United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-016

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3100:

Earthworm Subchronic 

Toxicity Test
Page i of 17

NOTICE

This guideline is one of a series of test guidelines established by the United States 

Environmental Protection Agency’s Office of Chemical Safety and Pollution Prevention 

(OCSPP) for use in testing pesticides and chemical substances to develop data for 

submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, 

et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et 

seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a). 

Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic 

Substances (OPPTS). To distinguish these guidelines from guidelines issued by other 

organizations, the numbering convention adopted in 1994 specifically included OPPTS as 

part of the guideline’s number. Any test guidelines developed after April 22, 2010 will use 

the new acronym (OCSPP) in their title.

The OCSPP harmonized test guidelines serve as a compendium of accepted scientific 

methodologies and protocols that are intended to provide data to inform regulatory decisions 

under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the 

test, and is also used by EPA, the public, and the companies that are subject to data 

submission requirements under TSCA, FIFRA, and/or the FFDCA. As a guidance 

document, these guidelines are not binding on either EPA or any outside parties, and the 

EPA may depart from the guidelines where circumstances warrant and without prior notice. 

At places in this guidance, the Agency uses the word “should.” In this guidance, the use of 

“should” with regard to an action means that the action is recommended rather than 

mandatory. The procedures contained in this guideline are strongly recommended for 

generating the data that are the subject of the guideline, but EPA recognizes that departures 

may be appropriate in specific situations. You may propose alternatives to the 

recommendations described in these guidelines, and the Agency will assess them for 

appropriateness on a case-by-case basis. 

For additional information about these test guidelines and to access these guidelines 

electronically, please go to http://www.epa.gov/ocspp and select “Test Methods & 

Guidelines” on the left side navigation menu. You may also access the guidelines in 

http://www.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
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OCSPP 850.3100: Earthworm subchronic toxicity test.

(a) Scope—

(1) Applicability. This guideline is intended to be used to help develop data to submit to 

EPA under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, et seq.), the 

Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and 

the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

(2) Background. The source materials used in developing this harmonized OCSPP test 

guideline include the OPPT guideline under 40 CFR 795.150 Earthworm Toxicity Test 

(proposed in the Federal Register of June 25, 1991 (56 FR 29155)) and ASTM E 1676, 

Standard Guide for Conducting Laboratory Soil Toxicity or Bioaccumulation Tests with 

the Lumbricid Earthworm Eisenia fetida. This guideline was formerly Public Draft 

OCSPP 850.6200 (April, 1996).

(b) Purpose. This guideline is intended for use in developing data on the toxicity of chemical 

substances and mixtures (“test chemicals” or “test substances”) subject to environmental effects 

test regulations. This guideline sets forth the procedures for a toxicity test using earthworms 

which are maintained in direct contact with an artificial (formulated) soil, allowing earthworms 

to ingest contaminated soil ad libitum. The Environmental Protection Agency will use data from 

this test in assessing the hazard of a chemical to earthworms in the soil environment.

(c) Definitions. The definitions in OCSPP 850.3000 apply to this guideline. In addition, the 

more specific definitions in this paragraph also apply to this guideline:

Artificial (formulated) soil means a defined dry weight mixture of 70 percent (70%) of 

number 70 mesh silica sand, 20% kaolin clay, 10% sphagnum peat moss, and calcium 

carbonate (to adjust the pH). These ingredients are weighed and mixed in the stated 

proportions and moistened to 35–45% (by weight) with reagent water.

Behavioral symptoms are indicators of toxicity to earthworms such that a distinct 

difference in position in the test container can be identified, e.g., below surface or on the 

surface; writhing on the surface; stiffened and shortened on the surface or elongated and 

pulsing; or inactive below surface in a ball.

Clitellum means a glandular portion of the anterior epidermis, appearing as saddle-shaped

or annular, usually differentiated externally by color. It is the most visible feature of an 

adult earthworm and secretes the cocoon into which eggs and sperm are deposited. 

Mature or adult worms mean a condition of the worm exhibiting a clitellum in the 

anterior of the body.

Mortality means the lack of movement by the test organism in response to a definite 

tactile stimulus to the anterior end. Also, because earthworms tend to disintegrate rapidly 

after death, the absence of organisms in the enclosed soil test container is considered to 

mean death has occurred.

Pathological symptoms mean toxic effects, such as surface lesions and mid-segmental 

swellings or general ulcerated areas on the surface of the earthworm.
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Test mixture means the test substance/artificial soil mixture(s) which the earthworms are 

exposed to during the test. 

(d) General considerations―

(1) Summary of the test. This toxicity test is conducted by placing acclimated 

earthworms in test chambers containing artificial (formulated) soil which has been spiked 

with the test substance. Earthworms are allowed to ingest this test mixture soil ad 

libitum. Mortality and other effects are examined on a weekly basis for 28 days. The 

results are expressed as the 28-d LC50 (median lethal concentration). Sublethal effects 

may also be determined, and expressed as EC50, (experimentally derived concentration of 

a test substance in a test matrix (e.g., growth medium, soil sediment, feed) that would be 

expected to cause a defined adverse effect in 50% of a group test organisms under 

specified exposure conditions), NOEC (no observed effect concentration), LOEC (lowest 

observed effect concentration), and MATC (maximum acceptable toxicant concentration) 

values.

(2) General test guidance. The general guidance in OCSPP 850.3000 applies to this 

guideline except as specifically noted herein. 

(3) Range-finding test. A range-finding test is usually conducted to establish the 

appropriate test concentrations to be used in the definitive test. The earthworms should 

be exposed (for at least 28 days) to a series of widely spaced concentrations of the test 

substance (e.g., 0.1, 1.0, 10, 100, 1,000 milligrams per kilogram (mg/kg) dry weight 

artificial soil). Treatment replication is not needed and nominal concentrations of the 

chemical are acceptable. The number of test organisms used and details of observations 

do not have to be the same as definitive testing. However, the range-finding test will be 

more useful the greater the similarity between the range-finding test and the definitive 

test. Results of range-finding tests should be reported along with the results of the 

definitive test, if range-finding tests are conducted.

(4) Definitive test. The goal of the definitive test is to determine the concentrationresponse curve for mortality and the LC50 (95% confidence limits and standard error) at 

28 days, as well as the slope of the concentration-response curve, its associated standard 

error and 95% confidence interval. Where sufficient data warrant, these values are also 

calculated for the 7, 14, and 21-day concentration response-curves for mortality. An 

additional desired endpoint is the weight loss EC50 (95% confidence interval) and the 

LOEC, NOEC and MATC values at each sampling time. For this determination, a 

minimum of five concentrations of the test chemical, plus appropriate controls, are tested. 

Analytical confirmation of test concentrations is performed as described in OCSPP

850.3000. A summary of test conditions is provided in Table 1 and validity elements for 

an acceptable definitive test are listed in Table 2 of this guideline.

(5) Limit test. In some situations, it is only necessary to ascertain that the 28-d LC50 is 

above a certain limit, and at the limit there is also no observable adverse growth effect. 

In a limit test, at least 20 earthworms are exposed to a single “limit concentration,” with 

the same number of organisms in appropriate control(s). The multiple-concentration 

definitive test may be waived if the following conditions are met. First, if one or fewer 

mortalities occur at the limit concentration (i.e. the LC50 > limit concentration), and 
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second, the limit treatment responses are not significantly reduced (or inhibited) as 

compared to the control response (i.e., NOEC > limit concentration). For most 

chemicals, 1,000 mg test substance (based upon 100% active ingredient for pesticides) 

per kg dry weight of artificial soil is considered appropriate as the limit concentration. 

Except for the number of treatment groups and number of test organisms, an acceptable 

limit test follows the same test procedures and is the same duration as the definitive test 

(see Table 1 of this guideline).

(e) Test standards―

(1) Test substance. The substance to be tested should be technical grade unless the test 

is designed to test a specific formulation, mixture, or end-use product. For pesticides, if 

more than one active ingredient constitutes a technical product, the technical grade of 

each active ingredient should be tested separately, in addition to the combination, if 

applicable. OCSPP 850.3000 lists the type of information that should be known about 

the test substance before testing, and discusses methods for preparation of test substances. 

(2) Test duration. The test duration is 28 days. 

(3) Test organism―

(i) Species. The test species is the earthworm Eisenia fetida andrei (Bouche). 

Some taxonomists consider these to be two separate species, Eisenia fetida 

(Savigny 1826) and Eisenia andrei (Andre 1963); see the references under 

paragraph (j)(1), (j)(5) and (j)(11) of this guideline for additional information 

about the taxonomy of the Eisenia fetida complex. These organisms naturally 

inhabit high organic environments such as compost and manure piles. They are 

easily bred in the laboratory and have been used commonly in laboratory 

experiments. The species identity of the test organism should be verified using 

appropriate taxonomic keys as described by Fender (paragraph (j)(4) of this 

guideline), or an equivalent method.

(ii) Source. 

(A) Earthworms used in toxicity tests should be purchased from a 

commercial source that can verify the species. Once verified, cultures 

should be maintained at the test facility. Information about culturing 

procedures can be found in the reference in paragraph (j)(1) of this 

guideline. 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 (culture).

(B) All newly acquired earthworms should be quarantined and observed 

for at least 14 days prior to use in a test. 

(iii) Age and size. Adult earthworms are used to start the test. The weight of the 

individual adult earthworms should be between 300 mg and 600 mg each. All 

organisms used in a test should be as uniform as possible in the state of maturity 

and weight. 
Page 4 of 17

(iv) Acclimation. It is recommended that earthworms be cultured at the test 

temperature. In any event, earthworms should be held for a minimum of 7 days in 

uncontaminated soil at the test temperature prior to testing. Any changes in soil 

temperature should not exceed 3 degrees Celsius (oC) per day or 1 oC per hour.

(v) Health status. Earthworms should not be used if they have been under stress 

from too much moisture or a lack of moisture as described by Reinecke and 

Venter (see paragraph (j)(13) of this guideline); excessive or inadequate food or 

temperature as described by Tomlin and Miller (paragraph (j)(17) of this 

guideline); pH variation as described by Satchell and Dottie (paragraph (j)(14) of 

this guideline); or crowding. Any of these conditions will produce earthworms 

that may not be healthy.

(vi) Care and handling. Adult earthworms should be handled with care. 

Sufficient numbers of earthworms should be harvested and sorted to insure that 

healthy individuals are used for the test. For a satisfactory test, any animals that 

appear to be injured are discarded and not used in the test. Additionally, any 

organisms that touch dry surfaces or are dropped or injured during handling are 

discarded.

(vii) Diet and feeding―

(A) Culture Period. Substrate food for culturing Eisenia fetida andrei

should be commercial alfalfa (Medicago sativa) pellets, saturated with 

reagent water at a ratio of approximately 1 gram (g) of dry pellets per 2 

milliliters (mL) water and aged for two weeks in a covered container. 

Worms are fed once or twice per week and the bedding turned by hand to 

inspect the general condition of the culture.

(B) Test Period. The earthworms are not fed during the test period.

(4) Administration of test substance. The test is started by introducing adult 

earthworms, which have been acclimated to the test conditions, into test chambers which 

contain artificial (formulated) soil (see paragraph (e)(7)(vii) of this guideline) to which 

the test substance has been added. Earthworms should be introduced into test soils on the 

surface of the test medium so as to evaluate burrowing behavior. The jar capped and 

secured without making an airtight seal.

(i) Preparation of treated soil. 

(A) Appropriate portions of artificial soil are mixed thoroughly with 

appropriate amounts of test substance (see paragraph (e)(4)(i)(B) of this 

guideline). Hand mixing or mechanical mixing may be used. Test 

concentrations should be prepared on day -1 and allowed to equilibrate 

overnight before adding test organisms.

(B) Test substances may be added directly to the artificial soil on a dry

weight basis, but are typically dissolved in reagent water (preferably) or a 

vehicle to form a stock solution. Aliquots of the stock solution, or 
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dilutions thereof, are then added to the artificial soil. If a solvent is used, 

the opened chambers are placed in a hood for 24 hours to evaporate the 

solvent prior to adding the earthworms.

(ii) Test concentrations. For the definitive test, a minimum of five 

concentrations of the test chemical, plus appropriate controls, are tested. Test 

concentrations should be chosen in a geometric series in which the ratio is 

between 1.5 and 2.0 (e.g., 2, 4, 8, 16, 32, and 64 mg/kg). All test concentrations 

should be based on milligram of test chemical (100% active ingredient for 

pesticides) per kilogram of artificial soil (air-dry weight).

(5) Controls. 

(i) Every test should include a negative control consisting of the same artificial 

(formulated) soil, conditions, procedures, and earthworms from the same group as 

used with the test substance, except that none of the test substance is added. In 

addition if a vehicle is used, a vehicle control is tested, using the same vehicle 

batch as used to make the stock solution and at the highest concentration used to 

prepare test concentrations. See OCSPP 850.3000 for additional information on 

preparation of test substances using a vehicle.

(ii) A test is not acceptable if average survival of either the negative or solvent 

control earthworms was less than 80% at the end of the test or the total mean 

weight of the earthworms in either the negative or solvent control containers 

declined significantly during the test (i.e., by 30%).

(6) Number of test organisms and replicates―

(i) Definitive test. In the definitive test, there is a minimum of three replicates for 

each treatment concentration and control group, with a minimum of 10 

earthworms per replicate. The earthworms are selected from the culture randomly 

into groups of 10. These groups are then randomly assigned to the test chambers 

and then weighed to verify they do not differ more than ± 10% among the 

replicates. Each test chamber contains 200 g (dry weight) of artificial soil. Each 

test chamber should contain an equal amount of test soil and an equal number of 

earthworms. For a satisfactory test, replicate test chambers are to be physically 

separated, since the test chamber is the experimental unit. In addition, test 

chambers within the testing area are positioned in a random manner or in a way in 

which appropriate statistical analyses can be used to determine the variation due 

to placement.

(ii) Limit test. In a limit test, at least 20 earthworms are exposed to a single 

“limit concentration,” with the same number of organisms in appropriate 

control(s).

(iii) Loading. The number of earthworms placed in a test chamber should not be 

so great as to affect the results of the test.
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(7) Facilities, apparatus and supplies. Normal laboratory equipment and supplies, and 

items especially listed in (e)(7)(i) through (e)(7)(vii).

(i) Facilities. 

(A) Facilities should be well ventilated and free of fumes and disturbances 

that may affect the test organisms.

(B) Ventilation hood for evaporating solvent from test soil when a solvent 

is used.

(ii) Instruments and general equipment. 

(A) Suitable balances to measure soil mixtures.

(B) A properly calibrated balance with sufficient accuracy should be used 

to weigh the worms.

(iii) Culturing. Polyethylene containers (e.g., rectangular dish pans measuring 

32.5 by 27.5 by 12.5 centimeters) for culturing earthworms.

(iv) Environmental control equipment.

(A) Apparatus for providing continuous lighting.

(B) A mechanism (e.g., environmental chamber) for controlling and 

maintaining the artificial soil temperature and relative humidity during the 

culturing, holding, acclimation, and test periods. Relative humidity should 

be maintained above 85%. An open pan of water can be used for this 

purpose to prevent moisture loss from the containers.

(v) Test containers and exposure system. 

(A) Chambers for exposing test earthworms to the test substance. Glass 

canning jars, 1-pint capacity, or their equivalent, should be used for 

testing. The lids should be reversed (i.e., turned upside down), loosely 

capped and secured without making an airtight seal to reduce evaporation 

and permit air exchange. 

(B) Construction materials and equipment that contact test mixtures 

should not contain substances that can be leached or dissolved into 

artificial soil in quantities that can affect the test results. Construction 

materials and equipment that contact test mixtures should be chosen to 

minimize sorption of test substances. 

(vi) Cleaning. The test chambers should be cleaned before each test following 

standard laboratory procedures. Hard glass jars are preferable and should be 

heated in an ashing oven between tests. For a satisfactory test, if soft glass is used 

it should be used only once and then be discarded. Refer to OCSPP 850.3000 for 

additional information.
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(vii) Artificial soil. 

(A) For each test substance concentration and control tested, enough 

artificial soil is prepared by recipe to yield 270 g of artificial soil (wet 

weight) per replicate. The constituents identified in this paragraph are 

mixed together on a dry weight basis: 10% Canadian sphagnum peat moss 

(that portion passing through a 2.36 millimeter (mm) screen); 20% kaolin 

clay (97% kaolinite with a particle size under 40 micrometers (μm)); and 

70% silica sand (grade 70, 97.1% particle size of 0.053 to 0.3 mm).

(B) After these materials are mixed together, an amount of calcium 

carbonate (99% purity) is added to the mixture to adjust the soil pH to 6.5 

± 0.5. The amount of calcium carbonate used will depend on the pH of 

the peat moss used.

(C) An appropriate amount of reagent water (e.g., 70 g per 200 g of dry

soil) is added to the artificial soil and mixed with the artificial soil to raise 

the artificial soil moisture level to 35% by weight.

(8) Environmental conditions. Environmental parameters during the test should be 

maintained as follows:

(i) Temperature. The test soil temperature should be 22 oC and it should be 

constant within ± 2 oC during the test, as described by Edwards (paragraph (j)(3) 

of this guideline), or using an equivalent method.

(ii) Lighting and photoperiod. Replicates should be illuminated continuously 

with incandescent or fluorescent lights as described by Edwards (paragraph (j)(3) 

of this guideline), or using an equivalent method. Light intensity should be about 

5.7 micromoles per square meter per second (μmol/m2/s) (for cool-white lights, 

this is equivalent to approximately 420 lux) measured at the artificial soil surface.

(9) Observations. Earthworms in the test chambers are observed periodically during the 

test; the dead worms are removed and the findings recorded. Soil temperature and pH, 

and the concentration of test substance in the treated soil are measured at specified 

intervals. Data collected during the test are used to develop concentration-response 

curves and LC50 values for the test substance.

(i) Measurement of test substance. Analytical confirmation of test 

concentrations in artificial soil is to be performed using validated analytical 

methods, as described in OCSPP 850.3000. A 10-g sample should be removed 

from each replicate prior to addition of earthworms to measure pH and test 

substance concentrations. The homogeneity of the test substance in treated soil 

should be determined.

(A) For stable test substances, monitor test concentrations at the beginning 

and at the end of the test. However, for volatile, unstable substances, 

those subject to ready degradation, or where there is uncertainty in the 
Page 8 of 17

analytical measurements, it may be necessary to measure test 

concentrations more often during the test, e.g. every seven days.

(B) In addition to analyzing samples of artificial soil, at least one reagent 

blank, containing all reagents used, should also be analyzed.

(C) The measured concentration of the test substance in artificial soil in 

any chamber during the test should not vary more than 50% from the 

measured concentration prior to initiation of the test; concentration 

measurements should be as described by Neuhauser et al. (paragraphs 

(j)(9) and (j)(10) of this guideline), or an equivalent method.

(D) The mean measured concentration of test substance in artificial soil 

(dry weight) should be used to plot all concentration-response curves and 

to calculate all LC50, EC50, LOEC, and NOEC values.

(ii) Environmental conditions―

(A) Temperature. Temperature should be measured and reported at the 

beginning of the test and on days 7, 14, 21, and 28 in all test chambers. 

The temperature should be measured at least hourly in one test container.

(B) Light intensity. Light intensity should be measured at least once 

during the test at the surface of the container and checked weekly in the 

test chambers.

(C) Total organic carbon (TOC) concentration. The TOC 

concentration in soil at test initiation should be determined as measured by 

the method of Plumb (paragraph (j)(12) of this guideline), or an equivalent 

method.

(D) Soil pH. The pH should be measured at test initiation and 7 day 

intervals (days 7, 14, 21, 28) thereafter in each test chamber.

(iii) Organisms―

(A) Mortality, behavior, and other adverse effects. At test initiation

burrowing behavior of earthworms placed on the surface of the test 

medium is recorded for each test chamber. Each test and control chamber 

should be checked for dead or affected earthworms and observations 

recorded 7, 14, 21, and 28 days after the beginning of the test or within 1 

hour of the designated times in all test chambers. Missing earthworms 

should be considered to have died. Mortality is assessed by emptying the 

test medium on a glass or other inert surface, sorting earthworms from the 

test mixture and testing their reaction to a gentle mechanical stimulus. 

Any adverse effects (e.g., behavioral or pathological symptoms) are noted 

and should be reported. The medium is returned to each container.
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(B) Weight loss. Total biomass of test organisms per test chamber is 

measured at the beginning and end of the test. The worms should be 

washed and purged before weighing. To accomplish this, the bedding is 

rinsed from the worms with reagent water; the worms are placed in petri 

dishes with wet filter paper, and allowed to purge the gut contents for 24 

hours. Before weighing worms, excess surface water may be removed by

placing the worms between layers of an absorbent towel. However, it is 

very important not to dry the surface of the worms which might stress the 

worms unduly. 

(f) Treatment of results—

(1) Descriptive summary statistics—

(i) Environmental conditions. The soil characteristics and TOC content should 

be described. Temperature and light intensity results should be summarized in 

tabular form, showing the range, mean and standard deviation during the test. 

The pH in each test chamber should be summarized in tabular form, showing the 

range, mean, and standard deviation.

(ii) Test substance soil concentration. Soil concentrations should be 

summarized in tabular form by treatment and sample time, showing mean 

concentrations and standard deviations. The treatment mean soil concentration 

should be calculated.

(iii) Mortality. Death is the primary effect used in this test guideline to evaluate 

the toxicity of the test substance. The number of initial earthworms at each 

treatment and control and the number of dead earthworms should be summarized 

in tabular form by observation time (e.g. day 7, 14, 21, and 28) treatment, and 

replicate.

(iv) Sublethal effects and growth. The sublethal effects and growth (i.e., fresh 

weight) data should be used to determine concentration-response curves, calculate 

EC50 values, and determine LOEC, NOEC, and MATC values. Number of 

earthworms with abnormal appearance or behavior symptoms should be 

summarized in tabular form by symptom, time of observation, treatment, and 

replicate. Mean total biomass at the beginning and end of the test for each 

treatment replicate should be summarized in tabular form and the mean and 

standard deviation in weight loss or gain calculated by treatment.

(2) Percent—

(i) Mortality. Calculate the percent mortality at each treatment level and control 

by observation time.

(ii) Appearance and behavior. Calculate the percent effected at each treatment 

level and control by symptom, and observation time.
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(3) Limit test—

(i) LC50 value. At test termination (28 days), if one or fewer earthworms are 

dead at the limit concentration, the LC50 is considered to be greater than the limit 

concentration. This is because the Binomial Theorem predicts that when 20 

earthworms are tested, the probability of seeing <1 dead earthworm if the true 28-

d LC50 is at or below the limit concentration is <0.001. Conversely the 

probability of seeing 2 or more dead earthworms if the true 28-d LC50 is at or 

below the limit dose is >0.999. Therefore, if <1 mortality occurs among the 20 

earthworms tested, the 28-d LC50 is reported as greater than the limit 

concentration (i.e., 28-d LC50 >1,000 mg/kg dry wt in soil).

(ii) Proportion of mortality ( pˆ ). The Binomial Theorem also can provide both 

an estimate of the true proportion of mortality ( pˆ ) in the population as well as 

confidence bounds on that estimate (see Table A4 of the reference in paragraph 

(j)(2)). For small sample sizes the interval may be large. For no mortalities in 20 

earthworms ( pˆ = 0), the upper 99% confidence bound on the estimate of pˆ is 

0.24 (95% upper confidence bound is 0.17), this means that the estimate of the 

true proportion of mortality may actually be as high as 0.24 when no earthworms 

die out of 20 tested (or as high as 0.17 for the 95% confidence estimate). For one 

mortality in 20 earthworms ( pˆ = 0.05), the upper 99% confidence bound on the 

estimate of pˆ is 0.32 (95% upper confidence bound is 0.29). For assessing risks, 

the confidence in the proportion impacted is considered in determining acute 

effects at environmental exposure doses.

(iii) NOEC. To ascertain that there is no observable adverse growth effect at the 

limit treatment (i.e., NOEC > limit dose), the limit treatment response is 

compared to the control treatment response using a one-sided two-sample 

parametric or nonparametric test for a reduction in growth from the control (see 

OCSPP 850.3000). The minimum significant difference detectable by the test or 

a similar estimate of the sensitivity of the test should be determined and reported.

(iv) Multiple-concentration definitive testing. 

(A) At test termination, if two or more mortalities occur among the 20 

earthworms tested or growth was significantly reduced from the control, a 

definitive dose-response test should be conducted.

(B) Multiple-concentration definitive testing may be waived if at test 

termination (28 days), one or fewer earthworms are dead at the limit 

concentration, and at the limit treatment there is also no observable 

adverse growth effect.

(4) Multiple-concentration definitive test―

(i) Concentration-response curve, slope and LC50. Statistical procedures are 

employed to calculate the 28-d LC50 (standard error and 95% confidence interval) 

based upon mortality. If a concentration-response model (e.g., probit) was fit to 
Page 11 of 17

the data to determine the LC50, the model parameters (e.g., slope) and their 

uncertainty estimates (e.g., standard error) should be recorded.

(ii) Concentration-response curve, slope and EC50. Statistical procedures are 

employed to calculate the 28-d EC50 (standard error and 95% confidence interval) 

based upon biomass weight loss. If a concentration-response model was fit to the 

data to determine the EC50, the model parameters (e.g., slope) and their 

uncertainty estimates (e.g., standard error) should be recorded.

(iii) NOEL. Appropriate statistical methods (e.g., one-way analysis of variance 

and multiple comparison tests) should be used to test for significant differences 

between treatment means and determine LOEC, NOEC and MATC for mortality, 

biomass weight loss, and other sublethal effects.

(iv) Statistical methods. Statistical procedures for modeling quantal and 

continuous data are available and should be used. Additional discussion about 

measurement endpoints and statistical procedures is found in OCSPP 850.3000.

(g) Tabular summary of test conditions. Table 1 lists the important conditions that should

prevail during the definitive test. Except for the number of test organisms per concentration, 

number of replicate chambers, and test concentrations, Table 1 also lists the important conditions 

that should prevail during a limit test. Meeting these test conditions will greatly increase the 

likelihood that the completed test will be acceptable or valid.
Page 12 of 17

Table 1.—Summary of Test Conditions for Earthworm Subchronic Toxicity Test

Test type Spiked formulated soil

Test duration

28 days

Temperature 22 °C (constant during test within ± 2 °C)

Light quality Incandescent or fluorescent lights

Light intensity 5.7 μmol/m2/s (approximately 420 lux)

Photoperiod Continuous light

Test chamber size Glass canning jars, 1 pint, or equivalent 

Substrate Artificial (formulated) soil

Substrate amount per replicate 270 g wet weight (200 g dry weight)

Age/size of test organisms per replicate Adult earthworms, 300 - 600 mg

Number of test organisms per concentration 30 (minimum)

Number of organisms per test chamber 10

Number of replicate chambers per concentration 3 (minimum)

Loading 300 - 600 mg per 270 g of soil

Feeding regime No feeding during test

Test concentrations Minimum of 5 test concentrations chosen in a 

geometric series plus a control, and a vehicle

control if a vehicle is used

Measures of Effect or Measurement Endpoints LC50 based upon mortality at 28 days at a minimum; 

EC50, NOEC, LOEC and MATC based upon 

sublethal effects (weight change)

(h) Test validity elements. This test would be considered to be unacceptable or invalid if one or 

more of the conditions in Table 2 occurred. This list should not be misconstrued as limiting the 

reason(s) that a test could be found unacceptable or invalid. However, except for the conditions 

listed in Table 2 and in OCSPP 850.3000, it is unlikely a study will be rejected when there are 

slight variations from guideline environmental conditions and study design unless the control 

organisms are significantly affected, the precision of the test is reduced, the power of a test to 

detect differences is reduced, and/or significant biases are introduced in defining the magnitude 

of effect on measurement endpoints as compared to guideline conditions. Before departing 

significantly from this guideline, the investigator should contact the Agency to discuss the reason 

for the departure and the effect the change(s) will have on test acceptability. In the test report, all 

departures from the guideline should be identified, reasons for these changes given, and any 

resulting effects on test endpoints noted and discussed.
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Table 2.—Test Validity Elements for Earthworm Subchronic Toxicity Test

1. All test chambers were not identical.

2. Treatments were not randomly assigned to individual test chamber locations or individual test 

organisms were not impartially or randomly assigned to test chambers.

3. The negative and vehicle control [when a vehicle was used] were not included in the test.

4. Average survival of either the negative or solvent control earthworms was less than 80% at the end of 

the test.

5. The total mean weight of the earthworms in the control containers declined significantly during the test 

(i.e., by 30%).

(i) Reporting―

(1) Background information. Background information to be supplied in the report 

consists at a minimum of those background information items listed in paragraph (j)(1) of 

OCSPP 850.3000.

(2) Guideline deviations. Provide a statement of the guideline or protocol followed. 

Include a description of any deviations from the test guideline or any occurrences which 

may have influenced the results of the test.

(3) Test substance. 

(i) Identification of the test substance: common name, IUPAC and CAS names, 

CAS number, structural formula, source, lot or batch number, chemical state or 

form of the test substance, and its purity (i.e. for pesticides, the identity and 

concentration of active ingredient(s)). If radiolabeled substance was used provide 

the radio purity and location(s) of the label.

(ii) Storage conditions of the test chemical or test substance and stability of the 

test chemical or test substance under storage conditions if stored prior to use.

(iii) Methods used for preparation of artificial soil, its age (i.e., prepared 1 day or 

7 days prior to use), and storage conditions if stored prior to use. If artificial soil 

is not used describe the source, collection method, handling and preparation of 

soil used in the test, its composition (percent sand, silt, and clay), and textural 

classification.

(iv) Source and amount of hydration water added to test soil and the moisture 

content for the test mixture at test initiation.

(v) Methods of preparation of the test substance and the treatment concentrations 

used in the range-finding and definitive test, or limit test: description of test 

chemical introduction into the test medium (e.g., as a powder, stock solution), the 

mass and volume of soil and test substance used for each treatment, how test 
Page 14 of 17

substance was mixed into the soil, number of treatments and nominal test 

substance concentration in soil in each treatment at the beginning of the test. 

(vi) If a vehicle (e.g., solvent) is used to prepare stock or test substance provide: 

the name and source of the vehicle, the nominal concentration(s) of the test 

substance in the vehicle, in stock solutions, or mixtures, and the vehicle

concentrations used in the treatments.

(vii) Description of stock solution preparation.

(4) Test organisms. 

(i) Scientific and common names and method of verification, and source.

(ii) Culture method and conditions, and feeding history.

(iii) Health status of culture used for collection of test earthworms (e.g., any 

prophylactic or preventative treatments, quarantine period).

(iv) Collection method and date of collection.

(v) Acclimation period.

(vi) Age and weight (by treatment and replicate) in tabular form at test initiation.

(5) Test system and conditions. Description of the test system and conditions used in 

the definitive or limit test, any preliminary range-finding tests, and any positive control 

tests.

(i) Description of the test chambers: type, size, volume, material, and conditioning 

method.

(ii) Statement verifying that earthworms were not fed during the test.

(iii) Volume and weight of soil used in each test container.

(iv) Time between introduction and mixing of the test substance with the soil and 

introduction of the earthworms.

(v) Number of worms per test container.

(vi) Number of test containers per concentration.

(vii) Methods used for test container and treatment randomization as well as 

methods for impartial assignment of earthworms to test containers.

(viii) Method of introducing earthworms to test containers.

(ix) Test duration.
Page 15 of 17

(x) Methods and frequency of environmental monitoring performed during the 

definitive or limit study for lighting, soil temperature and soil pH.

(xi) Method, type and frequency of observations made on earthworms during the 

test.

(xii) For the definitive or limit test, all analytical procedures and preservation 

methods should be described. The accuracy of the method, method detection 

limit, and limit of quantification should be given.

(6) Results. 

(i) Environmental monitoring data results (lighting, soil temperature and pH) in 

tabular form (provide raw data for measurements not made on a continuous 

basis), and descriptive statistics (mean, standard deviation, minimum, maximum).

(ii) For preliminary range-finding test(s), if conducted, the number of dead and 

intoxicated earthworms at each concentration level and in the control(s).

(iii) For the definitive and limit test, test substance concentration in the soil at test 

initiation (day 0), during (if needed, at day 7, 14, 21) and test conclusion (day 28), 

in tabular form and descriptive statistics (time weighted average, standard 

deviation, minimum, maximum).

(iv) The total organic carbon (TOC) content of the soil mixture.

(v) For the definitive and limit test, the number of dead earthworms at each 

observation period at each treatment level and control(s) (provide the raw data).

(vi) For the definitive and limit test, the burrowing behavior of earthworms 

observed at test initiation: description of behavior, duration, severity, and number 

affected at each treatment level and control(s) (provide the raw data).

(vii) For the definitive and limit test, a description of all observations made on the 

test earthworms that are suggestive or predictive of toxicity and all concomitant 

gross toxicological manifestations include time of onset, duration, severity, and 

number affected at each treatment level and control(s) (provide the raw data).

(viii) For the definitive and limit test, the weight, in tabular form, at each test 

concentration (including controls), in each test chamber at test initiation and 

termination.

(ix) For the definitive test, the LC50 value, and its standard error and 95% 

confidence interval should be reported for 7, 14, 21, and 28-day periods. The 

slope of the concentration-response curve, its standard error, and 95% confidence 

interval should also be reported.

(x) For the definitive test, the EC50 for weight with its standard error and 95% 

confidence interval, and the LOEC, NOEC, and MATC for day 28.
Page 16 of 17

(xi) Description of statistical method used, including software package, for 

determining LC50 EC50, LOEC, NOEC, and MATC values and the concentrationresponse model parameters and the basis for the choice of method. Provide 

results of any goodness-of-fit tests and determination of minimum significant 

differences.

(j) References. For additional background information on this test guideline, the references in 

this paragraph should be consulted:

(1) American Society for Testing and Materials. ASTM E 1676-04, Standard guide for 

conducting laboratory soil toxicity or bioaccumulation tests with the lumbricid 

earthworm Eisenia fetida and the enchytraeid potworm Enchytraeus albidus. Current 

edition approved April 1, 2004. In Annual Book of ASTM Standards, Vol. 11.06, West 

Conshohocken, PA. 

(2) Conover, W. 1980. Practical Nonparametric Statistics, 2

nd Edition. John Wiley &

Sons, Inc., New York, NY. 493 pp.

(3) Edwards, C.A., 1984. Report of the second stage in development of a standardized 

laboratory method for assessing the toxicity of chemical substances to earthworms. The 

Artificial Soil Test. DG X1/AL/82/43, Revision 4.

(4) Fender, W.M., 1985. Earthworms of the Western United States. Part 1. Lumbricidae, 

Megadrilogica 4: 93-129.

(5) Jaenicke, J., 1982. “Eisenia foetida” is two biological species. Megadrilogica 4:6-8.

(6) Greene, J.C., Bartels, C.L., Warren-Hicks, W.J., Parkhurst, B.R., Linder, G.L., 

Peterson, S.A., and Miller, W.E., 1989. Protocols for Short-Term Toxicity Screening of 

Hazardous Waste Sites, EPA/600/3-88/029, U.S. Environmental Protection Agency, 

Environmental Research Laboratory, Corvallis, OR. 

(7) Hartenstein, R. et al., 1979. Reproductive potential of the earthworm Eisenia foetida. 

Oecologia 43:329-340. 

(8) Meier, J.R., Chang, L.W., Jacobs, S., Torsella, J., Meckes, M.C. and Smith, M.K., 

1997. Use of plant and earthworm bioassays to evaluate remediation of soil from a site 

contaminated with polychlorinated biphenyls. Environmental Toxicology and Chemistry

16(5): 928-938. 

(9) Neuhauser, E.F. et al., 1986. Contact and artificial soil tests using earthworms to 

evaluate the impact of wastes in soils, In: Hazardous and Industrial Soil Waste Testing: 

Fourth Symposium, ASTM STP 886. J.K. Petros, Jr. and R.A. Conway, eds., American 

Society for Testing and Materials. Philadelphia, PA pp. 192-203.

(10) Neuhauser, E.F. et al., 1985. The toxicity of selected organic chemicals to the 

earthworm Eisenia fetida. Journal of Environmental Quality 14: 383-388.
Page 17 of 17

(11) Oien, N. and J. Stenerson, 1984. Esterases of earthworm - III. Electrophoresis 

reveals that Eisenia foetida (Savigny) is two species. Comparative Biochemistry and 

Physiology 78c (2):277-282. 

(12) Plumb, R.H., Jr., 1981. Procedures for handling and chemical analysis of sediment 

and water samples. Technical Report EPA/CE-81-1, prepared by Great Lakes 

Laboratory, State University College at Buffalo, Buffalo, NY., for the U.S. 

Environmental Protection Agency/Corps of Engineers Technical Committee on Criteria 

for Dredged and Fill Material. U.S. Army Engineer Waterways Experiment Station, CE, 

Vicksburg, MS.

(13) Reinecke, A.J. and Venter, J.M., 1987. Moisture preferences, growth and 

reproduction of the compost worm Eisenia fetida (Oligochaeta). Biology and Fertility of 

Soils 3: 135-141.

(14) Satchell, J.E. and Dottie, D.J., 1984. Factors affecting the longevity of earthworms 

stored in peat. Journal of Applied Ecology 21: 285-291.

(15) Stafford, E.A. and Edwards, C.A., 1985. Comparison of heavy metal uptake by

Eisenia faetida with that of other common earthworms. Final Technical Report. 

Entomology Department, Rothamsted Experiment Station, Harpenden, Herts. ALS 2JQ, 

U.K. U.S. Army Contract DAJA 45-84-0027. 

(16) Spurgeon, D.J. and Hopkin, S.P., 1995. Extrapolation of the laboratory-based OECD 

earthworm toxicity test to metal-contaminated field sites. Ecotoxicology 4:190-205. 

(17) Tomlin, A.D. and Miller, J.J., 1980. Development and fecundity of the manure 

worm, Eisenia faetida (Annelida:Lumbricidae), under laboratory conditions. In: D.L. 

Dindal (ed.), Soil Biology as Related to Land Use Practices. Proc. 7th Internat. Soil Zool. 

Coll. of ISSS. EPA, Washington, DC., pp. 673-678.

(18) vanGestel, C.A.M., and van Dis, W.A., 1988. The influence of soil characteristics on 

the earthworm Eisenia fetida andrei (Oligochaeta). Biology and Fertility of Soils 6:262-

265.