C. H. Högger, Swiss Federal Research Station for Agroecology and Agriculture, Reckenholz, CH-8046 Zürich, Switzerland, chhoegger@gmx.net

TUBIFEX TEST

The Tubifex Test according to Ammon (1985) is a highly sensitive, but simple and inexpensive laboratory test with the annelid Tubifex tubifex Müll. for pesticides and other substances, which may escape to the environment. It provides maximum exposure in a water-soil medium. It determines a general toxicity level (LC50) of the whole formulation including admixtures, if any.

Materials and Methods of the Tubifex Test

Three milliliters of an 1:1 sand: sandy loam mixture are put into 14 ml pill vials and 8 ml of the test solution are added. Generally the following dilution series is used: 0.1, 0.32, 1.0, 3.2, 10, 32, 100, 320, and 1000 pprn active ingredient. Granular materials are ground in a mortar and pestle before making an appropriate suspension. Some organic substances which are insoluble in water are first dissolved in a small amount of acetone. Dechlorinated tap water is used for dilutions. The vial is shaken and the contents are again allowed to settle for 1 h. Five Tubifex tubifex,  bought at a pet store or from laboratory cultures (Kosiorek, 1974, Marian et al., 1989), are added to each vial. Four replications per concentration are prepared. The test is conducted at room temperature. Routinely the live worms are counted after 2 and 7 days. For formulations and compounds like benomyl which are transformed by the soil microflora to more toxic metabolites, the test may last two to three weeks. The percentage of surviving worms is plotted against the concentrations (on a log scale) and the LC50 is determined graphically. The test is repeated at least once. New batches of worms are tested with a series of concentrations of a standard substance with a known LC50.
 

FUNNEL TEST

The Daniel Funnel Test according to Bieri et al. (1989) is a behavioral test and measures the effect of pesticides on the activity of agriculturally important, vertically tunneling (anécique) species such as Lumbricus terrestris L. in a laboratory setup, which simulates field conditions.

Materials and Methods of the Daniel Funnel Test

L. terrestris is collected from permanent grassland after extraction with a 0.3% mustard flour suspension in water (Högger, 1993). The worms are kept in aerated water for two days before further use. Polyethylene funnels of 12 cm diameter are fitted with a silicon rubber tube of 1.2 cm diam. and 30 cm length. The end of the tube is closed with a plug. The tube and the stem of the funnel are darkened with a removable black polyvinylchloride pipe. The funnel is filled with about 400 grams non-sterile moist field soil with known physical and chemical properties, preferably from the same location as the worms. Pesticides may be mixed evenly into the soil before filling it into the funnel. With a screwdriver the soil is pierced vertically to make an artificial earthworm tunnel. When a juvenile L. terrestris of about 2 grams (1.5-2.5 g) weight is added, it retreats into the hole. Five wheat kernels, small clover leaves or bait granules are put on the surface of the soil in each funnel. To simulate exposures in the field more closely, spray or granulate treatments are applied only to the surface at normal field dosage or a range of dosages. Each treatment is replicated 20 times. The funnel is covered with a mesh screen to prevent the escape of the worms. The funnels are kept in a growth chamber at 12oC for 21 days. After 4, 6, 8, 14, and 21 days the numbers of moved and withdrawn wheat kernels are recorded. The location of the worm is also recorded as either in the soil, about in the middle of the tube or at the bottom of the tube. After 21 days the worms are weighed again and the number of dead worms is counted, if any. The worms are watered in a shallow container for 48 h. Then they are weighed again. The numbers of moved and disappeared wheat kernels, the position of the worms are plotted for each day as well as the weight changes of the worms at the end of the experiment (Högger et al., 1992).
Bieri (1992) described a more standardized version of the funnel test using china clay as test medium.
 

FIELDTEST

The trial is performed in plots of 10 x 10 m on grassland with the intent to have a high population of earthworms available in a natural environment (Edwards and Brown, 1982) according to international usage. Alternatively, tests may be performed in well-settled crop fields. Earthworm extraction with expellants works better four to six weeks after a tillage operation.

Materials and Methods in the Field Test

Earthworm populations are determined in each plot shortly before pesticide applications by expelling them with three applications of five litres each of 0. 1 % formaldehyde solution or 0.3 % mustard flour suspension in metal rings of 1/4 m² area (56 cm diam. and 15 cm height) and which are pressed into the soil about 5 cm with a strong plywood (Högger, 1993). There are three sampling units per plot. The appearing earthworms are collected in water, identified, counted and weighed. Pesticides are applied at their normal application time and rate for field crops and four times this amount, preferably shortly before rainy weather is expected. Untreated plots serve as control. A toxic standard, e.g. DNOC at 20 litres/ha in 400 L water, serves as a second control to see whether an effect of the test pesticides may be observable. If no rain follows the application of pesticides, overhead irrigation may be useful. Dead worms should appear at least in the toxic control on the following morning. Three to four weeks and three to four months after the application earthworms are expelled again in three rings per plot. Each treatment is replicated at least twice, which requires 1600 m² per experiment in which 3 pesticides are tested. To avoid measuring any effects of migration, plots of 20 x 20 m may be necessary. But, this may often be considered impractical.

REFERENCES:

Ammon, H.U. 1985. Worm toxicity-test using Tubifex tubifex . FAO Workshop. Versailles. Les Colloques de I'INRA
    31: 303-317.
Bieri, M. 1992. Guidelines for the Daniel funnel test - a laboratory test to measure side effects of pesticides on the
     earth-worm Lumbricus terrestris L.  IOBC wprs Bulletin 1992/XV/3: 139-144.
Bieri, M., Schweizer, H., Christensen, K. and Daniel, 0. 1989. The effect of Metaldehyde and Methiocarb slug
    pellets on Lumbricus terrestris. p. 237-244 in: Henderson, I. (Ed.) Slugs and Snails in World Agriculture. BCPC
    Monograph No. 41.
Edwards, P.J. and Brown, S.M. 1982. Use of grassland plots to study the effect of pesticides on earthworms.
    Pedobiologia 24:145-150.
Hogger C.H. & Ammon H.U. 1994. Testing the toxicity of pesticides to earthworms in laboratory and field tests.
    IOBC wprs Bull. 17 (10): 157-178.
Hogger, C.H. 1993. Mustard flour instead of formalin for the extraction of earthworms in the field.
    Bull. Bodenkundl. Ges. Schweiz 17: 5-8.
Hogger, C.H., Ammon, H.U. and Bieri, M. (1992) The Daniel funnel test in a sequence of earthworm tests of
    agricultural pesticides. p. 230-224 in: Greig-Smith et al. Ecotoxicology of Earthworms. Intercept, Andover. 269 p.
Kosiorek, D. 1974. Development cycle of Tubifex tubifex Müll. in experimental culture. Pol. Arch. Hydrobiol.
    21: 411-422.
Lehmann, F.E. 1941. Die Zucht von Tubifex für Laboratoriumszwecke. Rev. Suisse Zoologie 48 (20): 559-561.
Marian, MY, Chandran, S. and Pandian, T.J. 1989. A rack culture system for Tubifex tubifex.  Aquacultural
    Engineering 8: 329-337.