5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, lumbricid earthworms and enchytraeid potworms have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Earthworms may ingest large quantities of soil, have a close relationship with other soil biomasses (for example, invertebrates, roots, humus, litter, and microorganisms), constitute up to 92 % of the invertebrate biomass of soil, and are important in recycling nutrients (1, 2).⁴ Enchytraeids contribute up to 5.2 % of soil respiration, constitute the second-highest biomass in many soils (the highest in acid soils in which earthworms are lacking) and effect considerably nutrient cycling and community metabolism (3-5). Earthworms and potworms accumulate and are affected by a variety of organic and inorganic compounds (2-10, 11-14). In addition, earthworms and potworms are important in terrestrial food webs, constituting a food source for a very wide variety of organisms, including birds, mammals, reptiles, amphibians, fish, insects, nematodes, and centipedes (15, 16, 3). A major change in the abundance of soil invertebrates such as lumbricids or enchytraeids, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.

5.2 A number of species of lumbricids and enchytraeid worms have been used in field and laboratory investigations in the United States and Europe. Although the sensitivity of various lumbricid species to specific chemicals may vary, from their study of four species of earthworms (including E. fetida) exposed to ten organic compounds representing six classes of chemicals, Neuhauser, et al (7) suggest that the selection of earthworm test species does not affect the assessment of a chemical's toxicity markedly. The sensitivity of various enchytraeid species has not been investigated in a comparable way so far, but ecological importance and practicability reasons favor strongly the selection of a species belonging to the genus Enchytraeus.

5.2.1 E. fetida is a species whose natural habitats are those of very high organic matter such as composts and manure piles. It was selected as the test species because it (1) is bred in the laboratory easily; ( 2) is the earthworm species used most commonly in laboratory experiments (17); (3) has been studied extensively, producing a data pool on the toxicity and bioaccumulation of a variety of compounds (2, 7, 8, 18-23); (4) has been approved for use in toxicity testing by the European Union (EU) and the Organization for Economic Cooperation and Development (OECD); and (5) has been used by the Environmental Protection Agency (EPA) for the toxicity screening of hazardous waste sites (24).

5.2.2 The recommended enchytraeid test species is Enchytraeus albidus Henle 1837 (white potworm). E. albidus is one of the biggest (up to 15 mm) species of the oligochaete family Enchytraeidae and it is distributed world-wide (25, 26). E. albidus is found in marine, limnic, and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows (4, 26). This broad ecological tolerance and some morphological variations might indicate that there are different races for this species. E. albidus is commercially available, sold as food for fish, can be bred easily in a wide range of organic waste materials and has a short life cycle (33 to 74 days; 27, 28). E. albidus was studied in various tests, which covered a wide range of compounds (28-30). In addition, it is currently under investigation for use in toxicity testing and soil quality assessment by the European Union (EU), the Organization for Economic Cooperation and Development (OECD), and the International Organization for Standardization (ISO). Other species of the genus Enchytraeus are also suitable, for example, E. buchholzi Vejdovsky 1879 or E. crypticus Westheide and Graefe 1992 (see Annex A4). Those species are true soil inhabitants and are smaller in size. Other species of Enchytraeus may be used, but they should be identified clearly and the rationale for their selection should be reported.

5.3 Results from soil toxicity tests might be an important consideration when assessing the hazards of materials to terrestrial organisms.

5.4 Information might also be obtained on the bioaccumulation of chemicals associated with soil by analysis of animal tissues for the chemicals being monitored. These results are useful for studying the biological availability of chemicals.

5.5 The soil toxicity test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity.

5.6 Results of soil toxicity tests could be used to compare the sensitivities of different species.

5.7 An understanding of the effect of these parameters on toxicity and bioaccumulation may be gained by varying soil characteristics such as pH, clay content, and organic material.

5.8 Results of soil toxicity tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations.

5.8.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites.

5.8.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation can be improved and costs reduced if subsamples of soil for laboratory toxicity tests, geochemical analyses, and community structure are taken simultaneously from the same grab of the same site.

5.9 Soil toxicity and bioaccumulation tests can be an important tool for making decisions regarding the extent of remedial action necessary for contaminated terrestrial sites.

1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of contaminants (for example, chemicals or biomolecules) associated with soil to earthworms (Family Lumbricidae) and potworms (Family Enchytraeidae) from soil toxicity or bioaccumulation tests. The methods are designed to assess lethal or sublethal toxic effects on earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-term tests (14 to 42 days) in terrestrial systems. Soils to be tested may be (1) reference soils or potentially toxic site soils; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Eisenia fetida (see Annex A1) and for the species Enchytraeus albidus (see Annex A4). Methods described in this guide may also be useful for conducting soil toxicity tests with other lumbricid and enchytraeid terrestrial species, although modifications may be necessary.

1.2 Modification of these procedures might be justified by special needs. The results of tests conducted using atypical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity and bioaccumulation tests with terrestrial worms.

1.3 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints (see Section 14). These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge and oils. These methods might also be useful for conducting bioaccumulation tests.

1.4 The results of toxicity tests with (1) materials (for example, chemicals or waste mixtures) added experimentally to artificial soil, reference soils, or site soils, (2) site soils diluted with reference soils, and (3) site or reference soils diluted with artificial soil, so as to create a series of concentrations, may be reported in terms of an LC50 (median lethal concentration) and sometimes an EC50 (median effect concentration). Test results may be reported in terms of NOEC (no observed effect concentration), LOEC (lowest observed effect concentration) or as an ECx (concentration where x % reduction of a biological effect occurs. Bioaccumulation test results are reported as the magnitude of contaminant concentration above either the Day 0 tissue baseline analysis or the Day 28 tissues from the negative control or reference soil (that is, 2x, 5x, 10x) (see A3.9).

1.5 This guide is arranged as follows:

Scope

 1

Referenced Documents

 2

Terminology

 3

Summary of Guide

 4

Significance and Use

 5

Interferences

 6

Apparatus

 7

Safety Precautions

 8

Soil

 9

Test Organism

10

Procedure

11

Analytical Methodology

12

Acceptability of Test

13

Calculation of Results

14

Report

15

Annexes

 Annex A1. Eisenia fetida

 Annex A2. Artificial Soil Composition

 Annex A3. Bioaccumulation Testing Utilizing Eisenia fetida

 Annex A4. Enchytraeid Reporduction Test (ERT)

References

1.6 The values stated in SI units are to be regarded as the standard.

1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. While some safety considerations are included in this guide, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct soil toxicity tests. Specific precautionary statements are given in Section 8.