Environmental Science & Engineering - www.esemag.com - May 2003
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Using earthworm behaviour to assess contaminated soil

by Natalie Feisthauer,
ESG International Inc.

Placement of earthworms into an avoidance response test unit filled with contaminated and control soil.

Researchers are using earthworm behaviour as an economical method to assess contaminated soils. These new test methods will assist decision-makers in addressing clean-up options.

Working with Environment Canada, researchers at ESG International are in the final stages of the development of national toxicity test methods that will be used to assess the toxicity of contaminated soils to terrestrial plants, arthropods (springtails) and earthworms. Three different types of earthworm tests have been developed and include acute (lethal), behaviour (sublethal) and chronic (reproduction) tests.

The acute toxicity test is used to measure the acute, or short-term, effects of contaminated soils on adult earthworm survival. Earthworm survival is assessed following 7 and 14 days of exposure to the contaminated soil. This acute test is useful as a screening tool; however, it cannot detect sublethal effects of contaminated soils.

The reproduction test is a 56 to 63 day test that is used to measure the effects of chronic or long-term exposure to contaminated soil on the reproductive ability of earthworms. At the beginning of a test, two reproductively mature earthworms are placed in a test unit (glass 500 mL wide-mouthed mason jar) that contains between 350 to 400 mL of test soil. After 28 or 35 days, the adults are removed from the test units, and their survival rate measured. At the end of the test, the number of juveniles produced, juvenile wet and dry mass, and the number of hatched and unhatched cocoons are measured.

The behaviour test is used to measure the avoidance-response of earthworms to potentially contaminated soils. It is a short test, with a 72-hour duration, and has been shown to be predictive (i.e., as sensitive) of the results of the much longer-term (56 to 63 day), reproduction tests. The behaviour test method has been evaluated with two different earthworm species, five different soil types, and with organic (petroleum hydrocarbons, pesticides) and inorganic (heavy metals) contaminants. The soils tested were either uncontaminated field-collected soils amended with a toxicant, or were site-specific contaminated soils collected from the field.

The behaviour test unit has a unique circular design, and consists of six equally-sized, pie-shaped compartments that surround a central hollow chamber (Figure 1). Each compartment is connected to the central chamber and adjacent compartments by two or three holes at the bottom of the compartment walls. As a result, earthworms can move freely among all compartments.

At the beginning of a test, 350 mL of soil is added to each compartment. Soil treatments (i.e., soils with different levels of contamination) can be assigned to compartments in a random, alternating or stratified pattern. A control treatment (soil with no contamination) is always included. Ten earthworms are added to the test unit one at a time in order to avoid a potential “clustering” effect. The compartment entered by each worm is recorded at the beginning of the test (e.g., t = 0 hours). The test temperature is controlled at 20 ± 3° C. After 72 hours, at the end of the test, compartments are isolated by inserting metal sheets to prevent worms from moving among compartments. The distribution of the earthworms among the soil treatments is recorded.

Figure 1. Avoidance-response test unit constructed of stainless steel.

The experimental design of the avoidance behaviour test has been under development for the last five years, and, depending on the design, the results have been analyzed either using Chi-square distribution analysis or, more recently, by estimating a 50% avoidance effect concentration (EC50). An EC50 for avoidance is the concentration of soil that causes an avoidance response in 50% of the worm test population.

The relationship between earthworm avoidance and adverse effects on reproduction was directly compared using the earthworm species Eisenia andrei, or compost worm, a commonly used invertebrate species for soil toxicity assessments. The contaminants assessed included the fungicide benomyl, copper sulphate, crude oil, amines, and condensates.

For each contaminant, the highest concentration at which E. andrei did not display a significant avoidance response (“avoidance threshold”) was compared with the EC20s or NOAECs determined for each of the five reproductive endpoints. EC20s are soil concentrations at which there is a 20% reduction in the reproduction endpoint relative to the control value, and NOAECs are no-observable-adverse-effect concentrations. The results illustrate how closely related the avoidance- response test results are to those of the reproduction tests. It is important to note that the concentrations did not cause direct mortality to adult worms in either acute or chronic tests.

The practical significance of the relationship between earthworm avoidance and reproduction is that the sensitive avoidance-response test can be used as a quick and economical substitute for reproduction tests in the initial screening of contaminated site soils. One significant limitation with application of the avoidance–response test method, however, is the fact that the relationship between earthworm avoidance and effects on reproduction must be empirically determined until a large enough database of different types of contaminants and soil types is sufficiently large to minimize uncertainty.

ESG International, Inc., recently represented Environment Canada at the International Standards Organisation (ISO) Meetings on Soil Quality in Olso, Norway. Researchers in Germany have also developed an earthworm avoidance–response test method that is quite different in design from the Environment Canada method. Both the Canadian and German methods are scheduled by the ISO Technical Committee 190 (Soil Quality) to become new international test methods. The Environment Canada earthworm acute, avoidance and reproduction test methods are currently undergoing scientific review and should be publicly available sometime in 2003.

Natalie Feisthauer is Manager of the Soil Ecotoxicity Laboratory at ESG International Inc.

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