INVERTEBRATE TESTS

ERDC TN-DOER-C22

September 2001

were not entered because the program's database did not contain information on vanadium. The

PUP procedure predicted shoot metal concentrations that were outside the currently measured range.

Discrepancies were attributed to the use of a highly organic soil instead of a reference soil with the

same characteristics as the test soil (Folsom, Davis, and Houghton 1988; Folsom and Price 1989;

Folsom and Houck 1990), and to the procedure's assumed linear relationship between the total metal

concentration in the soil and in the plant shoots.

INVERTEBRATE TESTS:

Experimental Design, Animal Material, Culture Conditions, and Analyses. Three

separate experiments were conducted: two using the lumbricid compost worm Eisenia fetida and

one using the enchytraeid worm Enchytraeus crypticus as a test organism. E. fetida is the standard

test organism used in terrestrial ecotoxicology and is widely used in North America and Western

Europe. The latter worm has an epigean, litter-dwelling life strategy, and reproduces via cocoons.

E. crypticus is an organism whose potential use as a bioassay organism for ecotoxicological testing

within North America and Western Europe is being explored. It is believed to feed on decomposing

plant material and associated microorganisms. The animals were surface-fed with the same food

as used for E. fetida. Toxicological effects in these test species stem largely from direct skin contact

with the toxic compounds in the interstitial water (Lokke and Van Gestel 1998).

Experiment I. A bifactorial experiment, using a completely randomized design with four replicates

per treatment, was conducted with the following treatments: (1) substrate type, and (2) test duration.

Four durations were tested, 12, 28, 42, and 56 days. The experiment was started on 14 July 2000,

with 20 adult E. fetida specimens per replicate cylinder (15-cm-diameter, 15-cm-high Plexiglas;

1,040 g soil fresh weight per cylinder), and four replicates per treatment. Testing occurred at 20 oC

under continuous fluorescent illumination, as described in American Society for Testing and

Materials (ASTM) (1998). E. fetida specimens were taken from the Environmental Laboratory

culture, originally purchased from Carolina Biological Supply Company, Burlington, NC. Food

was supplied regularly as needed. It was composed of rolled oats, purchased locally, and

powered earthworm food from Magic Products, Inc., Amherst Junction, WI. Each harvest

time, >12 specimens were removed from their cylinders, weighed, and deep-frozen until further

processing. Subsequently, the animals were dried in an oven to constant weight (24 hr at 70

oC). Growth response was expressed in g dry weight per 20 individuals, and reproductive potential

as number of cocoons per cylinder. Metal concentrations were determined in all samples by digesting

0.5 g dry animal material in a microwave oven for lead, vanadium, nickel, and zinc (USEPA Method

3050 (USEPA 1996)), or cold-vapor determination for mercury (USEPA Method 7471A (USEPA

1994)).

Experiment II. This experiment was similar to the former one, except that it was started with

12 juvenile E. fetida specimens to explore the youngest possible age at which toxic effects became

apparent. It lasted 65 days. Animal biomass and reproductive potential were determined, but

bioaccumulation was not measured.

Experiment III. This experiment was performed to explore culture conditions and culture duration

required to harvest sufficient biomass for analysis using a test organism new to the Environmental

Laboratory. Plastic petri dishes were filled with a preweighed quantity of substrate to be tested, and