sieved prior to test initiation.
(j) Reporting. In addition to information meeting the general report-
ing requirements of a toxicity test, a report of the results of a sediment
toxicity test for estuarine and marine amphipods should include the follow-
ing:
(1)	Name of test and investigators, name and location of laboratory,
and dates of start and end of test.
(2)	Source of control or test sediment, method for collection, han-
dling, shipping, storage, and disposal of sediment.
(3)	Source of test material, lot number if applicable, composition
(identities and concentrations of major ingredients and impurities if
known), known chemical and physical properties, and the identity and con-
centrations of any solvent used.
(4)	Source and characteristics of overlying water, description of any
pretreatment, and results of any demonstration of the ability of an orga-
nism to survive or grow in the water.
(5)	Source, history, and age of test organisms; source, history and
age of brood stock, culture procedures; and source and date of collection
of the test organisms, scientific name, name of person who identified the
organisms and the taxonomic key used, age or life stage, means and ranges
of weight or length, observed diseases or unusual appearance, treatments,
holding procedures.
(6)	Source and composition of food, concentrations of test material
and other contaminants, procedure used to prepare food, feeding methods,
frequency and ration.
(7)	Description of the experimental design and test chambers, the
depth and volume of sediment and overlying water in the chambers, light-
ing, number of test chambers an number of test organisms/treatment, date
and time test starts and ends, temperature measurements, DO concentration
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(as percent saturation) and any aeration used before starting a test and
during the conduct of a test.
(8)	Methods used for physical and chemical characterization of sedi-
ment.
(9)	Definitions of the effects used to calculate LC50s or EC50s, bio-
logical endpoints for tests, and a summary of general observations of other
effects.
(10)	A table of the biological data for each test chamber for each
treatment including the controls in sufficient detail to allow independent
statistical analysis.
(11)	Methods used for statistical analyses of data.
(12)	Summary of general observations on other effects or symptoms.
(13)	Anything unusual about the test, any deviation from these proce-
dures, and any other relevant information.
(k) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	U.S. Environmental Protection Agency. Methods for Assessing
the Toxicity of Sediment-Associated Contaminants with Estuarine and Ma-
rine Amphipods. EPA 600/R-94/025 (1994).
(2)	[Reserved]
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