United States Prevention, Pesticides EPA712-C-96-354
Environmental Protection and Toxic Substances April 1996
Agency (7101)
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1735
Whole Sediment Acute
Toxicity Invertebrates,
Freshwater
'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
(7U.S.C. I36,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 850.1735 Whole sediment acute toxicity invertebrates,
freshwater.
(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 sediments in freshwater in-
vertebrates. Natural sediment is spiked with different concentrations of test
chemical and the results from the sediment toxicity tests can be used to
determine causal relationships between the chemical and biological re-
sponse. Reported endpoints from whole sediment toxicity tests may in-
clude the LC50 (median lethal concentration), EC50 (median effective
concentration), NOEC (no-observable-effect-concentration), 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 (1C). 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.
ppt. ppt is parts per thousand.
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.
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Spiked sediment. Spiked sediment is a sediment to which a material
has been added for experimental purposes.
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
the amphipod, Hyalella azteca and the midge, Chironomus tentans. Dura-
tion of whole sediment tests is 10 to 28 days and is accomplished in 300-
mL test chambers containing 100 mL of sediment and 175 mL of overlying
water. The overlying water may be renewed daily or a flow-through sys-
tem may be used. Test organisms are fed during the toxicity test. The
endpoint for H. azteca is survival, and for C. tentans, survival, growth
and/or emergence.
(2) A range-finding test to establish a suitable range of test concentra-
tions is recommended. A definitive test will not be required if no toxicity
is observed at concentrations of 100 mg/kg dry weight of sediment.
(e) Water, formulated sediment, reagents, and standards—(1)
Water, (i) Testing and culture water must be of uniform quality, and is
acceptable if it allows satisfactory survival, growth, and reproduction of
the test organisms. Disease or apparent stress (e.g. discoloration, unusual
behavior) should not be prevalent. If problems occur during testing or cul-
turing, water characteristics should be analyzed.
(ii) Natural water is considered to be of uniform quality if the ranges
of hardness, alkalinity, and specific conductance are within 10 percent of
the respective averages. The monthly pH range should be <0.4 units.
Sources of natural water should be uncontaminated well or spring or sur-
face water. Special considerations for surface water include minimizing
quality and contamination variables, maximizing the levels of DO, and
confirming that sulfides and iron levels are low. Chlorinated water should
not be used for testing or culturing because chlorine-produced oxidants
and residual chlorine are toxic to aquatic organisms. Tap water is accept-
able if it is dechlorinated, deionized, and carbon filtered, but its use is
not encouraged.
(iii) If source water is contaminated with facultative pathogens, it
should be UV-irradiated using intensity meters and flow-controls, or fil-
tered through 0.45 (im pore size.
(iv) The DO concentration of source water should be between 90 and
100 percent saturation. In some cases aeration may be required using air
stones, surface aerators, or column aerators.
(v) High-purity distilled or deionized water may be reconstituted by
adding specified amounts of reagent grade chemicals. The deionization
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system should produce water with a resistance of 1 MQ. For each batch
of reconstituted water, the following parameters should be measured: Con-
ductivity, pH, hardness, DO, and alkalinity. Aeration should be employed
to maintain acceptable levels of pH and DO.
(vi) The preparation of 100 L of reconstituted water was developed
at the USEPA EMSL-Cincinnati and has been tested with H. azteca, C.
tentans, and Chironomus riparius in round-robin tests and is given as fol-
lows:
(A) Add approximately 75 L of deionized water to a properly cleaned
container capable of holding 100 L.
(B) Add 5 g of CaSO4 and 5 g of CaCk to a 2-L aliquot of deionized
water and mix (e.g., on a stir plate) for 30 min or until the salts dissolve.
(C) Add 3 g of MgSO4, 9.6 g NaHCO3, and 0.4 g KC1 to a second
2-L aliquot of deionized water and mix on a stir plate for 30 min.
(D) Pour the two 2-L aliquots containing the dissolved salts into the
75 L of deionized water and fill the carboy to 100 L with deionized water.
(E) Aerate the mixture for at least 24 h before use.
(F) The water quality of the reconstituted water should be approxi-
mately the following: Hardness, 90 to 100 mg/L as CaCOs, alkalinity 50
to 60 mg/L as CaCO3, conductivity 330 to 360 (iS/cm, and pH 7.8 to
8.2.
(vii) Synthetic seawater may be prepared by adding commercial sea
salts to deionized water. H. azteca may be cultured or tested at salinities
up to 15 ppt.
(2) Artificial sediment. Artificial sediments consist of mixtures of
materials designed to mimic natural sediments. Because artificial sedi-
ments have not been used routinely to assess the toxicity of contaminants
in sediment, the use of uncontaminated natural sediment is recommended.
If the use of artificial sediment is necessary, detailed information may be
found in paragraph (1)(1) of this guideline.
(3) 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.
(4) 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 contaminants 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 microorganisms,
unless an excessive number of oligochaetes are present. Because
oligochaetes may inhibit the growth of the test organisms, it may be advan-
tageous to remove them as well as other macroorganisms, rocks, wood,
and the like 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 artificial sediments. Additional research is needed
before artificial sediments may be used routinely. The responses of spiked
sediment may be affected by mixing time and aging. Spiked sediment may
be aged for at least 1 month to achieve equilibrium with the spiked chemi-
cals, if the chemical is known to be persistent. Sediments spiked with in-
dustrial chemicals should be used as soon as possible. Point estimates of
toxicity or minimum concentrations at which toxic effects are observed
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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 information required for all test materials includes 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 the analytical method at the planned concentrations of test
material.
(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, and should not be used.
(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. Precision, accuracy, and bias should be determined in
sediment, water, and tissue for each analytical method. Analysis should
include analytical standards and reagent blanks as well as recovery calcula-
tions.
(iii) Concentrations of spiked chemicals may be measured in sedi-
ment, interstitial water, and overlying water at the beginning and at the
end of the test if so required. Measurement of degradation products may
also be required. Sediment chemistry should be monitored during and at
the end of a test. Separate replicates resembling the biological replicates
and containing organisms should be specified for chemical sampling. The
concentration of test material in water is measured by pipetting water sam-
ples 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
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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
siphoning (without disturbing sediment) the overlying water. Appropriate
samples of sediment can then be removed for chemical analysis. The sug-
gested method for isolation of interstitial water is by centrifugation without
filtration.
(g) Collection, culture, and maintainence of test organisms—(1)
Hyalella azteca—(i) Life history. (A) H. azteca are found throughout
North and South America in permanent lakes, ponds, and streams. They
are commonly found in mesotrophic or eutrophic lakes that are capable
of supporting aquatic plants and that remain warm (20 to 30 °C) for most
of the summer months. Densities may exceed 10,000 M2 in optimal habi-
tats. H. azteca are epibenthic detritivores that burrow into the sediment.
They may be found in saline waters up to 29 percent, but are sensitive
to hardness (e.g. they are not found in waters with calcium at
< 7 mg/L and DO at < 2 mg/L).
(B) H. azteca reproduce sexually, averaging 18 eggs per brood and
approximately 15 broods every 152 days. Hatching occurs approximately
5 to 10 days after fertilization at 24 to 28 °C. They proceed through a
minimum of 9 instars, which are separated into 5 to 8 prereproductive
instars and an indefinite number of postreproductive instars. Instars 1
through 5 form the juvenile life stage, instars 6 and 7 form the adolescent
stage of development, instar 8 is the nuptial life stage, and later instars
form the adult stages of the amphipod.
(C) H. azteca may be cultured under illumination of 500-1,000 Ix.
They feed during daylight and avoid bright light by hiding under litter.
(D) H. azteca is tolerable of a wide range of temperatures (0-33 °C),
but are immobile at temperatures < 10 °C and die at temperatures
>33 °C. Reproduction can occur at temperatures of 10-18 °C, but the
highest rate of reproduction occurs at temperatures between 26 and
28 °C.
(E) H. azteca can tolerate a wide range of substrates. Survival and
growth of have not been shown to be negatively affected by either particle
size (> 90 percent silt and clay particles to 100 percent sand-sized particles)
or grain size and organic matter in 10-day tests. In tests where organisms
were not fed, survival decreased.
(ii) Culturing procedures. (A) To start a sediment test, 7- to
14-day-old amphipods must be produced. If growth is an endpoint, a nar-
rower range, such as 1- to 2-day-old amphipods should be used. Details
and further discussion of acceptable culture procedures for H. azteca are
presented in paragraph (1)(1) of this guideline.
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(B) H. azteca should be held and fed under the same conditions as
the mass culture for at least 2 days prior to test initiation.
(2) Chironomus tentans—(i) Life history. (A) C. tentans are found
in eutrophic ponds and lakes. In soft bottoms, approximately 95 percent
of chironomid larvae are found in the upper 10 cm. Chironomid larvae
are generally not found in sediments with hydrogen sulfide concentrations
>0.3 mg/L.
(B) The aquatic phases of C. tentans include the larval and pupal
stages. Female chironomids can oviposit eggs within 24 h of emergence,
releasing a single gelatinous egg mass containing roughly 2,300 eggs.
Hatch occurs in 2 to 4 days at 23 °C. The emergence of pupae as adults
occurs after 21 days at 23 °C.
(C) C. tentans are able to tolerate a wide range of grain sizes and
percentage organic matter. However, low percentage organic matter in con-
junction with no feeding may result in decreased survival. Survival is best
above pH 6.5. Poor control survival occurs at pH <6.5. Growth may also
be impacted by coarser sediment.
(ii) Culturing procedures. (A) The third instar chironomids must be
used to start a sediment test. Larvae should develop to the third instar
within 9 to 11 days at a temperature of 23 °C. The instar stage of midges
must be confirmed by head capsule width (-0.38 mm). Weight and height
of midges should be monitored at the beginning of a sediment test. Details
and further discussion of acceptable culture procedures are presented in
paragraph (1)(1) of this guideline.
(B) The time to first emergence and the success of emergence should
be recorded for all culture chambers. Growth may be monitored by peri-
odically measuring the midge head capsule width.
(h) Test method: Hyalella azteca 10- to 28-day sediment toxicity
test—(1) Test conditions. General test conditions required for a 10-day
sediment toxicity test with H. azteca are presented in the following table
XX. The 10-day sediment toxicity test must be conducted at 23 °C with
a 161ight:8dar photoperiod. Illumination should be approximately 500 to
1,000 Ix. The recommended test chambers are 300-mL high-form beakers
without lips containing 100 mL of sediment and 175 mL of overlying
water. The test is started using 10 7- to 14-day-old amphipods. Eight
replicates/treatment are recommended for routine testing. Because of po-
tential impacts on study results, feed added to the test chamber should
be kept to a minimum. Thoroughly mix food prior to removing aliquots.
In order to prevent bacterial and fungal growth, feeding should be sus-
pended for 1 to 2 days if food collects on sediment. Feeding should also
be suspended if DO falls below 40 percent of saturation. When feeding
is suspended in one treatment it should be suspended in all treatments.
Feeding rates and appearance of sediment surface should be observed daily
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and detailed records manitained. Each chamber should receive 2 volume
additions per day or flow-through of overlying water. Sources of overlying
water can be culture water, well water, surface water, site water, or recon-
stituted water.
Table XX.—General Test Conditions for 10-day Sediment Toxicity with H. azteca
Parameter
Conditions
1. Test type
2. Temperature
3. Light quality
4. Illuminance
5. Photoperiod
6. Test chamber
7. Sediment volume
8. Overlying water volume
9. Renewal of overlying water
10. Age of organisms
11. Number of organisms/chamber
12. Number or replicate chambers/
treatment.
13. Feeding
14. Aeration
15. Overlying water
16. Test chamber cleaning
17. Overlying water quality
18. Test duration
19. Endpoints
20. Test acceptability
Whole-sediment toxicity test with renewal of overlying water
23± 1 °C
Wide-spectrum fluorescent lights
500 to 1000 Lux
16L8D
300-mL high-form lipless beaker
100 mL
175 mL
2 volume additions/d
7-to 14- d old at start of test
10
Feed 1.5 mL daily to each test chamber
None (unless D.O. drops below 40% of saturation)
Culture water, well water, surface water, site water or reconstituted water
Gently brush outside of screen when clogged
Hardness, alkalinity, conductivity, pH, and ammonia at beginning and end of test; tem-
perature and D.O. daily
10-28d
Survival (growth optional)
Minimum mean control survival of 80% and above conditions
(2) Sediment into test chambers, (i) Sediment should be thoroughly
mixed and added to test chambers the day before (day—1) the start of
the test. The degree of homogeneity should be inspected visually. Homo-
geneity may be quantified by taking replicate subsamples and analyzing
for TOC, chemical concentration, and particle size.
(ii) Equal amounts of sediments should be added to each test chamber
on the basis of volume or dry weight. To minimize disturbance of sedi-
ment, overlying water should be poured gently along the sides of the test
chambers or poured over a Teflon baffle (with handle) positioned above
the sediment. The renewal of overlying water should commence on day-
1. The test begins once organisms are added to the test chambers (day-
0).
(3) Renewal of overlying water. Renewal or flow-through of over-
lying water is recommended during a test. Flow rates through any two
test chambers should not differ by more than 10 percent at any time during
the test. Each water-delivery system should be calibrated prior to test initi-
ation to verify that the system is functioning properly. Renewal of over-
lying water is started on day—1 before the addition of test organisms
or food on day-0.
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(4) Acclimation. Test organisms must be cultured and tested at
23 °C. The same water used for culture should be used for testing. Accli-
mation of test organisms to the test water is not required.
(5) Placement of organisms in test chambers. Handle test organisms
as little as possible. Amphipods may be placed into test chambers by
pipetting the organisms directly into the overlying water just below the
air-water interface or by placing the organisms into 30-mL counting cups
and floating them in the test chamber for 15 min prior to placement into
the overlying water. Measurements of length or weight should be made
on a subset of 20 organisms prior to test initiation.
(6) Monitoring a test. All test chambers should be checked daily.
Test organisms should be observed for abnormal behavior, such as sedi-
ment avoidance. The exposure system should also be monitored daily to
assure proper operation.
(7) Measurement of overlying water-quality characteristics, (i)
Conductivity, hardness, pH, alkalinity, and ammonia should be measured
in all treatments at the beginning and end of a test, and during any test
should not vary more than 50 percent. Samples should be removed with
a pipet from 1 to 2 cm above the sediment surface without disturbance.
Caution is required to avoid removing test organisms when sampling.
(ii) DO should be measured daily, and should be maintained between
40 percent and 100 percent saturation. Both DO and pH may be measured
in overlying water using a probe.
(iii) Temperature should be measured daily in one test chamber from
each treatment. The mean and instantaneous temperatures should not vary
from the desired temperature by more than 1 °C and 3 °C, respectively.
(8) Feeding. H. azteca may be fed with a mixture of yeast, Cerophyl,
and trout chow (YCT) at a rate of 1.5 mL daily per test chamber. Food
is required for proper maintenance of the test organisms but should be
kept to a minimum to prevent alteration of contaminant availability or the
growth of microbials such as fungus and bacteria. Collection of food on
the bottom of the test chamber or reduced concentration of DO are indica-
tors of possible overfeeding. Should either of the above conditions occur,
feeding should be suspended in all test chambers until conditions have
readjusted. Detailed records and observations should be made daily.
(9) Ending a test. Surviving amphipods may be pipetted from the
test chamber prior to sieving the sediment. Immobile organisms isolated
from either sediment or sieved material are considered dead. Sediment may
be sieved by pouring one-half of the overlying water volume followed
by one-half of the sediment through a #50 sieve (300 (im) into an examina-
tion pan. The coarser sediment remaining in the test chamber should be
washed through a #40 (425 (im) sieve into a second examination pan.
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Surviving organisms should be isolated and preserved (e.g. 8 percent sugar
formalin) and measured for growth. The amount of time taken to recover
test organisms should be consistent (e.g. 10 min per replicate). A recovery
rate of 90 percent of organisms from the sediment is acceptable.
(10) Test data, (i) The primary endpoint for 10-day sediment toxicity
test with H. azteca is survival.
(ii) Amphipod body length should be measured from the base of the
first of antenna to the tip of the third uropod along the curve of the dorsal
surface.
(iii) To determine dry weight of surviving amphipods:
(A) Pool all surviving organisms from a replicate.
(B) Dry the sample to constant weight at 60 to 90 °C.
(C) Bring sample to room temperature in a desiccator.
(D) Weigh the sample of organisms to the nearest 0.01 mg. This
measure will give the mean weight of surviving organisms per replicate.
(11) Interpretation of results—(i) Age sensitivity. The relative sen-
sitivity of H. azteca is comparable up to 24- to 26-day-old organisms.
Amphipods 7- to 14-day-old represent sensitivity of H. azteca up to adult
life stage.
(ii) Grain size. H. azteca tolerate a wide range of substrates. Neither
grain size nor TOC correlate with the toxic response in sediment toxicity
tests.
(iii) Isolating organisms at the end of a test. Quantitative recovery
of amphipods <7-days-old is difficult. Starting testing with
7-day-old amphipods facilitates recovery.
(iv) Influence of indigenous organisms. The presence of
oligochaetes does not reduce the survivability of amphipods in 28-day
sediment tests. However, high density of oligochaetes does reduce the
growth of amphipods. The number of oligochaetes and presence of preda-
tors in test sediment should be determined to improve the interpretation
of growth data.
(i) Interferences. (1) Interferences are defined as those characteristics
of sediment or sediment test systems that are unrelated to sediment-associ-
ated contaminants, but have the potential to affect the survival of test orga-
nisms. Interferences may lead to both Type I (false-positive) and Type
II (false-negative) errors.
(2) Interferences may result from sediment characteristics that affect
survival independently of chemical concentration, altered bioavailability
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(e.g. sediment manipulation, storage, etc.), or when indigenous species are
present.
(3) Test procedures and organism selection criteria were designed to
minimize impacts due to interferences, and are suitable for providing direct
measure of contaminant effects on benthic organisms.
(4) Several noncontaminant factors have the potential to affect sedi-
ment toxicity. These factors include but are not limited to avoidance, light-
ing, and geomorphological and physicochemical characteristics. Although
laboratory sediment toxicity tests results may be used to predict effects
in the field, extrapolations to the field may not prove valid in cases where
motile organisms are able to avoid exposure.
(5) Toxicological responses of some chemicals may be altered by UV
radiation contained in natural sunlight. Sediment testing with some chemi-
cals, which are photoinduced by UV light, may not provide results useful
for predicting field effects, because typical lighting (i.e. fluorescent) does
not emit UV radiation.
(6) Natural geomorphological and physicochemical characteristics of
sediment should be within the tolerance limits of the test organism. Factors
such as texture, grain size, and organic carbon may influence the toxic
response of the test organism.
(7) Sediment toxicity tests were designed to predict anticipated con-
taminant-related effects in the field or under natural conditions. However,
sediment toxicity is related to bio availability, which can be altered by
physical manipulation, temperature, adjuncts, and organism uptake.
(8) In some cases bioavailability may differ between the laboratory
and in situ. Sediment collection, handling, and storage are critical to pre-
serving the integrity of contaminant equilibrium. The manipulation of sedi-
ment may disrupt the equilibrium with organic carbon and the pore water/
particle system, resulting in the increased availability of organic com-
pounds.
(9) The testing temperature is important to bioavailability. Tempera-
ture affects contaminant solubility, the partitioning coefficient, as well as
the physical and chemical characteristics of sediment. Bioavailability may
also be altered by interactions between sediment and overlying water.
(10) Adjuncts such as food, water, or solvents may alter
bioavailability and promote the growth of microorganisms. While food ad-
dition is necessary, the quantity and composition of food added must be
carefully considered.
(11) Uptake of contaminants by the test organisms or test chambers
may influence bioavailability. Test organisms are sinks for contaminants,
but to a lesser degree than sediments.
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(12) The routes of exposure for sediment contaminants are not always
known. In some cases, it may desirable to normalize sediment concentra-
tions of contaminants to factors other than dry weight, such as organic-
carbon for nonionic organic compounds or acid volatile sulfides for certain
metals.
(13) The Agency recommends using natural sediments for spiking in
sediment toxicity tests. However, indigenous species sometimes exist in
field-collected sediments and their presence could negatively effect the
growth rates of test organisms. Biological activity may be inhibited by
gamma radiation, heat, sieving, mercuric chloride, or antibiotics, and their
impact on sediment characteristics must be determined prior to the com-
mencement of testing.
(j) Test method—Chironomus tentans 10-day survival and
growth test for sediments—(1) Test conditions. The 10-day sediment
toxicity test with C. tentans should be conducted at a temperature of 23
°C and photoperiod of 16 h lights h dark at 500 to 1,000 Ix. The rec-
ommended test chambers are 300-mL high-form beakers without lips con-
taining 100 mL of sediment and 175 mL of overlying water. Each test
chamber is filled with 10 third-instar midges to begin the test. All orga-
nisms must be third-instar (50 percent of organisms) or younger. For rou-
tine testing, eight replicates are recommended. Midges should be fed 1.5
mL of a 4 g/L suspension of Tetrafin daily. Overlying water in each test
chamber should receive two volume changes per day and can be culture
water, well water, surface water, site water, or reconstituted water.
(2) Sediment into test chambers. Test sediment should be mixed
thoroughly and placed into test chambers one day (day—1) before com-
mencement of the test. Sediment should be checked for homogeneity vis-
ually and quantitatively by analyzing TOC, chemical concentrations, and
particle size. Equal volumes of sediment should be added to each test
chamber, and on day - 1 overlying water should be added by pouring water
along a baffle to avoid any disturbance of the sediment. The test begins
once the test organisms are added to the test chambers (day-0).
(3) Renewal of overlaying water. The renewal of overlying water
is required and should be conducted on day—1 prior to the addition of
test organisms or food on day-0. Flow rates should not vary by more
than 10 percent between any two test chambers at any time during the
test. Proper system operation should be verified by calibration prior to
initiation of the test.
(4) Acclimation. The required culture and testing temperature is
23 °C. The test organisms should be cultured in the same water to be
used for testing. Acclimation of the test organisms to the test water is
not required.
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(5) Placing organisms in test chambers. Handle test organisms as
little as possible. Midges may be placed into test chambers by pipetting
the organisms directly into the overlying water just below the air-water
interface or by placing the organisms into 30-mL counting cups and float-
ing them in the test chamber for 15 min prior to placement into the over-
lying water. Measurements of length or weight should be made on a subset
of 20 organisms prior to test initiation. Head capsule widths should be
measured on midges to determine the instar used at test initiation.
(6) Monitoring a test. All test chambers should be checked daily.
Test organisms should be observed for abnormal behavior, such as sedi-
ment avoidance. The exposure system should also be monitored daily to
assure proper operation.
(7) Measurement of overlying water-quality characteristics, (i)
Conductivity, hardness, pH, alkalinity, and ammonia concentration should
be measured in all treatments at the beginning and end of a test, and during
any test should not vary more than 50 percent. Samples should be removed
with a pipet from 1 to 2 cm above the sediment surface without disturb-
ance. Caution is required to prevent removing test organisms when sam-
pling.
(ii) DO should be measured daily, and should be maintained between
40 and 100 percent saturation. Both DO and pH may be measured in over-
lying water using a probe.
(iii) Temperature should be measured in one test chamber from each
treatment daily. The mean and instantaneous temperatures should not vary
from the desired temperature by more than 1 and 3 °C, respectively.
(8) Feeding. Food is required for proper maintenance of the test orga-
nisms but should be kept to a minimum to prevent alteration of contami-
nant availability or the growth of microbials such as fungus and bacteria.
Collection of food on the bottom of the test chamber or reduced concentra-
tion of DO are indicators of possible overfeeding. Should either of the
these conditions occur, feeding should be suspended in all test chambers
until conditions have readjusted. Detailed records and observations should
be made daily.
(9) Ending a test. Surviving amphipods may be pipetted from the
test chamber prior to sieving the sediment. Immobile organisms isolated
from either sediment or sieved material are considered dead. Surviving
organisms should be preserved (e.g. 8 percent sugar-formalin) and meas-
ured for growth. Specific sieving instruction may be found in paragraph
(1)(1) of this guideline.
(10) Test data, (i) The endpoints measured in 10-day sediment tests
with C. tentans are dry weight and survival. At the end of the test, C.
tentans in control sediment should have an average size of 0.6 mg. Head
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capsule width should be measured prior to dry weight. To determine dry
weight of surviving midges:
(A) pool all surviving organisms from a replicate.
(B) Dry the sample at 60 to 90 °C to constant weight.
(C) Bring sample to room temperature in a desiccator.
(D) Weigh the sample of organisms to the nearest 0.01 mg. This
measure will give the mean weight of surviving organisms per replicate.
(iv) Pupae and adults should be excluded from dry weight determina-
tions. Length measurement is optional, but measurements should be from
the anterior of the labrum to the posterior of the last abdominal segment.
(11) Interpretation of results—(i) Age sensitivity. First and second
instar midges are more sensitive than third and fourth instar midges. Sedi-
ment tests should be initiated with midges of uniform size and age to
avoid changes in sensitivity. Sediment tests are conducted with the third-
instar midges because the greater size facilitates handling and isolation
from sediment at test termination.
(ii) Grain size. C. tentans are tolerant of a wide range of substrates.
The sensitivity of midges does not correlate with TOC or grain size. How-
ever, sensitivity may be influenced by artificial sediment when test orga-
nisms are not fed during the test.
(iii) Isolating organisms at the end of a test. Isolation and recovery
of midges at the end of the test is not difficult. The midges are typically
red and greater 5-mm in length.
(iv) Influence of indigenous organisms. There are no reports on the
influence of indigenous organisms on C. tentans survival and response
in sediment toxicity tests. However, survival of a congener, Chironomus
riparius, was not reduced in the presence of oligochaetes, but growth was
reduced in the presence of high numbers of oligochaetes. The number of
oligochaetes and presence of predators in test sediment should be deter-
mined to improve the interpretation of growth data.
(k) Reporting. In addition to information meeting general reporting
requirements, a report of the results of a whole sediment toxicity test
should also include the following:
(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.
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(3) Source of test material, lot number if applicable, composition
(identities and concentrations of major ingredient and impurities if known),
known chemical and physical properties, and the identity and concentra-
tions 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 and number of test organisms/treatment, date
and time test started and ended, temperature measurements, DO concentra-
tion (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 LC50 or ECSOs, 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.
(14) Published reports should contain enough information to clearly
identify the methodology used and the quality of the results.
(1) References. The following references should be consulted for ad-
ditional background material on this test guideline.
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(1) U.S. Environmental Protection Agency. Methods for Measuring
the Toxicity and Bioaccumulation of Sediment-Associated Contaminants
with Freshwater Invertebrates. EPA 600/R-94/024 (1994).
(2) [Reserved]
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