OECD 207 Earthworm Acute Toxicity Test: Principle, Methods, and Applications

The OECD 207 Earthworm Acute Toxicity Test is an internationally recognized laboratory method for evaluating the short-term harmful effects of chemicals on soil-dwelling organisms. This guide explains the test principle, sample types, test items, procedural methods, and regulatory applications of OECD TG 207. It is essential reading for manufacturers, regulatory affairs professionals, and environmental laboratories involved in chemical and pesticide registration.

Technical Overview

OECD Test Guideline 207, titled "Earthworm, Acute Toxicity Tests," was adopted by the Organisation for Economic Co-operation and Development in April 1984. It remains one of the foundational methods in terrestrial ecotoxicology for assessing the acute toxicity of chemical substances to earthworms, which are widely regarded as representative soil macroorganisms and key indicators of soil health.

The guideline describes two complementary procedures:

• A filter paper contact toxicity test, used as a rapid screening or range-finding method in which earthworms are exposed to the test substance on moistened filter paper.
• An artificial soil test, which serves as the definitive method in which earthworms are exposed to the test substance mixed into a standardized, defined artificial soil substrate.

The standard test species is Eisenia fetida (or the closely related Eisenia andrei), commonly known as the redworm or compost worm. This species is easily cultured in the laboratory, has well-documented sensitivity to a broad range of chemicals, and its biology and husbandry are thoroughly established.

The primary endpoint of the artificial soil test is mortality after 7 and 14 days of exposure, from which the median lethal concentration (LC50) and the no observed effect concentration (NOEC) are derived. The test is closely harmonized with international standard ISO 11268-1, and its chronic counterpart is OECD Test Guideline 222 (Earthworm Reproduction Test).

Testing Samples

OECD 207 can be applied to a wide variety of test materials encountered in regulatory and research contexts:

Industrial and Commercial Chemicals

New and existing chemical substances submitted for notification or registration under programs such as REACH typically require acute earthworm toxicity data to characterize hazards to the soil compartment.

Plant Protection Products

Active substances, formulated pesticide products, and metabolites or degradation products are tested to support environmental risk assessment for non-target soil organisms.

Biocides

Biocidal products intended for outdoor use or with potential soil exposure pathways are evaluated using earthworm acute toxicity data under biocidal product regulations.

Contaminated Soils and Environmental Samples

Soils from potentially contaminated sites, as well as waste-derived materials such as composts, sewage sludge, and dredged sediments, can be screened for ecotoxicological hazard.

Novel and Special Materials

Nanomaterials, fertilizers, soil amendments, and other emerging substances may be assessed, with appropriate adjustments to exposure preparation and characterization.

Testing Items

The core parameters measured and reported in an OECD 207 study include:

• Acute mortality of earthworms at each test concentration after 7 and 14 days of exposure.
• LC50 values (median lethal concentration, expressed in mg test substance per kg dry artificial soil) with 95% confidence intervals, where obtainable.
• NOEC (no observed effect concentration) and LOEC (lowest observed effect concentration) for mortality.
• Observations of sublethal effects and abnormal behavior, such as lesions, swelling, discoloration, stiffening, thinning, excessive mucus production, lethargy, or failure to burrow.
• Changes in body weight or biomass where recorded as supporting observations.
• Physicochemical characterization of the artificial soil, including pH, moisture content, and organic carbon content.
• Verification of test substance concentration and stability in the substrate, where analytically feasible.

Testing Methods

Test Organism and Husbandry

Adult Eisenia fetida or Eisenia andrei worms with a well-developed clitellum, aged at least two months and typically weighing between 300 and 600 mg each, are used. Worms are obtained from synchronized cultures and acclimatized in artificial soil under test conditions for at least 24 hours prior to exposure. Only healthy, undamaged worms are selected.

Filter Paper Contact Test

This simplified screening test is performed in flat-bottom glass vials lined with filter paper. A defined volume of test solution in a volatile solvent is applied to the paper, the solvent is evaporated, and the paper is re-moistened with water. One worm is placed in each vial, with at least 10 worms per concentration, and the vials are incubated in darkness at 20 ± 2°C for 48 hours, followed by a 24-hour observation period. Mortality and clinical signs are recorded, and the results are used to select concentration ranges for the definitive artificial soil test.

Artificial Soil Test

The definitive test uses a standardized artificial soil prepared from the following dry-weight constituents:

• 70% quartz industrial sand (dominant particle size 50 to 200 micrometers).
• 20% kaolinite clay (content of kaolinite preferably above 30%).
• 10% sphagnum peat, finely ground and air-dried.

The pH of the mixture is adjusted to 6.0 ± 0.5 with finely ground calcium carbonate, and deionized water is added to achieve a moisture content of approximately 35% of the dry substrate weight, corresponding to roughly 50 to 60% of the water holding capacity.

The test substance is mixed homogeneously into the artificial soil, typically via treatment of quartz sand or dissolution in a carrier. Test vessels each contain approximately 500 to 1000 grams of treated substrate, into which ten worms are introduced. A geometric series of at least five concentrations, spaced at a factor not exceeding 2.2, is tested alongside a negative control and, if needed, a solvent control. At least three, and preferably four, replicates per treatment are employed. Worms are not fed during the 14-day exposure, and vessels are held at 20 ± 2°C under continuous illumination of approximately 400 to 800 lux to keep the worms in the substrate.

Observations and Endpoint Determination

Mortality and clinical observations are recorded after 7 days and again after 14 days. At each assessment, the soil is emptied onto a tray and hand-sorted to recover live worms; animals not recovered are counted as dead, since dead earthworms decompose rapidly in soil. A worm is considered dead when it does not respond to gentle mechanical stimulation.

Validity Criteria

The study is considered valid only if mortality in the control group does not exceed 10% at the end of the test.

Data Analysis and Reporting

LC50 values at 7 and 14 days are calculated using appropriate statistical methods such as probit analysis, logit analysis, or graphical interpolation, each with 95% confidence limits where data permit. NOEC and LOEC are determined using statistical hypothesis testing, such as Fisher exact tests with appropriate corrections. The final report documents test conditions, organism source and condition, soil characterization, analytical verification, concentration-response data, calculated endpoints, and all observed abnormalities.

Applications

Data generated under OECD 207 are used across numerous regulatory and scientific contexts:

• Chemical registration and notification under REACH and comparable national chemical inventories, where acute toxicity to soil macroorganisms addresses the terrestrial compartment.
• Environmental risk assessment of agricultural pesticides and their transformation products for non-target soil invertebrates, including the derivation of toxicity-to-exposure ratios and trigger values.
• Support for biocidal product authorization where soil exposure is anticipated.
• Hazard classification and labeling of substances with respect to hazards to the soil environment.
• Screening of contaminated land, biowastes, composts, and soil amendments for ecotoxicological quality.
• Research applications, including comparative toxicity studies, mixture toxicity evaluation, and site-specific ecological risk assessment.
• As a range-finding and methodological foundation for higher-tier or chronic studies conducted under OECD 222.

Because OECD 207 is harmonized with ISO 11268-1 and embedded in the mutual acceptance of data framework among OECD member countries, results generated in a GLP-compliant laboratory are accepted by regulatory authorities worldwide without the need for repeat testing.

FAQ

What is the difference between OECD 207 and OECD 222?

OECD 207 is an acute test with a 14-day exposure that measures mortality and derives an LC50. OECD 222 is a chronic reproduction test lasting eight weeks that measures reproductive output in addition to mortality, providing a more sensitive long-term endpoint. Many regulatory programs request both studies, with OECD 222 often required at higher tiers.

Why is Eisenia fetida used as the test species?

Eisenia fetida is easy to culture in large numbers year-round, has a well-documented sensitivity profile across many chemical classes, exhibits relatively uniform responses, and its laboratory husbandry is standardized. These attributes make it a practical and reproducible surrogate for soil-dwelling oligochaetes.

What is the test duration and key assessment time point?

The definitive artificial soil test runs for 14 days, with mortality assessments performed at day 7 and day 14. The 14-day LC50 is the principal reported endpoint.

Is the filter paper test sufficient for regulatory submission?

No. The filter paper contact test is intended for screening and range-finding only. Regulatory authorities require the artificial soil test, because it provides a realistic soil matrix exposure and is the formally accepted definitive method under the guideline.

What quality criteria must a valid study meet?

The principal validity criterion is control mortality no greater than 10% at test termination. In addition, the reporting must confirm compliance with the prescribed soil composition, pH, moisture, temperature, and replication requirements, and GLP compliance is expected for regulatory use.

How are poorly soluble or volatile substances handled?

Poorly water-soluble substances are mixed into the substrate with quartz sand or a minimal amount of suitable carrier, while volatile substances may require sealed or minimized-headspace vessels with brief ventilation. Justification of the dosing approach must be documented in the study report.

What sample information should a sponsor provide before testing?

The sponsor should supply the test substance with a certificate of analysis, water solubility, vapor pressure, stability data, purity, and any available physical-chemical information. A typical screening program requires approximately 50 to 200 grams of test item depending on the concentration range and analytical plan.