United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA712-C-96-313
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1790
Chironomid Sediment
Toxicity Test
I

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 850.1790 Chironomid sediment toxicity test.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is 40 CFR 795.135 Chironomid Sediment
Toxicity Test (proposed in the Federal Register of June 25, 1991 (56
FR 29149)).
(b)	Purpose. This guideline may be used to develop data on the tox-
icity and bioavailability of chemical substances and mixtures ("chemi-
cals") in sediments subject to environmental effects test regulations under
TSCA. This guideline prescribes tests to be used to develop data on the
toxicity of chemicals present in sediments to chironomid larvae (midges).
The EPA will use data from these tests in assessing the hazard of a chemi-
cal to the environment.
(c)	Definitions. The definitions in section 3 of TSCA and 40 CFR
part 792, Good Laboratory Practice Standards (GLPS), apply to this test
guideline. In addition, the following definitions also apply:
Bioconcentration factor (BCF) is the quotient of the concentration
of a test substance in tissues of the chironomids at or over a specific time
period of exposure divided by the concentration of test substance in the
overlying water, interstitial water, or in the sediments at or during the
same time period.
Cation exchange capacity (CEC) is the sum total of exchangeable
cations that a sediment can absorb. The CEC is expressed in
milliequivalents of negative charge per 100 g or milliequivalents of nega-
tive charge per gram of sediment (dry weight).
COD is chemical oxygen demand.
EC50 is an experimentally-derived concentration of test substance in
the sediment that is calculated to affect 50 percent of a test population
during continuous exposure over a specified period of time.
Flow-through is a continuous or intermittent passage of dilution water
through a test chamber or culture tank with no recycling of water.
Geometric mean is the calculated mean between the highest test con-
centration with no statistically significant effects and the lowest concentra-
tion showing significant effects.
Interstitial water is liquid which is found in or directly adjacent to
sediments and can be extracted from these sediments by several processes.
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Loading is the ratio of chironomid biomass (grams wet weight) to
the volume (liters) of test solution in a test chamber at a specified time
or passing through the test chamber during a specific interval.
Lowest-observed-effect-concentration (LOEC) is the lowest treatment
(i.e., test concentration) of a test substance that is statistically different
in adverse effect on a specific population of test organisms from that ob-
served in controls.
MATC (maximum acceptable toxicant concentration) is the maximum
concentration at which a chemical may be present and not be toxic to
the test organism.
No-observed-effect-concentration (NOEC) is the highest treatment
(i.e., test concentration) of a test substance that shows no statistical dif-
ference in adverse effect on a specific population of test organisms from
that observed in controls.
Overlying water is liquid which is found above or placed over sedi-
ments. For purposes of this guideline, overlying water is equivalent to the
term water column.
Partial life-cycle toxicity test is one which uses a sensitive portion
of the life of a test organism (second instar of midges) to assess the effects
of test substances.
Redox potential (Eh) means the oxidizing or reducing intensity or
condition of a solution expressed as a current, referenced against a hydro-
gen electrode. Zero or negative Eh values may be exist due to reducing
conditions within wet sediments.
Sediment is matter that settles to the bottom of a liquid in natural
situations or a substrate prepared from a combination of natural sediments
and artificial components. Sediment is equivalent to the term solid-phase
sediments in this guideline.
Sediment partition coefficient is the ratio of the concentration of test
substance on the sediment to the concentration in the overlying water. For
the purposes of this guideline, this term is identical to soil-water partition
coefficient.
Spiking is the addition of a test substance to a negative control and/
or reference sediment so that the toxicity of a known quantity of test sub-
stance can be determined in a known nontoxic sediment. Often a solvent
carrier is needed for low-water soluble test substances.
Subchronic toxicity test is a method used to determine the concentra-
tion of a test substance in water and for sediment which produces an ad-
verse effect on chironomids over a partially extended period of time. In
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this guideline, mortality and growth (expressed as change in wet weight
of midges) are the criteria of toxicity.
TOC is total organic carbon.
(d) Test procedures—(1) Summary of test, (i) This flow-through
test consists of three parts. Part 1. is a 14-day aqueous exposure test, with
minimal sediments, with food, and with the test substance added to the
overlying water. Part 2. is a 14-day sediment exposure test, with one or
more sediments (4 to 6 cm in thickness) which may have varying amounts
of organic carbon, with food, and with the test substance added to sedi-
ments. Part 3. is a 14-day interstitial exposure test, with one or more sedi-
ments (4 to 6 cm in thickness) which may have varying amounts of organic
carbon, with food, and with the test substance added to overlying water.
The flow-through test is illustrated in the following Table 1.
Table 1.—Experimental Design for the Chironomid Sediment Flow-Through Toxicity Test
Test system
Test sub-
stance con-
centrations
(2 replicates
each) 1
Number of
sediments
(2 replicates
each)
Number of Samples Analyzed (2 replicates each)
Overlying
water P/C 2
Interstitial
water P/C 2
Sediments
Midges 3
Part 1






14-Day Aqueous Exposure 	
5(10)
na 4
5(10)
na
na
5(10)
Control (2 reps) 	
na
na
1 (2)
na
na
1 (2)
Solvent Control (2 reps)	
na
na
1 (2)
na
na
1 (2)
Part 2




14-Day Sediment Exposure 	
5(10)
1-3 s (2-6)
5(10)
na
5(10)
5(10)
Control (2 reps) 	
na
1 (2)
1 (2)
na
1 (2)
1 (2)
Solvent Control (2 reps)	
na
1 (2)
1 (2)
na
1 (2)
1 (2)
Part 3






14-Day Interstitial Water/Sediment Expo-






sure 	
5(10)
1-3 s (2-6)
5(10)
5(10)
5(10)

Control (2 reps) 	
na
1 (2)
1 (2)
1 (2)
1 (2)
1 (2)
Solvent Control (2 reps)	
na
1 (2)
1 (2)
1 (2)
1 (2)
1 (2)
1	Test substance concentration in all replicates measured at days 0 and 14 (reps = replicates)
2	P/C = physical/chemical measurements (dissolved oxygen, temperature (in °C), and pH) on days 0, 4, 7, 10 and 14.
3	Midges are observed throughout the test, dead chironomids recorded, removed, and weighed on days 4, 7, and 10. At end of
each test, remaining midges from each replicate are removed, counted, and weighed.
4	na = not applicable
5	Number of sediment types tested will depend on range of TOC content tested; 1 to 3 types (low, medium, and high TOC lev-
els) are recommended.
(ii) The day before the test is to be started, sediments (in treatments,
and reference and negative controls) should be screened to remove large
particles and endemic animals (especially midge predators) and added to
the test chambers. The amount of sediments to be added to each test cham-
ber will depend on the experimental design and test species. Only a mini-
mum amount (to a depth of 2 mm) should be added in the aqueous expo-
sure portion of the test. Each replicate test chamber should contain the
same amount of sediments. Overlying water should be added to each test
chamber.
(iii) In this flow-through test, the flow of dilution water through each
chamber is begun and adjusted to the rate desired. The test substance
should be introduced into each test chamber. The addition of test substance
in the flow-through system should be done at a rate which is sufficient
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to establish and maintain the desired concentration of test substance in
the test chamber.
(iv) At the initiation of the test, chironomids which have been cul-
tured or acclimated in accordance with the test design are randomly placed
into the test chambers. Midges in the test chambers are observed periodi-
cally during the test. Immobile or dead larvae should be counted, removed,
and weighed, and the findings recorded. "Floating" larvae are nonviable
and should be replaced. Dissolved oxygen (DO) concentration, pH, tem-
perature, the concentration (measured) of test substance, and other water
quality parameters should be measured at specified intervals in selected
test chambers, during all three parts of this test. (See Table 1 under para-
graph (d)(l)(i) of this guideline.) Data should be collected during the test
to determine any significant differences (P<0.05) in mortality and growth
as compared to the controls. BCFs should be calculated at the end of the
test based on route of exposure.
(2)	Range-finding test, (i) A range-finding test should be conducted
prior to beginning each of the three parts of the test to establish test solu-
tion concentrations for the three definitive parts of the test.
(ii)	The chironomids should be exposed to a series of widely spaced
concentrations of the test substance (e.g., 1, 10, 100 mg/L).
(iii)	A minimum of 10 chironomids should be exposed to each con-
centration of test substance for a period of time which allows estimation
of appropriate test concentrations. No replicates are required and nominal
concentrations of the chemical are acceptable.
(3)	Definitive test, (i) The purpose of the definitive portion of the
test is to determine concentration-response curves, EC50 values, effects
of a chemical on mortality and growth, and the determination of BCFs
during subchronic exposure.
(ii)	A minimum of 30 midges per concentration (15 midges per rep-
licate test chamber) should be exposed in each part of the test to five
or more concentrations of the test substance chosen in a geometric series
in which the ratio is between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, 64 mg/
L). An equal number of chironomids should be placed in two replicates.
The concentration ranges should be selected to determine the concentra-
tion-response curves, EC50 values, and MATC. Solutions should be ana-
lyzed for chemical concentration prior to use and at designated times dur-
ing the test.
(iii)	Each test should include controls consisting of the same dilution
water, sediments, conditions, procedures, and midges from the same popu-
lation (same egg mass in culture container), except that none of the test
substance is added.
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(iv)	The test duration is 14 days for each of the three parts of the
test. The test is unacceptable if more than 20 percent of the control orga-
nisms die or are stressed or diseased during the test. A test period longer
than 14 days may be necessary for high log Kow chemicals.
(v)	The number of dead chironomids in each test chamber should
be recorded on days 4, 7, 10, and 14 of the test. At the end of the test,
surviving midges are removed from the test chambers and weighed after
being blotted dry. Concentration-response curves, EC50 values, and associ-
ated 95 percent confidence limits for mortality should be determined for
days 4, 7, 10, and 14 in the aqueous exposure portion of the test. MATC,
NOEC, and LOEC values should be determined for midge survival and
growth.
(vi)	In addition to survival and growth, any abnormal behavior or
appearance of the chironomids should be reported.
(vii)	Distribution of midges among the test chambers should be ran-
domized. In addition, test chambers within the testing area should be posi-
tioned in a random manner or in a way that appropriate statistical analyses
can be used to determine variation due to placement.
(viii)	A control sediment and/or a reference sediment should be used
in each part of this test. Use of these controls/references will help deter-
mine if the test is acceptable, serve to monitor the health of the
chironomids used in the testing and the quality and suitability of test condi-
tions, parameters and procedures, and aid in analyzing data obtained from
this test. A negative control should be run in the test, using a sediment
known to be nontoxic to the midges. A reference sediment can be run
in the test in addition to or in place of the negative control. The reference
sediment should be obtained from an area that is known to have low levels
of chemical contamination and which is similar to or identical to the test
sediments in physical and chemical characteristics.
(ix)	In the first part of this test, the aqueous exposure, a minimal
amount of sediment (<2mm) is placed in the test chambers. The presence
of sediment is necessary to allow the midges to construct tubes, to reduce
stress to the chironomids, and to reduce cannibalism.
(x)	BCFs should be calculated at the end of each part of the test.
(4) Analytical measurements—(i) Water quality analysis. (A) The
hardness, acidity, alkalinity, conductivity, TOC or COD, and particulate
matter of the dilution water serving as the source of overlying water should
be measured on days 0 and 14. The month-to-month variation of these
values should be less than 10 percent and the pH should vary less than
0.4 units.
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(B) During all three parts of the flow-through test, DO, temperature,
and pH should be measured in each chamber on days 0, 4, 7, 10, and
14.
(ii) Analysis of test substance. (A) Deionized water should be used
in making stock solutions of the test substance. Standard analytical meth-
ods should be used whenever available in performing the analyses of water
and sediments. Radiolabeling of the test substance (e.g., by use of 14C)
may be necessary to measure quantities present in the sediments accu-
rately. The analytical method used to measure the amount of test substance
in the sample should be validated by appropriate laboratory practices be-
fore beginning the test. An analytical method is not acceptable if likely
degradation products of the test substance, such as hydrolysis and oxida-
tion products, give positive or negative interference which cannot be sys-
tematically identified and corrected mathematically. When radiolabeled
test substances are used, total radioactivity should be measured in all sam-
ples. At the end of the test, water, sediments, and tissue samples should
be analyzed using appropriate methodology to identify and estimate any
major (at least 10 percent of the parent compound) degradation products
or metabolites that may be present.
(B)	The overlying water from each test chamber should be sampled
for the test substance on days 0, 7, and 14 for all three aqueous exposure
parts of this test.
(C)	For the nonaqueous exposure parts of the test, the interstitial
water from each test chamber should be analyzed for the test substance
on days 0, 7, and 14. Interstitial water can be sampled by using a variety
of methods, such as removal of overlying water and centrifugation, filtra-
tion of sediments, pressing the sediments, or using an interstitial water
sample. Care should be taken during these measurements to prevent the
biodegradation, transformation, or volatilization of the test substance.
(D)	For the nonaqueous exposure portion of the test, the sediments
from each test chamber should be analyzed for the test substance on days
0, 7, and 14.
(E)	The sediment partition coefficient or soil-water partition coeffi-
cient is determined by dividing the average test substance concentration
in sediment by the respective average concentration in the water column.
Concentrations of test substance in the sediments to be used in this test
can be chosen by measuring these partition coefficients. This sediment par-
tition coefficient should be determined in triplicate by placing a quantity
of a sediment with a known TOC content and spiked with the radiolabeled
test substance into a quantity of dilution water. The ratio of sediment to
dilution water should simulate the ratio present in the test. The sediment/
dilution water mixture is shaken periodically, and the radiolabeled test sub-
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stance measured. This shaking and sampling procedure is repeated until
equilibrium is reached, as defined by the stage on the desorption curve.
(F)	Overlying water samples should be filtered through a 0.45 (im
filter to determine the concentration of dissolved test substance.
(G)	BCFs should be calculated by determining the amount of test
substance in the midge tissue and dividing by the concentration of test
substance in the water column, interstitial water, and sediments. At test
termination, the midges remaining in each test concentration are analyzed
for test substance. Suitable methods are available, such as radiolabeling
(14C) the test substance, combusting the midges, trapping and counting
the resulting radioactivity and the BCF calculated. If insufficient
chironomid biomass is present at the conclusion of the test replicates may
be pooled. BCFs cannot be calculated if after pooling there is insufficient
biomass or if the accumulated test substance concentration is lower than
the detection limit for the test substance.
(iii) Numerical. (A) The number of dead midge second instars should
be counted during each definitive test. Appropriate statistical analyses
should provide a goodness-of-fit determination for mortality concentration-
response curves calculated on days 4, 7, 10, and 14. A 4-, 7-, 10-, and
14-day LC50 value based on second instar mortality, and with correspond-
ing 95 percent confidence intervals, should be calculated. The methods
recommended for calculating EC50s include probit, logit, binomial, and
moving average.
(B)	Appropriate statistical tests (e.g., analysis of variance and mean
separation tests) should be used to test for significant chemical effects on
growth (measured as wet weights) on days 4, 7, and 14. An MATC should
be calculated using these test criteria.
(C)	In no case should any analytical measurements be pooled except
when calculating BCFs when there is insufficient biomass available for
individual measurements as described under paragraph (d)(4)(ii)(G) of this
guideline.
(e) Test conditions—(1) Test species—(i) Selection. (A) The midge,
Chironomus tentans or C. riparius, should be used in this test. Both spe-
cies are widely distributed throughout the United States, and the larvae
and adult flies can be cultured in the laboratory. The larval portion life
cycles of both species is spent in a tunnel or case within the upper layers
of benthic sediments of lakes, rivers, and estuaries. Feeding habits of both
species include both filter feeding and ingesting sediment particles.
(B) Second instar chironomids (<10 days) of the same age and size
are to be used in this test. Third and fourth instar are less desirable, as
some evidence indicates they are less sensitive, at least to copper. Each
instar is 4 to 7 days in duration.
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(ii)	Acquisition. (A) Chironomids to be used in this test should be
cultured at the test facility. Adult flies are collected from the chironomid
cultures and allowed to mate and lay egg masses. Two egg masses are
collected and allowed to hatch. The larvae are fed daily. When the second
instar stage (about 10 days after hatching) is reached, larvae are removed
and placed in the test chambers. Records should be kept regarding the
source of the initial stock and culturing techniques. All organisms used
for a particular test should have originated from the same population (cul-
ture container) and be the same age and size.
(B) Chironomids should not be used in a test if:
(7) During the final 48 hours of midge holding, obvious mortality
is observed.
(2) The larvae are not in the second instar.
(iii)	Feeding. (A) During the test, the chironomids should be fed the
same diet at the same frequency as that used for culturing and acclimation.
All treatments and controls should receive, as near as reasonably possible,
the same amount of food on a per-animal basis.
(B) The food concentration depends on the type used and the nutri-
tional requirements of the midges. The latter in turn is dependent upon
their developmental stage.
(iv)	Loading. The number of test organisms placed in a test chamber
should not affect the test results. Loading should not exceed 30
chironomids per liter per 24 hours in the flow-through test. Loading should
not affect test concentrations or cause the DO concentration to fall below
the recommended level.
(v)	Care and handling of test organisms. (A) Chironomids should
be cultured in dilution water under similar environmental conditions as
those in the test. Food such as Tetra Conditioning Food has been dem-
onstrated to be adequate for chironomid cultures.
(B)	Organisms should be handled as little as possible. When handling
is necessary, it should be done as gently, carefully, and as quickly as pos-
sible. During culturing and acclimation, midges should be observed for
any signs of stress, physical damage, and mortality. Dead and abnormal
individuals should be discarded. Organisms that are damaged or dropped
during handling should be discarded.
(C)	Wide-bore, smooth glass tubes or pipets equipped with a rubber
bulb can be used for transferring midges.
(vi)	Acclimation. (A) Midges should be maintained in 100 percent
dilution water at the test temperature for at least 4 days prior to the start
of the test. This is easily accomplished by culturing them in the dilution
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water at the test temperature. Chironomids should be fed the same food
during the test as is used for culturing and acclimation.
(B) Midges should be maintained in facilities similar to those of the
testing area during culturing and acclimation to the dilution water.
(2) Test system—(i) General. (A) Facilities needed to perform this
test include:
(7) Containers for culturing and acclimating the chironomids.
(2)	A mechanism for controlling and maintaining the water tempera-
ture during the culturing, acclimation, and test periods.
(3)	Apparatus for straining particulate matter, removing gas bubbles,
or aerating the water as necessary to ensure that the test solution flows
regularly into and out of the test chamber.
(4)	Test chambers can be small aquaria capable of holding 3 L of
water or test solution, 5.7-L clear glass battery jars, or 1-L beakers made
of borosilicate glass. Each chamber should be equipped with screened
overflow holes, standpipes, or U-shaped notches covered with Nitex
screen.
(B)	Construction materials and commercially purchased equipment
that may contact dilution water should not contain substances that can be
leaked or dissolved into aqueous solutions in quantities that can alter the
test results. Materials and equipment that contact test solutions should be
chosen to minimize sorption of test substances.
(C)	Test chambers should be loosely covered to reduce the loss of
test solution or dilution water by evaporation, and to minimize the entry
of dust or other particulates into the solutions.
(ii)	Test substance delivery. (A) In the flow-through test, propor-
tional diluters, metering pump systems, or other suitable systems should
be used to deliver the test substance to the test chambers.
(B) The delivery system should be calibrated before and after each
test. Calibration includes determining the flow rate through each chamber
and the concentration of the test substance in each chamber. The general
operation of the test substance delivery system should be checked twice
daily during the test. The 24-h flow rate through a test chamber should
be equal to at least 5x the volume of the test chamber. During a test,
the flow rates should not vary more than 10 percent from any one test
chamber to another or from one time to any other.
(iii)	Cleaning of test system. All test equipment and test chambers
should be cleaned before each test following standard laboratory proce-
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dures. Cleaning of test chambers may be necessary during the testing pe-
riod.
(iv) Dilution water. (A) Surface or ground water, reconstituted water,
or dechlorinated tap water are acceptable as dilution water if chironomids
will survive in it for the duration of the culturing, acclimation, and testing
periods without showing signs of stress. The quality of the dilution water
should be constant and should meet the specifications in the following
Table 2.:
Table 2.—Specifications For Dilution Water
Substance	Maximum Concentration
Particulate matter		20 mg/L
TOC or COD 		2 mg/L or 5 mg/L, respectively
Boron, fluoride 		100|^g/L
Un-ionized ammonia 		10 ^g/L
Aluminum, arsenic, chromium, cobalt, copper,	1 ^g/L
iron, lead, nickel, zinc..
Residual chlorine 		3 ^g/L
Cadmium, mercury, silver		100 ng/L
Total organophosphorus pesticides		50 ng/L
Total organochlorine pesticides and poly-	50 ng/L or 25 ng/L respectively
chlorinated biphenyls (PCBs) or organic chlo-
rine..
(B)	The water quality characteristics listed in Table 2. should be
measured at least twice a year or when it is suspected that these character-
istics may have changed significantly. If dechlorinated tap water is used,
daily chlorine analysis should be performed.
(C)	If the diluent water is from a ground or surface water source,
conductivity, hardness, alkalinity, pH, acidity, particulate matter, TOC or
COD, and particulate matter should be measured. Reconstituted water can
be made by adding specific amounts of reagent-grade chemicals to
deionized or distilled water. Glass distilled or carbon filtered deionized
water with conductivity of less than 1 jiohm/cm is acceptable as the diluent
for making reconstituted water.
(D)	If the test substance is not soluble in water, an appropriate carrier
such as triethylene glycol (CAS No. 112-27-6), dimethylformamide (CAS
No. 68-12-2), or acetone (CAS No. 67-64-1) should be used. The con-
centration of such carriers should not exceed 0.1 mL/L.
(v) Sediments. (A) Preparation and source. (7) Sediments used in
this test may contain low (<1 percent) to high (>15 percent) amounts of
organic carbon because they are derived from variable natural sediments.
Prior to use, the sediments should be sieved to remove larger particles.
The should be characterized for particle size distribution (sand, silt, clay
percentages), percent water holding capacity, total organic and inorganic
carbon, total volatile solids, COD, BOD, cation exchange capacity, redox
10

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potential (Eh), oils and greases, petroleum hydrocarbons, organophosphate
pesticide concentrations, organochlorine pesticide and polychlorinted
biphenyl (PCB) concentrations, toxic metal concentrations, and pH.
(2)	The source of the sediments used in this test should be known
and the characteristics listed above should be measured every time addi-
tional sediments are obtained. The sediments should not contain any en-
demic organisms, as these may be chironomid predators.
(3)	Sediments should not be resuspended during the test.
(3) Test parameters, (i) Environmental conditions of the water con-
tained in test chambers should be maintained as specified below:
(A)	Temperature of 20 ±1 °C for C. tentans and 22 ±1 °C for C.
riparius.
(B)	DO concentration of the dilution water should be 90 percent of
saturation or greater. The DO concentrations of the test solutions should
be 60 percent or greater of saturation throughout the test. Aeration may
be necessary, and if this is done, all treatment and control chambers should
be given the same aeration treatment.
(C)	A photoperiod of 16 h light/and 8 h dark with a 15- to 30-minute
transition period.
(ii) Additional measurements include:
(A)	The concentration of dissolved test substance (that which passes
through a 0.45 (im filter) in the chambers should be measured during the
test.
(B)	At a minimum, the concentration of test substance should be
measured as follows:
(7) In each chamber before the test.
(2)	In each chamber on days 7 and 14 of the test.
(3)	In at least one appropriate chamber whenever a malfunction is
detected in any part of the test substance delivery system.
(C)	Among replicate test chambers of a treatment concentration, the
measured concentration of the test substance should not vary by more than
20 percent at any time or 30 percent during the test.
(D)	The dissolved oxygen concentration, temperature, and pH should
be measured at the beginning of the test and on days 7 and 14 in each
chamber.
(f) Calculated values—(1) Sediment partition coefficient. (A) The
sediment or soil-water partition coefficient (Kp) is defined as the ratio of
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the concentration of the test substance in the sediment (Cs) to the con-
centration in the water or interstitial water (Cw) as given in the follwing
expression:
Kp = Cs/Cw
The resultant Kp values for the sediment or sediments tested are used to
select test substance concentrations for the sediment test.
(B)	The Kp value is equivalent or related to the sediment organic
carbon sorption coefficient multiplied by the percent organic carbon con-
tent of the sediment.
(C)	The sediment partition coefficient should be determined in trip-
licate for each sediment type at equilibrium by spiking with the
radiolabeled test substance and shaking. The test substance concentration
in the water is measured radiometrically at intervals and the data used
to create a desorption curve. The process is repeated until an equilibrium
is reached, as defined by the shape of the curve.
(2) BCFs. BCFs should be calculated for each part of the test. These
values are computed as the amount of test substance present in the midge
tissues divided by test substance concentrations in the water column, inter-
stitial water, and sediments. At test termination, the chironomids remaining
in each test concentration are analyzed for radiolabeled test substance.
(g) Reporting. The sponsor should submit all data developed by the
test that are suggestive and predictive of toxicity and all associated
toxicologic manifestations to the Agency. In addition to the reporting re-
quirements prescribed in the GLPS, the reporting of test data should in-
clude the following:
(1)	The name of the test, sponsor, testing laboratory, study director,
principal investigator, and dates of testing.
(2)	A detailed description of the test substance including its source,
lot number, composition (identity and concentration of major ingredients
and major impurities), known physical and chemical properties, and any
carriers or other additives used and their concentrations.
(3)	The source of the dilution water, its chemical characteristics (e.g.,
conductivity, hardness, pH, TOC or COD, and particulate matter) and a
description of any pretreatment.
(4)	The source of the sediment, its physical and chemical characteris-
tics (e.g., particle size distribution, TOC, pesticide and metal concentra-
tions), and a description of any pretreatment.
(5)	Detailed information about the chironomids used as a stock, in-
cluding the scientific name and method of verification, age, source, treat-
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ments, feeding history, acclimation procedures, and culture methods. The
age (in days) and instar stage of the midges used in the test should be
reported.
(6)	A description of the test chambers, the volume of solution in the
chambers, and the way the test was begun (e.g., conditioning and test sub-
stance additions). The number of test organisms per test chamber, the num-
ber of replicates per treatment, the lighting, the test substance delivery
system, flow rates expressed as volume additions per 24 hours for the
flow-through subchronic test, the method of feeding (manual or continu-
ous), and type and amount of food.
(7)	The concentration of the test substance in the water, interstitial
water, and sediments in test chambers at times designated in the flow-
through tests.
(8)	The number and percentage of organisms that show any adverse
effect in each test chamber at each observation period, and wet weights
of midges in each test chamber at days 7 and 14.
(9)	BCFs for all three parts of the test (i.e., overlying water or water
column, sediment, and interstitial water modes of exposure).
(10)	All chemical analyses of water quality and test substance con-
centrations, including methods, method validations, and reagent blanks.
(11)	The data records of the culture, acclimation, and test tempera-
tures. Information relating to calculation of sediment (or soil-water) parti-
tion coefficients (Kp).
(12)	Any deviation from this test guideline, and anything unusual
about the test (e.g., diluter failure and temperature fluctuations).
(13)	An LC50 value based on mortality and an EC50 value based
on adverse effects on growth (wet weights), with corresponding 95 percent
confidence limits, when sufficient data are present for days 4, 7, and 14.
These calculations should be made using the average measured concentra-
tion of the test substance.
(14)	Concentration-response curves utilizing the average measured
test substance concentration should be fitted to both number of midges
that show adverse effects (mortality) and effects on growth or wet weights
of midges at days 4, 7, and 14. A statistical test of goodness-of-fit should
be performed and the results reported.
(15)	The MATC to be reported is calculated as the geometric mean
between the lowest measured test substance concentrationthat had signifi-
cant (P<0.05) effect and the highest measured test substance concentration
that had no significant (P>0.05) effect on days 4, 7, and 14 of the test.
The criterion selected for MATC computation is the one which exhibits
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an effect (a statistically significant difference between treatment and con-
trol groups (P<0.05) at the lowest test substance concentration for the
shortest period of exposure. Appropriate statistical tests (analysis of vari-
ance and mean separation tests should be used to test for significant test
substance effects. The statistical tests employed and the results of these
tests should be reported.
(h) References. The following references should be consulted for fur-
ther background information on this test guideline.
(1)	Adams, W.J. et al. Aquatic safety assessment of chemicals sorbed
to sediments. R.D. Cardwell, R. Purdy, and R.C. Bahner, eds. In: Aquatic
Toxicology and Hazard Assessment. ASTM STP 854. American Society
for Testing and Materials, Philadephia, PA (1985).
(2)	Nebeker, A.V. et al. Relative sensitivity of Chironomus tentans
life stages to copper. Environmental Toxicology and Chemistry 3:151-158.
(1984).
(3)	Nebeker, A.V. et al. Biological methods for determining toxicity
of contaminated freshwater sediments to invertebrates. Environmental
Toxicology and Chemistry 3:617-630. (1984).
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